key: cord- -q m srvp authors: nakagawa, pablo; gomez, javier; grobe, justin l.; sigmund, curt d. title: the renin-angiotensin system in the central nervous system and its role in blood pressure regulation date: - - journal: curr hypertens rep doi: . /s - - - sha: doc_id: cord_uid: q m srvp purpose of the review: the main goal of this article is to discuss how the development of state-of-the-art technology has made it possible to address fundamental questions related to how the renin-angiotensin system (ras) operates within the brain from the neurophysiological and molecular perspective. recent findings: the existence of the brain ras remains surprisingly controversial. new sensitive in situ hybridization techniques and novel transgenic animals expressing reporter genes have provided pivotal information of the expression of ras genes within the brain. we discuss studies using genetically engineered animals combined with targeted viral microinjections to study molecular mechanisms implicated in the regulation of the brain ras. we also discuss novel drugs targeting the brain ras that have shown promising results in clinical studies and trials. summary: over the last years, several new physiological roles of the brain ras have been identified. in the coming years, efforts to incorporate cutting-edge technologies such as optogenetics, chemogenetics, and single-cell rna sequencing will lead to dramatic advances in our full understanding of how the brain ras operates at molecular and neurophysiological levels. the physiological relevance of the renin angiotensin system (ras) in blood pressure regulation and electrolyte homeostasis is well established and undisputable. the ras is traditionally described as a hormone system, which promotes arterial blood pressure elevation primarily by inducing vasoconstriction, sodium retention, and aldosterone release. the sustained overactivation of the ras could lead to hypertension, a disease affecting almost half of us american adults [ ] . the activation of the endocrine ras is initiated upon the release of renin from juxtaglomerular cell granules into the circulation. by catalyzing the cleavage of angiotensinogen to release angiotensin i peptide, renin acts as the rate limiting enzyme of the ras, at least in humans. thus, it is not surprising that there are a number of complex mechanisms regulating renin expression and secretion [ ] . the subsequent conversion of angiotensin (ang)-i to ang-ii is catalyzed by angiotensin converting enzyme (ace) which is localized to endothelial cells and is abundant in the lungs. most of the functions inducing blood pressure elevation are mediated through binding of ang-ii to angiotensin type receptor (at r), whereas, binding of ang-ii to angiotensin type ii receptors (at r) has been reported to generally oppose the actions of at r. other peptides of the ras, such as ang-( - ) and alamandine, also act to counter regulate the action of ang-ii at at r [ , ] . drugs targeting the ras are effective as treatments for hypertension and other diseases including heart failure, chronic kidney disease, diabetic nephropathy, marfan's syndrome, and some autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] . however, it is unclear why these drugs are effective even in patients exhibiting low or normal circulating renin activity [ , ] . the answer to this observation may lie in the existence of an independent autocrine/paracrine ras acting locally within several tissues, including the brain. the existence of the brain ras, which was initially proposed by bickerton and buckley in , has changed the traditional view of the ras [ ] . since the discovery that central ang-ii induces a potent pressor response, several new functions of the brain ras have been identified. central administration of ang-ii elicits potent dipsogenic responses, induces sodium intake, triggers sympathetic outflow to the kidney and other organs, and recently, evidence has established that the brain ras modulates metabolic function primarily through distinct nuclei within the hypothalamus [ ] [ ] [ ] [ ] . most of these effects can be attenuated by administration of ras blockers or by genetically ablating at r in specific brain regions or cell types [ ] [ ] [ ] . resistant hypertension, in which high blood pressure remains above / mmhg despite use of or more antihypertensive drugs (including a diuretic), accounts for approximately % of patients with essential hypertension [ ] . resistant hypertension and sympathetic overactivity have been linked to brain ras overactivation [ ] . thus, novel drugs targeting the brain ras might be useful to treat resistant hypertension and/or diseases associated with elevated sympathetic outflow such as heart failure [ ] . this article aims to bring the reader up-to-date on the important new findings and the currently controversial topics in the field. then, novel translatable strategies to attenuate the upregulation of brain ras activity in human resistant hypertension will be also discussed. although more than years of research supports the important role of the brain ras in modulating several physiological functions, it is not completely clear how angiotensin peptides are generated within the central nervous system (cns). there is extensive evidence indicating that angiotensinogen is highly expressed in astrocytes and in some specific populations of neurons, which suggests that the extracellular space of the cns has abundant renin substrate [ , [ ] [ ] [ ] [ ] [ ] . the distribution of the two main ang-ii receptors, at r and at r, was mapped initially by autoradiography and subsequently confirmed by either in situ hybridization or utilizing transgenic mice expressing reporter genes under the control of either at r or at r promoters [ , , •] . at r is highly expressed in most of the circumventricular organs such as the subfornical organ, the organum vasculosum laminae terminalis (ovlt), and the area postrema. however, the elevated expression of at r in some regions behind the bloodbrain barrier such as the paraventricular nucleus of the hypothalamus (pvn), the nucleus tractus solitarius (nts), the rostral and caudal ventrolateral medulla (rvlm and cvlm, respectively), the medial preoptic nucleus (mnpo), and some neurons within the arcuate nucleus (arc) suggests a role for ang-ii as a neuromodulator. expression of at r in the brain predominates over at r expression during fetal development [ ] . however, recent studies from de kloet et al. utilizing bacterial artificial chromosome transgenic at r-enhanced green fluorescent protein (egfp) reporter mouse confirmed the presence of at r in adult brains particularly in neurons and/or fiber terminals in circumventricular organs, hypothalamic nuclei, and the hindbrain [ ] . although most of the components of the ras have been identified in the brain, the lack of a reliable method to detect renin in small neuroanatomical structures had led some to question whether the central generation of ang-ii requires renin. indeed, renin-independent ang-ii generating biochemical pathways involving tonin and cathepsin d have been proposed [ ] [ ] [ ] [ ] . others have performed experiments which they interpret as a refutation of the brain ras [ ] . the difficulties in measuring renin stem from several factors. first, considering that renin is the rate-limiting enzyme for the generation of ang-i, and given the elevated bioavailability of angiotensinogen in the extracellular space of the brain, it is expected renin must be tightly controlled and secreted from specific cells within selected neuroanatomical nuclei. supporting this, minimal elevation of ang-ii levels in discrete neuroanatomical regions is expected to elicit extremely profound effects. indeed, evidence indicates that ablation or stimulation of angiotensinergic signaling within a few cells in the brain leads to extremely prominent cardiovascular and metabolic phenotypes [ ] [ ] [ ] . second, we and others have described the existence of an alternative renin isoform termed renin-b, which is the predominant transcribed renin isoform in the brain, but is absent in other tissues [ , ] . renin-b lacks the signal peptide and the first third of the pro-segment which implies that ( ) renin-b protein is catalytically active and ( ) is predicted to remain in an intracellular compartment [ ] . this observation was particularly compelling as an intracrine ras had been previously proposed [ , ] . notably, evidence that ang-ii can be generated intracellularly in presynaptic neurons and subsequently released to the synaptic terminal upon presynaptic depolarization would provide a strong argument to define ang-ii as a neurotransmitter. to test the hypothesis that renin-b is involved in the generation of intracellular ang-ii in the brain, we generated transgenic mice lacking the alternative renin-b, while renin-a was preserved [ ] . paradoxically, mice lacking renin-b exhibited a mild increase in blood pressure during the light cycle, which was attenuated by intracerebroventricular infusions of at r blockers or ace inhibitors [ •] . these data indicate that intracellular renin-b might play a regulatory role on the brain ras rather than generating intracellular ang-ii as initially proposed [ ] . even though our data do not support the existence of an intracellular ang-ii generating mechanism, they neither disprove it. the brain is one of the most vascularized systems because it receives . - . % of total cardiac output; thus, distinguishing renin levels generated within brain tissues (autocrine/paracrine) from the circulating renin (endocrine) is extremely challenging [ ] . despite evidence that renin activity and ang-ii have been identified in the cerebrospinal fluid (csf) and brain tissues from nephrectomized animals, those who resist the existence of the brain ras postulated that samples must be contaminated with traces of trapped blood [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . following this rationale, van thiel and colleagues attempted to provide evidence that the brain lacks the capacity to generate ang-ii by measuring renin activity (angiotensin-generating activity) in brain structures before and after organ buffer perfusion [ ] . it is not surprising that there was a significant decrease in renin activity within several brain structures after buffer perfusion because ( ) the brain has blood, and ( ) neurons unlikely store renin within granules as do specialized renal juxtaglomerular cells. indeed, the most direct and accurate conclusion from this study was that the brain indeed contains blood. it is surprising the authors decided to ignore their own data that renin activity remained at detectable levels in some buffer-perfused tissues including the brainstem. this is particularly interesting because in a double-transgenic mouse in which enhanced green fluorescence protein (gfp) is expressed under the control of the renin promoter and β-galactosidase is controlled by the angiotensinogen promoter, we identified unique reninexpressing cells in close proximity to angiotensinogenexpressing cells specifically within the rvlm, a brainstem nucleus [ , ] . it is of utmost importance that one contrasts a classical endocrine system and views the brain ras as a neuroendocrine, autacoid, or neurotransmitter system where single neurons or small collections of neurons mediate output from the cns. thus, cellular, neuroanatomical, and molecular specificities are the key aspects to be considered when neuroendocrine systems, such as the brain ras, are studied. it remains unfortunate that equivocal interpretations from experiments lacking sensitivity and neuroanatomical selectivity, those which are not required when studying the classical ras, re-emerge from time to time to question the existence of the brain ras [ ] . in the final section of this article, we will discuss how the development of state-of-the-art technology to study precise molecular signaling and neuronal circuits within the cns are appropriate to elucidate the key mechanisms regulating generation and action of angiotensin peptides within different neuroanatomical regions. the proteolytic activation of prorenin that normally occurs in secretory granules in renal jg cells is unlikely to occur in extrarenal tissues, including the brain [ ] . thus, it has been proposed that activation of renin in extrarenal tissues requires its binding to atpase h(+)-transporting lysosomal accessory protein , also known as the prorenin receptor (prr), encoded by the atp ap gene. prr has the unique capability to bind prorenin and induce its activation without prosegment removal [ ] . prr is highly expressed in neurons and some microglia cells and has been detected in several brain regions implicated in cardiovascular and autonomic control including the subfornical organ (sfo), paraventricular nucleus (pvn), nucleus of the solitary tract (nts), and the rostral ventrolateral medulla (rvlm) [ •, ] . despite initial reports indicating that prr might be involved in hypertension through local generation of ang-ii, other roles that are independent of ras activation have been described (reviewed in [ ] ). for instance, the transmembrane domain of the prr interacts with the vacuolar h(+)-atpase and plays an important role in lysosomal function and neuronal development [ ] . in humans, a unique mutation (c. c>t, p.d d) in the prr locus is associated with x-linked mental retardation and epilepsy [ ] . although many ras-independent functions of prr are well defined in kidney, the ras-independent functions of prr in the brain are not completely understood [ , , [ ] [ ] [ ] . in mice where neuronal prr was ablated at embryonic daỹ . (neuron filament promoter-cre crossed with prrfloxed mice; nefh-prrko), central generation of ang-ii in response to intracerebroventricular administration of recombinant prorenin was attenuated. to study the role of neuronal prr in the pathogenesis of hypertension, feng's lab utilized the model of low-renin hypertension induced by deoxycorticosterone acetate (doca) infusion and high dietary sodium. intracerebroventricular infusion of ace inhibitor prevents and reverses high blood pressure, demonstrating that production of ang-ii in the brain is required for doca-salt hypertension even though circulating ras activity is suppressed [ ] . moreover, two separate studies have shown that central infusion of an at r blocker mimics the effects of central delivery of ace inhibitors [ , ] . thus, it is well accepted that the blood pressure elevation in doca-salt hypertension is strongly driven by a neurogenic mechanism involving activation of the brain ras [ ] . li et al. reported that the selective ablation of prr in neurons attenuated the elevation of blood pressure in doca-salt hypertension when . m nacl was the only fluid offered [ ] . in contrast, experiments where doca-treated nefh-prrko mice were exposed to two-bottle choice paradigm for the assessment of sodium preference revealed that the ablation of prr in neurons is insufficient to decrease blood pressure but suppressed doca-induced saline intake [ ] . these data suggest expression of prr in specific brain regions controlling drinking behavior, such as the sfo is of physiological relevance. recently, souza and colleagues examined the role of prr in the pvn, a key integratory nucleus involved in blood pressure control [ ] . in this study, pvntargeted ablation of prr was induced in prr-floxed mice by bilateral stereotactic microinjection of adeno-associated virus (aav)-cre-gfp. the reduction of prr expression in the pvn by less than % was sufficient to attenuate doca-salt induced blood pressure elevations, cardiac and vasomotor sympathetic over-activity, and improved cardiac parasympathetic function [ • ]. in addition, live-cell calcium recordings utilizing a novel calcium biosensor (gcamp ) revealed pvntargeted ablation of prr attenuates calcium influx in response to ang-ii in doca-salt hypertension. despite the growing evidence indicating the importance of prr in the cns to control cardiovascular function, there is no generalized consensus whether the underlying mechanisms require local ang-ii generation. it is likely that both ang-ii-dependent and ang-ii-independent mechanisms might occur simultaneously at different degrees depending on the neuroanatomical localization and cell types, as well as different physiological and pathological circumstances [ ] . there is a growing interest in the soluble prr fragment (sprr) which arises from the proteolytic cleavage of prr by furin or site- protease to generate a -kd transmembrane/ cytoplasmic fragment and a -kd soluble prr form [ , ] . growing evidence supports that elevation of circulating sprr levels are associated with high blood pressure, chronic kidney disease, preeclampsia, and obstructive sleep apnea [ ] [ ] [ ] [ ] . however, the biological function and the physiological relevance of sprr were completely unknown until recently. many functions of sprr controlling renal function have been reported. for instance, sprr exerts antidiuretic actions in part by inducing frizzled -dependent stimulation of aquaporin expression in the collecting duct [ ] . studies specifically aiming to elucidate the role of sprr in the cns have not been reported. recently, gatineau et al. demonstrated that the selective deletion of adipose tissue prr elevates systolic blood pressure concomitant with increased circulating sprr levels in high fat diet-fed mice [ •] . in males, systemic infusion of recombinant mouse epitope tagged sprr resulted in blood pressure elevation and this increase was attenuated by ganglionic blockade, but not administration of at r blockers, indicating that autonomic dysfunction, but not circulating ras overactivation, is a key mechanism underlying sprr-mediated blood pressure elevation in obese male mice. in contrast, infusion of sprr in females failed to induce autonomic dysfunction but it induced elevated vasopressin levels and plasma renin indicating the existence of sex differences in sprr-mediated responses [ ] . given that the source of vasopressin is exclusively from the pvn and supraoptic nucleus (son), these observations support that sprr is biologically active in the cns and elevations of sprr in the brain might be implicated in certain forms of neurogenic hypertension linked to obesity. it has been proposed that many of the effects of ang-( - ) in the cns oppose many of the at r-mediated actions of ang-ii in the brain [ ] . ang-( - ) induces its effects mainly through mas receptor activation, although it has been reported to also act through at r [ ] . other studies proposed that mas receptors form heterodimers with at r in astrocytes [ ] . teixeira et al. recently reported that ang-( - ) binds to at r but fails to engage its canonical g protein signaling [ ] . instead, ang-( - ) triggers β-arrestin recruitment and intracellular signaling, suggesting it may have the properties of a biased agonist for at r. several synthetic at r biased agonists have been designed, but the existence of an endogenous functional biased ligand for at r has not been reported. ang-( - ) can be generated from the direct cleavage of ang-ii by angiotensin converting enzyme (ace ), but ace can also catalyze the conversion of angiotensin i to angiotensin-( - ), which subsequently is converted to ang-( - ) by ace or neutral endopeptidase [ ] . downregulation of ace and suppression of central ang-( - ) levels are thought to be one of the underlying mechanisms causing low renin hypertension [ , ] . in recent years, significant progress has been achieved particularly on the molecular mechanisms controlling brain ace activity. lambert et al. described a process termed "ace shedding" in which a disintegrin and metalloprotease (adam ) catalyze the cleavage of membrane anchored ace [ ] . however, advances on the physiological and pathophysiological role of adam -mediated ace shedding in the brain have only been recently demonstrated. xia and sriramula et al. demonstrated that neuron-targeted overexpression of ace is sufficient to ameliorate elevated blood pressure, autonomic dysfunction, and vasopressin release in response to doca-salt hypertension [ ] . moreover, braintargeted ablation of adam utilizing central infusions of sirna suppressed doca-salt induced hypertension concomitant with blunted reduction of ace activity in the hypothalamus and cerebrospinal fluid, indicating that ace shedding by adam in the brain is a relevant mechanism contributing to neurogenic hypertension. new evidence indicates that activation of at r is required for adam -mediated ace shedding possibly via reactive oxygen species and phosphorylation of extracellular signal-regulated kinase in neurons [ ] . it has been previously shown that a reduction of ace expression in the rvlm is a contributing factor in the development of hypertension in spontaneously hypertensive rats [ ] . mukerjee et al. provided evidence of the importance of ace /adam pathway in pre-sympathetic neurons within the pvn [ •] . interestingly, ace is expressed in gabaergic inhibitory neurons projecting onto the hypothalamus. thus, ace is thought to maintain a normal gabaergic inhibitory tone to the presympathetic neurons in the pvn in normal physiological conditions, while disinhibition of this pathway might lead to hypertension. in contrast, adam is expressed in single-minded family basic helixloop helix transcription factor (sim )-positive excitatory neurons within the pvn and promotes excitatory activity. ablation of adam in the pvn blunts pressor responses to acute pvn-targeted microinjection of ang-ii. this accumulating evidence suggests the importance of the opposing roles of ace and adam in modulating of sympathetic activity and central control of blood pressure. finally, it has been recognized that adam has a role in processing the proinflammatory cytokine, tumor necrosis factor alpha (tnf-α). indeed, upregulation of the brain adam was associated with elevated tnf-α implicating that attenuation of tnf-α-related mechanisms could be mediating part of the phenotype observed in mice lacking hypothalamic adam . numerous reports suggest elevation of tnf-α and activation of inflammatory cells (microglia) within certain brain regions triggers hypertensive responses [ ] [ ] [ ] . therefore, future studies are expected to clarify whether tnf-α plays a regulatory role in ace activity and other ras components. a quarter of hypertensive patients exhibit low-renin hypertension [ ] . it has been suggested that low-renin hypertension is in part driven by elevated angiotensinergic signaling in the brain [ , ] . therefore, the development of novel drugs modulating the brain ras might represent an effective solution to treat resistant hypertension coincident with elevated sympathetic activity and suppressed circulating renin activity. two decades ago, llorens-cortes's laboratory demonstrated that the conversion of ang-ii into angiotensin iii (ang-iii) in the brain is catalyzed by aminopeptidase a (apa), a zinc metalloprotease [ ] . importantly, ang-iii has been hypothesized to be the major biologically active peptide of the brain ras. this is based on the observation that inhibition of the brain apa completely prevents elevated blood pressure in models of neurogenic hypertension with elevated brain ras [ ] . it took years to translate the observation that pharmacological inhibition of apa can be used as a therapeutic tool to treat resistant hypertension in humans. recently, a new multicenter, open-label, phase ii study was released evidencing the efficacy of a brain penetrating inhibitor of apa, firibastat (previously named rb ), in reducing blood pressure in overweight patients of multiple ethnic origins without angioedema [ •] . moreover, an additional pilot double-blinded randomized placebo-controlled study in hypertensive patients demonstrated that blood pressure in firibastat-treated patients trended to be decreased compared to placebo controls without affecting systemic ras activity [ ] . firibastat is the first oral medication that may target the brain ras with promising clinical application. current efforts in designing new brainpenetrating apa inhibitors led to a -fold more potent new prodrug, ni /qgc , which has been shown to exert powerful antihypertensive effects in rats treated with doca-salt [ ] . in recent years, multiple pleiotropic roles of the brain ras, namely neuroinflammation, autophagy, er stress, and mitochondrial dysfunction, have emerged. these findings resulted in considerable advances in utilizing brain ras blockade or ras modulation as a therapeutic strategy to treat diseases beyond neurogenic hypertension. this is specifically relevant in stroke and cerebrovascular diseases, alzheimer's disease, cognitive dysfunction, parkinson disease, aging, and others [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . numerous other new molecules or administration routes to target the brain ras are currently under investigation. for instance, new classes of at r biased agonists, which can selectively activate β-arrestin without activating the classical g protein-coupled signaling, might represent potential tools to modulate angiotensin signaling within the brain. for example, carvalho-galvão et al. demonstrated that intracerebroventricular infusion of trv , a β-arrestin-biased at r-agonist effectively attenuated autonomic dysfunction and decreased arterial pressure in spontaneously hypertensive rats (shr) [ ] . thus, the development of brain-penetrating at r-biased agonists would be a promising strategy to treat resistant hypertension. finally, torika et al. demonstrated that intranasal administration of telmisartan is effective at reducing brain inflammation and ameliorating the progression of alzheimer's disease in mice indicating that novel routes of administration can also be employed to inhibit the brain ras without systemic off targets effects [ ] . the development of several cutting-edge technologies to study the cns predicts that we might witness a profound advance in this field in the near future. several laboratories are currently utilizing novel in situ hybridization techniques with significantly higher specificity and sensitivity. these are powerful tools to identify the anatomical and cellular localization of cells expressing components of the ras within the cns and to query the molecular and/or neural significance of these cells by multiplexing with different probes. we used rnascope® technology to confirm the abundance of agt and the presence of at r and prr in discrete cells within the brain. using this same technique, we and others have identified the distribution of ras genes in specific neun+ neurons, gfap+ astrocytes, and iba- + microglia cells in the sfo, pvn, arc, and the rvlm (unpublished). in addition, the development of an extensive array of transgenic mice carrying conditional alleles of ras genes as well as mice expressing tamoxifen-inducible and/or cell-specific cre recombinase expression facilitates further exploration of novel molecular and physiological functions of the ras within specific cell types within the brain. novel and previously generated animals expressing cre recombinase under the control of specific promotors including at r-cre or ren -cre mice, which can be crossed with mice expressing cre-dependent tdtomato (or other reporter genes) as well as mice expressing a fluorescent reporter gene under the control of ras genes such as at r-egfp or at a r-egfp (nz ), allow fluorescent labelling of cells expressing ras genes [ , •, , ] . significant progress has been made in developing techniques to identify the distinct roles of specific neurons in the brain. these techniques include optogenetics and designer receptors exclusively activated by designer drugs (dreadd), a chemogenetic technique, in which neuronal activity can be stimulated or suppressed in specific brain nuclei utilizing light or designer drugs, respectively. using these techniques, several laboratories recently showed the distinct roles of specific cells within selected nuclei controlling cardiovascular, metabolic, and autonomic function. for instance, de kloet el al. recently reported that optogenetic stimulation of at r expressing neurons in the pvn promotes blood pressure elevation and activation of the hypothalamic-pituitary-adrenal axis [ •] . similarly, nation et al. utilized dreadds to study the role of sfo neurons in thirst and salt appetite [ •] . stimulation of neuronal firing and activation of gq signaling in mice receiving sfo-targeted microinjection of a virus (aav -camkii-ha-hm d(gq)-ires-mcitrine) to induce selective neuronal expression of gαq via a designer receptor (hm d) that is exclusively activated by clozapine n-oxide, resulted in strong dipsogenic responses and preference to . m saline. the combination of these novel techniques with the array of transgenic mice described above are powerful tools to inquire the responses to stimulating or inactivating different neuronal circuits in the cns. finally, emerging "omics" techniques are becoming more accessible and reliable to study the transcriptome profile in different neuronal populations. there is particular interest in single-cell and single-nuclear rna sequencing technology to identify different clusters expressing ras genes to evaluate the molecular signature of cells. although studies utilizing these techniques to specifically evaluate the brain ras are not yet available, sapouckey recently reported an in silico re-analysis of hypothalamic single-cell rna sequencing datasets revealing that at r is expressed in a specific cluster of neurons expressing both agouti-related peptide (agrp) and leptin receptors [ • ]. this seminal discovery may illuminate the underlying mechanisms by which the brain ras controls resting metabolic rate and sympathetic activity in obesity-related hypertension. although the ras in the brain has been studied for decades, interesting and seminal discoveries continue to be made to this day. these include assessing the functional significance of newly identified components of the ras (such as prorenin receptor), the action of unconventional ras peptides (such as ang iii), enzymes which modify ras components (such as adam ), and new therapeutic tools to combat neurogenic hypertension. new technologies are making it easier to answer old questions-what is the localization of angiotensin receptors-and investigate new ones-what are function of specific subsets of at r-containng neurons? new genomic technologies such as single cell sequencing will provide novel platforms to understand the diversity of neuronal types which respond to ras activation or mediate downstream ras signaling. conflict of interest the authors declare no conflicts of interest relevant to this manuscript. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance heart disease and stroke statistics- update: a report from the classical renin-angiotensin system in kidney physiology angiotensin-( - ) counterregulates angiotensin ii signaling in human endothelial cells discovery and characterization of alamandine: a novel component of the renin-angiotensin system the use of renin-angiotensin system inhibitors in patients with chronic kidney disease heart failure drug treatment treatment of diabetic nephropathy with angiotensin ii 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alzheimer's disease memory is preserved in older adults taking at receptor blockers cognitive benefits of angiotensin iv and angiotensin-( - ): a systematic review of experimental studies angiotensin ii-and alzheimer-type cardiovascular aging central administration of trv improves baroreflex sensitivity and vascular reactivity in spontaneously hypertensive rats intranasal telmisartan ameliorates brain pathology in five familial alzheimer's disease mice distribution of angiotensin type a receptorcontaining cells in the brains of bacterial artificial chromosome transgenic mice renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened demonstrated that dreadd-mediated stimulation of neurons within the subfornical organ leads to prominent dipsogenic responses and salt appetite in silico re-analysis of hypothalamic single-cell rna sequencing datasets reveals that at r is expressed in a specific subcluster of neurons expressing both agouti-related peptide (agrp) and leptin receptors publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- - ic twhy authors: ding, dan; du, yimei; qiu, zhihua; yan, sen; chen, fen; wang, min; yang, shijun; zhou, yanzhao; hu, xiajun; deng, yihuan; wang, shijia; wang, liangping; zhang, hongrong; wu, hailang; yu, xian; zhou, zihua; liao, yuhua; chen, xiao title: vaccination against type angiotensin receptor prevents streptozotocin-induced diabetic nephropathy date: - - journal: j mol med (berl) doi: . /s - - - sha: doc_id: cord_uid: ic twhy abstract: recently, our group has developed a therapeutic hypertensive vaccine against angiotensin (ang) ii type receptor (at r) named atrqβ- . to explore its potential effectiveness on streptozotocin-induced diabetic nephropathy, male sprague dawley rats were randomly divided into two groups: a control and a diabetic model. after week, the diabetic rats were divided into four subgroups (each with rats) for -week treatments with saline, olmesartan, atrqβ- , and qβ virus-like particle (vlp), respectively. in addition to lower blood pressure, atrqβ- vaccination ameliorated biochemical parameter changes of renal dysfunction, mesangial expansion, and fibrosis through inhibiting oxidative stress, macrophage infiltration, and proinflammatory factor expression. furthermore, atrqβ- vaccination suppressed renal ang ii-at r activation and abrogated the downregulation of angiotensin-converting enzyme -ang ( – ), similar to olmesartan treatment, while no obvious feedback activation of circulating or local renin-angiotensin system (ras) was only observed in vaccine group. in rat mesangial cells, the anti-atr- antibody inhibited high glucose-induced transforming growth factor-β (tgf)-β /smad signal pathway. additionally, no significant immune-mediated damage was detected in vaccinated animals. in conclusion, the atrqβ- vaccine ameliorated streptozotocin-induced diabetic renal injury via modulating two ras axes and inhibiting tgf-β /smad signal pathway, providing a novel, safe, and promising method to treat diabetic nephropathy. key messages: overactivation of ras plays a crucial role in the development of the dn. our aim was to verify the effectiveness of atrqβ- vaccine in stz-induced dn. the atrqβ- modulated two ras axes and inhibited tgf-β /smad signal pathway. the vaccine therapy may provide a novel, safe, and promising method to treat dn. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. diabetic nephropathy (dn) is the most common cause of endstage renal diseases, responsible for over % of all cases in the usa, and this number is likely to increase unabated [ ] . overactivation of the renin-angiotensin system (ras) plays a crucial role in the development of the disease [ ] . current treatment, aimed at slowing progression, concentrates on two inter-related therapeutic strategies: blood pressure reduction and blockade of the ras [ ] [ ] [ ] . such treatments have been shown to reduce the functional changes seen in dn dan ding, yimei du and zhihua qiu contributed equally to this work. electronic supplementary material the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. and also to attenuate the structural abnormalities that characterize this disease [ ] [ ] [ ] . at present, blockade of the ras is achieved by two major drug classes: angiotensin-converting enzyme (ace) inhibitors and angiotensin ii (ang ii) receptor blockers (arbs), agents that also lower systemic blood pressure [ ] . however, they are not completely effective in preventing or reversing the progress of dn, in part due to a compensatory increase in plasma renin activity (pra) or ang ii [ ] . current therapies are insufficient, necessitating the search for new therapeutic strategies in dn. recently, we developed a therapeutic hypertensive vaccine atrqβ- , a peptide (atr- ) derived from human ang ii receptor type (at r) conjugated with qβ bacteriophage virus-like particles, which decreased the blood pressure of hypertensive animals effectively through diminishing the pressure response and inhibiting signal transduction initiated by ang ii with no obvious feedback activation of circulating or local ras [ ] . in our previous study, we demonstrated that an epitope from the rat at r, designated as atr , could decrease sbp of spontaneously hypertensive rats (shrs) and provide excellent protections in target organs [ ] . following our work, hiroshi itoh and his colleagues emphasized vaccination against at r for the prevention of l-name-induced nephropathy in shrs, not only for the attenuation of hypertension [ ] . ang ii and other components of ras also have a central role in the pathogenesis and progression of diabetic renal injuries. accordingly, this study was designed to explore the possibility of the atrqβ- vaccine in ameliorating of experimental dn and the potential mechanisms. the study was undertaken in sprague dawley rats treated with streptozotocin that developed renal injury similarities to human dn [ ] . detailed methods are available in the online supplement. male sprague dawley rats weighing - g were purchased from the experimental animal research center (hubei province, china). all animals were kept in the pathogen-free room in the experimental animal center (tongji medical college of huazhong university of science and technology, wuhan, china), and all experiments were carried out in accordance with guidelines for the care and use of laboratory animals (science and technology department of hubei province, china, ). experimental diabetes was induced by intraperitoneal injection of the β cell toxin streptozocin ( mg/kg) dissolved in fresh sodium citrate buffer (ph . ) following an overnight fast. animals with plasma glucose concentrations in excess of . mmol/l, week postinduction of diabetes, were included in the study. sham-injected control animals (sodium citrate buffer, ph . ) were followed concurrently. diabetes rats were then randomized into four groups (n= ), receiving one of the following treatments: ( ) dn group: equal volume saline injection subcutaneously (s.c); ( ) om group: olmesartan, mg/kg/day via oral gavage; ( ) atrqβ- group: the atrqβ- vaccine, which was immunized s.c μg on days , , and , formulated in aluminum hydroxide gel; ( ) virus-like particle (vlp) group: μg qβ vlp injected as group . each week, rats were weighed and their blood glucose levels were measured. atr- -specific antibody titers were detected in every weeks. every weeks, systolic blood pressure (sbp) was determined in preheated conscious rats via tail-cuff plethysmography using a non-invasive blood pressure controller and powerlab system. the rats were decapitated between am and am. blood samples were collected and divided into two parts: one was mixed with the enzyme inhibitor mixture ( ml blood in μl inhibitor mixture including μl . mol/l edta, μl . mol/l dimercaprol, and μl . mol/l -ohquinoline sulfate) for pra and ang ii concentration measurement by radioimmunoassay (ria) according to the assay kit instruction (nibt, beijing); the other was used for biochemical experiments. for tissue ang ii and ang ( - ) measurement, immediately after harvesting and weighing, the kidney cortex were immersed in ice-cold methanol, minced, and homogenized with tissue homogenizers. the homogenates were centrifuged ( rpm, °c, min), and the supernatants were dried overnight in a vacuum centrifuge. the dried residue was reconstituted in ml ria buffer and then was subjected to hplc to separate ang ii from other substances. the tissue ang ii concentration was detected according to the assay kit instruction (nibt, beijing), and ang ( - ) was measured by hplc. the plasma samples were used for the measurement of glucose, lipid level, creatinine (cr), and blood urea nitrogen (bun). urine samples were collected using metabolic cages, and the supernatant was used for examination of the -h urinary protein. parts of fresh left renal cortex were immediately fixed in . % glutaraldehyde for transmission electron microscopy ( t e m ) . t h e o t h e r p a r t s w e r e f i x e d w i t h % paraformaldehyde overnight, embedded in paraffin for histopathology. sections were stained with hematoxylin-eosin (h&e), masson's trichrome, and periodic acid-schiff (pas). frozen sections were stained with dihydroethidium (dhe). immumohistochemical staining was performed to detect the expression of tgf-β ( : , everest biotech) and macrophages (cd , : , abd serotec) in glomeruli. immunofluorescence staining of nephrin and podocin expression was performed on the paraffin sections of kidneys using monoclonal anti-nephrin ( : , abcam) and anti-podocin antibodies ( : , abcam). rat mesangial cells (rmcs) were cultured in dulbecco's modified eagle's medium supplemented with % fetal bovine serum, u/ml penicillin, and mg/ml streptomycin at °c in % air and % co . cells plated on -mm dishes were cultured to % confluence and divided into five groups: control group, in which cells were incubated in mmol/l d-glucose dmem, and mmol/l d-mannitol was added to the medium in order to take into the account of the effect of high osmolarity in other cell groups; hg group, in which cells were stimulated with a high concentration of glucose ( mmol/l) only for h; los group, in which cells were pretreated with − mol/l losartan for h; anti-atr- group, in which cells were pretreated with anti-atr- for h; anti-natr- group, in which cells were pretreated with anti-natr- for h. all cells except control group received stimulation with a high concentration of glucose ( mmol/l) for h after treatment. cell protein and messenger rna (mrna) were extracted for western blot and qrt-pcr analysis. data were shown as the mean±sem. statistical analyses of the data were performed with one-way anova using spss . . p< . was considered statistically significant. in comparison with control animals, diabetic rats had significantly reduced body weight, which was unaffected by treatment. the ratio of kidney weight/body weight was increased, while atrqβ- vaccine and olmesartan treatments decreased the level. blood glucose and lipid levels were elevated to a similar extent in all diabetic rat groups, irrespective of treatment (table ) . no evidence of skin damages at the site of subcutaneous injection was noted in vaccine-treated animals. less activities and poorer hair were shown in diabetic rats, but the reactivity was the same as normal rats. atrqβ- vaccination effectively reduced blood pressure and reversed biochemical parameters of renal dysfunction in stz-induced diabetic rats to examine antibody production, we measured the amounts of anti-atr- antibodies produced in response to vaccination with atrqβ- . after the second injection of the vaccine, the atr- -specific antibody titer was : , to : , , and it rose after the third injection, then peaked on day ( : , ) and gradually decreased thereafter (fig. a) . to investigate the efficacy in blood pressure, systolic blood pressure (sbp) levels were measured by the tail-cuff method. over the course of the study, sbp was elevated in dn compared to normal rats, while rats immunized with the atrqβ- vaccine were decreased compared to the vlp group, with a maximum decrease of . mmhg ( . + . vs. . + . mmhg, p< . , fig. b) . to evaluate the biochemical parameters of renal function, h urine volume, and total protein excretion, blood urea nitrogen and creatinine were measured. diabetic rats exhibited higher levels in all these parameters. atrqβ- vaccine and olmesartan-treated rats significantly reduced these parameters compared to dn and vlp groups, and no significant differences were between these two groups ( fig. c-f ). to confirm the effect of vaccination, we evaluated renal pathological changes. podocytes form the filtration slit diaphragms that prevents the escape of plasma protein from the glomerular circulation. the disruption of podocytes contributes to proteinuria and further development of glomerular sclerosis [ ] . reduced nephrin and podocin immunostaining in glomeruli were showed in diabetic rats, and atrqβ- vaccination prevented the reduction (fig. ) . in addition to podocyte injury, glomerular mesangial expansion is also a hallmark of dn. extracellular matrix deposition was detected by periodic acid-schiff staining, and glomerular sclerosis index (gsi) was calculated. compared with dn and vlp groups, gsi was significantly decreased in atrqβ- -vaccinated group, similar with olmesartantreated group, which was also confirmed by transmission electron microscopy (tem) (fig. ) . to further investigate the mechanisms, renal fibrosis and inflammation parameters were measured. the extent of interstitial expansion was quantified by masson's trichrome-stained sections. the interstitial expansive index was higher in dn and vlp groups, and atrqβ- vaccination blocked the increase (fig. ) . glomerular and interstitial inflammation was quantified by the cell numbers of macrophages stained positively with cd antibody, and atrqβ- vaccination successfully prevented the increased inflammation (fig. ) . besides, we detected transforming growth factor-β (tgf-β ) expression and reactive oxygen species, which was also decreased by atrqβ- vaccine treatment. further, the mrna expressions of profibrotic and proinflammatory factors in kidney cortex were measured. as shown in fig. f , g, the increased mrna levels in these factors were significantly suppressed by atrqβ- vaccination. there were no significant differences between atrqβ- -and olmesartan-treated groups. atrqβ- vaccination suppressed renal ang ii-at r activation and abrogated the downregulation of ace -ang ( - ) with no feedback of ras to determine whether blockade of at r lead to feedback activation of circulating ras, we detected the pra and ang ii concentration. the pra in atrqβ- vaccinated group was ± pmol/h/l, which had no significant difference with vlp group ( , ± pmol/h/l, p = . ). however, a distinct increase was observed in olmesartan group ( ± pmol/h/l, p= . ; fig. a) . similarly, the plasma concentration of ang ii in olmesartan group was higher than vlp group ( . ± . vs. . ± . pmol/l, p = . ), whereas no significant difference was observed between vlp and vaccine groups ( . ± . vs. . ± . , p= . ; fig. b ). to further examine the local ras, kidney ang ii concentration was measured. the concentration of ang ii in kidneys was lower in olmsartan-and vaccine-treated groups compared to dn and vlp groups (fig. d) . to investigate whether the protection effect was a result of increase ang ( - ) activation with simultaneous ang ii suppression, we subsequently measured plasma and kidney ang ( - ). the decline of ang ( - ) in diabetic rats was improved in atrqβ- vaccine and olmesartantreated groups (fig. c, e) . activation of ras in streptozotocin (stz)-induced diabetic kidneys was further confirmed by western blot and quantitative real-time pcr (fig. ) . the mrna expression of kidney renin in olmesartan group was obviously higher than vlp group (p< . ), while no difference was found between atrqβ- vaccine and vlp groups (fig. g) . ace and at r expressions were increased in diabetic rats, and both atrqβ- vaccine and olmesartan groups attenuated the increasing expressions. ace -ang ( - )-mas axis counteract ang ii-mediated effects in various organs, including the kidneys, and we observed reduced expression of ace -ang ( - )-mas in the diabetic kidneys. atrqβ- vaccine and olmesartan displayed upregulation of ace -ang ( - )-mas expression compared with vlp group. further, we also detected the downstream signal transduction and the activation of erk / , and p mapk phosphorylation was inhibited by atrqβ- vaccination, similar to olmesartan treatment (fig. e-f ). to further explore the mechanisms, cell experiment was undertaken by anti-atr- stimulation. glomerular mesangial cells are believed to be responsible for overproduction of ecm proteins in various pathologic conditions, and tgf-β has been proposed to play an important role. the tgf-β / smad signal pathway regulates the hypertrophic and prosclerotic changes in diabetic renal injury [ ] . anti-atr- and losartan treatment significantly decreased the expression of tgf-β and the phosphorylation levels of smad in rmcs stimulated by high glucose. additionally, we examined the expression of collagen iv and fibronectin, which showed similar change as tgf-β (fig. ) . for safety consideration, normal sd rats were immunized with the atrqβ- vaccine, and the histological changes of kidney were observed by light microscopy and tem. no evidence of skin damages at the site of subcutaneous injection was noted in vaccine-treated animals. compared with control group, no obvious cell proliferation and pathological changes in the mesangial area were shown in vaccine group. also, tem demonstrated that no immune complexes were observed in the basement membrane, and the structure of the glomerulus was intact (fig. ). in this study, we demonstrated for the first time that in a rat model of dn, a vaccine targeting at r named atrqβ- the rat dn model was induced by an intraperitoneal injection of stz that has been widely used to create diabetes models in rodents with pathological features similar to human diabetic nephropathy [ ] . the nephropathy subcommittee of the animal models of diabetic complications consortium (amdcc) has published the following validation criteria for rodent models of dn based on the clinical and pathological features of human dn: ( ) > % decrease in renal function, ( ) > -fold increase in albuminuria, and ( ) pathological features including advanced mesangial matrix expansion (±nodules), thickening of the glomerular basement membrane, arteriolar hyalinosis, and tubulointerstitial fibrosis [ ] . an ideal model of dn would display all of these criteria; however, no current model entirely satisfies them. that is the limitation in animal experiments. as the rodent models share many similarities with human disease, we cannot ignore the potential therapeutic value of the vaccine in the treatment of human diseases. our results showed that rats presented severe hyperglycemia and renal dysfunction manifested as increased plasma creatinine level, bun, h urinary volume, and protein excretion, indicating that a dn animal model was successfully established. the ratio of kidney weight/body weight in diabetic groups was significantly increased compared with that in control group, substantial to the presence of renal hypertrophy in diabetic rats. during the course of the study, systolic blood pressure was elevated in diabetic rats, notwithstanding the limitations of tail-cuff sphygmomanometry, atrqβ- vaccination lowered blood pressure (bp) smoothly, and olmesartan decreased to a greater extend. it is worth noting that our animal experiments showed many times that the antihypertensive effect of atrqβ- vaccine was dependent on the baseline of the bp level and exhibited no decreasing effect in normotensive rats. oppositely, arbs decreased bp irrespective of the basic level and had an increase risk of hypotension. therefore, atrqβ- vaccine may be more practical in diseases where the bp was not very high, such as dn. glomerular mesangial matrix expansion, renal fibrosis, and inflammation are typical pathological features of dn. atrqβ- vaccination ameliorated these morphologic changes of renal injury as well as decreased expression of profibrotic and proinflammatory factors. to further confirm the effects, rmcs were used to investigate the mechanism in vitro. the anti-atr- antibody treatment effectively inhibited high glucose-induced extracellular signal-regulated kinase phosphorylation and tgf-β /smad signal pathway. these results strengthened the renoprotection of atrqβ- vaccination in addition to the antihypertensive effect. the inhibition of oxidative stress and macrophage infiltration as well as proinflammatory factor expression may contribute to the amelioration of renal pathological changes. furthermore, compared with the obvious ras feedback of arbs, circulating or local ras were not elevated in diabetes animals immunized with atrqβ- vaccine. nevertheless, kidney ang ii concentration and at r expression were decreased in both groups. the recent development of ace -ang ( - )-mas concept has provided new insights into the effects of ang ii in animal experimental models. this novel concept states that ras has two axes: the ace-ang ii-at r axis and ace -ang ( - )-mas axis. the former axis induces vasoconstriction, proliferation, and proinflammatory functions through ang ii, and the latter axis counteract the effects of the former axis through the major heptapeptide effector ang ( - ) [ , ] . ace cleaves ang ii to produce abundant levels of ang ( - ) in the proximal tubule, which produces vasodilation and antiproliferative, natriretic, and diuretic effects [ ] . despite its significant role, the exact role of ace and ang ( - ) in kidney disease is not clearly understood. it has been reported that inhibition of ace function accelerates diabetic injury and human recombinant ace reduces the progression [ , ] . moreover, ang ( - ) attenuates the progression of diabetic nephropathy in animal models [ , ] . our study showed a downregulation of ace -ang ( - )-mas axis in diabetic rats, and the beneficial effects of atrqβ- vaccine and olmesartan treatment may partly due to the upregulation of this axis. although the exact mechanism of how these two axes counteract with each other remains unknown, we can conclude that the vaccine regulates the two ras axes, not only inhibiting at r overactivation. safety considerations are paramount when developing any vaccine but are particularly important when targeting a condition for which many safe and effective alternative therapies are available. four key factors define the safety of therapeutic vaccine: ( ) the targeted molecule, ( ) the reversible of antibody response, ( ) antibody-dependent cell-mediated cytotoxicity (adcc) and complement-mediated cytotoxicity, and ( ) fig. no imumune-mediated injury was observed in vaccinated animals. a representative staining of hematoxylin-eosin (h&e), masson's trichrome, and periodic acid-schiff (pas) and transmission electron microscopy (tem). b normal sd rats were immunized on days and , and the atr- -specific antibody titers were screened on days , , , , , , , and activation of t cells against self-molecules [ ] . combined with our previous studies, vaccination-targeted at r showed no immune-mediated injuries [ , ] and was also confirmed in this study. the antibody response was reversible as the possibility of halting antibody production. for adcc and complement-mediated cytotoxicity, there are still several controversies in terms of its onset and regulation. nevertheless, the potential effects caused by the vaccine needed further investigation. as the target peptide of the vaccine was only eight amino acids in length, then it was smaller than the minimal size of a t cell epitope and therefore should not be able to induce a t cell response [ ] . several studies had estimated that vaccines comprising self-molecules coupled to vlps present optical candidates capable of achieving efficacious antibody levels while fulfilling the necessary safety criteria [ , ] . examples like the ang ii vaccine cyt- -angqb, which showed effectively reduced bp without any uncontrolled immune stimulation in both animals and patients [ , ] . similar to the composition, the kidney damage caused by immune complexes was not detected and no visible pathological changes were observed by light microscopy and tem in atrqβ- -vaccinated animals. from the results above, the atrqβ- vaccine was found to be basically safe, although further assessments are needed to confirm this conclusion. compared with chemical drugs, the vaccine therapy has potentially superior advantages. the half-life of the anti-atr- antibody was . days [ ] , longer than any other chemical drugs presently used. no obvious feedback activation of circulating or local ras was observed. additionally, long-term treatment of chronic diseases is costly, tedious, and at the population level rather unsuccessful. monoclonal antibodies specific for host proteins have proven to be highly effective, and the movement from the passive administration of monoclonal antibodies to active vaccination against selfmolecular could provide affordable medicines and broader patient acceptance and compliance. the question today in many patient populations is not whether to block the ras but rather how best to inhibit its activity. from a theoretical perspective, vaccination has much to recommend it as 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cyt -angqb on ambulatory blood pressure: a double-blind, randomised, placebocontrolled phase iia study open access this article is distributed under the terms of the creative commons attribution . international license (http:// creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord- -iccaqz u authors: namsolleck, pawel; moll, gert n. title: does activation of the protective renin-angiotensin system have therapeutic potential in covid- ? date: - - journal: mol med doi: . /s - - - sha: doc_id: cord_uid: iccaqz u infection of lung cells by the corona virus results in a loss of the balance between, on the one hand, angiotensin ii-mediated stimulation of the angiotensin ii type receptor and, on the other hand, stimulation of the angiotensin ii type receptor and/or the mas receptor. the unbalanced enhanced stimulation of the angiotensin ii type receptor causes inflammation, edema and contributes to the pathogenesis of severe acute respiratory distress syndrome. here we hypothesize that stable, receptor-specific agonists of the angiotensin ii type receptor and of the mas receptor are molecular medicines to treat covid- patients. these agonists have therapeutic potential in the acute disease but in addition may reduce covid- -associated long-term pulmonary dysfunction and overall end-organ damage of this disease. recent publications highlight ace as a cell-entry receptor for sars-cov and sars-cov- . less attention is given to other, in particular protective, components of the renin angiotensin system (ras) (unger et al. ) . ras has a double nature, like the two-faced ancient roman god janus, which simultaneously looks in opposite directions. the detrimental arm of ras is formed by the ace-angiotensin ii (ang ii)-angiotensin ii type receptor (at r) axis. limiting the detrimental effects of at r by at r blockers (arbs) or by inhibiting ras via ace inhibitors (acei) is generally well-established. however, the use of arbs and acei in coronavirus disease- (covid- ) has been subject of debate. on the other hand, as part of the protective arm of ras, ang ii also stimulates the angiotensin ii type receptor (at r) and this octapeptide can be further cleaved by the carboxypeptidase ace to yield angiotensin-( - ) (ang-( - )), an agonist of the mas receptor (masr). the protective effects of at r and masr agonists are usually opposite to the detrimental effects of at r, but their clinical use, in cases of unbalance between the two arms of ras, is insufficiently explored. endogenous ligands of the ras receptors are rapidly degraded and lack receptor specificity. here we consider therapeutic perspectives of stable and specific at r and masr agonists in the balance between the detrimental and protective arm of ras is in several aspects seriously disturbed in covid- , thus causing a potentially lethal disease (fig. ) . after the sars-cov cell-entry following ace -interaction, subsequent down-regulation of cell surface ace is observed (kuba et al. ) . since sars-cov- also targets ace , likewise downregulation of ace is expected. reduced membrane expression of ace enhances the inflammatory response to the virus. covid- infection furthermore causes an increase in the decapeptide ang i and the octapeptide ang ii, whereas ang-( - ) levels decrease. thereby detrimental ang ii-mediated stimulation of at r is enhanced whereas protective ang-( - )-mediated stimulation of masr is decreased. at r stimulation reduces alveolar cell survival. it also causes inflammation and an increase in vascular permeability (huertas et al. ) . as a result, edema is accumulating in the alveoli which hampers gas-exchange leading to lower oxygen levels. taken together this adds to the severity of the acute respiratory distress. reduction of the unbalance in the ras by inhibition of the detrimental arm might be reached by either an arb or an acei. the combined use of arbs and acei is prohibited, but their single use is applied. arbs block the at r and thus ang ii can activate the unopposed protective receptor at r and further, after ace -mediated conversion of ang ii into ang-( - ), also the masr. unfortunately, arbs exert only limited therapeutic effect in tissue injury (unger et al. ) . moreover, arbs may reduce blood pressure, which in case of critically ill patients may lead to unwanted hypotension. acei block the acemediated cleavage of ang i and thereby block the formation of ang ii. pros and cons of the use of arbs and acei in covid- have been discussed (d'ardes et al. ). continuation of the use of an arb or an acei in covid- has been recommended (vaduganathan et al. ; ingraham et al. ; park et al. ; sanchis gomar et al. ) and has been suggested to be beneficial in cardiovascular disease (wang et al. ) . fear for induction of upregulation of the cov- -receptor ace leading to enhanced infection (sommerstein and gråni ) has not been supported by clinical data (gupta and misra ; kai and kai ) . in fact a clinical investigation demonstrated that no arb or acei-induced upregulation of ace takes place (sriram and insel ) . on the other hand, benefits with respect to reducing covid- itself have not (yet) been demonstrated in the clinic either (gupta and misra ; kai and kai ; rico-mesa et al. ). instead of blocking at r or inhibiting ace, here we focus on the potential benefits in covid- of stimulating the at r or masr. restoration of the balance in the ras after corona virus infection might be pursued by direct and specific stimulation of the protective arm via at r or via the ace -ang-( - ) -masr axis. in a subchronic lung injury model a cyclized at r-specific peptide agonist, with a half-life of > h in man, reduced inflammation and hypertrophy (wagenaar et al. ) . in an animal model of monocrotaline-induced pulmonary hypertension, a small molecule at r agonist c reversed pulmonary fibrosis and prevented right ventricular fibrosis. furthermore c improved right heart function, decreased pulmonary vessel wall thickness, and reduced pro-inflammatory cytokines (bruce et al. ) . in a bleomycin-induced lung injury model prolonged administration of the at r agonist c prevented and attenuated pulmonary fibrosis, collagen deposition and lung remodeling. in addition c reduced inflammation, improved lung pressure and reduced muscularization of the pulmonary vessels (rathinasabapathy et al. ) . currently the safety and efficacy of this agonist is tested in a phase trial with patients with covid- infection (clinicaltrials.gov identifier: nct ). recombinant human ace , which is not membrane bound, still binds to the corona virus and thereby limits the cell entry (fig. ) . furthermore recombinant ace converts ang ii into ang-( - ). in patients with pulmonary arterial hypertension a single dose of recombinant human ace resulted in a decreased level of pro-inflammatory cytokines and markers of oxidative stress accompanied by decreased pulmonary vascular resistance and increased cardiac output (hemnes et al. ) . to elucidate the molecular mechanisms leading to the observed effects, rnaseq on pulmonary arteries treated ex vivo with masr agonist ave was performed. significant changes in pressure regulation, inflammatory responses and cell migration pathways were observed indicating therapeutic effects of masr activation (hemnes et al. ) . stimulation of the masr reduces in vitro ang ii-or bleomycin-induced apoptosis of alveolar epithelial cells (uhal et al. ) . a recent review speculates on potential benefits of masr stimulation in covid- based on data obtained from animal models of asthma, lung fibrosis, ards, and pulmonary emphysema. the anti-inflammation effects, such as decreased cytokine and chemokine synthesis, migration of inflammatory cells to the lung and the resulting functional improvement of the lungs would be key benefits of masr stimulation (fig. ) . in addition, prolonged treatment might fig. anti-inflammatory and anti-fibrotic pathways mediated by activated at r and/or masr. the at r and masr are expressed in the cell as monomers, homodimers and at r-masr heterodimers (leonhardt et al. ) and their downstream pathways are largely similar, making it often impossible to distinguish between them. during infection the at r becomes activated initiating inflammatory processes via nfκb and mapk. prolonged activation of at r may initiate pro-fibrotic processes with tgfβ as a key molecule. agonist-mediated stimulation of at r or masr inhibits activation of nfκb and mapk resulting in anti-inflammation. for the anti-fibrotic action the inhibition of receptor tyrosine kinase activity by dephosphorylation on the one hand, and activation of cgmp on the other hand, plays a crucial role. in addition, heterodimerization between at r and at r or masr inhibits detrimental effects mediated by at r. blue lines: pro-fibrotic pathways; red lines: pro-inflammatory pathways; green lines: anti-inflammatory or antifibrotic pathways. at r / masr: angiotensin ii type receptor or mas receptor or at r-masr heterodimers; tgfbr : transforming growth factor beta receptor ii; at r: angiotensin ii type receptor; rtks: receptor tyrosine kinases; akt: protein kinase b; ptp: protein tyrosine phosphatase; psp: protein serine/threonine phosphatase; enos: nitric oxide synthase ; no: nitric oxide; cgmp: cyclic guanosine monophosphate; mmp : matrix metallopeptidase ; tgfβ: transforming growth factor beta; pdgf: platelet-derived growth factor; fgf: fibroblast growth factor; ctgf: connective tissue growth factor; vegf: vascular endothelial growth factor; ecm: extracellular matrix; erk: extracellular signal-regulated kinases; mapk: mitogen-activated protein kinase; nox: nadph oxidase; ros: reactive oxygen species; nfκb: nuclear factor kappa b; gαi: g protein alpha i subunit; atip: at r-interacting proteins/microtubuleassociated scaffold proteins; pp a: protein phosphatase a; shp- : src homology region domain-containing phosphatase- ; mkp- : mapk phosphatase . the pathways are based on: unger et al. ; sumners et al. ; zhang et al. ; leonhardt et al. ; chappell and al zayadneh result in anti-fibrotic effects in lung tissue (magalhaes et al. ) . the potential of the ace -ang-( - ) -masr axis has furthermore been recognized as witnessed by registered clinical trials of ang-( - ) in covid- (clinical-trials.gov, identifiers: nct ; nct ; nct ) . however, endogenous ang-( - ) lacks receptor specificity. ang-( - ) stimulates in vivo the masr but in vitro studies reported biased agonism at the at r (galandrin et al. ). in addition, ang-( - ) is very rapidly degraded resulting in a half-life of less than a minute in man. in contrast, specific and stable cyclic ang-( - ) exerts multiple therapeutic effects in lung tissue of animal models of acute and chronic lung injury (wagenaar et al. ; wösten-van asperen et al. ) . in an animal model of ards, cyclic ang-( - ) reduced lung injury and inflammation while improving blood oxygenation (fig. ) . cyclic ang-( - ), which is fully ace-resistant, did not change the blood pressure (wösten-van asperen et al. ) . in addition to the acute and sub-chronic effects in covid- , stable at r agonists (bruce et al. ) may reduce covid- associated long term pulmonary dysfunction. besides the lungs, covid- also affects heart, kidney, liver, gastrointestinal and the central nervous systems (gan et al. ) . in view of the demonstrated general therapeutic potential of the protective arm of ras in these organs and systems (unger et al. ) , treatment of severe ards in covid- with at r and masr agonists may concomitantly confer beneficial effects that reduce the overall end-organ damage of this disease. in conclusion, available data indicate the perspective of an effective strategy for treatment of ards and covid- by direct and selective stimulation of the protective arm of ras by at r-or masr-specific, peptidase-resistant agonists. the data converge to further investigations in viral pneumonia-mediated ards models. abbreviations ras: renin angiotensin system; sars: severe acute respiratory syndrome; cov- : coronavirus ; covid- : coronavirus disease ; ards: acute respiratory distress syndrome; at r: angiotensin ii type receptor; masr: mas receptor; arb: angiotensin ii type receptor blocker; ace: angiotensin converting enzyme; acei: angiotensin converting enzyme inhibitor; ang ii: angiotensin ii; ang-( - ): angiotensin-( - ) selective activation of angiotensin at receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis angiotensin-( - ) and the regulation of antifibrotic signaling pathways covid- and ras: unravelling an unclear relationship cardioprotective angiotensin-( - ) peptide acts as a natural-biased ligand at the angiotensin ii type receptor covid- as a viral functional ace deficiency disorder with ace related multi-organ disease contentious issues and evolving concepts in the clinical presentation and management of patients with covid- infection with reference to use of therapeutic and other drugs used in co-morbid diseases a potential therapeutic role for angiotensin converting enzyme in human pulmonary arterial hypertension endothelial cell dysfunction: a major player in sars-cov- infection (covid- )? understanding the renin-angiotensinaldosterone-sars-cov-axis: a comprehensive review interactions of coronaviruses with ace , angiotensin ii, and ras inhibitors-lessons form available evidence and insights into covid- a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury evidence for heterodimerization and functional interaction of the angiotensin type receptor and the receptor mas activation of ang-( - )/mas receptor is a possible strategy to treat coronavirus (sars-cov- ) infection is the use of ras inhibitors safe in the current era of covid- pandemic? the selective angiotensin ii type receptor agonist, compound , attenuates the progression of lung fibrosis and pulmonary hypertension in an experimental model of bleomycin-induced lung injury outcomes in patients with covid- infection taking acei/arb angiotensin-converting enzyme and antihypertensives (angiotensin receptor blockers and angiotensin-converting enzyme inhibitors) in coronavirus disease rapid resonse: preventing a covid- pandemic: ace inhibitors as a potential risk factor for fatal covid- risk of ace inhibitor and arb usage in covid- : evaluating the evidence anti-fibrotic mechanisms of angiotensin at -receptor stimulation regulation of alveolar epithelial cell survival by the ace- /angiotensin - /mas axis the protective arm of the renin angiotensin system. functional aspects and therapeutic implications renin-angiotensin-aldosterone system inhibitors in patients with covid- agonists of mas oncogene and angiotensin ii type receptors attenuate cardiopulmonary disease in rats with neonatal hyperoxia-induced lung injury good or bad: application of raas inhibitors in covid- patients with cardiovascular comorbidities acute respiratory distress syndrome leads to reduced ratio of ace/ace activities and is prevented by angiotensin-( - ) or an angiotensin ii receptor antagonist ace /ang-( - ) signaling and vascular remodeling publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions pn wrote the first version of the manuscript which has been extended by gnm. the author(s) read and approved the final manuscript. no funding for this work has been received.availability of data and materials not applicable.ethics approval and consent to participate not applicable. both authors read and agreed to the content of the final manuscript, and consented on its publication. the authors disclose that their employer, lanthiopep b.v., is owner of patents on angiotensin variants. gnm is director of lanthiopep b.v.. key: cord- -c fu jv authors: lu, chen-chen; wang, gui-hua; lu, jian; chen, pei-pei; zhang, yang; hu, ze-bo; ma, kun-ling title: role of podocyte injury in glomerulosclerosis date: - - journal: renal fibrosis: mechanisms and therapies doi: . / - - - - _ sha: doc_id: cord_uid: c fu jv finding new therapeutic targets of glomerulosclerosis treatment is an ongoing quest. due to a living environment of various stresses and pathological stimuli, podocytes are prone to injuries; moreover, as a cell without proliferative potential, loss of podocytes is vital in the pathogenesis of glomerulosclerosis. thus, sufficient understanding of factors and underlying mechanisms of podocyte injury facilitates the advancement of treating and prevention of glomerulosclerosis. the clinical symptom of podocyte injury is proteinuria, sometimes with loss of kidney functions progressing to glomerulosclerosis. injury-induced changes in podocyte physiology and function are actually not a simple passive process, but a complex interaction of proteins that comprise the anatomical structure of podocytes at molecular levels. this chapter lists several aspects of podocyte injuries along with potential mechanisms, including glucose and lipid metabolism disorder, hypertension, ras activation, micro-inflammation, immune disorder, and other factors. these aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries. the glomerular filtration membrane constituted by three components, porous endothelial cells, glomerular basement membrane (gbm), and epithelial cells in the gbm, which also called podocytes. podocytes are highly differentiated epithelial cells consist of three distinct parts: cell body, major processes, and foot processes (fps). podocytes have a voluminous cell body, which is at the central position of the cell and lies in the urinary space. the cell body contains a nucleus, abundant endoplasmic reticulum, a well-developed golgi system, lysosomes, and mitochondria. the densely distributed organelles in the cell body suggest a high level of anabolic and catabolic activity. microtubules and intermediate filaments, such as vimentin and desmin, are the dominated cytoskeleton components in cell body to accounts for the unique shape of podocytes (pavenstadt et al. ) . from the cell body, podocytes give rise to primary processes that reach to glomerular capillary, forming an affixation by fps. podocytes are polarized epithelial cells which contain a apical/luminal and a basal cell membrane. the apical surface domain forms a few fingerlike protrusions which bulge into bowman's space. the apical domain is negatively charged, which limits the passage of albumin (also negatively charged) and maintain the separation of adjacent podocytes by anion charge. the basal cell membrane mediates the affixation to the gbm by α β integrin and αand β-dystroglycans, which play the function by connecting to certain matrix proteins within the gbm (kreidberg et al. ; raats et al. ) . both of apical and basal membranes contain numerously distributed cholesterol-rich domains, and it was found that specific membrane proteins of podocytes are obviously arranged in rafts simons et al. ) . fps functionally consist of a luminal or apical membrane domain and a basal cell membrane domain. fps are characterized by a podosome-like cortical network of short, branched actin filaments and by the presence of highly ordered, parallel contractile actin filament bundles, which are thought to modulate the permeability of the filtration barrier through changes in fp morphology (greka and mundel ) . the foot processes of neighboring podocytes are bridged by slit diaphragm (sd), which is the site of convective fluid flow through the visceral epithelium and the final barrier to urinary protein loss. similar to the apical membrane domain of podocytes, the sd is also covered by a thick surface coat mainly constituted by sialoglycoproteins, including podoendin, podocalyxin, and others, which are responsible for the high negative surface charge of the podocytes. in addition, several molecules, including zo- (zonula occludens protein), nephrin, cd ap (cd -associated protein), fat, and p-cadherin, have all been shown to be expressed within the sd, and some of those molecules play a major role for its integrity (pavenstadt et al. ) . the unique shape of podocyte and the maintenance of its processes are owing to a well-developed cytoskeleton, which serves as the podocyte's "backbone." and also, the actin-rich cytoskeleton makes podocytes to be able to alter shape continually and dynamically. the cytoskeleton is comprised by microfilaments ( - nm diameter), intermediate filaments ( nm diameter), and microtubules ( nm diameter), which are mainly defined by their diameter. microtubules and intermediate filaments are predominant cytoskeletal constituents in the cell body and the primary processes. in the fps, microfilaments are the main cytoskeletal component, which consist of a network with densely accumulated f-actin and myosin. fp actin cytoskeleton is extensively distributed in all three domains of fps, resulting to an important role of actin for the function and dysfunction of podocytes. fp effacement requires the activation of actin filaments reorganization, a process which is regulated by multiple signaling events involving integrin activation, g protein-coupled receptor (gpcr) and growth factor receptor, and calcium (ca + ) influx pathways as upstream modulators of the actin cytoskeleton (takeda et al. ) . the complex architecture of podocytes, in particular on the maintenance of highly ordered, parallel, contractile actin filament bundles in fps, is required for the highly specialized functions of podocytes, which include (i) a size barrier to protein; (ii) charge barrier to protein; (iii) maintenance of the capillary loop shape; (iv) counteracting the intraglomerular pressure; (v) synthesis and maintenance of the gbm; (vi) production and secretion of vascular endothelial growth factor (vegf) required for gen integrity (shankland ) . podocyte is the most differentiated cell type in the glomerulus, which plays a crucial role in the glomerular filtration barrier. podocyte foot processes with the interposed sd represent the last filtration barrier of gbm. the sd is a subtle signal transduction unit characterized by a modified adherens junction that bridges the - -nm-wide filtration slits (reiser et al. ) . transmembrane proteins such as nephrin and fat constitute the rod-like units of sd which are connected by numerous linear bar, forming a network with pores the same size as or smaller than albumin (mundel and kriz ) . meanwhile, negatively charged apical domain of sd works as a charge barrier to prevent the albumin loss. thus, the podocyte is the important size and charge barrier of gbm, and podocytes' damage leads to the disruption of gbm integrity and proteinuria. podocytes stabilize glomerular architecture owing to fps counteract distensions of the glomerular basement membrane, which is regulated by vasoactive hormones. in this regard, they are responsible for % of the hydraulic resistance of the filtration barrier (pavenstadt ) . ang ii regulates the contractile state of their foot processes by activating a cl − conductance and increasing [ca + ] i, camp in podocytes, thereby modulating the ultrafiltration coefficient kf. other agonists such as avp, oxytocin, norepinephrine, and parathormone have also been reported to modulate [ca + ] i in podocytes. vasoactive hormones may also alter charge properties of the podocyte and thereby enhance urinary protein excretion (pavenstadt ) . vegf family consists of five secreted homodimeric glycoproteins: vegf-a, vegf-b, vegf-c, vegf-d, and placental growth factor. in human and murine kidneys, vegf-a isoform is constitutively expressed in podocytes, while playing its role mainly by contact with vegfr- and vegfr- predominately localized on the glomerular endothelial cells. it was assumed that vegf-a is critical for the regulation of endothelial cell survival, proliferation, differentiation, and migration as well as endothelium-dependent vasodilatation and vascular permeability (advani ) . the complex paracrine signaling pathway between podocytes and glomerular endothelial cells plays a central role in maintaining the structure and integrity of the kidney filtration barrier. podocyte injury is the common pathological process in many glomerular diseases such as minimal change disease, membranous glomerulopathy, focal segmental glomerulosclerosis (fsgs), diabetic nephropathy (dn), and lupus nephritis. physiological stresses or pathological stimuli like mechanical stress, oxidative stress, and immunologic stress disrupt the homeostasis of glomerular filtration barrier. transcapillary pressure increment is induced by glomerular hypertension/hyperfiltration, and podocyte processes' elongation is induced by capillary expansion contribute to cytoskeletal dysregulation and intrinsic stress (neal et al. ) . pathological factors, such as ischemia-reperfusion, chemical/toxic substances from the primary urine, usually cause reactive oxygen species (ros) production in podocytes . it was also reported that aldosterone and angiotensin ii promoted receptormediated ros generation in podocytes (liu et al. ) . immunologic stress is induced by cytokine/complement, such as cc chemokine receptor , tumor necrosis factor, and sublytic c b- -mediated intracellular stress in podocytes (nagata ) . typical electron microscopy manifestations of podocyte injury include microcystic, pseudocystic changes, vacuolization, the presence of cytoplasmic inclusion bodies, and detachment from the gbm. besides those changes, foot process effacement is the most characteristic change in podocyte injury. the damage of sd proteins contributes to cytoskeleton disorganization, leading to podocyte effacement and proteinuria (shankland ) . sd between adjacent podocytes is constituted predominantly by sd proteins including nephrin, podocin, cd ap, neph , and fat . mutations/abnormalities of those proteins result proteinuria and kidney disease. studies have shown that sd proteins regulate cytoskeleton organization and podocyte shape by interacting with proteins associated with actin cytoskeleton. fat- is an organizer of actin polymerization. cd ap connects the nephrin complex with the actin-modifying proteins wasp, capz, cortactin, and the arp / complex. reduced podocyte number causes proteinuria and glomerulosclerosis. podocyte detachment, podocyte apoptosis, and the lack of adequate podocyte proliferation are three main reasons leading to the decrease in podocyte number also called "podocytopenia." the lack of charge-and size-selective barriers induced by podocyte loss leads to proteinuria. studies have demonstrated the correlation of podocytes reduction with the onset and progression of glomerulosclerosis. because podocytes counteract the outward forces of glomerular pressures and maintain capillary loop shape, podocyte loss results to local bulging of the gbm when glomerular pressures increase in many renal diseases. the denuded gbm tends to form a synechia attachment by contacting with the parietal epithelial cells and bowman's capsule, which is thought to be the first "committed step" of focal segmental glomerular sclerosis (fsgs) (kriz et al. , a . podocytes maintain a healthy intraglomerular environment by cross talk with glomerular endothelial cells. endothelial cell swelling and attenuation of fenestrae are observed in podocyte injury models by ultrastructural study (kriz et al. ) . it was illustrated that podocyte injury disrupts intracapillary homeostasis, causing thrombotic micro-angiopathy and mesangial abnormalities by reducing vegf signaling (eremina et al. ; kobayashi et al. ) . glomerulosclerosis is a terminal consequence of podocyte injury. the classic type of glomerulosclerosis, as defined by segmental obliteration of glomerular capillaries by the extracellular matrix, has been believed to progress to complete sclerosis without regression (nagata ) . in early stage of fsgs, cellular lesions including transformed podocytes were accompanied by segmental sclerosis, supporting the fact that podocyte damage might be an early event of glomerulosclerosis. in a recent elegant review by kim js and his colleagues, the essential steps of glomerulosclerosis were suggested as follows: ( ) increased glomerular capillary pressure and filtration flow through podocyte slits, ( ) foot process effacement as an adaptive response, ( ) podocyte hypertrophy and glomerulomegaly, ( ) mismatch between glomerular tuft growth and podocyte hypertrophy, ( ) stretching and attenuation of podocyte cell body, ( ) pseudocysts formation by hindered flow of filtrates beneath the podocyte that is partially detached on bare areas of gbm, ( ) complete podocyte detachment by enlarged pseudocysts and adhesion to bowman's capsule, ( ) glomerular tuft's adhesion to bowman's capsule, ( ) spreading of filtrates to interstitium out of nephron through adhesion structure, and ( ) interstitial proliferation and nephron degeneration (kim et al. ). podocytes' injury and depletion was a crucial step in the development of albuminuria in dn. in dn, the number of podocyte-specific markers and podocytes number is decreased, which leads to the occurrence of albuminuria and further develops into glomerulosclerosis. hyperglycemia is the main pathological change of diabetes and plays an important role in promoting the occurrence and development of dn. increased intracellular glucose could induce multiple cell and molecular events in podocyte: ( ) generation of reactive oxygen species (ros) and advanced glycation end products (ages), ( ) increased flux of polyols and hexosamines, ( ) activation of protein kinase c (pkc), ( ) increased cytokines and growth factors, ( ) aberrant notch signaling, and ( ) activate the renal ras. these abnormal molecular pathological changes mediate the functional and morphological changes of podocytes in a direct or indirect way, including podocyte hypertrophy, epithelial mesenchymal transition (emt), podocyte detachment, and podocyte apoptosis. podocyte injury is a key factor in the development of dn. recent studies in both type and type diabetes have proposed that a reduction in the number of podocytes may lead to the development of proteinuria. it is reported that the structure and function of podocytes are abnormal under high glucose conditions, such as podocyte fusion, septal injury, and podocyte loss. there has been evidence that podocytes pos-sess a completely functional system for glucose uptake (lewko et al. ). coward et al. have revealed that the cultured human podocytes express glucose transporter (gluts) in two forms: glut and glut , which participate in insulin-dependent glucose transport to the cell (coward et al. (coward et al. , . in addition, the podocyte split protein, such as nefin, is also involved in glucose transport. schiffer et al. have demonstrated that podocytes also express another insulin-sensitive glucose transporter, glut (schiffer et al. ) . glut is the primary glucose transporter in most cells as well as in podocytes (coward et al. (coward et al. , . in diabetes, hyperglycemia and alteration of glucose transporter cause increased intracellular glucose concentration in podocyte and lead to severe impairment of the glomerular filtration barrier. conversely, zhang et al. found that enhancement of glut expression in diabetic podocyte significantly reduced the mesangial expansion and fibronectin accumulation by inhibiting the expression of vascular endothelial growth factor (vegf) (zhang et al. ) . similarly to other cells, under high glucose condition, podocyte can undergo many pathological changes induced by aberrations in various cellular and molecular events. high glucose induces generation of advanced glycation end products (ages) and reactive oxygen species (ros), increased flux of polyols and hexosamines, increased activity of protein kinase c (pkc), upregulated expression of cytokines and growth factors including vascular endothelial growth factor (vegf), and transforming growth factor-beta (tgf-β), induces aberrant notch signaling, and activates the renal ras (anil kumar et al. ) . pathomechanism of podocyte injury in dn mainly includes podocyte hypertrophy, emt, podocyte detachment, and podocyte apoptosis. podocyte hypertrophy is the initial stage of podocyte injury in early dn. hyperglycemia upregulated the expression of cyclin-dependent kinase p kip , which leads to further cell cycle arrest and hypertrophy. it was found that p kip -/-mice had significantly improved renal damage in dn (wolf et al. ) . several studies suggested high glucoseinduced podocyte hypertrophy by activating mtorc pathway (fantus et al. ). in addition, hyperglycemia increased expression of nuclear stat via the activation of the upstream signal transduction element gp , which eventually leads to podocyte hypertrophy. excessive hypertrophy could result in degenerative changes in podocyte structure and functions, leading to its detachment from glomerular basement membrane (gbm). previous studies have shown that phenotype conversion of podocyte was involved in the early stage of podocyte deletion in dn by inducing podocyte detachment or podocyte apoptosis. podocyte emt is a manifestation of podocyte phenotype conversion and one of the initiating factors leading to a variety of glomerular diseases. when emt occurs, the cells lose their original characteristics, resulting in disappearance of intercellular contact, impaired cell polarity, and expression of mesenchymal markers such as alpha smooth muscle actin (alpha-sma) and fibroblast-specific protein (fsp ). emt is also an explanation for podocyte depletion in dn (yamaguchi et al. ). emerging evidence suggested that podocytes could undergo emt in dn, characterized by loss of epithelial features such as nephrin and p-cadherin, while expressing mesenchymal markers such as fsp- , type i collagen, and fibronectin (reidy and susztak ). xing et al. ( ) demonstrated that stimulation with high glucose for h could activate the pi k/akt pathway in podocyte, and thereby induce the protein expressions of α-sma and desmin. dai et al. ( ) suggested that connective tissue growth factor (ctgf) and integrinlinked kinase (ilk) were involved in high glucose-induced phenotypic alterations of podocytes. lv et al. ( ) findings elaborated that rac /pak signaling contributed to high glucose-induced podocyte emt via promoting β-catenin and snail transcriptional activities, which could be a potential mechanism involved in podocytes injury in response to stimuli under diabetic conditions. guo et al. indicated high glucose can also activate β-catenin and snail expressions by upregulating gsk- β. in addition, hyperglycemia-induced podocyte detachment by decreasing the expression of key proteins involved in the foot process actin cytoskeleton, split diaphragm (sd) integrity, and podocyte-gbm interactions. a b integrins are the important transmembrane protein involved in anchoring foot processes in the gbm. high glucose regulates the expression of integrin subunits and inhibits the synthesis of agrin. therefore, high glucose affects not only the structure of podocytes, but also their ability to adhere to gbm (chen et al. ; han et al. ; yard et al. ) . it is found that high glucose can alter podocyte adhesion by decreasing expression of integrin α β v which was an important receptor that could tightly connect podocyte with the gbm and participated in the adhesion function of podocyte. in addition, α-actinin, an actin filament for protein crosslink, is also an important factor required for podocyte adhesions (dandapani et al. ) . high glucose and age treatment resulted in α-actinin- expression changes and induces cytoplasmatic translocation in podocyte (ha ) . there are some evidences that podocyte apoptosis played a role in reduction in density and number of glomerular in dn. high glucose led to podocyte apoptosis by increased production of ros, activation of poly(adp-ribose) polymerase, nf-kb, and p map kinase szabo et al. ) . in diabetes, the surface receptors of the ages are upregulated in the podocytes (tanji et al. ) . binding ages to receptors activates activated transcription factor foxo , which also induced podocyte apoptosis via p protein kinase signaling pathways (cohen et al. ). in addition, high glucose increased the protein expression of nestin, which is a vi intermediate filament protein-related cell cytoskeleton, thereby increased podocyte apoptosis rate . high glucose increased the expression of tgf-β in podocyte. tgf-β could induce podocyte apoptosis by directly activate smad , p map kinase, and notch pathway (li et al. ) . in addition to its direct effects, elevated glucose may act indirectly, via the proinflammatory response, ang ii-dependent pathways, and lipid accumulation. under high glucose conditions, secretion of the mcp- protein by cultured podocytes was increased rapidly (han et al. ) , and similar effect was observed in podocyte stimulated with ages (gu et al. ) . podocyte can also express tnf-α, a cytokine produced by various immune cells, in response to high glucose stimulation and in diabetic conditions (ikezumi et al. ; ruster et al. ). high glucose could stimulate activity and expression of the local ras components in podocyte, including ang ii and its at receptors (yoo et al. ). following that, it was recently demonstrated that local ras activation would lead to podocyte injury through a variety of pathways. ang ii could induce podocyte apoptosis through activation of nadph oxidase and production of ros, and upregulate the expression of glut transporters (gill and wilcox ; nose et al. ). in addition, ma et al. found that lipid accumulation in podocytes was increased under the high glucose stimulation, which is mediated through the disruption of low-density lipoprotein receptor (ldlr) pathway (zhang et al. a ). interestingly, reducing lipid accumulation in podocytes decreased the protein expression of sma and increased the expression of nephrin in podocyte. these studies reveal that high glucose-induced lipid accumulation is involved in the podocyte injury in dn. therefore, the above shows that high glucose could induce various other metabolic disorders and indirectly lead to podocyte injury. lipid metabolism disorder is commonly observed in patients with chronic kidney disease (ckd), accompanied by increased fasting triglyceride levels and decreased high-density lipoprotein cholesterol (hdl-c) (bianchi et al. ) . it is increasingly recognized that dysregulation of lipid metabolism is involved in the development and progression of ckd, such as obesity-related renal disease and dn (de vries et al. ) . podocytes, as specialized cells of glomerulus, play an important role in the pathologist of ckd when they are injured (fiorina et al. ) . and excessive lipid accumulation in podocytes can lead to cellular dysfunction and death, which is called lipotoxicity. between neighboring podocytes, there is a unique interdigitating structure bridged by sd, maintaining the proper glomerular filtration (ruotsalainen et al. ) . researches have revealed that sd is a lipid raft structure containing multiple podocyte-specific proteins, such as podocin and nephrin (schermer and benzing ) . in particular, podocin can recruit and bind to cholesterol to form sd, and this binding can influence the composition of lipid membrane, allowing cholesterol to contact with the ion-channel transient receptor potential canonical (trpc ) (huber et al. ). this suggests that cholesterol homeostasis is essential for glomerular functions. however, excessive cholesterol can also negatively disrupt the mutual binding of podocyte sd proteins, or interfere with the binding between podocyte sd proteins and caveolin- , a lipid raft-associated protein, binding nephrin, and cluster of differentiation (cd )-associated protein (sorensson et al. ) it is regulated by some functional proteins such as atp-binding cassette transporter a (abca ) involving cholesterol efflux, -hydroxy- -methyl-glutaryl coa reductase (hmg-coa reductase, hmgcr) regulating cholesterol synthesis and lowdensity lipoprotein receptor (ldlr) mediating cholesterol influx. the expression of hmgcr and ldlr is regulated by some transcription factors, such as the sterol regulatory element-binding protein (srebp), under negative feedback loops. when cells are rich in cholesterol or its derivatives, the transcription of ldlr gene or other genes necessary to lipid synthesis are suppressed. as a result, the cells are not able to generate and uptake cholesterol, and then establish cholesterol homeostasis. in contrast, when intracellular sterols are exhausted, the transcriptions of srebp target genes will be activated, increasing intracellular cholesterol (zhang et al. ). this enables cellular cholesterol homeostasis despite physiological fluctuations in cholesterol requirements and exogenous supply. however, it is demonstrated that the cellular cholesterol imbalance of podocytes can induce proteinuric glomerular diseases (merscher et al. ) . it is demonstrated that human glomerular podocytes express abca , hmgcr, and ldlr (merscher-gomez et al. ). ma et al. found that some pathogenic factors such as inflammation can disrupt ldlr pathway (zhang et al. b ). thus, excessive lipid accumulates in podocytes, resulting in effacement of the foot processes and epithelial mesenchymal transition of podocytes (zhang et al. b) . it is recently demonstrated that human podocytes treated with the sera from diabetic kidney disease (dkd) patients had increased cholesterol accumulation compared with human podocytes exposed to the sera of patients with diabetes, but no dkd. this was associated with a reduction of abca and an impairment of cholesterol efflux (merscher-gomez et al. ) . besides, it is showed that c-x-c motif ligand (cxcl ) is the main scavenger receptor for oxidized ldl (oxldl) in human podocyte (gutwein et al. ). the expression of glomerular cxcl was increased in patients with membranous nephropathy, accompanied with higher levels of oxldl (gutwein et al. ). and in diabetic db/db mice, cxcl pathway was activated, in parallel with increased cholesterol accumulation in kidney (hu et al. ) . in vitro, oxldl can induce loss of nephrin expression from cultured podocytes (bussolati et al. ) . in summary, cholesterol metabolism disorder can destroy the structure and function of podocytes, leading to the progression of ckd. in addition to hypercholesterolemia, free fatty acids (ffas) can also affect podocyte function in kidney disease. the essential role of fatty acids is to form the phospholipid bilayers of the cell membranes and act as phospholipid messengers, transmitting vital intracellular signals (lee ) . normal cellular fatty acid homeostasis reflects a balance between generation or delivery and utilization. srebp- c is involved in fatty acid and tg synthesis, targeting lipogenic enzymes including acetyl-coa carboxylase (acc) and fatty acid synthase (fas) (horton et al. ) . ffas can be transported into cells by the scavenger receptor platelet glycoprotein (also called as cd ) or via the assistance of vascular endothelial growth factor b (vegf-b) (hagberg et al. ; masuda et al. ). cellular ffas are esterified or transported into the mitochondria for oxidation and subsequent energy production (lee ) . palmitic and stearic acids, belonging to saturated ffas (sfas), and oleic acid, belonging to monounsaturated ffas (mufas), account for - % of plasma ffas (raclot et al. ) . sfas can induce insulin resistance and cell death, involving the pathogenesis of diabetes mellitus type (t dm) (lennon et al. ; sieber et al. ) . in contrast, mufas can prevent sfa-induced lipotoxicity (sieber et al. ) . in human podocytes, insulin resistance can be induced by palmitic acid (lennon et al. ). it is observed that insulin sensitivity in glomeruli of obese and diabetic rats is reduced (mima et al. ) . podocyte-specific insulin receptor knockout mice develop albuminuria and glomerulosclerosis, indicating that normal insulin signaling is critical for podocyte function and survival (welsh et al. ) . these findings imply that ffas play potential roles in insulin resistance, promoting the development and progression of obesity-related renal disease and dn. in the tubulointerstitial and glomerular segment of renal biopsies obtained from patients with dn, endoplasmic reticulum (er) stress is observed (sieber et al. ) . importantly, in a t d mouse model, the progression of dn can be attenuated by ameliorating er stress (qi et al. ) . er dyshomeostasis can decrease the er folding capacity, thereby leading to accumulation of unfolded and misfolded proteins. this in turn initiates the unfolded protein response (upr), adaptively maintaining proper er function (ma and hendershot ) . but if er stress persists, apoptosis will be induced by the proapoptotic transcription factor c/ebp homologous protein (chop) (rasheva and domingos ) . in podocytes, er stress induced by palmitic acid results in the upregulation of several upr markers/effectors, such as the er chaperone heavy chain-binding protein (bip), and chop, while monounsaturated palmitoleic and oleic acids only upregulated bip but not chop (sieber et al. ) . as bip can attenuate palmitic acid-induced apoptosis (laybutt et al. ) , the beneficial effect of mufas may own to the upregulation of bip. in addition to the unfolded proteins, alterations in er membrane lipid composition can also sensitively affect the expression of the er stress sensor inositol requiring enzyme (ire- ) (promlek et al. ) . it is shown that small molecule compound m c, specific ire- inhibition, can attenuate palmitic acid-induced podocyte death (sieber and jehle ) . enhanced ffa uptake by podocytes is induced by increased expression of cd and a decrease in fatty acid β-oxidation, leading to excessive intracellular lipid accumulation (soetikno et al. ). in animal model of type diabetes (t d), increased expression of srebp- in renal results in upregulation of enzymes responsible for ffa synthesis and as a consequence of a high level of triglyceride (tg) in renal (hashizume and mihara ) . accumulated lipids in podocytes limited mitochondrial fatty acids β-oxidation. it induced mitochondrial damage and inhibition of amp kinase (ampk) activity, leading to endoplasmic reticulum (er) stress, autophagy, and apoptosis in podocytes. as a result, mitochondrial dysfunction caused decreased podocyte density and increased in foot process width, together with inflammation (szeto et al. ) . renal accumulation of tg is associated with reduced expression of the ultrasensitive energy sensor ampk strongly. this suggests that the imbalance between energy-generating and energy-consuming pathways might be related to podocyte dysfunction in dkd and other disorders in ckd, due to lipid accumulation (wahl et al. ) . and hypertriglyceridemia can also increase podocytic de novo expression of desmin, which represents podocyte injury (joles et al. ) . since the first description that glycosphingolipid accumulation in the renal results in glomerular hypertrophy in streptozotocin (stz)-induced diabetic mice, several studies have highlighted the role of sphingolipids and gangliosides in podocyte biology (merscher-gomez et al. ) . analysis of kidney biopsy compartments from patients with fabry disease using unbiased quantitative stereology indicated age-dependent accumulation of globotriaosylceramide (gb ) in podocytes (najafian et al. ) . in vitro, globotriaosylsphingosine (known as lysoglobotriaosylceramide) acts as a profibrotic metabolite in cultured human podocytes (sanchez-nino et al. ) . ganglioside gm (gm ) is a receptor for soluble flt , locating in lipid raft domains in the sd of podocytes. binding of soluble flt to gm plays essential roles in autocrine preservation of the podocyte actin cytoskeleton and in prevention of proteinuria (jin et al. ) . o-acetylated disialosyllactosylceramide (gd ), a sialic-acid-containing lipid, was identified as a podocyte-specific ganglioside in rat (reivinen et al. ) . treating mice with an antibody against gd caused nephrin phosphorylation and dislocation from the podocyte sd . it is an emerging concept that sphingolipids act as modulators of podocyte function in fsgs and other glomerular diseases. patients with fsgs are more likely to have recurrence of proteinuria after kidney transplantation. and the number of acid sphingomyelinase-like phosphodiesterase b (smpdl b) positive podocytes is decreased in patients with recurrent proteinuria (fornoni et al. ) . to sum up, lipid metabolism disorder is involved in the pathogenesis of podocyte injury. cholesterol helps form sd between podocytes, maintaining the proper glomerular filtration. ldl-cholesterol uptake is mediated via the ldlr or cxcl and may cause er stress. cholesterol metabolism is regulated by several nuclear receptors and transcription factors, including srebp. excessive cholesterol accumulation in podocytes may contribute to kidney disease. free fatty acids are primarily transported via cd , causing oxidative and er stress based on the degree of saturation. sphingolipids and gangliosides also play a role in podocyte biology. binding of soluble flt to gm plays essential roles in autocrine preservation of the podocyte actin cytoskeleton and in prevention of proteinuria ( fig. . ). hypertension has become the second leading cause of end-stage renal disease (esrd) after diabetes mellitus (udani et al. ) . high blood pressure can affect renal vessels, glomeruli, and tubulointerstitium. recently, more and more studies have indicated that podocyte damage play an important role in hypertensive nephrosclerosis. decreased intrarenal podocyte and increased urinary podocyte were observed in hypertensive nephrosclerosis ). as terminally differentiated cells, podocyte loss leads to denudation of the glomerular basement membrane (gbm) and focal adhesion of the tufts to bowman's capsule, which finally results in glomerulosclerosis and reduced filtration (cellesi et al. ) . podocyte loss in hypertension includes detachment of viable cells and apoptosis (kriz et al. ) . the major factor for podocyte loss in hypertension is the capillary hypertension, which cause glomerular hypertrophy and hyperfiltration (kriz and lemley ) . glomerular hypertrophy results in relatively decreased podocyte density. puelles et al. ( ) examined the effect of hypertension on podocyte depletion using kidneys obtained from autopsy, and they did not observe a difference in total podocyte number solely driven by hypertension, while the relative podocyte depletion is associated with glomerular hypertrophy which resulted in the reductions in podocyte density. hyperfiltration gives rise to increased shear stress by elevating driving force and augmenting gbm area. podocytes cultured in vitro are sensitive to shear stress, which induces reorganization of cytoskeleton (friedrich et al. ) , and this helps them to cover an expanding gbm which further leads to foot process effacement. in desoxycorticosterone-trimethylacetate (doca) hypertensive mice, chloride intracellular channel a, which is highly enriched in podocytes foot process, protects against hypertension-induced podocyte injury through weakening the tensile strength of the actin cytoskeleton in rac -dependent manner (tavasoli et al. ). this has been considered to be the protective response for podocyte to escape detachment. however, this strategy is not always successful and finally results in podocyte detachment from gbm, as seen in progressive stage of fawnhooded hypertensive (fhh) (kriz et al. c) and doca hypertensive rat model . apoptosis is another cause for podocyte loss under shear stress in hypertension, which is before or in conjunction with cell detachment (kriz et al. ; ying et al. ) . besides mechanical stress, renin-angiotensin-aldosterone system (raas) plays central role in the pathogenesis of hypertensive nephrosclerosis, mainly through its actions on the subtype receptor. mechanical strain increased angiotensin ii production and upregulation of angiotensin receptor (at ) in cultured podocytes, while the increased apoptosis induced by mechanical strain was also in an angiotensin ii-dependent manner (durvasula et al. ) . increased angiotensin ii results in decreased expression of podocin and integrin β , which are both vital in viable podocytes adhesion to the gbm and interaction of podocytes with other gbm components. this might elucidate that the elevated intraglomerular pressure is translated into a maladaptive response in podocyte probably due to the activation of local tissue angiotensin system. furthermore, angiotensin ii is also considered to be associated with the rearrangement of the actin cytoskeleton (macconi et al. ) . aldosterone, an important mediator of the effect of angiotensin, has become a hot spot concern in hypertensive nephropathy. using only the inhibition of aldosterone by eplerenone dramatically alleviated podocyte injury in dahl salt-hypertensive rats, an animal model inclined to hypertensive glomerulosclerosis (nagase et al. ) . in a doubleblind, randomized, placebo-controlled trial, additional use of low-dose eplerenone to renin-angiotensin system inhibitors has renoprotective effects in hypertensive patients with non-diabetic chronic kidney disease (ando et al. ). these findings suggested that aldosterone plays an important role in hypertension-induced podocyte injury. the underlying mechanism is primarily due to aldosterone-induced mitochondrial dysfunction, which increased oxidative stress. in uninephrectomized rats infused with aldosterone and fed with high-salt diet, podocyte-associated proteins nephrin and podocin were dramatically decreased, along with reduced nicotinamideadenine dinucleotide phosphate oxidase activation, increased oxidative stress, and enhanced aldosterone effector kinase sgk . thus, podocyte is the prominent target for aldosterone by inducing oxidative stress and sgk ). selective mineralocorticoid receptor (mr) antagonist eplerenone also ameliorated the saltinduced proteinuria and podocyte injury in hypertensive rat model ). after detachment from gbm, podocyte moves through meshes of bowman's capsule to the urine and might keep alive. unfortunately, the detection of viable podocytes in the urine is a complex procedure, which is still unavailable in all laboratories. however, elevated mrna levels of podocin and nephrin can be examined in urine of hypertensive patients (kelder et al. ) . recent studies suggested that increased podocyte-derived extracellular vesicles may predict podocyte stress and subsequent podocyte loss in hypertensive patients, which might provide a novel non-invasive detective method (kwon et al. ) . podocytes, as the gatekeepers of protein in glomerular filtration barrier, are major targets of high blood pressure. in all, hypertension could cause mechanical stress and the activation of raas (mentioned below). mechanical stress further induces capillary hypertension, promoting glomerular hypertrophy and hyperfiltration. these changes would lead to reduced podocyte density and the reorganization of cytoskeleton in podocytes, resulting in detachment of viable podocyte and podocyte apoptosis, progressing to final glomerulosclerosis. more studies are needed to prove that podocytes can be the detective marker for hypertensive nephrosclerosis and find the more specific method for early diagnosis and treatment. hemodynamic changes and ras of the glomeruli are key factors of ckd patients' persistent proteinuria and disease progression. many investigations suggest that local intrarenal ras activation contributes to kidney tissue injury (gurley et al. ) , and ras activation accelerates renal injury by various mechanisms. angiotensinogen (agt), the original of ras, transforms into ang ii through the conversion of ang i as a result of the enzymatic cleavage process by renin and ace. as the most active peptide of ras, ang ii was demonstrated to induce tgf-β expression and provoke oxidative stress and inflammation, which are main factors in the initiation, development, and progression of ckd (ruggenenti et al. ) . under a condition of continuous glomerular hypertension in ckd, podocytes may undergo actin cytoskeletal reorganization, compensatory hypertrophy, weakened local adhesion ability due to downregulation of adhesion molecules of basement membrane cells, and apoptosis of podocytes induced by local ang ii activation. the continuous increase of ang ii caused by mechanical stress further affects the capillary intraglomerular pressure, resulting in a vicious circle and contributing to the pathogenesis of glomerulosclerosis (ruster and wolf ) . in addition to causing podocyte lesions by altering glomerular hemodynamics, ang ii also has a direct effect on the structure and functions of podocytes, which is mentioned later in this section. podocytes, in possession of a complete ras (marquez et al. ) , can produce functional ras elements themselves and participate in local ras systems as well, playing an important role in not only its own physiological process but pathological status ( fig. . ) . it has been reported that mechanical stress and high glucose could increase the production of local ang ii and at receptor (at r) in podocytes (durvasula et al. ; durvasula and shankland ) , with inducing the expression of other ras elements (sakoda et al. ) . there are also important elements of the ras system expressing in human differentiated podocytes, including angiotensin, renin, ace, at r, and at r subtype mrna, but the related proteins were not detected (liebau et al. ) . therefore, podocytes could not only be a target of the damage caused by ang ii, but a source of localized ang ii as well. however, it has been found that ang ii secreted by podocytes was not blocked by renin inhibitors, acei, and chymase inhibitors (liebau et al. ) , suggesting that there might be an unknown pathway for ang ii formation in podocytes. velez et al. ( ) used matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi-tof-ms) to quantify the presence of rasrelated peptide chains in rat podocytes, in order to further explore the role of podocytes in the metabolism of ras elements. as a result, after co-incubated with ang i, mesangial cells mainly produced ang ii while the main product of podocytes was ang ( - ) with almost no ang ii. furthermore, it was confirmed that podocyteproducing ang ( - ) is mainly through the neprilysin pathway, as ace-mediated ang ii production did not result in an increase of ang ii concentration in podocytes, which might be related to podocytes' degradation of ang ii through ace and aminopeptidase a pathways. as a new member of ras, ace might have a negatively regulatory effect on ace-produced ang ii of traditional ras, mainly by accelerating the degradation of ang ii to attenuate its effect, and through the generation of ang ( - ), which has the most expression in podocyte ras. there has been no evidence that podocytes express the receptor of ang ( - ), i.e., mas, yet ang ( - ) and its receptor seem to be involved in the renal protection for dn, such as regulating inflammation, oxidative stress, and retaining the progression of renal fibrosis. therefore, podocytes probably play an essential role in maintaining the balance of local ras system in the kidney, similar to that between systemic ang ii and intrarenal ras system, by degrading the systemic ang ii filtered from the glomeruli, and/or promoting the conversion of glomerular-filtrated ang i and agt to ang ( - ), thereby regulating the damage caused by the whole systemic ang ii to the kidney. the pathways of ang ii signaling mediating podocyte injury can be generally divided into the following aspect: ( ) to damage the function of the pore membrane and structure of the cytoskeleton the sd is an essential structure of the glomerular filtration barrier, which is connected to the foot processes adjacent to podocytes. nephrin and zonula occludens (zo)- are main proteins of sd, preventing macromolecules from entering the urine. it has been found that sd is susceptible to damage, leading to decreased expression of nephrin and zo- , and cytoskeletal reorganization of podocytes. it has been found that the expression of nephrin in renal biopsy specimens from patients with t dm-induced dkd was significantly reduced compared with healthy volunteers, and the patient's urinary concentration of nephrin was significantly positively correlated with their urinary protein level (jim et al. ) . ren et al. ( ) have found in vitro that ang ii could directly cause the downregulation and dephosphorylation of nephrin, which mediates podocyte injury. besides, application of acei and arb has been reported to inhibit the rearrangement of cytoplasmic zo- and reduced the degree of proteinuria (macconi et al. ) . ( ) to induce podocyte apoptosis one of the main causes of podocyte loss in ckd patients is podocyte apoptosis, and the occurrence of urinary podocyte plays an important role in glomerular sclerosis. ang ii reportedly could induce the apoptosis of rat podocytes cultured in vitro in a dose-and time-dependent manner, and this process required cells to be exposed to tgf-β and tgf-β antibody could inhibit apoptosis of podocytes (ding et al. ) . after activation of tgf-β in diabetic glomeruli, the nuclear factor κb might be inhibited via the gene smad , resulting in podocyte apoptosis. the advanced glycation end products (ages) were also found to activate the ras system of podocytes, upregulate ang ii levels, and induce podocyte apoptosis via a ages receptor-pik /protein kinase b (akt)-dependent signaling pathway; arb could attenuate ang ii-induced podocyte apoptosis. ( ) to cause cell phenotypic transformation and hypertrophy p kip encodes a protein which belongs to cyclin-dependent kinase (cdk) inhibitor proteins, which could control the cell cycle progression at g phrase, thereby inhibiting cell proliferation. it was found that ang ii could directly increase the levels of p kip mrna and protein in podocytes cultured in vitro and in vivo in dn, which was inhibited by arb. ang ii-induced upregulation of p kip expression might lead to podocyte hypertrophy (xu et al. ) . it was also observed that ang ii can upregulate the expression of p kip protein, causing pathological podocyte hypertrophy similar to that in a dn text (romero et al. ) . as an essential factor promoting the progression to renal fibrosis, emt in podocytes will result in loss of epithelial markers with de novo expression of emt markers; in more severe cases, it may lead to podocyte detachment from the glomerular basement membrane, thereby aggravating proteinuria and glomerulosclerosis loeffler and wolf ) . a recent study reported that a high concentration of glucose and ang ii promoted emt in podocytes, which could be reversed by silencing tcf (bai et al. ). ( ) to induce podocyte membrane depolarization and damage the charge barrier studies by using patch clamp recording technique in isolated glomeruli in vitro have demonstrated that ang ii could cause sustained and irreversible depolarization of podocyte membranes. stimulation of ang ii resulted in an immediate calcium influx of cultured podocytes (greka and mundel ) . studies have confirmed that trpc colocalized with podocyte nephrin and podocin, and its functional mutation could disrupt the integrity of the pore membrane, leading to proteinuria and fsgs (reiser et al. ; winn et al. ) . numerous studies have found that abnormal calcium signaling may be the main cause of related podocyte diseases. for example, calcium increases evoked by ang ii are primarily mediated via trpc channels and this pathway could be pharmacologically targeted to abate the development of dkd (nijenhuis et al. ; sonneveld et al. ). ( ) to induce podocyte autophagy as terminally differentiated cells, podocytes mainly reduce intracellular accumulation of damaged dna and macromolecular substances through autophagy rather than cell division (pan et al. ) . in vitro experiments, animal experiments, and human kidney biopsy indicate that podocytes have a high-level basis of autophagy, which plays an important role in maintaining the stability of podocytes. recent research using a ckd animal model has demonstrated that autophagy is an essential intracellular process to encourage the survival of renal cells (huber et al. ) , while excessive and dysfunctional autophagy might result in podocyte injury (de rechter et al. ) . it has been found that ang ii could enhance the ros production and increase oxidative stress in the renal system by enhancing the activity of systematic nadph, leading to detrimental podocyte autophagy (de rechter et al. ; yadav et al. ) , the underlying pathways of which is dependent or independent on mtor (mao et al. ) . a recent study has found that autophagy could enhance the cell viability of ang ii-treated podocytes, suggesting improving autophagy may become a new targeted therapy to relieve ang ii-induced podocyte injury . traditionally concerned solely as an inactive precursor of renin, prorenin actually participates in the functional regulation of body through the hydrolysis of agt to produce ang i and can also bind to prorenin/renin receptor (prr) (non-proteolytic pathway) to activate, like mitogen-activated protein kinases (mapks), initiating intracellular signal transductions. the plasma prorenin/renin ratio in diabetic patients was significantly higher, and the prorenin levels began to increase before the appearance of micro-albuminuria without changes in renin levels (sakoda et al. ) , suggesting that prorenin itself exerts somewhat important effects on dn. immunofluorescence double-labeling studies have showed that prorenin activated by non-proteolytic pathway coexisted with the podocyte marker nephrin, and electron microscopy also displayed that prr was distributed on podocyte foot processes (ichihara et al. ) . handle region peptide (hrp) is a polypeptide blocker of prorenin receptor. ichihara et al. ( ) have found that gene deletion of at r or using acei inhibitor could to some extent delay the occurrence of proteinuria and glomerular sclerosis in streptomycin-induced dn rats, while continuous instillation of hrp could almost completely block the progression of dn. it is noteworthy that the mapk signaling pathway was activated in at r-deficient mice, and hrp could significantly inhibit mapk, indicative of an equally important role of prorenin coupling with prr-induced angiotensin-independent pathway in diabetic kidney injuries. besides, sakoda et al. ( ) have confirmed that adding prorenin to human podocytes cultured in vitro could increase the intracellular level of ang ii and activate the mapk intracellular signal transduction pathway, resulting in podocyte damage. in mammalians, the acute-phase reaction is beneficial for eliminating acute insults for protection against microorganisms, limiting tissue damage, and maintaining homeostasis. this reaction would become disadvantageous under a chronic condition called micro-inflammation. micro-inflammation is a state with low-intensity, chronic persistent and dominant inflammation caused by the infection of non-pathogenic microorganisms, which is characterized by mild persistent elevation of inflammatory cytokines in the systemic circulation (kaysen ; schomig et al. ) . micro-inflammation is a continuous and relatively secretive action, the essence of which is immune inflammation. micro-inflammation state has no obvious clinical symptoms, there is no specific diagnostic criteria, and the diagnosis of micro-inflammation relies mainly on the examination of circulating inflammatory biomarkers such as c-reactive protein (crp) and serum amyloid a (saa), tumor necrosis factor alpha (tnf-α), and interleukin- (il- ). the acute-phase reactants including the above proteins are mainly synthesized by hepatocytes, such as complement components, coagulation proteins, and metal-binding proteins. it is important to note that when we are in the diagnosis of micro-inflammatory state, other causes and diseases of increased inflammatory markers must first be ruled out, such as connective tissue disease and recent microbial infection. during acute-phase reaction, the concentration of crp may increase over fold compared with normal levels (kaysen ). in addition, crp follows the course of a disease with little delay due to its short half-life. crp is supposed to bind multiple other binding specificities such as opsonin of bacteria, immune complexes, and chromatin. crp reflects not only the activity of inflammation, is also a sign of cytokine activation, its levels was positively associated with the degree of infection. the diagnosis of state of micro-inflammation based on crp is the level of crp > mg/l but not more than - mg/l. saa is a sensitive acute-phase reactant in micro-inflammatory state. the level of saa obviously rises before other acute-phase reaction proteins. a variety of inflammatory cytokines have emerged as being closely involved in the micro-inflammation state. immune cells and intrinsic renal cells such as podocytes secrete proinflammatory cytokines including interleukin- (il- ), il- , tnf-α, and monocyte chemoattractant protein- (mcp- ), which may contribute to the inflammatory process and aggravate diseases progression. for dn as an example, a strong induction of mcp- and keratinocyte chemoattractant (kc) by fetuin-a (feta) or lipopolysaccharide (lps) is associated with exacerbated palmitic acid-induced podocyte death. moreover, the prevention of mcp- and kc secretion and inhibition of il- attenuates the inflammatory and ultimate cell death response elicited by feta alone or combined with palmitic acid. the study offers evidence that inflammation aggravates palmitic acid-induced podocyte death and the il- β signaling might be novel potential therapeutic targets for prevention and treatment of dn (orellana et al. ) . infiltrating macrophages/monocytes are associated with chronic, low-grade inflammation. the macrophages can interact with resident renal cells to generate a proinflammatory micro-environment that amplifies tissue injury and promotes scarring. macrophage-derived tnf-α had a direct role in the progression of dn. conditional deletion of tnf-α from macrophages markedly reduced albuminuria, lessening the increase of plasma creatinine and histopathologic lesions (awad et al. ) . likewise, tonicity-responsive enhancer-binding protein (tonebp) in macrophages promotes hyperglycemia-mediated proinflammatory activation and chronic renal inflammation leading to dn and ckd (choi et al. ). lipids such as triglycerides and cholesterol may accumulate ectopically in the kidney, which contributes to a lipotoxicity process. palmitic acid-treated podocytes had intracellular lipid accumulation and abnormal lipid metabolism, accompanied by the process of inflammation, insulin resistance, and rearrangements of the sd and actin cytoskeleton of podocyte. thus, lipotoxicity accelerated podocyte damage through lipid accumulation related inflammation (martinez-garcia et al. ) . lipoproteins including ldl, vldl, and idl might act as proinflammatory mediators, which promote the production of inflammatory cytokines, such as tgf-β, platelet-derived growth factor (pdgf), and il- secreted from human mesangial cells. lipoprotein-mediated cytokine production may cause recruitment of monocytes, lipid-mediated cell proliferation and apoptosis, and extracellular matrix production, thus contributing to podocyte injuries and glomerulosclerosis. chronic inflammation can reduce podocyte insulin sensitivity. nucleotide-binding oligomerization domain-containing (nod ) is a subtype of intracellular pattern recognition receptor (prr), playing functions in innate immunity. of particular interest, increased levels of nod were observed in dn patients and high fat diet (hfd)/stz-induced mice models. furthermore, hfd/stz-induced diabetes mice models with nod knock-out showed reduced podocyte injury and proteinuria compared with wild-type diabetic mice (du et al. ) . in vitro, nod which was activated by bacterial component muramyl dipeptide in podocytes reduced insulininduced glucose uptake and inhibited serine phosphorylation of irs- . another study has explored the role of other prr toll-like receptors (tlrs) in the db/db mice model of dn. administration of a selective tlr / / inhibitor git improved insulin sensitivity, reduced albuminuria and urinary nephrin levels, indicative of reduced podocyte damage. tlr expression in podocytes was found to be highest expressed (cha et al. ) . given the links between some specific prrs activation and insulin stimulation in podocytes, how podocyte insulin responses are altered following prrs activation and inhibition may need specifically investigated. ikb/nf-κb is another important pathway of insulin resistance in podocyte, and nf-κb expression was increased in kidney tissues of patients with type diabetes. nf-κb can increase the level of irs serine phosphorylation and the expression of inflammatory mcp- , il- , and tnf-α. moreover, the increased expressed inflammatory factors can further activate the nf-κb. the inflammatory cytokines and the activation of nf-κb pathway form positive feedback to induce insulin resistance. xu et al. reported that chronic systemic inflammation exacerbates lipid accumulation in the kidney of apoe knockout mice by diverting lipid from the plasma to the kidney via the scap-srebp -ldlr pathway and causing renal injury (xu et al. ) . consisted with this, il- β stimulation in vitro increased the lipid accumulation in the podocytes by increasing the expression of lipid metabolism related proteins, for instance, ldlr, sterol regulatory element-binding protein- (srebp- ) and srebp cleavage-activating protein (scap), and through promoting translocation of the scap/srebp- complex from the endoplasmic reticulum to the golgi in the podocytes (zhang et al. b) . compared with db/db mice, podocyte injury was more severe in db/db mice with subcutaneous casein injections, which are supposed to induce inflammatory stress in vivo. altogether, inflammation may be associated with high risk for chronic renal fibrosis. activation of intrinsic proinflammatory signaling in podocytes such as nf-κb signal pathway aggravates podocyte injury and proteinuria. in stz-induced diabetic mice models with ccr knock-out, transgenic ccr overexpression in the podocytes resulted in significantly increased albuminuria and podocyte loss, without concurrent increase in kidney macrophage infiltration or inflammatory cytokine production. these findings support that activation of ccr signaling cascade in podocytes mediates diabetic renal injury, which is independent of macrophage recruitment (you et al. ). il- , a proinflammatory cytokine which is upregulated by high glucose and tgf-β , can increase mcp- and tgf-β expression in podocytes and induce apoptosis in podocytes through activating caspase- . in stz-induced early dn mice models, anti-il- monoclonal antibody ( e) treatment or il- r -deficiency led to lower blood glucose and improved renal functions, and il- is proved to be expressed in podocytes. collectively, intrinsic proinflammatory signaling in podocytes contributes to podocyte damage ( fig. . ). immune injuries are common causes of podocyte damage. processes interfering with podocyte's structural or functional integrity lead to disruption of the glomerular filtration barrier. primary-cultured human podocytes synthesize and secrete complement c physiologically, and the stimulation of inflammatory factor inf-γ could increase the production of c . under physiological conditions, c produced by glomerular podocytes can resist the invasion of foreign pathogens and protect local tissues. c activation can lead to decreased immune complex formation and increased disintegration. on the other hand, c activation leads to increased production of vasoactive molecules and chemokines, which in turn recruits more inflammatory mediators into the glomerulus. the activation of complement would produce proinflammatory components of complement, i.e., c a. in immune complex diseases and ischemia-reperfusion injury, c a is an important mediator that triggers an inflammatory cascade (heller et al. ) . the kidney is one of the organs that are most susceptible to abnormally activated complement, which can be seen in various glomerulonephritis. the main pathogenesis of idiopathic membranous nephropathy (imn) is caused by the binding of igg to the intrinsic antigen on the basement membrane side of glomerular podocytes, which combine to form an antigen-antibody complex, thereby activating the complementforming membrane attack complex (takano et al. ) . in imn, the concentrations of complement cleavage products such as c a, c a and c b- are significantly increased. c b- is the final product of complement activation in three pathways of complement activation, causing podocyte injury not through conventional lysis, but probably via the mechanism related to the activation of corresponding intracellular signaling pathways in a subdissolved form. ronco and debiec have confirmed that the podocyte surface antigen megalin binded to the corresponding antibody underwent an immune complex reaction, activated the complement system, and promoted the formation of the membrane attack complex c b- (ronco and debiec ) . as a stimulant of podocytes, c b- could destroy podocyte cytoskeletal proteins, inserting in the membrane to increase cell permeability, and activating a series of transduction pathways, resulting in the diffuse thickening of gbm and defects in glomerular filtration barrier, clinically leading to significant proteinuria. in addition, podocytes begin to express complement receptor (cr , or c br, or cd ) during the capillary synthesis stage of renal development and are evenly distributed on the cell membrane and the membrane of foot processes. cr is expressed as a cofactor of the complement factor i and expressed in most circulating cells. cr is the only physiological blocker of complement synthesis in podocytes and inactivates the lysate of complement to promote the clearance of immune complexes, protecting podocytes from complement-mediated damage (alexander et al. ). it has been reported that the production of cr was reduced in several glomerular diseases, making podocytes vulnerable to complement attacks. complement regulatory proteins include crry, cd , and decay acceleration factors (daf or cd ), which are vital to limiting the activation of podocyte complement (cheng et al. ) . podocyte expression of crry and cd could inhibit c invertase and the synthesis of c b- , thus to protect podocytes from injuries induced by antibody-complement activation. in addition, podocytes both in vitro and in vivo could be detected of daf. in a mouse model of nephritis, deficiency of daf resulted in serious podocyte foot fusion, indicating that daf might protect podocytes from complement-mediated injury (bao et al. ). in both physiological and pathological texts, podocytes of humans, rats, and mice all express the receptors of cytokines interleukin (il- ), il- , and il- . after stimulating podocytes cultured in vitro with il- and il- , the skeletal structure and intercellular-link protein of podocytes were damaged and the permeability increased (ha et al. ; kim et al. ) , suggesting that il- and il- could damage podocytes by binding to its receptors. in early minimal change disease (mcd), fsgs, and mn, podocytes increasingly express inflammatory mediators il- α/β along with il- type receptor (il- ri), and il- ri is decreasingly expressed at late stage of the disease when glomerular cell hyperplasia and sclerosis appear (brahler et al. ) , indicating that these molecules participate in podocyte damage and repair, glomerular local inflammation. in addition, both podocytes cultured in vitro and renal tissue express receptors of functional cc chemokine receptor (ccr) and cxc chemokine receptors (cxcr), which could couple with corresponding chemokines to promote the production of cytoplasm ca + and ros and be involved in podocyte injuries (huber et al. ) . moreover, it has been found that podocytes themselves could produce il- (ligand of cxcr /cxcr ), thus podocytes could be activated via autocrine. cxcl might play an important role in the inflammatory response of kidney diseases. podocytes overexpress cxcl under the stimulation of proinflammatory factors. soluble cxcl plays a chemotactic role in inflammation and immune response, while transmembrane cxcl removes oxldl (gutwein et al. ), which is harmful to the kidney. therefore, abnormal expression of cxcl in podocytes might cause renal damage due to excessive immune-inflammatory reaction or an accumulation of oxldl. it has been found that the expression of cxcl and oxldl in the glomeruli of mn patients increased not only significantly but consistently as well (gutwein et al. ). the inflammatory factor ifn-γ is the strongest stimulator of cxcl , which upregulates several forms and overall cell expression levels of cxcl , consequently promoting podocyte damage (wang et al. ) . under physiological conditions, podocytes of humans and mice could express tlr . stimulating cultured murine podocytes in vitro with the ligand of tlr -like lps, lipid a, and fibrins (endogenous ligand), resulted in an increasing expression of ccl and cxcl. in the mouse model of cryoglobulinemia membrane proliferative glomerulonephritis, podocytes expressed more tlr , promoting the synthesis and secretion of chemokines and further leukocyte recruitment and glomerular injury (banas et al. ) . it has been shown that under the stimulation of endogenous tlr ligand, podocytes upregulate tlr , promote the production of proinflammatory chemokines, and actively participate in the recruitment of inflammatory cells, all leading to glomerular injuries (banas et al. ) . apart from tlr , other members of the tlr family have also been proved to participate in podocyte injury. a recent study has pointed out that the overexpression of tlr- correlates with the progression of podocyte injury in glomerulonephritis, suggesting that altered levels of urinary tlr mrna might reflect the degree of podocyte injury in murine autoimmune gn (kimura et al. ) . tlr- and tlr- expressed by b cells and dendritic cells have been considered as important molecules involved in the pathogenesis of systemic lupus. recent study demonstrated that active ln onset in childhood expressed more tlr- , accompanied by weakened expression of podocyte sd protein nephrin, podocin, and synaptopodin; in the meantime, patients showed proteinuria and high ds-dna antibody and low complement (machida et al. ) . therefore, under pathological conditions, tlrs link the innate immune system with podocyte and glomerular injuries. b - (cd ) belongs to the immunoglobulin superfamily, mainly expressed in antigen-presenting cells, and provides a costimulatory signal by coupling with corresponding molecular receptors expressed on t cells, i.e., cd and ctla , regulating the immune responses induced by activated t cells. it has been found that b - was expressed on podocytes of lupus nephritis (ln) (reiser et al. ) , and the expression of podocyte b - in ln patients and ln mouse models is positively correlated with the degree of proteinuria. however, new evidence has stricken up a discordant tune (baye et al. ), leading to further mandatory studies of the application of b - blockers in treating proteinuric patients (novelli et al. a) . studies have shown that under the induction of hypoxia, high glucose, or bacteriocin lipopolysaccharide (lps), the expression of b - would be induced in podocytes which does not occur under physiological conditions, and participate in podocyte cytoskeletal reorganization and the pathogenesis of proteinuria (chang et al. ; fiorina et al. ; shimada et al. ) . in the glomerulus of nephritis, podocyte-expressed b - may also recruit t cells to where gbm is damaged and promote further inflammation. podocytes from necrotic crescentic nephritis rat model and cultured rat podocytes in vitro could express both mhc i/ii molecules and intercellular adhesion molecule (icam- ) after stimulation of ifn-γ, suggesting that cytokines could present the antigen to infiltrating t cells (goldwich et al. ) . recently, it has been pointed that compared to normal people, mcd patients but not fsgs patients excreted more urinary b - , while podocytes of relapsed mcd patients and fsgs patients did not express b - , thus b - might be used to identify mcd and fsgs (novelli et al. b ). the glomerulus is a well-recognized target of miscellaneous immune-mediated injuries, and the pathogenesis of immune-mediated glomerular disease is multifactorial ( fig. . ) . in mn, the surface molecules of glomerular podocyte act as antigens and trigger systematic immune responses, resulting in the formation of in situ immune complexes. the classic animal model of mn, heymann nephritis, reproduces typical mesangial lesions by eliciting auto-antibodies against the podocyte membrane protein megalin in rats (ronco and debiec ) . it has been reported in vivo that the occurrence of human newborns mn was due to the production of auto-antibodies against glomerular podocyte membrane proteins. neutral endopeptidase (nep) is a membrane protein expressed on the surface of human podocytes. studies have shown that neonatal mn occurs due to the presence of anti-nep auto-antibodies in children (herrmann et al. ). its origin is due to the mother's carrying the relevant mutation gene and lacking nep. if the mother bred a normal healthy fetus, the mother will produce an anti-nep antibody against the fetus during pregnancy and the antibody enters the fetus through the placenta. anti-nep antibodies react with nep antigens on fetal podocytes, forming an immune complex on the epithelial side, leading to neonatal mn. although the incidence of this type of patients is very low, its pathogenesis confirms the role of anti-podocyte antigen antibodies in the development of human mn (pozdzik et al. ) . t cell dysfunction and the release of cytokines (circulatory factors) causing podocyte injury are associated with the formation of proteinuria in mcd patients. it is currently believed that the cytokines produced by th and th cells in t cells are involved in the occurrence of mcd, but the cytokines produced by th cells (il- , il- , il- ) might be more important (mack ). animal experiments have found that the injection of il- to rats can reduce the content of heparin sulfate on the surface of podocytes, weaken the membrane filtration barrier of charge, and trigger proteinuria. there are also receptors for il- and il- on the podocyte, and the increase of circulating or local il- and il- can directly damage the podocyte through the receptors on the podocyte and increasing the permeability of the filtration membrane. shimada et al. ( ) have proposed that mcd is the result of a "two-hit" attack from podocyte immune dysfunction: the first hit is the effects of bacterial products, viruses, and various cytokines on podocytes, resulting in an abnormal expression of cd in podocytes, and further cytoskeleton reorganization and morphological changes of podocytes, increasing the permeability of gbm which might bring about proteinuria. however, due to the self-regulation of the body, podocytes can downregulate the expression of cd . if the auto-regulatory function of podocytes and the body is defective, the sustained expression of cd would lead to proteinuria and even mcd. moreover, ishimoto et al. ( ) also observed increasing expression of cd in the urine of mcd patients; in view of the fact that the expression of cd in podocytes can be induced by il- and bacterial products through the tlr pathway and regulated by ctla , suggesting that defective immune functions of podocytes is an essential cause of mcd. certain exogenous antigens (small molecular weight, positively charged) are implanted on the epithelial side and can also lead to the formation of in situ immune complexes. hepatitis b virus (hbv)-associated nephropathy is often manifested as mesangial lesions (especially in children), and hbeag plays an important role in its occurrence. the hbeag molecule is of small mass and negatively charged and can be implanted across the glomerular basement membrane (gbm) on the epithelial side, triggering the formation of in situ immune complexes (gupta and quigg ) . under inflammatory conditions, podocytes would inhibit the expression of mhc class ii molecules, promoting the remove of immune complexes from the gbm. in some cases, podocytes might act as antigen-presenting cells themselves, taking up and processing antigens to initiate specific t cell responses. there has been evidence that transgenic mice with a loss of mhc class ii exclusively in podocytes developed only a very moderate degree of nephrosclerosis and glomerular crescent formation compared to the control animals, indicative of their defective capacity to activate cd + t cells (goldwich et al. ). viral infection, such as human immunodeficiency virus (hiv)- , parvovirus b , cytomegalovirus (cmv), hepatitis b virus (hbv), and hepatitis c virus (hcv), is associated with podocyte injury. hiv-associated nephropathy (hivan) mostly manifests collapsing glomerulopathy or classic fsgs (chandra and kopp ) . podocyte infection is associated with podocyte injury and dedifferentiation and rapid loss of renal function. studies have reported that hiv virus can be internalized by podocytes in vitro, which might be associated with receptors, such as viral coat protein gp , and subsequent endocytosis, phagocytosis, or pinocytosis (bruggeman ) . although the transmission of virus in vitro has been well documented, further studies are needed to demonstrate the definite mechanism by which the virus enters podocyte in vivo. structural viral proteins, gag and pol, and non-structural proteins, vpr, nef, and tat, have been considered to be associated with hivan (conaldi et al. ; reid et al. ; zuo et al. ). hbv is a major cause for membranous nephropathy and fsgs scarcely, which can be diagnosed by evidence of hbv antigen or antibodies on kidney biopsy. the possible mechanisms of hbv-induced podocyte injury might be as follows: detective infection of the cells by hbv, deposition of circulating immune complex in renal cells, effects of hbv-induced immunological mediators (bhimma and coovadia ; sakai et al. ) . podocytes are also targets of some toxicity drugs, which may further progress to glomerulosclerosis. for example, gold, bucillamine, and d-penicillamine, which are used for the treatment of rheumatoid arthritis, are confirmed to cause mn. the possible mechanism might be closely related to stress, energy metabolism, and inflammation (fujiwara et al. ; seguin et al. ) . other drugs, like non-steroid anti-inflammatory drugs and interferon, also can be inductor of podocyte injury. organic solvents, like gasoline, dimethylbenzene, and formaldehyde, can induce podocyte injury including foot process fusion and decreased expression of nephrin and podocin (qin et al. ) . hypoxia can be induced by various pathogenic conditions including hypertension and diabetes. chronic hypoxia can trigger endoplasmic reticulum (er) stress, which result in increased ros. nephrin and alpha-actin- , the structural components of sd, are subject to mutations, which cause defective protein folding in the er of podocytes. the underling mechanism might include transient receptor protein and complement complex and increased expression of mcp- cybulsky ; maekawa and inagi ) . targeting hypoxia and er stress and the possible signal networks might be the novel target for intervention of podocyte injury in ckd. living in an environment of a variety of pathological stresses and stimuli, podocytes adapt to maintain the integrity and stability of the glomerular basement membrane, depending on their highly differentiated characteristics which also reflect the vulnerability of this barrier. the different responses of podocytes to injury are associated with the pathology and prognosis of glomerular diseases. as a vital type of renal intrinsic cells, podocyte damage is an important cause of nephrotic proteinuria and glomerular sclerosis. however, as a highly differentiated terminal cell, podocyte has no proliferative potential, and loss of podocyte is associated with poor renal outcomes such as increased proteinuria, glomerulosclerosis, and renal disease progression. podocytes have different responses to injuries, including endoplasmic reticulum stress and autophagy reactions caused by abnormal energy metabolism. this chapter lists several aspects of podocyte injuries along with potential underlying mechanisms, including glucose and lipid metabolism disorder, hypertension, ras activation, micro-inflammation, immune disorder, and other factors. these aspects are not technically separated items, but intertwined with each other in the pathogenesis of podocyte injuries. injured podocytes would undergo a series of morphological changes: fp disappearance, cellular shrink, pseudocysts form, cell hypertrophy, cytoplasmic lysosomal enrichment, etc. these changes eventually cause podocytes to detach from the gbm. moreover, due to the lack of proliferative capacity, the number of glomerular podocytes would become less and less, until reduced by more than % when glomerulosclerosis occurs. glomerulosclerosis is not a specific disease but a state representing podocyte injury which is mediated by diverse causes. podocytes interact with gbm and capillary loops tightly, dysfunction of which is an early event leading to glomerulosclerosis. glomerulosclerosis seems like a station to stay in just before arriving to destination. unanswered questions in the pathogenesis of podocyte injury and glomerulosclerosis are still ill-defined, and the causing list will continue to grow. uncovering the selective targeting to pathogenesis and underlying mechanism of podocyte injury and glomerulosclerosis is bound to provide clues to answer for treatment and prevention of the disease in the future. vascular endothelial growth factor and the kidney: something of the marvellous mouse podocyte complement factor h: the functional analog to human complement receptor anti-albuminuric effect of the aldosterone blocker eplerenone in non-diabetic hypertensive patients with albuminuria: a double-blind, randomised, placebo-controlled trial molecular and cellular events mediating glomerular podocyte dysfunction and depletion in diabetes mellitus macrophage-derived tumor necrosis factor-alpha mediates diabetic renal injury knocking down tcf inhibits high glucose-and angiotensin ii-induced epithelial to mesenchymal transition in podocytes tlr links podocytes with the innate immune system to mediate glomerular injury focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in t cells the costimulatory receptor b - is not induced in injured podocytes hepatitis b virus-associated nephropathy lipid metabolism abnormalities in chronic kidney disease intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria hiv- infection of renal cells in hiv-associated nephropathy statins prevent oxidized ldl-induced injury of glomerular podocytes by activating the phosphatidylinositol -kinase/akt-signaling pathway podocyte injury and repair mechanisms renal protective effects of toll-like receptor signaling blockade in type diabetic mice viruses and collapsing glomerulopathy: a brief critical review b - expression regulates the hypoxia-driven cytoskeleton rearrangement in glomerular podocytes altering expression of alpha beta integrin on podocytes of human and rats with diabetes calcium entry via trpc mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes role of nadph oxidase-mediated reactive oxygen species in podocyte injury complement regulatory proteins in kidneys of patients with anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis tonicity-responsive enhancer-binding protein mediates hyperglycemia-induced inflammation and vascular and renal injury evidence linking glycated albumin to altered glomerular nephrin and vegf expression, proteinuria, and diabetic nephropathy human immunodeficiency virus- tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells the human glomerular podocyte is a novel target for insulin action nephrin is critical for the action of insulin on human glomerular podocytes the intersecting roles of endoplasmic reticulum stress, ubiquitin-proteasome system, and autophagy in the pathogenesis of proteinuric kidney disease the roles of connective tissue growth factor and integrin-linked kinase in high glucose-induced phenotypic alterations of podocytes alphaactinin- is required for normal podocyte adhesion autophagy in renal diseases fatty kidney: emerging role of ectopic lipid in obesity-related renal disease angiotensin ii induces apoptosis in rat glomerular epithelial cells nod promotes renal injury by exacerbating inflammation and podocyte insulin resistance in diabetic nephropathy activation of a local renin angiotensin system in podocytes by glucose activation of a local tissue angiotensin system in podocytes by mechanical strain vegf inhibition and renal thrombotic microangiopathy roles of mtor complexes in the kidney: implications for renal disease and transplantation role of podocyte b - in diabetic nephropathy rituximab targets podocytes in recurrent focal segmental glomerulosclerosis podocytes are sensitive to fluid shear stress in vitro proximal tubules and podocytes are toxicity targets of bucillamine in a mouse model of drug-induced kidney injury angiotensin ii induces calcium-mediated autophagy in podocytes through enhancing reactive oxygen species levels nadph oxidases in the kidney podocytes are nonhematopoietic professional antigen-presenting cells balancing calcium signals through trpc and trpc in podocytes cell biology and pathology of podocytes role of receptor for advanced glycation end-products and signalling events in advanced glycation end-productinduced monocyte chemoattractant protein- expression in differentiated mouse podocytes glomerular diseases associated with hepatitis b and c at a angiotensin receptors in the renal proximal tubule regulate blood pressure cxcl is expressed in podocytes and acts as a scavenger receptor for oxidized low-density lipoprotein high glucose and advanced glycosylated end-products affect the expression of alphaactinin- in glomerular epithelial cells montelukast improves the changes of cytoskeletal and adaptor proteins of human podocytes by interleukin- vascular endothelial growth factor b controls endothelial fatty acid uptake effect of retinoic acid in experimental diabetic nephropathy high glucose and angiotensin ii increase beta integrin and integrin-linked kinase synthesis in cultured mouse podocytes atherogenic effects of tnf-alpha and il- via up-regulation of scavenger receptors selection of a c a receptor antagonist from phage libraries attenuating the inflammatory response in immune complex disease and ischemia/reperfusion injury membranous nephropathy: the start of a paradigm shift srebps: activators of the complete program of cholesterol and fatty acid synthesis in the liver inflammation-activated cxcl pathway contributes to tubulointerstitial injury in mouse diabetic nephropathy expression of functional ccr and cxcr chemokine receptors in podocytes podocin and mec- bind cholesterol to regulate the activity of associated ion channels emerging role of autophagy in kidney function, diseases and aging prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptor-deficient mice activated macrophages down-regulate podocyte nephrin and podocin expression via stress-activated protein kinases minimal change disease: a cd podocytopathy dysregulated nephrin in diabetic nephropathy of type diabetes: a cross sectional study soluble flt binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function early mechanisms of renal injury in hypercholesterolemic or hypertriglyceridemic rats the microinflammatory state in uremia: causes and potential consequences quantitative polymerase chain reaction-based analysis of podocyturia is a feasible diagnostic tool in preeclampsia secondary focal segmental glomerulosclerosis: from podocyte injury to glomerulosclerosis b cell-derived il- acts on podocytes to induce proteinuria and foot process effacement overexpression of toll-like receptor correlates with the progression of podocyte injury in murine autoimmune glomerulonephritis podocyte injury-driven intracapillary plasminogen activator inhibitor type accelerates podocyte loss via upar-mediated beta -integrin endocytosis alpha beta integrin has a crucial role in kidney and lung organogenesis podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat a potential role for mechanical forces in the detachment of podocytes and the progression of ckd the role of podocytes in the development of glomerular sclerosis progression of glomerular diseases: is the podocyte the culprit? from segmental glomerulosclerosis to total nephron degeneration and interstitial fibrosis: a histopathological study in rat models and human glomerulopathies development of vascular poleassociated glomerulosclerosis in the fawn-hooded rat the podocyte's response to stress: the enigma of foot process effacement elevated urinary podocyte-derived extracellular microvesicles in renovascular hypertensive patients endoplasmic reticulum stress contributes to beta cell apoptosis in type diabetes mechanisms and consequences of hypertriglyceridemia and cellular lipid accumulation in chronic kidney disease and metabolic syndrome saturated fatty acids induce insulin resistance in human podocytes: implications for diabetic nephropathy characterization of glucose uptake by cultured rat podocytes advanced glycation end products activate smad signaling via tgf-beta-dependent and independent mechanisms: implications for diabetic renal and vascular disease enhanced epithelial-to-mesenchymal transition associated with lysosome dysfunction in podocytes: role of p /sequestosome as a signaling hub functional expression of the renin-angiotensin system in human podocytes nestin protects mouse podocytes against high glucose-induced apoptosis by a cdk -dependent mechanism roles of na(+)/h(+) exchanger type and intracellular ph in angiotensin ii-induced reactive oxygen species generation and podocyte apoptosis epithelial-to-mesenchymal transition in diabetic nephropathy: fact or fiction? rac /pak signaling promotes epithelialmesenchymal transition of podocytes in vitro via triggering beta-catenin transcriptional activity under high glucose conditions the unfolding tale of the unfolded protein response effect of angiotensin-converting enzyme inhibition on glomerular basement membrane permeability and distribution of zonula occludens- in mwf rats expression of toll-like receptor in renal podocytes in childhood-onset active and inactive lupus nephritis podocyte antigens, dendritic cells and t cells contribute to renal injury in newly developed mouse models of glomerulonephritis stress signal network between hypoxia and er stress in chronic kidney disease ginsenoside rg inhibits angiotensin ii-induced podocyte autophagy via ampk/mtor/pi k pathway renin-angiotensin system within the diabetic podocyte renal lipotoxicity-associated inflammation and insulin resistance affects actin cytoskeleton organization in podocytes chylomicron remnants are increased in the postprandial state in cd deficiency metabolism, energetics, and lipid biology in the podocyte-cellular cholesterol-mediated glomerular injury cyclodextrin protects podocytes in diabetic kidney disease glomerular-specific protein kinase c-beta-induced insulin receptor substrate- dysfunction and insulin resistance in rat models of diabetes and obesity structure and function of podocytes: an update podocyte injury underlies the glomerulopathy of dahl salt-hypertensive rats and is reversed by aldosterone blocker salt-induced nephropathy in obese spontaneously hypertensive rats via paradoxical activation of the mineralocorticoid receptor: role of oxidative stress podocyte injury and its consequences progressive podocyte injury and globotriaosylceramide (gl- ) accumulation in young patients with fabry disease glomerular filtration into the subpodocyte space is highly restricted under physiological perfusion conditions angiotensin ii contributes to podocyte injury by increasing trpc expression via an nfat-mediated positive feedback signaling pathway regulation of glucose transporter (glut ) gene expression by angiotensin ii in mesangial cells: involvement of hb-egf and egf receptor transactivation another piece of the puzzle of podocyte b - expression: lupus nephritis any value of podocyte b - as a biomarker in human mcd and fsgs? fetuin-a aggravates lipotoxicity in podocytes via interleukin- signaling presecretory oxidation, aggregation, and autophagic destruction of apoprotein-b: a pathway for late-stage quality control roles of the podocyte in glomerular function cell biology of the glomerular podocyte anti-nep and anti-pla r antibodies in membranous nephropathy: an update membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor ire in different ways human podocyte depletion in association with older age and hypertension attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response mixed organic solvents induce renal injury in rats expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies selective release of human adipocyte fatty acids according to molecular structure cellular responses to endoplasmic reticulum stress and apoptosis an hiv- transgenic rat that develops hiv-related pathology and immunologic dysfunction epithelial-mesenchymal transition and podocyte loss in diabetic kidney disease the glomerular slit diaphragm is a modified adherens junction induction of b - in podocytes is associated with nephrotic syndrome trpc is a glomerular slit diaphragm-associated channel required for normal renal function a cell-type specific ganglioside of glomerular podocytes in rat kidney: an o-acetylated gd angiotensin ii induces nephrin dephosphorylation and podocyte injury: role of caveolin- parathyroid hormone-related protein induces hypertrophy in podocytes via tgf-beta( ) and p (kip ): implications for diabetic nephropathy molecular pathomechanisms of membranous nephropathy: from heymann nephritis to alloimmunization target antigens and nephritogenic antibodies in membranous nephropathy: of rats and men mechanisms and treatment of ckd nephrin is specifically located at the slit diaphragm of glomerular podocytes angiotensin ii as a morphogenic cytokine stimulating renal fibrogenesis angiotensin ii upregulates rage expression on podocytes: role of at receptors focal segmental glomerulosclerosis as a complication of hepatitis b virus infection aliskiren inhibits intracellular angiotensin ii levels without affecting (pro)renin receptor signals in human podocytes podocytes as a target of prorenin in diabetes globotriaosylsphingosine actions on human glomerular podocytes: implications for fabry nephropathy lipid-protein interactions along the slit diaphragm of podocytes localization of the glut glucose transporter in murine kidney and regulation in vivo in nondiabetic and diabetic conditions the microinflammatory state of uremia podocin, a raft-associated component of the glomerular slit diaphragm, interacts with cd ap and nephrin gene expression profiling in a model of d-penicillamine-induced autoimmunity in the brown norway rat: predictive value of early signs of danger the podocyte's response to injury: role in proteinuria and glomerulosclerosis podocyte as the target for aldosterone: roles of oxidative stress and sgk minimal change disease: a "two-hit" podocyte immune disorder? toll-like receptor ligands induce cd expression in human podocytes via an nf-kappab-dependent pathway free fatty acids and their metabolism affect function and survival of podocytes regulation of podocyte survival and endoplasmic reticulum stress by fatty acids involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm curcumin decreases renal triglyceride accumulation through ampk-srebp signaling pathway in streptozotocin-induced type diabetic rats glucose specifically regulates trpc expression in the podocyte in an angii-dependent manner glomerular endothelial fenestrae in vivo are not formed from caveolae glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy poly(adp-ribose) polymerase inhibitors ameliorate nephropathy of type diabetic leprdb/db mice protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury complement-mediated cellular injury loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton expression of advanced glycation end products and their cellular receptor rage in diabetic nephropathy and nondiabetic renal disease the chloride intracellular channel a stimulates podocyte rac , protecting against hypertension-induced glomerular injury epidemiology of hypertensive kidney disease characterization of reninangiotensin system enzyme activities in cultured mouse podocytes systemic and renal lipids in kidney disease development and progression podocyte loss in human hypertensive nephrosclerosis oxldl-induced lipid accumulation in glomerular podocytes: role of ifn-gamma, cxcl , and adam insulin signaling to the glomerular podocyte is critical for normal kidney function a mutation in the trpc cation channel causes familial focal segmental glomerulosclerosis (kip ) knockout mice are protected from diabetic nephropathy: evidence for p (kip ) haplotype insufficiency pten inhibits high glucose-induced phenotypic transition in podocytes angiotensin ii receptor blocker inhibits p kip expression in glucose-stimulated podocytes and in diabetic glomeruli inflammatory stress exacerbates lipid-mediated renal injury in apoe/cd /sra triple knockout mice ang ii promotes autophagy in podocytes epithelialmesenchymal transition as a potential explanation for podocyte depletion in diabetic nephropathy decreased glomerular expression of agrin in diabetic nephropathy and podocytes, cultured in high glucose medium induction of apoptosis during development of hypertensive nephrosclerosis activation of the renin-angiotensin system within podocytes in diabetes podocytespecific chemokine (c-c motif) receptor overexpression mediates diabetic renal injury in mice podocyte-specific overexpression of glut surprisingly reduces mesangial matrix expansion in diabetic nephropathy in mice dysregulation of low-density lipoprotein receptor contributes to podocyte injuries in diabetic nephropathy inflammatory stress exacerbates lipid accumulation and podocyte injuries in diabetic nephropathy dysregulation of the low-density lipoprotein receptor pathway is involved in lipid disorder-mediated organ injury hiv- genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis acknowledgements this study was supported by grants from the national key research and development program of china ( yfc ). key: cord- - he sjpf authors: goldstein, benjamin; trivedi, malav; speth, robert c. title: alterations in gene expression of components of the renin-angiotensin system and its related enzymes in lung cancer date: - - journal: lung cancer int doi: . / / sha: doc_id: cord_uid: he sjpf objectives: the study assessed the existence and significance of associations between the expression of fifteen renin-angiotensin system component genes and lung adenocarcinoma. materials and methods: ncbi's built-in statistical tool, geo r, was used to calculate student's t-tests for the associations found in a dna expression study of adenocarcinoma and matched healthy lung tissue samples. the raw data was processed with genespring™ and then used to generate figures with and without sidak's multiple comparison correction. results: ten genes were found to be significantly associated with adenocarcinoma. seven of these associations remained statistically significant after correction for multiple comparisons. notably, agtr , which encodes the at( ) angiotensin ii receptor subtype, was significantly underexpressed in adenocarcinoma tissue (p < . ). agtr , ace, enpep, mme, and prcp, which encode the at( ) angiotensin ii receptor, angiotensin-converting enzyme, aminopeptidase n, neprilysin, and prolylcarboxypeptidase, respectively, were also underexpressed. agt, which encodes angiotensinogen, the angiotensin peptide precursor, was overexpressed in adenocarcinoma tissue. conclusion: the results suggest an association between the expression of the genes for renin-angiotensin system-related proteins and adenocarcinoma. while further research is necessary to conclusively demonstrate a link between the renin-angiotensin system and lung cancers, the results suggest that the renin-angiotensin system plays a role in the pathology of adenocarcinoma. lung cancer continues to be the leading cause of cancer deaths, and lung adenocarcinomas account for approximately % of all lung cancers (https://www.cancer.org/cancer/ lungcancer-non-smallcell/index (accessed / / )). to assess factors associated with lung adenocarcinomas, a gene expression study comparing normal lung tissue and lung tumor was undertaken using the hg-u a affymetrix gene chip [ ] . while such associations do not establish causation, they characterize the underlying molecular marks or tumors and identify potential target sites for treatments. to ascertain if the renin-angiotensin system (ras) is altered in lung adenocarcinomas we evaluated the expression of genes of the classical ras ( figure ) and extended components of the ras as reported in a previous study [ ] . the ras (table ) functions primarily to maintain fluid and electrolyte homeostasis and regulate the cardiovascular system; however, the ras is pleiotropic and is now recognized to have effects that extend beyond its primary functions. for example, angiotensin ii (ang ii), the primary hormone of the ras, promotes the proliferation of mouse fibroblast t cells (but not sv transformed t cells in culture) [ ] , dna synthesis and proliferation of adrenal cortical cells in culture [ ] , and the hypertrophic and hyperplastic growth of vascular smooth muscle cells [ ] . both myointimal hyperplasia and vascular smooth muscle dna synthesis are increased in rats infused with ang ii [ ] . all at receptor figure : pathways of the renin-angiotensin system (ras) cascade. the classical ras cascade is shown with light shading and includes the two major angiotensin ii receptor subtypes, at and at . nonclassical components of the ras cascade would include the ability of the (pro)renin receptor to activate prorenin, as well as enhance the activity of renin to metabolize angiotensinogen to ang i, the ability of chymase to convert ang i to ang ii, metabolism of ang ii to iii and iii to iv by aminopeptidases a and n, respectively, and the novel at receptor for ang iv which is also known as insulin-regulated aminopeptidase. in addition, there is the ace /ang - /mas axis of the ras which is considered to be a counterregulator of the actions of the classical ras. this includes the enzyme angiotensin-converting enzyme (ace ) which primarily forms ang - from ang ii, but can also form ang - from ang i, prolylcarboxypeptidase which also forms ang - from ang ii, and the enzymes neprilysin and prolylendopeptidase which forms ang - directly from ang i, and mas, the putative receptor for ang - . the names of the genes that encode the various proteins of the ras and related proteins are shown in italics after the protein names. these proliferation-promoting effects of ang ii suggest that the ras might play a critical role in cancer [ ] [ ] [ ] [ ] . canonically, most studies suggest that ang ii acting via the at receptor can promote cancer by a variety of mechanisms. ang ii is angiogenic [ , ] and can promote blood flow to tumors by stimulating vascular endothelial growth factor (vegf) release; see reviews [ , ] . moreover, the tumor vessels are less responsive to ang ii such that the systemic vasoconstrictor actions of ang ii shunt blood to the tumor vasculature [ , ] . stimulation of the at receptor by ang ii transactivates the egf receptor [ , ] , a receptor that is overexpressed in many cancers [ , ] . activation of the at receptor promotes the release of a host of growth factors that also promote cell proliferation [ ] . additionally, in several animal models of cancer, both angiotensinconverting enzyme (ace) inhibitors and at angiotensin receptor subtype blockers (arbs) inhibit tumor growth [ ] . since both ace inhibitors and arbs are widely used antihypertensive agents, meta-analyses have been conducted to identify any correlation for cancer incidence in patients taking these drugs and/or the effects of these drugs on cancer risk. surprisingly, there is not universal agreement on the effects of ace inhibitors and arbs on cancer incidence. for example, a meta-analysis reported in [ ] found a slight increase in cancer risk in individuals that were being treated with arbs. however, a subsequent meta-analysis published the following year [ ] found no association between the use of arbs and cancer risk. however, they did observe an increase in cancer risk in patients taking both an arb and an ace inhibitor. of note the previous study [ ] compared arb + ace inhibitor treated groups compared to ace inhibitor only groups to show the association of arb use with increased cancer risk. an epidemiological study of ace inhibitor and/or arb usage reported an increased risk of cancer deaths [ ] ; however, a study from the united states food and drug administration [ ] found no evidence for an increased cancer risk from arbs. a recent meta-analysis of ras blockade with ace inhibitors and/or arbs suggested that ras blockade reduces cancer risk [ ] . another recent meta-analysis focusing on lung cancer risk in patients taking arbs indicated that arbs reduce lung cancer risk [ ] with all but one [ ] of studies showing a reduced risk of cancer with of the studies showing a significant reduction in lung cancer risk on their own [ , ] . thus the preponderance of clinical data suggests that arbs reduce lung cancer risk but that there may be individual or population differences in lung cancer risks associated with ras blockers. to further explore this putative relationship this study investigates changes in gene expression of ras and related protein in a population of lung adenocarcinoma patients [ ] . this could help to identify specific genes that could be investigated further to determine the exact role of ras and extended ras pathway genes in lung adenocarcinomas. the study is an in silico analysis of the expression of reninangiotensin system genes in connection with lung cancer. a total of renin-angiotensin system related genes were studied (see table and figure ). the gene expression analysis was conducted on data sourced from ncbi's geo database as log values. the specific dataset used, gse , profiled the gene expression of lung adenocarcinoma tissue samples and paired noncancerous tissue samples [ ] . the dataset was analyzed through the geo database's integrated statistics utility, geo r, in addition to prism (v. ), genespring (v. . ), and microsoft excel. more specifically, geo r calculated values in order to identify significant differences in gene expression while genespring extracted and normalized the raw expression data using provided algorithms to allow for further statistical calculations using prism (graphpad software), which was also used to generate graphs and other figures from tables of raw data created in excel. one value for anpep in the tumor group was excluded based upon the criterion of being more than standard deviations from the mean. since the number of samples in this specific data set differed between the tumor ( = ) and the normal tissue ( = ) groups, comparisons were made using an unpaired student's -test with welch's correction for heterogeneity of variance. data is presented with and without sidak's correction for multiple comparisons: = . , = − ( − . ) ( / ) ( genes were studied) to provide a relative estimate of significance ( table ) there were a number of profound differences in the expression of the genes encoding the proteins comprising and closely associated with the renin-angiotensin system (ras), between normal lung tissue and the lung tumor tissue (figures and and table ). additionally, there were substantial differences in the level of expression of the mrna for the different proteins. among the proteins that comprise the classical ras, the gene for the at subtype of the ang ii receptor was the most highly expressed in normal lung tissue (figures and s , and table ). the mrna for the other major ang ii receptor subtype at was expressed at less than half the intensity as the at receptor subtype. the gene expression for both ang ii receptor subtypes was dramatically reduced, and %, respectively, ( figure s ), for at and at in the lung tumor tissue ( < . , corrected for multiple comparisons, table ). the agt gene which encodes angiotensinogen was also highly expressed in the normal lung tissue, and its mrna was nearly doubled in the lung tumor tissue ( < . , corrected for multiple comparisons table ). ace, the gene that encodes the enzyme that converts the inactive precursor angiotensin i (ang i) to the active hormone, ang ii, was expressed at a lower level and its expression was reduced by about half in the lung tumor tissue ( < . corrected for multiple comparisons table ). ren, the gene for renin, the enzyme which cleaves angiotensinogen to make ang i, was the least highly expressed gene of the classical ras proteins and its expression level was unchanged in the lung tumor tissue. genes of the proteins that have ancillary interactions with the ras also showed a considerable variation in expression level in normal lung tissue, and the expression of several of these genes were altered in the lung tumor tissue (figures and s and table ). atp ap which encodes the (pro)renin receptor, but which may have a more important cellular function in promoting hydrogen ion transport and wnt signaling via the frizzled receptor [ ] was the most highly expressed gene in the normal figure : expression of genes of proteins that directly interact with renin-angiotensin system. these genes encode proteins that can alter the function of ras but have many other functions. atp ap encodes the prorenin receptor (atpase h(+)-transporting accessory protein ), cma encodes chymase, lnpep encodes the ang iv (at ) receptor (insulin-regulated aminopeptidase), enpep encodes aminopeptidase a, and anpep encodes aminopeptidase n. one value for anpep was excluded from the data analysis because it was > standard deviations apart from the rest of the dataset. * < . by unpaired test with welch's correction for heterogeneity of variance and sidak's correction for multiple comparisons. lung tissue. there was a small, %, increase in atp ap expression ( figure s ) that was not statistically significant after correction for multiple comparisons. ren expression in lung tumor tissue was also % higher than in normal lung lung cancer international tissue ( figure s ) and also was not statistically significant after correction for multiple comparisons (table ) . cma , the gene for chymase, which can facilitate the ras by converting ang i to ang ii, was expressed at a low level and its expression did not differ between normal lung tissue and lung tumor tissue. lnpep, the gene which encodes the at receptor, but which may have a more important cellular function as insulin-regulated aminopeptidase [ ] , was also expressed at a low level and its expression did not differ between normal lung tissue and lung tumor tissue. expression of enpep, the gene which encodes aminopeptidase a, a major ang ii metabolizing enzyme, was expressed at a moderate level in normal lung tissue with a statistically significant ( < . , corrected for multiple comparisons, table ) % reduction in its expression in lung tumor tissue ( figure s ). anpep, the gene which encodes aminopeptidase n, a major ang iii metabolizing aminopeptidase, was also expressed at a moderate level in normal lung tissue and at a slightly lower level in lung tumor tissue, but did not retain statistical significance ( = . ) after correction for multiple comparisons (table ). the genes for the primary component proteins of the ace /ang - /mas axis of the ras, ace , and mas were expressed at low levels in normal lung tissue and expression levels did not differ between normal lung tissue and lung tumor tissue (figures and s and table ). however, the genes for two enzymes that have ancillary functions associated with the ace /ang - / mas axis; mme and prcp, which encode neprilysin and prolyl carboxypeptidase, respectively, were highly expressed in normal lung tissue. their expression was significantly decreased in lung tumor tissue ( < . , corrected for multiple comparisons, table ) by % and %, respectively (figure s ) . prep, which encodes prolyl endopeptidase, another enzyme that can promote the functionality of the ace / ang - /mas axis, was expressed at a moderate level in normal lung tissue, but its expression was unaltered in lung tumor tissue. in view of the potential involvement of the ras in promoting lung as well as other cancers (see reviews [ , ] ) the extent and changes in the expression of genes encoding proteins of the ras, as well as proteins whose actions include alteration of classical ras activity, are of considerable interest. the high level of expression of the gene encoding the at receptor in the lung suggests that ang ii acting upon the at receptor plays an important role in lung function and cell growth. consistent with an oncogenic activity of the ras in lung, at receptor inhibition decreases the metastasis of lung cancer cells [ ] . see also reviews - . an additional mechanism whereby the ras could increase tumor growth by activating at receptors is by selectively increasing blood flow in newly formed vessels of tumors which, in contrast to the systemic vasculature, do not constrict in response to ang ii [ ] . corollary to such a mechanism, inhibitors of ang ii formation, agents that increase metabolism of ang ii, and at receptor blockers that lower bp may decrease tumor growth by disproportionately reducing blood flow to the tumor. also consistent with the ability of the at receptor to promote cancer cell growth, the at receptor can transactivate the egf receptor, a known oncogenic stimulus [ ] . however, the reduced expression of the at receptor gene in lung tumor cells, suggests that at receptor stimulation of the tumor cells may not be a driving force for their oncogenic activity. conversely, the slightly greater reduction in at receptor gene expression could contribute to the oncogenic activity of lung tumor cells by reducing the inhibitory actions on cell growth that ang ii causes by activating the at receptor. indeed, when at receptors were overexpressed in lung adenocarcinoma cells, their growth was inhibited [ ] . the increased expression of the precursor protein of the ras, angiotensinogen, in lung tumor tissue suggests that there may be an increased activation of the ras in lung tumor cells. angiotensinogen can be rate limiting in the production of ang ii, although the concentrations of renin, prorenin, and the (pro)renin receptor are the primary determinants of the rate of formation of ang i and ang ii. of note, however, there is a non-ras functionality of angiotensinogen to interact with dna, in particular to reverse formation of carcinogenic dna adducts generated by the contents of tobacco smoke [ ] . in this case the increase in angiotensinogen gene expression in lung tumor cells could be a compensatory response of the cells to normalize their dna by removing the adducts formed by tobacco smoke carcinogens. additionally, angiotensinogen, as well as des-angiotensin i angiotensinogen, has antiangiogenic effects lung cancer international and reduces metastasis of b -f melanoma cells to the lungs of mice [ ] . consistent with the reduced expression of ace in the lung tumor tissue, serum levels of ace are decreased in lung cancer patients to a greater extent than in other cancers [ ] . while ace inhibitors are heavily used as blockers of the ras actions on the at receptor, ace is not the rate limiting step in ang ii production. moreover, ang ii can also be generated by chymase, and the mrna for chymase was not altered in the lung tumor cells. another consideration is that the mrna transcribe from the gene enpep which encodes aminopeptidase a, also known as angiotensinase a, a major metabolic enzyme of ang ii, is reduced and this could indicate a prolongation of the stimulation of at and at receptors by ang ii. while ang iii the product of aminopeptidase a metabolism of ang ii is a full agonist at at and at receptors, it is known to be rapidly metabolized following its formation, such that any slowing of the conversion of ang ii to ang iii will increase the stimulation of at and at receptors. moreover, early studies of the proliferative effects of ang ii suggested that ang iii lacked the ability to stimulate cell proliferation [ ] . the low level and apparent lack of differential expression of the genes encoding components of the ace /ang - /mas axis in normal lung tissue and lung tumor tissue are surprising in view of the large body of literature suggesting that the ace /ang - /mas axis plays an important role in lung function [ ] , susceptibility to the sars virus [ ] , and inhibition of angiogenesis in lung cancer [ ] . the reductions in the high level of expression of the genes for two enzymes capable of cleaving ang i and ang ii to form ang - ; neprilysin and prolyl carboxypeptidase, respectively, may cause a reduction in the shunting of ang i and ang ii away from their roles in the classical ras and reduce the antagonistic action of the ace /ang - /mas axis. this would promote the actions of ang ii at the at receptor while simultaneously reducing the stimulation of the mas receptor by ang - . it is well established that not all cancers are the same and it is not surprising that alterations in the ras in different cancers are not consistent. for example, a large-scale study of estrogen receptor-positive breast cancer tumors revealed an increase in agtr mrna expression [ ] , which is the opposite of what we observed in the lung cancer study from which this data is derived. equally contradictory, agtr as well as ace mrna expression is upregulated in hormoneindependent prostate cancer [ ] . while the changes observed in mrna expression in some of the components of the ras in lung adenoma tumor cells are substantial, there are three caveats that must be acknowledged. first, it is uncertain whether these changes in mrna are contributing to the phenotypic changes in lung cells to become cancer cells, or if they are a result of the phenotypic changes associated with the transformation of normal lung cells to cancer cells. second, alterations in mrna expression do not always reflect alterations in protein expression. to assess this second caveat it will be necessary to determine the level of expression of the proteins encoded by the mrnas that showed significant changes. the third caveat relates to possible changes in signal transduction by the at , at , and mas receptors. it is possible that signal transduction in response to activation of these receptors by ang ii, ang iii, and ang - could be increased or decreased. it is known that the at receptor transduces responses to ang ii via g protein-coupled and g protein independent pathways [ ] and that peptide antagonists of the at receptor can signal exclusively through the g protein independent pathway [ ] , so it is not unreasonable for at receptor activation to have differential effects on cell proliferation due to differences in the efficiency of these two signaling pathways in cancer cells. while it would be of interest to assess the mrna expression of these signal transduction proteins, it is not possible to specify that their changes are specifically linked to these angiotensin receptors since they mediate transduction by a variety of hormones, cytokines, and neurotransmitters. the current study emphasizes the involvement of components of the ras (both direct and extended) in the development and progression of lung cancer. in spite of the several limitations pertaining to the dataset, this study provides a novel therapeutic targeting modality for treatment of lung cancer. moreover, the current study also provides a platform to mimic our approach of data mining previously conducted genome-wide expression studies to identify novel therapeutic targets for a variety of cancers and other diseases. this will decrease the cost burden of investigating new drug entities and possibly allow for the repurposing of already approved drugs. the authors declare that there are no conflicts of interest regarding the publication of this paper. gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival renin-angiotensin system gene expression and neurodegenerative diseases proceedings: influence of angiotensin ii and angiotensin ii antagonist (p ) in vitro on cell growth in t and virus transformed sv t cells angiotensin stimulation of bovine adrenocortical cell growth molecular mechanisms of vascular 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/angiogenesis-( - )/mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension angiotensin-converting enzyme is a functional receptor for the sars coronavirus angiotensin-( - ) inhibits tumor angiogenesis in human lung cancer xenografts with a reduction in vascular endothelial growth factor agtr overexpression defines a subset of breast cancer and confers sensitivity to losartan, an agtr antagonist renin-angiotensin system is an important factor in hormone refractory prostate cancer activation and targeting of extracellular signal-regulated kinases by -arrestin scaffolds differential kinetic and spatial patterns of -arrestin and g proteinmediated erk activation by the angiotensin ii receptor the authors thank homood as sobeai for assistance in using genespring software. key: cord- -dgnbfzli authors: diamond, betty title: the renin–angiotensin system: an integrated view of lung disease and coagulopathy in covid- and therapeutic implications date: - - journal: j exp med doi: . /jem. sha: doc_id: cord_uid: dgnbfzli the renin–angiotensin system (ras) has long been appreciated as a major regulator of blood pressure, but has more recently been recognized as a mechanism for modulating inflammation as well. while there has been concern in covid- patients over the use of drugs that target this system, the ras has not been explored fully as a druggable target. the abbreviated description of the ras suggests that its dysregulation may be at the center of covid- . engagement of the second angiotensin receptor, at , also expressed fairly ubiquitously, by angiotensin ii leads to vasodilation and to suppression of inflammation (crowley and rudemiller, ) . under both acute and chronic inflammatory conditions, the level of at is increased and the level of at is decreased (crowley and rudemiller, ; koka et al., ; tikellis and thomas, ) . a second ace, ace , exists as a membrane-bound protease on numerous cell types (clarke et al., ) . ace cleaves angiotensin ii to produce a small peptide, ang - , which binds to a g protein-coupled receptor mas to induce an anti-inflammatory and anti-apoptotic program and vasodilation (simões e silva et al., ) . ace is the cellular receptor for the sars-cov- virus (lan et al., ) , the causative agent for covid- , and permits viral entry into ace -expressing cells. when the viral spike protein binds ace , however, another protease termed tace, or adam , is activated; this causes ace to be shed from the cell membrane, leading to decreased degradation of angiotensin ii and decreased production of ang - (shah and catt, ) . interestingly, a high concentration of aldosterone reduces mas expression, also impairing this arm of the ras (stoll et al., ) . so how does this relate to covid- ? sars-cov- infection can cause a particularly severe disease in hypertensive and obese individuals (kenyon, ; richardson et al., ) . hypertensive individuals generally have high levels of angiotensin and high levels of tf, leading to the hypercoagulable state that is seen in hypertension (dielis et al., ) . it is important to note that renin levels are often low in african americans, but there is evidence in rodent models that tissue levels of angiotensin may be high even in the face of low renin (williams et al., ) . obesity is associated with high levels of ace and at (barton et al., ) . both of these comorbidities will, therefore, enhance the pro-inflammatory, pro-apoptotic, procoagulant arms of the ras. a model of covid- might be that sars-cov- infects lung alveolar epithelial cells, the source of surfactant, causing a cytopathic effect. in addition, angiotensin ii binding to alveolar epithelial cells induces their death through apoptosis. because ace is shed when bound by spike protein, we speculate that ang - is not generated. with diminished ang - to bind mas, there may be no activation of the arm of the ras pathway that protects against apoptosis. first, therefore, we suggest there is a collapse of alveolae with ensuing hypoxia. because angiotensin ii increases vascular permeability, there may be extravasation of neutrophils into alveolae. moreover, angiotensin ii acting on endothelial cells induces il- leading to neutrophil recruitment (nabah et al., ) . these neutrophils can release ros and form neutrophil extracellular traps (khan et al., ) . indeed, neutrophils in the alveolae have been seen in lung pathology of infected patients (barnes et al., ) . this accumulation of neutrophils, we propose, will lead to netosis and hypercapnia with inadequate diffusion of co through the alveolae if our model is correct. surfactant, which usually protects against netosis (rodriguez et al., ) , will be sparse due to the loss of alveolar epithelial cells. this may lead to an impairment in lung function that appears to be like what is seen in acute respiratory distress syndrome, but in sars-cov- infection, the destruction of lung function may progress through a different pathway. in acute respiratory distress syndrome, the loss of lung function is a consequence of cytokine storm due to the activation of the innate and adaptive immune systems. we propose that, in covid- , the lung is destroyed by a combination of the cytopathic effect of the virus and an imbalanced ras with too much at and insufficient ace (fig. ) . this is the crux of the issue and would explain the greater disease severity in the elderly as well as in hypertensive and obese individuals, rather than in young and healthy individuals. in this model, the key issue is the loss of ace due to ace engagement of the spike protein and activation of tace. indeed, ace deficiency exacerbates disease in mouse models of methicillin-resistant staphylococcus aureus and two strains of influenza (khan et al., ; yang et al., ; zou et al., ) . in patients infected with h n bird flu, angiotensin ii levels rise, with high levels associated with greater disease severity. similarly, in mice infected with h n , angiotensin ii levels predict outcome, and ace deficiency leads to greater mortality (zou et al., ) . in mice infected with h n , ace deficiency also leads to more severe disease. angiotensin, not cleaved by ace , binds the at receptor, contributing to pathology; an at receptor inhibitor, losartan, improves disease outcome (yang et al., ) . interestingly, in a mouse model of pseudomonas aeruginosa infection, a deficiency of ace function leads to increased neutrophil accumulation in the lung through an il- -dependent pathway (sodhi et al., ) . the increased tf in endothelial cells leads to thrombosis, which is present in numerous hospitalized covid- patients (bikdeli et al., ; klok et al., ) . the increased aldosterone may be responsible for the hypokalemia seen in many covid- patients. more speculatively, while the inflammatory arm of the ras produces tnf, il- , and other pro-inflammatory cytokines without producing interferon, ace cleaves bradykinin, a known suppressor of interferon, and so might lead to increased interferon in some individuals (seliga et al., ) . some individuals with a particular genetic predisposition may produce high levels of interferon, and these may be the individuals who develop "covid toes" (landa et al., ; zhang et al., ) . this predisposition may include those genes with risk alleles in systemic lupus erythematosus (sle) that have a propensity for high interferon production (ghodke-puranik and niewold, ) . interestingly, low interferon is seen in many covid- patients and is more consistent with activation of the ras pathway than activation of tlrs (hadjadj et al., preprint) . tlr activation induces interferon; activation of at by angiotensin ii, in contrast, leads to proinflammatory cytokines but not interferon (meng et al., ) . there are implications of this model. first, covid- patients will have a high level of angiotensin ii, perhaps even african americans, and a low level of ang - due to the diminished activity of ace . second, those with covid toes may have risk alleles in the interferon pathway, similar to some sle risk alleles, as similar pathology is seen in sle and interferonopathies (kolivras et al., ) . high interferon may lead to an effective antiviral response but also have the negative consequence of leading to interferon-induced microangiopathy (massey and jones, ) . ace inhibitors might be beneficial rather than harmful. angiotensin receptor blockers, which preferentially target at , might also be useful. while there was initial concern over the use of these drugs, as they increase ace expression when used not in the context of covid- (vaduganathan et al., ) , it is now clear they are not harmful in the context of the virus and may indeed be beneficial (ghosal et al., preprint; robertson, ) . finally, the use of the ang - peptide to counter the effects of the angiotensin-at interaction might be a novel therapeutic to pursue. figure . the ras in health and in sars-cov- infection. left: in a healthy individual, there is a balance of at , at , and membrane-bound ace with production of ang - . aldosterone increases mas expression, and ang - binds mas to suppress inflammation. right: in a sars-cov- -infected individual, there is an imbalanced ras, with viral spike protein causing ace shedding, diminished production of ang - , and high at with low at . microbes infect ace inhibtors appear to be safe and beneficial for covid- , say researchers this work was supported by grants from the national institutes of health (nih p ai and nih r -ar ). key: cord- -dvl pud authors: gan, rosemary; rosoman, nicholas p.; henshaw, david j.e.; noble, euan p.; georgius, peter; sommerfeld, nigel title: covid- as a viral functional ace deficiency disorder with ace related multi-organ disease date: - - journal: med hypotheses doi: . /j.mehy. . sha: doc_id: cord_uid: dvl pud sars-cov- , the agent of covid- , shares a lineage with sars-cov- , and a common fatal pulmonary profile but with striking differences in presentation, clinical course, and response to treatment. in contrast to sars-cov- (sars), covid- has presented as an often bi-phasic, multi-organ pathology, with a proclivity for severe disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is usually related to respiratory collapse, autopsy reveals multi-organ pathology. chronic pulmonary disease is underrepresented in the group with severe covid- . a commonality of aberrant renin angiotensin system (ras) is suggested in the at-risk group. the identification of angiotensin-converting-enzyme (ace ) as the receptor allowing viral entry to cells precipitated our interest in the role of ace in covid- pathogenesis. we propose that covid- is a viral multisystem disease, with dominant vascular pathology, mediated by global reduction in ace function, pronounced in disease conditions with ras bias toward angiotensin-converting-enzyme (ace) over ace . it is further complicated by organ specific pathology related to loss of ace expressing cells particularly affecting the endothelium, alveolus, glomerulus and cardiac microvasculature. the possible upregulation in ace receptor expression may predispose individuals with aberrant ras status to higher viral load on infection and relatively more cell loss. relative ace deficiency leads to enhanced and protracted tissue, and vessel exposure to angiotensin ii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il- ) resulting in inflammation. additionally, there is a profound loss of the “protective” angiotensin ( - ), a vasodilator with anti-inflammatory, anti-thrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity. our model predicts global vascular insult related to direct endothelial cell damage, vasoconstriction and thrombosis with a disease specific cytokine profile related to angiotensin ii rather than “cytokine storm”. our proposed mechanism of lung injury provides an explanation for early hypoxia without reduction in lung compliance and suggests a need for revision of treatment protocols to address vasoconstriction, thromboprophylaxis, and to minimize additional small airways and alveolar trauma via ventilation choice. our model predicts long term sequelae of scarring/fibrosis in vessels, lungs, renal and cardiac tissue with protracted illness in at-risk individuals. it is hoped that our model stimulates review of current diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is usually related to respiratory collapse, autopsy reveals multiorgan pathology. chronic pulmonary disease is underrepresented in the group with severe covid- . a commonality of aberrant renin angiotensin system (ras) is suggested in the at-risk group. the identification of angiotensin-converting-enzyme (ace ) as the receptor allowing viral entry to cells precipitated our interest in the role of ace in covid- pathogenesis. we propose that covid- is a viral multisystem disease, with dominant vascular pathology, mediated by global reduction in ace function, pronounced in disease conditions with ras bias toward angiotensin-converting-enzyme (ace) over ace . it is further complicated by organ specific pathology related to loss of ace expressing cells particularly affecting the endothelium, alveolus, glomerulus and cardiac microvasculature. the possible upregulation in ace receptor expression may predispose individuals with aberrant ras status to higher viral load on infection and relatively more cell loss. relative ace deficiency leads to enhanced and protracted tissue, and vessel exposure to angiotensin ii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il- ) resulting in inflammation. additionally, there is a profound loss of the "protective" angiotensin ( - ), a vasodilator with anti-inflammatory, antithrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity. our model predicts global vascular insult related to direct endothelial cell damage, vasoconstriction and thrombosis with a disease specific cytokine profile related to angiotensin ii rather than "cytokine storm". our proposed mechanism of lung injury provides an explanation for early hypoxia without reduction in lung compliance and suggests a need for revision of treatment protocols to address vasoconstriction, thromboprophylaxis, and to minimize additional small airways and alveolar trauma via ventilation choice. our model predicts long term sequelae of scarring/fibrosis in vessels, lungs, renal and cardiac tissue with protracted illness in at-risk individuals. it is hoped that our model stimulates review of current diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. sars-cov- , the agent of covid- , shares a lineage with sars-cov- disease (sars), and a common fatal pulmonary profile but with striking differences in presentation, clinical course, and response to treatment [ ] [ ] [ ] [ ] . in contrast to sars, covid- has presented as an often bi-phasic, multi-organ pathology, with a proclivity for severe disease in the elderly and those with hypertension, diabetes and cardiovascular disease. whilst death is almost universally related to respiratory collapse with multiple systems revealed to be failing at autopsy, the co-morbidity of chronic pulmonary disease is surprisingly relatively underrepresented in the group with severe covid- [ ] . covid- has not responded consistently to immunosuppressive therapy, antimicrobial agents or invasive ventilation [ ] . there is evolving evidence for a mechanism of lung injury that is not always typical of acute respiratory distress syndrome (ards) [ ] . there is a need to revise the disease model for covid- away from historical pulmonary-centric septic disease states typically characterised by lymphopaenia and cytokine storm with secondary bacterial sepsis. the diagnosis, assessment and management of covid- cannot be based entirely on previous sars, mers or influenza pandemics. our disease hypothesis suggests a revision of our use of diagnostic tools (to better capture atypical disease presentations) and consider early treatment and/or prevention of microvascular thrombosis and constriction in identified groups at high risk of severe or fatal disease. our hypothesis provides a mechanism of pulmonary insult, that warrants revision of indications for invasive ventilation particularly in view of poor outcomes [ , ], with higher mortality for invasively ventilated patients than that recorded in sars [ , , ]. whilst the respiratory system is almost universally involved in clinical presentation and dominates current therapy in covid- , those with more severe disease have been observed to have comorbidities in the vascular, renal, and cardiovascular systems [ , ] . appreciating the clear differences between sars and covid- in presentation, poor prognostic indicators related to individuals' co-morbid status, and biochemical and radiologic profiles, a novel disease model may assist in: ) the early recognition of atypical (non-respiratory) presentations of disease; ) early prophylactic treatment intervention for individuals at risk of severe and critical disease which could take place in the community; ) revised management of pulmonary complications including those related to prone posturing and ventilation protocols; ) allowing better utilisation of data collated at a global level in the absence of an evidence-based disease model at this time; ) identification of different markers of disease progression in at-risk individuals. the hypothesis of covid- as a viral functional angiotensin-converting-enzyme (ace ) deficiency disorder, with ace related multi-organ cell loss, comes some way to addressing this need. this model is supported by global data and observations, including that gathered phenotypically, biochemically, radiologically, and at autopsy. it evolved from the early observation of hypoxic pulmonary insult occurring without reduction in pulmonary compliance and often in the absence of fever. this is not consistent with ards, documented in historical pulmonary septic disease states with cytokine storm complicated by hypoxia. a pulmonary vascular pathology was sought in parallel with a possible disruption of surfactant function. thrombosis and vasoconstriction were implied, with the former supported by consistent, but not specific radiologic findings of ground glass change. thrombosis has now been confirmed in late disease and at autopsy [ ] . the commonality of an aberrant renin angiotensin system (ras), was identified in the group at risk of severe disease, rather than the presence of underlying lung disease. the biochemical markers in early disease suggested coagulation, renal, cardiac, and respiratory pathology in evolution. with the identification of ace as the receptor for sars-cov- entry, the role of and distribution of ace in the human body was investigated. the hypothesis evolved, providing an explanation for pathophysiologic commonality in the at-risk group in covid- , a mechanism for hypoxic pulmonary pathology and the unexpected degree of lung injury consequent to ventilation as well as vascular, renal and cardiac complications. it also predicts that presentation may be atypical and that long term sequelae related to endothelial, renal, pulmonary and cardiac involvement may occur. the hypothesis also predicts a cytokine profile driven by ace deficiency in the ras, rather than viral-related immune modulation. the immunologic effects of the ras are well documented [ ] , favouring il- production and affecting cd + and cd + cell populations. ace is a membrane-associated aminopeptidase expressed in the endothelium, renal and cardiovascular tissue, and epithelia of the small intestine and testes [ ] . ace is also expressed in the lung, kidney, and gi tract according to pcr analysis -tissues shown to harbor sars-cov- [ , ] . the entry receptor utilized by sars-cov- in covid- is ace , and the enzyme utilized for s protein priming is transmembrane protease serine (tmprss ) [ ] or the controversial poly-basic furin cleavage site. we propose that covid- is a multiorgan pathology with dominant morbidity related to respiratory insult that is compounded by a more generalised disease driver that leads to a poorer prognosis and higher mortality in an identified at-risk group. we propose that the disease driver is relative ace deficiency related to viral destruction of ace expressing tissues, with more severe disease manifesting in a cohort with abnormality in the ras favouring the angiotensin-converting-enzyme (ace) arm. this is supported by the observed frequency of hypertension, cardiovascular disease and diabetes as co-morbidities in the group with severe and fatal disease [ , ] . these disease entities have been well studied, with documentation of ras abnormality characterised by increased ace and angiotensin ii (atii) levels (fig. ) . we hypothesise that in these conditions, there is a greater dependence on ace to abrogate the effects of atii and increase angiotensin ( - ) (at ( - ) ). an upregulation of ace expressing cells related to chronic atii elevation [ ] or treatment with aceinhibitors [ ] , may increase the infective potential of sars-cov- in this group as a consequence of the duality of ace functioning as both a receptor for viral entry to cells and as an enzyme. with infection related cell damage and loss, enzymatic ace activity would be globally compromised. organ specific loss of ace expressing cells (endothelial, alveolus in lungs, proximal tubule and glomerulus in kidneys, pericytes in heart) is likely to also contribute to the pathophysiology of covid- . these conditions are by no means the only ones that may confer escalated risk of severe or critical disease. we suggest that the pathogenesis is mediated by enhanced and protracted tissue, and vessel exposure to atii, characterised by vasoconstriction, enhanced thrombosis, cell proliferation and recruitment, increased tissue permeability, and cytokine production (including il- ) resulting in inflammation. a recent study noted serum levels of atii were elevated in covid- patients and correlated with viral load and lung injury [ ] . atii is generated by both ace and non-ace pathways, but is the dominant substrate for ace , being converted to at( - ) [ ] , a vasodilator with antiinflammatory, anti-thrombotic, antiproliferative, antifibrotic, anti-arrhythmic, and antioxidant activity [ , ] . the viral destruction of ace expressing cells may lead to the profound loss of the "protective" at( - ) effects in an environment of atii effect dominance (fig. ) . enhanced thrombosis may occur via several atii driven pathways, including increased thrombin [ ] , procoagulant endothelial cell activity via bradykinin [ , ] , immune effects via t cells (cd +, cd +) [ , ] , platelet aggregation [ ] , and reduced fibrinolysis via bradykinin and endothelin. the prothrombotic effects of atii appear to extend to the microvasculature in murine models [ ] . platelet aggregation and endothelial damage in the septic setting may contribute further to thrombosis. we hypothesise that ace expressing cells are rapidly infected by sars-cov- . initially, this is by ingestion and/or aspiration. early respiratory symptomatology is consistent with previously described sars-cov- infection via ace receptors in the ciliated respiratory epithelial cells, with apical release of replicated virus [ ] rapidly infecting the downstream ace expressing cells -including type alveolar cells. as opposed to sars-cov- infection, significant hypoxia in covid- is not accompanied in early disease by a reduction in pulmonary compliance [ , ] . this and colleagues reported that abnormal coagulation parameters, particularly d-dimer and fibrin degradation products (fdps), were associated with poor prognosis in covid- [ ] . an association with anticoagulation and improved survival after adjustment for ventilation was noted in a large united states cohort [ ] . possible atii driven thrombosis and vasoconstriction are likely to be compounded by alveolar damage via the viral loss of ace -rich type alveolar cells. these cells in health are tasked with surfactant production, maintenance of alveolar homeostasis, and transdifferentiation into type alveolar cells, which comprise % of the lining of the alveolus. the demise of these cells is likely to lead to hypoxia via surfactant loss, alveolar flooding, and apoptosis of type alveolar cells without replacement via transdifferentiation from type alveolar cells (fig. ) . our hypothesis suggests that as disease progresses, the destruction of alveoli leads to dad, rather than ards. the destruction of alveoli is evident at autopsy, with dad [ ] and radiologic appearances consistent with dad are documented in advanced disease [ , ] . our hypothesis supports a possible mechanism for disappointing outcomes in ventilated patients, particularly related to those requiring or receiving high positive end expiratory pressure (peep) and the observed autopsy findings [ , , ] . the advantage of prone posturing and peep application is well documented in ards [ ] . advantage in ards was felt to be largely related to the distribution of oedema, where the mass of the diseased lung may be increased to % of the normal [ ] . in normal subjects, prone posturing improved alveolar ventilation, however, the addition of cmh o peep in ventilated patients reduced v/q matching via perfusion changes in dependent lung [ ] . it is possible that prior to the evolution of pulmonary oedema in covid- (when lung compliance is near normal), escalating peep in response to deteriorating hypoxia may be self-defeating. in addition to this, our disease model identifies that the at-risk group characteristic of aberrant ras status favouring ace activity may be more susceptible to volume induced lung injury related to peep. decreased ace activity with increased ace activity contributed to lung injury [ ] in in vitro studies of cyclical stretch of human lung epithelial cells and to volume induced lung injury in animal models [ , ] . viral spread to the vascular system may occur via pulmonary capillaries given the intimacy of the pulmonary capillary bed to the infected alveoli. we postulate the viraemia to lead to vascular, renal and myocardial infection at this time, supported in a remote manner by the somewhat unpredictable timing of fever, and the reports of biphasic clinical decline that involves shortness of breath, hypoxia (pao /fio < mmhg), transaminitis, low-normal procalcitonin, and abnormal chest imaging ( fig. ) [ ]. in hypertension and renal disease, particularly diabetic nephropathy, an aberrant ras is well documented, with a bias to higher ace/ace in renal biopsy [ ] [ ] [ ] . we suggest that ace deficiency leads to increased and unabating atii activity resulting in vasoconstriction, water and sodium retention, cell proliferation, thrombosis, inflammation, oxidative stress, and fibrosis. in addition to the damaging effects of atii, the deficiency in ace leads to a reduction in at( - ), with loss of its vasodilatory, diuretic, antiproliferative, antioxidant, and antithrombotic effects (via bradykinin) [ ] . in the acute setting, renal injury is likely due initially to the sustained effects of atii, including vasoconstriction that favours the efferent over afferent vessels, resulting in increased glomerular pressures, water and sodium retention, albuminuria, and possible microvascular thrombosis (fig. ) . this is supported by documentation of albuminuria and elevated creatine kinase [ , ] . the physical loss of ace expressing glomerular cells may further add to the renal insult. as disease progresses, a more diffuse renal impairment develops with the absence of overt septic inflammation in autopsy specimens [ ] . an abnormal renal ace/ace ratio is also seen in diabetics without established nephropathy, iga nephropathy, and subtotal nephrectomy [ ] . this group of patients may well carry higher risk for covid- renal disease. the hypothesis also predicts that renal scarring is likely to occur in survivors particularly related to vessel and glomerular damage. myocardial damage is suggested by the elevation in hypersensitive troponin i (tni) and dysrhythmia in a small series ( . % and . %, respectively) [ ] . with the disease model proposed here, elevated atii activity with suboptimal at( - ) activity may lead in the predisposed heart to microvascular thrombosis, vasoconstriction related hypoxia, and cell/fibroblast proliferation, with both acute and intermediate term sequelae of progressive heart failure, and adverse remodelling and fibrosis (fig. ) . right ventricular dysfunction is likely to reflect the pulmonary vascular burden of thrombosis and vasoconstriction. ace is expressed by cardiac myocytes, fibroblasts, and endothelial cells. ace expression is increased in human heart failure and may reflect a predisposition to cardiac disease susceptibility in the context of covid- [ , ] . chen and colleagues identified that the pericyte, with very high ace expression, may be the entry point for myocardial infection [ ] . in the autopsy series from new orleans, fox and colleagues acknowledged that the finding of scattered individual myocyte necrosis without evidence of viral myocarditis would be consistent with microvascular dysfunction related to pericyte infection [ ] , as hypothesized by chen and colleagues [ ] . chen's findings are consistent with our belief that cardiac infection follows viraemia, with a delayed declaration of pathological involvement. all subjects had gross right ventricular dilatation. none of the autopsy subjects had known cardiac disease. more recently, peng and colleagues described echocardiographic features of severe covid- [ ] . this included left ventricular segmental contraction abnormalities (takotsubo cardiomyopathy), right ventricular dilatation and systolic dysfunction, and, finally, global systolic and diastolic heart failure. these findings are not inconsistent with cardiac microvascular dysfunction and ischaemia with, as described previously, the right ventricular failure reflecting the pulmonary vascular burden of thrombosis and vasoconstriction. tni elevation and echocardiographic assessment remain useful tools for assessing disease progression or prognosis, particularly in subjects with existing cardiac failure [ , [ ] [ ] [ ] . the hypothesis predicts adverse remodelling of the myocardium as a consequence of microvascular cardiac disease. our hypothesis supports the revision of use of diagnostic tools with reference to the possibility of atypical (non-respiratory) disease presentations. tissues with high ace expression may be targeted for sampling in early or protracted infection to minimise community spread and false negative results. faecal or urine pcr testing for viral infection may be reconsidered if respiratory sampling is unhelpful. we propose that identified high risk groups be considered for early treatment or prevention of microvascular thrombosis and constriction, given the likelihood of progression to multisystem failure and the possibility of vascular, pulmonary, myocardial, and renal scarring sequelae in survivors. [ , ] . we suggest consideration of low molecular weight heparin (lmwh) or unfractionated heparin as these agents are likely to have both adequate anti-coagulant and additional antiinflammatory effects (via reduction in il- ). in a recent retrospective analysis of lmwh anticoagulation, shi and colleagues reported a reduction in levels of d-dimer, fdps, and il- , with an improvement in lymphocyte percentages when compared to a control group [ ] . sildenafil, with a number of studies in progress, may be considered as a vasodilator for efficacy of administration and cost in parallel with long term safety data and welldefined recommendations for monitoring of side effects. sildenafil may also have positive effects on cilial function in covid- [ ] . reconsideration of parameters for invasive ventilation and multitargeted therapy may be in order given poor outcomes [ , ], with higher mortality for invasively ventilated patients than that recorded in sars [ , , ] , and the possible alternative mechanism of pulmonary insult proposed -thrombosis, vasoconstriction, surfactant loss, and alveolar destruction via viral loss of type alveolar cells. peep < cmh o with prone posturing and supplemental oxygen should be considered in patients requiring respiratory support in the absence of ards. with respect to individuals with only mild or moderate disease, we propose that future treatment study protocols consider prophylactic level anticoagulation in at-risk individuals with a low threshold for escalation to full anticoagulation and vasodilation if disease progression is supported by measures of clinical state (particularly hypoxia), and well documented pathology parameters (including d-dimer, fdps, crp, neutrophil, lymphocyte and platelet levels, tni, and liver and renal function profiles, including albuminuria and il- ). prevention of thrombosis may be commenced in the community in symptomatic infected patients and at-risk populations with mild disease or positive exposure, given their increased risk of venous thromboembolism and the widespread safety data for lmwh and heparin use to establish efficacy. whilst thrombosis may not be universal, the intervention is low risk and may abrogate one of the irreversible elements of this disease. in areas with high disease prevalence, prophylactic-level anti-coagulation could be administered and monitored by local medical officers in the outpatient setting and in residential care facilities. emerging results of single-target therapies will necessarily impact on the evolution of disease models like ours, with autopsy data unfortunately offering our best evidence for end stage disease description, but little guidance for early therapeutic options. in conclusion, we present a covid- disease hypothesis with the intention of stimulating discussion and further research on diagnostic and therapeutic intervention protocols, particularly with respect to early anticoagulation, vasodilatation and revision of ventilatory support choices. this may benefit a large group of people who are at risk . cardiac effects of ace deficiency in an ace/ace biased renin-angiotensin system as a mechanism for thrombosis, adverse remodelling, and fibrosis. audit/audits/cmp/reports. covid- does not lead to a typical acute respiratory distress syndrome management of critically ill patients with severe acute respiratory syndrome (sars) presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the clinical evidence does not support corticosteroid treatment for -ncov lung injury covid- pneumonia: different respiratory treatments for different phenotypes? intensive care medicine a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study correction to: clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china pulmonary and cardiac pathology in covid- : the first autopsy series immunologic effects of the renin-angiotensin system high expression of ace receptor of -ncov on the epithelial cells of oral mucosa tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss clinical characteristics of hospitalized patients with novel coronavirus infected pneumonia in wuhan, china clinical characteristics of patients who died of coronavirus disease in china chapter -ace cell biology, regulation, and physiological functions in: the protective arm of the renin angiotensin system (ras). t unger, um steckelings and ras dos santos are patients with hypertension and diabetes mellitus at increased risk for covid- infection? clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - acute respiratory distress syndrome leads to reduced ratio of ace/ace activities and is prevented by angiotensin-( - ) or an angiotensin ii receptor antagonist roles of coagulation and fibrinolysis in angiotensin ii-enhanced microvascular thrombosis angiotensin ii, tissue factor and the thrombotic paradox of hypertension bradykinin enhances in vitro procoagulant and antifibrinolytic properties of rat vascular endothelial cells role of t lymphocytes in angiotensin iiâmediated microvascular thrombosis physiological and pathophysiological functions of the at subtype receptor of angiotensin ii ace receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia covid- pneumonia: ards or not? abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia association of treatment dose anticoagulation with in-hospital survival among hospitalized patients with covid- covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal mechanical stress and the induction of lung fibrosis via the prone positioning in severe acute respiratory distress syndrome prone positioning: beyond physiology positive end-expiratory pressure redistributes regional blood flow and ventilation differently in supine and prone humans ace and ace in kidney disease renal histopathological analysis of postmortem findings of patients with covid- in china synergistic expression of angiotensin-converting enzyme (ace) and ace in human renal tissue and confounding effects of hypertension on the ace to ace ratio kidney disease is associated with in-hospital death of patients with covid- the ace expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov- clinical characteristics of deceased patients with coronavirus disease : retrospective study using echocardiography to guide the treatment of novel coronavirus pneumonia covid- and the heart clinical management of severe acute respiratory infection when covid- is suspected: interim guidance the potential of low molecular weight heparin to mitigate cytokine storm in severe covid- patients: a retrospective clinical study sildenafil in the treatment of pulmonary hypertension marcus cremonese, medical illustration (http://www.medicalillustration.com.au/), and danielle santarelli, medical writing (genesis research services). no conflicts of interest to report. no sources of funding to report. key: cord- -rxmpi o authors: guang, cuie; phillips, robert d.; jiang, bo; milani, franco title: three key proteases – angiotensin-i-converting enzyme (ace), ace and renin – within and beyond the renin-angiotensin system date: - - journal: arch cardiovasc dis doi: . /j.acvd. . . sha: doc_id: cord_uid: rxmpi o the discovery of angiotensin-i-converting enzyme (ace ) and a (pro)renin receptor has renewed interest in the physiology of the renin-angiotensin system (ras). through the ace /angiotensin-( – )/mas counter-regulatory axis, ace balances the vasoconstrictive, proliferative, fibrotic and proinflammatory effects of the ace/angiotensin ii/at axis. the (pro)renin receptor system shows an angiotensin-dependent function related to increased generation of angiotensin i, and an angiotensin-independent aspect related to intracellular signalling. activation of ace and inhibition of ace and renin have been at the core of the ras regulation. the aim of this review is to discuss the biochemistry and biological functions of ace, ace and renin within and beyond the ras, and thus provide a perspective for future bioactives from natural plant and/or food resources related to the three proteases. angiotensine ; inhibiteurs de l'enzyme de conversion ; inhibiteur de l'enzyme de conversion ii ; rénine ; récepteur de la prorénine résumé la découverte de l'enzyme de conversion de l'angiotensine (ac ) et un récepteur à la prorénine est une avancée récente dans la compréhension de la physiologie du système rénine-angiotensine. au sein de l'axe inhibiteur de l'enzyme de conversion de l'angiotensine /angiotensine /mas, l'ac contrebalance l'effet vasoconstricteur, prolifératif, fibrosant et pro-inflammatoire de l'axe ace/angiotensine /at . le récepteur à la prorénine a une fonction angiotensine dépendante, liée à l'augmentation de la production d'angiotensine , et un aspect indépendant de l'angiotensine, lié aux signaux intracellulaires. l'activation de l'ac et l'inhibition de l'ace de la rénine ont été considérées comme au centre de la régulation du système rénine-angiotensine. l'objet de cette revue générale est de discuter les fonctions biochimiques et biologiques de l'ace, de l'ac et de la rénine au sein et au-delà du système rénine-angiotensine et ainsi de proposer une perspective de développement d'agents actifs extraits de plantes naturelles ou d'alimentation, produits liés à ces trois protéases. © elsevier masson sas. tous droits réservés. the renin-angiotensin system (ras) is not only an endocrine but also a paracrine and an intracrine system [ ] . in mammals, the intravascular ras plays a key role in maintaining blood pressure homeostasis and fluid and salt balance, and the tissue or local ras is involved in physiological and pathological processes, such as tissue growth and remodelling, development and inflammation [ ] . in a classical ras, the substrate angiotensinogen (agt), which is released into the circulation from the liver, is degraded by the enzyme renin that originates in the kidney, generating the inactive angiotensin i (ang i). when this decapeptide comes into contact with angiotensin-i-converting enzyme (ace) at the endothelial surface of blood vessels, the c-terminal dipeptide is cleaved, giving rise to angiotensin ii (ang ii), the main effector molecule of the ras. through interactions with specific receptors, particularly its type or at receptor, ang ii stimulates a wide variety of signalling pathways in the heart, blood vessels, kidneys, adipose tissue, pancreas and brain, initiating most of the physiological and pathophysiological effects that have been attributed to the ras [ ] . due to the function of directly generating the main effector ang ii, ace -together with the classical axis ace/ang ii/at -has been at the core of ras studies since its discovery. in , a homologue of ace, known as angiotensin-iconverting enzyme (ace ), was cloned by two independent research groups [ , ] . evidence indicates that ace negatively regulates the activated ras by degrading ang ii to the heptapeptide ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) . moreover, through the mas receptor (a g protein-coupled receptor), the resulting ang-( - ) counterbalances the cardiovascular effects of ang ii by opposing many at receptor-mediated actions [ ] . ace and the axis ace /ang-( - )/mas are becoming the focus of intense research regarding the ras [ ] . the discovery of a (pro)renin receptor ([p]rr) and the introduction of renin inhibitors have also brought (pro)renin back into the spotlight [ ] . far from being a straightforward cascade containing one substrate (agt), two proteases (renin and ace), two peptides (ang i and ang ii) and one receptor (at ), the ras currently consists of several axes upstream and downstream of the classical cascade, which include more than two dozen peptidases, nearly a dozen ang fragments and at least six different receptors [ ] . here, we review three critical proteases (ace, ace and renin) within and beyond the ras and thus intend to find new connections between natural plant and/or food resources and the ras. occurrence, gene encoding and structure of ace ace (ec . . . ) is a monomeric glycoprotein that is distributed in many tissues and biological fluids. there are two isoforms of ace in humans: somatic ace (sace) and germinal ace (gace). somatic ace is found in many types of endothelial and epithelial cells [ ] . germinal ace or testicular ace is present exclusively in germinal cells in the male testis. although ace is a type i integral membrane protein, it can also be released as a soluble enzyme into extracellular fluids, such as plasma and seminal and cerebrospinal fluids, following post-translational proteolytic cleavage by a membrane protein sheddase or secretase [ ] [ ] [ ] . somatic ace and gace are encoded by a single gene containing exons. the promoter for sace is situated in the flanking region of the first exon, whereas that for gace is within intron , which results in different lengths for the two isoforms. the longer sace ( - kda) is transcribed from exon to exon , excluding exon , whereas the shorter gace ( - kda) is transcribed from exon to exon . exon encodes a unique sequence for the n-terminus of gace, whereas downstream exons encode a common sequence for both isozymes [ ] . somatic ace and gace both consist of a -residue hydrophilic c-terminal cytoplasmic domain, a -residue hydrophobic transmembrane domain that anchors the protein in the membrane and an n-terminal ectodomain ( fig. ) that is heavily glycosylated with mannose, galactose, fructose, n-acetylneuraminic acid and n-acetylglucosamine [ ] . the ectodomain of sace is further divided into two similar domains (n domain and c domain) encoded by the homologous exons - and - , respectively, and each domain contains an active his-glu-x-x-his (hexxh) sequence [ ] . somatic ace is the only known metallopeptidase with two homologous active sites [ ] , which implies that there has been a gene duplication event during evolution [ ] . except for a unique sequence constituting its n-terminus, gace is identical to the c-terminal half of sace [ ] . due to cleavage of the membrane-bound residues by ace secretase, soluble circulating ace lacks a transmembrane portion and a cytosolic domain [ ] . the three-dimensional x-ray crystallographic structure of a deglycosylated truncated version of gace (c domain of sace), reveals a preponderance of ␣-helices with a zinc ion and two chloride ions incorporated. a deep narrow channel separates the molecule into two subdomains and the active site is located toward the bottom of this channel. an n-terminal 'lid' on the top of molecule appears to allow only small peptide substrates access to the active site cleft. in fact, the structure bears little similarity to that of carboxypeptidase a (m family) on which the initial drug development of ace inhibitors was based. instead, it resembles rat neurolysin (m family) and pyrococcus furiosus carboxypeptidase (m family), despite sharing little sequence similarity with these two proteins [ ] . corradi et al. [ ] reported the crystal structure of the n domain of sace. similarly, it has an ellipsoid shape with a central groove dividing it into two subdomains, one of which contains the n-terminal region that covers the central binding cavity. but the structure reveals differences in the active site and it contains only one chloride ion, equivalent to chloride ii of gace. the three-dimensional structures of c domains (based on gace) and n domains provide an opportunity to design domain-selective ace inhibitors that may exhibit new pharmacological profiles [ , ] . according to the catalytic mechanism and the critical amino acid residue involved, peptidases are classified into four major types: serine, cysteine, aspartic and metallo [ ] . ace is an m family metallopeptidase: ma(e), the gluzincins [ ] . two histidine residues of the functional motif hexxh and a third distant glutamate positioned - residues further towards the c-terminus are the ligands for the zinc cofactor [ ] . an activated water molecule complexed to zn + serves as the nucleophile to attack the carbonyl group of the targeted peptide bond [ ] . the activity of ace is also chloride dependent. chloride primarily activates the active sites of ace and enhances the binding of substrates [ ] . each active domain of ace displays differences in sensitivity to chloride activation [ ] . the activity of the c domain of sace depends highly on chloride ion concentration and is inactive in its absence, whereas the n domain can be completely activated at relatively low concentrations of this anion and is still active in the absence of chloride [ , ] . germinal ace depends on chloride to a lesser extent compared with the c domain of sace [ ] . cushman and cheung [ ] reported an optimal in vitro ace activity of rabbit rung acetone extract in the presence of mm nacl at ph . - . . the two active domains of sace are also subtly different in substrate specificity. they hydrolyze bradykinin almost equally but the c domain active site can hydrolyze ang i, substrate p [ ] and hippuryl-his-leu [ ] more efficiently, while the n domain active site preferentially hydrolyzes ang-( - ) [ ] , luteinizing hormone-releasing hormone (lh-rh) [ ] , the haemoregulatory peptide n-acetyl-ser-asp-lys-pro (acsdkp) [ ] and alzheimer amyloid ␤-peptide (a␤) [ ] . fuchs et al. [ ] proved that the c-terminal catalytic domain was the main site of ang i cleavage in mice. the differentiation of catalytic specificity might be due to very subtle variation in substrate-specific amino acids [ ] and chlorideinduced conformational alteration of active sites [ ] . ace acts as an exopeptidase to cleave dipeptides from the free c-termini of two typical substrates, ang i and bradykinin. for certain substrates such as cholecystokinin [ ] , substrate p [ ] and lh-rh [ ] , which have amidated c-termini, ace not only displays exopeptidase activity but also acts as an endopeptidase [ ] . the most prominent example of endopeptidase activity is ace hydrolyzing the synthetic a␤-( - ) peptide into four fragments: an a␤- peptide and the others corresponding to products of a␤-( - ) hydrolysis [ ] . thus, ace might have a more general impact on the metabolism of biologically active peptides than previously recognized [ ] . the two substrates used most often for measuring ace activity and inhibition in vitro -hippuryl-his-leu and n-[ -( -furyl)acryloyl]-lphenylalanylglycylglycine (fapgg) -only have the n-termini blocked and substrates with two termini blocked have been developed [ ] . ace was originally isolated in as a 'hypertensinconverting enzyme' [ ] . in the ras, ace cleaves the decapeptide ang i-( - ) (asp-arg-val-tyr-ile-his-pro-phe-his-leu) into the octapeptide ang ii-( - ) by removing the c-terminal dipeptide his-leu. when the ras is overactive, ang ii exerts its harmful effects primarily via the at receptor, whereas the at receptor may oppose and counterbalance those effects mediated by at receptor to exert protective actions [ ] . ang ii is a potent vasoconstrictor, stimulates the release of aldosterone and antidiuretic hormone or vasopressin and increases the retention of sodium and water. these effects act directly in concert to raise blood pressure. a nonapeptide derivative of ang i, des-asp -ang i-( - ), prevents infarction-related and non-infarction-related cardiac injuries and disorders. his-leu can be cleaved from the peptide by ace to produce ang iii-( - ) [ ] , which has % of the vasoconstriction activity of ang ii. ang iii exerts its effects, in principle, in a similar manner to ang ii, and may be equally or even more important in mediating the release of vasopressin [ ] . ace also degrades ang-( - ) to ang-( - ) then further degrades this peptide to the inactive ang-( - ) (fig. ). in addition, ace (also termed kininase ii) inactivates the vasodilators bradykinin-( - ) (arg-pro-pro-gly-phe-ser-pro-phe-arg) and kallidin (lys-bradykinin) in the kallikrein-kinin system, by cleaving the c-terminal dipeptide phe-arg. ace eventually cleaves its primary metabolite bradykinin-( - ) into the shorter fragment bradykinin-( - ) [ ] . through ang ii and aldosterone, ace may also be implicated in the impairment of nitric oxide bioavailability and cell oxidative stress, augmenting the generation of reactive oxygen species and peroxynitrite [ , ] . with the ability to hydrolyze neuropeptides such as enkephalin [ , ] , substrate p, neurotensin [ ] and lh-rh, ace may be involved in the functioning of the brain and nervous system. ace may affect the digestive system by hydrolyzing the peptide hormone cholecystokinin and gastrin [ ] . the in vivo experiment conducted by azizi et al. [ ] proved that acute ace inhibition could increase the level of the natural stem cell regulator acsdkp in plasma. acsdkp substantially inhibits cell cycle entry of normal haematopoietic stem cells and protects haemopoiesis against damage caused by cycleactive cytotoxic agents [ ] . acsdkp can also inhibit the proliferation of hepatocytes [ ] and lymphocytes [ ] and stimulate angiogenesis [ ] in vivo. the in vivo antifibrotic effect of ace inhibition is partially mediated by acsdkp [ ] . ace may also affect susceptibility to alzheimer's disease by degrading a␤ and preventing the accumulation of amyloid plaques in vivo [ ] . in the brains of amyloid precursor protein swedish mutation transgenic mice, ace converts a␤ - to a␤ - and degrades a␤, and chronic inhibition of ace with captopril enhances predominant a␤ - deposition [ ] . however, through the inhibition of brain ace activity in the a␤ - -injected mice, perindopril ameliorates cognitive impairment and may therefore have a beneficial effect on alzheimer's disease as well as hypertension [ ] . using the assumed mechanistic analogy to other zinc metallopeptidases, plus the knowledge that several snake-venom peptides potentiate the action of bradykinin by inhibiting ace, efforts were undertaken to develop orally-active peptide analogues for potential use in the treatment of hypertension [ ] . the first such compound, captopril or d- -mercapto- -methylpropanoyl-l-proline, is an analogue of the ala-pro sequence, with sulphydryl as a strong chelating group for the zinc ion. its adverse effects, which were the same as those caused by mercapto-containing penicillamine, prompted the design of non-sulphydryl ace inhibitors [ ] . the results were two active inhibitors: enalaprilat and lisinopril. they are both essentially tripeptide analogues with a zinc-co-ordinating carboxyl group and a phenylalanine that occupies the s groove in the enzyme. lisinopril is a lysine analogue of enalaprilat but it is hydrophilic, with greater affinity than enalaprilat. the later compounds are all variations of the first three inhibitors, with most of the differences residing in the functionalities that bind the active site zinc and the s pocket. in addition to phosphonates, ketones are also useful as chelators [ ] . currently, there are more than ace inhibitors marketed that are widely used as first-line therapy for cardiovascular diseases, including hypertension, heart failure, heart attack and left ventricular dysfunction. according to the functional moiety, they are divided into three types: thiol (captopril), carboxylate (benazepril, enalapril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril) or phosphate (fosinopril). some ace inhibitors are now administrated clinically as ethyl-ester prodrugs, which have good bioavailability but are inactive in their own right. they are then converted to the active diacid molecules in vivo by esterases. as a drug class, ace inhibitors are very effective, have a relatively low incidence of side effects and are well tolerated. a common side effect of ace inhibitors is a dry cough, which appears in - % of patients and may result in the discontinuation of treatment. another serious problem is angioedema, which affects . - . % of patients and can be life-threatening. the two side effects have generally been attributed to altered concentrations of bradykinin [ ] . use of ace inhibitors during the second and third trimesters of pregnancy is contraindicated because of their association with an increased risk of foetopathy, a group of conditions that includes oligohydramnios, intrauterine growth retardation, hypocalvaria, renal dysplasia, anuria, renal failure and death. exposure to ace inhibitors during the first trimester of pregnancy may place the infant at increased risk for major congenital malformations [ ] . the initial drug development of clinical ace inhibitors was based on the assumption of an active site related to that of carboxypeptidase a but organized to remove a dipeptide rather than a single amino acid from the cterminus of its substrate. it is now known that sace has two active sites, neither of which resembles that of carboxypeptidase a, and that these sites are not identical. clinical ace inhibitors, however, show little discrimination between these two active sites [ ] . ang i is hydrolyzed predominantly by the c domain of sace in vivo [ ] but bradykinin is hydrolyzed by both active sites [ ] ; therefore a c domain-selective inhibitor would allow some degradation of bradykinin by the n domain and this degradation could be enough to prevent the accumulation of excess bradykinin that has been observed during attacks of angioedema. that is, the c domain-selective inhibition could possibly result in specialized control of blood pressure with fewer vasodilatorrelated adverse effects [ ] . a structure-activity study has proved that the group substitution involving the phenyl ring and carbon chain at the sulphonyl and propionyl moieties of captopril is essential for better activity towards the c domain of ace [ ] . there is increasing evidence that the n domain of sace is responsible for the in vivo degradation of the natural haemoregulatory hormone acsdkp [ , , ] . so, n domain-selective inhibition might open up novel therapeutic areas. two phosphinic tetrapeptides, rxpa and rxp , have been found to be highly selective inhibitors of the c and n domains of sace, respectively [ ] . the availability of the three-dimensional structures of the c and n domains of sace makes the structure-based design of active site-specific inhibitors possible [ , ] . ace (ec . . .-) is also a type i transmembrane glycoprotein and its expression has now been recognized as being ubiquitous. it appears to be susceptible to cleavage that releases the catalytical active ectodomain. a disintegrin and metalloprotease (adam ; also known as tumour necrosis factor-alpha cleavage enzyme [tace]) is a major protease for soluble ace shedding, while phorbol ester, ionomycin, endotoxin and the proinflammatory cytokines interleukin- ␤ and tumour necrosis factor-alpha can also acutely induce ectodomain release [ ] . calmodulin binding sites have been identified in the cytoplasmic tail of ace and calmodulin inhibitors are proved to increase ace shedding [ ] . the cleavage site resides between amino acids and near the predicted transmembrane domain and residue lys in the ectodomain plays an important role in dictating ace ectodomain shedding [ ] . ace has amino acids encoded from exons and shares about % sequence identity with the n and c domains of sace [ ] . ace also belongs to the m family of metalloproteases but consists of a single active site domain that, by sequence comparison, more closely resembles the n domain than the c domain of sace ( fig. ) [ ] . in addition to the conserved zinc metallopeptidase consensus sequence hexxh (amino acids - ), there is a conserved glutamate residue residues c-terminal to the second histidine of the zinc motif, which serves as the third zinc ligand [ ] . the threedimensional structure of a truncated extracellular region of human ace shows that the active site domain (residues - ) can be further divided into two subdomains i and ii, which form two sides of a long deep cleft and are connected only at the floor of the active site cleft by a prominent ␣helix. the deeply recessed and shielded proteolytic active site of ace is a common structural feature of proteases and can avoid hydrolysis of correctly folded and functional proteins. the zinc is co-ordinated by his , his , glu and one water molecule in the subdomain i near the bottom, whereas a chloride ion is co-ordinated by arg , trp and lys in the subdomain ii [ ] . the ace transmembrane c-terminal domain shares % sequence identity with collectrin (fig. ) , a non-catalytic protein shown to have a critical role in amino acid reabsorption in the kidney [ ] , pancreatic beta cell proliferation and possibly insulin exocytosis [ ] . unlike sace and gace, which are primarily dipeptidylcarboxypeptidases, ace functions predominantly as a monocarboxypeptidase, with a substrate preference for hydrolysis between proline and a hydrophobic or basic cterminal residue [ ] . it is like carboxypeptidase a in its action model but is different in active structure because the latter has an hxxe zinc-binding motif with the third ligand (histidine) positioned - residues further towards the c-terminus [ ] . ace efficiently cleaves a single residue phenylalanine from ang ii to generate ang- ( - ) , with about -fold higher catalytic efficiency than the conversion of ang i to ang-( - ) by removing the c-terminal leucine residue (fig. ). other substrates with high catalytic efficiency are apelin- , dynorphin a-( - ) and des-arg bradykinin [ ] . arg of ace has been identified as a residue critical to substrate selectivity [ ] . ace activity is also regulated by chloride ions; it has been proposed that chloride binding induces subtle changes in the conformation of the active site, which either facilitate or hinder substrate binding [ ] . for the substrate ang i, ace activity increases with increased [cl − ] and a plateau is reached at approximately mm cl − . for the substrate ang ii, an increase in ace activity is observed as [cl − ] increases from - mm but any further increase in [cl − ] decreases ace activity until a plateau is reached at mm cl − ; ace activity at mm cl − is even lower than that in the absence of cl − ( mm). consequently, an increase in [cl − ] above mm, which is the physiological concentration in human plasma, increases ang i and decreases ang ii cleavage by ace . this has an effect on the localized concentration of ang ii in the kidney, where ace has a high level of expression and extracellular chloride ion levels fluctuate. thus, in vivo cl − sensitivity may serve as a homeostatic regulatory mechanism [ ] . ace activity is unaffected by inhibitors of ace (captopril, lisinopril and enalaprilat) or carboxypeptidase a (benzylsuccinate and potato carboxypeptidase inhibitor) [ ] . the major function of ace is to counter-regulate ace activity by reducing ang ii bioavailability and increasing ang-( - ) formation. as a result, ace plays a crucial role in maintaining the balance between the two axes ace /ang-( - )/mas and ace/ang ii/at of the ras; a chronic and sustained imbalance may lead to pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems [ ] . studies have shown that ace overexpression and recombinant ace treatment can attenuate hypertension in animal models [ , ] , while in humans, there is a strong association between ace polymorphisms and hypertension in han chinese [ ] . in addition to the ang ii system, ace may regulate blood pressure through other peptide systems, such as bradykinin and/or apelin [ ] . ace gene delivery [ ] and ace-( - ) infusion [ ] also have beneficial effects on atherosclerosis, whereas ace deficiency accentuates vascular atherosclerosis and inflammation [ ] in animal models. regarding heart function, ace null mice display impaired cardiac contractility [ ] and the loss of ace in wild-type mice accelerates adverse ventricular remodelling by potentiation of ang ii effects by means of the at receptors [ ] . ang-( - ), through interaction with the mas receptor, can improve atrial tachyarrhythmias [ ] , myocardial performance, cardiac modelling and survival [ , ] in rodent heart failure models. in humans, ace gene variants might be involved in modulation of left ventricular mass in men [ ] and soluble ace activity is increased in patients with heart failure and correlates with disease severity to exert cardioprotective actions [ ] . deletion of the ace gene in mice leads to the development of glomerulosclerosis and increased albuminuria [ ] , while treatment with the ace inhibitor mln can worsen renal damage in streptozotocin-induced diabetic mice [ ] . chronic treatment with ang-( - ) improves renal endothelial dysfunction via the mas receptor in apolipoprotein e-deficient mice, by increasing levels of endogenous nitric oxide [ ] , whereas genetic deletion of the mas receptor in mice leads to a reduction in urine volume, sodium retention, microalbuminuria and reduced renal blood flow, which are associated with upregulation of the at receptor and transforming growth factor-beta messenger ribonucleic acid [ ] . this evidence indicates the protective role of ace /ang-( - )/mas in renal function. ace has also been shown to regulate cardiovascular functions in brain regions. overexpression of ace in the rostral ventrolateral medulla reduces high blood pressure and heart rate in spontaneously hypertensive rats (shrs) [ ] . in the nucleus tractus solitarius of shrs, ace gene transfer improves baroreceptor heart rate reflex [ ] . in the mouse subfornical organ, ace overexpression inhibits at receptor expression and prevents ang ii-mediated pressor and drinking responses [ ] . in addition, ace /ang-( - )/mas can exert cerebroprotective functions in endothelin- -induced ischaemic stroke in rodent models [ ] . ace exerts a host of actions on the cardiopulmonary system, which include prevention of endothelial dysfunction, reduction in pulmonary oxidative stress, attenuation of vascular impairment, anti-inflammatory effects and anticardiac remodelling effects. all these properties are responsible for the protective role of ace against pulmonary arterial hypertension [ ] . structure-based drug screening has identified two ace activators: a xanthenone ( -[( -diethylamino)ethyl-amino]- -(hydroxymethyl)- -[( -methylphenyl)sulphonyloxy]- h-xanthene- -one; xnt) and resorcinolnaphthalein. xnt hydrogen bonds with ace residues lys , tyr , gly and his , and resorcinolnaphthalein is involved in three hydrogen bonds with residues gln , gln and gly . xnt and resorcinolnaphthalein modulate ace activity possibly by two mechanisms. logically, the closed conformation of the enzyme cannot allow the substrate into its active site. in the presence of compound, the free enzyme may be shifted to the open form, effectively increasing the activity coefficient of the enzyme. alternatively, product release is a rate-limiting step in ace turnover. ace activity may be enhanced in the presence of compound, as the enzyme-product complex empties more quickly and ace becomes available to start another cycle. xnt and resorcinolnaphthalein enhance in vitro ace activity in a dose-dependent manner and show no significant effects on ace activity. administration of xnt to shrs can result in a decrease in blood pressure, improvements in cardiac function and reversal of myocardial, perivascular and renal fibrosis [ ] . the protective role of xnt against hypertension-induced cardiac fibrosis is associated with activation of ace , increases in ang-( - ) and inhibition of extracellular signal-regulated kinases [ ] . furthermore, ace activation by xnt attenuates thrombus formation and reduces platelet attachment to vessels [ ] . xnt also prevents pulmonary vascular remodelling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension [ ] . development of xnt is being pursued to translate the vasoprotective concept of ace into an effective cardiovascular therapy [ ] . diminazine aceturate, a small molecule antiprotozoal agent that shares structural similarity with xnt, has recently been demonstrated to be a potent activator of ace and to decrease mean arterial pressure and myocardial fibrosis in shrs [ ] . intracerebroventricular infusion of diminazine aceturate, prior to and following endothelin- -induced middle cerebral artery occlusion, significantly attenuates cerebral infarct size and neurological deficits in a rat model [ ] . these data suggest that diminazine aceturate may serve as a lead compound for the discovery of the next generation of drugs for the treatment of cardiovascular disease and hypertension [ ] . interestingly, peptidase-independent actions of ace have also been elucidated. ace has been identified as an essential receptor for the coronavirus (cov) that causes severe acute respiratory syndrome (sars). the spike protein of sars-cov attaches the virus to its cellular receptor ace with a binding domain on the spike protein mediating the interaction [ ] . this ace -spike interaction leads to endocytosis of virus particles through internalization with ace , induces the fusion of virus with host cells and establishes sars-cov infection with the release of viral rnas into cytoplasm [ ] . the spike protein of sars-cov also induces tace-dependent shedding of the ace ectodomain, with the involvement of the ace cytoplasmic domain. cellular tace and the ace cytoplasmic tail promote viral entry and infection. these observations indicate that ace shedding and its causative cellular signals may be attributable to sars-cov-induced tissue damage [ ] . an ace inhibitor, n-( -aminoethyl)- aziridine-ethanamine, has been identified as being effective in blocking sars-cov spike proteinmediated cell fusion [ ] . the sars-cov receptor function of ace is independent of its catalytic activities for ang ii degradation but ace -mediated ang ii degradation is still important for lung protection from sars pathogenesis [ , ] . so, the consequence of long-term activation of ace must be determined and the effects of ace activators on the immune competence of animals and their vulnerability to sars-cov infection must be tested before these molecules are ready for preclinical trials [ ] . ace is also the receptor for the human coronavirus nl that was discovered in in the netherlands and was shown to be globally distributed [ ] . additionally, due to the high homology with collectrin at its transmembrane, ace binds to b at -family amino acid transporters and contributes to the absorption of neutral amino acids in animal intestines [ , ] . thus, ace may be involved in multiple biological functions. renin (ec . . . ) is a key enzyme of the ras. since its discovery years ago, an impressive quantity of information about renin has been compiled. it is generally accepted that the active renin found in the circulation of mammals almost exclusively originates from the kidney. in addition to systemic renin, there are a number of extrarenal tissues that express renin as part of the local or tissue-specific rass. within the kidney, renin is predominantly produced by the juxtaglomerular cells. (pro)renin gene transcription in these cells is controlled through several mechanisms, among which cyclic adenosine monophosphate (camp)/protein kinase a/camp response element binding protein (stimulatory) and calcium/protein kinase c (inhibitory) cascades are employed by physiological cues, whereas signal transducers, activators of transcription and nuclear factor b transcription factors (inhibitory) and members of the nuclear receptor superfamily probably become relevant in pathological situations. prorenin is stored in vesicles, activated to renin and then released upon demand. the release of renin is under the control of the intracellular camp (stimulatory), ca + (inhibitory) and cyclic guanosine monophosphate signalling pathways. meanwhile, a great number of intrarenally generated or systemically acting factors have been identified that control renin secretion directly at the level of renin-producing cells, by activating the camp or ca + signalling pathways [ ] . renin is an aspartyl protease with a typical structure made of two lobes. the cleft between the lobes contains the active site, characterized by two catalytic aspartic residues. renin is a highly specific enzyme and has only one known substrate (agt) [ ] . prorenin is the 'inactive' precursor of renin. in addition to the main organ, the kidney, other tissues, such as the brain, the adrenal gland, the submandibular gland, the glands of the reproductive system and adipose tissue, are also able to secrete prorenin in the surrounding milieu and in plasma. in contrast to renin, prorenin is released constitutively; renin and prorenin levels are usually well correlated. under some physiopathological circumstances, such as pregnancy and diabetes, prorenin levels exceed by far those of renin [ ] . prorenin has a prosegment of amino acid residues attached to the n-terminus of mature renin; the prosegment folds into an active site cleft of mature renin to prevent catalytic interaction with agt [ ] . prorenin-renin conversion occurs in the kidney before renin is released from the juxtaglomerular cells. several enzymes, including proconvertase and cathepsin b, have been proposed to be responsible for this irreversible cleavage of the prosegment from prorenin to form mature renin. a (reversible) non-proteolytic activation of prorenin may also occur. at acidic ph and/or low temperature, prorenin is capable of undergoing a conformational change, involving the unfolding of the prosegment from the enzymatic cleft. this non-proteolyticallyactivated prorenin is fully enzymatically active [ ] . under physiological conditions, approximately % of plasma prorenin is in the open form and can also display enzymatic activity [ ] . the traditional assumptions of renin being just an enzyme responsible for the cleavage of agt and prorenin being just an 'inactive' proenzyme were challenged by the cloning of a human (p)rr in [ ] . the (p)rr is a single transmembrane-domain protein of amino acids, with a large unglycosylated and highly hydrophobic n-terminal domain responsible for renin and prorenin binding and a short cytoplasmic tail of about amino acids involved in intracellular signalling [ ] . the (p)rr was first identified on cultured human mesangial cells and bound well to renin and prorenin with a k d in the nanomolar range [ ] . binding of prorenin induced a conformational change in the molecule, increasing its enzymatic activity from virtually zero to values similar to those of active renin in solution without proteolytic removal of the prosegment [ , ] . two regions in the human prorenin segment, namely t p fkr p (a gate that is not accessible by its specific antibodies until it is loosened from the active site cleft) and i p flkr p (a handle, a protruding pentameric segment), have been identified as being crucial for the non-proteolytic activation [ ] . renin bound to the (p)rr displays a -to -fold increase in the catalytic efficiency of agt conversion to ang i compared with free renin [ , ] . in addition to the ang-dependent function on the cell surface related to the increased catalytic activity of receptor-bound (pro)renin (renin activity of activated prorenin and increased activity of renin) that leads to the formation of ang i from agt, the (p)rr system also has ang-independent intracellular effects that are not necessarily related to the ras [ ] . pathological conditions like high blood pressure upregulate the receptor, whereas elevated (pro)renin concentrations downregulate the receptor via translocation of the transcription factor promyelotic zinc finger protein to the nucleus [ ] . in the ang-independent signalling pathways, prorenin-induced activation of mitogen-activated protein kinase p and subsequent phosphorylation of heat shock protein result in actin polymerization, while (pro)renin-induced activation of the extracellular signal-regulated kinase / (p /p ) signalling cascade leads to the intracellular expression of profibrotic genes, giving rise to the synthesis of transforming growth factor-␤, plasminogen activator inhibitor- , collagen and fibronectin. these effects potentially increase cardiac and renal hypertrophy and fibrosis [ , [ ] [ ] [ ] . the discovery of the (p)rr has confirmed the hypothesis that renin is also a hormone. it has been suggested that blocking the (p)rr may be a new target for renal and cardiac end-organ protection [ ] . additionally, the mannose- -phosphate/insulin-like growth factor ii receptor can bind both renin and prorenin with high affinity and is believed to serve as a clearance receptor for renin/prorenin. an intracellular renin-binding protein has been also cloned and found to be an inhibitor of renin activity but its deletion affected neither blood pressure nor plasma renin [ ] . after the discovery of the receptor, a (p)rr antagonist was designed, based on the idea that the prosegment contains a handle region that binds to the receptor, allowing prorenin to become catalytically active in a non-proteolytic manner [ ] . the antagonist (also known as hand region peptide, hrp), consisting of amino acids (nh -rillkkmpsv-cooh), resembles the handle region (i p llkk p ) of rat prorenin prosegment and will thus competitively bind to the (p)rr as a decoy peptide and inhibit the receptor-mediated activation of prorenin [ ] . treatment with the hrp in diabetic mice and rats decreased the renal content of ang i and ii and inhibited the development of nephropathy without affecting hyperglycaemia [ , ] . in stroke-prone shrs, continuous subcutaneous administration of the hrp inhibited activation of the tissue ras without affecting the circulating ras or arterial pressure and significantly attenuated the development and progression of proteinuria, glomerulosclerosis and cardiac fibrosis [ , ] . infusion of the hrp in human (p)rr transgenic rats significantly inhibited the development of glomerulosclerosis, proteinuria, mitogen-activated protein kinase activation and transforming growth factor-␤ expression in the kidneys [ ] . future research should be able to determine to what degree the beneficial in vivo effects of hrp are due to prorenin blockade [ ] and are related to interference with renin binding and the ras [ ] . decoy effects of the hrp, however, were not confirmed by other research groups [ ] [ ] [ ] [ ] . ace inhibitors and at receptor blockers (arbs) are proven to be effective therapeutic agents in the treatment of cvd. however, both ace inhibitors and arbs lead to a substantial compensatory rise in circulating active renin and ang peptides that may eventually limit their therapeutic potential [ ] . moreover, the increased ang i seen with ace inhibitors can subsequently be converted to ang ii by non-ace pathways, mediated by chymase and chymotrypsinlike ang-generating enzyme [ ] . in addition to the side effects of ace inhibitors, such as cough and angioedema, a meta-analysis of randomized controlled trials in suggested that arbs are associated with a modestly increased risk of new cancer diagnosis, although conclusions about the exact risk of cancer associated with each particular drug have not been drawn [ ] . based on this study, the united states food and drug administration is conducting a review to assess the possible link between the use of arbs and cancer. therefore, direct renin inhibition may be an alternative pharmacological approach to ras inhibition. renin is the rate-limiting step of the ras and the concept of blocking the ras at its origin by inhibiting renin has existed for at least years [ ] . the first-generation renin inhibitors were peptide analogues of agt and the secondgeneration compounds were peptidomimetic agents that are dipeptide transition-state analogue inhibitors of the active site. but these renin inhibitors had limited clinical use due to poor metabolic stability and oral bioavailability, short duration of action, weak antihypertensive activity and high cost of synthesis [ ] . a combination of molecular modelling and x-ray crystallographic analysis of the active site of renin led to the development of aliskiren, a new non-peptide low-molecular-weight orally-active renin inhibitor. aliskiren has a high binding affinity for renin and appears to bind to both the hydrophobic s -/s -binding pocket and a large, distinct subpocket that extends from the s -binding site toward the hydrophobic core of the enzyme. aliskiren is a potent competitive inhibitor of renin but very poorly inhibits related aspartic peptidases. it shows no effects on cytochrome p isoenzyme activities and is not bound extensively to blood proteins, therefore having a low potential for drug interactions [ ] . compared with the earlier renin inhibitors, aliskiren is more resistant to intestinal degradation and possesses significantly improved oral bioavailability. the terminal half-life ranges from to hours, which makes it suitable for once-daily dosing. aliskiren monotherapy displays an antihypertensive efficacy similar if not superior to that of other first-line antihypertensive drugs, with a safety and tolerability profile similar to that of arbs. combined with various antihypertensive agents, aliskiren exhibits synergistic effects [ ] . from , aliskiren was approved by regulatory bodies in both europe and the united states, for use alone and with other agents in the treatment of hypertension [ ] . recent studies showed that aliskiren treatment also markedly increased the plasma concentration of (pro)renin in patients and failed to inhibit the non-catalytic effects of (pro)renin [ , ] . the pharmaceutical company novartis reported that the addition of aliskiren to standard therapy for patients recovering from a heart attack did not provide the anticipated effect of limiting adverse changes to the heart's left ventricle. in december , novartis announced the early termination of the aliskiren trial in type diabetes using cardio-renal endpoints (altitude) and advised that aliskiren should not be used in combination with ace inhibitors or arbs in patients with diabetes. the study was conducted in type diabetic patients at high risk of fatal and non-fatal cardiovascular and renal events; aliskiren mg was given in addition to an ace inhibitor or an arb. the overseeing data monitoring committee concluded that study patients were unlikely to benefit from aliskiren and that there was a higher incidence of adverse events related to non-fatal stroke, renal complications, hyperkalaemia and hypotension in this highrisk population after - months of combined therapy (www.novartis.com). aliskiren has been shown to be a very safe antihypertensive after more than , patient-years of data. however, as we have seen in ongoing telmisartan alone and in combination with ramipril global endpoint trial (ontarget) that the combination of telmisartan (an arb) and ramipril (an ace inhibitor) is associated with more adverse events without an increase in benefit in patients with vascular disease or high-risk diabetes without heart failure [ ] , the combination of multiple safe drugs in this class may no longer be a promising strategy. over the last decade, a number of food-derived compounds have been shown to have in vitro ace inhibitory activity. some of them display significant antihypertensive activity in rats and humans. among these compounds, foodderived ace inhibitory peptides have been most widely studied [ , ] . a quantitative structure-activity relationship study indicated that a potent ace inhibitory dipeptide should have a large and hydrophobic amino acid at the c-terminus and a non-polar amino acid or possibly a positively charged amino acid at the n-terminus. for tripeptides, the most favourable residues for the c-terminus are aromatic amino acids, while hydrophobic amino acids are preferred for the n-terminus and positively charged amino acids are preferred for the middle position [ ] . the in vitro half-maximal inhibitory concentration (ic ) values of food-derived ace inhibitory peptides are about fold higher than that of synthetic captopril but they have higher in vivo activities than would be expected from their in vitro activities. it has been suggested that food-derived peptides might act via different antihypertensive mechanisms, possess higher affinities for tissues and be more slowly eliminated than synthetic captopril [ ] . until now, only two lactotripeptides (vpp and ipp) have been successfully commercialized. data from van mierlo et al., however, did not support a blood pressure-lowering effect of the two tripeptides in caucasians [ ] . given the discrepancy between in vivo and in vitro results, further investigation into the in vivo and clinical antihypertensive effects and mechanisms of food-derived ace inhibitors is necessary. three dipeptides (ir, kf and ef) in pea protein hydrolate have been identified as inhibiting renin activity in vitro [ ] . oleic acid and linoleic acid isolated from rice and some cereals also have in vitro renin inhibitory activity [ , ] . a series of studies have shown that saponins from different food/plant sources, primarily from soybean, can inhibit in vitro renin activity [ ] [ ] [ ] [ ] ; among them, orally administered soybean saponin can lead to a reduction of blood pressure in shrs but the direct evidence for saponin suppressing plasma renin activity in vivo is still lacking [ ] . experiments are required to further investigate the biochemical and biological properties of these plant/food-derived non-peptides that are related to renin inhibition. significant conceptual progress made in the last few years leads us to conclude that ace could serve as a new direction for improved therapeutics for cardiovascular disease. compared with ace inhibitor therapy, ace is an endogenous regulator of the ras. targeting ace would not only produce the vasoprotective/antiproliferative peptide ang-( - ) but would also influence the vasoconstrictive/proliferative effects of the ace/ang ii/at axis. as a part of the vasoprotective/antiproliferative axis, ace can effectively control fibrosis and structural remodelling and is extremely beneficial for pulmonary hypertension. ace activation may provide improved protection and reversal of ischaemia-induced neural damage. thus, direct activation of ace could result in better outcomes in cardiovascular disease. additionally, ace is a multifunctional enzyme with many biologically active substrates. the effects of ace on substrates other than ang i and ang ii may hold relevance for the treatment of cardiovascular disease [ ] . xnt analogues exist in many natural plant resources and have shown various physiological functions. using these resources to search for in vitro ace activators would be a good starting point for developing plant food-derived ace activating agents. ongoing experiments would characterize these ace activators. compared with ras regulatory drugs, these plant/food-derived bioactives appear more natural and safer to the consumer. as part of a daily diet, bioactives from food sources may result in a much lower healthcare cost. plant/food-derived ras regulators could be applied in the prevention of hypertension and as initial treatment in mildly hypertensive individuals [ ] . the authors declare that they have no conflicts of interest concerning this article. renin-angiotensin system revisited physiology and pharmacology of the (pro)renin receptor an ace in the hole alternative pathways of the renin angiotensin system and their potential role in cardiac remodeling a novel angiotensinconverting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters prorenin and (pro)renin receptor: a review of available data 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a structural basis for domain-specific inhibitor design evolutionary families of peptidases the angiotensin converting enzyme (ace) the two homologous domains of human angiotensin i-converting enzyme are both catalytically active peptidase specificity characterization of c-and n-terminal catalytic sites of angiotensin i-converting enzyme differences in the properties and enzymatic specificities of the two active sites of angiotensin i-converting enzyme (kininase ii). studies with bradykinin and other natural peptides spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung the structure of testis angiotensin-converting enzyme in complex with the c domain-specific inhibitor rxpa n-domain-specific substrate and c-domain inhibitors of angiotensin-converting enzyme: angiotensin-( - ) and keto-ace the hemoregulatory peptide n-acetyl-ser-asp-lys-pro is a natural and specific substrate of the n-terminal active site of human angiotensinconverting enzyme the n-terminal active centre of human angiotensin-converting enzyme degrades alzheimer amyloid beta-peptide angiotensin-converting enzyme c-terminal catalytic domain is the main site of angiotensin i cleavage in vivo novel activity of angiotensin-converting enzyme. hydrolysis of cholecystokinin and gastrin analogues with release of the amidated c-terminal dipeptide hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase novel activity of human angiotensin i converting enzyme: release of the nh -and cooh-terminal tripeptides from the luteinizing hormone-releasing hormone molecular basis of exopeptidase activity in the c-terminal domain of human angiotensin i-converting enzyme: insights into the origins of its exopeptidase activity the n-domain of angiotensin-converting enzyme specifically hydrolyzes the arg- -his- bond of alzheimer's abeta-( - ) peptide and its isoasp- analogue with different efficiency as evidenced by quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry the preparation and function of the hypertensin-converting enzyme potentiation of bradykinin effect by angiotensin-converting enzyme inhibition does not correlate with angiotensin-converting enzyme activity in the rat mesenteric arteries addition of eplerenone to an angiotensin-converting enzyme inhibitor effectively improves nitric oxide bioavailability angiotensin-converting enzyme i inhibitory activity of phlorotannins from ecklonia stolonifera degradation of metenkephalin by hemolymph peptidases in mytilus edulis the enzymatic degradation and transport of leucine-enkephalin and -imidazolidinone enkephalin prodrugs at the blood-brain barrier acute angiotensinconverting enzyme inhibition increases the plasma level of the natural stem cell regulator n-acetyl-seryl-aspartyl-lysylproline the synthetic tetrapeptide acsdkp protects cells that reconstitute longterm bone marrow stromal cultures from the effects of mafosfamide (asta z ) in vivo effect of the tetrapeptide, n-acetyl-ser-asp-lys-pro, on the g -s transition of rat hepatocytes the tetrapeptide acsdkp, a negative regulator of cell cycle entry, inhibits the proliferation of human and chicken lymphocytes the tetrapeptide acsdkp, an inhibitor of primitive hematopoietic cell proliferation, induces angiogenesis in vitro and in vivo prevention of myocardial fibrosis by n-acetyl-seryl-aspartyl-lysyl-proline in diabetic rats angiotensin-converting enzyme degrades alzheimer amyloid beta-peptide (a beta) a beta aggregation, deposition, fibril formation; and inhibits cytotoxicity angiotensin-converting enzyme converts amyloid beta-protein - (abeta( - )) to - ), and its inhibition enhances brain abeta deposition effect of a centrally active angiotensin-converting enzyme inhibitor, perindopril, on cognitive performance in a mouse model of alzheimer's disease a new class of angiotensin-converting enzyme inhibitors ace revisited: a new target for structure-based drug design major congenital malformations after first-trimester exposure to ace inhibitors roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin i and bradykinin: insights from selective inhibitors d-qsar studies on angiotensinconverting enzyme (ace) inhibitors: a molecular design in hypertensive agents rxp , a selective inhibitor of the n-domain of angiotensin i-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-ser-asp-lys-pro with no effect on angiotensin i hydrolysis ectodomain shedding of angiotensin converting enzyme in human airway epithelia tumor necrosis factoralpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndromecoronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis kidney amino acid transport hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ace and ace ) identified by site-directed mutagenesis therapeutic implications of the vasoprotective axis of the reninangiotensin system in cardiovascular diseases transgenic angiotensinconverting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme : prevention of angiotensin ii-dependent hypertension correlation of angiotensin-converting enzyme gene polymorphisms with stage hypertension in han chinese angiotensin converting enzyme- confers endothelial protection and attenuates atherosclerosis genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse candesartan ameliorates cardiac dysfunction observed in angiotensin-converting enzyme -deficient mice loss of angiotensin-converting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction the angiotensin-( - )/mas receptor axis is expressed in sinoatrial node cells of rats association of angiotensinconverting enzyme (ace ) gene polymorphisms with parameters of left ventricular hypertrophy in men. results of the monica augsburg echocardiographic substudy detection of soluble angiotensin-converting enzyme in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system loss of angiotensinconverting enzyme- leads to the late development of angiotensin ii-dependent glomerulosclerosis ace inhibition worsens glomerular injury in association with increased ace expression in streptozotocin-induced diabetic mice chronic treatment with angiotensin-( - ) improves renal endothelial dysfunction in apolipoproteine-deficient mice genetic deletion of the angiotensin-( - ) receptor mas leads to glomerular hyperfiltration and microalbuminuria overexpression of angiotensin-converting enzyme in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats gene transfer of angiotensin-converting enzyme in the nucleus tractus solitarius improves baroreceptor heart rate reflex in spontaneously hypertensive rats angiotensin-converting enzyme overexpression in the subfornical organ prevents the angiotensin ii-mediated pressor and drinking responses and is associated with angiotensin ii type receptor downregulation cerebroprotection by angiotensin-( - ) in endothelin- -induced ischaemic stroke ace , a promising therapeutic target for pulmonary hypertension structure-based identification of small-molecule angiotensinconverting enzyme activators as novel antihypertensive agents angiotensin-converting enzyme activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases ace activation promotes antithrombotic activity evidence for angiotensin-converting enzyme as a therapeutic target for the prevention of pulmonary hypertension diminazene aceturate is an ace activator and a novel antihypertensive drug structure of sars coronavirus spike receptor-binding domain complexed with receptor modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace induces tnf-alpha production and facilitates viral entry structure-based discovery of a novel angiotensin-converting enzyme inhibitor a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations physiology of kidney renin prorenin and the putative (pro)renin receptor inhibition of diabetic nephropathy by a decoy peptide corresponding to the ''handle'' region for nonproteolytic activation of prorenin prorenin and the (pro)renin receptor-an update pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin the (pro)renin receptor: a new addition to the renin-angiotensin system? human prorenin has ''gate and handle'' regions for its non-proteolytic activation binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system renin-stimulated tgf-beta expression is regulated by a mitogen-activated protein kinase in mesangial cells renin increases mesangial cell transforming growth factor-beta and matrix proteins through receptor-mediated, angiotensin ii-independent mechanisms prorenin induces intracellular signaling in cardiomyocytes independently of angiotensin ii prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptor-deficient mice regression of nephropathy developed in diabetes by (pro)renin receptor blockade contribution of nonproteolytically activated prorenin in glomeruli to hypertensive renal damage nonproteolytic activation of prorenin contributes to development of cardiac fibrosis in genetic hypertension slowly progressive, angiotensin ii-independent glomerulosclerosis in human (pro)renin receptor-transgenic rats prorenin is the endogenous agonist of the (pro)renin receptor. binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor prorenin and renininduced extracellular signal-regulated kinase / activation in monocytes is not blocked by aliskiren or the handle-region peptide the putative (pro)renin receptor blocker hrp fails to prevent (pro)renin signaling pro)renin receptor peptide inhibitor ''handle-region'' peptide does not affect hypertensive nephrosclerosis in goldblatt rats aliskiren: a new renin inhibitor as antihypertensive new class of agents for treatment of hypertension: focus on direct renin inhibition angiotensinreceptor blockade and risk of cancer: meta-analysis of randomised controlled trials prorenin engages the (pro)renin receptor like renin and both ligand activities are unopposed by aliskiren telmisartan, ramipril, or both in patients at high risk for vascular events plant food-derived angiotensin i converting enzyme inhibitory peptides structural requirements of angiotensin i-converting enzyme inhibitory peptides: quantitative structure-activity relationship study of di-and tripeptides lactotripeptides do not lower ambulatory blood pressure in untreated whites: results from controlled multicenter crossover studies identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate the occurrence of renin inhibitor in rice: isolation, identification, and structure-function relationship renin inhibitory activity in rice and cereals reduction of blood pressure by soybean saponins, renin inhibitors from soybean, in spontaneously hypertensive rats inhibition of human renin activity by saponins human renin inhibitory activity in legumes isolation of human renin inhibitor from soybean: soyasaponin i is the novel human renin inhibitor in soybean this work was supported partly by fundamental research funds for the central universities (jusrp a ) and the self-determined research program of jiangnan university (no. & no. ). key: cord- - lkzcslx authors: Álvarez-aragón, luis miguel; cuesta-muñoz, antonio luis; Álvarez-lópez, inmaculada title: inquiring into benefits of independent activation of non-classical renin-angiotensin system in the clinical prognosis and reduction of covid- mortality date: - - journal: clin infect dis doi: . /cid/ciaa sha: doc_id: cord_uid: lkzcslx nan a c c e p t e d m a n u s c r i p t we have read with great interest the elegant manuscript by hanff et al [ ] proposing a very interesting association between the classical renin-angiotensin system (ras) and angiotensinconverting enzyme (ace ) dysregulation present in cardiovascular disease (cvd) and the high mortality index in patients with cvd and coronavirus disease (covid- ). the authors state that pharmacological inhibition of classical ras could have two simultaneous and incompatible outcomes. on the one hand, it will decrease the proinflammatory effect of angiotensin ii with its subsequent benefit on decreasing the risk of acute respiratory distress syndrome (ards) observed in these patients, and on the other hand, it will increase ace expression and therefore the virulence of sars-cov . it has been shown that coronaviruses, in order to access the inside of their host cells, first must bind to a host receptor to be able to fuse both viral and host membranes [ ] . in humans, the host receptor is ace and, like sars coronavirus (sars-cov), sars-cov- also needs ace to attack human alveolar epithelial cells [ ] . therefore, this also makes ace crucial in the infectivity and pathogenesis of sars-cov- . in addition to the heart and kidneys, the classical ras and ace are also present in the lungs [ ] . it is pertinent to evoke that ace is also a fundamental component in the ace -angiotensin( - )-masr axis, also known as non-classical ras, indicating, therefore, the existence of non-classical ras also in the lungs. non-classical ras is a counter-regulatory system of the classical ras in that its end product, angiotensin( - ), which after binding to the mas receptor, presents important antiinflammatory, anti-proliferative, anti-fibrotic, natriuretic and vasodilator effects [ ] , actions completely opposed to those promoted by the end product of the classical ras angiotensin ii. while pharmacological exclusion or initiation of classical ras inhibition as an adjuvant treatment for sars-cov in patients with cvd is elucidated, and considering that covid- patients present downregulation of ace [ ] , and hence low angiotensin( - ), the direct pharmacological activation of the non-classical ras would be an attractive and plausible approach to tackle the reduction of angiotensin( - ) to lessen the unwanted effects of angiotensin ii. sodium-glucose co-transporter- (sglt ) inhibitors are a group of oral medications used to treat type diabetes, however, large epidemiological studies have demonstrated that slgt inhibitors present strong nephro-and cardiovascular-protective effects. in addition, in vitro studies in human renal cells treated with slgt inhibitors have shown an increment in angiotensin( - ) due to the independent activation of the non-classical res, leading to important anti-inflammatory and anti-m a n u s c r i p t fibrotic effects [ , ] . by analogy, it is reasonable to assume that slgt inhibitors could also activate the non-classical res in the lungs. a vast majority of diabetic patients also present cvd and many of them are treated with slgt inhibitors to both lower blood glucose and protect the kidney and heart. hence, would diabetic patients with cvd and treated with slgt inhibitors present a milder ards as compared to those with a different treatment? would they have a better clinical prognosis? and, would the use of slgt inhibitors in non-diabetic patients improve clinical prognosis as well? these are interesting questions since the answers might open a new door to counteract the devastating consequences of the proinflammatory cytokine storm present in covid- patients. we declare no conflict of interest. is there an association between covid- mortality and the renin-angiotensin system-a call for epidemiologic investigations receptor recognition by the novel coronavirus from wuhan: an analysis based on decadelong structural studies of sars coronavirus structure analysis of the receptor binding of -ncov renin-angiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation counterregulatory renin-angiotensin system in cardiovascular disease mechanical forces in diabetic kidney disease: a trigger for impaired glucose metabolism beat it early: putative renoprotective haemodynamic effects of oral hypoglycaemic agents a c c e p t e d m a n u s c r i p t key: cord- -zznm ik authors: van den eynde, jef; de groote, senne; van lerberghe, robin; van den eynde, raf; oosterlinck, wouter title: cardiothoracic robotic assisted surgery in times of covid- date: - - journal: j robot surg doi: . /s - - - sha: doc_id: cord_uid: zznm ik the coronavirus disease (covid- ) pandemic poses an immense threat to healthcare systems worldwide. at a time when elective surgeries are being suspended and questions are being raised about how the remaining procedures on covid- positive patients can be performed safely, it is important to consider the potential role of robotic assisted surgery within the current pandemic. recently, several robotic assisted surgery societies have issued their recommendations. to date, however, no specific recommendations are available for cardiothoracic robotic assisted surgery in covid- positive patients. here, we discuss the potential risks, benefits, and preventive measures that need to be taken into account when considering robotic assisted surgery for cardiothoracic indications in patients with confirmed covid- . it is suggested that robotic assisted surgery might have various advantages such as early recovery after surgery, shorter hospital stay, and reduced loss of blood and fluids as well as smaller incisions. however, electrosurgical and ultrasonic devices, as well as co insufflation should be managed with caution to prevent the risk of aerosolization of viral particles. the coronavirus disease (covid- ) pandemic poses an immense threat to healthcare systems worldwide. its repercussions are also felt in multiple branches of surgery, with the majority of elective surgeries being suspended to prioritize the use of means, operating rooms, and intensive care beds for covid- positive patients. questions have been raised as to which surgical procedures can still take place and if they do, how they can be performed safely. in response to this situation, various surgical societies have already issued their recommendations on adequate patient selection and preparation, as well as measures that can be taken to minimize the spread of viral particles. more recently, the european society of urology-robotic urology section (erus) [ ] , the american association of gynecologic laparoscopists (aagl) [ ] , and the society of european robotic gynaecological surgery (sergs) [ ] have published their statements on robotic assisted surgery (ras) in response to the current pandemic. however, to date no specific recommendations are available for cardiothoracic ras in covid- positive patients. it has to be noted that most guidelines recommend to suspend all elective procedures, first to create capacity for the care of victims of the pandemic but second to prevent exacerbation of the cytokine storm associated with covid- infection. as all surgical procedures induce a considerable amount of inflammation, this should always be weighed against the benefits of timely intervention. once a decision has been made after a thorough selection, several measures need to be taken into account during ras, as summarized in table . as pointed out by erus and aagl, electrosurgical and ultrasonic devices can produce large amounts of smoke. the low-temperature aerosol from ultrasonic scalpels seems to be ineffective in deactivating the molecular components of viruses and other microbial agents [ ] . among others, activated corynebacterium, papillomavirus, and hiv have been detected in surgical smoke. gloster et al. [ ] reported the transmission of a rare papillomavirus to several healthcare workers after exposure to surgical smoke. to decrease the production of surgical smoke, the power setting of the electrocautery should, therefore, be as low as possible and long dissecting times at the same spot should be avoided. furthermore, it has been demonstrated that min of electrocautery creates smoke with higher particle concentrations during laparoscopic surgery when compared to open surgery [ ] . a possible explanation might be the relatively low gas mobilisation within a pneumoperitoneum. moreover, the increased intracavitary pressures associated with pneumoperitoneum might represent an additional risk for aerosolization of viral particles with potential exposure of the operating staff. because similar principles are applied for co insufflation of the thorax, it is important to also be aware of this theoretical risk of viral spread in cardiothoracic ras. viral load might potentially even be higher in operating fields close to the lungs, especially because covid- lesions are mostly seen at the basal and peripheral parts of the lungs [ ] . insufflation pressures should, therefore, be reduced to the lowest level possible without compromising the surgical field exposure; research has demonstrated that robotic vision remains stable and optimal up to mmhg [ ] . care should also be taken to prevent sudden release of trocar valves or non-air-tight change of instruments. additionally, it is advised that an integrated flow system is used with continuous smoke evacuation through an ultra-low penetrating air (ulpa) filter or water lock to avoid escape of gases and particles [ ] . if an incision needs to be made after the robotic assisted part of the procedure, such as is the case in robotic assisted minimally invasive coronary artery bypass grafting, co insufflation should be stopped and intrathoracic pressures should have decreased sufficiently before the incision can be made. importantly, cardiothoracic ras may require one-lung ventilation to create more space within the thoracic cavity, hence providing adequate visualization during robotic surgery. however, the use of one-lung ventilation may constitute several dangers for both the patient and healthcare workers. first, airway instrumentation, including in-and extubation maneuvers as well as bronchoscopy, for the placement of a double lumen tube (dlt) or endobronchial blocker represents additional risk of aerosolization of viral particles [ ] . second, one-lung ventilation may be associated with intolerable hypoxemia or hypercapnia in covid- -injured lungs [ ] . third, one-lung ventilation is associated with additional lung injury, even when protective one-lung ventilation strategies are used [ ] . to date, no clinical studies have addressed these dangers, but it may be advisable to avoid one-lung ventilation in covid- diseased lungs if possible. lowering the positive end-expiratory pressure (peep) in patients with acute respiratory distress syndrome (ards) in an attempt to create more space and better surgical visualization is also not a good option, as this will result in hypoxemia [ ] . provided that the above discussed risks are taken into account and met with these preventive measures, cardiothoracic ras might on the other hand have various benefits to offer during the current covid- pandemic when compared to conventional open surgery. first, ras allows for earlier mobilization of the patient and a shorter hospital stay, resulting in lower chances of patients contracting complications such as pneumonia or spreading covid- at the ward. in addition, more hospital beds will be preserved to meet the increased need of capacity for the treatment of non-surgical covid- positive patients. furthermore, blood and fluid loss are considerably lower after ras than after open surgery and incisions are smaller, thus implying less routes through which the virus can spread to healthcare professionals, other patients, and the hospital environment. offering a type of surgery which includes less direct tissue contact, which is performed in a closed system, and which is characterized by early postoperative recovery, ras might be an option with regard to patients requiring cardiothoracic surgery in times of covid- , although only after thorough selection based on patient characteristics and severity of the condition that requires treatment. caution table measures during cardiothoracic robot assisted surgery (adapted and modified from table all surgery during the covid- pandemic should be regarded as high-risk, and, therefore, adequate preventive measures should be taken even in patients who tested negative or who have not been tested for covid- during cardiothoracic robotic assisted surgery, take steps to minimize co release close the taps of ports before inserting them to avoid escape of gas during insertion attach a co filter (ulpa or similar) or water lock to one of the ports for smoke evacuation. do not open the tap of any ports unless they are attached to a co filter or being used to deliver the gas minimize introduction and removal of instruments through the ports as much as possible. for introduction of material (such as bags, meshes) or specimen retrieval (such as biopsies), deflate the thorax with a suction device before entering or removing the material into or from the thorax or use an air-lock system. re-insert the port before turning co on again at the end of the procedure turn co off, deflate the thorax with a suction device and via the port with co filter, before removal of the ports avoid the use of ultrasonic sealing and use lowest possible electrocautery power. if possible use electrothermal bipolar vessel sealing one-lung ventilation should not be used in patients with covd- diseased lungs and peep should not be lowered in an attempt to improve surgical visualisation is advised with the use of electrosurgical and ultrasonic devices, co insufflation, and the use of trocar valves, all of which carry a potential risk of aerosolization into the operating theatre. adequate measures including filtration systems should, therefore, at any time be respected when performing ras in covd- positive patients. furthermore, one-lung ventilation should not be used in covid- diseased lungs. finally, general measures recommended by surgical societies such as personal protective equipment, optimal patient selection, and limitation of operating room staff evidently remain applicable in ras and should be adhered to strictly. funding none. conflicts of interest wo is proctor for minimally invasive multivessel midcab at medtronic. jvde, sdg, rvl, and rvde declare that they have no conflict of interest. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. erus (eau robotic urology section) guidelines during covid- emergency amercian association of gynecologic laparoscopists (aagl). ( ) covid- : joint statement on minimally invasive gynecologic surgery robot assisted surgery during the covid- pandemic, especially for gynecological cancer: a statement of the society of european robotic gynaecological surgery (sergs) minimally invasive surgery and the novel coronavirus outbreak: lessons learned in china and italy risk of acquiring human papillomavirus from the plume produced by the carbon dioxide laser in the treatment of warts characterization of smoke generated during the use of surgical knife in laparotomy surgeries essentials for radiologists on covid- : an update-radiology scientific expert panel influence of pneumoperitoneum pressure on surgical field during robotic and laparoscopic surgery: a comparative study anesthetic issues for robotic cardiac surgery thoracic anesthesia of patients with suspected or confirmed novel coronavirus infection: preliminary recommendations for airway management by the eacta thoracic subspecialty committee lung injury after one-lung ventilation: a review of the pathophysiologic mechanisms affecting the ventilated and the collapsed lung covid- does not lead to a "typical" acute respiratory distress syndrome key: cord- -iggkxbbf authors: phillips, m. ian; de oliveira, edilamar menezes title: brain renin angiotensin in disease date: - - journal: j mol med (berl) doi: . /s - - - sha: doc_id: cord_uid: iggkxbbf a brain renin angiotensin system (ras) and its role in cardiovascular control and fluid homeostasis was at first controversial. this was because a circulating kidney-derived renin angiotensin system was so similar and well established. but, the pursuit of brain ras has proven to be correct. in the course of accepting brain ras, high standards of proof attracted state of the art techniques in all the new developments of biolo gy. consequently, brain ras is a robust concept that has enlightened neuroscience as well as cardiovascular physiology and is a model neuropeptide system. molecular biology confirmed the components of brain ras and their location in the brain. transgenic mice and rats bearing renin and extra copies of angiotensinogen genes revealed the importance of brain ras. cre-lox delivery in vectors has enabled pinpoint gene deletion of brain ras in discrete brain nuclei. the new concept of brain ras includes ace- , ang – , and prorenin and mas receptors. angiotensin ii (ang ii) generated in the brain by brain renin has many neural effects. it activates behavioral effects by selective activation of ang ii receptor subtypes in different locations. it regulates sympathetic activity and baroreflexes and contributes to neurogenic hypertension. new findings implicate brain ras in a much wider range of neural effects. we review brain ras involvement in alzheimer’s disease, stroke memory, and learning alcoholism stress depression. there is growing evidence to consider developing treatment strategies for a variety of neurological disease states based on brain ras. of drinking obviously requires neural circuits in the brain. it was reasoned that the effects of ang ii in the brain were isolated from circulating ras by the blood-brain barrier (bbb). however, circumventricular organs (cvos) lack a bbb and the circulating ras and the endogenous brain ras appear to have a complex interface in the cvos [ , ] . to prove a brain ras existed, neurophysiological techniques were required. felix and phillips [ ] showed that ang ii activated neurons in the brain and in the subformical organ (sfo), and a peptide antagonist of ang ii, saralasin, inhibited ang ii-induced activity in those neurons. this suggested that ang ii is a nonclassical neurotransmitter. taking the concept a step further, it was reasoned that if ang ii, formed by brain renin, was involved in hypertension, then inhibiting brain ras would lower blood pressure. simply injecting saralasin alone into the brain of stroke prone hypertensive rats (spshr) temporarily reduced high blood pressure [ ] . this occurred even in the absence of kidney renin when the spshr rats were bilaterally nephrectomized [ ] . the finding was later confirmed by molecular inhibition of ang ii synthesis in the brain [ ] . it meant that brain ras plays a role in neurogenic hypertension. immunocytochemistry with renin antibodies showed staining in neurons [ , ] . renin-like activity was evident in the hypothalamus, pituitary, and pineal glands. antibodies to ang ii revealed a clear distribution of ang iilike staining in the hypothalamus and cvos and in higher concentration in shr [ ] . what was needed were actual measurements of the renin and ang ii proteins. this was the achieved with high-performance liquid chromatography [ , ] and direct protein assay [ ] . brain levels of ang ii were higher than circulating levels of ang ii, suggesting independence of the two systems. having shown that ang ii was in the brain, the next level of proof required demonstrating that it had been synthesized in the brain. molecular biology was used to measure and locate mrna for agt and renin in the brain [ , ] . stornetta et al. [ ] identified glial cells as the site of synthesis of agt using in situ hybridization mrna. mendelsohn et al. [ ] , using autoradiography with pseudocolor imaging, were able to map the entire distribution ang ii receptor binding sites in the brain. this proved that there were sites within the brain that had evolved to be activated by ang ii made in the brain. minute injections of ang ii into key brain nuclei showed that ang ii could differentially produce pressor effects, elicit drinking, release vasopressin, inhibit the baroreflex, and increase sympathetic nervous system activity [ , ] . the confirmation that there was a separate brain ras, independent of the circulating ras, gave rise to the more generalized concept of local or tissue angiotensins in most other tissues including heart, blood vessels, and, ironically, the kidney [ ] whitebread et al. [ ] and chiu et al. [ ] reported that there were two subtypes of the ang ii receptor, at and at . from this has sprung not only insight into genes involved in brain function but also the highly successful angiotensin receptor blockade class of antihypertensive drugs. the development of the transgenic mice [ ] was relevant to brain ras with transgenic mice overexpressing agt by a metallothionein promoter [ ] . transgenic mice, overexpressing agt in brain and liver, led to high blood pressure [ , ] . but, what about renin? renin exists in only one form in humans, ren- , but in two forms in mice, ren- and ren- . after inserting a mouse ren- gene into a transgenic rat [tgr(mren- ) ], mullins et al. [ ] reported fulminant hypertension in transgenic rats, even though circulating ang ii levels were low. the homogenous offspring developed malignant hypertension and had high mortality. the heterozygous offspring had end organ damage in the heart and kidney with extreme hypertension. hypertension disappeared when the brains of these mice was anesthetized. curt sigmund's group [ ] developed transgenic mice that overexpressed human renin and human agt. with either a glial fibrillary acidic protein promoter or a synapsin- promoter, to stimulate gene expression in neurons or glial cells, hypertension and increased drinking resulted. transgenic animals must undergo embryonic development. if the inserted gene or the deleted gene of the transgenic animal is not embryonically lethal, it may be compensated for by other genes duplicating its role. this may account for the surprisingly benign effects of at knockout transgenic mouse [ , ] . to overcome this possibility, we developed gene suppression methods in adult animals using in vivo rna-based inhibition. antisense inhibition of agt mrna or at r mrna oligonucleotides delivered to the brain reduces hypertension in shr [ ] . antisense inhibition of at r specifically diminishes at receptors in the paraventricular nucleus and organum vasculosum of the lamina terminalis [ ] . functionally, at antisense inhibited the effects of ang ii in the brain including the pressor response, vasopressin release, and drinking [ ] . in the double transgenic mice developed by sigmund and colleagues [ ] , to overexpress both human renin and human agt so that they constantly have high ang ii levels, antisense to at r mrna dramatically reduced the blood pressure. [phillips et al., unpublished] . in an elegant study, davisson et al. [ ] , using a combination of at a -and at b -deficient mice, showed that at a receptors are mainly involved in central blood pressure (bp) control, while at b receptors mediate the drinking response. the question of which genes and exactly where in the brain cardiovascular effects are regulated may now be answered with greater precision. davisson and colleagues, who previously demonstrated uptake of vectors into specific location of the brain, reported an approach to gene deletion in mice with the cre-lox system [ ] . cre, a bacteriophage p -derived dna recombinase, serves to recombine precisely defined dna sequences that have been "floxed" by loxp sequences prior to embryonic development. because cre recombines the loxp sequences, the floxed segment is deleted and rendered nonfunctional. sinnayah et al. [ ] microinjected cre with a promoter into selective targets, such as the sfo and supraoptic nucleus, delivered by adenovirus vectors or feline immunodeficiency virus. with this technique, the sigmund group have demonstrated the presence of brain ras and its physiological and blood pressure effects [ , ] . the classical brain ras components must now include ang iii, ang iv, ang ( - ), ace , prorenin, and mas receptors (fig. ) . ang iii is a metabolite of ang ii found in brain and cerebrospinal fluid. it appears to act on the at receptor. ang iv is an endogenous agonist that has been identified in the brain in areas distinct from those where at and at are located and probably acts on an at receptor. an action of brain ras is implied in memory and learning by the finding that stimulation of at receptors potentiates the release of acetylcholine from hippocampal brain slices [ ] . ace generates ang ( - ) from ang i [ , ] ; ace is a human ace homolog that differs greatly from ace in substrate specificity and its activity is not modified by ace inhibitors [ ] . ace is widely expressed in a variety of tissues including brain [ ] [ ] [ ] [ ] . mice lacking the ace gene showed elevation in plasma ang ii level [ ] and enhanced pressor response to ang i. ang ( - ) is formed from ang ii via the action of several tissue-specific endopeptidases or from ang i via ace . ang ( - ) can be metabolized by ace to ang ( ) ( ) ( ) ( ) ( ) ; it is a peptide with no known biological activity. ang ( - ) produces its biological effects such as vasodilatation, diuresis, natriuresis, and antitrophic via the mas receptor [ ] . mas receptor stimulation also induces to production of nitric oxide (no) and prostacyclin. this new [ , ] . this points to a complex interface between the brain ras and the systemic ras in controlling vital cardiovascular functions. prorenin receptors were first cloned by nguyen et al. [ ] . they bind prorenin with higher affinity than renin [ ] . tissue prorenin [ , ] is active and renin per se has cellular effects independent of ang ii generation [ , ] . prorenin receptors may help to explain the presence of tissues ras, where the generation of ang ii is independent of systemic ras. there are two forms of renin expressed in the brain of rodents and humans: secreted prorenin and nonsecreted renin. this suggests that renin is constitutively active [ ] . using transgenic mice that express nonsecreted active renin or secreted prorenin in the brain, bp and drinking volume were increase similarly in both models. bp was normalized by an intracerebral ventricular injection of losartan in both models, whereas the same dose given systemically had no effect. these data support the concept of an intracellular form of renin in the brain, which may promote functional changes in fluid homeostasis and bp regulation [ ] . ace , ang ( - ), and mas receptors ace is a carboxypeptidase that counterregulates the vasoconstrictors effects of ang ii to degrade it to the vasodilator peptide ang ( - ) [ ] . the first functional evidence of the ang ( - ) in the brain showed that the peptide stimulates the release of vasopressin, equivalent to that obtained with ang ii [ ] . in the central nervous system, ang ( - ) acts as neuromodulator, especially in areas related to tonic and reflex control of arterial pressure. its effects are blocked by a mas receptor antagonist a- [ ] . becker et al. [ ] showed mas receptor in cardiovascular and hydroelectrolytic control areas of the rat brain. ang-( - ) appears to act through the mas receptor. the distribution of ace is widespread in the mouse brain, predominantly in neurons. ace mrna is found in rat medulla oblongata, and ace overlaps with compo-nents of the ras in the same areas [ , ] . ace- , ang ( - ), and mas receptors are all localized in brain areas related to the control of cardiovascular function. the physiological effects of central ang ii are mainly mediated by at r, including vasoconstriction effects, vasopressin release, retention of salt and water, cell growth, and stimulation of aldosterone from the adrenal gland [ ] . however, via at , the ang ii mediates a wide variety of actions, including vasodilatation, inhibition of cellular growth, cell differentiation, and apoptosis. in addition to the brain ras role in neurogenic hypertension discussed above, evidence is accumulating that brain ras is involved in alzheimer's disease, stroke, memory and learning, alcoholism, depression and, emotional stress. alzheimer's disease (ad) is an age-related, neurodegenerative disease with a prevalence of - % in individuals over years [ ] . the dementia presents as primary indicator with memory loss following progressive cognitive impairment and personality changes [ ] . two hypotheses have been proposed to explain the pathogenesis of ad [ ] . the amyloid plaque (ap) hypothesis postulates that ap is generated by proteolytic activity of the βand γ-secretase processing the amyloid precursor protein (app). however, although ap was observed in transgenic mice with mutations of app and presenilin- (ps ) genes, the mice did not exhibit neurodegeneration or memory loss observed in ad patients. supporting the presenilin hypothesis, it postulates the neurodegenerative and behavioral effects independent of ap. an increase in the ace activity and alteration in others components of brain ras has been demonstrated in ad [ ] [ ] [ ] [ ] . the beneficial effects of ace inhibitors and at blockers have been shown in animals and humans suggesting reducing brain ras activity is important. there are many effects of decreased ang ii activity including reduced blood pressure, less acetylcholine release, and increase of substance p-a substrate of ace which is reported to increase neprilysin activity, a recognized amyloid β degrading enzyme [ ] . treatment with ace inhibitors and at antagonists has been shown to prevent stroke in spshr and in salt-loaded dahl salt-sensitive rats [ ] . some clinical trails (syst-eur, systolic hypertension in europe; progress, peridopril protection against recurrent stroke) and randomized studies using antihypertensive treatments have been performed using different combinations of ace inhibitors: at receptor antagonists, β-blockers, and diuretics treatments [ ] [ ] [ ] [ ] . at receptor antagonists normalized the expression of cerebrovascular nitric oxide synthase, cerebrovascular compliance, and protected cerebrovascular flow [ ] . similarly, cerebral blood flow was increased by ang ( - ) and reduced by a selective ang ( - ) antagonist a- [ ] . these data suggest that ang ( - ) could have protective effects in ischemia. in general, the results of treatment show a reduction of bp, stroke, vascular dementia, and cognitive decline [ ] . zhou et al. [ ] showed evidence of an active local ras in brain microvessels influenced by circulating prorenin and ang ii with a predominant localization to the microvessel endothelium. kagiyama et al. [ ] induced stroke with middle cerebral artery occlusion in rats and found that local angii levels and at receptor concentrations increased. they concluded that brain ras and at receptors increase apoptosis in cerebral ischemia. several studies have indicated brain ras in memory function. at antagonist treatment reduced anxiety and improve learning, spatial working memory, and motor performance in the aged rat [ ] [ ] [ ] [ ] . denobel et al. [ ] reported that intracerebroventricular infusion of renin disrupts passive avoidance learning. coadministration of ace inhibitor (captopril) or at antagonist attenuates this deficit induced by renin. at receptor antagonist was not effective. ace inhibitors have been shown to enhance memory learned by fear or habituation [ ] . recently, bonini et al. [ ] showed that the intrahippocampal infusion of ang ii in rats induces amnesia. the effect was completely reversible and did not alter locomotion activity, exploratory behavior, or anxiety state. this effect of ang ii was blocked by the at antagonist but not by losartan. long-term potentiation (ltp) is a correlation of learning and memory processes. ang iv and ang ( - ) increase the excitability and ltp of neurons of the hippocampus [ ] . furthermore, mas receptor knockout mice do not show alteration of hippocampal ltp [ ] . a picture emerges of inhibitory influence by ang ii acting at the at receptor, and a facilitatory role by ang iv acting at the at to increase excitability, ltp, associative and spatial learning, and memory. although, the ang ii participation in memory consolidation is controversial, results suggest that ang iv and ang - via mas receptor in the brain are involved in cognitive behavior. alcohol intake ang ii induces drinking, but does it induce alcohol drinking? losartan, the at receptor blocker, has been reported to block toxic effects of low doses of ethanol [ ] . maul et al. [ ] showed that in at knockout mice, ang ii affected alcohol consumption, but at receptor and bradykinin receptors are not involved [ ] . furthermore, ethanol-induced inhibition of hippocampal ltp is mediated by at receptors [ ] . recently, sommer et al. [ ] , using rats treated with spirapril-a blood-brain-penetrating inhibitor of ace-or transgenic rats [tgr(asraogen) ] with reduced central agt expression, showed that increased ethanol consumption was associated with an upregulation of agt transcript levels and a downregulation of local at mrna. taken together, these data suggest that reducing brain ras could be a strategy for treating alcoholism. the etiology of depression involves several neurotransmitter systems. the first evidence that brain ras may be important in depression was observed in hypertensive patients undergoing captopril treatment. depressed patients in the group noted reduced depression [ ] . in this context, ras polymorphisms have been investigated for their association with depression, including the agt t allele, ace d allele, and at r c allele, which are associated with markedly higher plasma agt and ace activity and with greater responsiveness to ang ii at lower concentrations. the ace d allele was more prevalent in depressed patients of japanese population [ ] . agt m allele was associated with increased susceptibility for bipolar affective disorder in brazilian patients [ ] . recently, saab et al. [ ] observed that at c allele was prevalent in a depressed lebanese population. ras polymorphisms associated with depression are, in some cases, associated with suicidal behavior [ ] . among males, the risk for suicide in i/i homozygotes was about % higher than in subjects with other genotypes. ace i/d polymorphism may also increase the vulnerability to suicide [ ] . however, not all studies have found associations of these polymorphisms with depression, so the jury is still out. brain ras distribution of at receptors follows the hypothalamic-pituitary-adrenal axis. during stress, both the peripheral and the central ang ii systems are stimulated, with increases in at receptors expression and ang ii levels. different types of stress have been associated with peripheral sympathetic nerve stimulation, increased plasma renin activity, and higher circulating ang ii [ ] . at r are increased in brain by isolation stress induced by short periods of isolation and to -h isolation stress. at receptor blocker prevented this response and the ulcerations of the gastric mucosa produced by isolation stress [ ] . peng and phillips [ , ] demonstrated that in cold-induced hypertension, agt and ang ii in the brain predominate and in different brain areas, at r are upregulated and at r downregulated. at antisense treatment reduced the cold-stress-induced high blood pressure [ ] . experimental studies also showed that pretreatment with losartan provide protection from stress induced by immobility or forced swimming [ ] . ang ii is an important stress hormone, and blockade of at receptors might be considered for stress-induced disorders. to inhibit central at receptors may be impractical, but the hypothesis of paton suggests inhibition of at r in brain capillaries could be an alternative if no is involved [ , ] . detlev ganten's bold hypothesis in has proven correct. the brain ras is a model for neuropeptide processing and action in the brain. brain ras has neuronal effects far beyond cardiovascular and fluid homeostasis and may be developed for therapies for neurological dysfunctions the discovery of renin years ago angiotensin-forming enzyme in brain tissue a micromethod for the measurement of renin in brain nuclei: its application in spontaneously hypertensive rats the cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin subfornical organ: a dipsogenic site of action of angiotensin ii angiotensin ii in central nervous system physiology specific angiotensin ii receptive neurons in the cat subfornical organ lowering of hypertension by central saralasin in the absence of plasma renin antisense inhibition of at receptor mrna and angiotensinogen mrna in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin wide distribution of immunoreactive renin in nerve cells of human brain renin-like immunocytochemical activity in the rat and mouse brain angiotensin-like immunoreactivity in the brain of the spontaneously hypertensive rat angiotensin synthesis in the brain and increased turnover in hypertensive rats angiotensin ii in rat brain comigrates with authentic angiotensin ii in blood pressure liquid chromatography presence of renin in primary neuronal and glial cells from rat brain localization of angiotensinogen messenger rna sequences in the rat brain identification of renin and angiotensinogen messenger rna sequences in rat brain astrocytes synthesize angiotensinogen in brain autoradiographic localization of angiotensin ii receptors in rat brain visualization of specific angiotensin ii binding sites in the brain by fluorescent microscopy functions of angiotensin in the central nervous system preliminary biochemical characterization of two angiotensin ii receptor subtypes identification of angiotensin ii receptor subtypes germ-line transformation of mice generation of transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes studies on the regulation of renin genes using transgenic mice high blood pressure in transgenic mice carrying the rat angiotensinogen gene fulminant hypertension in transgenic rats harbouring the mouse ren- gene the brain reninangiotensin system in transgenic mice carrying a highly regulated human renin transgene behavioural and cardiovascular effects of disrupting the angiotensin ii type- receptor in mice effects on blood pressure and exploratory behaviour of mice lacking angiotensin ii type- receptor antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate genes a decrease in angiotensin receptor binding in rat brain nuclei by antisense oligonucleotides to the angiotensin at receptor antisense oligonucleotide to at receptor mrna inhibits central angiotensin induced thirst and vasopressin the brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes divergent functions of angiotensin ii receptor isoforms in the brain targeted viral delivery of the cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain localization of renin expressing cells in the brain using a ren-egfp transgenic model efficient liver-specific deletion of the floxed human angiotensinogen transgene by adenoviral delivery of the cre recombinase in vivo potentiation of cholinergic transmission in the rat hippocampus by angiotensin iv and lvv-hemorphin- a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - distinct roles for ang ii and ang-( - ) in the regulation of angiotensin-converting enzyme in rat astrocytes new mass spectrometric assay for angiotensin-converting enzyme activity angiotensin-converting enzyme is an essential regulator of heart function angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas detection of angiotensin ii mediated nitric oxide release within the nucleus of the solitary tract using electron-paramagnetic resonance (epr) spectroscopy pivotal role of the renin/prorenin receptor in angiotensin ii production and cellular responses to renin binding properties of rat prorenin and renin to the recombinant rat renin/prorenin receptor prepared by a baculovirus expression system tissue activity of circulating prorenin in vivo enzymatic assay reveals catalytic activity of the human renin precursor in tissues specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor- antigen evidence supporting a functional role for intracellular renin in the brain effects of reninangiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors release of vasopressin from the rat hypothalamoneurohypophysial system by angiotensin-( - ) heptapeptide cardiovascular effects of angiotensin-( - ) injected into the dorsal medulla of rats immunofluorescence localization of the receptor mas in cardiovascular-related areas of the rat brain differential expression of neuronal ace in transgenic mice with overexpression of the brain reninangiotensin system is inhibition of the reninangiotensin system a new treatment option for alzheimer's disease transcriptional regulation of the presenilin- gene: implication in alzheimer's disease effects of brain-penetrating ace inhibitors on alzheimer disease progression angiotensin-converting enzyme inhibitors and incidence of alzheimer's disease in japan prevention of dementia: lessons from syst-eur and progress prevention of dementia in randomised double-blind placebo-controlled systolic hypertension in europe (syst-eur) trial stroke prevention by losartan in stroke-prone spontaneously hypertensive rats brain angiotensin ii: new developments, unanswered questions and therapeutic opportunities systemic and regional. hemodynamic effects of angiotensin-( - ) in rats at receptor blockade regulates the local angiotensin ii system in cerebral microvessels from spontaneously hypertensive rats expression of angiotensin type and type receptors in brain after transient middle cerebral artery occlusion in rats non-peptide angiotensin ii receptor antagonist and angiotensin-converting enzyme inhibitor: effect on a renin-induced deficit of a passive avoidance response in rats angiotensin ii blocks memory consolidation through an at receptor-dependent mechanism angiotensin ii disrupts inhibitory avoidance memory retrieval angiotensin-( - ) enhances ltp in the hippocampus through the gprotein-coupled receptor mas losartan reduces ethanol intoxication in the rat central angiotensin ii controls alcohol consumption via its at receptor ethanol and diazepam inhibition of hippocampal ltp is mediated by angiotensin ii and at receptors plasticity and impact of the central renin-angiotensin system during development of ethanol dependence captopril treatment of major depression with serial measurements of blood cortisol concentrations frequency of the fragile x syndrome in japanese mentally retarded males angiotensinogen and angiotensin converting enzyme gene polymorphisms and the risk of bipolar affective disorder in humans renin-angiotensin-system gene polymorphisms and depression association between a functional polymorphism in the renin-angiotensin system and completed suicide angiotensin ii-an important stress hormone reduction of cold-induced hypertension by antisense oligodeoxynucleotides to angiotensinogen mrna and at -receptor mrna in brain and blood the predominant role of brain angiotensinogen and angiotensin in environmentally induced hypertension brain and peripheral angiotensin ii play a major role in stress differential baroreflex control of sympathetic drive by angiotensin ii in the nucleus tractus solitarii acknowledgements em oliveira is recipient of a brazil-cnpq fellowship. mip was supported by nih merit award r -hl and nih r , r -hl . key: cord- - uog j authors: mabillard, holly; sayer, john a. title: electrolyte disturbances in sars-cov- infection date: - - journal: f res doi: . /f research. . sha: doc_id: cord_uid: uog j the global pandemic secondary to the severe acute respiratory syndrome coronavirus (sars-cov- ) is leading to unprecedented global morbidity and mortality. with a bewildering array of complications, renal involvement in various forms is common, including serum electrolyte derangements. hypokalaemia secondary to sars-cov- was common in a reported chinese cohort. here we review the emerging evidence on hypokalaemia and sars-cov- infection, the potential pathophysiological mechanisms based on early clinical and histopathological data and important clinical implications. mechanisms of hypokalaemia are multifactorial and so the electrolyte disturbance can be difficult to avoid. we provide further support to the theory of renin-angiotensin-aldosterone (ras) activation, discuss the strengths and weaknesses of implicating ras involvement and highlight the importance of calculating the transtubular potassium gradient to identify those at risk of hypokalaemia and its complications. the global pandemic secondary to the severe acute respiratory syndrome coronavirus (sars-cov- ) is leading to unprecedented global morbidity and mortality. common symptoms include fever ( % of patients), cough ( %) and dyspnoea ( %) but other features such as myalgia ( %), diarrhoea ( %), anosmia and hypogeusia ( %) are also common . the most frequent serious manifestation of infection is pneumonia with % of patients developing serious manifestations such as hypoxia, dyspnoea, extensive pulmonary involvement and acute respiratory distress syndrome (ards) - . a recent meta-analysis including , patients with sars-cov- infection described an ards incidence of . % . anecdotal data and clinical observation from patients with the world health organization named disease coronavirus disease (covid- ) has highlighted a bewildering array of presentations and pathologies in those infected with sar-cov- many of which, it should be stressed, are rare. these include additional respiratory complications (pulmonary fibrosis -reported in % of those hospitalised with sars-cov- months post-discharge in one study ) , cardiovascular complications (acute cardiac injury ( % ), cardiomyopathy ( / patients ), cardiac tamponade, heart failure, dysrhythmias ( % ) and venous thromboembolic events ( % )) , neurological complications (myopathy, acute stroke ( . % of those with severe infection ), guillain-barre syndrome ( . % hospitalised patients ) and encephalopathy) , acute liver and/or pancreatic injury ( % and % respectively in one cohort) , cytokine storm syndrome, septic shock, dic, diarrhoea, kawasaki-like disease and renal complications (acute tubular injury, rhabdomyolysis, proteinuria, secondary focal segmental glomerulosclerosis and possible renin-angiotensinaldosterone system activation) . a few small studies have demonstrated that sars-cov- can affect the kidneys directly but it is unclear if this is a key mechanism in acute kidney injury seen in patients with sars-cov- infection. involvement of the renin-angiotensin-aldosterone (ras) pathway has been speculated as a mechanism to lead to more severe covid- disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (ckd), diabetes mellitus and cardiovascular disease than those with milder disease. this is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor . here we aim to review the evidence, potential clinical implications and possible mechanisms of electrolyte disturbances and kidney injury in patients with sars-cov- infection. firstly, it is important to highlight how transmission of the virus occurs in humans. the sars-cov- spike protein enters host cells via the angiotensin-converting enzyme (ace ) receptor on the surface of pulmonary type alveolar cells , . ace is crucial to counter-regulate ras and shares approximately % homology with ace. ras pathway components are co-expressed in most organs and tissues in the body and communicate via both paracrine and autocrine signalling. ace converts angiotensin-ii into angiotensin-( - ), which acts on the mas receptor expressed on a variety of tissue cell lineages relevant to cardiovascular disease in addition to type alveolar epithelial cells. it lowers blood pressure through vasodilation and promotion of renal sodium and water excretion and it also attenuates inflammation by producing nitric oxide . meanwhile, ace converts angiotensin-i to angiotensin-ii which has directly opposing effects to signalling mediated by ace . angiotensin-ii acts at the type angiotensin receptor (at r) to increase blood pressure by the induction of vasoconstriction and renal sodium and water reabsorption. this also creates oxidative stress which promotes inflammation and fibrosis. the balance between these two opposing pathways determines potential tissue injury, predominantly in the heart and kidneys , . not only has the ace receptor found to be used for host cell entry by the sars-cov virus, but the affinity for sars-cov- is - -fold higher . during host cell entry, proteolytic cleavage of ace by transmembrane serine protease (tmprss ) occurs which is thought to suppress ace expression. this in theory would lead to a reduction in angiotensin-( - ) generation and angiotensin-ii levels would increase resulting in oxidative-stress mediated tissue damage and hypertension , . this mechanism resulting in lung parenchymal injury has already been demonstrated in mice injected with sars-cov virus . involvement of the ras pathway has therefore been speculated as a mechanism to lead to more severe covid- disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (ckd), diabetes mellitus and cardiovascular disease than those with milder disease. this is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor . hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition . in fact, the incidence of ventricular fibrillation has been shown to be fivefold higher in patients with hypokalaemia compared to those with hyperkalaemia . furthermore, inadequate management of hypokalaemia has been identified in % of hospitalised patients , reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce. what is the evidence for hypokalaemia in sars-cov- infection so far? the release of a pre-print retrospective chinese study initially sparked interest into hypokalaemia as a potentially prevalent biochemical disturbance in sars-cov- infection . this was released around a similar time to concerns around ras inhibitors and sars-cov- where speculation that ras inhibitors may increase risk of sars-cov- infection and its severity due to the knowledge that ace is the viral binding site for host cell entry. it has been stressed by multiple organisations globally that ras inhibitors should be continued unless otherwise medially indicated as the risks of stopping these important drugs have numerous serious complications to the patient. this study claimed that hypokalaemia was prevalent amongst sars-cov- infected patients, affecting of patients ( %) that had a serum potassium of < . mmol/l and only patients had a serum k > . mmol/l which is the value required for those with myocardial dysfunction. of the patients, % had severe hypokalaemia (serum potassium < . mmol/l). in total, % of all patients and % of those with severe hypokalaemia displayed metabolic alkalosis (ph > . ) compared to % with normokalaemia suggesting the possibility of ras activation. the study suggested that the hypokalaemia was not due to gastrointestinal (gi) loss of potassium as only a small proportion of patients had gi symptoms, there was no significant difference between serum potassium in those with or without diarrhoea and significant urinary potassium wasting was shown. a total of patients had a spot urine potassium/ creatinine ratio and there were significantly higher urinary potassium/creatinine ratios in those with hypokalaemia compared with normokalaemia. the study reported that the degree of hypokalaemia correlated with severity of sars-cov- symptoms and they suggested that hypokalaemia can be difficult to correct as seen in two patients because the renal potassium wasting persists until clinical recovery from the virus. this study is relatively small, limited to a chinese population and did not disclose medications or diuretic use which could be a significant confounding factor here. as diuretics (especially "loop" diuretics) are used to improve oxygenation in patients with ards, some of these patients may well have been prescribed them. it was postulated that hypokalaemia in these patients is due to the effects of increased angiotensin- resulting from the proteolytic cleavage of ace from virion invasion of the host although this conclusion cannot be made without measuring components of the ras system in these patients. further studies including declaration of medications/diuretic use and measurement of components of the ras pathway are necessary to make conclusions about aetiology of hypokalaemia in sars-cov- infection although hypokalaemia was clearly a clinical risk in these patients that should be communicated to the medical community. a study performed in hong kong during the sars-cov epidemic compared symptoms and laboratory findings in sars-cov rt-pcr swab-positive patients to negative patients . hypokalaemia was present in . % of rt-pcr positive patients and was reported as a 'common laboratory finding'. it was also suggested in this paper that the hypokalaemia was due to the increased effects of angiotensin- but, again, the ras components were not measured in patients. in contrast, the largest sars-cov- case series so far ( patients included) did not demonstrate any major difference in serum potassium between those with mild and those with severe disease . serum potassium was mostly reported as normal in this cohort. sars-cov was found in the urine in one patient but urine was not routinely tested for the virus in this cohort. a small chinese, retrospective cohort of patients who were hospitalised with varying degrees of severity of sars-cov- infection were studied to comprehensively assess renal function, risk factors for, and incidence of early renal injury. % of patients had hypokalaemia and % also had hyponatraemia . however, this study did not acknowledge if any of the patients were taking diuretics or other medications that could alter serum potassium or sodium concentration. the theory of ras activation causing hypokalaemia is popular, however it is recognised that hyperaldosteronism does not always cause hypokalaemia. this is explained by the 'renal potassium switch' mechanism ( figure ), which involves the paradoxical actions of aldosterone on potassium; for example, aldosterone increases sodium reabsorption in states of low circulating volume without significantly altering potassium balance, but it also increases serum potassium excretion in high potassium states without affecting sodium balance. this paradoxical action all depends on the sodium delivery to the aldosterone-sensitive distal nephron (collecting duct) which is generated by the sodium-chloride co-symporter (ncc) in the early distal convoluted tubule. activation of this 'switch' is by phosphorylation of ncc by the wnk/spak/osr pathway , and is done by aldosterone in response to low or high serum potassium i.e. ncc is maximally phosphorylated when serum potassium is < . mmol/l and not at all phosphorylated when serum potassium is > . mmol/l . in states of decreased circulating volume, enac activity is increased due to the effects of excess aldosterone (which generates a sufficient intracellular electrochemical gradient to cause active transport of potassium from blood via na + k + -atpase which would be passively excreted into the tubular lumen by romk and bk channels in the principal cell). whether this generates an effect on potassium or not depends on the integrated activity of the entire distal nephron. in a state of low circulating volume, ncc in the early dct is 'switched on' which results in increased reabsorption of sodium and chloride in the early dct and, along with sodium reabsorption in the pct, a net increase of sodium reabsorption from the kidney occurs. as a result, sodium delivery to the aldosterone sensitive distal nephron (collecting duct) is decreased which offsets the effects of enac and potassium excretion is therefore unchanged. this results in a state of hyperaldosteronism without hypokalaemia . it should be noted that, although the renal potassium 'switch' mechanism is a popular theory, it has never been proven although there are case reports of patients with normokalaemic primary hyperaldosteronism that develop hypokalaemia with a thiazide diuretic . in states of hyperaldosteronism and volume depletion the renal potassium switch is 'active'. angiotensin-ii (ang-ii) stimulates kir . / . to hyperpolarise the basolateral membrane potential and lowers intracellular chloride. this action activates wnk (with-no-lysine-kinase- ) and wnk (with-no-lysine-kinase- ) by phosphorylation which subsequently phosphorylates sps -related proline / alanine-rich kinase (spak) and oxidative stress-responsive kinase (osr ) which, again, phosphorylates the sodium chloride co-symporter (ncc). ncc then avidly reabsorbs sodium (and chloride) back into the body in the early part of the distal convoluted tubule (dct ). this results in reduced tubular sodium delivery to the collecting duct. subsequently there is less sodium for the epithelial sodium channel (enac) to reabsorb and its action is offset. enac usually reabsorbs sodium and, via an electrochemical gradient made by sodium-potassium-atpase, potassium is normally excreted into the tubular lumen by romk (renal outer medulla potassium channel) and bk (big potassium/maxi-k channel) and eliminated from the body. when enac is less active, as a consequence of less tubular sodium delivery, less potassium is excreted. this results in a state of hyperaldosteronism with a normal serum potassium. in states of hyperaldosteronism and normal cardiovascular volume (in response to high serum potassium) the switch is 'inactive'. ncc is not phosphorylated which results in more distal sodium delivery to the collecting duct and subsequent enac activation resulting in excretion of potassium into the tubular lumen and a state of hypokalaemia , . arrows represent the direction of movement of electrolytes. red arrows reflect a reduction in movement and green arrows represent an increase in movement. clc-kb, chloride voltagegated channel kb; kir . / . , inwardly rectifying potassium channel . ; at r, type angiotensin receptor. other electrolyte disturbances various case reports describe the onset of syndrome of inappropriate anti-diuretic hormone secretion (siadh) in some patients with sars-cov- infection . although siadh is frequently associated with atypical pneumonia and pneumonia is a frequent complication of sars-cov- infection, the literature does describe this phenomenon in the absence of respiratory symptoms, fever and any alternative explanation for siadh . it is therefore prudent to consider testing for sars-cov- infection in cases of siadh without a clear aetiology. additional studies are required to ascertain incidence and pathogenesis of siadh in sars-cov- infection. one retrospective cohort study of laboratory-confirmed covid- patients without a history of chronic kidney disease have shown a high incidence of novel acquired incomplete renal fanconi syndrome, often preceding acute kidney injury and/or resolving during the clinical recovery phase of the infection . % of patients admitted to hospital for mild, moderate or severe respiratory failure due to sars-cov- infection had acute incomplete renal fanconi syndrome. proteinuria was always associated and often with severe renal phosphate leak (in addition to frequent hypophosphataemia, hyperuricosuria and glycosuria). renal associated hypokalaemia and renal tubular acidosis was not analysed in this study due to many patients requiring potassium supplementation in addition to the effects of mechanical ventilation, gfr alterations and other factors. the authors suggest that the development of incomplete renal fanconi syndrome could be used as a predictor of acute kidney injury (and a prognostic marker) as % of patients with severe stage and kdigo acute kidney injury experienced proximal tubule injury prior to acute kidney injury onset. none of these patients had significant haemodynamic instability or had high mean sofa scores to suggest an alternative pre-renal aetiology for their acute kidney injury. a quarter of the patients were given lopinavir/ ritonavir but this is rarely associated with acute kidney injury and not associated with renal fanconi syndrome. there are two studies so far (one in pre-print form) to look at renal histopathology on post-mortem findings in sars-cov- patients who died of the disease ( of the had pre-morbid clinical kidney injury signs) , . the first demonstrated prominent proximal tubule injury (observed in all patients) with frank necrosis observed in some specimens and three patients had high creatinine phosphokinase and pigmented casts on light microscopy, probably representing rhabdomyolysis. there was occasional distal tubule and collecting duct swelling with some interstitial oedema and virus particles were seen in seven of the specimens on electron microscopy in proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ace expression . the second post-mortem case series in (currently in pre-print) examined six post-mortem renal histopathology samples of patients who died with sars-cov- infection . all specimens had histopathological evidence of acute tubular necrosis, two patients had severe lymphocytic infiltration of the tubulointerstitium and sars-cov- nucleocapsid antigens were observed in renal tubule cells. transmission electron microscopy was used in samples from two different patients, both of which demonstrated the presence of virions and virus-like particles in renal cells and these cells were markedly swollen with mitochondrial, lysosomal and endoplasmic reticulum expansion. these finding suggest that sars-cov- directly infects kidneys and specifically infects and damages kidney tubules. furthermore, strong cd + macrophage presence and c b- deposition seen in the tubulointerstitium of all samples (yet absent in the rest of the kidney tissue and very little in the glomeruli and capillaries in two patients) suggest that further tubular damage occurs as a consequence of macrophage recruitment and c b- activation and deposition. it would be logical to suggest that histopathological evidence of damage to tubular cells would result, clinically, as a tubulopathy, of which we know urine electrolyte wasting is a recognised complication and these case series are histopathological evidence to support this suggestion. finally, we know that tmprss primes sars-cov- to gain cellular entry and tmprss is only detectable (in low levels) at the s section of the proximal convoluted tubule in the kidney . it is therefore assumed that this is one way in which the kidney is 'infected' by the virus and reiterates the probability of direct virus-induced tubular damage. various case series have demonstrated the virus in urine , and it is unclear as to how it enters the tubular lumen and whether this is via the apical membrane of tubule cells. however, one study did look at the presence of sars-cov- in bodily fluids over time and did not detect the virus in urine in any of the nine patients studied . a couple of case reports have demonstrated that the virus may affect the kidney in other ways which may be an alternative explanation for the entry of virus into the urine , . collapsing focal segmental glomerulosclerosis (fsgs) has been described twice in the literature so far in patients of african or african american origin and both of these patients demonstrated severe tubular injury. it was noted that severe acute tubular necrosis (atn) was observed in the absence of haemodynamic compromise and severe pulmonary involvement suggesting that the tubulopathy was not ischaemic and more likely to be because of either direct viral toxicity or cytokine-mediated damage. various clinical and histopathological studies have demonstrated evidence of hypokalaemia, hyponatraemia, siadh, incomplete fanconi syndrome and tubulopathy in patients with sars-cov- infection. the data from china and evidence from the sars-cov case series provide good clinical justification to monitor potassium levels in patients infected with sars-cov- . we do need to see this clinical finding replicated in multi-centres to be able to make definitive conclusions about this clinical sequela. although hyperkalaemia can occur for many reasons in patients with sars-cov- infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia . based on evidence of significant urine potassium wasting which can be prolonged, we suggest checking the transtubular potassium gradient (ttkg) in patients with sars-cov- infection. the ttkg adjusts the urinary potassium for the concentrating effects that occur in the collecting duct where water is removed from urine. this would help identify those at risk of severe or prolonged hypokalaemia and prompt preemptive cautious electrolyte monitoring and replacement which should help reduce the risk of hypokalaemic complications which can be fatal. the ttkg is easily measured and only requires measurements of serum and urine osmolality and potassium (urine potassium/serum potassium)/(urine osmolality/ serum osmolality). a high ttkg in the setting of hypokalaemia suggests that potassium wasting is of renal origin. this, for example, may be due to hyperaldosteronism, pseudohyperaldosteronism or a potassium wasting tubulopathy. the validity of this measurement depends on three assumptions: ( ) few solutes are reabsorbed in the medullary collecting duct, ( ) potassium is neither secreted or reabsorbed in the medullary collecting duct and ( ) the osmolality of fluid in the terminal collecting duct is iso-osmolar to plasma. as water is reabsorbed in the medullary collecting duct, urine potassium concentration is not an accurate index evaluating distal potassium secretion because the effect of water is not taken into account. the urine to plasma osmolality ratio adjusts for the degree of medullary water reabsorption which increases urine potassium concentration as more water is reabsorbed. therefore, ttkg is relatively accurate providing the urine is not dilute, that the osmolality of urine is greater or equal to the osmolality of plasma (because vasopressin is required for optimal potassium excretion in the distal nephron) and that urine sodium concentration is > mmol/l so that distal sodium delivery is not limiting . the main premise underlying the ttkg is the absence of significant solute transport in the collecting duct so that any change in urinary potassium concentration only occurs due to medullary water reabsorption and the ttkg does not overestimate the gradient for collecting duct potassium secretion. however, we now know that some urea is reabsorbed in the late cortical collecting duct and this urea recycling aids the tubular secretion of potassium so it should be noted that the ttkg should not be used as a diagnostic tool in hyperkalaemia aetiology as it is not reliable . in situations of dilute urine and high urine flow rate, the ttkg also underestimates potassium secretory capacity in the hyperkalaemic patient . however, we believe that the utility of the ttkg is still of value in hypokalaemic patients to differentiate between renal and extra-renal potassium wasting providing the aforementioned criteria necessitating its accuracy is adhered to. measuring components of the renin-angiotensin-aldosterone pathway would help us to identify if there really is involvement of the sars-cov- virus; however, measuring these components in the acute setting has its limitations. the ras system is heavily influenced by a multitude of factors. many of the patients with sars-cov- infection are critically unwell and often present to hospital in states of low cardiovascular volume. it has been recommended that patients with ards also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation. both of these factors would typically result in ras activation and could be very misleading when determining virus-related ras pathway involvement. some studies have shown that low urine ph (causing acidic urine) can be a predictor of ras activation so future studies determining ras system involvement of sars-cov- infection should, as well as ttkg, also consider urine ph as an early marker of ras system activation. potassium levels < . mmol/l can be arrhythmogenic and specifically can cause qtc interval prolongation, torsade de pointes, ventricular fibrillation and sudden cardiac death . this is particularly relevant for a couple of reasons. first, many of the drugs currently undergoing clinical trials in sars-cov- patients prolong the qtc interval (hydroxychloroquine , azithromycin , favipiravir, lopinavir/ritonavir and fingolimod: nct ) and some drugs also risk hypokalaemia which may worsen pre-existing electrolyte disturbance (e.g. methylprednisolone: nct , nct ; thalidomide: nct , nct ; and bevacizumab: nct ). second, cardiac involvement in sars-cov- is high ( . % of infected patients admitted to icu experienced an arrhythmia). induction of arrhythmias can be due to multi-factorial aetiologies of cardiac injury in sars-cov- patients such as hypoxia-mediated, direct tissue damage, cytokine-storm syndrome, worsening coronary perfusion and the direct effects of medications . both of these factors really emphasise the importance of maintaining normokalaemia in these patients to reduce morbidity and mortality. in addition to the arrhythmogenic effects of both the sars-cov- cardiac sequelae and various clinical trial drugs, many patients are being given diuretics to improve the oxygenation in ards, which also risks hypokalaemic complications. loop diuretics can induce hypokalaemia by blocking the na + -k + - clcotransporter (nkcc ) in the thick ascending limb of the loop of henle which results in failure of sodium, potassium and chloride to be reabsorbed into the concentrated medullary interstitium (this transporter normally reabsorbs about % of the sodium load). this enhances distal tubular concentration of sodium, reduced hypertonicity of the surrounding interstitium and less water reabsorption in the collecting duct. distal sodium delivery increases potassium loss via the na+/k+-atpase pumps at the apical membrane of the principal cell of the collecting duct as there is more sodium to be exchanged with potassium for excretion. thiazide diuretics can cause hypokalaemia by the same principle of enhanced distal sodium delivery as they block the sodium-chloride co-symporter (ncc) in the distal convoluted tubule. furthermore, loop diuretics also block nkcc at the macula densa which (along with the ras-activation response to initial volume reduction) induces renin secretion, making ras system measurement inaccurate if patients are on diuretics. if diuretics are to be used, it would be wise to consider potassium-sparing agents in hypokalaemic patients to reduce the cardiac complications of worsening hypokalaemia that can occur with those that are not potassium sparing. it would be logical that with clear histopathological data suggesting tubular injury, hypokalaemia is to be expected in patients infected with sars-cov- . tubular injury seems to be multifactorial in these patients; atn from sirs/cytokine storm syndrome, peritubular congestion, virion invasion, drug toxicity and pigment-cast nephropathy from rhabdomyolysis and therefore difficult to avoid. the data on outcomes in cardiac arrest for sars-cov- patients is unfortunately poor. chinese data suggests that in a series of patients who underwent resuscitation efforts, return of spontaneous circulation was only achieved in ( . %) and only four patients were alive at days . the strong association of hypokalaemia with arrhythmia and sudden cardiac death reiterates the importance of vigilance for detection and treatment of this electrolyte disturbance during and after admission given such poor outcomes when spontaneous circulation is lost. to truly know the extent of involvement of the ras pathway as a potential cause of hypokalaemia in sars-cov- patients, further studies require measurement of all components of the ras pathway. until we know this, we cannot make definite conclusions about the mechanisms of hypokalaemia in these patients outside of the histopathological evidence of tubulopathy published, of which multiple aetiological factors are likely. no data are associated with this article. "hypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition". please rephrase -hypokalaemia is important, but at the same time rare (does not occur with 'any disease'). ○ "furthermore, inadequate management of hypokalaemia has been identified in % of hospitalised patients , reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce". please reference, or clarify. management of hypokalaemia is not usually resource-intensive, and i have not seen evidence that potassium replacement is difficult to come by in the pandemic. ○ "this study claimed that hypokalaemia was prevalent amongst sars-cov- infected patients, affecting of patients ( %) that had a serum potassium of . mmol/l which is the value required for those with myocardial dysfunction." hypokalaemia affected % of patients with a k less than . ? or hypokalaemia as defined by k< . affected % of the cohort. ○ "only patients had a serum k > . mmol/l which is the value required for those with myocardial dysfunction". please clarify what is meant by this phrase. ○ "principal cell" does not warrant capitalisation. ○ "although hyperkalaemia can occur for many reasons in patients with sars-cov- infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia [ ] ." this data is from the setting of acute myocardial infarction, not sars-cov- , and therefore may not be applicable. please add a qualifying statement. ○ you recommend measuring the transtubular potassium gradient. does this significantly alter management in these patients, outside of a research context? ○ "it has been recommended that patients with ards also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation." this advice has since been dropped in the context of sars-cov- -please make this clear. coronavirus disease case surveillance - united states covid- and the cardiovascular system its occurrence in types and rta despite sustained correction of systemic acidosis pubmed abstract | publisher full text | free full text in-hospital cardiac arrest outcomes among patients with covid- pneumonia in wuhan, china. resuscitation fetal programming and the angiotensin-( - ) axis: a review of the experimental and clinical data pubmed abstract | publisher full text | free full text renal ace expression in human kidney disease some aspects of the writing were difficult to follow please broaden abstract to include electrolyte disturbances in addition to hypokalaemia, reflecting the article as a whole. in the first paragraph, there is quite a lot of text dealing with the many complications of covid infection, which is a bit irrelevant.○ "…proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ace expression" this is not true; see the human protein atlas or for example .○ "a few small studies have demonstrated that sars-cov- can affect the kidneys directly, but it is unclear if this is a key mechanism in acute kidney injury seen in patients with sars-cov- infection"? reference. it would be useful to include a comment on the prevalence of acute kidney injury in sars-cov- patients. reviewer expertise: renal tubular physiology.we confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.reviewer report august https://doi.org/ . /f research. .r © welling p. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. johns hopkins university, school of medicine, baltimore, md, usa i have read the revised review. it is much improved. i would accept without further revision. competing interests: no competing interests were disclosed. johns hopkins university, school of medicine, baltimore, md, usa as pointed out by this review, the evidence for hypokalemia in covid- is weak, promulgated by a single unpublished article that was not corroborated by larger series studies. my chief concern about the review, is that it amplifies the myth. furthermore, the frequent use of loop diuretics in ards, which cause hypokalemia, makes a causal connection between the development of hypokalemia and covid- precarious.at the very least, the title should be changed to minimize the connection between hypokalemia and covid- .paragraph is somewhat misleading. although covid- does have an array of different sequela, acute respiratory distress syndrome (ards) seems to be the most common serve form of the disease. the first paragraph should provide an incidence of this and the other sequela. reference to large studies from china, europe and north american should be cited.paragraph the statement "sars-cov- may affect the kidneys directly and indirectly by both renal and renin-angiotensin-aldosterone system (ras) involvement," has no factual basis. there are a few small studies that demonstrate that sars-cov- can infect the kidney but it is presently unclear if this is a key mechanism of aki in covid .paragraph provide a reference to mas receptor on type alveolar epithelial cells as stated. paragraph last sentence. this is evidence for sars-cov, not sars-cov provide a reference to "hyperaldosteronism does not always cause hypokalaemia." although "switch" activation has been a popular explanation for this, it has never been proven. perhaps, the authors could cite clinical studies, demonstrating the development of severe hypokalemia in normokalemic pa patients following thiazide administration as evidence.the limitations of ttkg should be articulated. are all factual statements correct and adequately supported by citations? partly are arguments sufficiently supported by evidence from the published literature? partly competing interests: no competing interests were disclosed.reviewer expertise: i am an expert on molecular underpinnings of potassium balance and electrolyte disorders. i coined the "potassium switch"i confirm that i have read this submission and believe that i have an appropriate level of expertise to state that i do not consider it to be of an acceptable scientific standard, for reasons outlined above.the benefits of publishing with f research:your article is published within days, with no editorial bias • you can publish traditional articles, null/negative results, case reports, data notes and more • the peer review process is transparent and collaborative • your article is indexed in pubmed after passing peer review • dedicated customer support at every stage • for pre-submission enquiries, contact research@f .com key: cord- - h vvim authors: chen, xiang-fan; li, xiao-li; liu, jin-xin; xu, jing; zhao, yan-yan; yang, min; zhang, yan title: inhibition on angiotensin-converting enzyme exerts beneficial effects on trabecular bone in orchidectomized mice date: - - journal: pharmacol rep doi: . /j.pharep. . . sha: doc_id: cord_uid: h vvim background: this study aimed to study the osteo-preservative effects of captopril, an inhibitor on angiotensin-converting enzyme (ace), on bone mass, micro-architecture and histomorphology as well as the modulation of captopril on skeletal renin-angiotensin system (ras) and regulators for bone metabolism in mice with bilateral orchidectomy. methods: the orchidectomized (orx) mice were orally administered with vehicle or captopril at low dose ( mg/kg) and high dose ( mg/kg) for six weeks. the distal femoral end, the proximal tibial head and the lumbar vertebra (lv) were stained by hematoxylin and eosin, safranin o/fast green and masson-trichrome. micro-computed tomography was performed to measure bone mineral density (bmd). results: treatment with captopril increased trabecular bone area at distal metaphysis of femur, proximal metaphysis of tibia and lv- , moreover, high dose of captopril significantly elevated trabecular bmd of lv- and lv- . the mrna expressions of renin receptor, angiotensinogen, carbonic anhydrase ii, matrix metalloproteinase- , and tumor necrosis factor-alpha were significantly decreased in tibia of orx mice following treatment with captopril. the administration with captopril enhanced the ratio of opg/rankl mrna expression, the mrna expression of transforming growth factor-beta and the protein expression of bradykinin receptor- . conclusions: the inhibition on ace by captopril exerts beneficial effects on trabecular bone of orx mice. the therapeutic efficacy may be attributed to the regulation of captopril on local ras and cytokines in bone. osteoporosis is a disease that thins and weakens bones to the point that they become fragile and easily break. it is well-known that osteoporosis-related fractures due to low-trauma or fragility result in heavy health-related costs, substantial disability and mortality among postmenopausal women and older men [ ] . osteoporosis is now recognized as a major threat to health in aging people. men sustain bone loss of approximately . - % per year from the sixth decade despite not undergoing a menopausal transition as women do [ ] . overall, the trend of age-adjusted prevalence of osteoporosis was similar between women and men [ , ] . however, the mortality and morbidity caused by osteoporotic fractures are greater in men with testosterone deficiency-induced osteoporosis than those in women with postmenopausal osteoporosis [ , ] . bone metabolism is normally modulated by regulators and cytokines in bone micro-environment. the expression ratio of osteoprotegerin (opg) and receptor activator of nuclear factor-kb ligand (rankl), both of which are secreted by osteoblasts, determines the maturation of osteoclasts. matrix metalloproteinase (mmp)- and carbonic anhydrase (ca)ii produced from osteoclasts are responsible for resorbing organic proteins and inorganic minerals, respectively. bradykinin manages bone metabolism through modulating osteogenesis and osteoclastogenesis by binding to its receptor. recently, various cytokines like tumor necrosis factor (tnf)-α and transforming growth factor (tgf)-β are found to play major roles in bone health. the renin-angiotensin system (ras) components exist in tissues and organs, namely tissue ras, participating in kinds of pathophysiologic processes [ , ] . recent animal studies indicated the existence of ras components in trabecular bone [ , ] , and cell studies found the expression of angiotensin receptors in primary osteoblasts [ ] , suggesting there locally exist ras components in bone micro-environment. the previous functional studies indicated that the skeletal ras displays key biological actions on mediating bone metabolism [ ] [ ] [ ] . angiotensin ii, key effector in ras, is produced from angiotensin i by catalyzation of angiotensin-converting enzyme (ace), a vital molecule in ras. the clinical studies found that ace inhibitor (acei) was capable of elevating bone mineral density (bmd) and reducing fracture risk in patients [ , ] . in line with the clinical observations of the bone-preservative properties following ace inhibition, animal studies showed that treatment with acei repressed estrogen deficiency-induced decrease in bone density [ , ] and accelerated bone healing [ , ] . while, in a contrary to the beneficial effects of acei on bone health, the recent emerging evidences indicated that the use of acei might potentially accelerate bone loss [ ] [ ] [ ] . therefore, we are keen to know whether the inhibition on ace could preserve bone tissue of mice with testosterone deficiency-induced osteoporosis. this study investigated the osteo-preservative effects of captopril on bone density, histology and micro-structure of trabecular bone as well as the modulation of captopril on skeletal ras and regulators for bone metabolism in mice with bilateral orchidectomy. thirty-two three-month-old male icr mice (slac laboratory animal, shanghai, china) received commercial diet and distilled water ad libitum during experimental period. the acclimatized mice underwent either bilateral laparotomy (sham, n = ) or bilateral orchidectomy (n = ). after recovery for week, the orchidectomized (orx) mice were randomly divided into three groups: orx mice with vehicle treatment (n = ), orx mice with orally administration of captopril with low dose (orx-l, mg/kg, n = ) or high dose (orx-h, mg/kg, n = ) by intragastric gavage. the dosage for captopril used in this study was referred as described previously [ , ] . six weeks after drug administration, tibias, femurs and lumbar vertebras were immediately collected for a variety of analyses. the animal experiment complied with the arrive guidelines, and the study protocol was approved by the animal ethics committee. the femurs, tibias, and lumbar vertebras were fixed in % formaldehyde in pbs (ph . ), decalcified in edta ( . m, ph . ), and embedded in paraffin. the bone sections with mm were cut on a microtome. safranin o (sigma-aldrich)/fast green staining was performed on femurs and tibias. additionally, both the hematoxylin and eosin staining and the masson-trichrome staining were performed on lumbar vertebra (lv)- . the images for all stained slides were captured under microscope (leica dm- ). trabecular bone area (%) and thickness were measured using an osteomeasure system (osteometrics inc., decatur, ga, usa) as described previously [ ] and the whole process was performed in a blind manner. the lv- and lv- without decalcification were scanned with micro vivact system (scanco medical, bassersdorf, switzerland). the detecting parameters were set as previously described [ ] . trabecular bone was determined by a fixed threshold. after images were captured ( ma), slices were established as the volume of interest. trabecular bmd was obtained. tibias were crushed under liquid nitrogen condition and total rna was isolated by trizol (invitrogen, carlsbad, ca, usa). agarose gel electrophoresis was performed to verify rna integrity. moloney murine leukemia virus reverse transcriptase (invitrogen, usa) was used to synthesize cdnas by reverse transcription reactions with mg of total rna. dna engine (abi) was applied for regular pcr with cdnas as the template. glyceraldehyde- phosphate dehydrogenase (gapdh) was applied as a reference control to measure the amount of pcr products. the primer sequence utilized in this study was referred as previously described [ ] . the femur was homogenized and extracted in laemmli buffer (boston bioproducts, worcester, ma, usa), followed by min boiling and centrifugation to obtain the supernatant. protein concentration was determined by bio-rad protein assay kit. mg protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which was blocked with % (w/v) nonfat dry milk in tbst containing . % (w/v) tween , and incubated with one of the following primary antibodies (santa cruz biotechnology, usa) at c overnight: mouse anti-renin monoclonal antibody, goat anti-bradykinin receptor polyclonal antibody. after washes with tbst, the membranes were incubated with secondary antibody with dilution of : , . the protein bands were captured by odyssey infrared imaging system (li-cor, usa), quantified (odyssey application software version . ) and normalized to β-actin signal using mouse monoclonal anti-β-actin antibody (sigma, usa). the results were expressed as mean ae standard error of mean (sem). the analysis for statistical difference was carried out with prism version . (graphpad). inter-group differences were analyzed by one-way anova, and followed by tukey's multiple comparison tests which were applied to compare the values between groups and the difference with p value of less than . was considered statistically significant. the affection of captopril on trabecular bone was evaluated by safranin o/fast green staining at the distal end of femur (fig. a ) and the proximal metaphysis of tibia (fig. b) . the loss of bone mass and the deterioration of network connection were clearly shown at spongiosa zones at both femur (fig. c , p < . ) and tibia (p < . ) in orchidectomized (orx) group as compared to those in sham group. treatment with low dose of captopril (orx-l) increased the trabecular bone area at proximal tibial metaphysis (p < . ), and treatment with high dose of captopril (orx-h) significantly elevated (p < . ) the area of trabecular bone at both femur and tibia in orx mice. histological staining on trabecular bone at lumbar vertebra great amount of trabecular bone exists at spine bone, thus, hematoxylin and eosin (he) staining ( fig. a) and massontrichrome staining (fig. b) were performed on lumbar vertebra (lv)- . similarly as observed at femur and tibia, the orchidectomy induced the loss of bone mass and the impairment of cancellous bone at lv- as demonstrated by the decrease of trabecular bone thickness (fig. c, p < . ) and trabecular bone area (fig. d , p < . ). captopril with low dose (p < . ) and high dose (p < . ) dramatically increased the trabecular bone area of lv- in orx mice. captopril dose-dependently enhanced the thickness of trabecular bone even though there was no statistical difference between captopril-treated groups and orx group. the measurement by micro-computed tomography (fig. ) showed that the orchidectomy alone induced significant decrease in bmd at both lv- (p < . ) and lv- (p < . ). the treatment with high dose of captopril dramatically (p < . ) increased bmd at both lv- and lv- , which was well consistent with the histological result at lumbar vertebra. to clarify that tissue ras was involved in the action of captopril on bone, the mrna expressions of ras components in tibia, such as angiotensinogen (agt) and renin receptor (renin-r), were determined (fig. ) . the expressions of renin-r (fig. b , p < . ) and agt (fig. b , p < . ) were significantly up-regulated in tibia of vehicle-treated orx mice. gene expression of renin-r was down-regulated by captopril with both doses (p < . ) and mrna expression of agt was markedly reduced only in orx-h group (p < . ) when compared to those in orx group. the expression ratio of osteoprotegerin (opg) and receptor activator of nf-kb ligand (rankl) plays vital role in formation and maturation of osteoclasts, thus, the expression of opg and rankl and the ratio of opg/rankl were determined (fig. ) . the mrna expression of opg was lower in orx group than that in sham group (fig. c, p < . ) , and captopril significantly increased opg mrna expression as compared to that of orx group (p < . ). the statistical difference for rankl mrna expression was not found among groups. while, the expression ratio of opg/rankl was decreased in orx group (p < . ) and this ratio in orx mice after treatment with low and high dose of captopril was recovered to the level in sham group (p < . ). the mrna expressions of markers for bone resorption were measured in tibia for further evaluating the potential effect of captopril on bone resorptive activity (fig. ) . the orchidectomy significantly increased (fig. d , p < . ) the mrna expression of caii. the captopril treatments with low dose and high dose dramatically reversed this change (p < . ). the captopril treatment at high dose significantly down-regulated the mrna expression of mmp- as compared to that in vehicle-treated orx group (p < . ). thus, this study indicated the potential regulation of captopril on osteoclastic activity. in addition, the mrna expressions of two cytokines, tnf-α and transforming tgf-β, were determined in tibia (fig. ) . the mice after orchidectomy showed the increased expression of tnf-α (fig. e , p < . ) and the decreased expression of tgf-β (p < . ). treatment with captopril reduced the tnf-α expression by % (p < . ) and enhanced the tgf-β expression by % (p < . ) as comparison with those in orx group. orchidectomy significantly induced the down-regulation of bradykinin receptor- (b r) protein (fig. b, p < . ) , but did not alter the protein expression of renin in femur. treatment with low dose and high dose of captopril in orx mice markedly increased the expression of b r (p < . ). of noted, the protein expression of renin was higher (p < . ) in orx-h group than that in vehicletreated orx group. angiotensin-converting enzyme inhibitors (aceis) are classically applied for anti-hypertension treatment [ ] , and are currently in wide use for the treatment of diabetic complications [ , ] . given the key role of tissue renin-angiotensin system (ras) in maintaining bone health, it is vital to study the effect of aceis on bone injury induced by testosterone deficiency. in the present study, we investigated the therapeutic effect of captopril, one typical acei, on trabecular bone histology and bone mass at long bone and spine bone as well as studied the modulation of captopril on regulators for bone metabolism in orchidectomized (orx) mice. this study demonstrated that the treatment with captopril effectively attenuated the orchidectomy-induced pathological alterations of micro-structure of trabecular bone at lumbar vertebra (lv)- , distal metaphysis of femur and proximal metaphysis of tibia as observed by histological staining, moreover, bone mineral density (bmd) at both lv- and lv- was significantly enhanced in orx mice in response to captopril treatment for weeks. these results revealed the preventive effects of inhibiting angiotensin-converting enzyme (ace) on decrease of bone mineral density and impairment of trabecular bone structure at axial and appendicular bones. the beneficial effect of captopril on bone of orx mice shown in this study was well consistent with previous animal studies, which demonstrated that the intervention of tsukuba hypertensive mice with acei enalapril attenuated osteoporosis [ ] as well as the ovariectomized (ovx) rats treated with captopril showed the increased area of trabecular bone at lv- and the improved mechanical properties at lv- [ ] . additionally, the patients after treatment with aceis had an increase in bmd and a reduced risk in fracture [ ] . thus, the preclinical and clinical studies fully suggested the potential of acei in exerting osteoprotective efficacy. of noted, the recent studies demonstrated that captopril repaired cortical morphometric features [ ] and improved cortical bone thickness [ ] in ovx rats, while, the potential effect of captopril on cortical bone in orx animals, such as the middle-shaft of tibia and femur, require further investigation. it is evident that the activation of skeletal ras would result in bone injuries [ , ] . this study revealed that orchidectomy alone increased the mrna expressions of renin receptor (renin-r) and angiotensinogen (agt) in bone, and the similar experimental results were found in tibia of rats [ ] and mice [ ] with diabetes. upon to the treatment with captopril, the abnormal expression of agt and renin-r in orx mice was almost reduced to the level of sham group. thus, this study indicated the biological role of tissue ras in development of testosterone deficiency-induced osteoporosis and suggested that the skeletal ras may a candidate target in drug discovery for anti-osteoporosis of elderly males. our results clearly showed that the expressions of genes involved in bone resorption (caii, mmp- ) were significantly induced and the ratio of opg/rankl expression (a marker for osteoclastogenesis) was decreased in tibia of orx mice, a typical feature of bone turnover associated with testosterone deficiency. treatment of orx mice with captopril significantly suppressed these changes of caii, mmp- and opg/rankl. similar results were reported by others that captopril increased the ratio of opg/rankl in bone [ ] and serum [ ] of ovx rats. both mmp- and caii, produced from osteoclasts, are enzymes to dissolve organic component and inorganic substance of bone, respectively [ ] . thus, our results clearly indicated that captopril exerted the suppression on bone resorption by which captopril protected against bone deteriorations induced by testosterone deficiency in male mice. besides the local ras in bone tissue, bradykinin, the major active peptide in the kallikrein-kinin system (kks), is able to regulate bone metabolism through acting on bradykinin receptor- (b r) [ ] . the expression of bradykinin receptor was decreased in osteoblasts with exposure to high glucose level and in bone of ovx mice [ , ] . this study demonstrated that captopril effectively reversed orchidectomy-induced down-regulation of b r protein expression in mice, indicating that bradykinin receptor was involved in management of captopril on bone metabolism in orx mice. tnf-α, one of inflammatory markers, could activate osteoclasts, consequently stimulating bone resorption and resulting in osteoporosis. in vivo studies showed that the administration of animals with ras inhibitors could reduce tnf-α expression in tissue [ , ] , similarly, in this study captopril dramatically decreased mrna expression of tnf-α in bone of orx mice. in addition, tgf-β is an important modulator for bone formation and repair. the present study showed the down-regulation of tgf-β in bone of orx mice, which was in consistent with early study [ ] . the increase in tgf-β expression in response to captopril treatment was found in orx mice in this study, and the similar action of captopril on tgf-β was also reported in human lung fibroblasts [ ] . our study indicated that the regulation of captopril on tnf-α and tgf-β might be involved in its protective effects on bone tissue of orx mice. although treatment with captopril attenuated bone injury in orx mice, we found that captopril at high dose in this study induced the elevation of renin protein expression in bone. the compensatory increase in renin expression is a common problem for aceis due to the disruption of the feedback inhibitory loop in renin production [ ] . the increase in ras activity might ultimately limit the therapeutic effectiveness of ace inhibition. thus, the clinical practice currently suggests the application of ras blockers with aliskiren, one renin inhibitor which could suppress the first and rate-limiting step within ras [ ] . while, whether the combination of acei with aliskiren could synergistically ameliorate bone impairments induced by testosterone deficiency need to be further clarified. taken together, this study clearly showed the beneficial effect of captopril on trabecular bone in orchidectomized mice with testosterone deficiency, and the underlying mechanism may, at least partially, be attributed to the regulation of captopril on skeletal ras and local cytokines. pyrroloquinoline quinone prevents testosterone deficiency-induced osteoporosis by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption effects of body size and skeletal site on the estimated prevalence of osteoporosis in women and men prevalence of osteoporosis and low bone mass in older chinese population based on bone mineral density at multiple skeletal sites prevalence of osteoporosis and related lifestyle and metabolic factors of postmenopausal women and elderly men: a cross-sectional study in gansu province male osteoporosis: new trends in diagnosis and therapy pathophysiology of age-related bone loss and osteoporosis the effect of renin angiotensin system on tamoxifen resistance role of angiotensin-converting enzyme in cardiac hypertrophy induced by nitric oxide synthase inhibition angiotensin ii type receptor blockade increases bone mass role of the local bone renin angiotensin system in steroid induced osteonecrosis in rabbits activation of renin-angiotensin system induces osteoporosis independently of hypertension involvement of the skeletal renin-angiotensin system in age-related osteoporosis of ageing mice early molecular responses of bone to obstructive nephropathy induced by unilateral ureteral obstruction in mice effects of angiotensinconverting enzyme inhibitor, captopril, on bone of mice with streptozotocininduced type diabetes evolution of the bone mass of hypertense menopausal women in treatment with fosinopril angiotensin converting enzyme inhibitor use is associated with higher bone mineral density in elderly chinese prevention of osteoporosis by angiotensin-converting enzyme inhibitor in spontaneous hypertensive rats ace- / ang - /mas cascade mediates ace inhibitor, captopril, protective effects in estrogen-deficient osteoporotic rats inhibition of angiotensin-converting enzyme stimulates fracture healing and periosteal callus formation -role of a local renin-angiotensin system the effects of the angiotensin converting enzyme inhibitor enalapril and the angiotensin ii type receptor blocker losartan on fracture healing in rats does the use of ace inhibitors or angiotensin receptor blockers affect bone loss in older men the effect of angiotensin-converting enzyme inhibitor use on bone loss in elderly chinese differential response of bone and kidney to acei in db/db mice: a potential effect of captopril on accelerating bone loss effect of ace inhibitors and at receptor antagonists on pentylenetetrazole-induced convulsions in mice the angiotensin converting enzyme inhibitor lisinopril improves muscle histopathology but not contractile function in a mouse model of duchenne muscular dystrophy involvement of skeletal renin-angiotensin system and kallikrein-kinin system in bone deteriorations of type diabetic mice with estrogen deficiency renin inhibitor aliskiren exerts beneficial effect on trabecular bone by regulating skeletal reninangiotensin system and kallikrein-kinin system in ovariectomized mice ace phenotyping as a guide toward personalized therapy with ace inhibitors effect of angiotensinconverting enzyme inhibitors and angiotensin ii receptor blockers on all-cause mortality, cardiovascular deaths, and cardiovascular events in patients with diabetes mellitus: a meta-analysis targeting inflammation: new therapeutic approaches in chronic kidney disease (ckd) captopril improves osteopenia in ovariectomized rats and promotes bone formation in osteoblasts antihypertensive medications, bone mineral density, and fractures: a review of old cardiac drugs that provides new insights into osteoporosis a comparative study between the effect of -β estradiol and angiotensin converting enzyme inhibitor on osteoporosis in ovariectomized rats local bone interaction between renin-angiotensin system and kallikrein-kinin system in diabetic rat bradykinin regulates osteoblast differentiation by akt/erk/nfkb signaling axis bradykinin receptors and ephb /ephrinb pathway in response to high glucose-induced osteoblast dysfunction and hyperglycemia-induced bone deterioration in mice inhibition of aneurysm progression by direct renin inhibition in a rabbit model effect of angiotensin-converting enzyme inhibitor on cardiac fibrosis and oxidative stress status in lipopolysaccharide-induced inflammation model in rats orchiectomy markedly reduces the concentration of the three isoforms of transforming growth factor beta in rat bone, and reduction is prevented by testosterone profibrosing effect of angiotensin converting enzyme inhibitors in human lung fibroblasts rationale for combining a direct renin inhibitor with other reninangiotensin system blockers. focus on aliskiren and combinations clinical role of direct renin inhibition in hypertension this work was supported in part by jiangsu specially-appointed professor program and university of shanghai for science and technology ( kjfz ). the authors declare no conflict of interest. key: cord- -p msabl authors: obukhov, alexander g.; stevens, bruce r.; prasad, ram; li calzi, sergio; boulton, michael e.; raizada, mohan k.; oudit, gavin y.; grant, maria b. title: sars-cov- infections and ace : clinical outcomes linked with increased morbidity and mortality in individuals with diabetes date: - - journal: diabetes doi: . /dbi - sha: doc_id: cord_uid: p msabl individuals with diabetes suffering from coronavirus disease (covid- ) exhibit increased morbidity and mortality compared with individuals without diabetes. in this perspective, we critically evaluate and argue that this is due to a dysregulated renin-angiotensin system (ras). previously, we have shown that loss of angiotensin-i converting enzyme (ace ) promotes the ace/angiotensin-ii (ang-ii)/angiotensin type receptor (at r) axis, a deleterious arm of ras, unleashing its detrimental effects in diabetes. as suggested by the recent reports regarding the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov- ), upon entry into the host, this virus binds to the extracellular domain of ace in nasal, lung, and gut epithelial cells through its spike glycoprotein subunit s . we put forth the hypothesis that during this process, reduced ace could result in clinical deterioration in covid- patients with diabetes via aggravating ang-ii–dependent pathways and partly driving not only lung but also bone marrow and gastrointestinal pathology. in addition to systemic ras, the pathophysiological response of the local ras within the intestinal epithelium involves mechanisms distinct from that of ras in the lung; however, both lung and gut are impacted by diabetes-induced bone marrow dysfunction. careful targeting of the systemic and tissue ras may optimize clinical outcomes in subjects with diabetes infected with sars-cov- . infection. studies indicate that - % of individuals with severe infections have diabetes ( , ) . however, a recent meta-analysis of six clinical studies involving , covid- patients provided evidence that individuals with diabetes exhibited a similar prevalence of being infected with sars-cov- as the overall population, but presence of diabetes was a critical comorbidity that increased the risk of a poor outcome ( ) . certain racial groups such as african americans and native americans are highly prone to developing diabetes and experience disparities in health care making them particularly vulnerable to covid- ( ) . however, to date there is a paucity of data regarding comorbidities, covid- outcomes, and mechanisms that modulate viral pathogenesis. in this perspective, we bring attention to specific factors that may complicate covid- in individuals with diabetes including ) the presence of bone marrow changes (myeloidosis) that predispose those with diabetes to an excessive proinflammatory response (cytokine storm) and contribute to insulin resistance and reduced vascular repair, and worsening function of the heart, kidney, and systemic vasculature as a whole; ) increased circulating furin levels that could cleave the spike protein and increase infectivity of sars-cov- ; ) dysregulated autophagy that may promote replication and/or reduce viral clearance; and ) gut dysbiosis that leads to widespread systemic inflammation, increased gut glucose and sodium absorption, and reduced tryptophan and other key amino acid absorption needed for incretin secretion and glucose homeostasis. central to each of these dysfunctions is the dysregulated ras, in particular, the global loss of ace , which we propose is a unifying mechanism that could lead to the increased risk of morbidity and mortality in individuals with diabetes presenting with covid- . the ras is a key hormonal circuit tasked in regulating extracellular fluid volume and blood pressure in mammals. if blood pressure falls, the juxtaglomerular cells in the kidneys produce and secrete renin, which cleaves serum angiotensinogen to produce angiotensin i (ang-i). then, angiotensin-i converting enzyme (ace) further converts ang-i into angiotensin ii (ang-ii) in the lungs by removing the c-terminal dipeptide from ang-i (fig. ) . ang-ii in turn activates the g-protein-coupled angiotensin type receptor (at r) on adrenal zona glomerulosa cells to produce aldosterone, which causes sodium retention, an increase in blood volume, and blood pressure stabilization. besides its beneficial function in regulating extracellular fluid volume, a dysregulated ras, as seen in diabetes, could lead to an increase in serum levels of ang-ii that could cause a plethora of potentially harmful effects including vasoconstriction, inflammation, and increased oxidative stress. ace converts ang-ii into ang- - , and ang- - acts through the mas receptor (masr) to oppose the effects of ang-ii. this ability of ace to convert the serum vasopressor ang-ii into the vasodilating ang- - identifies it as a "negative" regulator of ras. notably, covid- patients exhibit increased serum levels of ang-ii ( ) , which would support less cleavage by ace and thus potentially less ace activity. in addition to systemic ras, "local" ras exists within each tissue including a lung ras, intestinal ras, and bone marrow ras. interestingly, the ras system in the intestinal mucosa significantly contributes to the regulation of glucose, salt, and water uptake. emerging covid- gastrointestinal disturbances implicate a central role of intestinal pathophysiology in exacerbation of hyperglycemia and blood pressure in individuals with diabetes infected with sars-cov- . ace protein: function, interaction between ace and adam , and ace as the receptor for sars-cov and sars-cov- ace was discovered in ( ), just - years before the first wave of sars-cov coronavirus pandemic, when ace was identified as the major sars-cov "receptor" on host cells. ace functions as a metallocarboxypeptidase, a plasma membrane-bound proteolytic enzyme ( fig. a ) that removes a single carboxy-terminal amino acid from specific bioactive oligopeptides, such as ang-i and ang-ii to form ang- - and ang- - , respectively ( fig. ). unlike ace, which is a peptidyl dipeptidase removing two carboxy-terminal amino acids from ang-i and ang- - , ace is not inhibited by typical ace inhibitors, such as captopril. the ectodomain of ace can be shed into the systemic circulation as a soluble protein, preserving the catalytic activity of ace (soluble ace ) and its ability to generate in the circulation the vasoprotective peptide, ang- - . shedding of the ace ectodomain occurs after proteolytic cleavage by plasma membrane-anchored endopeptidases, enzymes capable of breaking nonterminal peptide bonds, such as a disintegrin and metallopeptidase domain protein (adam ) ( ) or the type ii transmembrane serine proteases (tmprss ) ( ) . human ace (hace ) is predominantly expressed in the nasal epithelium, airways, lungs, heart, adipose tissue, kidneys, small intestine, and colon ( , ( ) ( ) ( ) . the high density of hace is found in human nasal epithelium goblet cells, human ciliated cells of the airways, the type alveolar (at- ) epithelial cells, and bronchial transient secretory cells ( , ) . high hace expression in the nasal epithelium is consistent with clinical observations that symptomatic individuals with covid- present with a higher viral load in the nasal cavity compared with the throat ( ) and that some covid- patients complain of inability to smell ( ) . the hace -expressing nasal epithelium may provide an "intermediate site" for viral replication before its invasion of the lungs to cause symptomatic covid- and may serve as a sanctuary niche for sars-cov- survival without spreading to the lungs in human subjects, thus perhaps permitting asymptomatic human-to-human transmission. notably, the sars-cov- -positive individuals may shed the virus for up to days ( ). the ace protein contains two cofactors: zn and cl ions ( fig. a) . the zinc binding site, coordinating zn , is critical for the catalytic activity of ace and consists of h -e -x-x-h in hace . the cl binding site regulates the efficacy of ace to cleave its substrates (ang-i and ang-ii) in an extracellular cl -dependent manner ( ) . the spike proteins of sars-cov and sars-cov- bind the membrane-bound ace to enter the host cells ( ) ( ) ( ) . the interface of the sars-cov- receptor binding domain located on the s subunit of the spike protein and the n-terminal segments of hace was mapped using cryo-em and x-ray crystallography (fig. b ) ( ) ( ) ( ) , and the structure data shed light on the underlying mechanisms. there are several virus binding hotspots on the surface of hace that are critical for virus infectivity; these include hotspot (lysine ), hotspot (lysine ), and the hydrophobic interaction site (tyrosine y ) ( ) . compared with sars-cov, sars-cov- forms additional hydrogen bonds, dipole-dipole interactions, and salt bridges ( ) , suggesting stronger interaction. the affinity binding data indicate that the receptor binding domain of sars-cov- has a greater affinity to hace compared with that of the sars-cov virus ( ) , potentially explaining the enhanced ability of sars-cov- to quickly spread and infect a great number of hosts. while membrane-bound hace is the major cellular receptor for sars-cov- binding and internalization, the soluble form of hace is efficient at preventing the coronaviruses attachment to the membrane-bound hace ( ) . proteolytic cleavage of the homotrimeric spike protein ectodomains at the s /s subunit junction is critical for entry of coronaviruses into host cells. the ectopeptidase tmprss and endosomal peptidases cathepsin b/l are the major cellular enzymes that mediate coronavirus "priming" in sars-cov- ( ) by cleaving the spike protein at the s / s cleavage site (iay↓tms) (fig. ) . the sars-cov- spike protein has an additional canonical furin cleavage site (prrar↓sv) located upstream of the conserved iay↓tms cleavage site ( , ) . this is a unique property of sars-cov- because the furin site is not present in sars-cov ( , ) . the presence of a furin-like cleavage site in viral spike-shaped hemagglutinin proteins has been associated with an increased virulence and pathogenicity in avian and human influenza viruses. consistently, the current pandemic epidemiological data confirm the increased transmissibility and pathogenicity of sars-cov- as compared with sars-cov ( ) . renin is produced and secreted in the juxtaglomerular cells of the kidney when plasma nacl decreases or blood pressure falls. renin cleaves angiotensinogen to produce ang-i, which is further converted into ang-ii by ace in the lungs. ang-ii induces aldosterone secretion from adrenal zona glomerulosa cells, which in turn promotes sodium and water retention in the kidneys, increasing blood pressure. thus, initial serum ang-ii levels are set by renin. however, the steady-state serum ang-ii level is also markedly affected by the rate of its conversion to ang- - by ace . therefore, ace activity contributes to regulating the steady-state levels of ang-ii. if we consider an example of a rainwater barrel and assume that renin is the actual rainfall amount, ang-ii is the rainwater, and ace activity is the barrel's outlet spigot, then the rainfall amount (renin) would always determine the rainwater (ang-ii) inflow rate and level into the barrel. but if we would keep the barrel outlet spigot always open (ace is active) during and after rainstorms, the final level of rainwater would not be as high as in the case if the barrel's outlet spigot were closed (ace is inactive). aldo, aldosterone; zg, zona glomerulosa. the structure of the sars-cov- spike protein provides insight on why the addition of the furin cleavage site may increase transmissibility of the virus. according to the structure, the tmprss cleavage site (iay↓tms) is located in a shallow pocket on the lateral surface of the sars-cov- spike protein ( fig. ) , whereas the short solvent-exposed protein loop harboring the furin-cleavage site (not solved in the structure and shown as the dotted lines in fig. ) appears to hang over the tmprss cleavage site, obstructing access. the newly biosynthesized sars-cov- viral particles are likely released by budding in a golgi compartment-dependent manner. since furin is a ca -dependent endopeptidase, which is present and active only in the golgi compartment (fig. ) , the complete cleavage of the furin site is expected in golgi compartment-processed sars-cov- spike proteins and is experimentally confirmed ( ). furin-cleaved s /s subunits remain noncovalently bound in the homotrimeric spike protein assembly. it is possible that in the furin precleaved sars-cov- spike protein, the tmprss cleavage site is no longer obstructed and is more accessible for tmprss and/or cathepsins. however, experimental confirmation will be needed for this hypothesis. the sars-cov- spike protein can be in the closed (folded) or open conformation when the viral receptor binding domain unfolds and extends above the trimeric spike protein structure (fig. ). whether furin cleavage affects the equilibrium between the two spike protein conformations also remains unclear and awaits experimental evidence. as covid- progresses, sars-cov- may also involve the lytic release pathway for newly produced viral particles, bypassing the budding process utilizing the furin-containing golgi compartments (fig. ). in such cases, the spike protein of sars-cov- may remain at least partially uncleaved by intracellular furin. at this stage of covid- , extracellular furin may be utilized to complete the cleavage of spike protein's furin cleavage sites, facilitating the virus spread in the infected host. notably, circulating levels of furin are elevated in patients with diabetes ( ), and patients with diabetes infected with sars-cov- present with increased mortality ( ) and delayed recovery from sars-cov- infection. also, individuals with high plasma furin concentration typically have a pronounced dysmetabolic phenotype and elevated risk of diabetes. there is increasing evidence that dysregulated autophagy contributes to the pathogenesis of diabetes and its complications. autophagy is primarily recognized for its essential role in cellular housekeeping and homeostasis through the sequestration and transfer of intracellular components to lysosomes for degradation. however, the endocytic pathway and autophagy are key processes affecting virus infection and replication, including the coronavirus family ( ) . viral rna replication in coronavirusinfected cells occurs in double membrane vesicles that resemble autophagosomes (fig. ) . additionally, nonstructural protein (nsp ) of sars-cov- can generate autophagosomes, and an associated mutation in nsp is identified in covid- patients ( , ) . interestingly, inhibition of the canonical autophagy pathway, using in vitro approaches, does not appear to have an effect on sars-cov replication, suggesting a noncanonical process. however, a key autophagy protein, lc , colocalizes with viral replication-transcription complexes, and an s-phase kinase-associated protein (skp ) reduces autophagy protein beclin levels in coronavirus infections ( , ) . in both cases, fusion between autophagosomes and lysosomes is blocked, leading to an accumulation of autophagosomes favoring replication of the virus. inhibiting skp or enhancing autophagy flux has been shown to reduce the replication of coronaviruses ( , ) . ras can be an important regulator of autophagy. ang-ii activation of angiotensin type receptor (at r) attenuates autophagy, whereas ang-ii activation of at r induces autophagy through ampk/mtor signaling. ang- - induces autophagy via the cofilin receptor ( ) . activation of intestinal ras promotes paneth cell autophagy leading to bowel inflammation and arrested release of antimicrobial factors including defensin , which inhibits sars-cov- infection by cloaking ace ( ) . given the strong association between the ras and autophagy, both may serve as therapeutic targets to ablate sars-cov- infection and replication, and this may further explain the possible beneficial effects of ace inhibitors/atr blockers in the treatment of covid- , discussed further below. several mechanisms may contribute to increased severity of covid- progression in subjects with diabetes. individuals with diabetes are more vulnerable to most infections and may exhibit decreased viral clearance due to reduced neutrophil chemotaxis, phagocytosis, and intracellular killing of microbes. under noninfectious conditions, chronic diabetes in both human and rodent models was associated with myeloidosis ( ), with monocytes expressing higher levels of proinflammatory cytokines that may, in patients with acute respiratory distress syndrome (ards), contribute to cytokine storm. once bound to ace , sars-cov was shown to downregulate cellular expression of ace , and the unopposed action of ang-ii was deemed responsible for worsening lung injury ( ) . whether this is the case with sars-cov- is not known. ang-ii receptor blockers, ace inhibitors, thiazolidinediones, incretin glp- agonists, and statins figure -block diagram of the homotrimeric sars-cov- spike protein assembly. "rbd" stands for receptor binding domain, "fp" stands for fusion peptide, and the ipf block depicts the location of internal fusion peptide. s and s are two segments of sars-cov- ectodomain that can be cleaved with the indicated endopeptidases. the cryo-em structure ( ) of the spike protein is shown in the center of the figure. the proteolytic sites are shown in green. in the structure, the residues preceding and following the furin cleavage site are colored in brown. the inset shows a magnifying view of the tmprss /cathepsin cleavage site. the structure of the homotrimeric sars-cov- spike protein complex was replotted from pdb id: vyb ( ) . each spike protein in the homotrimer is color coded for better identification. this is a positive-sense single-stranded rna virus. sars-cov- viral rna serves to code the viral genome and as mrna for direct protein translation by the host cell ribosomes. indeed, viral rna contains a poly-a tail at the end and a typical mrna cap structure at the end. sars-cov- viral rna is nonsegmented. viral rna genome translation starts with the production of two replicase polyproteins, pp a and pp ab, which consist of or covalently linked nonstructural proteins (nsp), respectively. these two large polyproteins are subject to proteolytic cleavage by proteases resulting in the formation of individual nsp -nsp . viral nsp functions as a papain-like protease and is important for cleaving the interdomain junctions between nsp and nsp , whereas nsp is a chymotrypsin-like protease, which is also named "main protease" because it is responsible for cleaving interdomain junctions between nsp and nsp . nsp can induce small-diameter autophagosome formation in infected cells. nsp (rdrp) is an rna-dependent rna polymerase, which is critical for a largescale replication of viral rna. nsp requires several cofactors, such as nsp and nsp . the rna helicase nsp (hel) is important for are typical medications for diabetes that are known to increase ace expression. lack of evidence regarding the risk or benefit of ace inhibitors and angiotensin receptor blockers (arbs) has resulted in the american college of cardiology, american heart association, american society of hypertension, and european heart association recommendations that patients should continue treatment with their usual antihypertensive therapy ( ) . however, we would propose that drug-induced increases in ace expression would potentially be beneficial in subjects with diabetes by increasing ang- - and shifting the ras axis away from the profibrotic, proinflammatory arm of ras. thus, in subjects with diabetes, infection with sars-cov- would potentially result in additional loss of ace expression in blood vessels and could exacerbate the already compromised vasculature ( ) . the existence of specific ras systems in organs including the bone marrow has been well established. local ras is active in primitive embryonic hematopoiesis ( ) and continues to regulate each stage of physiological and pathological blood cell production in the adult via autocrine, paracrine, and intracrine pathways. local ras peptides directly regulate myelopoiesis, erythropoiesis, thrombopoiesis, and the development of other cellular lineages ( ) . the bone marrow plays a critical role in the pathogenesis of diabetic complications. individuals with diabetes with vascular complications typically have reduced numbers and migratory function of bone marrow-derived vascular reparative cells, called circulating angiogenic cells (cacs or cd cells). ang- - improved migration, restored bioavailable nitric oxide, and reduced reactive oxygen species in diabetic cacs. ang- - gene modification of cacs restored the cells in vivo vasoreparative function ( ) . a unique set of individuals with diabetes that remained free of microvascular complications, despite . years of poor glycemic control, had higher mrna levels for ace and masr in their cacs compared with age-, sex-, and glycemia-matched individuals with diabetes with microvascular complications ( ) . in akita mice, global loss of ace (ace /y -akita mice) was associated with a reduction of hematopoietic stem/progenitor cells (hs/ pc), a shift of hematopoiesis toward myelopoiesis in bone marrow, and an impairment of hs/pc migration and proliferation. migratory and proliferative dysfunction of these cells was corrected by exposure to ang- - ( ) . these data support that activation of the protective ras is beneficial for the dysfunctional diabetic bone marrow. diabetes-associated bone marrow dysfunction and loss of vascular reparative cells, such as cacs, may contribute to vascular dysfunction in covid- patients that can be manifested as cardiac disease including arrhythmias, viral myocarditis, heart failure, and cardiac arrest ( ) ( ) ( ) . the impact of global loss of ace in cardiac dysfunction is supported by preclinical studies showing that hearts from akita mice exhibit marked systolic dysfunction and that ace /y -akita mice show impaired flow-mediated dilation of the femoral artery in response to ischemia/reperfusion injury, indicative of endothelial dysfunction. in contrast, gain-of-function studies using ace overexpression, via adenoviral gene delivery, in type diabetic rats decreased collagen accumulation and improved left ventricular remodeling and function ( ). the impact of dysregulated ras is seen in obesity and type diabetes models. heart failure with preserved ejection fraction (hfpef) is a proinflammatory state closely linked to obesity-related cardiovascular dysfunction. loss of ace increases epicardial adipose tissue macrophage polarization to proinflammatory m -like phenotype and worsens hfpef in response to diet-induced obesity. ang- - has potent anti-inflammatory effects in adipose tissue of obese type diabetic mice and protects against diabetic cardiomyopathy and nephropathy. importantly, ang- - decreased macrophage m polarization and preserved cardiac function in diet-induced obese ace knockout mice ( ) . in covid- patients, the prevalence of kidney disease on admission and the development of acute kidney injury during hospitalization is high and associated with in-hospital mortality ( ) . patients with diabetes with nephropathy have reduced ace . global loss of ace replication. nsp is a viral n -methyltransferase ensuring the fidelity of replication. the viral rna also encodes four structural proteins: the spike protein (s), envelop protein (e), membrane protein (m), and nucleocapsid protein (n). in sars-cov- virions, viral rna is enveloped with a membrane that is stabilized by the imbedded structural proteins, including s, e, m, and n proteins. the s or spike protein is a homotrimer that gives the viral particles a characteristic appearance of spiky corona. the s protein is critical for the viral entry into the host cells. the s subunit of the sars-cov- spike protein utilizes the hace protein as its cellular receptor. the tmprss protein is the key endopeptidase that is important for priming the spike protein of sars-cov- , allowing viral entry into host cells. cathepsin b/l is an endosomal protease that can substitute tmprss activity during spike protein priming before viral rna gains access into the cellular cytosolic compartments. sars-cov- replication takes place in double membrane vesicles (dmv) that are associated with the specific areas of the rough endoplasmic reticulum or other intracellular membranes, including autophagosomal membranes. the m, e, and n structural proteins together with the s protein are important for formation and stabilization of the sars-cov- viral particles. viral structural protein modification takes place in the golgi compartment before viral particles are ready for budding. furin is a ca -dependent endopeptidase enriched in the golgi compartments that precleaves the spike protein at a specific cleavage site in the golgi compartments, with s and s subunits remaining noncovalently bound in budding virions. adam proteolytic activity generates soluble hace . exacerbates diabetic kidney injury while potentiating ang-ii-mediated cardiorenal fibrosis and oxidative stress in the heart and kidney ( ) . in akita mice, recombinant hace (rhace ) treatment for weeks resulted in decreased glomerular mesangial matrix expansion, which was associated with increased ang- - levels and lowered ang-ii levels, along with reduced nadph oxidase activity. the loss of ace via adam proteolytic cleavage, which is strongly activated in covid- patients, will likely promote further injury to the cardiovascular system and kidneys in patients with diabetes ( , ) . importantly, ace overexpression increases the antihypertensive components of the ras and pretreatment with rhace prevents ang-ii-induced hypertension in preclinical experimental models. however, these results have yet to be validated in human hypertension. while the lung is not considered a target tissue for diabetic complications, covid- patients with diabetes experience worse pulmonary disease than those without diabetes. ace knockout mice exhibit ards pathology. ards triggers multiple pulmonary diseases and is observed in covid- patients. importantly, ace deficiency or treatment with at r blockers of ace /y mice rescues them from ards ( ) . taken together, these studies support that in individuals with diabetes with vascular complications, the loss of the protective ras would serve to intensify sars-cov- -induced pathology. the recent demonstration of sars-cov- actively infecting human enterocytes and the mounting gastrointestinal symptomology implicate gastrointestinal tract pathophysiology in covid- infection ( , ). the digestive system possesses the body's site of greatest relative expression of ace , which in the gut exists as a tetramer with b at (fig. ) . while b at is not expressed in lung pneumocytes, ace :b at complex in the gut acts as a central player in local gut ras and regulates uptake of glucose, sodium, water, and amino acids ( ) ( ) ( ) . however, ace : b at complex internalization by sars-cov- (fig. ) destabilizes the gastrointestinal tract's role in diabetes and blood pressure regulation (fig. ) . b at (slc a ) is the intestine's primary epithelial apical membrane transporter serving na -coupled uptake of neutral amino acids, such as tryptophan. b at was originally discovered and functionally characterized by stevens et al. ( ) , and the transporter was initially named nbb, b, b , or b( ) in the literature but was subsequently called b at . ace chaperones the trafficking of b at to form the stabilized dimer of ace :b at ( ) in the apical membrane (fig. ) . importantly, b at substrates, notably tryptophan and glutamine, signal downregulation of lymphoid proinflammatory cytokines, promote tight junction formation, activate the release of antimicrobial peptides, and modulate mucosal cell autophagy as defense mechanisms. in the models shown in figs. and , binding of sars-cov- s to ace ( ) (fig. ) results in downregulating both intestinal ace and b at , with consequences of disrupting sodium and glucose transport, promoting leaky gut syndrome, elevating plasma bacterial lipopolysaccharide, and enhancing inflammation (figs. and ) . intestine, lumen-facing ace, and ace participate in the food digestion process but are also intertwined in cross talk with gut microbiome metametabolomics of bioactive peptides. such peptides include a balance of agonists and antagonists of enterocyte apical membrane masr and at r, which are physiologically tasked with regulating uptake of dietary na via nhe and glucose absorption via sglt and glut (fig. ). intestinal ace :b at dimer of heterodimers promotes enterocyte na -coupled uptake of phenylalanine, glutamine, tryptophan and its microbiome-generated metabolites, and other neutral amino acid agonists of nutrient-sensing receptors. these stimulate release of glp- and gip into the blood from gut mucosal enteroendocrine l cells (fig. ) ( ) . these incretins circulate to activate pancreatic b-cells, suppress a-cells, and afford brain satiety. sars-cov- infection of gut mucosa results in endocytosis of apical ace , thereby downregulating its activity ( ) , resulting in gut luminal accumulation of at r agonist peptides and disrupting all functions of b at . the dysregulated ras in the bone marrow with its accompanying myeloidosis promotes chronic inflammation that can contribute to both lung and gut pathology (fig. ). an extensive literature supports the concept of communication between the gut and bone marrow. the gut microbiota is a critical extrinsic regulator of hematopoiesis ( ) , as very low concentrations of microbial antigens set the size of the bone marrow myeloid cell pool, and the size of this pool correlates strongly with the complexity of the intestinal microbiota. in turn, bone marrow cells migrate to the gut and impact gut function via changes in blood flow, gut immunity, and epithelial and endothelial tight junction integrity. recruitment of bone marrow-derived immune cell to the gut is necessary for host defense and contributes to inflammation resolution and tissue healing. loss of ace in diabetes results in phylogenetic differences in the gut bacterial community composition with increases in bacteria that have been associated with peptidoglycan generation, which promotes systemic inflammation ( ) . overactivation of bone marrow-derived immune cells including proinflammatory monocytes results in secretion of a large number of harmful cytokines into the circulation that promotes insulin resistance. in the patient with diabetes infected with covid- , developing pneumonia can be devastating, as preexisting systemic inflammation can rapidly lead to multiple organ failure. inflammatory cytokine storm is a notable cause of death in critically ill covid- patients and may be driven as much by gutinduced inflammation as lung injury. thus, imbalance in the bone marrow ras system (fig. ) may represent a central mechanism to not only initiate but also propagate lung and gut injury. from the perspective of gut enterocyte local ras, orally delivered ace inhibitors upregulate expression of both intestinal ace and b at with their attending nutrientsignaled release of glp- , gip, and mucosal antimicrobial peptides ( ) (fig. ) . in a preclinical colitis model, the arb irbesartan restored intestinal b at and ace expression and tryptophan homeostasis with concurrent reduction of intestinal inflammasome activity through an mtor s kinase pathway ( ) . irbesartan further shifted the gut microbiota composition toward favorable taxa and away from stress-related dysbiosis ( ) . activation of enterocyte at r signaled apoptosis with reduced mucosal villus height, while losartan-mediated blockage of gut at r resulted in increased mucosal cell proliferation and reduced apoptosis. increasing gut ace by engineering probiotic species such as lactobacillus paracasei (lp) to express this recombinant protein was a strategy used to prevent microvascular complications in diabetic mice. lp expressing the secretable ace fused with the nontoxic subunit b of cholera toxin (which acts as a carrier to facilitate transmucosal transport), showed increased ace activities in serum and tissues, and reduced diabetic complications ( ) . these results provide proof of concept for feasibility of using engineered probiotic species as a live vector for delivery of decoy hace for possible treatment of enteric covid- infection. rhace given as intravenous medication may be explored as beneficial to covid- patients with pulmonary complication, as it increases pulmonary blood flow and oxygenation in a pig model of lipopolysaccharide-induced ards. supplementation with ace or inhibition of ang-ii improves outcomes in acute lung injury. a pilot trial demonstrated that rhace is well-tolerated in ards patients and showed the anticipated changes in ras peptides. taken together, evidence unequivocally supports the concept that ace is critical in pulmonary function and its imbalance in covid- infection contributes to the devastating lung consequences. an ace activator, diminazene aceturate (dize) is a known antiprotozoal drug used in humans, but it has additional benefits including potent anti-inflammatory the complex comprises a dimer of heterodimers formed by two b at subunits (red) contacting with two ace subunits (green), with each ace interfacing with a single sars-cov- spike (brown). the complex was stabilized using na cotransporter b at transport substrate leucine within the center membrane-spanning domain, known to serve tryptophan, glutamine, and other neutral amino acids in addition to leucine. intestinal apical membranes express the b at :ace complex, which does not occur in lung pneumocytes ( ) . b: side view showing charged moiety interactions in the extracellular region of the gut lumen (top inset) and also hydrophobic interactions of b at tm and tm interacting with the single long transmembrane domain of ace within the apical membrane (bottom inset). data coordinates were obtained from pdb id: m ( ) . ( ) . luminal agonist and antagonist bioactive peptides are derived from interactions of gut digestive enzymes intertwined with microbiome metabolism. oral arbs and ace inhibitor drugs impact gut ras. gut ras governs sodium and glucose uptake via nhe , sglt , and glut . the ace :b at complex dimer of heterodimers ( ) serves the na -coupled transport of neutral amino acids, including tryptophan. in enteroendocrine l cells, basolateral tryptophan stimulates glp- and gip secretion. these incretins maintain gut tight junctions, preventing dysbiosis, stimulate pancreatic b-cells, and blunt a-cells, thereby modulating plasma glucose levels. sars-cov- binding to ace disrupts this homeostasis. and antifibrotic activity. dize has been used in type diabetes to prevent nephropathy and gastric inflammation. dize modulated the ras by reducing serum ang-ii and the expression of at r, but it increased ang- - ( ). dize not only increased ace activity but also increased the expression of ace in select cell types where dize inhibited the expression of il- , il- , and mcp- at both mrna and protein levels following stimulation with lipopolysaccharide. collectively, these results show that dize downregulates proinflammatory cytokine production by many distinct cell types and suggest that this drug may provide benefit to covid- patients by reducing pulmonary inflammation and fibrosis, gut inflammation, and cytokine storm. as the global pandemic unfolds and rapidly spreads, there is an urgent need for basic and clinical studies to address the many unanswered questions posed by covid- . this perspective has directed attention to the disruption of ras in the lung, gastrointestinal tract, and bone marrow as possible mechanisms of sars-cov- disease pathogenesis. the dysregulated ras can potentially impact clinical outcomes in individuals with diabetes resulting in increased morbidity and mortality. ace has emerged as the pleiotropic regulator of the ras, by metabolizing ang-ii into the beneficial peptide ang- - , while being harmful as the sars-cov- receptor. loss of ace indirectly via proteolytic processing, autophagy, and shedding partly could not only drive lung pathology but also gut disease in individuals with diabetes infected with covid- . sars-cov- , by downregulating intestinal ace -b at , could promote leaky gut syndrome with elevated plasma bacterial lipopolysaccharides and/or peptidoglycans enhancing systemic inflammation. careful targeting of the ras axis may represent a strategy for improving clinical outcomes in subjects with diabetes infected with covid- . ace , a pleiotropic regulator of the ras, is hijacked as a receptor for sars-cov- to promote viral infection. loss of ace indirectly via proteolytic processing, autophagy, and adam -mediated shedding (not shown) partly drives not only lung but also gut disease in individuals with diabetes with covid- . sars-cov- s binding to ace initiates internalization of ace :b at complex (gut) or ace (outside of gut). thus, sars-cov- by downregulating intestinal ace -b at would promote leaky gut syndrome with elevated plasma bacterial lipopolysaccharides and/or peptidoglycans enhancing systemic inflammation. in the lung, virus internalization also promotes a reduction in ace that results in pulmonary pathology. careful targeting of the ras axis will likely optimize clinical outcomes in subjects with diabetes infected with sars-cov- . wbc, white blood cell. china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china clinical characteristics of patients infected with sars-cov- in wuhan clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study prevalence and impact of diabetes among people infected with sars-cov- covid- testing, hospital admission, and intensive care among , , united states veterans aged - years clinical and biochemical indexes from -ncov infected patients linked to viral loads and lung injury sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) tmprss and adam cleave ace differentially and only proteolysis by tmprss augments entry driven by the severe acute respiratory syndrome coronavirus spike protein hca lung biological network. sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov- receptor ace and tmprss are primarily expressed in bronchial transient secretory cells digestive system is a potential route of covid- : an analysis of single-cell coexpression pattern of key proteins in viral entry process sars-cov- : olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov- spike glycoprotein structural and functional basis of sars-cov- entry by using human ace structural basis of receptor recognition by sars-cov- structural basis for the recognition of sars-cov- by full-length human ace structure of the sars-cov- spike receptor-binding domain bound to the ace receptor characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov the spike glycoprotein of the new coronavirus -ncov contains a furin-like cleavage site absent in cov of the same clade quantifying sars-cov- transmission suggests epidemic control with digital contact tracing plasma levels of the proprotein convertase furin and incidence of diabetes and mortality targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid- skp attenuates autophagy through beclin -ubiquitination and its inhibition reduces mers-coronavirus infection autophagy in metabolic syndrome: breaking the wheel by targeting the renin-angiotensin system human intestinal defensin inhibits sars-cov- invasion by cloaking ace a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury renin-angiotensin system blockers and the covid- pandemic: at present there is no evidence to abandon renin-angiotensin system blockers angiotensin-converting enzyme (cd ) marks hematopoietic stem cells in human embryonic, fetal, and adult hematopoietic tissues a role for the renin-angiotensin system in hematopoiesis activation of the ace / angiotensin-( - )/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors loss of angiotensin-converting enzyme exacerbates diabetic retinopathy by promoting bone marrow dysfunction cardiac involvement in a patient with coronavirus disease (covid- ) association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) kidney disease is associated with in-hospital death of patients with covid- angiotensin converting enzyme : a doubleedged sword sars-cov- productively infects human gut enterocytes human intestine luminal ace and amino acid transporter expression increased by ace-inhibitors imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in ibd: a novel therapeutic target? the meaning of mas multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles glutamine triggers and potentiates glucagon-like peptide- secretion by raising cytosolic ca and camp renin-angiotensin-aldosterone system inhibitors in patients with covid- microbiota-derived compounds drive steady-state granulopoiesis via myd /ticam signaling bone marrow-derived cells restore functional integrity of the gut epithelial and vascular barriers in a model of diabetes and ace deficiency angiotensin receptor blocker irbesartan reduces stress-induced intestinal inflammation via at a signaling and ace -dependent mechanism in mice expression of human ace in lactobacillus and beneficial effects in diabetic retinopathy in mice ace x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis key: cord- -gakayv e authors: bian, jingwei; li, zijian title: angiotensin-converting enzyme (ace ): sars-cov- receptor and ras modulator date: - - journal: acta pharm sin b doi: . /j.apsb. . . sha: doc_id: cord_uid: gakayv e the coronavirus disease (covid- ) outbreak is caused by severe acute respiratory syndrome coronavirus (sars-cov- ). angiotensin-converting enzyme (ace ) was rapidly identified as the critical functional receptor for sars-cov- . ace is well-known as a counter-regulator of the renin-angiotensin system (ras) and plays a key role in the cardiovascular system. given that ace functions as both a sars-cov- receptor and a ras modulator, the treatment for covid- presents a dilemma of how to limit virus entry but protect ace physiological functions. thus, an in-depth summary of the recent progress of ace research and its relationship to the virus is urgently needed to provide possible solution to the dilemma. here, we summarize the complexity and interplay between the coronavirus, ace and ras (including anti-ras drugs). we propose five novel working modes for functional receptor for sars-cov- infection and the routes of ace -mediated virus entering host cells, as well as its regulatory mechanism. for the controversy of anti-ras drugs application, we also give theoretical analysis and discussed for drug application. these will contribute to a deeper understanding of the complex mechanisms of underlying the relationship between the virus and ace , and provide guidance for virus intervention strategies. the coronavirus disease outbreak is caused by a novel coronavirus, which was named severe acute respiratory syndrome coronavirus (sars-cov- ). globally, as of may , there have been , , confirmed covid- cases and , deaths, which has already posed a great threat to global public health security . although respiratory symptoms are predominant, multi-organ dysfunction occurs in response to sars-cov- infections, including acute cardiac and kidney injuries, arrhythmias and liver function abnormalities . at present, most patients with covid- have a good prognosis; however, patients with underlying disorders have greater severity and higher mortality. . % of all patients had at least one underlying disease, particularly, hypertension ( . %) and coronary heart disease ( . %) . therefore, integrated treatment for covid - patients with underlying diseases is key to reduce mortality. sars-cov- has been shown to share the same functional receptor, angiotensin-converting enzyme (ace ), with severe acute respiratory syndrome coronavirus (sars-cov) , . ace is a carboxypeptidase and a negative regulator of the renin-angiotensin system (ras) through balancing its homology angiotensin-converting enzyme (ace). ace mediates angiotensin (ang) ii production to activate ras that plays a key role in cardiovascular diseases, especially hypertension . thus, ace inhibitor (acei) is used widely for treatment of hypertension, which reduces ang ii levels. since ace is a homologue of ace, disputes have arisen about whether acei can upregulate ace and thus the risk and severity of coronavirus infection increase. it calls into question whether to continue using of acei in virus-infected patients with hypertension. at present, some experts suggest that covid- patients with hypertension should stop using acei , . on the other hand, other experts believe that not only does acei not increase the risk of sars-cov- infection, but acei could reduce lung injury and cardiovascular damage in covid- patients . thus, a comprehensive was the functional receptor of sars-cov- . excitingly, the structures of sars-cov- and ace were obtained by cryo-electron microscopy (cryo-em) quickly. firstly, the cryo-em structure of the sars-cov- s-protein was determined in the prefusion conformation. the predominant state of the s-protein trimer has one of the three rbds rotated up in a receptor-accessible conformation. the biophysical and structural evidence showed that the sars-cov- s-protein bound ace with higher affinity than the sars-cov s-protein (approximately -to -fold) , which might explain why sars-cov- is more contagious than sars-cov. then, the cryo-em structure of full-length human ace in complex with a neutral amino acid transporter b at was revealed . immediately, the crystal structure of the rbd of sars-cov- s-protein bound with ace was determined . this series of work strongly confirmed that ace is the functional receptor of sars-cov- . notably, the structure of full-length human ace in complex with b at revealed a novel mode that showed sars-cov- bound to ace in a homodimer manner (fig. b) . in this mode, b at might be a key factor in determining the formation of an ace homodimer. thus, these results suggest that sars-cov- binds with ace by two working modes: i) sars-cov- binds directly with an ace monomer without the b at protein (fig. a) and ii) sars-cov- binds with ace homodimer when with the existing of b at protein (fig. b) . however, the binding affinity and biological function of these two different modes are still unclear. ace is widely expressed across a variety of organs and its expression is higher in many organs (such as heart, kidney, etc.) than that in lung. however, the lung is the major organ infected by sars-cov- . in addition, although ace knockout mice showed a significant decrease in sars-cov infection, it did not completely prevent virus infection from occurring . those data suggested that there could be other receptors involved in a viral invasion. more recently, cd , a transmembrane glycoprotein that belongs to the immunoglobulin superfamily, was identified as a receptor for sars-cov- , . interestingly, previous studies have shown that cd played a key role in sars-cov invasion into host cells, while cd antagonistic peptides have an inhibitory effect on sars-cov . these results further suggested that cd might be a novel receptor for sars-cov- . another possible receptor is cd l (l-sign), a type transmembrane glycoprotein in the c-type lectin family , , which has been identified as the receptor of sars-cov from in vitro studies . thus, given that sars-cov- is similar to sars-cov, cd l could be another potential receptor for sars-cov- . therefore, besides ace , there are several other potential receptors for sars-cov- . sars-cov- might invade cells through an alternative j o u r n a l p r e -p r o o f receptor mode (fig. c ) or a co-receptors mode (fig. d) . interestingly, viruses are very tricky, as they can also infect host cells in a detour or bait-and-switch strategy. for example, sars-cov can first attach to the surface of dendritic cells (dc) through the dc-sign receptor, which is a dc-specific intercellular adhesion molecule- -grabbing non-integrin, a homologue of cd l. then the virus is presented to host cells by the dc cells and binds to ace of host cell, which eventually leads to infection . similarly, sars-cov- might also use this subterfuge to infect a host cell. since the dc-sign receptor plays only a transmitting role, it could be called the "transmissive receptor" (fig. e ). in summary, we propose five distinctive working modes of sars-cov- receptors: monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. the five modes are distinct but also interconnected. ace was discovered as a homologue of ace in , . it plays a physiological and pathological role in cardiovascular, renal, intestinal and respiratory systems [ ] [ ] [ ] [ ] . ace is a type i transmembrane protein, which consists of amino acids with an extracellular n-terminal domain and a short to be internalized into cytoplasm upon virus binding , . in addition to the membrane-bound form, the soluble form of ace can be detected in the plasma and urine through its extracellular domain shedding , . a disintegrin and metallopeptidase domain (adam ) and type ii transmembrane serine proteases (tmprss ) are considered as the proteases for ace proteolytic cleavage - . identifying the distribution of ace in organs has major implications for understanding the pathogenesis and treatment options for sars-cov- . ace was initially only detected in the heart, kidneys and testes , , . then, ace mrna expression was revealed in virtually all organs ( human tissues) by real-time pcr . in , hamming et al. investigated the localization of the ace protein in human organs by immunohistochemistry and ace protein expression was found in various organs, including oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain. more recently, single-cell rna-seq datasets revealed that the abundance of ace expression from high to low is in the ileum, heart, kidney, bladder, respiratory tract, lung, esophagus, stomach and liver . here, we present a map of ace distribution and expression in major organs based on the previous results and the biogps website (http://biogps.org) (fig. , left panel). given that ace is the functional receptor of sars-cov- , its expression is associated with the organic vulnerability to sars-cov- . indeed, the tissue expression of ace shows some correlation with the sites of sars-cov- infection. for example, ace protein is abundantly expressed in the lung and heart which are the vulnerable organs for sars-cov- . however, the level of ace expression is not completely related with the vulnerability to sars-cov- . here, we present a comparative map of ace expression and vulnerability to sars-cov- in different organs (fig. ). based on the comparative map, the ace expression is the highest in ileum, but the ileum is not the most vulnerable organ, suggesting other complicated mechanisms might be involve in virus infection. the possible mechanisms for inconsistency between ace expression and virus vulnerability include: i) there might be other unknown receptor-mediated virus infection; ii) ace alone is not enough to mediate virus infection, which needs another co-receptor's assistance (such as angiotensin ii type receptor (at r), a potential receptor for sars-cov- ); iii) the function of the ace receptor is regulated by some protein as yet unidentified. for example, b at , a neutral amino acid transporter also expressed in the ileum , can bind ace and promotes ace to form a homodimer. the formation of ace homodimer hides the cleavage-sites for ace ectodomain shedding, which j o u r n a l p r e -p r o o f reduces ace endocytosis and further decreases virus infection in the ileum at least ; and/or iv) some proteases, notably tmprss , are also involved in the virus infection. tmprss can promote the membrane fusion between virus and host cell upon ace engagement and sars-cov- s-protein activation . in summary, the inconsistency between ace expression and virus vulnerability reflects the complex mechanisms involved in virus infection and also further supports the proposed working modes shown in fig. . understanding how ace -mediated sars-cov- entering cell will provide valuable information for virus pathogenesis and drug target. after sars-cov- binds to ace , there are two routes that sars-cov- enters host cell: endocytosis and membrane fusion. coronaviruses, such as sars-cov and mers-cov, have been shown to enter host cells via receptor-mediated endocytosis , . recently, using sars-cov- s-protein pseudovirus system, it was found that, after binding with ace , sars-cov- was also endocytosed into cell (fig. a) . receptor-mediated endocytosis, including both clathrin-dependent pathways and caveolae-dependent pathways, is the most important mechanism for virus internalization , . clathrin-dependent endocytosis is considered the primary endocytic route for the coronavirus. the infection of both sars-cov and mers-cov have been shown to occur by clathrin-dependent endocytosis , . as an alternative to clathrin, caveolae-dependent endocytosis has been also described as the endocytic route for some viruses, such as simian virus (sv ) . however, the caveolae-dependent endocytosis does not seem to involve ace endocytosis after sars-cov infection. additionally, lipid raft-dependent endocytosis was found in sars-cov as a novel pathway, which is both clathrin-and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluids , . therefore, it is reasonable to assume that sars-cov- binds to ace and might be able to either initiate clathrin-dependent and/or non-caveolae lipid raft-dependent endocytosis to enter host cell (fig. a ), but these need to be further verified by biological experiments. in addition to ace -mediated virus endocytosis, tmprss -mediated direct membrane fusion is another important way for sars-cov- entry. unlike the route that ace binds with sars-cov- j o u r n a l p r e -p r o o f to mediated virus endocytosis together, during the process of tmprss -mediated membranes fusion, ace plays a role in arresting and fixing the sars-cov- at the surface. after the viruses arrested and fixed, tmprss induces direct membrane fusion between virus and host cell (fig. b) . notably, the route of tmprss -mediated membrane fusion is also crucial for sars-cov and mers-cov entry . the proteolytic shedding of a transmembrane protein like ace can result in release of the soluble extracellular domain (ectodomain) from the membrane and a fragment that remains bound to the membrane. the shedding is an important regulatory mechanism to control the function and distribution of membrane proteins, which can terminate the function of a full-length membrane protein or release the biologically active ectodomain to activate the membrane protein. in addition, the shedding contributes to membrane protein endocytosis , . ace shedding can increase sars-cov entry [ ] [ ] [ ] , but the exact molecular mechanism responsible for increased virus entry is unclear at present. some researchers have suggested that ace shedding promoted ace -mediated virus internalization and increased virus uptake into target cells . here, we summarize two distinct modes for ace shedding: adam -dependent shedding and tmprss -dependent shedding (fig. ). adam , a disintegrin and metalloproteinase, has an important established role in the regulation of ace shedding . adam functions in the shedding of ace via arginine and lysine residues within ace amino acids to (fig. ) . sars-cov s-protein binding facilitates adam -dependent ace shedding and has been shown to induce viral entry into the cell . on the other hand, there is also another research has suggested that only type ii transmembrane serine proteases tmppss , but not adam- , promotes sars-cov entry via ace shedding . different from adam , tmppss requires arginine and lysine residues within ace amino acids to for ace cleavage . notably, the neutral amino acid transporter b at might inhibit tmprss -dependent ace shedding (fig. b) since b at binds ace to form a protein complex. the structure of the complex of ace /b at reveals that the tmprss cleavage sites (residues of - ) are hidden in the dimeric interface of ace . it indicates that b at may block the access of tmprss to the cleavage sites on ace which would result in decrease of ace shedding. in addition, the proprotein convertase furin has been also identified as an important factor for regulation of ace -mediated virus entering host cells. furin mainly preactivates sars-cov- s-protein during viral packaging, and enhances virus entry into target cells , . however, recent study showed that furin reduced sars-cov- entry into vero cells shows that the increase of adam- -dependent ace shedding is associated with myocardial hypertrophy and fibrosis . in addition, elevated soluble ace activity is associated with severer of myocardial dysfunction and is an independent predictor of adverse clinical events . however, the question whether soluble ace , produced by ace shedding, can bind with sars-cov- s-protein to clear away virus is confusing, which will be the future direction for research on soluble ace . ras plays a critical role in maintaining blood pressure homeostasis, as well as fluid and salt balance. therefore, ras is intimately connected the pathophysiology of heart and kidney diseases , . production of angiotensins from angiotensinogen requires the participation and coordination of many j o u r n a l p r e -p r o o f on ang ii, because the affinity of ace with ang ii is times higher than that with ang i . ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) binds to the mas receptor (masr), which is a seven transmembrane g-protein-coupled receptor, and forms an ace -ang ( - )-masr axis to mediate vasodilation and decreases blood pressure , . in summary, current knowledge of the ras has been transformed from a linear hormonal system to a complex counter-regulatory system. the counter-regulatory ras axis, ace -ang ( - )-masr axis, opposes the effect of the ace-ang ii-at r axis and has been show to reverse organ damage in renal or cardiovascular diseases (fig. ) . insert fig. led to significant down-regulation of ace expression . in post-mortem autopsy heart tissues from patients who succumbed to sars-cov, seven heart samples had detectable viral sars-cov genome. these patients were also characterized by reduced myocardial ace expression , which further confirmed that ace expression was decrease after virus infection. given that ace functions as a counter-regulator of ras, the decrease of ace expression leads to the weakened ace -ang ( - )-masr axis, mainly manifested as the increase of ang ii and decrease of vasodilator ang ( - ) level. at present, a cohort study of covid- patients have partly confirmed this view. it was found that the level of ang ii in the plasma sample from sars-cov- infected patients was significantly higher than that from uninfected individuals since ace has been identified as the functional receptor of sars-cov- , some disputes have at present, it is not clear whether acei and arb increase ace expression at the protein level. for example, perindopril (acei) was able to increase hepatic ace expression at the protein level under conditions of liver fibrosis . however, another acei drug ramipril decreased ace protein expression after myocardial infarction . for arb drugs, olmesartan up-regulated ace protein expression in the carotid arteries after balloon injury. but there were no changes in ace protein expression in uninjured carotid arteries in olmesartan-treated rats . therefore, from an ace ace functions as both a sars-cov- receptor and ras modulator, which presents the dilemma of j o u r n a l p r e -p r o o f how to limit virus entry while protecting its physiological function. therefore, it is of great significance to understand fully the function and mechanism of ace and the relationships among virus, ace and ras. although ace has been identified as a sars-cov- receptor, there might be other receptors or co-receptors for this virus that are yet to be discovered. in this review, we propose five working modes of functional receptors for sars-cov- : monomer receptor, homodimer receptor, alternative receptors, co-receptors and transmissive receptor. we summarize the routes of ace receptor-mediated virus entering host cells (ace -mediated virus endocytosis via clathrin-dependent pathways and non-caveolar lipid raft dependent pathways, and tmprss -mediated membrane fusion upon ace engagement) and its regulatory mechanism. in addition, a comparative map of ace expression and vulnerability to sars-cov- in different organs was described. moreover, the complex relationship among coronavirus, ace and ras (including anti-ras drugs) is also summarized and discussed. these will contribute to a deeper understanding of the complex mechanisms and intervention strategies for virus infection and target organ damage. these raise further important implications for therapeutic targets for sars-cov- . effective therapies against sars-cov- are urgently required due to the severity of the outbreak. based on the above theoretical summary, five steps are important anti-viral targets, including ) the binding between coronavirus and ace ; ) virus entry mediated by ace ; ) virus replication; ) virus assembly, and ) virus exit (fig. ) . one of important strategies to control viral infections is to block the initial binding of virus to its in addition, ace interference is another way to block the binding between the sars-cov- s-protein and ace . for example, chloroquine can impact terminal glycosylation of ace , thereby preventing it from binding to the s-protein and inhibiting the sars-cov- infection [ ] [ ] [ ] . furthermore, interfering with other receptors (such as cd ) proposed in fig. also can be considered as the therapeutic targets . for example, meplazumab, an anti-cd humanized antibody, significantly inhibited the viruses from invading host cells . j o u r n a l p r e -p r o o f ②). ace receptor-mediated endocytosis is a complex process regulated by a variety of proteins, which are potential target for interference. for example, ap -associated protein kinase (aak ) is a well-known positive regulator of receptor endocytosis. an inhibitor of aak (baricitinib) is predicted to reduce the ability of the sars-cov- entry by inhibiting ace receptor-mediated endocytosis . in addition, camostat mesylate, a tmprss inhibitor, has been shown to inhibit sars-cov- entry into cells . of course, in addition to the steps related to ace (the binding between coronavirus and ace , and virus entry mediated by ace ), virus replication has been the hotspot for antiviral drug research. some drugs have entered clinical practice (fig. ③) . covid- situation reports sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace clinical characteristics of coronavirus disease in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus angiotensin ii signal transduction: an update on mechanisms of physiology and pathophysiology are patients with hypertension and diabetes mellitus at increased risk for covid- infection? hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid- coronavirus disease (covid- ) and cardiovascular disease: a 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-o-ac-sia , nl acute lower respiratory tract infection in children ace severe acute respiratory syndrome ace , cd l, dc-sign - oc common cold -o-ac-sia ace , angiotensin-converting enzyme ; ddp , dipeptidyl peptidase ; cd l (also called l-sign), liver/lymph node-specific intercellular adhesion molecule- -grabbing integrin dendritic cell-specific intercellular adhesion molecule- -grabbing integrin the authors acknowledge funding support from the national natural science foundation of china ( , , , and ) and beijing municipal natural science foundation ( , china). all authors researched data for the article and discussed its content. jingwei bian wrote the manuscript. zijian li designed, reviewed and edited this manuscript before submission. on behalf of all authors, the corresponding author states that there is no conflict of interest. key: cord- -fuwp qt authors: lu, chen-chen; hu, ze-bo; wang, ru; hong, ze-hui; lu, jian; chen, pei-pei; zhang, jia-xiu; li, xue-qi; yuan, ben-yin; huang, si-jia; ruan, xiong-zhong; liu, bi-cheng; ma, kun-ling title: gut microbiota dysbiosis-induced activation of the intrarenal renin–angiotensin system is involved in kidney injuries in rat diabetic nephropathy date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: fuwp qt some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (dm). in this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (dn) and its underlying mechanisms. male sd rats were intraperitoneally injected with streptozotocin to induce dm. dm rats were orally administered compound broad-spectrum antibiotics for weeks. after the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. we found that compared with the control rats, dm rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. furthermore, the protein levels of angiotensin ii, angiotensin-converting enzyme, and angiotensin ii type receptor in the kidneys of dm rats were significantly increased. administration of broad-spectrum antibiotics in dm rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal ras activation, and attenuated kidney damage. finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin ii protein expression (r = . , p < . ). in conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early dn through activating intrarenal ras. as a chronic microvascular complication of diabetes, diabetic nephropathy (dn) has become one of the major causes leading to the death of diabetic patients. according to the th idf diabetes atlas, million adults were diagnosed with diabetes worldwide in , and this population has been expected to grow by % by [ ] . the rapid growth of diabetes has led to a dramatically increasing prevalence of dn. in china, due to the rapid development of the social economy and lifestyle changes, the prevalence of diabetes is also on the rise. according to the latest survey data in china, the prevalence of diabetes in adults is . %, and the total number of diabetic patients is as high as million, nearly half of which also have dn [ ] . however, due to the limited diagnosis of early dn, most of the newly diagnosed patients have already progressed to stage iii or iv, which is basically irreversible. therefore, it is of great importance to clarify the pathophysiological changes in the early stages of dn and formulate intervention strategies for clinical diagnosis and treatment. the activation of the intrarenal renin-angiotensin system (ras) has long been considered one of the initiators of dn. it has been reported that under diabetic conditions, the circulating ras is normal or decreased, while local ras is highly activated in the kidney. in addition, renal tissue is sensitive to angiotensin ii (ang ii), leading to renal vasoconstriction, higher resistance of the glomerular efferent artery, and increased sodium and water reabsorption, all resulting in increased blood pressure and glomerular hypertension [ ] . in addition, ang ii can also promote the phenotypic transformation of glomerular endothelial cells and podocytes, the deposition of extracellular matrix, and the secretion of inflammatory and profibrotic chemokines and factors, accelerating the progression of dn [ ] [ ] [ ] [ ] . in recent years, the effect of gut microbiota on diabetes and its complications has aroused great interest. the gut microbiota of humans weighs~ . - . kg, including~ trillion bacteria, the distribution density of which increases gradually from the proximal end to the distal intestine. the composition of gut microbiota in the host is associated with several factors, such as genetic and environmental influence and long-term dietary patterns, and this microbial community usually manifests as a state of equilibrium between different groups in the gut. while the host provides nutrients to the gut microbiota, the latter helps digest complex carbohydrates, producing immune molecules, short-chain fatty acids (scfas) and other metabolites that exert immune and metabolic functions [ , ] . studies have revealed notable differences in the gut microbiota between diabetic and healthy people [ ] . in the intestines of healthy people, there are rich butyrate-producing bacteria, such as escherichia coli, clostridium, etc. in patients with type diabetes (t dm), butyrateproducing bacteria are significantly reduced compared with increasing opportunistic pathogens [ ] . karlsson et al. [ ] have shown that postmenopausal women with t dm in europe have significant insulin resistance, partly because of a significant decrease in the abundance of faecalibacterium prausnitzii and roseburia, which are known as dwellers and butyrate producers of the human gut [ ] and have been linked to improved insulin sensitivity and diabetes [ , ] . larsen et al. [ ] found that the abundance of intestinal firmicutes in adult male patients with t dm decreased significantly, while the abundance of bacteroides and proteobacteria increased, and the ratio of bacteroides/ firmicutes was correlated with patients' glucose tolerance and blood-glucose (bg) levels. compared with healthy controls, the abundance of firmicutes increased in t dm patients, while the abundance of bacteroidetes decreased [ , ] . in animal studies, treatment with a prebiotic (oligofructose) in high-fat-fed diabetic mice not only increased the bifidobacterial content of their guts but also improved their glucose tolerance and insulin resistance as well [ ] . therefore, the gut microbiota might be closely related to the occurrence and development of diabetes. recent studies have found that under the stimulation of injurious factors, the gut microbiota could produce excessive scfas such as acetate, mediate immune disorders and chronic inflammatory reactions of the host, and promote the occurrence of diseases such as diabetes, obesity, and inflammatory bowel disease [ ] [ ] [ ] . the gut microbiota of high-fat-fed rats has been reported to promote insulin secretion and aggravate insulin resistance by synthesizing large amounts of acetate [ ] . studies have shown that scfas are involved in physiological pathways by binding to their receptors, g protein-coupled receptors (gpcrs) and olfactory receptors (olfr). reportedly, functional receptors of scfas include gpr , gpr , gpr , olfr [ , ] and so on. brown et al. [ ] found that the smooth muscle cells of renal arteries express scfa receptors, and among them, gpr and olfr are relatively abundant. intestinal-derived propionic acid can bind to the renal arteriolar olfr and activate intrarenal ras, further increasing the secretion of renin and angiotensin and thus regulating circulating and glomerular pressure. therefore, we speculated that in the development of diabetes, the gut microbiota was likely to produce excessive scfas, especially acetate, which could bind to renal-related signal receptors, thus activating intrarenal ras and mediating the early pathophysiological processes of dn. experimental animals and measurement of general parameters eight-week-old healthy male sprague-dawley rats were kept under the following conditions: a constant -h photoperiod, temperature of - °c, and free access to food and water. after weeks of adaptive feeding, these sd rats were randomly divided into three groups: the control group, diabetic mellitus (dm) group, and diabetic rats treated with antibiotics (dm + ab) (n = ). the latter two groups were intraperitoneally injected with streptozotocin at a dose of . mg/kg (sigma, usa), and bg levels were measured days after the injection to confirm the establishment of diabetic models. all rats of the three groups were normally fed, while the dm + ab group were orally given compound antibiotic solution, consisting of g/l ampicillin, g/l neomycin, . g/l vancomycin, and . g/l amphotericin b. all rats were sacrificed after weeks, with blood, urine, feces, and renal tissues harvested (kidney weights were measured at the time of sacrifice). the concentrations of bg were determined by a bg meter (shanghai johnson & johnson medical devices company). serum creatinine and blood urea nitrogen (bun) were measured by an automatic analyzer (hitachi tokyo, japan). the rats were kept alone in metabolic cages to collect their -h urine samples, of which the level of -h urinary protein was quantitatively analyzed by the lowry assay. the measurement of blood pressure in the three groups was performed at the animal core facility of nanjing medical university. the procedures for the animal experiments were approved by the ethical committee of southeast university and followed the latest version of the declaration of helsinki. after the kidneys were removed, the tissues were decapsulated and fixed in % paraformaldehyde and glutaraldehyde. after h, fixed tissues were embedded in paraffin for observation of the pathological changes under light microscopy (olympus, japan) or embedded in % lanthanum nitrate for ultramicrostructural observation of the podocytes and glomerular basement membrane (gbm) under electron microscopy (jem- , japan). the tissues were cut into -μm-thick slices and then stained with periodic acid-schiff (pas) solution and wheat germ agglutinin (wga) after removing the paraffin. immunofluorescent staining of the tissue slices was performed using primary antibodies against wilms' tumor (wt- ) and nephrin (santa cruz, usa) and examined by laser confocal microscopy (leica, germany). the levels of plasma acetate were measured by gas chromatography. briefly, mmol/l -methylvaleric acid (macklin, china) was used as an internal standard stock solution, while mm acetic acid (sigma-aldrich, usa) was used as the acetate standard stock solution. the diluent and extracting solvent were ethyl acetate. different concentrations of acetate standards were prepared with mmol/l internal standard. a total of μl of plasma was spiked with μl of mm stock solution of internal standard and acidified with μl of hydrochloric acid. after shaking for s, the mixture was sonicated for min and placed at °c until layering. after centrifugation for min at a speed of r/min , the organic phase was filtrated using a . -μm filter. a -μl injection of standards and filtrated samples was used for gas chromatography analysis with a db- column ( m × . mm, agilent, usa) at a speed of ml/min. the split ratio was : . the carrier gas was nitrogen. the initial temperature was °c, which increased to °c ( °c/min). it was then heated to °c ( °c/min), gradually increasing to °c and waiting for min. the standard curve was made according to the concentration and peak area of acetate standards to that of the internal standard. the plasma acetate concentration was then quantified. s ribosomal dna (rdna) sequencing analysis the gene sequencing of gut microbiota was performed using s rdna sequencing technology. fresh fecal specimens were collected using sterile tweezers and tubes and stored at − °c. the total dna of the fecal bacteria was extracted according to the manual of the qiaamp dna stool mini kit (qiagen, hilden, germany). briefly, feces were homogenized and lysed in asl buffer. the mixture was centrifuged at r/min for min. an inhibitex tablet was dissolved in the supernatant, followed by centrifugation. proteinase k and ethanol were added to the supernatant. lysates were then loaded onto the qiaamp spin column with a qiaamp membrane. the column was efficiently washed in two steps with repeated centrifugation. lastly, purified dna was eluted in low-salt buffer. pcr amplification of the variable region - of bacterial s rdna was conducted through double eight cycles to yield detectable products. the amplicons were mixed and purified to construct the gene library after quantification by real-time pcr. sequencing was performed on the illumina miseq × bp platform. operational taxonomic unit picking ( % nucleotide sequence identity) was assigned using the ribosomal database project classifier. chimeric sequences were removed using uchime. the alpha diversity analysis was performed with mothur (version . . ), while the beta diversity analysis was performed with r program (version . . ). the levels of renin, ang І, and ang ii in the plasma of the three groups were measured by a radioimmunoassay kit (beijing north institute of biological technology, china) in accordance with the instructions. the method for determining the level of plasma renin activity (pra) is as previously described [ ] : the optimal dose of trypsin (invitrogen, usa) added was confirmed by constructing a dose-response curve, which was used for subsequent determination. plasma samples were treated with trypsin and kept at °c, ph . , for min for activation, after which trypsin inhibitor was added for min at room temperature to terminate the activation. the levels of renin activation before and after the addition of trypsin were measured, and the pra level of each sample was the total renin level by trypsin activation minus the renin level before trypsin addition. western blot analysis proteins were extracted from kidney tissues, and the protein concentration of each sample was measured. by adding lysate, loading buffer, and ddh o, the protein concentration and volume of each sample were equal, and all samples were boiled at °c for min for storage. gel electrophoresis was performed in a sodium dodecyl sulfate-polyacrylamide system, and gel transfer was performed using polyvinylidene fluoride membranes. the membranes were then immersed in blocking buffer for h at room temperature and incubated overnight at °c in primary antibodies against angiotensin-converting enzyme (ace), angiotensinogen (agt), ang ii, and ang ii type receptor (at ) (santa cruz, usa). after washing with . % tris buffered saline and tween, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies for h at room temperature. protein expression was observed using the ecl system (bio-rad, usa). statistical analysis all data were processed using spss . (ibm, usa). measurement data were expressed as the mean ± standard deviation (sd), t-test was used for statistical comparisons among the data of three groups, and the spearman correlation test was used for correlation analysis. p values < . were considered statistically significant. general parameters of the three groups compared with the controls, the bg level of the dm group was significantly elevated, confirming that the stz-induced diabetic model was established. compared with the dm group, treatment with antibiotics caused an obviously reduced bg level in the dm + ab group (fig. a) , suggesting that gut microbiota might be closely related to the body's high bg levels under diabetic conditions. the plasma insulin level in the three groups showed an opposite trend compared with that of bg (fig. b) . changes in gut microbiota in the three groups the results of s rdna gene sequencing showed that there were significant differences in the bacterial composition and abundance of gut microbiota between the control and dm groups. after the application of broad-spectrum antibiotics, most of the gut microbiota in the dm + ab group was killed (fig. a) . the subgroup sequencing analysis results of gut microbiota in each group have shown that the abundance of blautia, roseburia, and paraprevotella in the colon of the dm group was significantly increased compared with that of the controls (fig. b-d) , while the abundance of bacteroides was relatively decreased, suggesting that the composition and abundance of gut microbiota have both changed under diabetic status. the results of the dm + ab group further confirmed the bactericidal effect of the treatment with broad-spectrum antibiotics (fig. e) . treatment with antibiotics significantly reduced the level of plasma acetate the results of gas chromatography analysis demonstrated significantly increasing plasma levels of acetate in the dm group, which might be due to the abnormalities of gut microbiota under diabetic conditions. antibiotic intervention significantly lowered the plasma acetate level in the dm + ab group (fig. ) . the effect of gut microbiota on renal injury of incipient dn compared with the controls, the ratio of kidney weight-to-body weight in both the dm group and the dm + ab group was significantly higher (fig. a) , indicative of renal hypertrophy under the state of diabetes. in terms of proteinuria, we observed a significantly increasing level of -h urine protein in the dm group compared with that of the control group. after the antibiotic intervention, the dm + ab group had a significant reduction in -h urine protein compared with that of the dm group (p < . ) (fig. b) . in addition, compared with the control group, the level of bun increased in both the dm group and the dm + ab group (p < . ), but there was no significant difference in blood creatinine among the three groups (fig. c, d) , suggesting that this abnormality of gut microbiota has not yet developed to a sufficient degree to exert obvious effects on renal functions. we next observed renal pathological changes to further evaluate the degree of renal injury in the three groups. the results of pas staining showed mild mesangial expansion among the kidneys in the dm group compared with that in the dm + ab group (fig. a, b) . under electron microscopy, ultramicrostructural changes in the glomerular filtration membrane in each group could be observed. the gbm in the dm group was thickened, and there was fusion of fenestrated endothelium, along with partly merged and missing podocyte foot processes, which corresponds to the degree of renal injury in early dn. after the antibiotic intervention, the basement membrane thickening, endothelial fusion, and podocyte injury in the dm + ab group recovered to some extent (fig. c) . we performed wga immunofluorescence staining to observe the changes in glomerular endothelium glycocalyx. under laser confocal microscopy, we found that compared with the control group, the thickness of the glomerular endothelium was significantly reduced in the dm group. in the dm + ab group, the reduced glycocalyx was repaired after the antibiotic intervention (fig. d) . the immunofluorescence staining results also showed significantly decreased expression of glomerular podocyte-specific protein wt- and nephrin in the dm group compared with that in the control group, which was relatively recovered in the dm + ab group (fig. e-g) , suggesting that unbalanced gut microbiota might be a key factor resulting in injuries to the glomerular filtration membrane in early dn. intrarenal ras is activated in early dn the measurement of circulating ras in the three groups showed no significant differences in pra and the level of ang І (fig. a, b) . however, compared with the dm group, the concentration of ang ii in the circulation was significantly reduced (fig. c) . the western blot results to evaluate the degree of intrarenal ras activation showed that compared with the control group, the protein expression of ace, ang ii, and at r in the kidney of the dm group was significantly increased, and antibiotic treatment showed a suppressing effect on these three ras-activating indicators (fig. d, e) . the expression of agt showed an opposite trend, which might be a result of negative-feedback adjustment considering its role as a rate-limiting enzyme. this result suggests that the dysbiosis of the gut microbiota may be involved in the intrarenal ras activation of early dn. correlation analysis between plasma acetate levels and intrarenal ang ii expression to investigate the causal relationship between the dysbiosis of gut microbiota and intrarenal ras activation, we further analyzed the correlation between the plasma acetate concentration and intrarenal ang ii protein expression as determined by western blot (fig. f) . a positive correlation was observed (r = . , p < . ). most kidney diseases are characterized by an initial injury, followed by compensatory growth and associated functional alterations, usually manifested as renal hypertrophy [ ] . stages І and ii of dn are mainly characterized by glomerular hypertension, hyperfiltration, and renal hypertrophy [ ] . renal hypertrophy is an important and significant marker of structural and functional changes in the kidney in early dn, mainly characterized by glomerular cell hypertrophy, thickening of the basement membrane, and an increase in the mesangial matrix. in this study, we confirmed that the experimental animals had developed corresponding renal injury under diabetic conditions by pathological observation. the ras can be divided into the circulatory and local ras depending on its location of synthesis and action. in circulating plasma acetate concentration (µ µmol/l) ras, ang ii is produced under the serial effects of renin secreted by the kidney, angiotensin from the liver, and ace located in vascular endothelial cells. in certain organs or cells, the synthesis of ras components is independent. ang ii can be produced in the intercellular space [ ] under the effect of enzymes other than renin and ace (such as chymase) and exerts many pathophysiological effects by activating at r [ ] . the activation of ras has always been considered an important factor in the development of dn, and local ras seems to be more involved compared with the circulating ras [ ] . the kidney itself contains all the ras components [ ] . high glucose has been reported to promote the production of ang ii, which in turn leads to glomerular hyperfiltration and high permeability, as well as extracellular matrix deposition [ ] . clinical application of ras inhibitors could significantly retard the development of proteinuria in dn patients and lower the incidence of esrd. as a current first-line treatment for dn, the efficacy of ras inhibitors in preventing dn is still very limited. the rising prevalence of dn has suggested that a deeper understanding of the molecular mechanisms underlying dns is required to seek better treatment. the relationship between gut microbiota and many metabolic diseases has been a hotpot for research in recent years. under normal circumstances, firmicutes and bacteroidetes take up a large proportion of the whole microbiota in the gut, while the composition of other phyla varies individually due to several factors, such as genetics, diet, and antibiotic use. however, under pathological conditions, the species and abundance of the host gut microbiota would change significantly, mainly characterized by decreasing normally dominant bacteria and increasing pathogenic bacteria. it has also been shown in this study that compared with the control group, there was microecological dysbiosis of gut microbiota in the intestinal tract of diabetic rats. the application of antibiotic intervention is a common method used in studies of gut microbiota and its role in the development of diseases. in this study, we observed that the intervention of antibiotics affected many aspects of the dn animal model, from basic functions and pathological changes in the kidneys to the amount of products released from gut microbiota. the use of mixed antibiotics has ensured the killing effect of the bacteria in the intestinal tract of experimental animals, and in the meantime, it could also attenuate the renal injury of early dn, including reducing the amount of -h urine protein and mitigating the injury to the glomerular filtration membrane and the pathological changes in the tubules. scfas are a major product of the fermentation of carbohydrates by gut microbiota, and there are three main types: acetate, d the changes in glomerular endothelium glycocalyx were assessed by wga staining (original magnification, × ). e wt- and nephrin protein expression was evaluated by immunofluorescent staining (original magnification, × ). the arrows indicate glomerular endothelium glycocalyx. f quantitation of immunofluorescence staining for wt- . *p < . ; compared with the control; ## p < . compared with dm. g quantitation of immunofluorescence staining for nephrin. ***p < . ; compared with the control; # p < . compared with dm. propionate, and butyrate. the butyrate-producing microbiota is essential in maintaining the balance of the intestinal environment in humans. however, the number of butyrate-producing microbiota in diabetic patients is significantly reduced, while the number of other opportunistic pathogens shows an increasing trend. there is evidence that a butyrate-producing clostridium genus could exert antidiabetic effects by increasing the production of butyrate and upregulating the expression of scfa receptors in the gut [ ] . as a main scfa product of gut microbiota, acetate has demonstrated an intricate effect on the internal environment. it has been reported that acetate is almost undetectable in the blood of germ-free mice [ ] , while the content of acetate in animals with a high-fat diet is significantly increased, indicating that the level of acetate might be a potential indicator of the activity of gut microbiota. this study has shown that antibiotic intervention significantly reduced the relatively higher plasma level of acetate in diabetic rats, considering the alleviated renal lesions after antibiotic intervention. it can be speculated that overproduction of acetate might be adverse to the development of early dn. however, the causal relationship between gut microbiota dysbiosis and the development of dn remains to be elucidated, and more explorations may provide a new perspective and therapeutic target for the future diagnosis and treatment of dn. ras activation has been considered one of the important initiating factors in the early development of dn, yet the exact associations between gut microbiota and ras activation remain to be elucidated. pluznick et al. [ ] found that signals from the gut microbiota, i.e., scfas, could be received by corresponding receptors expressed at renal small arterioles, further regulating the secretion of renin, which was involved in maintaining glomerular pressure. this process could be blocked by antibiotics or knockout of the scfa receptor. considering the characteristics of glomerular hypertension and hyperfiltration in the early stage of dn, the disordered gut microbiota is likely to generate excessive scfas, which bind to corresponding receptors in the kidney and regulate ras, thus promoting the pathological changes in early dn. in this study, we have shown that in the early stage of dn, the expression of ras in the kidney was significantly elevated, indicating ras activation at this stage. after antibiotic intervention, the level of circulating ang ii was lowered, and the expression of ras within the kidney was also significantly weakened, suggesting that there might be a causal relationship between the dysbiosis of gut microbiota and intrarenal ras activation in early dn. therapeutic intervention could be applied to change the composition of gut microbiota, focusing on the preservation of beneficial phyla, to create renoprotective prospects [ ] . intrarenal ras is activated in early dn. plasma renin activity level (a), ang i level (b), and ang ii level (c). the protein expression levels of ras were measured by western blotting (d). the histograms represent the mean ± sd of the densitometric scans of the protein bands normalized to β-actin (e). correlation analysis of plasma acetate levels and intrarenal ang ii expression levels (f) (r = . , p < . ). *p < . compared with the control group, # p < . compared with the dm group. in summary, we have established a dn model to observe the changes in gut microbiota and its metabolite acetate in early dn and to further investigate the association between these changes and ras activation in the kidney to elucidate the underlying mechanism of early renal injury in dn. further exploration of the causal relationship and intricate mechanism of gut microbiota and ras activation in the early development of dn are required to develop new prevention strategies for clinical early dn. idf diabetes atlas: global estimates of diabetes prevalence for and projections for prevalence and control of diabetes in chinese adults upregulation of renal renin-angiotensin system in mouse diabetic nephropathy diabetic nephropathy: the role of inflammation in fibroblast activation and kidney fibrosis immunity and inflammation in diabetic kidney disease: translating mechanisms to biomarkers and treatment targets oxidative stress in diabetic nephropathy with early chronic kidney disease role of the intrarenal renin-angiotensin system in the progression of renal disease signals from the gut microbiota to distant organs in physiology and disease unraveling the environmental and genetic interactions in atherosclerosis: central role of the gut microbiota microbiota and diabetes: an evolving relationship a metagenome-wide association study of gut microbiota in type diabetes gut metagenome in european women with normal, impaired and diabetic glucose control diversity of human colonic butyrateproducing bacteria revealed by analysis of the butyryl-coa:acetate coatransferase gene differential adaptation of human gut microbiota to bariatric surgery-induced weight loss: links with metabolic and low-grade inflammation markers transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome gut microbiota in human adults with type diabetes differs from nondiabetic adults gut microbiota in children with type diabetes differs from that in healthy children: a case-control study toward defining the autoimmune microbiome for type diabetes selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia the neuropharmacology of butyrate: the bread and butter of the microbiota-gutbrain axis? regulation of inflammation by short chain fatty acids short-chain fatty acids activate gpr and gpr on intestinal epithelial cells to promote inflammatory responses in mice acetate mediates a microbiome-brain-beta-cell axis to promote metabolic syndrome olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation immunocytochemical localization of na+ channels in rat kidney medulla plasma and renal renin concentrations in adult sheep after prenatal betamethasone exposure progression of renal disease and renal hypertrophy the stages in diabetic renal disease. with emphasis on the stage of incipient diabetic nephropathy the intracrine renin-angiotensin system pleiotropic at receptor signaling pathways mediating physiological and pathogenic actions of angiotensin ii angiotensinconverting enzyme amplification limited to the circulation does not protect mice from development of diabetic nephropathy paracrine regulation of the renal microcirculation the intrarenal renin-angiotensin system and diabetic nephropathy anti-diabetic effects of clostridium butyricum cgmcc . through promoting the growth of gut butyrate-producing bacteria in type diabetic mice gut microbiota in cardiovascular health and disease key: cord- - nkzp z authors: turk, can; turk, seyhan; temirci, elif sena; malkan, umit yavuz; haznedaroglu, İbrahim c. title: in vitro analysis of the renin–angiotensin system and inflammatory gene transcripts in human bronchial epithelial cells after infection with severe acute respiratory syndrome coronavirus date: - - journal: j renin angiotensin aldosterone syst doi: . / sha: doc_id: cord_uid: nkzp z introduction: severe acute respiratory syndrome coronavirus (sars-cov- ) is a recently identified coronavirus family member that triggers a respiratory disease similar to severe acute respiratory syndrome coronavirus (sars-cov). sars-cov and sars-cov- are very similar to each other in many respects, such as structure, genetics, and pathobiology. we hypothesized that coronaviruses could affect pulmonary tissues via integration with the critical immune genes after their interaction with renin–angiotensin system (ras) elements. the aim of the present bioinformatics study was to assess expression changes of the ras and non-ras genes, particularly immune response genes, in the lung epithelial cells after infection with sars-cov. methods: linear regression, hierarchical clustering, pathway analysis, and network analysis were performed using the e-geod- data set. results: the whole-genome expression data of the lung epithelial cells infected with sars-cov for , , and hours were analyzed, and a total of ras family and immune genes were found to be highly correlated with the exposure time to the virus in the studied groups. conclusion: ras genes are important at the initiation of the infections caused by coronavirus family members and may have a strong relationship with the exchange of immune genes in due course following the infection. coronaviruses (cov) are the enveloped rna viruses that can cause enzootic fatal infections primarily in birds and mammals. [ ] [ ] [ ] [ ] moreover, cov has the ability to infect humankind. [ ] [ ] [ ] the presenting symptoms and signs of the infection could be chills, night sweats, persistent cough, diarrhea, and fever. however, the viral infection could cause a wide variety of clinical presentations, ranging from upper respiratory infections (uri) to lower respiratory infections (lri), including bronchitis, pneumonia, and severe acute respiratory syndrome (sars). cov affects the respiratory system and specifically the lungs. , , furthermore, the viral infection can also affect the gastrointestinal tract, as well as the neurological and hepatic systems. the severe acute respiratory syndrome (sars) epidemic in and the middle east respiratory syndrome (mers) in revealed that cov can be fatal when they cross the species barrier and infect humans. a newly identified cov called sars-cov- (formerly entitled ncov) appeared within the last month of in the city of wuhan, china. very early findings suggested that the virus passed from a specific animal species to humans. however, some studies then revealed that sars-cov- could also be passed from person to person, leading to serious respiratory diseases such as acute respiratory distress syndrome similar to that seen in the sars-cov infection. , sars-cov- is an enveloped, singlestranded, positive-sense rna (beta-) cov. the same as sars-cov and mers-cov, the sars-cov- genome can encode structural proteins such as the spike glycoprotein, non-structural proteins, and accessory proteins. the spike glycoprotein, in which this virus has encoding functions, plays an important role in the interactions of the virus and the cell receptor during viral entry. more specifically, a spike protein that is associated with the envelope of sars-cov- binds first to the host receptor and then mediates the entry of the cov into the host cells by fusing viral and host membranes. [ ] [ ] [ ] once the fusion has taken place, the rna virus begins to replicate its genome in the cell and makes new virions that will be secreted to infect other cells. [ ] [ ] [ ] there is a clear pathobiological association between cov and the renin-angiotensin system (ras). sars-cov (a member of the cov family that emerged in ), which is very similar to sars-cov- , and angiotensin converting enzyme (ace ) interactions have been extensively studied. , there is a fundamental atomic interaction between the spike protein receptor binding domain of the virus and the host receptor of ace . sars-cov infections lead to ace down-regulation by the binding of the sars-cov spike protein to ace . thus, the sars-cov family, including sars-cov- , utilizes ace as a critical receptor to enter target host cells. , the main task of ace is to hydrolyze angiotensin ii (ang ii) to form angiotensin ( - ) (ang-( - )) that binds to the g-coupled protein receptor mas to antagonize ang ii-mediated cellular effects. this function is very important since the increased ang ii levels are estimated to cause increased ace activity. ace is one of the central ras enzymes that regulate blood pressure, fluid/electrolyte balance, and systemic vascular resistance in the tissue ras. ace is also critical for the ras, which functions for the regulation of cell growth/proliferation, inflammation/cytokine production, blood pressure, and as a homeostatic regulator of vascular function. the ras is highly active within the lungs where sars-cov- is estimated to be the primary target organ. ang ii can cause pulmonary vasoconstriction in response to hypoxia, which is important in patients with pneumonia or lung injury. locally increased ang ii production increases vascular permeability, which facilitates pulmonary edema. ace , which plays a role in regulating the ang ii as well as ang-( - ) levels, may be important for those pathobiological lung events. , , ace is an important negative directing factor for the severity of lung injury, and sars-cov spike protein-associated ace inhibition has been shown to contribute to the severity of lung pathologies. in addition, numerous genes are involved within the interactions of ace , the ras, and the pulmonary microenvironment. the activation of the local pulmonary ras can affect the genesis of lung damage through multiple mechanisms, such as increments in vascular permeability and alterations in alveolar epithelial cells. the pathogenesis of the cov is a rather complicated process, which may also include local tissue ras. , the main purpose of this present in silico genomic study was to assess how the expressions of the ras gene family changes after cellular infection with sars-cov in the lung epithelial cell culture. we hypothesized that cov could affect pulmonary tissues via integration with the critical immune genes of the affected cellular microenvironment after their interaction with ras elements. we also aimed to figure out whether these genomic alterations are related to the exposure time of the virus. in order to understand the pathobiology of sars-cov and sars-cov- infection better, since they share similarities within many aspects we examined the gene expression changes of other gene families as well. elucidation of the interactions between sars-cov- and ras genes and the description of the related genomic mechanisms are very important in the fight against this fatal viral global infection. gene expression data of human bronchial epithelial cells treated with sars-cov for , , and hours were obtained from array express (gse ). these data were generated by yoshikawa et al. to characterize the dynamic, spatial, and temporal changes of the gene expression induced by sars-cov using microarray technology. in order to use the obtained data in other targeted analyses, the raw data were normalized by robust multisequence analysis in accordance with the procedure in the affy package in r. these data consist of , genes ( , probe sets). in addition, each gene has three repeated expression data values for , , and hours, respectively. the whole normalized gene expression data of lung epithelial cells infected with sars-cov for , , and hours were compared between different groups in order to determine significantly and differentially expressed ras family genes. the mean value of ras gene transcripts was determined for each group infected with sars-cov for , , and hours. the comparison was made between the -and -hour groups as well as between the -and -hour groups, based on the ras gene transcripts. p-values were calculated for each gene using the t-test. the genes belonging to the ras family with a statistically significant difference among the groups were determined (p⩽ . ). in order to determine the genes whose expression is highly correlated with the exposure time to the virus, we performed linear regression analysis. genes with a standard deviation > . were identified between the groups using microsoft excel ( ). pearson product-moment correlation coefficient values (r) were calculated for these genes. genes with a pearson's r > . were selected for use in the subsequent analyses. in order to determine whether the identified genes could distinguish between the virus and infected groups, to identify the network connections between these genes and to demonstrate the pathways in which these genes were involved, we performed hierarchical clustering, pathway analysis, and network analysis. genes with the highest variation and pearson's r-value between groups were hierarchically clustered using the similarity metric parameter and the euclidean distance gene cluster v . program as a full link. using the david online tool, the pathways related to the genes were determined to elaborate on the pathway data resulting from the analysis further. , in addition, the genemania application in cytoscape was used to indicate the network and pathway relationship between the selected genes and other genes. , among the three groups of human bronchial epithelial cells with different exposure times to sars-cov ( -, -and -hour groups), we identified the changes in expression of the ras gene transcripts as well as the trend in expression of the most differentially expressed genes among groups other than the ras family. a total of seven ras signaling pathway genes (nine probe sets; alanyl aminopeptidase (anpep), ace , angiotensin converting enzyme (ace), insulinlike growth factor receptor (igf r), angiotensinogen (agt), epidermal growth factor receptor (egfr) and membrane metalloendopeptidase (mme)) showed significantly different expression values between the -hour group and the -hour group. comparing the -hour and -hour groups, eight genes (nine probe sets; arginyl aminopeptidase (rnpep), epidermal growth factor receptor (egfr), anpep, neurolysin (nln), ace , igf r, leucyl and cysteinyl aminopeptidase (lnpep) and cathepsin d (ctsd)) were statistically significantly expressed (table ) . four common genes (igf r, anpep, ace , and egfr) in these two separate groups were identified (figure ). figure shows the correlation of the expression and exposure time to the virus of the statistically significant table . the list of the ras family genes whose expression shows a significant difference between -hour vs -hour (a) and -hour vs -hour (b) is depicted. seven of the ras signaling pathway genes showed different expression values in -hour vs -hour group, while of the genes found significantly expressed in the -hour vs -hour group. four genes were found to be common to both groups. as mentioned, these genes consistently showed statistically meaningful expression differences according to exposure time to the virus. four common members of ras genes found in the two groups ( figure ). in order to figure out the role of the non-ras genes, we determined the most variant genes among these three groups. the standard deviation values of genes ( probe sets) were found to be > . . linear regression analysis revealed that all these genes had a pearson's r > . and were highly correlated with the exposure time to the virus (table ) . likewise, all of these genes showed a positive correlation with exposure time to the virus. the correlation figure for each gene that was found to be highly correlated to virus infection exposure time is presented in the supplemental material. in addition, as shown in figure , these genes were able to be clustered for the -, -and -hour groups separately. table shows the pathway analysis of all the significant genes. with the aim of discovering the relationship of these genes with each other and with other genes, a network analysis was performed ( figure ). sars-cov- was found to be mostly similar to the sars-cov at the amino-acid level, despite some differences. based on the phylogenetic analysis on all genomes of various viruses, sars-cov- is in the same beta-cov clone as sars-cov, sars-like bat cov, and mers-cov. sars-cov- has the highest similarity with sars-like bat cov and is less associated with mers-cov. the mechanism of sars-cov and its association with the renin-angiotensin pathway had been defined in previous studies. the first genetic proof of the ace and sars-cov receptor relationship was reported by kuba et al. our results showed that in lung epithelial cells, ace gene expression increased between and hours and remained at the same level between . hierarchical cluster of most variant genes between three groups. as shown, determined genes were able to classify the groups clearly. in the -hour group, these genes show low expression, while in the -hour group, they all show high expression. table . pathways related to non-ras differentially expressed genes. most of non-ras differentially expressed genes were found to be related immune the system and pathways which could be involved during the course of the viral infectious disease. oas ′- ′-oligoadenylate synthetase (oas ) related genes homo sapiens kegg_pathway hepatitis c, measles, influenza a, herpes simplex infection cxcl c-x-c motif chemokine ligand (cxcl ) related genes homo sapiens kegg_pathway cytokine-cytokine receptor interaction, chemokine signaling pathway, toll-like receptor signaling pathway, rig-i-like receptor signaling pathway, cytosolic dna-sensing pathway, tnf signaling pathway, influenza a cxcl c-x-c motif chemokine ligand ace protein level in the lungs of sars-cov-infected mice on day . they also reported that the addition of sars-cov spike into mice exacerbates acute lung failure in vivo that can be reduced by inhibiting the renin-angiotensin pathway. moreover, sars-cov- uses a similar mechanism as sars-cov. the structural analyses showed that like sars-cov, sars-cov- also utilizes ace as the host receptor. based on the evidence with regard to the similarity between sars-cov and sars-cov- , we used data from yoshikawa et al. in their study, all the genome expression data of the confluent b cells which were infected with sars-cov and grown in t- flasks for , and hours were used to characterize the dynamic, spatial and temporal changes of the gene expressions caused by sars-cov. the study was performed three times at each time point in order to meet the minimum number required for the application of statistical algorithms, and a total of nine arrays were given for sars-cov. our current study was carried out to understand the biological mechanism of sars-cov- infection better by focusing on the similarity of sars-cov- and sars-cov in terms of structure, biological function, and the pathology of the infection. we proposed a hypothesis based on the genomic results obtained in the present study under three key headings ( figure ). according to the results of the analysis, there was a significant up-regulation of ace and anpep genes in human bronchial epithelial cells within and hours of infection. many previous studies indicated that sars-cov also uses ace as a receptor to enter host cells, as in sars-cov- . the results of our present study showed the additional possible function of anpep ( figure ). the anpep gene acts as a receptor, especially for hcov- e, another member of the cov family. this virus is predicted to induce the infection by triggering conformational changes in the spike glycoprotein, which interacts with the host anpep receptor and activates membrane fusion. moreover, anpep (cd ) can act as a receptor in sars-cov and induce growth inhibition and apoptosis by infecting hematopoietic stem/progenitor cells. these findings are in concordance with our results demonstrating the up-regulation in the ace and anpep as exposure time to the virus increased. as suggested in many previous studies, ace forms ang-( - ) from ang ii and binds to the mas receptor, the specific receptor of ang- ( - ) , and then inhibits inflammatory, vascular and cellular growth mechanisms. in this case, it may be useful to use the agonistic peptides of ang-( - ) to change the functioning of ace in favor of the infected host in the initial phase of infection. based on our results, in this phase, as the exposure time to sars-cov increases, egfr and igf r, two receptors with key roles in the ras signaling pathway, were significantly down-regulated in the infected human bronchial epithelial cells. habib et al. proposed that apoptosis occurs in various cell types that require an active tyrosine kinase but do not require egfr autophosphorylation sites by experimentally increasing the level of egfr expression. the expression of a predominant negative ras mutant in cells that over-express the egfr leads to significantly enhanced egfr-induced apoptosis. on the other hand, igf r, acting as a g-coupled protein receptor, could lead to the activation of the mitochondria-mediated apoptosis pathway, when insulin-like growth factor-ii (igf ) binds to this receptor. thus, those two receptors, which are associated with cellular apoptosis, are expected to be downregulated as the infection time of virus-infected cells progresses. this event could give the chance for the virus to have sufficient time to continue replication and increase its copy number. this phase of the viral infection might be important, particularly for hypertensive subjects. in this in silico genomic study, genes ( probe sets) were detected which showed noteworthy up-regulation in -versus -hour groups. almost all of these are genes associated with the immune system, innate immune response, and adaptive immune response. the relevant biological analyzers of these genes whose expressions are concordant with cell exposure time to the virus were explored via the pathway analyzers of the genes. the genes involved in specific pathways related to the critical pathological events of cov infection, including sars-cov and sars-cov- , such as the toll-like receptor (tlr) signaling pathway. tlrs are essential sensor molecules of the host innate immune system. various tlrs are involved in early interactions of invasive viruses and host cells, which affect viral pathogenesis and regulate viral replication as well as host responses. initiation of antiviral immune responses with tlr agonists has been shown to provide protection from many different viruses, including hepatitis b virus, influenza virus, some hiv strains, and cov. furthermore, non-structural protein of the sars-cov interacts with irf through the papain-like protease domain. after that, the associations of the tlr and rlr pathways occur, and this binding prevents the nuclear translocation as well as the phosphorylation of irf . it has previously been observed that he tlr signal through the trif adapter protein protects mice from lethal sars-cov. thus, a balanced immune response that works both in trif and myd -guided pathways provides the most effective host-cell intrinsic antiviral defense responses against severe sars-cov, and the absence of any branch of tlr signaling leads to sars-cov being fatal. another important pathway is associated with the interferon beta (ifnb ) gene identified as a result of pathway analysis is the natural killer (nk) cell-mediated cytotoxicity pathway. interferons are considered as the screams of the affected cells during the active virus attack. ifnb deficiency results in partial suppression of the sterol pathway in macrophages during viral infections, thereby associating the regulation of the lipid metabolism pathway with interferon antiviral defense responses. furthermore, nk cells are important in immune defense against virus infections. enlarged virus replication and more serious diseases during encephalomyocarditis virus, coxsackie virus, and theiler's murine encephalitis virus infections are linked with the reduction of nk cells or low levels of nk cell cytolytic function. nk cells are correspondingly associated with direct inhibition of virus replication and stimulation of liver damage during mouse hepatitis virus (mhv) infection. it is not known whether nk cells play a direct antiviral role in human infections with picornaviruses or cov such as sars. lysosome, which has an important path in cov infection, is another pathway related to the pathway analysis performed. enveloped viruses must fuse with a host-cell membrane so as to transport their genomes to the host cell. although some viruses fuse with the plasma membrane, many viral infections are associated with endocytosis before fusion. in the endosomal microenvironment, a particular marker induces conformational changes in viral fusion proteins, leading to viral and host membrane fusion. according to the de haan et al. study, the proteins known to be important for late endosomal maturation and endosome-lysosome fusion deeply promoted infection of cells with mouse hepatitis cov (mhv). in another study of sars-cov, sars-cov accessory protein open reading frames (sars a) were oligomerized by dynamically inserting them into late endosomal, lysosomal and trans-golgi network membranes. furthermore, the chemotactic cytokines are engaged in a great deal of biological processes. besides microbial infection, the infected cells have strong chemokine signals. those signals could play significant roles in both innate and adaptive immune responses that control the growth of the invading pathogen. therefore, the presence of chemokine signaling pathways parallels the other results obtained. the main limitation of this study is the lack of uninfected control data, which is subject to further experimental studies. in conclusion, if the results obtained in the current study are validated by in vitro experiments and clinical samples, it can be suggested that the disruption of ras genes may be important for the initial management of the cov infections, particularly sars-cov- . the results of the current study help us to understand better the pathobiology of sars-cov and other similar cov family members such as sars-cov- . based on our results, the interactions between the sars-cov- and ras genes, resulting in immune-related genomic disruption, lead to acquired immune deficiency states. it is hoped that critical local ras-affecting drugs such as mas agonists, soluble ace and ang-( - ) will be used for the modulation of sars-cov- and ras pathological interactions for the improvement of immune genomic states in patients infected with sars-cov- in future trials. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. supplemental material for this article is available online. seyhan turk https://orcid.org/ - - - elif sena temirci https://orcid.org/ - - - umit yavuz malkan https://orcid.org/ - - - coronavirus infection in equines: a review bat coronaviruses in china coronaviruses: an overview of their replication and pathogenesis clinical features of patients infected with novel coronavirus in wuhan, china the novel coronavirus: a bird's eye view coronaviruses and the human airway: a universal system for virus-host 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gene function genome composition and divergence of the novel coronavirus ( -ncov) originating in china the novel coronavirus ( -ncov) uses the sars-coronavirus receptor ace and the cellular protease tmprss for entry into target cells. biorxiv. epub ahead of print diarrhea may be underestimated: a missing link in novel coronavirus. gut. epub ahead of print hematological findings in sars patients and possible mechanisms angiotensin-( - ) and mas receptor axis in inflammation and fibrosis epidermal growth factor receptor induced apoptosis: potentiation by inhibition of ras signaling creb negatively regulates igf r gene expression and downstream pathways to inhibit hypoxia-induced h c cardiomyoblast cell death toll-like receptors in antiviral innate immunity toll-like receptor signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection metabolic regulators nampt and sirt serially participate in the macrophage interferon antiviral cascade roles of natural killer cells in antiviral immunity physiological and molecular triggers for sars-cov membrane fusion and entry into host cells coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner sars-coronavirus open reading frame- a drives multimodal necrotic cell death chemokine cxcl and coronavirus-induced neurologic disease key: cord- -mwu q w authors: dent, paul title: cell signaling and translational developmental therapeutics date: - - journal: reference module in chemistry, molecular sciences and chemical engineering doi: . /b - - - - . - sha: doc_id: cord_uid: mwu q w the relationships between drug pharmacodynamics and subsequent changes in cellular signaling processes are complex. many in vitro cell signaling studies often use drug concentrations above physiologically safe drug levels achievable in a patient's plasma. drug companies develop agents to inhibit or modify the activities of specific target enzymes, often without a full consideration that their compounds have additional unknown targets. these two negative sequelae, when published together, become impediments against successful developmental therapeutics and translation because this data distorts our understanding of signaling mechanisms and reduces the probability of successfully translating drug-based concepts from the bench to the bedside. this article will discuss cellular signaling in isolation and as it relates to extant single and combined therapeutic drug interventions. this will lead to a hypothetical series standardized sequential approaches describing a rigorous concept to drug development and clinical translation. the field of cell signaling and signal transduction dates back to the late th century. in , epinephrine (adrenaline) was discovered. by the s, insulin and glucagon had been discovered. , collectively, these discoveries paved the way for researchers to explore how these hormones acted to regulate glucose metabolism in the liver and skeletal muscle. the laboratory of dr. carl cori played a seminal role in partially unravelling how glycogen could be broken down by glycogen phosphorylase. he, his wife gerty and bernardo houssay received the nobel prize in physiology or medicine for their work. although the cori laboratory had discovered and described glycogen phosphorylase, it was not until that leloir discovered the enzyme that made glycogen, glycogen synthase. during the s, fischer, krebs and sutherland not only discovered and characterized the kinase which a dedicated to professor sir philip cohen on the occasion of his th birthday. comprehensive pharmacology https://doi.org/ . /b - - - - . - regulated glycogen phosphorylase, phosphorylase kinase, but defined for the first time that the phosphorylation of proteins could regulate enzyme activity. [ ] [ ] [ ] [ ] further development of signal transduction up until the late s, however, no-one had been able to elucidate how insulin signaled to make a cell store glucose as glycogen nor how epinephrine and glucagon activated phosphorylase kinase/glycogen phosphorylase to break down glycogen. sutherland and colleagues during their investigations into glycogen phosphorylase discovered a heat-stable factor in liver sections whose levels were regulated by epinephrine and glucagon: cyclic amp, the first second messenger. [ ] [ ] [ ] subsequently, fischer and krebs isolated the kinase regulated by camp, protein kinase a (pka). for these discoveries, sutherland, as well as fischer and krebs, received the nobel prize. sutherland, fischer, and krebs during their studies also discovered an enzyme activity which could remove phosphate from glycogen synthase, i.e. a protein phosphatase. a postdoctoral researcher from the laboratory of fischer in the late s, philip cohen, focused their independent career upon characterizing the many protein phosphatases in cells and above all understanding how phosphatases regulated glycogen metabolism, naming the ser/thr protein phosphatases. over the or so years after the discovery of pka, multiple additional small molecule second messengers were discovered including: calcium ions, diacyl glycerol and ip ; and nitric oxide and cyclic gmp (cgmp). [ ] [ ] [ ] [ ] signaling by cgmp in the eye was shown to be essential for the perception of light and cgmp as well as with nitric oxide in the regulation of smooth muscle contractility resulted in the nobel prize being awarded to murad in . , during the s and s work by lefkowitz, gilman and johnson led to the discovery of serpentine plasma membrane receptors for hormones, e.g. the beta-adrenergic receptor for epinephrine, as well as receptor-associated large gtp binding protein complexes on the inner leaflet of the plasma membrane which transduced receptor signals to intracellular effectors such as: ( ) adenylyl cyclase leading to the generation of camp; ( ) activation of phospholipases leading to the generation of diacyl glycerol and inositol , , -trisphosphate (ip ), with ip triggering the release of calcium ions into the cytosol. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diacyl glycerol and calcium ion then activated multiple protein kinase c (pkc) isoforms. this resulted in the award of additional nobel prizes. serpentine g-protein coupled receptor (gpcr) signaling can be down-regulated by proteins called arrestins. [ ] [ ] [ ] arrestin proteins prevent both the ga gbg proteins interacting with the gpcr and cause the gpcr to be internalized. internalization can result either in receptor degradation or recycling back to the plasma membrane. thus, by the mid-to late- s a large body of literature existed which argued that signal transduction pathways consisted of a receptor linked to a large gtp-binding protein which in turn regulated an enzyme that generated "second messengers;" the second messengers would then diffuse throughout the cytosol activating cellular processes, predominantly for metabolism. in parallel to the study of serpentine receptors, other investigators were focused on the relatively few proteins who became phosphorylated on tyrosine. studies in this field were focused on the insulin receptor (metabolism) and the epidermal growth factor receptor (egfr, erbb ) (growth, cancer). [ ] [ ] [ ] [ ] insulin caused the insulin receptor to become tyrosine phosphorylated, and a substrate for the receptor, insulin receptor substrate (irs ), was discovered. for many years prior to the s, diagrams of insulin receptor signaling would include the receptor and irs , together with downstream insulin targets such as glycogen synthase. in-between the receptor and synthase was drawn a "black box" as the pathway by which insulin regulated glycogen synthase appeared to be intractable to investigation. studies by the laboratory of larner and villar-palasi argued that insulin caused the generation of a "mediator" second messenger which was an inositol phospholipid, that regulated glycogen synthase. [ ] [ ] [ ] although at the time this concept was not widely supported, subsequent studies over the following years demonstrated that insulin activated phosphatidyl inositol -kinase whose product, phosphatidylinositol ( , , )-trisphosphate (pi( , , )p ), caused activation of the membrane-associated kinase, phosphoinositide-dependent kinase- (pdk ). [ ] [ ] [ ] [ ] [ ] pdk was shown to phosphorylate akt t causing enzyme activation, and akt to phosphorylate and inactivate glycogen synthase kinase (gsk ). reduced gsk activity results in reduced glycogen synthase phosphorylation, leading to activation of synthase activity. one additional component within this process was activation of protein phosphatases to facilitate the dephosphorylation and activation of glycogen synthase. thus, after years of research, by the mid- s, the regulation of glycogen metabolism by epinephrine and insulin had largely been elucidated. for the egfr and other subsequently discovered membrane associated tyrosine kinases, e.g. the non-receptor scr family and the fibroblast growth factor receptor (fgfr) family, understanding how these enzymes signaled into the cell again initially rested on studies using traditional biochemical methods. [ ] [ ] [ ] [ ] in the mid- s, a postdoctoral researcher in the laboratory of dr. ora rosen, thomas sturgill, was given a project in which he was to identify a kilo dalton protein whose tyrosine phosphorylation was increased after exposing cells to insulin. as an independent investigator sturgill continued his studies into the enzyme he called map -kinase, microtubule associate protein (map ) being the substrate used to measure its kinase activity. it was subsequently renamed to be "mitogen activated protein kinase" (mapk) after it was discovered to not only be regulated by many growth factors, but also that it was an intermediary kinase in the regulation of another insulin-activated kinase p ribosomal s kinase (p rsk). this enzyme should not be confused with p s kinase with is a component of the pi k pathway. , by the end of the s, it had been determined that there was another mapk isoform (p ) and that these kinases were regulated by tyrosine/threonine joint phosphorylation. , at that time, kinases were considered to be specific for either serine/threonine or for tyrosine. the discovery of mek and mek (mitogen/extracellular regulated kinase), kinases that phosphorylated the mapks on both tyrosine and threonine was considered biochemically novel. [ ] [ ] [ ] [ ] from work in yeast (cerevisiae, pombe), however, was in parallel demonstrating that they also expressed mapk-like and mek-like enzymes, and that their mek-like enzymes phosphorylated the mapk-like enzymes on tyrosine and threonine. [ ] [ ] [ ] the mammalian mapk/renamed erk / (extracellular regulated kinase) pathway in yeasts regulates the yeast response to pheromones. this understanding facilitated the further characterization of mek and mek . the next question in the development of the "mapk pathway" was to define the kinase(s) upstream of mek / . based on data from yeasts, this kinase should have been similar to the mammalian map k, known as mekk (mitogen/extracellular regulated kinase kinase). however, in , two groups linked c-raf- and its truncated oncogenic variant v-raf as the kinase activity which enhanced mek / phosphorylation and activity; there are no yeast homologues of the raf family proteins. , of note, prior to those studies it was believed that raf- was downstream of erk / . the function of mekk subsequently, and with its family members, was linked in mammalian cells to the regulation of the c-jun nh -terminal kinase (jnk / ) and p mapk pathways. [ ] [ ] [ ] [ ] [ ] [ ] contemporaneously with these studies, researchers were determining how receptor tyrosine kinases regulated ras family small gtp binding proteins, and other groups determining how ras proteins signaled downstream off the plasma membrane and into the cytosol. [ ] [ ] [ ] [ ] [ ] it was demonstrated that the proteins grb (growth factor receptor-bound protein ) and sos (son of sevenless homolog ) linked receptor tyrosine phosphorylation to the exchange of gtp for gdp in ras proteins. within months of these discoveries being published, it was shown that gtp-bound ras would associate with the nh -terminal domain of raf- . - gdp-bound ras proteins did not associate with raf- . thus, within the period between and , the first of the "map kinase pathways" had been delineated. because of extant data from yeasts, other parallel mammalian map kinase pathways were rapidly discovered and delineated. for example, as mentioned previously, the p mapk pathway in mammalian cells is a stress-induced signaling pathway and was the equivalent of the hog osmo-sensing pathway in yeasts. the jnk pathway has similarities to several yeast and mammalian mapks, but only a % best-fit to erk and erk . it was discovered as a uv-activated kinase that bound to the nh -terminus of the transcription factor c-jun. a parallel mapk pathway, the erk "big map kinase pathway" was discovered and inhibitors of mek / also inhibit mek , demonstrating the close functional alignment of both pathways. hence, by the mid- s the basic structures of multiple map kinase as well as the pi k pathway were in place. broadly, over the past years, signaling by erk / and erk were most often linked to tumor cell growth whereas signaling by p mapk and jnk were linked to cell death. [ ] [ ] [ ] however, coordinated erk/jnk signaling strongly promoted growth and under prolonged high activity erk / signaling would cause growth arrest via the induction of cyclin dependent kinase inhibitor proteins or tumor cell death. [ ] [ ] [ ] these were also reflected at the level of receptor tyrosine kinases, comparing different ligands for the same receptor with different on-/off-rates, e.g. egf and tgfa, as well as associated with ligand concentration. high ligand levels permanently downregulate the receptor, and ligands such as egf that remain with the receptor in endosomes cycle the receptor for degradation. [ ] [ ] [ ] [ ] signaling by p mapk regulated chaperone functions but also could cause cell cycle arrest and dna damage repair. , what also became readily apparent was that activation of the same pathway to the same extent in different tumor cells could result in different changes in tumor cell biology, with some cells exhibiting growth/growth arrest and other cells becoming moribund either through apoptosis, necrosis or autophagy. [ ] [ ] [ ] some of these behaviors could in part be explained due to the differential expression of driving oncogenes such as mutation of p , ras proteins, receptor tyrosine kinases or the lipid phosphatase: phosphatase and tensin homologue on chromosome ten (pten). - the cellular process of autophagy was discovered in the s. , the primary purpose of the process is to recycle cellular components into their elemental building blocks during times of metabolic stress, permitting the cell to survive. materials are first encapsulated in a double membrane, called an autophagosome. [ ] [ ] [ ] autophagosomes fuse with lysosomes, the interior acidifies, and they become autolysosomes where materials are digested, ready for recycling. the regulation of autophagy and with it the sensing of nutrient and atp energy levels within a cell are regulated by mammalian target of rapamycin (mtor) and the ampdependent protein kinase (ampk), respectively. [ ] [ ] [ ] [ ] [ ] the regulation of mtor is complex as it integrates upstream signaling from akt in the pi k pathway, together with other signals that sense amino acid, lipid and carbohydrate levels. there are two complexes of proteins which associate with mtor, with the kinase being termed mtorc or mtorc based on the members of the protein complex. [ ] [ ] [ ] the ampk senses amp levels, which are high when the cell is depleted of atp; high amp levels cause allosteric activation of the ampk, and activated ampk then acts to phosphorylate and inactivate mtor. [ ] [ ] [ ] the ampk is itself regulated by phosphorylation, with the most notable regulators being liver kinase b (lkb ) and ataxia-telangiectasia mutated (atm). [ ] [ ] [ ] [ ] [ ] [ ] lkb is often mutated in tumor cells, leading to dysregulation of energy sensing and autophagy regulation. in the nucleus atm senses dna damage and cytosolic atm senses the levels of reactive oxygen species; atm at both cellular locations phosphorylates and activates the ampk. the key regulatory target for both mtor and the ampk is the kinase unc- like autophagy activating kinase (ulk / ). - ulk is a classic example of a protein whose function is regulated by multi-site phosphorylation. phosphorylation of ulk at specific sites by mtor inactivates the kinase. phosphorylation of ulk at different specific sites by the ampk activates the kinase. [ ] [ ] [ ] the primary substrate of ulk is the gate-keeper protein for autophagosome formation, atg . phosphorylation of atg leads to the formation of multi-protein complexes which act to form a double membrane around the cellular materials that will be digested. autophagic flux occurs where a fully-formed autophagosome fuses with an endosome/lysosome to form an autolysosome. , autolysosomes acidify their interior, activating a variety of proteases and other enzymes required to break down the vesicle's contents. many tumor cells exquisitely rely on autophagy to survive, which explains why drugs such as chloroquine, which prevent autophagosome lysosome fusion, have been trialed as cancer therapeutics. , alternatively, as tumor cells utilize autophagy for survival, drugs which profoundly stimulate autophagosome formation and autophagic flux cause the over-digestion of cellular proteins and cause the cytosolic release from the autolysosome of active proteases, which collectively leads to a multi-factorial form of tumor cell death. using our understanding of autophagy and cell signaling to therapeutically kill tumor cells in all scientific studies, experiments should be performed from an agnostic standpoint. that is, follow the data wherever it may lead, regardless of prior opinions or perceptions. twenty years ago, in collaboration with dr. paul fisher, we began to investigate the molecular mechanisms by which the cytokine il- acted to kill tumor cells. [ ] [ ] [ ] at that time, the mechanisms by which tumor cells died were not particularly sophisticated, with death receptor signaling via caspases / (the extrinsic apoptosis pathway) and mitochondrial dysfunction via caspase (the intrinsic apoptosis pathway) being the two pathways then considered most important in the causation of tumor cell death. because we had observed the cytokine was inactivating mtor, studies were performed to define if "autophagy" played any role in the cytokine's biology. molecular knock down of key autophagy regulatory proteins, atg or beclin , profoundly suppressed il- lethality. our studies with autophagy and il- resulted in other laboratory projects exploring the role of autophagy in their biology and killing mechanisms. for example, in hepatoma cells, the combination of the multi-kinase inhibitor sorafenib with the histone deacetylase (hdac) inhibitor vorinostat killed cells by activating the death receptor cd , and in hepatoma cells, knock down of atg or beclin enhanced drug combination lethality, i.e. autophagy was acting as a protective cellular response. however, in pancreatic cancer cells, knock down of atg or beclin significantly reduced the ability of this drug combination to kill, i.e. autophagy played a role in the killing process. subsequent studies in the laboratory over the past decade have almost invariably discovered that autophagosome formation was playing an essential role in the tumor cell killing process. one consideration when discussing the role of autophagy in causing cell death is whether the autophagic process caused killing directly, or indirectly by causing, e.g. mitochondrial dysfunction, followed by release of cytochrome c and apoptosis inducing factor (aif) into the cytosol. aif moves to the nucleus to cause dna fragmentation in a fashion similar to necrosis. cytochrome c binds to apoptotic protease activating factor (apaf- ) which together with atp causes the cleavage of pro-caspase . activated caspase cleaves and activates caspase , which moves to the nucleus to cause apoptotic dna fragmentation, with dna fragments encapsulated in membranes. alongside the apoptotic processes, cathepsin proteases released from autolysosomes can cleave and activate the pro-apoptotic protein bid that is upstream of mitochondria, and which will lead to mitochondrial dysfunction and death. however, it is possible that release of activated proteases by themselves into the cytoplasm can also cause death, without involvement of the mitochondria. we will now illustrate in more detail the role of autophagy in the development of anti-cancer therapeutics and in the development of anti-viral therapeutics. the multi-kinase inhibitor drugs sorafenib and pazopanib are approved for the treatment of liver/kidney cancers and soft tissue sarcoma, respectively. , for both drugs, we demonstrated that they synergized with hdac inhibitors to kill liver, kidney, pancreatic and sarcoma tumor cells. [ ] [ ] [ ] [ ] contemporaneously with these studies, we were also studying the celecoxib derivative developmental drug, osu- . originally osu- was proposed to inhibit pdk within the pi k/akt pathway. , osu- has an order of magnitude anti-cancer efficacy than the parent compound. the key, arguably single, mechanism by which we found osu- acted to kill tumor cells was by causing the generation of autophagosomes followed by autophagic flux and the cytotoxic actions of autolysosome localized proteases such as cathepsin b. ultimately, we determined that osu- was an inhibitor of chaperone proteins, in particular grp . grp is an endoplasmic reticulum (er) localized chaperone that plays an essential role in regulating er stress signaling during times of protein overload and protein denaturation. as we compared the chemical structures of osu- , pazopanib and sorafenib we realized that had many similarities. compared to osu- which had ic values of inhibiting the atpase activities of hsp and hsp in the and nm range, respectively, the chaperone inhibitory activities of sorafenib were found to be similar, and the inhibitory activity of pazopanib significantly stronger with ic values of and nm, respectively. [ ] [ ] [ ] [ ] [ ] thus, drugs that had been developed and marketed as "multi-kinase inhibitors" for many years also had multiple unknown chaperone targets. hence, just because a drug company states on their packaging that a drug inhibits enzymes a, b and c to cause a therapeutic effect, does not mean that the drug also inhibits unknown enzymes y and z. furthermore, it is probable that without inhibition of y and z, the inhibition of a, b and c together will only have a modest therapeutic effect. in the case of osu- , despite a phase i trial in cancer patients (nct ), further studies with drug took an unexpected turn away from cancer therapeutics, and towards infectious disease and the development of the drug as an anti-viral agent. , all human pathogenic viruses require cells express functional grp . , in a virus-dependent manner, different viruses also recruit other additional chaperone proteins to facilitate their replication and life cycle. , osu- is not a highaffinity inhibitor of a single chaperone or chaperone family, unlike many chaperone inhibitors developed for use in the cancer therapeutics field. , however, because the drug inhibits multiple hsp family and hsp family chaperones within its clinically relevant safe concentration range, osu- could potentially become a broad spectrum anti-viral drug. osu- prevented the reproduction of viruses including mumps, influenza, measles, coxsackie virus b , junín, rubella, west nile, yellow fever, hiv (wild type and protease resistant), and ebola, effects that were replicated by molecular knock down of multiple chaperone proteins, alone or in combination. very recently we discovered, to some extent not surprisingly, that osu- could also prevent synthesis of the sars-cov- spike protein. in three separate animal model systems, rabbit hemorrhagic fever virus, zika and dengue osu- prolonged animal survival and significantly reduced the negative sequelae of virus infection. [ ] [ ] [ ] subsequent studies using the fda approved cancer therapeutic drugs sorafenib and pazopanib also demonstrated that these fda approved drugs also have potent anti-viral properties. thus, a project which began as development of an anticancer drug became a project developing broad spectrum anti-viral drugs. the role of an activating point mutant in the egf receptor was first demonstrated in non-small cell lung cancer (nsclc). [ ] [ ] [ ] [ ] subsequently, as patient tumors carrying the activated egfr were treated for prolonged periods with egfr inhibitors such as gefitinib, it became evident that drug resistance, when it eventually evolved, was mediated by the evolution of a second point mutation in the egfr. , [ ] [ ] [ ] second and third generation egfr inhibitory drugs such as afatinib and osimertinib potently inhibit double mutant egfr and are in first-line clinical use. [ ] [ ] [ ] at the time of these discoveries we had several research projects determining whether we could combine afatinib with other agents to kill nsclc cells. [ ] [ ] [ ] as part of this work, we generated afatinib-resistant h nsclc cells by treating tumors in mice until the tumor completely regressed and then had begun to regrow. h cells already express a double mutant egfr, so we were expecting to discover novel evolutionary survival signals. initial characterization of the resistant cells demonstrated they had permanently up-regulated signaling by the receptors c-kit, c-met and erbb to survive during exposure to afatinib. additional characterization studies then delivered unexpected data; whilst afatinib-resistant h cells were resistant to the irreversible erbb receptor inhibitor afatinib, they were not resistant to the irreversible erbb inhibitor neratinib. furthermore, the ability of neratinib as a single agent or when combined with other drugs, including afatinib, was enhanced in the afatinib-resistant cells. ostensibly, both drugs should mechanistically "do" exactly the same thing to a tumor cell. thus, by implication, in addition to erbb family receptors, neratinib had to have additional "targets" to cause killing in the resistant cells. two molecular modeling manuscripts had stated neratinib, in addition to inhibiting erbb family tyrosine kinases could also inhibit map k and map k serine/threonine kinases. , in parallel to the studies described above, from our loading control data, we observed that neratinib but not afatinib, could rapidly reduce the protein expression of erbb family receptors in a wide variety of tumor cell types. , , we also included negative controls in our studies; c-met and c-kit. to our surprise, neratinib also reduced c-met and c-kit levels, albeit in a delayed fashion. to down-regulate the egfr required a ubiquitination step whereas to down-regulate c-met did not. growth factor receptors localize in large quaternary structures in the plasma membrane and we hypothesized that if neratinib was reducing the levels of the egfr, c-met and c-kit, could it also reduce the levels of an important signal transducer on the inner leaflet of the plasma membrane: ras. in pancreatic cancer cells neratinib not only caused internalization and degradation of the egfr, it also caused the degradation of the key oncogenic driver in this disease, mutant k-ras. subsequently, in melanoma cells expressing a mutant n-ras, similar findings with neratinib were obtained. , the convergence of the afatinib-resistance studies and the ras down-regulation studies was a project to define the roles of map k and map k enzymes in the biological actions of neratinib. from the modeling studies, two potential neratinib targets were mst and mst . this caught our interest because the dose-limiting sequela for neratinib is diarrhea, and mst and mst play important roles in regulating the integrity of the epithelial brush boarder in the gut. , because we did not know what effects would be observed, we agnostically examined the activities of multiple map k enzymes, as well as associated chaperone/docking proteins following neratinib exposure. as map k/map k enzymes are expressed in carcinoma cells which express high levels of erbb family receptors as well as in blood cancer cells that express none or very low levels of that receptor family, we performed studies in both tumor cell types. regardless of erbb family receptor expression, neratinib reduced the expression of ras proteins and reduced tumor cell viability. neratinib reduced the phosphorylation of mst / , mst and mst in carcinoma and blood cancer cells; this would a priori predict that phosphorylation of their downstream substrates such as lats / or the cytoskeletal protein ezrin, would be reduced. , as was a priori expected, the phosphorylation of ezrin was reduced. however, the phosphorylation of lats / was enhanced, as were the downstream substrates of these enzymes, the co-transcription factors yap and taz. yap and taz are hippo pathway effectors and when phosphorylated leave the nucleus which is followed by degradation in the cytoplasm. , as yap and taz cooperate with mutant k-ras to drive pancreatic cancer growth and metastasis, our data suggest that neratinib could be a useful drug to employ in the treatment of this disease. , this data also suggests that inhibition of the mst "map k" kinases probably caused a compensatory activation of another "map k" kinase(s) which phosphorylated lats / . thus, the key take-home messages from this section are that without a full appreciation and understanding of all potential targets of a particular drug, its mechanisms of action cannot be properly understood. because neratinib inhibits map k/map k enzymes besides erbb family receptors and particularly her /erbb , very few pre-clinical studies were performed in cells that did not over-express her /erbb and none in cells that express mutant ras proteins or in blood cancer cells. these findings emphasize that in developmental drug and therapeutics studies, a broad agnostic approach is essential so as not to miss potential unknown off targets. this is diametrically different to almost all cell biology research projects where intense focus on a particular pathway, or even a component of a pathway is a standard approach. similarly, studying the mechanisms of cell killing by a drug by their nature have to be conceptually broad because very frequently drug-induced killing is not "pure" with only one pathway to tumor cell death being engaged. the drug-induced killing mechanism, for example, could include death receptor signaling, mitochondrial dysfunction and autophagosome formation, all interacting in a contemporaneous fashion. again, this approach is diametrically different to almost all basic science cell biology research projects. developing a compound into a putative drug and eventually into an agent that can be tested in humans is a long process that generally costs in the region of $ - million dollars. to some extent, the high cost of all prescription drugs to the consumer is influenced by this math. the screening of millions of compounds may result in the discovery of a new agent with anti-cancer, antiviral or anti-bacterial properties. alternatively, compounds are screened against a specific target until molecules are defined that potently act to inhibit the target's biological activity. optimization of these compounds, either by computer aided design, or by traditional organic chemistry methods, results, hopefully, in a series of compounds all with a low nanomolar ic inhibitory activity. drug development companies will then determine which of the drugs has the greatest apparent bioactivity in a range of tumor cell lines, alongside determination of in-animal stability and bioactivity against tumors. these studies collectively will deliver one or two compounds that are considered worthy of further investigation and development. it is at this point where drug companies will often seek outside academic collaborators to assist in their drug development studies. the first thing the independent academic collaborator needs to know is what was the highest safe dose of the compounds used in prior mouse studies? and, ideally, if pharmacodynamic and pharmacokinetic studies were performed, what was the safest peak plasma concentration of the compound, termed the c max and often listed as ng/ml (which requires conversion into a molar value). thus, if the highest safe dose of a compound is mg per kg of animal, with a plasma c max of mm, then all preliminary in vitro cell-based investigative studies must use the compound at concentrations well below mm. to further understand the biology of the compound, preliminary in vitro dose-response studies against tumor cells are most often performed on a log-scale, e.g. , , , , and nm. the first question the academic investigator should ask is, in their hands, does the dose-response effect on tumor cell growth/viability correspond to the claimed inhibitory ic of the compound against its purified specific target? i.e. if the protein target has an ic inhibition of nm and an ic for growth inhibition and cell killing of nm, it suggests the compound may be binding tightly to the serum in the culture media, resulting in a very low concentration of free "active" drug. on the other hand, if the target inhibition ic is nm but the ic for growth arrest/killing is nm, the data implies the compound may have additional unknown higher affinity targets in addition to its primary target which all collectively contribute to the biological efficacy of the agent. in this article we have discussed the fda approved drugs sorafenib and neratinib. sorafenib was originally developed to inhibit raf- and b-raf. prior to the discovery that raf- phosphorylated mek / , it was noted that the catalytic site of the raf- serine/ threonine kinase most closely resembled the active sites of src family non-receptor tyrosine kinases. hence, it was no surprise that within a few years sorafenib was also shown to also inhibit class iii receptor tyrosine kinases, and investigators now considered the biology of drug to be as an "anti-angiogenic" agent rather than per se an inhibitor of raf- . , finally, sorafenib was shown to be an inhibitor within its physiological range of hsp and hsp chaperone proteins. , similarly, neratinib was developed solely with the intention of inhibiting the receptor tyrosine kinase her (erbb ) as a putative therapeutic for her + breast cancer. yet, within several years of neratinib entering the clinic, two groups demonstrated it could inhibit map k and map k serine/threonine kinases with low nanomolar ic values. , so, if the compound under investigation is considered by a drug company to be a "specific" inhibitor of a particular protein kinase, regardless as to whether the agent is also fda approved, the in vitro studies the academic investigator should perform are an agnostic wide-ranging series of assessments, over a clinically-relevant drug dose-response range and over a time course. these studies will define, in your own hands, the changes in phosphorylation of the proposed target but also of multiple other cellular signaling pathways. this involves studying components of each specific pathway, e.g. the regulatory phosphorylation and total expression of erbb , erbb , erbb , erbb , raf- , b-raf, mek / and erk / , as well as of downstream nuclear transcription factors whose functions are controlled by each pathway, such as camp response element-binding protein (creb). such wide-ranging data-intense screening studies are difficult to perform using traditional sds page and western blotting approaches, and more advanced methods such as dot-blots or staining fixed cells in situ and measuring the intensity fluorescent staining, using validated antibodies, which permit a high-throughput approach, are required. an old phrase in science is: "the data is what it is." thus, if signaling from the primary target in one signaling pathway is only partially inhibited by the drug under examination, but signaling through an unrelated pathway is almost abolished, one would therefore tentatively conclude that the compound has an unknown target in a different signaling pathway. or, if at a low concentration of the drug no inhibition of the primary target is observed, but that this occurs alongside changes in the activities of other pathways, an effect which is also associated with significant levels of growth arrest and tumor cell death, one would conclude that the biological primary target is not the key functional target which regulates tumor cell biology. these examples for drug actions are binary, and in reality, the differential effects upon signaling and tumor cell biology of any drug are more subtle and nuanced. a different set of concepts come into play when rationally combining fda approved drugs to develop a novel anti-cancer therapeutic approach. first, the safe c max values for both agents in patients should be determined alongside their plasma half-lives, c min at h values and serum binding properties. the c max/c min values alongside the drug's half-life should inform the researcher that, for example during a h in vitro time course assay, a drug concentration considerably less than the c max but above the c min should be used to approximate for a physiologic treatment concentration. many clinically relevant drugs are stated to be % protein bound in % serum; most in vitro studies are performed using % (v/v) fetal calf serum. what is self-evident, however from extant data, is that for a drug such as sorafenib, with a safe c max of mm and a stated % plasma protein binding, is that a free sorafenib concentration of nm in vitro has a very modest impact on altering tumor cell biology, i.e. the partitioning on-off rate for drug association with plasma proteins and with tumor tissue must also be taken into consideration when deciding the most in vitro physiologic drug concentration. thus, taking all of these parameters into account, studies in the author's laboratory, in vitro with cells in % (v/v) serum, and in an attempt to remain within the physiologic range, do not use sorafenib above mm. an additional consideration for drug combination studies is to determine from the literature the doselimiting toxicities of each drug. regardless of excellent laboratory-based data, if the two drugs being combined both have doselimiting toxicities (dlts) in the same tissue, e.g. the gastrointestinal tract (gi), the likelihood that both agents can be safely and successfully combined in a patient is considerably reduced. studies to define hormonal signaling and intracellular signal transduction are almost years old. although much essential biological information was gleaned from work performed in the s to the late s, it was only with the widespread use of more modern molecular biology approaches combined alongside classic biochemical approaches that the signal transduction landscape of the last years evolved. today, essentially all of the building blocks of all signal transduction pathways are known. what is still under investigation are the complex protein-protein interactions which define nuanced signaling during growth, development, and various pathologies. small molecule therapeutic interventions have been and are being developed using ever more sophisticated technologies, of which some have shown considerable clinical utility. however, many of the newly developed "specific" targeted drugs have had little to no testing to fully define off-target effectors of their biology. it is very probable that this on-target/off-target issue for all drugs will never be fully resolved. hence, the step-wise approaches described in this article will still be required to fully understand the use and application of all new drugs. the samuri chemist, and his work on adrenalin observations with insulin on department of soldiers' civil re-establishment diabetics aqueous extracts of pancreas iii. some precipitation 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autophagy wins the nobel prize in physiology or medicine: breakthroughs in baker's yeast fuel advances in biomedical research aminopeptidase i of saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction isolation and characterization of autophagy-defective mutants of saccharomyces cerevisiae so many roads: the multifaceted regulation of autophagy induction mtor at the nexus of nutrition, growth, ageing and disease two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury ampk: regulation of metabolic dynamics in the context of autophagy diverse signaling mechanisms of mtor complexes: mtorc and mtorc in forming a formidable relationship mtor as a central hub of nutrient signalling and cell growth distinct roles of mtor targets s k and s k in breast cancer allosteric regulation of amp-activated protein kinase by adenylate 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suppresses dengue virus replication by down-regulation of pi k/akt and grp exploring the in vitro potential of celecoxib derivative ar- as an effective antiviral compound against four dengue virus serotypes egfr mutation and resistance of non-small-cell lung cancer to gefitinib irreversible inhibitors of the egf receptor may circumvent acquired resistance to gefitinib activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib gefitinib-sensitizing egfr mutations in lung cancer activate anti-apoptotic pathways egf receptor mutations in lung cancer: from humans to mice and maybe back to humans presence of epidermal growth factor receptor gene t m mutation as a minor clone in non-small cell lung cancer epidermal growth factor receptor mutations in patients with non-small cell lung cancer third-generation epidermal growth factor receptor tyrosine kinase inhibitors for the treatment of non-small cell lung cancer afatinib 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expression in t mammary tumors and enhances m macrophage infiltration the levels of mutant k-ras and mutant n-ras are rapidly reduced in a beclin /atg -dependent fashion by the irreversible erbb / / inhibitor neratinib neratinib degrades mst via autophagy that reduces membrane stiffness and is essential for the inactivation of pi k, erk / , and yap/taz signaling risk of gastrointestinal complications in breast cancer patients treated with neratinib: a meta-analysis targeting neratinib-induced diarrhea with budesonide and colesevelam in a rat model yap/taz functions and their regulation at a glance yap/taz: drivers of tumor growth, metastasis, and resistance to therapy yap activation enables bypass of oncogenic kras addiction in pancreatic cancer downstream of mutant kras, the transcription regulator yap is essential for neoplastic progression to pancreatic ductal adenocarcinoma primary structure of v-raf: relatedness to the src family of oncogenes safety and pharmacokinetics of the dual action raf kinase and vascular endothelial growth factor receptor inhibitor, bay - , in patients with advanced, refractory solid tumors results of phase i pharmacokinetic and pharmacodynamic studies of the raf kinase inhibitor bay - in patients with solid tumors neratinib: an oral, irreversible dual egfr/her inhibitor for breast and non-small cell lung cancer bay - : early clinical data in patients with advanced solid malignancies key: cord- - yaol r authors: ryan, paul macdaragh; caplice, noel title: covid- and relative angiotensin-converting enzyme deficiency: role in disease severity and therapeutic response date: - - journal: open heart doi: . /openhrt- - sha: doc_id: cord_uid: yaol r nan severe acute respiratory syndrome coronavirus (sars-cov- ) is a novel coronavirus first identified in an outbreak of pneumonia in wuhan, hubei province, china in december . despite mitigation of the initial epidemic, the viral syndrome now named coronavirus disease (covid)- has since spread rapidly throughout the world, representing a pandemic with profound implications for human morbidity and mortality. in turn, the capacity of diverse healthcare and economic systems to cope with rising infections and associated intensive care requirements is strained. the most extensive clinical experience of this virus to date comes from the more than positive cases identified in hubei province. [ ] [ ] [ ] [ ] these early clinical reports clearly indicated that although most clinical manifestations of covid- requiring hospitalisation are respiratory, there is a substantial minority of patients who undergo progressive and severe cardiovascular compromise. subjects at highest risk of death appear to be more elderly patients with pre-existing cardiovascular disease and/or classical risk factors that accompany advanced cardiovascular illness. the goal of this viewpoint article is to examine whether the nature of sars-cov infection and the homeostatic status of highrisk cardiovascular patients can be linked in a mechanistic framework that provides insights into corrective therapy over and above current and emergent antiviral approaches to there are a number of reasons why covid- may be associated with cardiovascular complications that include inter alia-specific aspects of sars-cov- structure and receptor targeting, and target cell location and its relationship with cardiovascular disease homeostasis. in addition, there are potential risk amplifiers including hitherto unanticipated interactions between viral targets within the host and established homeostatic pathways that may already be perturbed in patients with advanced cardiovascular disease. in this viewpoint, we underscored specific alterations in renin angiotensin system-ace (ras-ace ) homeostasis that may contribute to more adverse covid- outcomes in patients with pre-existing cardiovascular disease. moreover, we proposed and developed a rationale for specific therapeutic interventions that might mitigate some of the more deleterious cellular pathology and cardiovascular effects of this syndrome. structure of sars-cov- , viral infection, cell targets and ace sars-cov- , a positive-strand rna virus, shares % genomic homology with sars-cov virus and initial infection is mediated through interaction of virion spike glycoprotein (s protein) with the ace receptor on target cells. the s protein is cleaved into s and s subunits that act cooperatively to allow ace receptor engagement and viral cell entry. s , via a receptor-binding domain (rbd), binds the peptidase domain of ace with the s subunit implicated in membrane fusion. s cleavage site activation by host transmembrane serine protease (tmprss ) is facilitated by a conformational change secondary to s -ace binding (figure ). comparative genomic analysis of sars-cov- suggested that it is optimised for binding to human ace and in this way it exploits the membrane-bound receptor in host cells to initiate and spread infection. the primary role of ace in health is maturation of angiotensin, a peptide implicated in vascular homeostasis, vasomotor tone and blood pressure regulation. importantly ace is expressed on diverse human cells including epithelial cells in the lung and small and large intestines, tubular cells of the kidney, vascular endothelial and smooth muscle cells and cardiomyocytes (figure ) and reduction in ace is well known to be associated with hypertension, diabetes, coronary artery disease, myocardial infarct repair and heart failure. although robust histopathological data are still unavailable for organ and tissue damage in covid- patients, it may be useful to look at the homologous sars-cov infection that shows similar ace viral tropism. pathological examination of sars-cov subjects has identified viral infection throughout the respiratory tract, spleen and lymph nodes, intestinal epithelium and mucosal lymphoid tissue, liver hepatocytes, tubular epithelium of the kidney, neural cells in the brain and cardiomyocytes and vascular cells within the heart. moreover, postviral cell apoptosis, necrosis, interstitial oedema, macrophage infiltration, lymphocyte depletion and early fibrosis have been documented to varying degrees in all of these tissues in sars-cov subjects. preliminary real-time pcr detection of sars-cov- in different clinical specimens shows similar widespread seeding of virus in respiratory, circulatory and gastrointestinal systems. although the exact mechanism by which sars-cov- induces cellular damage remains unknown, clinical and laboratory data from early cohort studies - would support a similar tissue distribution to sars-cov with a notable increase in clinical cardiovascular effects. in naïve humans subjects, sars-cov successfully evades the innate immune response and hijacks host cell metabolism through initial degradation of host mrna and modulation of ubiquitination. efficient viral replication within the host cell then ensues, leading to cell damage and viral-induced cytolysis. interestingly, comparative analysis of two successive sars epidemics in early s showed that increased affinity of the sars virus for human ace receptor strongly predicted severity of clinical disease suggesting that spike protein conformation is potentially a key determinant of virulence. moreover, the spike protein present on sars-cov and sars-cov- facilitates host cell-to-cell fusion to form syncytia that may have potential pathological consequences for tissues such as the heart. interestingly, in several wuhan cohorts cardiac injury and arrythmia were prominent in high-risk figure homeostasis of ras-ace under normal healthy conditions ; perturbation of ras-ace homeostasis in cardiovascular disease, hypertension and diabetes mellitus ; covid- may potentially further upregulate ras in cvd patients and deplete ace ; proposition that rhace replacement therapy improves ras-ace balance by augmenting ace and decreasing ras activation. cvd, cardiovascular disease; htn, hypertension; dm, diabetes mellitus; ras, renin angiotensin system; rhace , recombinanthuman ace . covid- subjects. previous sars-cov studies also indicated that clinical and pathological impacts of viral infection in various tissues and organs are determined not solely by viral load or tropism with respect to cell surface receptors but also by the counter-regulatory pathways within the host. these host mechanisms include inter alia complex elements of homeostatic and inflammatory pathways, anti-apoptotic responses and the compensatory repair power or functional reserve of tissues and organs within the infected subject. the balance between these countervailing forces in sars-cov- infection may in large part determine the severity of clinical manifestation and outcome of covid- in human subjects. ace function, receptor distribution and ras homeostasis ras as a signalling pathway acts as a homeostatic regulator of vascular function, and at a tissue level it regulates function within organs such as the kidney, lungs and heart. at a regional level, ras regulates blood flow to organs and controls trophic responses to a wide range of triggers including cell death, injury, inflammation, fibrosis and vascular repair. many of these regulatory elements involve opposing functions to accommodate rapidly coordinated responses to local stimuli. the dipeptide carboxypeptidase ace metabolises angiotensin i to form angiotensin ii (angii) and angii is subsequently metabolised by the carboxypeptidase ace into the vasodilator angiotensin ( - ) (ang - ). while ace and angii have been the focus of therapeutic targeting for more than four decades, ace has only recently come to prominence as a major player in ras imbalance, especially in the presence of disease. although ace shares sequence homology with the extracellular domain of ace, it functions quite differently as a carboxypeptidase and is not blocked by ace inhibitors. therefore, ace represents an endogenous counter-regulatory pathway with ras and acts in opposition to the ace axis ( figure ) . indeed, in the cardiovascular system ace may be more important than ace in regulating the local effects of angii and ang - with consequences for counter-balancing ras activation. angii contributes to systemic hypertension and locally promotes vasoconstriction, endothelial dysfunction, vascular inflammation, endothelial oxidative stress, smooth muscle cell migration, proliferation and contraction and in situ thrombosis. in the context of acute respiratory distress syndrome (ards), angii is regarded as a pivotal player in vascular permeability, cytokine amplification and inflammatory cell infiltration. in contrast, ang - opposes local vascular effects of angii, has antioxidative and anti-inflammatory effects and attenuates vascular disease in murine models. genetic knockout studies show that under normal conditions deficiency of ace has relatively modest effects on blood pressure, cardiovascular homeostasis and renal function when compared with ace gene deletion. however, under conditions of oxidative stress, sepsis, inflammation or experimental hypertension, diabetes, atherosclerosis, myocardial infarction or heart failure, ace deficiency greatly amplifies the rasassociated pathological phenotype in animal models and clinical specimens. furthermore, ace knockout models show increased tissue and circulating levels of angii and reduced ang - , with accelerated diabetic nephropathy and greater angii-induced cardiac hypertrophy, fibrosis and infarction, whereas ace deletion confers a modest reduction in circulating angii, but no effect on tissue levels. this would support multiple non-ace pathways for angii generation in the body, but limited pathways for angii degradation, which highlights the potential importance of ace disruption at a tissue level especially in states of ras activation. moreover, this underscores the potential of ace replacement as a more potent strategy in reducing angii and augmenting ang - compared with conventional ras inhibition. thus, in cardiovascular disease there exists an imbalance between increased ras activation and reduced ace counter-regulatory effects (figure ), which may render subjects with increased cardiovascular risk burden susceptible to conditions that worsen asymmetries in ras/ace . theoretical implications of ace -expressing cell destruction by sars-cov- in cardiovascular system and other organs given that sars-cov- specifically targets ace receptor and based on sars-cov homology and previous sars-cov pathology studies, it is reasonable to expect that during viremia host cells and tissues that express ace in the major organs such as lungs, heart, vascular system and kidneys will be susceptible to viral infection. it is also conceivable that relative loss of ace (through viral destruction) in these affected tissues at a time of increasing ras activation may act to amplify the pathophysiological consequences of viral infection in these organs. previous studies of sars-cov show viral induction of ace shedding in infected cells and downregulation of ace levels in tissues has been linked to viral replication efficiency and pathogenicity. therefore, extensive viral lysis of ace -expressing cells may act as a tipping point in tissues ability to adequately respond via ras/ace to ensuing inflammatory, oxidative stress and ongoing cytokine-mediated cellular insults (figure ). in contrast, in healthy adults and younger patients, as seen in genetic knockout models, it is conceivable that, with ras-ace homeostasis intact, the consequences of acute viral infection may not reach this threshold. two poorly prognostic elements may thus combine in sars-cov- infection of cardiovascular disease patients, namely ( ) a pre-existing imbalance in ras activity and ace counter-regulation that renders this cohort susceptible to more cardiovascular complications of covid- and ( ) destructive targeting by sars-cov- of ace -expressing cells in multiple cardiovascular and other tissues leading to further critical loss of ace reserve. this amplified ras-ace imbalance in covid- may greatly exacerbate tissue injury and inflammation in affected organs of cardiovascular patients. rationale for, and safety of ace replacement therapy in covid- subjects ace protein replacement has proven beneficial in multiple preclinical models such as hypertension, diabetic vasculopathy and renal disease, postmyocardial infarction, heart failure and especially in ards where ras-ace imbalance is prominent. the mechanism of ace benefit is likely increased metabolism of local tissue angii and increased bioavailability of ang - with reduction in reactive oxidation, cell death, inflammation and endothelial cell activation. these protective activities of ace may thus counteract the deleterious effects of tissue injury and associated activation of ras. in human subjects, this has already been exploited with completion of the first pilot phase / trial of recombinant human ace (rhace ) in ards. in this safety study, which was not powered for efficacy, rhace proved safe and was well-tolerated haemodynamically with a rapid reduction in angii and increase in ang - observed after intravenous infusion. pharmacokinetic evaluation of rhace in healthy volunteers supports a twice daily infusion, although given a relatively short half-life it is likely that continuous infusion if tolerated would be more effective in more severely ill subjects. angii and ang - responses to ace replacement are rapid (within min) so it may be feasible to identify responders and non-responders quickly, although angii assay variability may require further study and validation. moreover prescreening using angii levels, ace /ace activity ratios and prognostic ace gene polymorphisms may further assist in risk stratification of patients more likely to respond to ace therapy. we proposed that existing covid- subjects should have an ace deficiency state confirmed prospectively by analysis of ace , angii and ang - levels in lowrisk and high-risk subjects with further dichotomisation based on survival or non-survival. in severe covid- , with no current options to save these patients, there is also a rationale for testing the ace deficiency hypothesis in phase / randomised controlled trials of rhace protein in subjects at high risk of death. such risk stratification could be based on conventional intensive care unit prognostic scoring systems in addition to circulating factors that indicate poor outcomes such as elevated soluble urokinase plasminogen activator levels. we noted a recently planned open-label, randomised controlled trial of rhace for treatment of patients with covid- in wuhan. further studies will be required to identify whether ace therapy should be targeted only at high-risk cardiovascular patients or all patients at risk of progression to cardiorespiratory failure and death. administration of ace recombinant protein in covid- patients raises the question of whether this might exacerbate sars-cov- infection given viral tropism. all experimental data suggested that the opposite response is more likely. despite this early phase / clinical trials should evaluate potential side effects such as hypotension, tachycardia and worsening respiratory viewpoint failure with close monitoring of vital signs, respiratory parameters, electrocardiography and haematology and blood chemistry profiles. given that ace downregulation by sars-cov is linked to viral replication efficiency, it is conceivable that ace augmentation may reduce virulence. importantly, rhace is a soluble protein whereas sars-cov- binds to cell membrane-bound ace and requires the cooperation of the tmprss protease to achieve viral cell entry. therefore, soluble ace alone is likely insufficient to allow viral infection of cells. previous studies with sars-cov and more recently sars-cov- studies showed that soluble ace protein actually inhibits sars-cov- viral spike rbd binding to membrane ace receptor. for sars-cov pathogenesis, ace not only acts as a receptor for viral entry but also protects against local tissue injury. if the same holds true for sars-cov- , then soluble rhace may reduce ongoing sars-cov- access to membrane-bound ace receptor, alter favourably local angii/ang - levels and inhibit deleterious ras effects on remaining at risk tissues in covid- patients. in the current covid- pandemic, saving lives and increasing hospital capacity for ventilatory support are priorities for many already stretched and often overwhelmed healthcare systems. currently, we have supportive but very limited therapeutic options for highrisk covid- patients in intensive care settings. in this viewpoint we presented a theoretical framework by which ace replacement or augmentation appears a rational therapeutic option for severely ill, high cardiovascular risk, no-other option covid- subjects. if early phase / ace replacement proves promising, then expedited compassionate off-label use might also be considered. given that ace is synthesised using recombinant manufacturing, it is likely that a number of pharmaceutical companies would have the capacity to scale up production quickly for global use. clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of coronavirus disease in china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study association of cardiac injury with mortality in hospitalized patients with covid- in wuhan, china structural basis for the recognition of sars-cov- by full-length human ace structure of sars coronavirus spike receptor-binding domain complexed with receptor activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme (ace ) is a key modulator of the renin angiotensin system in health and disease pathology and pathogenesis of severe acute respiratory syndrome detection of sars-cov- in different types of clinical specimens molecular pathology of emerging coronavirus infections receptor and viral determinants of sars-coronavirus adaptation to human ace severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection role for nonstructural protein of severe acute respiratory syndrome coronavirus in chemokine dysregulation the inextricable role of the kidney in hypertension tissue renin-angiotensin systems. their role in cardiovascular disease a novel angiotensinconverting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - evaluation of angiotensinconverting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse ace deficiency modifies renoprotection afforded by ace inhibition in experimental diabetes prevention of accelerated atherosclerosis by angiotensin-converting enzyme inhibition in diabetic apolipoprotein e-deficient mice angiotensin ii induces smooth muscle cell proliferation in the normal and injured rat arterial wall vasoprotective and atheroprotective effects of angiotensin ( - ) in apolipoprotein e-deficient mice angiotensin-converting enzyme is an essential regulator of heart function characterization of renal angiotensin-converting enzyme in diabetic nephropathy angiotensin-converting enzyme (ace ) expression and activity in human carotid atherosclerotic lesions loss of angiotensin-converting enzyme- (ace ) accelerates diabetic kidney injury angiotensin-converting enzyme suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction differential ang ii generation in plasma and tissue of mice with decreased expression of the ace gene high expression of ace receptor of -ncov on the epithelial cells of oral mucosa differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl replication-dependent downregulation of cellular angiotensin-converting enzyme protein expression by human coronavirus nl angiotensin-converting enzyme protects from severe acute lung failure angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target a pilot clinical trial of recombinant human angiotensin-converting enzyme in acute respiratory distress syndrome pharmacokinetics and pharmacodynamics of recombinant human angiotensinconverting enzyme in healthy human subjects recombinant human ace : acing out angiotensin ii in ards therapy soluble urokinase plasminogen activator receptor (supar) as an early predictor of severe respiratory failure in patients with covid- pneumonia nct -recombinant human angiotensinconverting enzyme (rhace ) as a treatment for patients with covid- characterization of the receptorbinding domain (rbd) of novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine key: cord- -rcx b authors: khazak, vladimir; golemis, erica a.; weber, lutz title: development of a yeast two-hybrid screen for selection of human ras-raf protein interaction inhibitors date: journal: chemical genomics doi: . / - - - - _ sha: doc_id: cord_uid: rcx b a yeast two-hybrid screening system was developed to screen for small molecules that inhibit the interaction of the ras and the raf proteins. hyperpermeable yeast strains useful for high-throughput screening (hts) for the two-hybrid system were created. differential inhibition of the ras-raf vs the hsrpb -hsrpb interaction allowed the identification of selective inhibitors. with recent advances in genome-wide sequencing, studies of the function of individual proteins and protein complexes have become increasingly important for understanding biological functions, and for selection of novel targets for drug discovery applications. the yeast two-hybrid system was originally developed to identify and study protein-protein interactions ( , ). this method was later expanded to allow detection of interactions between proteins and rna ( ), proteins and nonprotein ligands ( ), proteins and peptides ( ), proteins and multiple partners ( , ), and whole-genome applications ( - ). in addition, some investigators have begun to study the potential of two-hybrid screens to detect protein-small-molecule interactions ( ) ( ) ( ) . however, limited progress has so far been made to adapt such yeast screening technologies for the identification of new clinical candidates in high-throughput screens (htss). one major barrier to the use of yeast for drug screening has been thought to be the relative impermeability of yeast cells to a broad spectrum of organic molecules. physical and chemical genetic techniques have been used to enhance the permeability of yeast membranes. permeabilizing agents, such as polymyxin b sulfate and polymyxin b nonapeptide, have been used to physically disrupt the integrity of yeast membranes ( ) . however, use of such chemical agents in drug screening is not ideal, because of the toxicity induced by polymyxin b treatment. as an alternative strategy, a number of yeast genes involved in the control of membrane permeability have been identified, which might be targeted to improve strain permeability ( ) . in particular, a network of regulators associated with the phenotype known as pleiotropic drug resistance (pdr), which closely resembles the mammalian multi-drug resistance phenotype (mdr) ( ), is known to affect cellular transport and drug resistance. pdrlp and pdr p, members of the c zinc cluster family of transcriptional regulatory proteins, redundantly modulate expression of abc transporter proteins by inducing their transcription ( , ) . disruption of pdri and pdr , the genes encoding pdrlp and pdr p, respectively, results in decreased expression of the abc transporter pdr , and thereby increases drug sensitivity of pdr − pdr − cells ( ) . further, it has been shown that overexpression of either the hxt p or hxt p hexose transporter proteins independently promotes drug sensitivity, by increasing uptake of certain drugs ( , ) . based on these earlier studies, we have exploited these properties of pdr and hxt mutants to create hyperpermeable yeast strains useful for hts for the two-hybrid system. we here describe the creation of these strains and their utilization for the selection of small-molecule inhibitors of interaction between ras and raf oncoproteins. the methods outlined below describe ( ) the construction of expression plasmids bearing conditionally regulated yeast hxt and hxt hexose transporter genes, ( ) integration of hxt -and hxt -containing dna fragments in the pdr and pdr chromosomal loci of yeast two-hybrid strains, ( ) analysis of the permeability of newly developed yeast strains with selected known smallmolecule inhibitors, and by screen of a large combinatorial library of compounds, and ( ) selection of inhibitors of the interaction between human ras and raf- by screening the combinatorial library of compounds in the obtained hyperpermeable yeast two-hybrid strain. in order to enhance the permeability of the yeast s. cerevisiae to small-molecular-weight compounds, the two yeast hexose transporters hxt and hxt were subcloned under control of the galactose-inducible gal promoter and subsequently integrated by homologous recombination into the genetic loci for pdr and pdr , thereby destroying the coding sequence of these genes. the cloning and integration strategies are presented in flow charts ( figs. and ) . additional maps for cloning intermediates are available upon request. all polymerase chain reaction (pcr) primer sequences are shown in table . primers vk and vk were used to amplify an -base-pair (bp) ' fragment of pdr , and primers vk and vk were used to amplify a -bp ' fragment of pdr , using pcr. the amplified pdr fragments were cloned into pgem-t/a (promega, madison, wi) to construct the pgem - -pdr plasmid, with a unique bamhi site between the fragments. next, the phiscada plasmid was constructed by replacing the salmonella hisg dna fragment in the pnky plasmid with the e. coli cadba gene operon. next, the hisg-ura -cada gene fragment from phis-cada was isolated, purified, and ligated into the bamhi site of the pgem - -pdr vector to construct the ppdr -hiscada plasmid ( fig. a ) (see note ). primers vk and vkl were used to amplify an -bp ' fragment of the yeast pdr gene, and vk and vk were used to amplify a -bp ' fragment of the yeast pdr gene. the amplified fragments were then cloned into pgem-t/a to create pgem - -pdr , which bears a unique bamhi site between the pdr fragments. the phisint plasmid was then constructed by replacing the hisg ' fragment of the pnky plasmid with a fragment from the human ) . the hisg-ura -int fragment was then isolated, purified, and ligated into the bamhi site of the pgem - -pdr vector to create the ppdr -hisint plasmid (fig. b) . primers vk and vk were used to pcr amplify the coding sequence of the yeast hxt gene. the hxt gene fragment was ligated downstream of the gal -inducible promoter and upstream of the cyc transcription terminator region (tt) in the pyes plasmid to create the pyes-hxt plasmid. the fragment containing the gal promoter region, the hxt gene fragment, and the cyc tt region from pyes-hxt were then ligated into phiscada (see above) to create phiscada-hxt ( fig. c ) (see note ). primers vk and vk were used to pcr amplify the coding sequence of the yeast hxt gene. the hxt fragment was ligated downstream of the gal -inducible promoter and upstream of the cyc tt region in the pyes plasmid to create the pyes-hxt plasmid. the fragment containing the gal promoter region, the hxt coding region, and the cyc tt region from pyes-hxt were then excised, purified, and ligated into phisint to create phisint-hxt (fig. d) . in order to integrate conditionally expressed hxt and hxt transporter genes in a yeast two-hybrid genetic background ( ), the expression plasmids from subheading . . were digested with restriction enzymes, and dna fragments containing the target genes were purified and used for subsequent sitespecific chromosomal recombination (see figs. and ) . yeast transformed with these plasmids were selected based on a ura + phenotype, and subsequently characterized by pcr of genomic dna to confirm that correct integrations had occurred. the plasmid ppdr -hisint was digested with aatii-saci and the purified integrative cassette, hisg-ura -pdr -int (see fig. c ) was transformed into parental strains sky and sky . positive cells were selected on yeast dropout media that lacked uracil. cells that grew on such media contained integrated copies of hisg-ura -int cassette and were designated sky and sky . this is the source of the hisg-hxt -int cassette (fig. strains sky and sky were used for the next round of integrative transformation. the goal of this transformation was to replace the ura gene at the pdr locus in the chromosome of sky and sky with a galactose-inducible copy of hxt . to this end, plasmid phishxt -int was digested with aatii and pshai, and the hisg-hxt -int cassette was purified and transformed into sky and sky . to select yeast with the hisg-hxt -int cassette integrated into the chromosome, cells were propagated on ypd plates for - h to decrease the amount of ura p enzyme, and then replica-plated on plates containing minimal do media containing . mg/ml -fluoro-orotic acid ( foa) and uracil at a concentration of . mg/ml (see note ). yeast that express the ura gene convert foa to a toxic metabolite and die. however, cells that have replaced the ura gene with hxt grow normally. several hundred transformants containing the hxt gene at the pdr chromosomal locus were obtained. two strains containing the inserted cassettes (fig. c) were selected, and named sky and sky . at the next round of integrative transformation, the hisg-ura -cada cassette in ppdr -hiscada was digested with aatii-saci and the purified cassette ( fig. c) was transformed into sky and sky . two resulting yeast strains, sky and sky , were selected on yeast dropout media that lacked uracil. the hisg-hxt -cada cassette was then purified and transformed into sky and sky , thereby replacing the hisg-ura -cada cassette with the hisg-hxt -cada cassette. another round of foa selection (see fig. d ) yielded two strains, sky and sky , with hxt substituted for ura at the pdr locus (see fig. e ). cells selected after this final round of integrative transformation have integrated copies of hxt and hxt at the pdr and pdr loci, respectively. the full genotype of sky is (matα trp ura his lexaop-leu ciop-lys pdr ::hxt pdr ::hxt ), and that of sky is (matα trp ura his lexaop-leu ciop-lys pdr ::hxt pdr ::hxt ). to confirm the proper insertion of hxt and hxt into the chromosomal loci of pdr and pdr , respectively, genomic dna was purified from sky and sky , and analyzed using pcr for the presence of hxt and hxt recombinant sequences within pdr and pdr loci. (figs. a,b and a,b) . figure a depicts the dna fragment pdr -hisg-hxt -cada-pdr , which was inserted into the pdr locus of sky and sky , showing the location and orientation of pcr primers. figure b shows an agarose gel stained with ethidium bromide to visualize the pcr products. the six bands correspond to the proper lengths between the specific primer pairs, and confirm the correct insertion and orientation of the cassettes. figure a depicts the dna fragment pdr -hisg-hxt -int-pdr that was inserted into the pdr locus of sky and sky , indicating the location and direction of pcr primers. fig. b shows an ethidium bromide-stained gel of the pcr products. the eight bands correspond to the proper lengths between the specific primer pairs indicated, and confirm the correct insertion and orientation of the cassettes. to test the new strains for enhanced permeability to small-molecular-weight inhibitors, sky and sky and their parental strains were incubated with various concentrations of selected antifungal compounds, and with a diverse combinatorial library of uncharacterized compounds. cycloheximide (cyh), -nitroquinoline-oxide ( -nqo), sulfomethuron methyl (smm) and zeocin (zeo) were used to evaluate the drug sensitivity of the yeast strains. yeast cells were seeded as a lawn on ypd or ypg/r plates by being dispersed in a "pad" of top agar poured over a normal plate ( μl of resuspended yeast [ cells] to ml of cooled % low-melt seaplaque agarose prepared with ypd or ypg/r growth media). immediately after seeding, cyh ( mg/ml), nqo ( . mg/ml), smm ( mg/ml), and zeo ( mg/ ml) stock solutions were diluted -, -, -, -, and -fold, and applied onto the top-agar growing yeast by means of a pronged replicator device that delivered -μl liquid aliquots of compound into the top-agar layer. sensitivity to compounds was scored by visualization of growth on this media, and the size of "death zones" around areas of compound application. the plates shown in fig. demonstrate the sensitivity of the parental and modified yeast strains to the test compounds. both parental sky and modified sky strains showed no sensitivity to smm at all concentrations tested. however, based on the size of the death zones, sky is significantly more sensitive to cyh and -nqo than sky on ypd media (fig. ) . further, when the sky yeast were incubated on gal/raff-containing media (thereby inducing expression of hxt and hxt genes), their sensitivity to cyh and -nqo increased. the estimated increase of death-zone diameters for -nqo for sky on ypg/r media in comparison with the parental strains grown on the same media was approx - %. after h of incubation on ypg/r media, sensitivity to cyh was estimated to be . μg/ml minimal inhibitory concentration (mic) in comparison to . μg/ml for the parental strain. the mic is defined as the lowest concentration (micrograms per milliliter of broth) that inhibited visible growth, disregarding a haze of barely visible growth. sensitivity to cyh of a yeast strain harboring pdr and pdr deletions, and with the hxt gene overexpressed from a multicopy plasmid, was previously reported as having an mic of . μg/ ml cyh ( ) . thus, integrating inducible copies of hxt and hxt into the chromosome while deleting pdr and pdr significantly increased the sensitivity of yeast cells to the tested compounds (see note ). a comparative analysis of parental strain sky and its derivative sky to , compounds from the diverse combinatorial chemical library is shown in table . sky was sensitive to compounds, while sky was sensitive to compounds. thus, sky was sensitive to . times as many compounds as the parental strain, sky . the sensitivity/permeability of modified yeast sky and sky was similar to the permeability of e. coli strains specifically designed for hts purposes, and screened with the same library of compounds (data not shown). this fact created a unique opportunity to use modified yeast two-hybrid strains sky and sky for screening of various protein-protein interaction inhibitors. the ras/raf/mek/erk signaling pathway controls fundamental cellular processes including proliferation, differentiation, and survival, and the altered expression or action of components of this pathway is commonly observed in human cancers ( ) ( ) ( ) . ras has suffered oncogenic mutations in nearly % of human cancers and mediates its action through interaction with downstream effector targets ( , ) . activated ras binds to and recruits raf- to the cell membrane, where raf- is activated by a complex mechanism that is not yet completely understood ( ) . agents capable of inhibiting the activating interaction between oncogenic ras and raf proteins have the potential to be valuable additions to the chemotherapy of multiple cancers. the human ras and raf proteins effectively interact in a yeast two-hybrid system ( ), which makes this interaction an attractive drug target in yeast strains with increased permeability. to screen for chemical compounds that would effectively block or diminish the interaction between a dna binding domain (dbd) fusion to ras (ci-h-ras) and an activation domain (ad) fused raf (ad-c-raf- ), while simultaneously testing the properties of the new permeable yeast strains, these two fusion proteins were expressed in sky , and in parallel in sky , and plated as described under subheading . . ., with the addition of x-gal. yeast containing an independent interacting protein pair, dbd-fused-hsrpb and ad-hsrpb , were similarly plated in parallel in both strains, as a control to be used for detection and subtraction of toxic compounds. these strains were used to screen a library of , compounds applied in microarray format. a representative panel demonstrating specific vs nonspecific inhibition of growth is shown in fig. . from compounds that produced growth-inhibiting effects in the sky ci-h-ras-ad-raf- strain, compounds also reduced β-galactosidase activity, with varying degrees of selectivity for ras-raf- vs hsrpb -hsrpb on plates. significantly fewer positive colonies were detected in the screen performed for ras-raf interaction inhibitors in the sky yeast strain (data not shown). further assessment of the selectivity of interaction inhibition by liquid beta-galactosidase assay ( ) identified compounds that produced a clear reduction (with a final range of - % of starting values) in lacz activity in sky expressing h-ras and raf- , but not lexa-hsrpb and ad-hsrpb , when included in culture medium at concentrations of μm. these compounds, reflecting a yield of approx . % from the starting library, were used for subsequent functional analyses in mammalian cells, that have extensively validated their mechanism of action, as described in ( ) . here, we have reported a general approach for creating a super-permeable yeast two-hybrid strain, and the application of this strain for hts for identifi- fig. . screening plates with yeast sky ras-raf, sky hsrpb -hsrpb , and sky ras-raf strains incubated with the compounds from combinatorial chemical library. the area with putative ras-raf inhibitors and individual antifungal compounds is indicated by frame or by arrow, respectively. note increased size of "death zones" for some compounds on sky vs sky plates. cation of novel protein-protein interaction inhibitors. the developed strains were successfully used for screening a diverse combinatorial library to select a novel class of clinically relevant protein-protein interaction inhibitors, and have the potential to be used in future efforts to search for new drugs in twohybrid hts. . our choice of the hisg dna fragment from salmonella, cadba gene from e. coli, and the int gene from the hiv virus was based on the lack of sequences homologous to these dna sequences in yeast. thus, the chromosomal integration of the expression cassettes was directed to the specific sites intended (pdr , pdr ) through homologous recombination. the specific dnas we have used can be successfully replaced in integration cassettes by other dna sequences that have no or low homology to yeast genomic dna. . gal was chosen as a promoter because of its strength and inducible nature. further, comparison of the yeast sensitivity to the given compound on media supplemented with glucose (ypd) vs galactose (ypg) allows unequivocal estimation of the contribution of the hxt gene to the reduction of resistance phenotype. . we found that the ura p enzyme synthesized from the first integration cassette is present in the yeast cells at the time of addition of foa compound even in the cells with a successful second cassette integration and elimination of an active copy of ura gene. the activity of these remnants of ura p causes the conversion of foa into a toxic metabolite, and subsequent cell death. additional propagation of the yeast on the ypd media, rich in exogenous uracil, is sufficient to significantly reduce the level of the earlier synthesized ura p enzyme in the cells with successful recombinations, and escape from foa-related toxicity. . the absence of improved sensitivity to smm and zeo in the modified sky strain indicates that the enhancement of permeability is not universal, but limited to some compound classes, likely reflecting differences in the uptake and efflux mechanisms for different drugs. a two-hybrid dual bait system to discriminate specificity of protein interactions a novel genetic system to detect protein-protein interactions a three-hybrid system to detect rna-protein interactions in vivo a family of human cdc -related protein kinases genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase the inositol '-phosphatase ship binds to immunoreceptor signaling motifs and responds to high affinity ige receptor aggregation a conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system toward a functional analysis of the yeast genome through exhaustive two-hybrid screens interaction mating reveals binary and ternary connections between drosophila cell cycle regulators ) a protein linkage map of escherichia coli bacteriophage t identification of a calcium channel modulator using a high throughput yeast two-hybrid screen reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and dna-protein interactions cytoskeletal protein abp- directs the intracellular trafficking of furin and modulates proprotein processing in the endocytic pathway effects of polymyxin b sulfate and polymyxin b nonapeptide on growth and permeability of the yeast saccharomyces cerevisiae a simple method for the isolation and characterization of thymidylate uptaking mutants in saccharomyces cerevisiae a major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport transcriptional control of the yeast pdr gene by the pdr gene product allelism of pleiotropic drug resistance in saccharomyces cerevisiae. can multiple-drug-resistance phenomenon in the yeast saccharomyces cerevisiae: involvement of two hexose transporters the hexose transporter family of saccharomyces cerevisiae analysis of the interaction of the novel rna polymerase ii (pol ii) subunit hsrpb with its partner hsrpb and with pol ii aspects of growth factor signal transduction in the cell cytoplasm signal transduction-a conserved pathway from the membrane to the nucleus the pathway to signal achievement ras effectors increasing complexity of ras signal transduction: involvement of rho family proteins meaningful relationships: the regulation of the ras/raf/mek/ erk pathway by protein interactions mammalian ras interacts directly with the serine/threonine kinase raf -present) current protocols in molecular biology inhibitors of ras/raf- interaction identified by two-hybrid screening revert ras-dependent transformation phenotypes in human cancer cells the authors thank dr. antonios makris for providing the cdna encoding the human h-ras and raf- genes. we also thank dr. alan hinnebusch for providing the pnky plasmid. eg was supported by nih core grant ca- , and an appropriation from the commonwealth of pennsylvania (to fox chase cancer center). key: cord- - p oeac authors: hasan, syed shahzad; kow, chia siang; hadi, muhammad abdul; zaidi, syed tabish razi; merchant, hamid a. title: mortality and disease severity among covid- patients receiving renin-angiotensin system inhibitors: a systematic review and meta-analysis date: - - journal: am j cardiovasc drugs doi: . /s - - - sha: doc_id: cord_uid: p oeac introduction: the use of renin-angiotensin system (ras) inhibitors, including angiotensin-converting enzyme inhibitors (aceis) and angiotensin receptor blockers (arbs), was alleged to cause a more severe course of novel coronavirus disease (covid- ). methods: we systematically reviewed the published studies to assess the association of ras inhibitors with mortality as well as disease severity in covid- patients. a systematic literature search was performed to retrieve relevant original studies investigating mortality and severity (severe/critical disease) in covid- patients with and without exposure to ras inhibitors. results: a total of original studies were included for qualitative synthesis. twenty-four studies that reported adjusted effect sizes ( studies reported mortality outcomes and studies reported disease severity outcomes), conducted in ras inhibitor–exposed and unexposed groups, were pooled in random-effects models to estimate overall risk. quality assessment of studies revealed that most of the studies included were of fair quality. the use of an acei/arb in covid- patients was significantly associated with lower odds (odds ratio [or] = . , % confidence interval [ci] . – . ; n = , ) or hazard (hazard ratio [hr] = . , % ci . – . ; n = , ) of mortality compared with non-use of acei/arb. however, the use of an acei/arb was non-significantly associated with lower odds (or = . , % ci . – . ; n = ) or hazard (hr = . , % ci . – . ; n = ) of developing severe/critical disease compared with non-use of an acei/arb. discussion: since there was no increased risk of harm, the use of ras inhibitors for hypertension and other established clinical indications can be maintained in covid- patients. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. cardiovascular diseases including hypertension remain one of the most significant comorbidities identified in patients with novel coronavirus disease (covid- ) [ ] [ ] [ ] [ ] [ ] [ ] . the presence of cardiovascular diseases including hypertension tends to portend a more severe course of illness in patients hospitalized with covid- [ ] [ ] [ ] [ ] [ ] [ ] . some researchers were quick to link such an association with the use of renin-angiotensin system (ras) inhibitors, including angiotensinconverting enzyme inhibitors (aceis) and angiotensin type i receptor blockers (arbs), among patients with cardiovascular disease [ , ] . the causative pathogen for covid- , namely severe acute respiratory syndrome coronavirus (sars-cov- ), binds to the ace receptor on host cells to gain entry [ ] . in several animal models and human studies, the expression of ace is increased with the chronic administration of ras inhibitors, which suggests a possibility for the increased susceptibility to sars-cov- infection and a more severe course of illness with the use of ras inhibitors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . conversely, there are studies [ ] [ ] [ ] [ ] [ ] [ ] that reported no association between increased expression of ace receptor and the use of ras inhibitors. interestingly, a protective role of ace has been suggested in the mitigation of coronavirus-induced lung injury [ , ] . in addition, it has been theoreticized that ras inhibitors can be used as a potential therapy for covid- as both ace and its product angiotensin ii, for which their downstream effects are inhibited by ras inhibitors, seem to promote lung injury [ ] . it has been demonstrated that the coronavirus spike protein binds to ace , leading to ace downregulation, which in turn results in the retention of angiotensin ii due to a lack of its clearance/conversion to angiotensin ( - ) by ace and leads to vasoconstriction of pulmonary vessels, increased pulmonary vascular permeability, and increased inflammation due to overactivity of the vasoconstrictive ace/angiotensin ii pathway [ , ] . therefore, users of ras inhibitors should, in theory, be protected from acute respiratory distress syndrome (ards), the primary cause of mortality in covid- [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nonetheless, such a lung-protective mechanism is not firmly established in human trials. several learned cardiovascular societies have discredited the association between ras inhibitors and the increased susceptibility to covid- or associated poor outcomes and encouraged clinicians and patients to continue the use of prescribed ras inhibitors in hypertension or other established indications [ ] . in addition, several observational studies have since reported clinical outcomes among patients with covid- receiving ras inhibitors . as this issue remains one of the most debated subjects among clinicians and researchers amid the covid- pandemic, we systematically reviewed the data from available studies to date to critically examine the association of ras inhibitors use with mortality and disease severity in covid- . this systematic review was performed with a protocol in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) statements [ ] . to systematically review the mortality and disease severity in covid- patients receiving ras inhibitors, a literature search was performed using scientific databases to identify original studies (prospective or retrospective in design) published in full between january and august . the eligibility criteria, which were defined prospectively, are provided in box . original, observational (prospective or retrospective) studies included patients with coronavirus disease (covid- ) documented use of either angiotensin-converting enzyme (ace) inhibitors or angiotensin receptor blockers (arbs) reported frequency, percentage, and/or adjusted estimate of mortality or disease severity and/or adverse clinical outcomes (septic shock, admission to intensive care units) associated with covid- from any region or language two authors (ssh and csk) independently performed a systematic literature search in pubmed, google scholar, and two preprint repositories (medrxiv and ssrn) without language restriction up to th august . the medical literature was searched using the following search terms: angiotensin-converting enzyme or ace or ace inhibitor or angiotensin receptor blocker or arb or renin-angiotensin-system or renin-angiotensin-system or ras inhibitor or renin-angiotensin-aldosterone or raa inhibitor or raas inhibitor) and covid- or novel coronavirus or severe acute respiratory syndrome or sars-cov- . the search was limited to original observational studies (prospective or retrospective), involving human subjects, and published in any language. however, studies in the chinese language were only assessed by csk (native chinese speaker). the titles and abstracts of the resulting articles were first examined to exclude irrelevant studies. subsequently, the full texts of the remaining articles were read to determine if studies met the eligibility criteria in full. bibliographies of retrieved articles were also reviewed to search for additional studies. differing decisions were resolved by mutual consensus. articles were excluded if they contained no original data (narrative reviews, letters, opinions, and comments) or reported a combined severity and mortality endpoint without individual presentation of severity and mortality data. one of the authors (csk) extracted data independently on a microsoft excel spreadsheet (xp professional edition; microsoft, redmond, washington, usa) that was verified by the second reviewer (ssh). in the case of disagreement, a third author was involved to resolve, by consensus, any discrepancies with respect to the relevance of the sources. the following data were collected for each study: the name of the first author; country; publication year; study design; the number of subjects; the age of the subjects; the presence of hypertension; the frequency of deaths; the frequency of severe/critical disease; adjusted estimates; and confounders. the methodological quality of the eligible studies was examined using the newcastle-ottawa scale for cohort studies [ ] . the newcastle-ottawa scale is easy to use with its star rating system and is considered reliable to measure biases in cohort studies. each of the selected cohort studies was evaluated for selection of study group ( - stars), comparability or quality of adjustment for confounding factors ( - stars), and ascertainment of the outcome of interest ( - stars), with a maximum of nine stars representing the highest methodological quality. studies with a newcastle-ottawa scale score of > were regarded as high quality. the reported odds ratios (ors) and hazard ratios (hrs) that had been adjusted for potential covariates in the respective original studies and the corresponding % confidence intervals (cis) were extracted and pooled in a random-effects model to estimate the association between the use of aceis/ arbs and the risk of mortality and severe/critical illness in covid- patients. if a study reported the estimates from different multivariable models, the most extensively adjusted estimate in terms of the number of covariates was extracted. however, in the presence of different multivariable models adjusted for the same number of covariates, the model containing the most clinically meaningful covariates was extracted for the pooled analysis. a random-effects model was employed since we assumed that the treatment effect was not the same across all the studies included in the analysis. cochran's q heterogeneity test (q test) and a related metric, the i , were used to evaluate heterogeneity. we used a p value of . and an i value of % as the cut-off points for statistically significant heterogeneity. publication bias was examined through visual inspection of the funnel plot for symmetry. four different subgroup analyses were conducted: ( ) the first subgroup analysis considered only studies that included, exclusively, hypertensive covid- patients in their analysis, to determine the effect of aceis/ arbs in this patient population; ( ) the second subgroup analysis was based on the region where the studies were performed to determine the presence of heterogeneity in the effect of aceis/arbs across different regions globally; ( ) the third subgroup analysis considered only studies that reported separate estimates for the use of aceis and arbs, to determine the presence of differential effects between the two classes of ras inhibitors; and ( ) the fourth subgroup analysis was based on the different definitions of severe/ critical outcome in covid- , to determine the presence of heterogeneity in the effect of aceis/arbs according to the definitions of severe/critical outcome. all meta-analytical calculations were performed using meta xl, version . (epigear international, queensland, australia). our literature search yielded , unique titles. a prisma flowchart [ ] of the literature search and study selection with the number of studies at each stage is presented in fig. . after deduplication and application of the eligibility criteria, relevant full-text articles were examined for inclusion. of these, studies were excluded for three reasons: no original data, combined mortality and severity endpoint, or retraction. a total of original studies that compared the mortality and/or severity outcomes between covid- patients receiving an acei/ arb and their counterparts not receiving an acei/arb were finally shortlisted for the qualitative synthesis. the characteristics of the shortlisted studies are summarized in table . among shortlisted studies, studies were from china , nine studies were from italy [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ten studies were from the united states [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , four studies were from south korea [ ] [ ] [ ] [ ] , three studies were from france [ ] [ ] [ ] , two studies were from spain [ , ] , one study each was from belgium [ ] , denmark [ ] , hong kong [ ] , kuwait [ ] , singapore [ ] , turkey [ ] , and the united kingdom [ ] ; and one study included data from countries [ ] . there were studies that included hypertensive patients exclusively ( %) [ , , , - , , , - , , , , , , , , , , - , ] . the comparison of mortality and clinical severity outcomes between acei/arb users and non-acei/arb users with covid- is summarized in table . there were studies [ - , - , - , - , , , - , - ] and studies [ , , - , - , , , - , , , , , , , - , - ] , respectively, that reported mortality outcomes and clinical severity outcomes among covid- patients with and without the use of aceis/ arbs. among studies that reported mortality outcomes, studies [ , , , , , , , , , , , , - , , , , , , , ] provided adjusted mortality estimates with the use of an acei/arb relative to the non-use of an acei/arb. out of studies that reported outcomes on clinical severity, studies [ , , , , , , , , , , , , , , , ] provided adjusted estimates for severe/critical disease with the use of an acei/ arb relative to the non-use of an acei/arb. there were studies [ , , , , , , , , , , , , - , , , , , , , ] that provided adjusted estimates on the risk of mortality among acei/ arb and non-acei/arb users. the newcastle-ottawa scale [ ] was used for the quality assessment of all studies that provided adjusted estimates and were included in the meta-analysis of the risk of mortality associated with the use of aceis/arbs (supplementary [ , , , , , , , , , - , , , , , ] . representativeness of the exposed cohort could not be established in studies [ , , , , , , , , , , , , , , , ] that included hospitalized patients only. in studies [ , , , , - , , , , , , ] , it was unclear whether the entire included cohort of patients was followed until discharge/death. since selçuk et al. [ ] provided mortality estimates with a very wide ci (or = . ; % ci . - . ), we excluded this study from our meta-analysis. in a pooled analysis of the remaining original studies that provided adjusted ors, with , covid- patients being analyzed [ , , , , , , , , , , , ] , the use of an acei/arb was significantly associated with lower odds of mortality compared to non-use of an acei/arb ( fig. ; pooled or = . , % ci . - . ). in a separate pooled analysis of studies that provided adjusted hrs, with , covid- patients being analyzed [ , , , , , , , , , , ] , the use of an acei/arb was significantly associated with lower risk of mortality . compared to the non-use of an acei/arb (supplementary figure s ; pooled hr = . , % ci . - . ). the funnel plot was used to detect the publication bias and revealed some degree of asymmetry (supplementary figure s and s ). subgroup analysis that was limited to studies that provided adjusted mortality estimates for exclusively hypertensive patients with covid- demonstrated a statistically non-significant association with lower odds of mortality (supplementary table s ; pooled or = . , % ci . - . ; six studies [ , , , , , ] ) and a statistically significant association with lower risk of mortality (supplementary table s ; pooled hr = . , % ci . - . ; five studies [ , , , , ] ) among users of aceis/arbs compared to the non-users. [ , , , ] in the users of aceis/arbs compared to the non-users. subgroup analyses that were limited to studies that provided respective mortality estimates (adjusted or) for acei and arb showed a statistically non-significant association [ ] defined severe/critical outcomes from covid- based on the requirement for admission into intensive care units or intubation. eleven studies did not classify patients into severe/critical cohort and non-severe/critical cohort; however, in our analyses, we considered patients with septic shock (for zhang et al. [ ] ) and those admitted into intensive care units (for the remaining ten studies [ , , , , , , , , , ] ) as having a severe/critical course of covid- . the newcastle-ottawa scale [ ] was used for the quality assessment of all studies that provided adjusted estimates and were included in the meta-analysis of severe/critical outcomes associated with the use of aceis/arbs (supplementary table s ). of the original studies included, only one study, by zhang et al. [ ] , was deemed 'good', with a score of points. ten studies [ , , , , , , , , , ] were regarded as 'fair', with scores of - points. five studies [ , , , , ] were considered 'poor' (i.e., scored less than points) in the quality assessment. the main quality issue noticed in studies [ , , , , , , , , , , , , , ] was the comparison of cohorts with inadequate adjustment for confounders that may influence the estimated risk of severe/critical disease associated with the use of aceis/arbs. another major issue detected during quality assessment in studies [ , , , , , , , , , , , ] was the inability to ascertain the exposure to aceis/arbs during the course of illness, where the possibility of discontinuation of aceis/arbs upon covid- diagnosis could not be ruled out based on the study design. similarly, studies [ , , , , , , , , , , , ] were not able to demonstrate that severe/critical outcome was not present at the start of the study based on their study design. in studies [ , , , , , , , , , , ] , it was unclear whether the entire cohort of patients was followed until discharge/death. the representativeness of the exposed cohort could not be established in ten studies [ , , , , , , , , , ] that included hospitalized patients only. in a pooled analysis of original studies that provided adjusted ors, with covid- patients being analyzed [ , , , , , , , , , , , , ] , the use of an acei/arb was non-significantly associated with lower odds of developing severe/critical disease compared to the non-use of an acei/arb ( fig. ; pooled or = . , % ci . - . ). in a separate pooled analysis of three studies that provided adjusted hrs, with covid- patients being analyzed [ , , ] , the use of an acei/arb was non-significantly associated with a lower risk of developing severe/critical disease compared to the non-use of an acei/ arb (supplementary figure s ; pooled hr = . , % ci . - . ). the funnel plot was used to detect the publication bias and revealed some degree of asymmetry. subgroup analysis that was limited to six studies that provided adjusted mortality estimates for exclusively hypertensive patients with covid- [ , , , , , ] subgroup analyses limited to the studies that provided respective estimates for severe/critical outcomes (adjusted or) for aceis and arbs observed a statistically significant association with higher odds of development of severe/ critical illness with the use of an acei (supplementary table s ; pooled or = . , % ci . - . ; five studies [ , , , , ] ), but a statistically non-significant association with lower odds of development of severe/critical illness with the use of an arb (supplementary table s ; pooled or = . , % ci . - . ; five studies [ , , , , ] ), compared to the non-use of an acei and an arb, respectively, among patients with covid- . subgroup analysis with regards to different definitions of severe/critical outcomes used across the studies reported a statistically non-significant association with lower odds of developing severe/critical illness according to the definitions of the chinese national health commission (supplementary table s ; pooled or = . , % ci . - . ; three studies [ , , ] ) and a non-significant association with lower odds of being admitted into intensive care units (supplementary table s ; pooled or = . , % ci . - . ; eight studies [ , , , , , , , ] ), with the use of aceis/arbs compared to the non-use of aceis/arbs. to the best of our knowledge, this systematic review and meta-analysis is the most comprehensive exploration and analysis of the existing literature in terms of mortality and clinical outcomes with the use of ras inhibitors to date. an in-depth analysis of study methods and findings for each original study is of utmost importance to prevent false assumptions about the safety of ras inhibitors in covid- . from our literature review, there have been a large number of observational studies [ - , - , - , - , , , - , - ] thus far evaluating the association between the use of aceis/arbs in covid- and mortality since concerns were raised with regard to the potential harms from the use of these drugs. however, conflicting results were observed among these studies. there were studies [ , - , , , , , , , , , - , , , - ] that reported a higher crude mortality rate; conversely studies [ , , , , , , , - , , , , , , , , ] reported a lower crude mortality rate among acei/arb users with covid- compared with their counterparts without receiving aceis/arbs. however, as mentioned earlier, there were only a few studies that adjusted covariates that may confound the association between the use of aceis/arbs in covid- and the risk of mortality. with different extents of adjustment for covariables, all [ , - , , , ] but one study [ ] (which reported a higher crude mortality rate among acei/arb users compared to non-acei/arb users) demonstrated no significant difference in the risk of mortality between acei/ arb users and non-acei/arb. the study by selçuk et al. [ ] reported a higher crude mortality rate and provided mortality estimates with a very wide ci (or = . ; % ci . - . ); therefore, the reliability of their findings is questionable. in fact, in our random-effects meta-analysis of studies that provided adjusted mortality estimates, we observed significantly lower risk of mortality with the use of aceis/arbs in covid- patients relative to non-use of aceis/arbs. our findings are in line with previously reported meta-analyses [ ] [ ] [ ] [ ] [ ] with unadjusted estimates that demonstrated no increased risk of mortality with the use of aceis/arbs in covid- patients. similarly, upon extensive literature review, we found that a vast number of observational studies [ , , - , - , , , - , , , , , , , - , - ] have thus far evaluated the association between the use of aceis/arbs in covid- and clinical severity of illness, albeit with conflicting results. there were studies [ , , , , , - , , , , , , - ] that reported a higher crude rate for the severe/critical outcomes; however, studies [ , , , , - , , , , , ] reported a lower crude rate for severe/critical clinical outcomes, in acei/arb users with covid- compared to non-users. nonetheless, only a few studies adjusted covariates that may have confounded the association between the use of aceis/ arbs in covid- and clinical severity. with different extents of adjustment for covariables, among seven studies [ , , , , , , ] that reported a higher crude rate for severe/critical outcomes, five studies [ , , , , ] demonstrated no significant difference with regards to the risk of severe/critical outcomes between acei/arb users and non-acei/arb users. conversely, liabeuf et al. [ ] and mehta et al. [ ] demonstrated significantly higher odds for admission into intensive care units with the use of an acei/arb relative to the non-use of acei/arb upon adjustment of covariates, but arguably this may be due to the residual confounding effect. in fact, the random-effects meta-analysis of studies with adjusted estimates observed no significant association between the use of aceis/arbs and severe/critical outcomes in covid- patients, which concurs with previously reported unadjusted meta-analyses [ , , ] that showed no increased risk of developing severe/ critical illness with the use of aceis/arbs in covid- patients. nevertheless, an unadjusted meta-analysis [ ] reported a significantly reduced risk of developing severe/ critical illness with the use of aceis/arbs in covid- patients. the subgroup analysis is important to determine the presence of heterogeneity on the effect of ras inhibitors based on different definitions of severe/critical outcomes. we observed no significant association with the use of aceis/ arbs and severe/critical outcomes based on the definition by the chinese national health commission. this is in line with another meta-analysis [ ] that pooled studies that defined the clinical severity based on the chinese national health commission. we also observed no significant association with the use of aceis/arbs and severe/critical outcomes based on admission into intensive care units. when studies that included hypertensive patients exclusively or provided a separate analysis for hypertensive patients were analyzed, we observed a greater number of studies (n = ) [ , , , , , , , , - , , , , , ] reporting a lower crude mortality rate than studies (n = ) [ , , , , , , , ] reporting higher crude mortality rates in covid- hypertensive acei/arb users relative to the non-acei/arb users. with the exception of the study by selçuk et al. [ ] , all studies that reported a higher crude mortality rate [ , , ] demonstrated no significant difference with regards to the risk of mortality between acei/arb users and non-acei/ arb users upon adjustment for covariables. we observed a significantly lower risk of mortality in covid- acei/ arb users in our subgroup meta-analyses that were limited to studies that provided adjusted mortality estimates for exclusively hypertensive patients. these findings are in line with two previously reported unadjusted meta-analyses in covid- hypertensive patients [ , ] . however, another meta-analysis [ ] reported no significant association between the use of aceis/arbs and mortality among covid- patients with hypertension where the authors pooled mortality estimates without adjustment of covariates. likewise, for studies that included hypertensive patients exclusively or provided a separate analysis for hypertensive patients, we also observed a greater number of studies (n = ) [ , , , , , , , , , , ] reporting a lower crude rate for severe/critical outcomes than studies (n = ) [ , , , , , , , ] reporting a higher crude rate for severe/critical outcomes, though the only study [ ] that reported a higher crude rate for severe/critical outcomes and provided an adjusted estimate demonstrated no significant difference with regards to the risk of severe/ critical outcomes between acei/arb users and non-acei/ arb users. indeed, our subgroup meta-analyses that were limited to studies that provided adjusted estimates for severe/ critical outcomes in exclusively hypertensive patients noted a significantly reduced risk of developing severe/critical disease among covid- acei/arb users. it is interesting to observe that acei/arb users with or without hypertension had a lower risk of death from covid- , but no difference with regards to the risk of developing severe/critical illness from covid- , compared to non-acei/arb users with or without hypertension. equally interesting is the observation that hypertensive acei/arb users had a lower risk of death and a lower risk of developing severe/critical disease from covid- compared to hypertensive non-acei/arb users. one explanation for such observations is that ras inhibitors may be able to protect covid- patients from angiotensin ii-related lung injury and subsequent ards, the major cause of mortality in covid- patients [ ] . however, since the definition of severe/critical illness in our meta-analysis did not specifically include ards, such a protective effect was not reflected in the risk of developing severe/critical illness in our analysis. in fact, a retrospective case-control study [ ] performed before the covid- pandemic reported better survival rates among patients with ards who received ras inhibitors compared to their counterparts who did not receive ras inhibitors. another plausible explanation for the observations is that hypertensive patients may be more sensitive to the protective effects of ras inhibitors against the development of a fatal or severe/critical course of illness from covid- since they have a higher baseline risk of a worse prognosis. in fact, the mortality benefits demonstrated in the meta-analysis were driven mainly by studies that provided adjusted mortality estimates for exclusively hypertensive patients (constituting % of the weight of the meta-analysis on mortality). in contrast, studies that provided adjusted estimates for severe/critical outcomes in exclusively hypertensive patients only constituted . % of the weight of the meta-analysis on severe/critical illness. since previous meta-analyses [ ] [ ] [ ] [ ] [ ] [ ] [ ] included a majority of studies from china, the generalizability of their findings to patient populations from other regions was also questioned. in the present study, we observed no regional difference with regard to mortality estimates, where separate pooled analyses of studies from east asian countries, european countries, and the united states, respectively, did not find an increased risk of mortality with the use of aceis/ arbs. nonetheless, our findings did suggest a potential regional difference in the risk of severe/critical outcomes with the use of aceis/arbs in which pooled analysis of studies from east asian countries demonstrated a significant association with lower odds of developing severe/critical disease compared to the studies from europe and the united states. since east asian populations have a genetically higher expression of ace compared to caucasians (europeans or americans) [ ] , the downregulation of ace upon a viral invasion of the host cells by sars-cov- may be consequently greater in east asian populations. therefore, east asian populations with covid- may be more sensitive to the protective effect of ras inhibitors against lung injuries from downregulation of ace and the subsequent development of severe/critical illness, though it does not appear to translate to any significant mortality benefits. nevertheless, these findings should be confirmed in a large pharmacogenetic study comparing the effects of aceis/ arbs in covid- patients of different ethnicities. while both aceis and arbs could prevent lung injuries induced by angiotensin ii in covid- , arbs can provide further protection due to their different mechanisms of action. in its native state, the angiotensin type i receptor (at r) binds to ace to form a receptor complex [ ] . arbs stabilize the ace -at receptor complex on the cell membrane and thus prevent the interaction of sars-cov- with the ace catalytic site. in addition, arbs are reported to possess anti-inflammatory effects due to their ability to utilize the ace /angiotensin ii type receptor/mas receptor pathway that could limit sars-cov- -mediated cytokine storm. it is therefore interesting to determine the respective effects of aceis and arbs in covid- patients. we demonstrated no significant difference in the risk of mortality with the use of either aceis or arbs in covid- patients compared to non-use of either of these two agents. surprisingly, we found that aceis were associated with higher odds of developing severe/critical illness from covid- relative to non-use of aceis, while no difference in the risk of developing severe/critical illness from covid- was found with the use of arbs relative to non-use of arbs. in the pooled analysis, the increased risk of developing severe/critical illness from covid- is largely driven by the findings in the study by mehta et al. [ ] (constituting % of the weight of the meta-analysis), which revealed higher odds of admission into intensive care units with the use of aceis compared to the non-use of aceis. it should be noted that there may be confounding by indication since mehta et al. [ ] compared the clinical outcomes between covid- users of aceis who had comorbidities and covid- non-users of aceis who may or may not have had comorbidities. in fact, the other studies [ , , , ] included in the subgroup pooled analysis reported no difference in the risk of severe/ critical illness with the use of aceis compared to non-use of the aceis. a key strength of this systematic review and meta-analysis was the pooling of adjusted estimates on the mortality and severe/critical outcomes from the use of ras inhibitors in covid- patients. previous meta-analyses were either unadjusted [ - , , ] or only pooled adjusted estimates for combined mortality and severity endpoints [ ] . inclusion of studies that provided adjusted estimates would reduce the confounding effects that could modify the association between the use of ras inhibitors and risk of adverse clinical outcomes in covid- . furthermore, independent analysis of the risk of mortality and the risk of severe/critical outcomes, instead of combining mortality and severity endpoints, enabled the evaluation if the effect of ras inhibitors on the risk of severe/critical outcomes will translate the same to the risk of mortality. as discussed above, we have observed on two occasions that the effect of the use of ras inhibitors on severe/critical outcomes did not translate the same to the risk of mortality. another key strength of our systematic review and meta-analysis lies in our comprehensive inclusion of a large number of studies across different countries and continents. previous metaanalyses included studies mainly from china and therefore were limited regarding the generalizability of their findings to other populations. inclusion of a large number of studies with adjusted estimates allowed different subgroup analyses to be performed in our study, including subgroup analysis on hypertensive cohort, which reduced confounding by indication, subgroup analysis stratified by the regions where the studies were conducted, to evaluate the regional difference in the effects of ras inhibitors on clinical outcomes in covid- , and subgroup analysis to determine the respective effects of aceis and arbs on clinical outcomes in covid- . nonetheless, this study has also limitations that should be considered while interpreting the findings. notably, although we pooled only studies that provided adjusted estimates, most of the studies did not adequately adjust for all confounders. other than demographic characteristics, major confounders include comorbidities, co-medications, and the therapies intended for treating covid- . it is acknowledged that patients with comorbidity, especially cardiovascular diseases and diabetes mellitus, are particularly vulnerable to the severe course of covid- [ , ] . moreover, some co-medications such as statins are reported to exert protective effects on mortality or the development of severe/ critical disease from covid- [ , ] . in addition, the varying proportion of patients receiving various potential therapies for covid- in the absence of an established treatment may also confound the association between the use of ras inhibitors and clinical outcomes from covid- . among the studies included in our meta-analysis, only two studies properly adjusted for major confounders (zhang et al. [ ] and zhou et al. [ ] ), and coincidentally these two studies also reported a significantly reduced risk of mortality from covid- with the use of ras inhibitors. despite these findings, residual confounding cannot be completely ruled out, like in any observational study. moreover, limited by the study designs, we could not establish with certainty whether ras inhibitors were continued during the course of the disease in covid- patients, as the use of ras inhibitors was only established via medical record review or medical database review in majority of the studies included. furthermore, there were some studies in which the duration of follow-up may not have been long enough for the outcomes of interest (mortality and/or severe/critical illness) to occur. for patients with or without hypertension regardless of the treatment setting, it can be concluded from our metaanalyses that the use of ras inhibitors would not increase the risk of mortality or severity in covid- . indeed, the risk, if any, is 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statins is associated with a reduced risk of mortality among individuals with covid- meta-analysis of effectiveness of statins in patients with severe covid- key: cord- -mmpox authors: izumi, yasukatsu; iwao, hiroshi title: angiotensin ii peptides date: - - journal: handbook of biologically active peptides doi: . /b - - - - . -x sha: doc_id: cord_uid: mmpox abstract much evidence supports the notion that angiotensin ii (ang ii), the central product of the renin–angiotensin system (ras), may play a central role not only in the etiology of hypertension but also in the pathophysiology of cardiovascular diseases in humans. ang ii, via the ang ii type receptor, directly causes cellular phenotypic changes and cell growth, regulates the gene expression of various bioactive substances, and activates multiple intracellular signaling cascades in cardiac myocytes and fibroblasts, as well as vascular endothelial and smooth muscle cells. recently, new factors have been discovered, such as angiotensin-converting enzyme , angiotensin-( - ), and its receptor mas. this section summarizes the current knowledge about the broad ras in the pathophysiology of cardiac hypertrophy and remodeling, heart failure, vascular thickening, and atherosclerosis. research on the renin-angiotensin system (ras) began with the discovery of renin from the kidney by tiegerstedt and bergman in . in , a peptide that had vasoconstrictive effects in the ras was discovered, and it was named hypertensin by braun-menendez in argentina, and angionin by page and helmer in the united states. these two terms persisted for about years, until it was agreed to rename the pressor substance angiotensin. in the s, two forms of angiotensin were recognized, the first a decapeptide (angiotensin i: ang i) and the second an octapeptide (angiotensin ii: ang ii). skeggs et al. in the united states reported that ang ii was formed by the enzymatic cleavage of ang i by another enzyme, termed angiotensin-converting enzyme (ace). schwyzer and bumpus succeeded in the synthesis of ang ii in . gross suggested, in , that the ras was involved in the regulation of aldosterone secretion, and then davis, genet, laragh et al. proved his hypothesis. in the early s, polypeptides either inhibiting the formation of ang ii or blocking ang ii receptors were discovered, but these were not orally active. cushman and ondetti succeeded in the development of captopril, the orally active ace inhibitor in . after that, many experimental and clinical studies with ace inhibitors uncovered additional roles for the ras in the pathophysiology of hypertension, heart failure, and vascular diseases. furthermore, losartan (dup ), an orally active, highly selective and potent nonpeptide ang ii receptor blocker (arb), was developed in , and the cloning of ang ii receptors, type (at r) and type (at r) was accomplished in the early s. angiotensin-( - ) [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] was discovered in by santos et al., and angiotensin-converting enzyme (ace ) was identified in , which catalyzes the conversion of ang i [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] to ang-( - ) by the removal of a single carboxy-terminal amino acid. ace is an essential regulator of heart function and a functional receptor for the sars coronavirus. the ras plays an important role in the regulation of arterial blood pressure. renin is an enzyme that acts on angiotensinogen to catalyze the formation of ang i. ang i is then cleaved by ace to yield ang ii. a representation of the biochemical pathways of ras is shown in fig. . the major element of the rate of ang ii production is the amount of renin released by the kidney. renin is synthesized, stored, and secreted into the renal arterial circulation by the granular juxtaglomerular cells. the secretion of renin is controlled predominantly by three pathways: the first mechanism controlling renin release is the intrarenal macula densa pathway and the second is the intrarenal baroreceptor pathway. the third mechanism is the β-adrenergic receptor pathway, which is mediated by the release of norepinephrine from postganglionic sympathetic nerve terminals. an increase in renin secretion enhances the formation of ang ii, and ang ii stimulates the at r on juxtaglomerular cells to inhibit renin release. the substrate for renin is angiotensinogen, an abundant α -globulin that circulates in the plasma. the primary structure of angiotensinogen has been deduced by molecular cloning. angiotensinogen is synthesized primarily in the liver, although mrna that encodes the protein is abundant in fat, certain regions of the central nervous system, and the kidney. the rate of ang ii synthesis can be influenced by changes in angiotensinogen levels. ace was discovered in plasma as the factor responsible for conversion of ang i to ang ii. the ace gene contains, in intron , an insertion/deletion polymorphism that explains % of the phenotypic variance in serum ace levels. individuals homozygous for the deletion allele have higher levels of serum ace. ang i is rapidly converted to ang ii, when given intravenously. ang iii [ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ] can be formed by the action of aminopeptidase on ang ii. ang iii ang ii cause qualitatively similar effects. ang iii is approximately as potent as ang ii in stimulating the secretion of aldosterone. however, ang iii is only % as potent as ang ii in elevating blood pressure. ang iv [ang- ( ) ( ) ( ) ( ) ( ) ( ) ] is generated by the sequential cleavage of two amino acid residues from the amino terminus of ang ii by aminopeptidases localized to the endothelial surface. there are bypass pathways in the ras to produce ang ii besides the main enzymes such as renin and ace. arakawa et al. showed that trypsin and kallikrein can produce ang ii. one of the most famous enzymes in such bypass pathways is a heart chymase that is secreted by mast cells. circulating renin of renal origin acts on circulating angiotensinogen of hepatic origin to produce ang i in the plasma, and circulating ang i is converted by plasma ace and pulmonary endothelial ace to ang ii. then, ang ii is delivered to its target organs via blood flow. this traditional pathway is called the circulating ras. however, heart, blood vessels, and several other tissues contain and/or synthesize components of the ras, called the tissue (local) ras. many tissues, including heart, blood vessels, brain, kidney, and adrenal gland, express mrnas for renin, angiotensinogen, and/or ace. furthermore, several other ang i cleavage products have been found in the cultured cell types from these tissues, for example, ang-( - ), ang-( - ), ang iii, and ang iv (fig. ) . ang i and ang ii have been localized in atria and ventricles. therefore, it seems that the tissue ras exists independently of the renal/hepatic-based system. several serine proteases, such as tonin and cathepsin g, have been shown to hydrolyze ang ii precursors. an aspartyl protease with cathepsin d-like properties was shown to convert angiotensinogen to ang i. conversion of ang i to ang ii in human and dog cardiac ventricles may occur by heart chymase. unlike ace, human heart chymase shows high specificity for ang i and does not degrade bradykinin or vasoactive intestinal peptide. it is likely that the relative contribution of ace and chymase to cardiac ang ii formation varies with the cardiac chamber in the human heart, because ace levels are the highest in the atria and chymase levels are the highest in ventricles. it is recently believed that chymase plays a crucial role in various tissues because chymase can not only produce ang ii but also convert precursors of transforming growth factor (tgf)-β and matrix metalloproteinase- to their active forms, whose increases damage various organs. the effects of angiotensins are exerted through specific cell surface receptors. there are two subtypes of ang ii receptors, types and (at r and at r). the successful cloning of the at r in and the at r in allowed the development of further research on the structure and function of this receptor. the at r consists of two subtypes, at a r and at b r, which have % homology with regard to amino acid sequence and have similar pharmacological properties and tissue distribution patterns. the at r is a member of the seven-transmembrane-spanning, g protein-coupled receptor (gpcr) family; it binds to heterotrimeric g proteins and lacks intrinsic tyrosine kinase activity. the human at r gene is mapped to chromosome , and the at a r and at b r genes in rats are mapped to chromosomes and , respectively. the at r is ubiquitously and abundantly distributed in adult tissues, including blood vessel, heart, kidney, adrenal gland, liver, brain, and the lung. the at r mediates all the classic well-known effects of ang ii, such as elevation of blood pressure, vasoconstriction, increase in cardiac contractility, aldosterone release from the adrenal gland, facilitation of catecholamine release from nerve endings, and renal sodium and water absorption. therefore, the at r has a role in atherosclerosis, congestive cardiac failure, and several acute and chronic inflammatory diseases, conditions in which inflammation is known to play a significant role (fig. ). numerous selective and potent nonpeptide at r antagonists, such as losartan, candesartan, valsartan, irbesartan, eprosartan, telmisartan, tasosartan, have been developed. in contrast, the at r has high affinity for pd , pd , cgp , l- , , l- , , and cgp a. the cdna and genomic dnas of human, rat, and mouse at rs have been cloned. the at r is mainly expressed in developing fetal tissues, and it is believed to have an essential role in physiological vascular development. the at r expression rapidly decreases after birth, and, in the adult, expression of this receptor is limited mainly to the uterus, ovary, certain brain nuclei, heart, and adrenal medulla. unlike the at r, which has been shown to have subtypes in rats and mice, there is no evidence for subtypes of the at r. although a comparison of amino acid sequences of at a r and at r in rats, deduced from nucleotide sequences, shows a low homology between these receptors ( %), the at r is also a seven-transmembrane domain receptor. in various cell lines, the at r-activated protein tyrosine phosphatase was shown to inhibit cell growth or induce programmed cell death (apoptosis). the at r inhibited at r-mediated cell growth, demonstrating an antagonistic action. it is believed that the expression balance of at and the at is important for cardiovascular diseases. there have also been conflicting findings regarding these receptors. for example, it is controversial whether cardiac hypertrophy is promoted by the at r. however, the at r plays a cardioprotective role on left ventricular function after myocardial infarction. in contrast to extensive data on the molecular and cellular functions and pathophysiological significance of the at r, the role of the at r in cardiovascular diseases remains to be defined. at present, the implications of overstimulation of at r by long-term use of arb are being investigated in clinical trials. it has been suggested that ang-( - ) mediates its effects by interacting with the gpcr mas, a prototypic seventransmembrane domain receptor, which is predominantly expressed not only in the brain and testis but is also found in the kidney, heart, and blood vessels. the ang at receptor was originally defined as the specific, high-affinity binding site for ang iv. therefore, it was suspected to be classically a gpcr. and then, albiston et al. have identified it as the transmembrane enzyme, insulinregulated aminopeptidase (irap), an abundant protein that is found in specialized vesicles containing the insulinsensitive glucose transporter glut . generally, pathological left ventricular hypertrophy is characterized not only by an increase in myocyte size (quantitative change) but also by myocyte gene reprogramming (qualitative change), as shown by enhanced expression of fetal phenotypes of genes such as β-myosin heavy chain (β-mhc), skeletal α-actin, and atrial natriuretic peptide (anp). in the cardiac ventricle of most mammalian species, mhc consists of two isoforms, α-and β-mhcs. in the rat, α-mhc is the predominant isoform in adult hearts, whereas β-mhc is the predominant isoform in fetal hearts. therefore, changes in the ratio of β-mhc to α-mhc in the cardiac ventricle significantly alter the contractile properties of the heart. cardiac sarcomeric actin is also composed of two isoforms: cardiac α-actin and skeletal α-actin. cardiac α-actin is predominantly expressed in adult rat hearts, whereas skeletal α-actin is normally expressed in fetal and neonatal rat hearts. the ratio of skeletal α-actin to cardiac α-actin in the ventricle plays a significant role in cardiac function, because skeletal α-actin has greater contractility than cardiac α-actin. furthermore, in addition to showing enhanced expression of β-mhc and skeletal α-actin, hypertrophic ventricular myocytes are also characterized by significant upregulation of anp, which is scarcely expressed in normal adult ventricular myocytes. another important property of pathological cardiac hypertrophy is increased accumulation of extracellular matrix (ecm) proteins such as collagen (particularly collagen types i and iii) and fibronectin in the interstitium and around blood vessels within the heart. these changes play a central role in ventricular fibrosis or remodeling. , increased interstitial collagen deposition in the heart enhances cardiac stiffness and results in diastolic dysfunction. fibronectin is localized on the surface of cardiac myocytes, connects cardiac myocytes to perimyocytic collagen and is believed to affect cardiac systolic and diastolic functions. thus, increased ecm accumulation, and the abovementioned ventricular myocyte gene reprogramming play critical roles in the impairment of cardiac performance and pathophysiology of cardiac failure. ecm proteins within the heart are predominantly produced by fibroblasts. unlike cardiac myocytes, cardiac fibroblasts proliferate and increase the production of ecm proteins when the heart is exposed to hypertrophic stimuli such as hemodynamic overload. thus, cardiac fibroblasts and cardiac myocytes play key roles in the development of pathological cardiac hypertrophy and dysfunction. accumulating in vitro and in vivo evidence supports the concept that ang ii is involved in all these important processes of pathological cardiac hypertrophy, including myocyte hypertrophy, myocyte gene reprogramming, fibroblast proliferation, and ecm protein accumulation. effect of at r on cardiac remodeling (fig. ) activated at r can activate multiple signaling pathways. the acute vasoconstrictor function of ang ii is primarily mediated through at r by classical g protein-dependent signaling mechanisms. investigations of the effects of ang ii on cardiac intracellular signaling cascades are essential to elucidate the molecular mechanism underlying ang iiinduced pathological cardiac hypertrophy. accumulating in vitro evidence on cultured cardiac myocytes or fibroblasts suggests that mitogen-activated protein (map) kinases, including extracellular signal-regulated kinase (erk), c-jun amino terminal kinase (jnk) and p map kinase (p mapk), may be responsible for myocyte hypertrophy and gene reprogramming or fibroblast proliferation. ang iiinduced cardiac activation of jnk occurs in a more sensitive manner than that of erk. the erk cascade activates platelet-derived growth factor (pdgf), epidermal growth factor receptor (egfr), insulin receptor pathways, and nonreceptor tyrosine kinases belonging to the c-src family, prolinerich tyrosine kinase , focal adhesion kinase, and janus kinases (jaks). besides the g protein-dependent effects of ang ii activated at r, the activated at r can also stimulate g protein-independent signal transduction mechanisms by directly associating with signaling molecules, such as β-arrestins, jak, cdc , and src. jnk activation by ang ii without erk activation is followed by activation of activator protein- (ap- ). importantly, ap- regulates the expression of various genes by binding the ap- consensus sequence present in their promoter regions. interestingly, fetal phenotypes of cardiac genes such as skeletal α-actin and anp, and cardiac fibrosisassociated genes such as tgf-β and collagen type i, have ap- responsive sequences in their promoter regions. indeed, ap- activation has been demonstrated to lead to increased promoter activity of skeletal α-actin and tgf-β . therefore, it is intriguing to postulate that jnk activation, in part through the activation of ap- , may be implicated in ang ii-induced cardiac hypertrophic response in vivo. an in vivo study showed that apoptosis signal-regulating kinase (ask ), one of the map kinase kinase kinases, plays an important role in ang ii-induced cardiac hypertrophy and remodeling, including cardiomyocyte hypertrophy, cardiac hypertrophy-related mrna upregulation, cardiomyocyte apoptosis, interstitial fibrosis, coronary arterial remodeling, and collagen gene upregulation. the ras, tgf-β, and β-adrenergic system network mediates this myocardial remodeling. tgf-β mrna and protein expression is augmented in cardiomyocytes and cardiac fibroblasts, which in these situations transdifferentiate to myofibroblast phenotype, following progressive diastolic dysfunction. ang ii caused a significant increase in tgf-β mrna. chronic administration of ang ii-induced tgf-β protein expression in myocardium. ang ii-induced hypertrophic growth of cardiomyocytes is mediated by tgf-β , which is downstream to ang ii in network. mice expressing constitutively hyperactive tgf-β had a chronic hypertrophy, fibrosis, and cardiac dysfunction that was resistant to the arb telmisartan but blocked by tgf-β antagonist. the ang ii-mediated cardiac remodeling mechanism is thus dependent on tgf-β . from all the studies performed so far, it is clear that ang ii/tgf-β induced autocrine-paracrine cellular responses in cardiac fibroblasts, in the myocardial interstitium, and cardiomyocytes cause cardiac hypertrophy. ang ii-induced tgf-β signaling also induces translocation of smad proteins into the nucleus to drive transcription of fibrotic marker proteins such as collagen, fibronectin, and connective tissue growth factor (ctgf). ctgf is a profibrotic factor that stimulates tgf-β responses mediating fibrosis and apoptosis. the inflammatory cytokines such as interleukin- , interleukin- , vascular cell adhesion molecule- , and monocyte chemoattractant protein- , are believed to have important roles in the coupling acute ischemic episodes and chronic myocardial remodeling with chronic angiotensin receptor stimulation. ang ii facilitates the release of norepinephrine from cardiac sympathetic nerve terminals. in ang ii-infused rats, surgical cardiac sympathectomy or treatment with β -adrenergic receptor blocker significantly prevented cardiac myocyte necrosis, showing that ang ii-induced cardiac damage is, at least in part, mediated by catecholamine release from cardiac sympathetic neurons. thus, the activation of cardiac sympathetic neurons by ang ii also contributes to pathological cardiac hypertrophy. ang ii, via the at r, is also known to be a potent mediator of oxidative stress and reactive oxygen species (ros)mediated signaling. a large body of evidence indicates that ros plays a major part in the initiation and progression of cardiovascular dysfunctions associated with diseases such as hyperlipidemia, diabetes mellitus, hypertension, ischemic heart disease, and chronic heart failure. in addition, ang ii signaling activates membrane nad(p)h oxidases in vsmcs to produce ros, which mediate the pleiotrophic effects of ang ii. ang ii signaling mediated activation of nad(p)h oxidases involves the upstream mediators src/ egfr/pi k/rac- and pld/pkc/p phox phosphorylation. , transcription factors such as nuclear factor-κb, ap- , and nrf that are implicated in the pathogenesis of atherosclerosis are activated by ros. ros may cause vessel inflammation by inducing the release of cytokines and causing an increase in the expression of the leukocyte adhesion molecules in the cell membranes. this may increase recruitment of monocytes to the area of endothelial damage. thus, activated at r-induced ros production can lead to changes in structural and functional properties of the vasculature and is the central aspect of vascular pathobiology in hypertension, diabetes, and cardiovascular diseases. numerous in vivo experiments have shown that ang ii can induce vsmc proliferation in vivo. ang ii infusion increased mesenteric vascular media width, media crosssectional area, and media/lumen ratio. despite detailed investigations into the molecular mechanism of ang ii-mediated vsmc growth in vitro, this process is poorly understood. ang ii infusion, at least in part independent of its blood pressure-elevating effect, increased aortic mrna and protein expression of fibronectin, which is an ecm protein that induces phenotypic change of vsmcs from a contractile to a synthetic phenotype. basic fibroblast growth factor (bfgf) may play a key role in ang ii-mediated vsmc replication in vivo, as shown by the observation that the injection of anti-bfgf antibody significantly inhibited the mitogenic effect of ang ii infusion on rat carotid arteries. ang ii infusion in rats doubled superoxide production in rat aorta by activation of nad(p)h oxidase. on the other hand, norepinephrine infusion did not increase vascular superoxide production, despite a hypertensive effect comparable to that of ang ii, suggesting that vsmc growth due to ang ii may be specifically mediated by increased superoxide generation. ang ii infusion in rats increased heme oxygenase- (ho- ) mrna and protein in the endothelium. because ho- is an oxidant-sensitive gene, it is possible that increased oxidative stress is a trigger for ho- mrna upregulation in the ang ii-infused rat aorta and that ho- may serve to abrogate this increased stress caused by ang ii. ang ii infusion stimulated aortic thrombin receptor mrna expression in rats, which was blocked by either arb or the heparin-binding chimera of human cu/zn superoxide dismutase but not by normalization of blood pressure with hydralazine treatment, suggesting that ang ii increases vascular thrombin receptor by at r-mediated superoxide production and may be implicated in the pathophysiology of atherosclerosis by thrombin cascade activation. the injection of ang ii resulted in increased oxidized low-density lipoprotein uptake by peritoneal macrophages and increased macrophage proteoglycan content, suggesting that ang ii may accelerate atherosclerosis by promoting foam cell formation and cholesterol accumulation in the vascular wall. endothelial dysfunction in the context of hypertension and cardiovascular diseases is partly dependent on the production of ros. at r-induced ros not only burns nitric oxide (no) but also reduces no formation in the endothelium by oxidizing tetrahydrobiopterin, a crucial cofactor for endothelial no synthase (enos). superoxide anion, a highly reactive ros, is known to cause breakdown of endothelial-derived relaxing factor and dysfunction of endothelium. regenerated endothelium has an impaired ability to release endothelial-derived relaxing factors, in particular no. activation of angiotensin receptors triggers an efflux of cytosolic calcium in endothelial cells (ecs). calcium efflux activates pla- to release arachidonic acid, which is metabolized by cyclooxygenase (cox) to generate various eicosanoids including endoperoxides and various prostaglandins that activate thromboxane a /prostanoid receptors located on the vsmcs causing contractions. endothelial cox activity also produces ros. in addition, uric acid, despite its known antioxidant effects, may cause human vascular ec dysfunction by local activation of ras, inducing oxidative stress. ros may, in turn, activate local ras of whole vasculature, including ec, smooth muscle cells, fibroblast-enhancing ang ii production and concomitant stimulation of at r to create a positive feedback loop between local ras and ros. thus, in hypertensive cardiovascular diseases, endothelial dysfunction is characterized by blunted endothelium-dependent vasodilations or enhanced endothelium-dependent contractions. , generally, at r is believed to promote apoptosis and inhibit proliferation and hypertrophy. unlike at r, at r contributes to maintenance of blood pressure by controlling the vascular tone through vasodilatation. available evidence indicates links between at r and bradykinin b receptor (b r) in no production in vasodilation. , at r stimulation by ang ii leads to an increase in cgmp levels through a mechanism involving b r, causing no release. at r activation stimulates bradykinin formation by activating kininogenases. chronic infusion of ang ii into at r overexpressing mice completely abolished the at r-mediated pressor effect, suggesting vasodilatation by at r. this was blocked by one of the inhibitors of b r and nos. in addition, aortic explants from these transgenic mice overexpressing at r showed greatly increased cgmp/no production and diminished at r-induced vascular constriction. removal of endothelium or treatment with the inhibitors of b r and nos abolished these at r-mediated effects, indicating that endothelium is the primary site of effect manifestation by at r. the at r in aortic smcs also stimulates the production of bradykinin, which stimulates the no/cgmp system to promote vasodilation. in the at r-deficient mice, bradykinin-induced dilation is seriously impaired. bradykinininduced dilation is inhibited by at r antagonist but not by losartan, an arb. thus, at r and b r may form functional heterodimers, as recently shown, and this might lead to increased no and cgmp production. the physical association between the dimerized at r and b r initiates changes in intracellular phosphoprotein signaling activities and enhanced production of no and cgmp. , growth and apoptosis are also two opposite components of tissue remodeling. cardiac remodeling happens in certain physiological or pathological circumstances, such as exercise, aging, hypertension, and myocardial infarction. it is known that although at r expression decreases sharply after birth, it can increase again in some pathophysiological conditions. stimulation of at r expression may inhibit neointima formation, cell proliferation, inflammation in vascular injury, myocardial infarction, and ischemic diseases, suggesting its protective role. conversely, in at r-deficient mice, recovery from acute myocardial infarction was reduced compared to the wild-type mice. although neointima formation and vsmc proliferation after vascular injury were exaggerated in at r-deficient mice, they were suppressed in at a r-deficient mice. at r possibly exerts antiproliferative effects and proapoptotic effects in vsmcs by controlling at a r. the number of apoptotic cells in the injured artery was increased significantly in at a r-deficient mice but decreased in at rdeficient mice, indicating that at r mediates apoptosis. selective at r stimulation also inhibited restenosis after balloon angioplasty. thus, experimental studies have shown a beneficial role for the reexpression of the at r after vascular injury leading to inhibition of cell growth and promotion of apoptosis. gene transfer studies in vivo have also supported that at r expression attenuated neointima accumulation in injured carotid arteries by antagonizing growth-promoting effects of at r. vascular injury studies using mice with disrupted at r expression showed enhanced neointimal formation compared with wild-type mice, suggesting a protective role for at r. at r expression is upregulated in several disease conditions. in the left ventricle-specific at r overexpression mice, elevated left ventricular endodiastolic pressure, increased systolic and diastolic dimensions, severely depressed left ventricular fractional shortening, and wall thinning were observed, suggesting that ventricular myocytespecific expression of at r promotes the development of dilated cardiomyopathy and heart failure in vivo. in vivo ectopic at r overexpression in postnatal left ventricular myocardium is thus detrimental, explaining the sharp decline of at r after birth. if prolonged arb treatment turns out to be completely beneficial through clinical trials, then it is either a dosage effect or it depends on the time and site of at r expression. the implications of overstimulation of at r by long-term use of arb are being investigated in clinical trials. ang-( - ) mediates its effects by interacting with the grcr, mas, a prototypic seven-transmembrane domain receptor, which is not only predominantly expressed in the brain and testis but is also found in the kidney, heart, and blood vessels. moreover, several studies have shown that the interaction of ang-( - ) with mas induces the protective cardiovascular actions such as no release, akt phosphorylation and vasodilation. ang-( - ) also functions as an antithrombotic, antiproliferative, and antioxidative agent. although the mechanisms of action in the vascular system has not yet been well established, several studies have focused on control of the no and o − balance, which regulates cardiovascular function. a reduction in superoxide dismutase and catalase activity in mas-deficient mice demonstrates impaired antioxidant properties in these animals. moreover, ros levels are increased in mas-deficient animals. the masdeficient mice exhibit impaired in vivo endothelial function and increased blood pressure. the transgenic rats overexpressing human ace in vsmcs, showed improved endothelial function and decreased vasoconstriction response to ang ii. furthermore, the infusion of ave , a mas agonist, increases the hypotensive effect of bradykinin in rats; one plausible mechanism for this effect involves an increase in no levels. ace protects endothelial cells from ang ii-mediated macrophage infiltration and oxidative stress in an ang-( - )-dependent manner in atherosclerosis. the long-term ang-( - ) treatment induces atheroprotective effects in apoe-deficient mice, an animal model for atherosclerosis. the improvement in endothelial function resulted from an increase in no bioavailability, as the heptapeptide increased nos expression and decreased o − production. the genetic deletion of ace significantly increases plaque formation in apoe-deficient mice, which is decreased by targeted vascular ace overexpression. consequently, these actions of the ace -ang-( - )-mas system may be exploited in the treatment of atherosclerosis and other vascular diseases. thus, the ace -ang-( 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ii-regulated apoptosis through distinct at and at receptors in neointimal formation new approaches to blockade of the reninangiotensin-aldosterone system: chymase as an important target to prevent organ damage endothelial dysfunction: a strategic target in the treatment of hypertension? reactive oxygen species in the vasculature: molecular and cellular mechanisms p phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells: role in nad(p)h oxidase regulation by angiotensin ii extracellular matrix remodeling in heart failure: a role for de novo angiotensin ii generation endothelial dysfunction and elevated blood pressure in mas gene-deleted mice key: cord- -o fo authors: arnold, ruth h. title: covid- – does this disease kill due to imbalance of the renin angiotensin system (ras) caused by genetic and gender differences in the response to viral ace attacks? date: - - journal: heart lung circ doi: . /j.hlc. . . sha: doc_id: cord_uid: o fo abstract debate continues in the medical literature on the role of the renin angiotensin system (ras) in coronavirus disease (covid- ) pathophysiology and the implications for the use of cardiovascular drugs acting on the ras. could these drugs – which include angiotensin converting enzyme inhibitors (aceis) and angiotensin receptors blockers (arbs) – be harmful or potential key therapeutic agents in covid- ? [abstract] debate continues in the medical literature on the role of the renin angiotensin system (ras) in coronavirus disease (covid- ) pathophysiology and the implications for the use of cardiovascular drugs acting on the ras. could these drugs -which include angiotensin converting enzyme inhibitors (aceis) and angiotensin receptors blockers (arbs) -be harmful or potential key therapeutic agents in covid- ? debate has arisen due to the finding that underlying cardiovascular disease and hypertension are associated with significantly increased risk of hospitalisation and death in covid- [ , ] , in addition to the viral receptor being angiotensin-converting enzyme- (ace- ) [ - ] . coronaviruses downregulate ace- anti-inflammatory actions leading to imbalance in ace:ace- [ ] which may be fundamental in covid- pathophysiology. key gender and genetic differences in the regulation of ace:ace- balance [ ] [ ] [ ] may explain differences observed in disease severity [ , , ] . social media amplification of concern about continuation of ras medications during the current pandemic prompted cardiovascular societies to publish position statements strongly advising continued use, given a lack of evidence that ras drugs were unsafe and the substantial risk of hospitalisation when these drugs are withdrawn from patients with valid indications for treatment [ , ] . three ( ) large observational studies published in the new england journal of medicine (nejm] now support the view that there is no evidence for harm due to ras medications in covid- [ , , ] . however, given the fundamental role of the (ras) in covid- , a broader therapeutic indication for acei and arbs, over and above treatment of patients with established cardiovascular indications has been suggested [ , ] but is yet to be tested in randomised trials. vitamin d status may also be linked to the ras impacts of covid- , as vitamin d is a negative regulator of renin synthesis, and a small study has reported high rates of vitamin d deficiency in covid patients requiring intensive care [ ] . both vitamin d and ace have a role in the immune system, which may add a complex dimension to the issue of ras drugs and vitamin d status in covid- [ , ] . covid- has a more severe course in patients with pre-existing cardiovascular disease [ , , ] and causes a significant rate of cardiovascular events [ ] . further research is required to define the pathological mechanisms and the basis for the observed gender, age, and racial differences in severity. ras medications can be used with greater confidence in covid- patients with valid indications for their use, but a broader role for manipulation of the ras in covid- will require randomised trial data. coronavirus- (sars-cov- ) is believed to gain cell entry via viral spike protein binding to the ace- receptor [ , ] . virology research for other similar coronaviruses indicates that viral cell infection is followed by rapid downregulation of ace- [ , ] . the ace- system is the yang of the yin-yang balance of the ras hypertension system. ace- is a zinc metalloprotease, discovered in [ ] , which mainly breaks down angiotensin ii (ang ii), the main effector of the ras system, to ang( - ), resulting in a decrease in blood pressure, vasoconstriction and decreased inflammation. ace- is usually membrane bound but is shed in covid infection, in acute lung injury and myocardial infarction [ ] . whilst the role of circulating ace- is unclear, conditions such as advanced heart failure have higher plasma ace- proportional to worsening clinical status [ , ] . serum ace- levels are also significantly higher in hypertensive patients [ ] and gender differences are described with up to % greater levels in male patients [ ] . there is debate as to whether the observed higher levels in male, hypertensive, and diabetic patients are related to increased ace- expression as a compensatory mechanism, or whether it is due to increased ace- shedding, limiting tissue ace- activity and causing excess tissue ang ii. the role of circulating, versus tissue, ace- levels in susceptibility to covid infection is unclear [ ] . in viral downregulation of ace- , unopposed ace generates high ang ii causing increased bp, vasoconstriction, inflammation and cell damage. to exacerbate matters, the active metabolite generated by ace- , ang( - ), can be broken down by ace itself, accentuating the ras imbalance caused by covid- [ ] (figure ). covid- is highly contagious spreading from one province, in december , to the whole of china in days, despite extreme shutdown measures [ ] . by may , the disease has infected , , people with , deaths worldwide [ ] and is causing enormous global economic and social impacts. epidemiology for , confirmed, suspected and asymptomatic cases in china suggested mild disease in %, with . % fatality overall [ ] . early mortality figures in different countries have shown striking differences, with greater mortality in europe, compared to asian countries [ ] , which may relate to fundamental differences in racial ace and ace- polymorphisms, impacting both susceptibility to the disease and subsequent pathological severity. population demographics clearly play a role with elderly patients at greatest risk of severe disease and death. key differences in the speed of government actions to implement extensive public health measures, as recommended by the world health organization (who), have also impacted mortality rates as hospitals in some countries have been overwhelmed by a rapid rise in cases. why do % of cases have a mild disease and yet % have a more severe disease? the chinese center for disease control and prevention (ccdc) epidemiology showed that there was an overall mortality of . % [ ] . the death rate increased with age and with co-morbidities, particularly cardiovascular disease ( . %), diabetes ( . %) and hypertension ( . %), all the risk factors typically seen in those with "metabolic syndrome". whilst it is clear that co-morbidities and age increase mortality, what is not clear is whether this excess is due to the impact of the co-morbid conditions on host inflammatory response alone, or to the impact of a treatment some of this patient group is taking [ ] . since the ras system is fundamentally involved in covid- pathogenesis, there was great speculation that drugs widely used in hypertension and heart failure treatment, acting on the ras, may impact disease severity, with some suggesting a protective effect [ , ] and others concerned about potential harm [ ] . these drugs include arbs, aceis and the combination drug valsartan/sacubitril (entresto) which combines an arb with a neprilysin inhibitor (also known as an angiotensin receptor-neprilysin inhibitor [arni]). whilst there is evidence that acei and arb treatment may increase expression of ace- in some tissues in animal models, data supporting or quantifying this effect in human subjects at clinical doses of ras medications is lacking [ ] . hence the relevance of possible medication-induced alteration in ace- levels, over and above the welldescribed significant differences in circulating levels of ace- in advanced heart failure, hypertension, males and diabetics, and the implications for covid- severity, is unclear [ , , ] . increased ace- was speculated to increase susceptibility to covid- by allowing more virus into cells which, in turn, may exacerbate disease severity. the counter argument is that, once infected, having more ace- could be protective against viral attack and downregulation of the ace- antiinflammatory system [ , ] . three ( ) large observational studies reported in the nejm have now examined this issue and found no evidence for increased risk of adverse outcomes in covid- patients taking acei or arbs [ , , ] adding weight to previously reported smaller retrospective clinical studies [ ] [ ] [ ] which also showed no excess mortality for acei/arbs. mehra et al. [ ] reported data for , patients admitted with covid- in hospitals in europe, asia and north america, and confirmed that age, coronary disease, heart failure, cardiac arrhythmia, chronic obstructive pulmonary disease and current smoking were all associated with increased in-hospital mortality but there was no increased risk of mortality associated with risk of acei or arbs. they did find that use of acei and statins was more common in survivors, however this was not randomised data. a multicentre chinese study of , hospitalised patients with covid- and pre-existing hypertension showed a death rate of . % in those on acei or arb versus . % in non-users (p= . ). the data was observational and not randomised, but even after adjustment for confounding variables a lower mortality, with a hazard ratio of . (p= . ) persisted for those receiving acei/arbs, with a hr of . (p= . ) compared to use of other antihypertensive agents [ ] . the study did not separate treatment with arbs from acei [ ] . the answer to observed differences in covid- death rates could lie in key differences in the ace:ace- system balance related to age, gender and racial variation in genetic ace and ace- polymorphisms and environmental factors influencing ace- expression [ , ) . there are ace gene polymorphisms that cause insertion (i) or deletion (d) of a sequence of the gene, and ace activity levels in dd carriers are approximately twice that found in ii genotypes [ ] . the possible impact of ace genotype in the covid- disease state, when the ability to breakdown ang ii is impaired, could be hypothesised to cause a greater quantity of circulating ang ii and a more marked imbalance of the ras and more severe disease. genetic and environmental factors influencing ace- expression could also result in differences in the risk of becoming infected in the first place by affecting the affinity of the ace- binding domain for the viral spike protein and by causing differences in the density of ace- receptors present in tissues such as the lungs. ace- polymorphisms may then contribute to significant differences in disease severity by determining the extent of the ace:ace imbalance, particularly in the endothelium of the lungs, where the virus attacks at the same time as disabling the ace -mediated repair systems. chen and co-workers ( ) [ ] have analysed tissues in thousands of individuals and found significantly higher tissue ace- in asian females compared to males and other ethnic groups. they have described an age-dependent decrease in ace- expression and a highly significant decrease in type ii diabetic patients. the loci for higher ace- expression is almost % in east asians and > % higher than other ethnic groups. ace- is suppressed by inflammatory cytokines, by diabetes and induced by oestrogen and androgen, both of which are decreased in the elderly. the work by chen and co-workers suggests a negative correlation between covid- mortality and tissue ace- levels, at both a population and molecular level [ ] . circulating ace- , however, has been described to increase in pathological states such as hypertension, diabetes and heart failure and perhaps differences in circulating, shed ace- and tissue-bound ace- are key in virus susceptibility and subsequent disease severity. gender differences in ace- ace- is on the x chromosome, hence men have only one allele and women have two [ , ] . further to this, oestrogen is believed to upregulate ace- expression [ ] , giving pre-menopausal women the advantage of two alleles and oestrogen upregulation, making a deficiency of ace- and its ability to rally less likely in the event of a viral attack. in support of this hypothesis, the chinese cdcc reported overall female mortality of . % including suspected cases as well as serologically confirmed cases versus . % in males. this difference was more marked in serologically confirmed cases, . % female versus . % male mortality. the overall ratio was . males to . females believed infected [ ] . hence the men were not significantly less likely to become infected, just more likely to have a more severe pathophysiological expression of the disease. whilst the who suggested [ ] that these gender differences may be driven by substantially greater rates of smoking in the chinese male population, the rates of smoking in the covid- cases were not provided in the epidemiological study. the x-linked nature of the ace gene, with females having a greater range of phenotypes, may have a greater role than has been yet been fully defined in coronavirus pathogenesis gender differences. the range of ace- phenotypes and their ability to upregulate compensatory mechanisms in the face of viral ace- downregulation may play a role. marked gender differences in disease have been confirmed in other large observational studies with marcia et al reporting % women in , patients with covid and mehra et al. reported % female patients in , covid- patients requiring hospital admission and furthermore reported improved survival in female patients, independent of older age [ ] . serum ace- levels are reported to be significantly lower in females [ ] and there are fundamental differences in immune responses to viral infections based on gender with stronger humoral and cellular immune responses described in females [ ] . smoking has been linked to upregulation of ace and downregulation of ace- [ ] , which could be hypothesised to make smokers less likely than the general population to become infected by virtue of having less ace- required for viral entry. however, if a smoker becomes infected, a pre-existing ace:ace- imbalance may result in more severe disease due to greater inflammation, higher risk of ards and multi-factorial issues including poor baseline lung function and bacterial superinfection. evidence supporting this picture is seen in data from wuhan. although smokers are said to comprise . % of the male population in china [ ] , the rate of current smokers reported in covid- series seems well below this; % by huang et al. [ due to the extreme complexity of the ras system, another component may be relevant in causing greater mortality in smokers with covid- . neprilysin is a metalloprotease that has a number of functions but can act to breakdown ang ii to ang( - ) , the role usually performed by ace- . it is highly expressed in airways, pulmonary interstitium and alveolar cells. a study examining circulating neprilysin levels in , community-based subjects > years, found that the lowest tertile group had the highest rates of smokers (p< . ) and higher rates of hypertension (p= . ) [ ] . if smokers have low lung neprilysin activity, they may lack an important safety mechanism that could overcome virally mediated loss of the ace- . this might be relevant in patients taking entresto (sacubitril/valsartan), a medication with proven benefit in the treatment of patients with heart failure. neprilysin inhibition by the sacubitril component of entresto may block the alternate method of generating the anti-inflammatory mediator ( - ) when ace- , under viral attack, is less able to do so. clearly this is a complex question, impossible to answer without more data and even recently reported observational studies did not have sufficient patients taking entresto to report on the safety of this drug in covid- [ , , ] . reports from china showed that patients with severe disease, leading to intensive care unit (icu) admission and death, had features consistent with unopposed ras activity, such as higher blood pressure (bp) on admission to hospital and higher bp on admission to icu in non-survivors [ , ] , with a mean systolic bp of mmhg in those requiring icu admission versus mmhg in milder cases (p= . ) [ ] . lower serum potassium levels, combined with high urinary potassium has also been reported in more severe patients [ , ] , suggesting ang ii-mediated activation of aldosterone. the presence of significant hypertension, instead of hypotension, in a group of patients admitted to icu versus those not requiring icu, is a particularly interesting clinical finding with implications for the pathogenesis of covid- . excess ras activity without ace- balance to break down ang ii and block adverse effects, such as cellular damage and fibrosis, may play a key role in determining disease severity, similar to the ras pro-inflammatory effects well known to contribute to heart failure ( figure ). autopsy data published for four covid- patients [ ] has described platelet rich clot formation occluding small vessels and a microangiopathic picture, consistent with virally mediated vascular damage. the ace:ace- imbalance caused by viral ace- downregulation understanding the ace:ace- imbalance in covid- , its role in age, gender and racial differences in disease severity, and the difference between tissue bound and circulating ace- , could all prove key in treating this disease. in support of the fundamental ace:ace- imbalance having clinical relevance, it has been reported that serum ang ii levels are indeed significantly elevated in covid- and correlate with both viral load and lung injury [ , ] . whilst ace formation of ang ii can be blocked by ace inhibitors, up to % of ang ii is formed via non-ace pathways such as chymase [ ] , making ace inhibitors potentially less effective than arb blockers in diminishing the problem of the excess ang ii, since the acei may only block % formation of the ang ii. on the other hand, the arbs are highly selective blockers of the main receptor for the ang ii, the angiotensin receptor type (at ) receptor. arbs do not decrease the amount of ang ii produced in the first place, but by blocking the at receptors they activate a key counter regulatory anti-inflammatory cascade to restore the ace:ace- balance. the excessive circulating ang ii, unable to bind blocked at receptors, binds at receptors which trigger counter-regulatory anti-inflammatory actions and upregulates ace- expression [ ] to remove excessive circulating ang ii. a protective role of increased tissue ace- expression seems plausible since groups with higher levels such as younger people, premenopausal women and possibly asian populations [ ] do seem to have lower disease severity and mortality [ , ] compared to european populations with an older population and high rates of co-morbidities such as diabetes, hypertension and cardiovascular disease, all of which have been described as having higher circulating ace- levels, due to increased ace- shedding. it has been pointed out that in another rna virus, human immunodeficiency virus (hiv), higher expression of binding sites such as cd actually protected from, rather than increased, virulence [ ] . males have only one x-linked ace- allele and could be more vulnerable to phenotypes causing impaired ace- regulation. patients with pre-existing hypertension may have a greater frequency of the ace polymorphisms that result in excessive ang ii levels, exacerbated by viral impairment of ang ii clearance. covid- is a disease where a major battlefront is at the endothelium, fought by the tissue ras. there have already been cases reported in the media of families tragically affected by very high rates of severe disease and mortality, in line with genetic factors playing a significant role in covid- pathophysiology [ ] . coronaviruses directly impact cellular systems, other than the ras, which are important in inflammatory pathways. rna viruses remodel host membranes and lipid metabolism creating a suitable environment for their replication [ ] . lipid metabolism pathways are impacted in corona virus infection, with glycerophospholipids and fatty acids significantly elevated in sars cov- e infected cells. up-regulated phosphatidylinositol (pi) may promote coronavirus entry [ ] . covid- is associated with cardiac events and significant serum troponin level elevation in a clinical setting [ ] and elevated lysophospholipids, including lp , have been reported in acute coronary syndromes from other causes, undergoing coronary angiography [ ] . hcov- e infection up-regulates fatty acids, believed to promote efficient coronavirus replication. however, yan and co-workers [ ] reported that exogenous addition of fatty acids may interfere with viral replication by upsetting the delicate balance of fatty acids, causing reversion of lysophospholipids into phospholipids, limiting viral replication. it is possible that the manipulation of fatty acids in a clinical setting may upset the balance needed for viral replication. use of drugs such as statins may also impact viral ability to harness cellular lipid pathways. clearly more data is needed to investigate the role of fatty acid and cholesterol manipulation. the finding that statins decrease in-hospital mortality, even in a non-randomised study [ ] , is of interest. long term sequelae of sars, caused by a related coronavirus (sars co-v), have been reported in follow-up studies in survivors out to years. these include sleep disturbance, characterised by increased stage sleep percentage, diffuse myalgia, chronic fatigue, depression, lung damage and avascular femoral head necrosis [ ] [ ] [ ] . deranged lipid metabolism has also been reported in sars survivors. lipid metabolites, including phosphatidylinositol (pi) and lysophosphatidylinositol (lpi), are associated with cellular entry and/or egress of respiratory viruses. lpis and pis were markedly upregulated in recovered sars patients. lpis are thought to have a critical role in glucose homeostasis. a large proportion of the recovered sars patients reported glucose metabolic disorders, including hyperinsulinaemia, insulin resistance, hyperglycaemia, and type or diabetes [ ] . patients who died of sars were found to have extensive lung damage as well as features of systemic vasculitis. autoantibodies against human epithelial and endothelial cells can develop after sars-cov infection and this could explain the severe deterioration in some patients in phase ii of the disease and may lead to post-infectious cellular injury and sars-induced immunopathology in survivors [ ] . this long-term immunopathology could also be a risk in covid- , a related virus which also acts to inflame the endothelium and, due to gender differences in immune function, female survivors may be at greater risk to autoimmune mediated sequelae [ ] . immune system role of vitamin d and ace vitamin d deficiency can be related to immune system dysfunction [ ] and inflammation, and can cause a pro-thrombotic state; it is common in europe in winter. vitamin d is a potent negative endocrine regulator of the ras and works predominantly by suppressing renin synthesis [ ] . vitamin d deficiency and genetic and geographical variation in its function may be another factor contributing to greater imbalance of the ace:ace- system leading to differences in disease severity in covid- . it has been suggested that vitamin d supplementation could reduce the risk of covid- infection and death [ ] . lau et al. [ ] reported a small study of vitamin d levels in patients which is yet to be peer reviewed. they described vitamin d deficiency in . % of covid- patients in icu and . % of patients admitted to a ward bed. they found that % of patients under age admitted to icu had vitamin d deficiency (n= ). ace is best known for its role in blood pressure regulation by converting angiotensin i to ang ii, but in fact it cleaves many peptides and has a role in innate and adaptive immune responses, with ace expressed on both neutrophils and macrophages. the role of ace in immune responses and inflammation is complex and includes defence against intracellular pathogens, mediated via actions that are not dependent on ang ii, as reviewed by bernstein and co-workers [ ] . these complex interactions mean that acei and arbs may not have the same impacts in covid- , a disease impacting both haemodynamic and inflammatory pathways. many agents are under investigation or in clinical trial for the treatment of covid- but so far no anti-viral drugs or vaccine has been officially approved for covid- treatment. hundreds of trials are listed in the who covid- trial data base (www.who.int international clinical trials registry). a small trial using hydroxychloroquine and azithromycin showed some promise in decreasing viral carriage to only to days [ ] , an improvement on days reported from china [ ] . this combination could, however, carry a risk of arrhythmia due to long qt, in a group of patients where abnormal troponins and endothelial dysfunction can be present, with some arrythmias reported in china [ ] . longer trials will be needed. preliminary clinical data for the investigational rna antiviral agent, remdesivir, did not show a survival benefit in a study of patients in china but did show a trend to earlier recovery [ ] , and the adaptive covid- treatment trial (actt) a study of , patients with lung involvement in the usa has not yet been peer reviewed but a press release from the national institutes of health (nih) [ ] reports a % faster recovery time (p< . ) of versus days and possibly a trend to a survival benefit with % versus . % mortality (p= . ). a variety of therapeutic approaches, in addition to a vaccine, may contribute to covid- treatment and prevention in the medium and long term. however, in the current more urgent situation there are , , active cases and , ( %) are serious or critical, with many countries still in the exponential rise phase of new cases. treatment that can be distributed in large quantities is needed if the economic impact of this pandemic is to be addressed. a meta-analysis of cardiac troponin results in covid patients reported that higher levels correlated with more severe disease [ ] . this is consistent with either myocarditis or an acute coronary syndrome, conditions for which manipulation of the ras with commonly available drugs, already have an indication, based on treating endothelial and myocardial inflammation. use of these drugs in covid- patients meeting the definition for an acute coronary syndrome or for another valid cardiovascular indication can now be given with more confidence in light of recent studies showing no detriment for these drugs in covid- [ , , ] .this approach may be one that is directed at fundamental viral vascular pathogenesis. a broader therapeutic role for ras inhibition in covid- is being investigated in two randomised trials examining the effects of the arb losartan on the severity and prognosis in covid- , in hospitalised covid- patients (nct ) and in non-hospitalised patients (nct ). in the current global covid- pandemic, many countries are in lockdown with social and economic paralysis. the impact of covid- has been described as a warzone with many patients dying a lonely death, isolated from loved ones in overcrowded and hastily expanded hospitals. currently, only supportive care is available. whilst many therapeutic agents are under investigation [ , ] , none have yet been approved for use in covid- , nor demonstrated a significant survival benefit. even if some agents are proven to be effective, it will take time for trials and for production on a large scale. thinking of covid- as a fundamentally cardiovascular disease that attacks the endothelium could provide momentum to use safe, established and widely available medications acting on the ras, where they are already indicated for acute coronary syndrome and cardiac dysfunction, present in many patients with covid- . whilst more data on the role of vitamin d in covid pathogenesis is needed, replacing an essential vitamin when it is deficient, seems a safe, cost effective and easily achieved therapeutic strategy. any successful shift to milder disease severity will ease the burden on health care systems. switching on endogenous anti-inflammatory pathways may also play a key role in decreasing the risk of longterm vascular and autoimmune sequelae in covid- survivors, which could pose a further 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shows remdesivir accelerates recovery from advanced covid- updated approaches against sars-cov- world health organization. overview of the types/classes of candidate key: cord- -e aw gkd authors: messerli, franz h.; siontis, george c.m.; rexhaj, emrush title: covid- and renin angiotensin blockers: current evidence and recommendations date: - - journal: circulation doi: . /circulationaha. . sha: doc_id: cord_uid: e aw gkd nan b oth angiotensin-converting enzyme inhibitors (aceis) and angiotensin ii receptor blockers (arbs) have repeatedly, but not consistently, been documented to slow progression of pulmonary complications in vulnerable patients. a previously published meta-analysis pointed toward a putative protective role of aceis, and to lesser extent of arbs, in risk of community acquired pneumonia. aceis in studies were associated with decreased pneumonia mortality compared with control treatment (odds ratio [or] . [ % ci, . - . ]). arbs in randomized controlled trial only showed a reduction of borderline significance (or . [ % ci, . - . ]). these seemingly beneficial findings of renin angiotensin system (ras) blockade on outcomes in pneumonia resurfaced in the recent literature in relation to severe acute respiratory syndrome coronavirus (sars-cov- ), also known as coronavirus disease (covid- ), infection. several potential therapeutic/preventive approaches to address angiotensin-converting enzyme (ace )-mediated covid- have been described, including the suggestion that arbs could be administered in the form of a nasal spray to treat covid- . because pneumonia is a common potentially fatal complication in covid- infection, we wondered whether ras blockade could exert a favorable effect on pneumonia-related outcomes. we systematically reviewed the literature for evidence from animal models and clinical data related to the role of aceis/arbs and viral infections. we searched pubmed and embase for original research on animals or humans investigating the impact of aceis/arbs on viral infections. the following search terms were used: viral, virus, infect, pneumonia, acute respiratory distress syndrome, acute lung injury, and angiotensin. overall, we identified studies - evaluating the role of aceis/ arbs in viral infections of respiratory system. in studies of animal models, - virusinduced lung injury and the role of losartan was tested; all reported a key role of decreased ras activation through losartan (table) . in retrospective study, lower rates of death and intubation were noted among patients who continued to be on aceis during hospitalization (or . [ % ci, . - . ]). we did not find evidence supporting the continuation, withdrawal, or de novo initiation of aceis/ arbs in patients with sars-cov- -infection. epidemiological data suggest that patients with cardiovascular disease and hypertension are more susceptible to sars-cov- -infection and that the course of pneumonia is more severe in hypertensive relative to normotensive subjects. covid- -associated pneumonia death cases were marked by more comorbidities, such as cardiovascular disease ( . %) and hypertension ( . %). considerably higher is the prevalence of these comorbidities in black patients. however, susceptibility for infection and complications may be explained by advanced age and multiple comorbidities in many patients. two large independent studies of influenza type a h n have documented a higher prevalence of hypertension and diabetes. thus, confounding is likely to be extensive. sars-cov- , the recently identified strain responsible for the current covid- epidemic, relies on ace protein as a cellular entry receptor by binding with its spike protein, similar to sars-cov. ace participates in a pathway that is counter-regulatory to the effects of angiotensin ii, and the cardiorenal protective effect of arbs may be attributable in part to increased metabolism of angiotensin ii by ace . importantly, ace expression protects from lung injury, an action which is downregulated by binding of sars-cov via its spike protein. experimentally and in humans, ras blockade has been shown to upregulate ace activity, thereby potentially antagonizing some effects of covid- . injection of sars-cov spike protein into mice worsens acute lung failure in vivo that can be attenuated by inhibiting the ras pathway. conversely, speculation has been put forward that the enhanced ace expression with ras antagonists might increase the number of binding sites thereby increasing the odds of infection with sars-cov- . given that ace is a cellular entry receptor for sars-cov- , careful evaluation of efficacy and safety of antihypertensive therapy with ras blockade is needed. there is experimental and clinical evidence that ras blockade can mitigate pulmonary outcome in some forms of viral pneumonia. presently, there are no data regarding a favorable effect of ras blockade on pulmonary outcome in sars-cov- -infected patients. whether or not infectivity to viral infection is increased in patients treated with ras blockers remains unknown. however, recent news media coverage of this issue has provoked concern and unfortunately even motivated some patients to discontinue ras blockers all together. of note, the ras effects among available blockers is markedly different. direct renin inhibitors ace indicate angiotensin-converting enzyme; acei, angiotensin-converting enzyme inhibitor; ali, acute-lung injury; arb, angiotensin ii receptor blocker; at , angiotensin ; pcr, polymerase chain reaction; ras, renin-angiotensin system; rsv, respiratory syncytial virus; and sars-cov, severe acute respiratory syndromecoronavirus. have been shown to diminish activity of the ras and also downregulate ace in animal models. thus with direct renin inhibitors, ras blockade and cardiopulmonary protection is maintained, but ace seems to be downregulated in general, ras blockers are efficacious and welltolerated drugs with few adverse side effects. however, once fever and dehydration set in, the ras will become upregulated to the extent that its blockade can result in profound hypotension, syncope, cardiovascular collapse, renal failure, and shock. more often than not, this sequence of events occurs unexpectedly and is abrupt. presently, all guidelines recommend continuing acei/ arbs in patients diagnosed with covid- infection. until the evidence in aggregate becomes firmer (and it will), we recommend the following with regard to co-vid- and ras blockade: . in noninfected patients and patients at risk, there is currently no valid reason to discontinue ras blockade. . in healthy subjects at risk, evidence is not (yet?) sufficient to prophylactically recommend ras blockade. sists, patients on aceis or arbs could temporarily be switched to a direct renin inhibitor. . in covid- -positive patients on ras blockers, the drugs should be continued. . in febrile patients with pulmonary symptoms on ras blockers, close monitoring of blood pressure and renal function is advisable; ras blockers should be discontinued only as clinically indicated. cardiovascular disease in general, and hypertension and diabetes mellitus specifically, are prevalent in sars-cov- -infected patients. ras blockers are extensively used for cardiovascular disease and hypertension therapy. there is inconsistent evidence to suggest that ras blockers exert a favorable effect on pulmonary outcome in viral pneumonia, but no data are available specifically for sars-cov- -infected patients. conversely, patients with cardiovascular disease and hypertension seem to be prone to sars-cov- -infection, although this most likely is attributable to confounding factors. ras blockade should be continued in infected patients as clinically indicated. present evidence is insufficient to recommend use of ras blockade prophylactically in subjects at risk or therapeutically in those infected with sars-cov- . risk of pneumonia associated with use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers: systematic review and meta-analysis a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury angiotensin-converting enzyme (ace ) mediates influenza h n virus-induced acute lung injury angiotensin ii receptor blocker as a novel therapy in acute lung injury induced by avian influenza a h n virus infection in mouse impact of angiotensin-converting enzyme inhibitors and statins on viral pneumonia on april , , the ahead-of-print version of this article was revised. the authors revised the first paragraph of the article with an update concerning the difference in the risk of pneumonia in patients who were taking arbs versus those who were not. also, the reference found in the second paragraph of page was been revised. none. key: cord- - pnm fn authors: lubel, john s; herath, chandana b; burrell, louise m; angus, peter w title: liver disease and the renin–angiotensin system: recent discoveries and clinical implications date: - - journal: j gastroenterol hepatol doi: . /j. - . . .x sha: doc_id: cord_uid: pnm fn the renin–angiotensin system (ras) is a key regulator of vascular resistance, sodium and water homeostasis and the response to tissue injury. historically, angiotensin ii (ang ii) was thought to be the primary effector peptide of this system. ang ii is produced predominantly by the effect of angiotensin converting enzyme (ace) on angiotensin i (ang i). ang ii acts mainly through the angiotensin ii type‐ receptor (at( )) and, together with ace, these components represent the ‘classical’ axis of the ras. drug therapies targeting the ras by inhibiting ang ii formation (ace inhibitors) or binding to its receptor (angiotensin receptor blockers) are now in widespread clinical use and have been shown to reduce tissue injury and fibrosis in cardiac and renal disease independently of their effects on blood pressure. in , two groups using different methodologies identified a homolog of ace, called ace , which cleaves ang ii to form the biologically active heptapeptide, ang‐( – ). conceptually, ace , ang‐( – ), and its putative receptor, the mas receptor represent an ‘alternative’ axis of the ras capable of opposing the often deleterious actions of ang ii. interestingly, ace inhibitors and angiotensin receptor blockers increase ang‐( – ) production and it has been proposed that some of the beneficial effects of these drugs are mediated through upregulation of ang‐( – ) rather than inhibition of ang ii production or receptor binding. the present review focuses on the novel components and pathways of the ras with particular reference to their potential contribution towards the pathophysiology of liver disease. most of us can recall the schema of the renin-angiotensin system (ras) taught in physiology lectures (fig. ) . the system is often depicted as a simple enzyme cascade starting with the degradation of angiotensinogen (derived from the liver) by circulating renin (secreted from the juxtaglomerular apparatus of the kidney) to form angiotensin i (ang i). subsequent enzymatic action by angiotensin converting enzyme (ace) in the capillaries of the lung yields the predominant effector peptide of the system, angiotensin ii (ang ii). [ ] [ ] [ ] two receptors for ang ii have been cloned and characterized, the angiotensin ii type- receptor (at ) is the abundant receptor in adult life, whereas the angiotensin ii type- receptor (at ) is present in the fetus and persists in the central nervous system of adults. [ ] [ ] [ ] binding of ang ii to the at receptor mediates a number of diverse effects including vasoconstriction and sodium hemostasis. ang ii also participates in inflammation and wound healing through the release of critical cytokines and production of extracellular matrix. the effect of ang ii on vascular tone and systemic blood pressure has been extensively studied and is mediated through direct effects on vascular smooth muscle cells or indirectly by increasing vascular sympathetic tone. sodiumconserving effects occur via reabsorption of sodium by the renal tubules as well as stimulating the adrenal gland to secrete aldosterone. the effect of ang ii to stimulate thirst is mediated through at receptors in the brain. figure illustrates the conventional view of the 'classical' ras. this schema is useful as it clarifies how drugs like ace inhibitors or at receptor blockers (arb) produce their beneficial therapeutic effects in cardiovascular and renal disease. however, there have been a number of major advances in our understanding of the ras which have made it clear that the system is far more complex than this 'classical' view would suggest (fig. ) . one key point of understanding is that ang ii is just one member of a family of angiotensin peptides produced by the ras. ang ii consists of eight amino acids, which, like other peptides, has a free amino group at one end (n-terminus) and a free carboxyl group (c-terminus) at the other. ang ii can also be denoted as ang- ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , where the first amino acid is at the n-terminus and the eighth amino acid is at the c-terminus. therefore, cleavage of amino acids from either end of the ang ii molecule can generate smaller peptide fragments (fig. ) . for example, removal of the n-terminus amino acid results in the generation of a peptide consisting of seven amino acids starting from the second amino acid of ang ii, and is denoted by ang-( - ) (historically also known as ang iii). of the fragments that can be generated from ang ii, only three are known to be physiologically relevant. two are derived from n-terminus cleavage, ang-( - ) (ang iii) and ang-( - ) (ang iv) and one formed by cleavage of a single amino acid from the c-terminus, angiotensin-( - ) (ang-( - )). ang iii is formed following cleavage of the aspartate-arginine bond of ang ii by aminopeptidase a, and ang iv can be formed by further cleavage of ang iii by aminopeptidase b or n. ang iii shares many of the properties of ang ii with % of the pressor activity and % of the aldosterone stimulating activity. ang iv has its own distinct receptor (at ) and has central nervous system effects together with some opposing actions to ang ii. ang-( - ) is generated from cleavage of either ang ii or ang i and has been the focus of much research since the discovery that it has biological functions that oppose those of ang ii. for decades, the 'classical' arm of the ras was recognized as being the only system of biological relevance. however, this interpretation was challenged in the late s with the discovery of ang-( - ) and description of its diverse biological functions. this was followed by studies which clearly demonstrated new components of the ras, such as ace , and the ang-( - ) receptor, mas. these new components, together with the effector molecule ang-( - ), form the axis which we now recognize as the 'alternative' arm of the ras. the discovery of these new ras components provided some missing connections to the hitherto complex biochemical pathways of the ras. the new components of the alternative arm of the ras are reviewed below with a major emphasis on their potential contribution towards the pathophysiology of liver disease. interest in alternative components of the ras was re-ignited in the year when two groups independently discovered an enzyme similar to ace in human tissue. , this homolog of ace was initially called hace but has subsequently been named angiotensin converting enzyme (ace ). although structurally similar to ace, ace has different substrate affinities and resists inhibition by ace inhibitors. ace is a zinc-metalloproteinase and, like ace, is a type- transmembrane protein. it consists of amino acids with a single transmembrane alpha-helical portion, an external n-terminus portion containing the catalytically active enzyme and an internal inactive c-terminus section. a transmembrane proteinase, adam acts as a 'sheddase' releasing the active enzyme into the extracellular environment (fig. ) . the released ace (soluble ace ) is a carboxypeptidase, capable of cleaving a single amino acid from the c-termini of its various substrates, including, ang ii, ang i, des-arg -bradykinin, neurotensin - and kinetensin (see review by burrell and colleagues). importantly, ace can generate ang-( - ) directly from ang ii or indirectly by cleaving ang i into an inactive intermediate fragment, ang-( - ), which is then cleaved by ace to produce ang-( - ) (fig. ). of these two ace pathways, the conversion of ang ii into ang-( - ) is kinetically favoured -fold compared to the conversion of ang i to ang-( - ). , in addition to its role in the 'alternative' ras, the ace transmembrane protein has, interestingly, been identified as a receptor site for spike proteins of the severe acute respiratory syndrome (sars) coronavirus, thereby facilitating infection of target cells. much of the work on ang-( - ) has been carried out in animals and, to date, this peptide has been shown to have antihypertensive, anti-arrhythmic, and cardioprotective properties [ ] [ ] [ ] as well as anti-trophic properties in vascular endothelial cells, smooth muscle cells, cardiac myocytes and cardiac fibroblasts. [ ] [ ] [ ] [ ] in contrast to ang ii, ang-( - ) also has anti-inflammatory, antifibrotic and anti-thrombotic properties. , as a result of these studies, ang-( - ) has been proposed to represent the effector peptide of a counterbalancing arm of the ras, capable of opposing the deleterious actions of ang ii. the putative receptor for ang-( - ) is the g protein-coupled receptor encoded by the mas proto-oncogene, although other receptors may well exist. thus, ace together with ang-( - ) and the mas receptor represent an 'alternative' arm or axis of the ras which may present a counter- figure 'classical' renin-angiotensin system (ras). the ras is depicted here as a linear cascade leading to the generation of angiotensin ii (ang ii) through the enzymatic action of renin on angiotensinogen and angiotensin converting enzyme (ace) on angiotensin i (ang i). there are two known receptors for angiotensin ii, angiotensin ii type- receptor (at ) and angiotensin ii type- receptor (at ). the at receptor is thought to play a more important role than the at receptor in human disease. balancing system to the deleterious ace/ang ii/at axis (fig. ) . clearly, ace holds a central role in the ras influencing both axes, as it is capable of simultaneously degrading ang ii and generating ang-( - ) (fig. ) . angiotensin converting enzyme is known to participate actively in the kallikrein-kinin system by degrading bradykinin (fig. ) . inhibitors of ace can therefore lead to the accumulation of bradykinin, which may contribute to the antihypertensive properties of these drugs, as well as to some of the observed side-effects, such as chronic cough and angioedema. in the liver, bradykinin binds to the b receptor and causes increases in hepatic resistance and elevation of portal pressure. in other vascular beds, bradykinin induces vasodilatation on binding to the b receptor, and ang-( - ) has been shown to induce bradykinin-mediated relaxation in porcine coronary arteries. a possible explanation for this is that ang-( - ) has ace inhibitory properties that prevent acemediated degradation of bradykinin. in recent years, scientists have departed from the traditionally held view of the ras being exclusively a circulating endocrine system and have realized that many organs, such as the heart, kidney, liver and pancreas, constitutionally express all the 'classical' ras components required for a functioning, autonomous intra-organ contemporary renin-angiotensin system (ras). angiotensin converting enzyme (ace) has a central role in the ras influencing both the 'classical' and 'alternative' axes, as it degrades angiotensin ii (ang ii) while simultaneously generating ang-( - ). ace is important in generating ang ii, but is also responsible for the degradation of ang-( - ) into the inactive peptide fragment ang- ( ) ( ) ( ) ( ) ( ) . the ras interacts with the kinin system through ace degradation of bradykinin. the two axes of the ras and the kinin system are shaded grey. enzymes are shown in yellow boxes and peptides in blue boxes. aminopeptidase a (apa) and aminopeptidase n (apn) sequentially cleave ang ii to form angiotensin iii and angiotensin iv, respectively. neprilysin (nep) is involved in both the ras and the kinin system. possible peptide-receptor interactions are shown by dashed lines. peptide structure and fragments of angiotensin i. angiotensin i is a decapeptide (ang-( - )) which can be fragmented by various enzymes into four peptides with biological activity; angiotensin ii (ang-( - )), angiotensin iii (ang-( - )), angiotensin iv (ang-( - )) and angiotensin - (ang-( - )). further enzymatic degradation of ang - yields the inactive fragment angiotensin - (ang-( - )). aminopeptidases are shown in blue and cleave amino acids from the n-terminus, whereas carboxypeptidases are shown in red and cleave amino acids from the c-terminus. amino acids are given numerical values, where , aspartic acid; , arginine; , valine; , tyrosine; , isoleucine; , histidine; , proline; , phenylalanine; , histidine; , leucine. ras. , these locally generated angiotensin peptide fragments have been demonstrated to have a multitude of actions, being implicated in cell growth, cell proliferation, apoptosis, reactive oxygen species generation, inflammation, and fibrogenesis. although conceptually separate, the local intra-organ ras and the systemic ras must interact and the final peptide products will depend on the interplay between the two. despite the discovery of ang-( - ) and the recognition that many of its actions oppose ang ii, the importance of this heptapeptide fragment of ang ii remained elusive until recently. it is now clear that in the diseased liver, not only are the 'classical' ras components such as renin, ace, ang ii and the at receptor overexpressed, but, importantly, components of the 'alternative' ras, such as ace , ang-( - ) and the mas receptor are also upregulated. , the implication from these studies is that the 'classical' components contribute to the fibrotic process whereas the 'alternative' components may be upregulated in an attempt to restore the status quo. in liver disease, architectural changes to the microscopic structure of the liver occur as a result of inflammation and fibrosis. these changes lead to capillarization of the hepatic sinusoids, increased extracellular matrix (ecm) formation and elevated hepatic resistance; the latter impedes liver blood flow and leads to portal hypertension. stretching of the portal vein (as with increased hepatic resistance to blood flow) and oxidative stress together cause release of vasodilators, including nitric oxide, which induce a number of compensatory mechanisms important for restoring the functional blood volume. these mechanisms are effected via sodium and water preservation and stimulation of the sympathetic nervous system, which together contribute to the development of ascites, edema, hepatorenal syndrome, and a hyperdynamic circulation, all of which are typically seen in patients with advanced liver disease. the ras is involved with all these processes. as the result, manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway could have potential therapeutic benefits. balanced against the possible benefits are the potential side effects of such therapy, as the compensatory mechanisms activated by the systemic ras are necessary to maintain an adequate circulation. counterbalancing effects of the two axes of the renin-angiotensin system (ras). the ras can be thought of as two counterbalancing axes. the angiotensin converting enzyme (ace)/angiotensin ii/at receptor axis causes vasoconstriction, salt retention, inflammation, fibrosis and thrombosis, whereas the ace /angiotensin - /mas receptor axis has opposing effects. hepatic stellate cells (hsc) are thought to play a pivotal role in fibrogenesis within the liver, and there is a large body of evidence to support the hypothesis that ang ii promotes activation, and dedifferentiation of these cells into myofibroblasts. furthermore, ang ii encourages myofibroblast contraction, proliferation and promotes release of inflammatory cytokines as well as the deposition of extracellular matrix (ecm). although both of the ang ii receptors (at and at ) are expressed in the liver, the at receptor is far in abundance and is thought to be responsible for most of the ang ii-mediated effects. studies using gene-deletion mice have demonstrated that at a receptor-deficient mice are protected from hepatic fibrosis whereas at receptor-deficient mice have worse fibrosis. a great deal of evidence supporting the role of the ras in hepatic fibrosis has come from animal studies using ace inhibitors and angiotensin receptor blockers (arb). numerous studies using a variety of animal models have demonstrated antifibrotic effects of these drugs. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, there appear to be some conflicting observations reported in the literature. for example, losartan treatment failed to influence either liver injury or progression of fibrosis in an animal model of non-alcoholic steatohepatitis (nash). [editor's note: a detailed review of animal models of nash has been written by larter and yeh for a later article in this basic science miniseries.] in contrast, a study with a similar model of nash but using the arb olmesartan, demonstrated a % reduction in fibrosis in the arb-treated group. the avid interest in ras-blocking drugs is, in part, related to their relative safety in humans and widespread use in cardiovascular and renal medicine. despite the large number of animal studies, there is a relative paucity of human data to support the use of these drugs in human liver disease. in part, this could be due to the need to perform multiple liver biopsies to histologically confirm resolution of fibrosis, which, outside the setting of posttransplantation recurrent hepatitis c, is rarely indicated in . in addition, the slow progression of fibrosis in most diseases such as hepatitis c and non-alcoholic fatty liver disease (nafld) make it difficult to detect possible beneficial effects of antifibrotic therapy, unless studies are conducted over a number of years. a pilot study examining the effects of months of losartan treatment on liver fibrosis in chronic hepatitis c demonstrated a significant decrease in fibrosis stage in the treated group compared to control patients. in support of this, a study using candesartan for weeks in compensated child a and b cirrhotic patients demonstrated a significant reduction of plasma hyaluronic acid levels, a surrogate marker for fibrogenesis. however, in this study, two of three serum markers of fibrosis used showed no improvement, and there were no histological data provided; this makes it difficult to evaluate any effects on architectural changes. a number of other studies have reported possible antifibrotic effects of ras blockers in patients with hepatitis c. in one study, hepatitis c virus (hcv)-infected patients with mild fibrosis were treated with losartan mg/day and ursodeoxycholic acid mg/day whereas controls received ursodeoxycholic acid alone. there were significant reductions in serum markers of hepatic fibrosis such as transforming growth factor b (tgf-b ) and type iv collagen in the losartan and ursodeoxycholic acid group, but no significant changes in fibrosis score between the groups. another report described outcomes in patients with hepatitis c treated with low-dose interferon (ifn alpha ¥ iu times a week for months) in combination with the ace inhibitor, perindopril ( mg/day). treatment was accompanied by significant improvement in serum markers of fibrosis (hyaluronic acid, type iv collagen s and procollagen iii-n-peptide), but histological analysis was not carried out. although this study did not have a perindopril monotherapy group, a subsequent study by the same group demonstrated that perindopril alone decreased serum fibrosis markers in patients with chronic hepatitis c. the addition of interferon significantly augmented the effect of perindopril monotherapy. finally, a retrospective review compared liver histology in liver transplant patients with recurrent hepatitis c who were taking ras-blocking drugs (n = ) with those who were not (n = ). the group taking ras blockers were less likely to develop severe hepatic fibrosis (bridging fibrosis or cirrhosis) at and years after transplantation than were the control group ( % vs % at year [p < . ], and % vs % at years, respectively). only small studies have looked at ras blockers and nash. one such study (n = ) found that giving losartan ( mg/day for weeks) in hypertensive patients with nash reduced serum tgf-b , serum ferritin and aminotransferase levels. five patients showed improvement in the grade of hepatic necroinflammation. the study design could have been improved had the investigators examined pre-and post-treatment histology and biochemical markers in a placebo group. in a subsequent study, the pre-and post-treatment biopsies of seven patients with nash treated with losartan ( mg/day for weeks) were compared with eight patients with nafld who acted as a control group. the treatment group showed a significant improvement in necroinflammatory grade, stage of fibrosis, significantly fewer activated hsc and a mild increase in quiescent hsc at the end of weeks. however, the lack of a proper randomized control group is a particular problem in studies of patients with nash, as the disease can improve in response to changes in lifestyle. fixed changes in hepatic architecture account for approximately % of the total resistance to portal blood flow in the cirrhotic liver. the remaining % results from a reversible or 'dynamic' resistance caused by the contraction of activated myofibroblasts positioned around the sinusoidal endothelial cells within the space of disse. as portal resistance increases, a number of factors, including distension of the portal venous system, endotoxemia and oxidative stress result in the release of mediators, including nitric oxide, which dilate the mesenteric and systemic vasculature. activation of compensatory mechanisms designed to restore functional blood volume results in sodium and water retention, stimulation of the sympathetic nervous system and the development of a hyperdynamic circulation. this cascade of events contributes to many of the key features and complications of advanced liver disease including development of ascites, edema and the hepatorenal syndrome. the ras is involved with all these processes. manipulation of the ras with either antagonists of the 'classical' pathway, or agonists of the 'alternative' pathway therefore has potential for therapeutic benefit. variceal bleeding is one of the most important causes of morbidity and mortality in patients with portal hypertension. a number of pharmacological approaches have been developed for the prevention and treatment of this problem. non-selective ß-adrenergic antagonists (beta-blockers) lower portal pressure by decreasing cardiac output and constricting the mesenteric vascular bed but have no direct effect on intrahepatic resistance to portal flow. these drugs have become the mainstay of treatment for the prevention of variceal bleeding. however, only % of patients achieve the target reduction in portal pressure of %, as measured by hepatic venous pressure gradient (hvpg), and they are poorly tolerated in patients with severe liver disease. as a result, there is a major interest in the development of other pharmacological therapies which can lower portal pressure. interestingly, betablockers interact with the ras by inhibiting renin release, but have not been shown to impact on the development or progression of hepatic fibrosis. in contrast, the use of either ace inhibitors or arb to reduce portal pressure is an attractive proposition, as these drugs have the additional potential benefit of slowing the progression of hepatic fibrosis. ang ii is a potent vasoconstrictor, and myofibroblasts derived from hsc express the at receptor and contract in response to ang ii. , additionally, cirrhotic rat livers are hyperresponsive to ang ii with an increased portal pressure compared to those from healthy rats as a result of increased expression of at receptors. this finding is of interest given that the relative importance of ang ii as a mediator of increased portal resistance has been questioned, based on a study of hepatic hemodynamics in isolated perfused cirrhotic rat livers which suggested that ang ii-mediated vasoconstriction is attenuated in the cirrhotic liver. following some persuasive animal studies, , the effects of at blockade on portal hypertension have been examined in a number of human studies. despite some encouraging initial studies showing a significant reduction of portal pressure by arb, subsequent well-designed studies have failed to confirm these findings. schneider and colleagues reported a dramatic reduction in hvpg with losartan in both moderate and severe portal hypertensive patients, but with only a mmhg drop in mean arterial pressure (map). these findings were markedly different to a subsequent randomized controlled trial comparing the hemodynamic effects of losartan with propanolol given for weeks following an index variceal bleed. losartan failed to reduce hvpg, yet resulted in a significant reduction of map by %. treatment tolerance was equivalent. the hemodynamic effect of losartan was further corroborated by a recent small study of pre-ascitic patients which also found that losartan had no affect on hvpg, but did cause a drop in map of . %. irbesartan, another arb, produced only modest reduction in portal pressures ( % Ϯ . %, p < . ) in a randomized, placebo-controlled, double-blind study. importantly, however, this was associated with significant arterial hypotension and significant renal impairment in % of patients. in this study, plasma renin activity before treatment was a predictor of patients that would not tolerate treatment. the explanation for this adverse effect is that the ras is known to play a central role in the homeostatic response to vasodilatation in patients with portal hypertension. the ras, together with other compensatory systems, the posterior pituitary (through vasopressin secretion) and the sympathetic nervous system, endeavors to restore circulatory volume and organ perfusion by inducing vasoconstriction and sodium and water retention. in patients with advanced cirrhosis, plasma renin, ang ii, ace and aldosterone levels are all increased and, within the kidney, ang ii is critical for maintenance of renal perfusion pressure and an adequate glomerular filtration rate (gfr). as liver disease progresses, the decrease in effective circulatory volume results in vasoconstriction of the glomerular afferent circulation, renal hypoperfusion and a fall in gfr. in response to renal hypoperfusion, ang ii selectively constricts the efferent glomerular arterioles; this restores glomerular perfusion pressure and gfr. the maintenance of adequate renal perfusion is therefore ace dependent. furthermore, ace inhibition results in a rapid fall of gfr. , this adverse effect of ras inhibition on renal function in patients with advanced cirrhosis represents a major disadvantage for the use of this class of drug for the treatment of portal hypertension. a recent study by debernardi-venon and colleagues examined the effects of candesartan treatment for weeks on compensated child a and b cirrhotic patients. treatment was well tolerated, with a mild but significant reduction in hvpg in more than % of those treated. furthermore, % of patients treated achieved a % reduction in their hvpg. interestingly, the changes in hvpg correlated well with those observed for plasma hyaluronic acid. however, the treatment group was preselected in that patients were excluded from analysis if they had large varices, evidence of significant arterial hypotension or renal impairment. angiotensin receptor blockers have also been studied in portal hypertensive gastropathy; at least one study has reported a positive benefit from their use. the effects of ace inhibitors on portal pressure have also been examined in a few small studies, but the results generally have been disappointing, with poor agreement between studies. - a number of explanations have been proposed to explain the lack of uniformity in results from clinical studies investigating the benefits and adverse effects of ras inhibitors. there are known genetic polymorphisms for the at receptor gene and genes responsible for cleaving angiotensin i, including ace; these may confer patient-to-patient variations in response to these drugs. this has led to the suggestion that genetic testing may help determine which patients are likely to have a positive response to therapy. in addition, chronic ace inhibition may not lead to sustained ang ii suppression because of increased renin activity and upregulation of alternative enzymes, such as hepatic chymase, which is capable of generating ang ii from ang i. , furthermore, chronic use of arb also results in hyper-reninemia and elevated ang ii levels; the latter increasingly compete with the at receptor antagonist for binding sites on the at receptor molecule. , finally, it has also been claimed that there is tissuedependent responsiveness to ace inhibitors and arb and, at current therapeutic dosing, both classes of drug may not completely inhibit their respective targets. to date, no studies have examined the effects on portal pressure of combined therapy with an ace inhibitor and arb; theoretically, this may overcome some of the possible issues of ang ii reactivation with use of ace inhibitors alone. in summary, the use of ras inhibitors (other than betablockers) to reduce portal pressure has been disappointing. at the doses used in clinical trials, these drugs appear to have only minor effects on portal pressure but very significant side-effects, includ-ing systemic hypotension and renal impairment. these complications are a useful reminder of the homeostatic role the ras plays in maintaining map and gfr in the vasodilated patient with severe liver disease. [ ] [ ] [ ] based on the current available evidence, the use of either ace inhibitors or arb for reducing portal pressure remains controversial and cannot be recommended outside clinical trials. as outlined above, there is increasing evidence that both the 'classical' and the 'alternative' ras are upregulated in chronic liver disease. , it has recently been suggested that the progression of liver fibrosis may be influenced by a balance between ace and ace activation. in both an animal model of secondary biliary fibrosis and in humans with hepatitis c, ace gene and activity are upregulated. , as fibrosis worsens, the progressive rise in ace and at gene expressions coincide with an increase in ace and mas expression, together with increased plasma levels of both ang-( - ) and ang ii. , cirrhotic livers have a greater capacity than healthy livers to convert ang ii to ang-( - ) because of upregulated ace gene and protein expression (fig. ). in addition, the hepatic production of ang-( - ) from ang ii is augmented by ace inhibition. , this increased ang-( - ) production in the presence of an ace inhibitor can be explained by the fact that ang-( - ) is cleaved by ace to produce the inactive peptide ang-( - ) (fig. ) . inhibition of ace therefore increases ang-( - ) half-life, leading to an increase in net production and accumulation of ang-( - ). [ ] [ ] [ ] evidence for a beneficial role of ang-( - ) in hepatic fibrosis has been provided by a study examining the effects of the mas receptor antagonist [ -d-ala]-ang-( - ) (a ). treatment with a worsened experimental liver injury with increases in tgf-b and hydroxyproline levels; this infers that mas receptor stimulation plays a protective role in liver fibrosis. further compelling evidence for a beneficial role of ang-( - ) has come from a recent rat study presented at aasld by our group. we demonstrated that ang-( - ) infusion in bile duct-ligated rats attenuated fibrosis as quantified using metavir fibrosis score, hydroxyproline content, and type collagen mrna expression. alpha-smooth muscle actin (a-sma) gene and protein expression were also reduced, indicating that hepatic stellate cell activation was inhibited by ang-( - ). interestingly, ang-( - ) infusion also inhibited ace gene and protein expression, and resulted in downregulation of mas receptor gene expression. the ang-( - ) infusion group also showed decreased mrna expression levels for connective tissue growth factor (ctgf, also known as ccn ) and vascular endothelial growth factor (vegf), two critical growth factors implicated in fibrosis and tissue repair. this is the first direct evidence showing that ang-( - ) can ameliorate hepatic fibrosis. evidence from studies in ace deletion mice further supports a central role of ace in regulating fibrosis in liver disease. despite a number of reports that ang-( - ) is a vasodilator, experiments on rat isolated perfused livers have failed to demonstrate any vasodilatory effect in normal or cirrhotic livers. , , likewise, experiments in isolated vessels from normal and cirrhotic rats also failed to show any direct vasodilatory effect of this peptide. conversely, ang-( - ) has been shown to enhance acetylcholine-mediated vasodilatation in aortic rings from cirrhotic rats. the vasodilatory effects of ang-( - ) are thought to be mediated through increased production of nitric oxide (no). , hence, the absence of a vasodilatory effect by ang-( - ) in the cirrhotic rat liver could be explained by the known general impairment of no-dependent vasodilatation in the cirrhotic liver due to endothelial dysfunction. , in summary, there is considerable evidence supporting the concept that opposing axes of the ras are involved in the pathogenesis of chronic liver injury. on one side, the ace/ang ii/at receptor axis promotes liver injury and deposition of extracellular matrix, on the other, ace /ang-( - )/mas receptor promotes collagen degradation and resolution of inflammation. both axes are upregulated in liver disease, but presumably the balance between the two systems is critical in determining the net effect. for many years researchers in the field of ras have concentrated on blocking components of the 'classical' system in an attempt to reduce fibrosis. however, both ace inhibitors and arb have an impact on other components of the 'classical' ras apart from ang ii, as plasma renin activity and ang i levels increase following chronic therapy. this, in part, explains the phenomenon of 'angiotensin ii reactivation' and 'aldosterone escape' whereby chronic administration of an ace inhibitor fails to completely suppress either plasma ang ii or aldosterone production. [ ] [ ] [ ] the actual mechanism underlying this phenomenon remains elusive, figure effects of angiotensin converting enzyme (ace) inhibitors and angiotensin type- receptor (at ) receptor blockers (arb) on the two axes of the renin-angiotensin system (ras). the ras is shown as a balance with 'classical' and 'alternative' axes counterbalancing each other. ace inhibitors (acei) cause an initial reduction in angiotensin ii (ang ii), but after chronic administration increases in plasma renin activity and plasma angiotensin i (ang i) levels occur. both ang ii and aldosterone levels can subsequently rise as a consequence of non-ace-dependent pathways facilitated by enzymes such as chymase. both acei and arb result in elevated levels of ace and ang-( - ) which possibly contribute to the effects of these drugs. manipulations of the ras aimed at tipping the balance in favour of 'alternative' components represents a potential target for antifibrotic therapies. liver and the renin-angiotensin system js lubel et al. although non-ace-dependent pathways involving enzymes like chymase, which is capable of generating ang ii, may play an important part. , interestingly, ace inhibitors and arb have a profound impact on the 'alternative' system by causing significant increases in ang-( - ). , , , it has been postulated that some of the beneficial effects observed with arb and ace inhibitors are mediated through ang-( - ). , , [ ] [ ] [ ] [ ] [ ] in support of this, ace activity and gene expression are both increased in the heart by arb or ace inhibitors. , the elevated ace activity in such tissues would result in both diminished levels of ang ii and simultaneous elevations in tissue ang-( - ), thus tipping the ras balance in favor of the 'alternative' axis. interestingly, our own studies and those of others have shown that ang-( - ) can inhibit ace activity and gene expression; this would further tend to alter the balance of the two axes towards the 'alternative' axis ( fig. ) . , conclusions and future directions our understanding of the ras has considerably expanded since the discovery of ace . emerging evidence supports the hypothesis that the ras consists of two opposing axes. manipulation of the ras, by either blocking the 'classical' ras or by stimulating the 'alternative' ras represents a potential target for antifibrotic and portal hypertension therapy. limitations to treatment may be the side-effects of such drugs, particularly their impact on arterial blood pressure and renal function. current therapies such as ace inhibitors and arb used in cardiovascular and renal fibrosis have been shown to impact on both the 'classical' and 'alternative' pathways. the elevated ang-( - ) plasma levels caused by these drugs may represent a mechanism by which these drugs exert some of their effects. new drugs which mimic the effects of ang-( - ) have been developed. this represents a novel set of agents that could be used for the treatment of hepatic fibrosis or portal hypertension. one such drug, ave is a non-peptide analog of ang-( - ). this orally active ang-( - ) receptor agonist represents an entirely new class of drug spawn from the latest insights into the complexities of the contemporary ras and has possible novel therapeutic applications in liver disease. purification and properties of angiotensin-converting enzyme from hog lung biochemistry and kinetics of the renin-angiotensin system renin-angiotensin system: a dual tissue and hormonal system for cardiovascular control isolation of a cdna encoding the vascular type- angiotensin ii receptor cloning and expression of a complementary dna encoding a bovine adrenal angiotensin ii type- receptor molecular cloning of a novel angiotensin ii receptor isoform involved in phosphotyrosine phosphatase inhibition signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin ii in vascular smooth muscle cells angiotensin ii receptors and functional correlates active fragments of angiotensin ii: enzymatic pathways of synthesis and biological effects angiotensin iv in the central nervous system release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-( - ) heptapeptide a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor ace , a new regulator of the renin-angiotensin system evaluation of angiotensin-converting enzyme (ace), its homologue ace and neprilysin in angiotensin peptide metabolism hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase 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sprague-dawley rats bradykinin, angiotensin-( - ), and ace inhibitors: how do they interact? fate of bradykinin on the rat liver when administered by the venous or arterial route angiotensin - induces bradykinin-mediated relaxation in porcine coronary artery angiotensin-( - ) augments bradykinin-induced vasodilation by competing with ace and releasing nitric oxide activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin ii a local pancreatic renin-angiotensin system: endocrine and exocrine roles upregulation of hepatic angiotensin-converting enzyme (ace ) and angiotensin-( - ) levels in experimental biliary fibrosis chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme modern management of portal hypertension hepatic stellate cells as a target for the treatment of liver fibrosis at a-deficient mice show less severe progression of liver fibrosis induced by ccl( ) anti-fibrogenic function of angiotensin ii type receptor in ccl -induced liver fibrosis angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis an angiotensin ii type receptor antagonist, olmesartan medoxomil, improves experimental liver fibrosis by suppression of proliferation and collagen synthesis in activated hepatic stellate cells acei attenuates the progression of ccl -induced rat hepatic fibrogenesis by inhibiting tgf-beta , pdgf-bb, nf-kappab and mmp- , anti-fibrogenic effect of an angiotensin converting enzyme inhibitor on chronic carbon tetrachloride-induced hepatic fibrosis in rats effect of angiotensin ii type receptor blockade on experimental hepatic fibrogenesis captopril reduces collagen and mast cell and eosinophil accumulation in pig serum-induced rat liver fibrosis anti-fibrogenic effects of captopril and candesartan cilexetil on the hepatic fibrosis development in rat. the effect of at -r blocker on the hepatic fibrosis effects of at receptor antagonist, 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chronic hepatitis: colocalization of chymase with fibrosis renin-angiotensin system inhibition: how much is too much of a good thing endogenous angiotensin ii levels and the mechanism of action of angiotensin-converting enzyme inhibitors and angiotensin receptor type antagonists aldosterone and renal haemodynamics in cirrhosis with ascites hepatic hemodynamics and the renin-angiotensin-aldosterone system in cirrhosis diagnostic significance of serum angiotensin-converting enzyme activity in biochemical tests with special reference of chronic liver diseases liver fibrosis: a balance of aces? the renin-angiotensin system in a rat model of hepatic fibrosis: evidence for a protective role of angiotensin metabolism of angiotensin-( - ) by angiotensin-converting enzyme converting enzyme determines plasma clearance of angiotensin-( - ) pathways for angiotensin-( - ) metabolism in pulmonary and renal tissues angiotensin - reduces bile duct proliferation and hepatic fibrosis in the bile duct ligated rat angiotensin-converting enzyme is a negative regulator of chronic liver injury advances in biochemical and functional roles of angiotensin-converting enzyme and angiotensin-( - ) in regulation of cardiovascular function upregulation of the ace /ang( - )/mas receptor axis in the bile duct ligation (bdl) model of hepatic fibrosis does not affect hepatic sinusoidal resistance hepatic conversion of angiotensin i and the portal hypertensive response to angiotensin ii in normal and regenerating liver angiotensin-( - )-stimulated nitric oxide and superoxide release from endothelial cells vascular endothelial dysfunction in cirrhosis influence of caveolin on constitutively activated recombinant enos: insights into enos dysfunction in bdl rat liver reactive hyperreninemia is a major determinant of plasma angiotensin ii during ace inhibition gradual reactivation of vascular angiotensin i to angiotensin ii conversion during chronic ace inhibitor therapy in patients with diabetes mellitus determinants of increased angiotensin ii levels in severe chronic heart failure patients despite ace inhibition how often are angiotensin ii and aldosterone concentrations raised during chronic ace inhibitor treatment in cardiac failure? expression of angiotensin ii type receptor in human cirrhotic livers: its relation to fibrosis and portal hypertension effects of renin-angiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors divergent regulation of circulating and intrarenal renin-angiotensin systems in response to long-term blockade effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme angiotensin-( - ) contributes to the antihypertensive effects of blockade of the renin-angiotensin system the role of ang ( - ) in mediating the chronic hypotensive effects of losartan in normal rats vasodepressor actions of angiotensin-( - ) unmasked during combined treatment with lisinopril and losartan evidence that prostaglandins mediate the antihypertensive actions of angiotensin-( - ) during chronic blockade of the renin-angiotensin system upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors pharmacological effects of ave , a nonpeptide angiotensin-( - ) receptor agonist angiotensin-converting enzyme and new insights into the renin-angiotensin system dr john lubel is a recipient of an australia national health and medical research council (nhmrc) scholarship, and peter angus and louise burrell hold an nhmrc project grant ( ). key: cord- - sh eksm authors: garg, m.; angus, p. w.; burrell, l. m.; herath, c.; gibson, p. r.; lubel, j. s. title: review article: the pathophysiological roles of the renin–angiotensin system in the gastrointestinal tract date: - - journal: aliment pharmacol ther doi: . /j. - . . .x sha: doc_id: cord_uid: sh eksm background: the renin‐angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. aim: to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. methods: an extensive online literature review including pubmed and medline. results: there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. conclusions: the gastrointestinal renin‐angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) is a homeostatic pathway widely known to regulate cardiovascular and renal physiology; however, little is known about its influence in gastrointestinal tissues. to elicit the anatomical distribution and physiological significance of the components of the ras in the gastrointestinal tract. an extensive online literature review including pubmed and medline. there is evidence for ras involvement in gastrointestinal physiology and pathophysiology, with all the components required for autonomous regulation identified throughout the gastrointestinal tract. the ras is implicated in the regulation of glucose, amino acid, fluid and electrolyte absorption and secretion, motility, inflammation, blood flow and possibly malignant disease within the gastrointestinal tract. animal studies investigating the effects of ras blockade in a range of conditions including inflammatory bowel disease, functional gut disorders, gastrointestinal malignancy and even intestinal ischaemia have been encouraging to date. given the ready availability of drugs that modify the ras and their excellent safety profile, an opportunity exists for investigation of their possible therapeutic role in a variety of human gastrointestinal diseases. the gastrointestinal renin-angiotensin system appears to be intricately involved in a number of physiological processes, and provides a possible target for novel investigative and therapeutic approaches. the renin-angiotensin system (ras) plays a central role in regulating cardiovascular and renal physiology. the contemporary view of the ras has evolved from that of a simple linear pathway involving the conversion of angiotensinogen to angiotensin ii (ang ii) via a two-step process facilitated by renin and angiotensin converting enzyme (ace), to a much more complex system involving homologues of ace and multiple angiotensin peptides which play supplementary and counter-regulatory roles ( figure ). the ras was, for many years, thought of as an endocrine system with enzymes and peptides released into the systemic circulation to act on target organs. more recently, it has been recognised that most organs including the brain, kidney, heart, liver, pancreas, reproductive organs, skin and the gastrointestinal tract constitutively express all the components required to allow autonomous function of a local intra-organ ras, where it performs both paracrine and autocrine functions. table summarises the current view of the ras, the key components and their physiological and clinical effects. essentially, the relative activity of two counterbalancing pathways determines the predominant tissue effect. the proinflammatory, profibrotic pathway includes the classical ras components ace and ang ii, and renin, prorenin, chymase and neutral endopeptidase (nep, also known as neprilysin). renin, a glycoprotein derived predominantly from the juxtaglomerular apparatus in the kidney, is an aspartyl protease that cleaves the liver-derived angiotensinogen to angiotensin i. both renin and its proenzyme prorenin, which was previously considered physiologically inactive, have now been demonstrated to have independent pro-inflammatory and pro-fibrotic effects via signalling through the pro(renin) receptor (prr). the classical ras comprising the zinc metalloproteinase ace and ang ii induces vasoconstriction, salt and figure | the contemporary renin-angiotensin system (ras). ace, angiotensin converting enzyme; nep, neutral endopeptidase; am, aminopeptidase; at r, angiotensin type receptor; at r, angiotensin type receptor; at r, angiotensin type receptor; prr, (pro)renin receptor. water retention, thirst response, cardiac hypertrophy, tissue inflammation and fibrosis through the g-protein coupled seven-transmembrane domain receptor angiotensin type i receptor (at r). ang ii also stimulates adrenal gland secretion of aldosterone resulting in renal sodium and water retention. inhibition of this pathway with either ace inhibitors or at r antagonists has beneficial effects in hypertension, cardiac failure, ischaemic heart disease, diabetic nephropathy and renal fibrosis. chymase expressed in the heart and vascular wall and secreted by activated mast cells, acts as an alternative enzyme to ace to generate ang ii from ang i. - nep, a membrane bound zinc metalloproteinase with a structure distinct from ace, was discovered in the s as a key enzyme involved in the cleavage of bradykinin. , in recent years, it has been shown to also have a role in the formation of ang ( - ) from ang i, as an inactivator of atrial natriuretic peptide and in the degradation of amyloid b peptide, a protein involved in the pathogenesis of alzheimer's disease. the net effect of nep inhibition is vasodilatation and natriuresis, a property encompassed by vasopeptidase inhibitors that target both ace and nep and may have additional anti-hypertensive effects to ace inhibitors. in contrast, the alternative ras, comprising ace and ang ( - ), acting via the g-protein coupled seventransmembrane receptor mas, , has vasodilatory, antihypertensive, anti-thrombotic, cardioprotective, antiinflammatory and anti-fibrotic effects in a variety of tissues. - ace is a zinc metalloproteinase and homologue of ace, which cleaves a single amino acid from ang ii to form the heptapeptide ang ( - ). indeed, part of the clinical benefit attributed to ace inhibitors and at r blockers (arbs) may be through the diversion of the classical ras components towards ang ( - ) with subsequent mas receptor activation. [ ] [ ] [ ] the complexity of the ras is further highlighted by recent findings regarding the actions of other angiotensin peptides including angiotensin iii [ang iii, also denoted as ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) ], angiotensin iv [ang iv, also known as ang ( ) ( ) ( ) ( ) ( ) ( ) ] and the at and at receptors. ang iii is formed by cleavage of ang ii by aminopeptidase a, and ang iv results from further conversion by aminopeptidase b or n ( figure ). the at receptor (at r) has affinity for ang ii, ang iii, ang iv and ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and is also thought to have effects counteracting the at r and analogous to those of the mas receptor, with vasodilatory, anti-inflammatory and anti-proliferative downstream actions. previously recognised largely for an important role in foetal development, more recently, the at r has been shown to be upregulated in atherosclerotic disease, cutaneous wounds and pancreatic fibrosis, and to stimulate neurite outgrowth, a marker of neuronal regeneration. , ang iii is believed to have actions analogous to ang ii. ang iv appears to have opposing effects to ang ii, and acts predominantly via the at r and the at r, formerly known as insulin regulated aminopeptidase (irap). the greatest role of ang iv is in the central nervous system (cns), where it has a positive effect on neuronal development, learning and memory. , concept of local ras there is considerable evidence that most or all of the components of the ras are present in a variety of organs, supporting the theory that local expression and modulation of the ras play important roles in tissue homeostasis. these roles may be summarised as involving ( ) fluid and electrolyte transport, ( ) regional blood flow regulation and ( ) promoting the wound healing response, including cell proliferation, inflammation and fibrosis. some of the regional effects of the ras are listed below: (i) in the kidney, local angiotensinogen is converted by renin to ang i, which in turn is cleaved by tubular brush border ace to ang ii to facilitate sodium and fluid absorption via luminal at r. this may influence blood pressure independent of systemic ang ii levels and vascular tone. , (ii) the heart expresses renin, prr, ace, chymase, angiotensinogen, at r and at r, and these components modulate myocyte proliferation and cardiac remodelling. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] (iii) the brain has been shown to express renin, angiotensinogen, ang ii, ang iii, ang iv, ang ( - ), at r, at r and at r, with these components regulating blood pressure, fluid and electrolyte balance, thirst, maintenance of the blood-brain barrier and neuronal development including learning and memory processes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] (iv) the liver expresses renin, angiotensinogen, ang ii, ace, at r, ang ( - ), ace and mas receptors, all of which are upregulated in the diseased liver. , furthermore, arbs and ang ( - ) have been demonstrated to reduce liver fibrosis in animal models. [ ] [ ] [ ] (v) in the pancreas, ang ii has been shown to inhibit glucose stimulated insulin secretion, and via at r and at r, regulates exocrine enzyme secretion and the microcirculation. [ ] [ ] [ ] (vi) a local ras has been identified and shown to be involved in tissue homeostasis in the reproductive organs, skin and even adipose tissue. [ ] [ ] [ ] [ ] [ ] [ ] detailed reviews of these and other local ras effects are published elsewhere. , , , , [ ] [ ] [ ] localisation and functionality of the ras in the gastrointestinal tract our understanding of the involvement of the ras in the gastrointestinal tract has gradually evolved over the past five decades since the formulation of the hypothesis that ang ii had a direct effect on intestinal smooth muscle in addition to an indirect effect via myenteric plexus cholinergic neurons. [ ] [ ] [ ] since then, many of the components of the ras have been identified throughout the gastrointestinal tract. an overview of the current state of knowledge is illustrated in figure . the regions of the gut are addressed separately here. most attention has been paid to the small intestine (figure a) , as outlined below: (i) ace, ace and neutral endopeptidase: ace has been shown in humans to be located in abundance on the brush border of epithelial cells and in the mesenteric microvascular endothelium. ace mrna and protein is present in large amounts in small intestinal epithelial brush border, muscularis mucosa and muscularis propria, as well as microvascular endothelium and vascular smooth muscle cells. remarkably, the highest tissue concentrations in the human body of ace and ace mrna are found in the terminal ileum, duodenum and colon. , expression of nep has been demonstrated in the rat intestinal wall, and is suppressible by administration of the combined ace/nep inhibitor omapatrilat. (ii) angiotensin receptors: at r has been localised to the epithelial brush border. the circular and longitudinal muscle layers and the myenteric plexus also strongly express at r, but the at r appears to be largely restricted to the myenteric plexus. , small vessels in the muscularis propria also express at r. , in early studies in the rat intestine, ang ii binding sites were reported to be confined to the muscularis, but subsequent reports have identified expression of at r and a lesser amount of at r in the muscularis mucosa and mucosa, including in epithelial cells. , (iii) renin: mrna for renin has been detected in the human small intestine. (iv) angiotensin peptides: ang ii has been detected in the crypt and crypt-villus junction epithelial cells. to date, the expression of angiotensinogen, ang i or ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) in the human small intestine has not been reported. however, angiotensinogen has been widely localised in the rat brush border, epithelial cells, lamina propria, muscularis mucosa, submucosal blood vessels and muscularis propria. angiotensinogen mrna has been isolated in concentrations of over one-third that of the liver in the rat mesentery, and a high level of proangiotensin- , a precursor of ang i, has been located in the rat intestine. thus, all of the required components for local production and action of ang ii appear to be present in small intestine. there is now evidence of important roles for the ras in a variety of intestinal processes: (i) bicarbonate secretion: this is stimulated by ang ii via at r and at r in the duodenum. (ii) sodium and water absorption: in the jejunum and ileum, this process appears to be modulated by ang ii in conjunction with the enteric sympathetic nervous system. [ ] [ ] [ ] when applied in low dose to rat jejunum, ang ii stimulates sodium and water absorption through at r, but in high dose, it unexpectedly inhibits absorption through at r. both ang ii and ang iii may also increase sodium and water absorption via stimulation of release of noradrenaline from sympathetic neurons, which in turn may act through adrenergic receptors on the basal surface of epithelial cells. , , (iii) glucose absorption: ang ii has also been shown to inhibit rat jejunal sodium-dependent glucose transporter (sglt )-mediated glucose uptake in vitro. (iv) digestion and absorption of peptides: both brush border ace and ace are thought to function as peptidases, allowing for mucosal digestion and absorption of peptides. , ace increases the activity of the neutral amino acid transporter b at , which is mutated in a rare amino acid deficiency disorder, hartnup disorder, clinically manifested by cerebellar ataxia and pellagra-like skin rash. (v) secretion: a role for ace in active secretion has been suggested by the observation that ace is the target for the coronavirus mediating severe acute respiratory syndrome, sars-cov. some patients with this infection suffer from watery diarrhoea, but the exact mechanism remains to be determined. there have been limited studies of ras components in the colonic wall (figure b) . by a combination of rt-pcr and immunohistochemistry, renin was found in the surface epithelium, lamina propria mesenchymal cells, microvascular walls and muscularis mucosa. at r was detected on surface epithelial cells and in crypt bases, lamina propria macrophages, myofibroblasts and mucosal vessel walls, and weak expression of at r has been found on surface epithelium, in crypts and in some mesenchymal cells. ace was also weakly expressed in parts of the surface epithelium, and more prominently in mesenteric microvascular walls, lamina propria and submucosal mesenchymal cells. ace appears to be localised to the mesenteric microvascular endothelium in the colon and is not present in the epithelium. angiotensinogen mrna has also been isolated in homogenised rat colon, but its expression has not been examined in the human colon. a more limited range of functional roles has been attributed to components of the ras in the colon than in small intestine. ang ii has been shown to increase sodium and water reabsorption in rats through nacl coupled transport. the response of circular and longitudinal muscle contraction to ang ii also suggests a role in normal colonic motility. as detailed below, the ras may also be involved in the inflammation associated with ibd, as mucosal levels of ang i and ang ii are higher in patients with active crohn's colitis compared with normal controls and patients with ulcerative colitis. components of the ras are present in the mucosal biopsy specimens of gastric antrum and body from healthy adults (figure c ). renin and angiotensinogen were both seen in lamina propria mesenchymal cells and vascular endothelial cells. at r and at r were both observed in gastric epithelium (mainly in the basal surface), lamina propria mesenchymal cells and vascular endothelium. at r were noted in a subgroup of endocrine cells in the base of antral mucosal glands, and ace and nep in vascular endothelial cells, but not in other parts of the mucosa. other investigators, however, have noted ace in fundic chief cells and mucin secreting cells of the antrum. longitudinal and circular muscle of the stomach has been demonstrated to respond in vitro to ang ii, suggesting the presence of appropriate receptors on gastric myocytes. to date, few functional or pathogenic roles have been attributed to the ras in the stomach. a role of local ras in gastric inflammation has been suggested by higher expression of at r expression in helicobacter pylori positive than h. pylori negative patients and the potentiation of ulceration in animal models by ang ii. , oesophagus immunoreactive ace, at r and at r have been found in the lamina propria microvascular walls, and at r and at r were identified in the superficial stratified epithelium and circular and longitudinal muscle of the oesophagus (figure d ). ang ii caused contraction of isolated oesophageal smooth muscle in vitro, and the at r antagonist candesartan inhibited swallow-induced peristaltic contractions in the distal oesophagus. the expression of other ras components has not been reported. the presence of the various components of the ras in the gastrointestinal tract raise the possibility that modification of this system locally may be a potential therapeutic target in a myriad of gastrointestinal diseases where current strategies are suboptimal. these include inflammatory bowel disease (ibd), gastrointestinal cancer, gut motility disorders and mesenteric ischaemia. although clinical data are sparse, results from animal models and pre-clinical studies provide support for further investigation. information relevant to ibd has arisen from studies in crohn's disease (cd) and ulcerative colitis (uc) and animal models of both ibd and other chronic inflammatory conditions. studies in patients with crohn's disease and ulcerative colitis. two components of the ras have been studied. the first is ace, which was subject to intense interest from the 's in its role in sarcoidosis and other granulomatous conditions. studies of serum ace concentrations yielded largely conflicting findings in ibd, with many studies showing reduced levels [ ] [ ] [ ] [ ] and some finding no difference. [ ] [ ] [ ] many of these studies have been limited by relatively small numbers of patients. serum ace levels are associated with ace gene insertion/deletion (i/d) polymorphisms, with higher levels seen with the dd polymorphism than id or ii. matsuda et al. showed that the ace gene polymorphism variation was similar in patients with cd and patients with uc to controls, but that serum ace levels were lower in patients with ibd after adjusting for polymorphisms. furthermore, they demonstrated that ace levels significantly increased in all of nine patients with active cd when they achieved clinical remission. a larger study involving uc patients and cd patients also found no difference in ace gene polymorphisms when compared with normal controls, but a subgroup analysis revealed a higher proportion of dd genotype in uc patients with extra-intestinal manifestations, with an odds ratio (or) of . . indeed, it is difficult to reconcile these findings into a unifying hypothesis regarding the role of ace in ibd pathogenesis. it is possible that inflammatory cytokines such as tnf-a and il- downregulate systemic endothelial ace production, whereas ang ii produced via local intestinal ace contributes to tissue inflammation. tissue ace levels have not been measured in active ibd. the other major area of investigation has focussed on angiotensin peptide levels in cd. genotype analyses have revealed a significant association of angiotensinogen- aa genotype (or . ) in a cohort of patients with ibd. this genotype results in increased production of angiotensinogen via a substitution in its gene promoter. indeed, mucosal levels of ang i and ang ii are elevated in rectosigmoid biopsies in patients with crohn's colitis compared with patients with uc or normal controls, and a significant correlation was noted between these levels and endoscopic grade of colitis. although these findings demonstrate that components of the local tissue ras probably play a part in inflammation, they do not prove any causal link in the pathogenesis of ibd. nonetheless, they suggest that inhibition of the local ras provides a potential avenue for targeting inflammation and fibrosis. studies in animal models. angiotensin converting enzyme inhibition or angiotensin receptor antagonism have been shown to produce a number of beneficial anti-inflammatory effects in rodent models of intestinal inflammation. ang ii and at a receptor expression are both upregulated in dextran sodium sulphate (dss)induced colitis, a widely studied mouse model of colitis, and inflammation was significantly ameliorated in at a receptor-deficient mice. the ace inhibitor enalaprilat, given parenterally, reduced inflammation and tnf-a, and topical enalaprilat reduced tgf-b expression and fibrosis in mice with dss-colitis. administration of the arb, valsartan, significantly reduced macroscopic inflammation, tnf-a, tgf-b and il- in mice with trinitrobenzene sulphonic acid (tnbs) induced colitis, reduced microscopic inflammation and raised il- (an anti-inflammatory cytokine secreted by regulatory t cells) in dss-colitis. , the ace inhibitor captopril reduced macroscopic and microscopic inflammation, fibrosis and tgfb mrna expression in mice with tnbs-induced colitis. homozygous deficiency of angiotensinogen protects against tnbs-induced colitis, with reduced il -b, ifn-c and greater il- and il- production than wild-type mice. animal studies have not been restricted to the classical ras pathway. nep knockout mice have been shown to have more severe colitis in response to dinitrobenzene sulphonic acid than wild-type mice, which is prevented by the administration of recombinant nep. other evidence to suggest that ras dysfunction may potentiate immune-based diseases such as ibd and provide a target for therapy comes from recent studies involving models for multiple sclerosis (ms). t helper type (th ) and type (th ) cells are strongly implicated in the pathogenesis of ibd, especially cd, and lisinopril and candesartan have been demonstrated to suppress th and th cytokine expression and induce foxp + regulatory t cells in experimental autoimmune encephalitis (eae), a mouse model of ms. other groups have shown a crucial role for ang ii and at r in eae , and murine autoimmune nephritis. the possible involvement of perturbed ras components in solid organ malignancies represents a fascinating expansion of our insight into local tissue ras. early epidemiological studies demonstrated a reduced risk of incident and fatal cancer in patients on ace inhibitors versus those on other anti-hypertensive medication. , in contrast, a recent meta-analysis reported a higher risk of lung cancer in patients taking arbs ; however, two large meta-analyses since then have reported no increased risk. , all these studies have been limited by their retrospective design. increasing in vitro and pre-clinical data suggest a protective effect of at r inhibition against cancer cell proliferation, invasion and metastasis in a variety of solid organs. in gastric cancer, at r and ang ii are expressed to a greater extent than in adjacent normal tissue. the at r and ace gene polymorphism d allele increase risk of nodal metastasis and tumour stage in patients with intestinal-type gastric cancer. in gastric cancer cell cultures, ang ii stimulates map kinase, nfjb and survivin activation, increasing proliferation. the most likely mechanism by which the ras may influence cancer biology is through increasing angiogenesis, , via increased expression of vascular endothelial growth factor (vefg) signalling. [ ] [ ] [ ] in mouse models, ace inhibition and arbs reduced colorectal cancer liver metastases and prolonged survival in peritoneal carcinomatosis. indeed, ace inhibitor use independently protected against distal metastasis in a single centre retrospective review of patients with colorectal carcinoma. also, patients treated with ace inhibitors had a non-significant trend towards reduction in risk of oesophageal adenocarcinoma in a retrospective study of the uk general practice research database. recently, there have been intriguing insights into the involvement of the ras in peutz-jeghers syndrome (pjs). shorning and colleagues have shown that in mice lkb gene deletion, which is associated with pjs, results in marked transcriptional upregulation of the renin gene ren , and also increased ace expression and ang ii production. in human pjs tumour tissue, at r was noted to be increased in stromal tissue, but reduced in the epithelium. gut motility disorders the functional role of the ras in smooth muscle contraction suggests that it might be a target in motility dysfunction. for example, the ability of ang ii blockade to inhibit contraction of oesophageal body and lower oesophageal sphincter (les) smooth muscle, together with the demonstrated reduction in the amplitude of contraction of primary peristaltic oesophageal waves and les on manometry in vivo, suggests a possible role in treatment of hypercontractile oesophageal disorders such as diffuse oesophageal spasm, nutcracker oesophagus and achalasia. furthermore, selective at r-mediated contraction of the les may be an option for treatment of gastro-oesophageal reflux disease, a condition that affects up to - % of the population. the contribution of at r to small and large intestinal muscle contractility also provides an opportunity to intervene in functional intestinal and motility disorders through at r agonism or blockade. furthermore, the role of ace and ace in intestinal fluid and electrolyte absorption suggests a potential mechanism for modulating fluid shifts across the brush border, with subsequent effects on stool consistency and frequency. the ras plays an important role in regulation of the smooth muscle tone of the mesenteric vasculature. in acute hypovolaemia and systemic sepsis, splanchnic vasoconstriction occurs as a homeostatic response to preserve cerebral and renal blood flow, predisposing the gut to ischaemia. this splanchnic response has been shown to correlate with a markedly increased expression of ang ii. furthermore, lower-body negative pressure induction in normal human volunteers has also been shown to raise serum ang ii and reduce intestinal mucosal nitric oxide production. the administration of candesartan maintained jejunal and mucosal perfusion during severe hypovolaemia in pigs and reduced mortality. , a further porcine study reported improved mucosal oxygen delivery, but not an improvement in intestinal mucosal acidosis in pigs administered candesartan during endotoxic shock. these results were replicated by tardos et al. in pigs with burn and endotoxin induced gut ischaemia. in this model, intestinal permeability and bacterial translocation were reduced with the administration of the ang ii inhibitor dup . the obvious limiting factor in applying ang ii blockade in humans at risk of mesenteric ischaemia is the potential for current arbs and ace inhibitors to cause further hypotension and kidney injury, although no adverse renal consequences were noted in one porcine study. manipulation of local gastrointestinal tract ras -potential targets and limitations as a ubiquitous system with a wide array of homeostatic roles, investigation into therapies that manipulate the ras has been extensive. apart from the well-established roles of at receptor blockade and ace inhibition in the treatment of hypertension, cardiovascular and kidney disease, new drugs targeting the mas receptor, at receptor, at r, renin and nep, as well as a recombinant ang ( - ), are under trial for various applications ( table ) . there is limited knowledge of the effect of currently available ace inhibitors and arbs on gastrointestinal function at doses employed for cardiovascular and renal effect at a molecular level. most of these drugs may result in adverse effects including nausea, diarrhoea or constipation in an idiosyncratic manner in between and per cent of patients, often noted in similar numbers of patients in the placebo arm in randomised con-trolled trials. it is likely that gastrointestinal tissue concentrations achieved by these medications at current doses are insufficient to note clinical effect. furthermore, the effect of these drugs in pathological states, like functional gut disorders or ibd, has not been described. for applicability to human gastrointestinal disease, drugs manipulating the ras will need to target the relevant areas of the gut to obtain satisfactory tissue effect without systemic adverse reactions. ideally, this will comprise delivery to the mucosa, absorption and binding of cellular receptors and sufficient first pass hepatic conversion to inactive metabolites to limit systemic effect. there is now a significant body of literature demonstrating the existence and pathophysiological relevance of local tissue renin-angiotensin systems. given the current evidence of involvement of the ras in gastrointestinal fluid and electrolyte homeostasis, smooth muscle control, inflammation and malignancy, it follows that manipulation of this system could be of benefit in a range of gi pathologies. therapies targeting the gastrointestinal ras are attractive, given their excellent safety and tolerability profile and confirmed benefits in other organs and diseases. it is hoped that the recent emergence of further experimental evidence supporting a role for the local ras in intestinal disorders will provide greater impetus for the initiation of well-conducted clinical trials in human disease. renin, (pro)renin and 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cardiac dysfunction recombinant human angiotensinconverting enzyme as a new reninangiotensin system peptidase for heart failure therapy effects of ace inhibition in the post-myocardial infarction heart comparison of vasopeptidase inhibitor, omapatrilat, and lisinopril on exercise tolerance and morbidity in patients with heart failure: impress randomised trial comparison of omapatrilat and enalapril in patients with chronic heart failure: the omapatrilat versus enalapril randomized trial of utility in reducing events (overture) blood-pressure reduction with lcz , a novel dual-acting inhibitor of the angiotensin ii receptor and neprilysin: a randomised, doubleblind, placebo-controlled, active comparator study inhibition of diabetic nephropathy by a decoy peptide corresponding to the "handle" region for nonproteolytic activation of prorenin involvement of receptor-bound prorenin in development of nephropathy in diabetic db/db mice prorenin receptor blockade inhibits development of glomerulosclerosis in diabetic angiotensin ii type a receptordeficient mice prorenin receptor blockers: effects on cardiovascular complications of diabetes and hypertension pathologic roles of prorenin and (pro)renin receptor in the eye role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization suppression of ocular inflammation in endotoxin-induced uveitis by inhibiting nonproteolytic activation of prorenin key: cord- -q fjx authors: okuno, keisuke; cicalese, stephanie; elliott, katherine j.; kawai, tatsuo; hashimoto, tomoki; eguchi, satoru title: targeting molecular mechanism of vascular smooth muscle senescence induced by angiotensin ii, a potential therapy via senolytics and senomorphics date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: q fjx cardiovascular disease (cvd) is a prevalent issue in the global aging population. premature vascular aging such as elevated arterial stiffness appears to be a major risk factor for cvd. vascular smooth muscle cells (vsmcs) are one of the essential parts of arterial pathology and prone to stress-induced senescence. the pervasiveness of senescent vsmcs in the vasculature increases with age and can be further expedited by various stressing events such as oxidative stress, mitochondria dysfunction, endoplasmic reticulum stress, and chronic inflammation. angiotensin ii (angii) can induce many of these responses in vsmcs and is thus considered a key regulator of vsmc senescence associated with cvd. understanding the precise mechanisms and consequences of senescent cell accumulation may uncover a new generation of therapies including senolytic and senomorphic compounds against cvd. accordingly, in this review article, we discuss potential molecular mechanisms of vsmc senescence such as those induced by angii and the therapeutic manipulations of senescence to control age-related cvd and associated conditions such as by senolytic. a discrepancy between lifespan and healthspan is becoming a global challenge as life-expectancy increases [ ] . aging is a major risk factor for cardiovascular diseases (cvd) such as hypertension, coronary and cerebral artery diseases [ ] . the vascular system is damaged with age, demonstrating several signatures of vascular disorders [ ] . accumulating evidence support that cellular senescence contributes to the progression of cardiovascular pathologies along with age-related disorders [ ] . it is important to note that the concept of early vascular aging has been developed to identify an individual with a high cvd risk who has a dissociation between chronological and biological vascular aging [ ] . as a major hormone of the renin angiotensin system (ras), angiotensin ii (angii) has been implicated in induction of premature cellular senescence in vessel wall, thus promoting early vascular aging [ , ] . cellular senescence was first defined in the s, where normal human fibroblasts lost the ability to replicate in culture at certain passages, indicating that cell senescence may be related to aging in vivo [ ] . this type of senescence, termed replicative cell senescence, is a consequence of the critical loss of telomere length that occurs once somatic cells have undergone a maximal number of cellular division cycles [ ] . replicative senescence is associated with a persistent dna damage response (ddr) [ ] and induces cell senescence through p /p and p ink a /the retinoblastoma protein (rb), two parallel tumor suppressor signaling pathways [ ] . premature cellular senescence, termed stress-induced premature senescence (sips), can be achieved with non-chronological stress conditions [ ] . without detectable ddr, sips can be induced by various type of stressors including oxidative stress and metabolic stress [ ] . regardless of the induction mechanisms, senescent cells have some common (but not exclusive) features, which include permanent growth arrest, increased cell size, induction of a senescence-associated β-galactosidase (sa-βgal) activity, expression of a cyclin-dependent kinase inhibitor p ink a , formation of senescence-associated heterochromatin foci (sahf), and senescence-associated secretory phenotype (sasp) [ ] . over time senescent cells accumulate from damage within tissue, most significantly at etiological sites of multiple diseases throughout the lifespan. specifically, this includes insults to the cardiovascular system such as, atherosclerosis, diabetes, and heart failure [ ] . sasp transmits persistent sterile inflammation that is associated with age-related chronic diseases and frailty, thus providing a novel therapeutic opportunity [ , ] . in this review, we focus on potential signaling and consequences of senescence in vascular smooth muscle cells (vsmcs) in response to angii. we also advocate that the senescence mechanism will provide novel therapeutic targets to maintain healthy aging and/or counteract against cvd development. it is hypothesized that chronic sasp is the major contributor to aging associated organ dysfunction induced by senescent cells [ , ] . cytokines, matrix remodeling enzymes, and extracellular vesicles have all been identified in senescent cell secretions, however the exact profile can change depending on the stressor [ , , ] . nuclear factor-κ b (nf-κb) and ccaat/enhancer-binding protein (c/ebp-β) are classical mediators of sasp. thus far varieties of sasp mediators have been reported which include toll-like receptors, an autocrine feed forward secretion of interleukin- α (il- α), and epigenetic mediators such as high mobility group box (hmgb ) and bromodomain-containing protein (brd ). in addition, the cellular nutrient sensing pathways, mammalian target of rapamycin (mtor) and nicotinamide adenine dinucleotide (nad + ) are involved in sasp regulation (reviewed in [ ] ). regarding sasp in vascular cell types, a prior study has demonstrated that senescent human aortic vsmcs secrete il- α and promote adjacent cells to a pro-adhesive and-inflammatory state in an autocrine manner [ ] . however, in this study the findings are limited to replicative senescence and only one kind of stress-induced senescence (bleomycin) [ ] . in another study using human coronary vsmcs, bleomycin-induced or replicative senescent cells were characterized including total cell lysates proteomics and qpcr, which demonstrated up-regulation of il- β, il- and high mobility group box- (hmgb ). this study, however, did not confirm these factors to be secreted upon senescence [ ] . in addition, arterial vsmcs isolated from children with chronic kidney disease showed elevated senescence and sasp, which include il- and calcification promoting bone morphogenetic protein- and osteoprotegerin [ ] . it is also important to note the paracrine mechanisms of sasp potentially occur in vsmcs via other senescent cell types such as endothelial cells [ ] , fibroblasts, immune cells and adipocytes as these are all cellular communicators of the vasculature. these recently recognized cell communications are considered as key parts of inflamm-aging; a chronic low-grade inflammation associated with aging and aging-related diseases [ ] . interestingly extracellular vesicles also termed exosome has been recently implicated as a part of sasp in some cell systems [ , ] including endothelial cells [ ] . in particular, functional as well as proteomic characterization of exosomes have been performed and compared in aortic vsmcs and endothelial cells. endothelial cell-derived exosome but not vsmc-derived exosome caused hmgb -dependent inflammatory responses and senescence in vsmcs [ ] suggesting a paracrine role of vascular exosomes in mediating senescence and sasp. regarding the angii-induced senescence, removal of senescent cells by a senolytic abt attenuated angii-induced leukocyte adhesion in cultured endothelial cells suggesting a contribution of sasp in endothelial dysfunction induced by angii [ ] . as only a limited percentage of endothelial cells undergo senescence upon angii exposure, these findings suggest potential paracrine role of sasp in angii-induced cardiovascular pathology [ ] . however, further research is needed to characterize vascular cell sasp and its consequences with more pathologically relevant conditions. our understanding of the molecular mechanism leading to senescence is becoming much clearer, particularly those in cvd [ , , ] . moreover, genetic or pharmacological removal (senolytics) or prevention (senomorphics) appears to be a powerful tool to investigate contribution of senescence in cvd [ ] . in addition, drugs to attenuate the sasp network have been proposed as senostatics, which may also be a therapeutic against age-related diseases [ ] . genetic removal of senescent cells in mice have been developed such as p ink a -attac mice in which selective apoptosis can be induced in senescent cells demonstrating improvements in aging related disorders [ ] and extension of healthy lifespan [ ] . similarly, p - mr mice with low density lipoprotein receptor (ldlr) −/− background as well as p ink a -attac ldlr−/− mice, removal of senescent cells prevented atherosclerosis development [ ] . pharmacological removal of senescent cells by abt which inhibits anti-apoptotic proteins bcl- and bcl-x and selectively kills senescent cells [ ] was also effective against atherosclerosis in ldlr−/− mice [ ] . in addition, endothelial p deletion prevented cardiac fibrosis and heart failure in response to pressure overload in mice [ ] and protected systemic metabolic disorders in mice fed with high caloric diet [ ] . systemic removal of senescent cells in aged mice by abt protected mice from cardiac hypertrophy and heart failure upon experimental myocardial infraction [ ] . it is important to note that immune system has an endogenous defense mechanism to remove excess senescent cells via immunosurveillance, which leads to a development of immunotherapies to eliminate senescent cells [ ] . the recent most exciting advancement in this field may be the development of engineered t cells expressing a chimeric antigen receptor (car t cells) to target senescence in vivo. the urokinase-type plasminogen activator receptor (upar) was identified as a selective cell surface marker expressed in senescent cells [ ] . car t cells targeting upar appears effective against senescent malignant cells in vivo as well as liver fibrosis in animal models of non-alcoholic steatohepatitis, a severe form of fatty liver disease [ ] . contrary to these finding suggesting detrimental roles of senescence, p /p ink a double knockout mice showed accelerated cardiac fibrosis upon pressure overload even while the induction of senescence was prevented [ ] . clinical trials utilizing senolytics are currently ongoing for several chronic diseases [ ] . although senolytic effects have been investigated in cvd models, limited information is available regarding the potential application of senomorphics or senostatistics in vascular cells or animal models of chronic diseases. a prior review article defined senomorphics as agents that should prevent either senescence induction or sasp without induction of senescent cell apoptosis [ ] . according to this definition, effects of several candidate compounds of senomorphics on senescence, sasp and age-related diseases have been described [ ] . however, none of these compounds were originally designed as senomorphics. among the potential targets to control sasp by senostatics, cgmp-amp synthase (cgas), a cytosolic dna sensor that activates innate immunity and stimulator of interferon genes (sting) signaling pathway has recently been identified as a critical sasp regulator [ ] . retrotransposons are a special class of parasitic genetic elements that can replicate their dna within our genes. one of the successful retrotransposons is the class of long interspersed nuclear element (line ). these kb, fully functional retrotransposons can replicate not only themselves and accumulates by age and aging-related diseases, but accumulation of line cdna triggers strong type i interferon response via activation of cgas-sting leading to sasp [ ] . this response can be antagonized by nucleoside reverse transcriptase inhibitors (nrtis) that inhibit the l reverse transcriptase. the nrti lamivudine was able to attenuate sasp in senescent cells and age-associated inflammation in mice [ , ] . thus, nrti could be a promising candidate for senostatics. at this point, the effects of the senomorphics and or senostatics on cvd remain unclear. further research on the potential application and mechanistic investigation with seno-modulatory interventions is strongly desired. general mechanisms contributing to arterial aging include mitochondrial dysfunction, oxidative stress, inflammation, and activation of the ras [ ] . chronic ras activation leads to damage in several organs, which is associated with aging and oxidative stress in cells or mitochondria [ ] . aging also causes enhancement in the activity of and response to the ras. [ ] . angii is the essential hormone acting through two receptor subtypes, angii type receptor (at r) and angii type receptor (at r). activation of at r promotes the majority of ras physiology and pathophysiology including vasoconstriction, cardiovascular hypertrophy and fibrosis, inflammation and oxidative stress [ ] . while there are some exceptions and the exact signaling mechanisms are still unclear, activation of at r generally counteracts the classical ras actions including cell growth, oxidative stress, inflammation and fibrosis [ , ] . moreover, enhancement of senescence markers induced by angii was observed in vsmcs cultured from at r deficient mice suggesting the anti-senescence role for at r [ ] . in addition, for the past two decades, significant advancement has been made in understanding the function and mechanisms utilized by new members of the angiotensin family ligands and receptors [ ] . among them, using systemic pro (renin) receptor transgenic mice, it was demonstrated that pro (renin) receptor mediates skeletal muscle atrophy and the feature of sarcopenia by inducing muscle senescence via wnt/β-catenin signal activation [ ] . in contrast, the angiotensin converting enzyme (ace )/ang( - )/mas receptor arm of ras generally counter-regulate the classical ras function ( figure ) [ ] . ace deficiency in mice accelerated age-related muscle weakness and the associated senescence phenotype as a model of sarcopenia, whereas ang( - ) treatment restored this [ , ] . protection against angii-as well as il- β-induced senescence by ang( - )-dependent activation of mas receptor was also shown in endothelial cells [ ] . research on the potential application and mechanistic investigation with seno-modulatory interventions is strongly desired. general mechanisms contributing to arterial aging include mitochondrial dysfunction, oxidative stress, inflammation, and activation of the ras [ ] . chronic ras activation leads to damage in several organs, which is associated with aging and oxidative stress in cells or mitochondria [ ] . aging also causes enhancement in the activity of and response to the ras. [ ] . angii is the essential hormone acting through two receptor subtypes, angii type receptor (at r) and angii type receptor (at r). activation of at r promotes the majority of ras physiology and pathophysiology including vasoconstriction, cardiovascular hypertrophy and fibrosis, inflammation and oxidative stress [ ] . while there are some exceptions and the exact signaling mechanisms are still unclear, activation of at r generally counteracts the classical ras actions including cell growth, oxidative stress, inflammation and fibrosis [ , ] . moreover, enhancement of senescence markers induced by angii was observed in vsmcs cultured from at r deficient mice suggesting the anti-senescence role for at r [ ] . in addition, for the past two decades, significant advancement has been made in understanding the function and mechanisms utilized by new members of the angiotensin family ligands and receptors [ ] . among them, using systemic pro (renin) receptor transgenic mice, it was demonstrated that pro (renin) receptor mediates skeletal muscle atrophy and the feature of sarcopenia by inducing muscle senescence via wnt/β-catenin signal activation [ ] . in contrast, the angiotensin converting enzyme (ace )/ang( - )/mas receptor arm of ras generally counterregulate the classical ras function (figure ) [ ] . ace deficiency in mice accelerated age-related muscle weakness and the associated senescence phenotype as a model of sarcopenia, whereas ang( - ) treatment restored this [ , ] . protection against angii-as well as il- β-induced senescence by ang( - )-dependent activation of mas receptor was also shown in endothelial cells [ ] . [ , ] . in contrast, at r protects against at r-mediated senescence [ ] . pro (renin) receptor (prr) is a new member of the ras receptors and mediates senescence [ ] . ace cleaves angii and ang( - ) to produce ang( - ) to initiate protective arm of novel ras (the ace /ang( - )/mas axis), which counter-regulates at rmediated senescence [ , ] . the roles of additional ang( - ) receptor mrgd and angiv receptor (at r) play in senescence remain unknown. [ , ] . in contrast, at r protects against at r-mediated senescence [ ] . pro (renin) receptor (prr) is a new member of the ras receptors and mediates senescence [ ] . ace cleaves angii and ang( - ) to produce ang( - ) to initiate protective arm of novel ras (the ace /ang( - )/mas axis), which counter-regulates at r-mediated senescence [ , ] . the roles of additional ang( - ) receptor mrgd and angiv receptor (at r) play in senescence remain unknown. previous reports have demonstrated that expression of at r increases with age, whereas at r decreases with age in rodents [ , ] . as lifespan can be extended with at r (at a r) knockout as well as treatment with at r blockers in rodents [ , ] , it is reasonable to speculate the critical role at r has in mediating cardiovascular aging. the mechanism of the lifespan extension seems to involve preservation of mitochondrial numbers [ , ] . accordingly, inhibition of the classical ras by an ace inhibitor appears effective in attenuating frailty and systemic inflammatory responses in aged mice [ ] . analyses on ace polymorphism [ ] and at r polymorphism [ ] further support the critical negative relationship between angii/at r and healthspan in humans. the angii antagonists are the most frequently used anti-hypertensives. however, only circumstantial information is available regarding the anti-aging actions of angii antagonists in humans [ , ] . while it is challenging, more translational research is encouraged on the ras system in humans, as there are new opportunities to design and evaluate the efficacies of these drugs against human aging or premature aging populations [ ] . angii-induced senescence of vsmcs appears to be mediated through several signaling mechanisms involving nicotinamide adenine dinucleotide phosphate (nadph) oxidase, reactive oxygen species (ros) and mtor ( figure and table ). in contrast, activation of autophagy, cyclic amp (camp)/protein kinase a (pka), sirtuins, nuclear factor erythroid -related factor (nrf ) and klotho counter-regulate angii-induced senescence via several distinct mechanisms ( figure and table ). the following section will aid in explaining the inter-relationship of these signaling events and whether they are required (needed factors) or sufficient (main driver of the alteration) for the phenotypes. previous reports have demonstrated that expression of at r increases with age, whereas at r decreases with age in rodents [ , ] . as lifespan can be extended with at r (at ar) knockout as well as treatment with at r blockers in rodents [ , ] , it is reasonable to speculate the critical role at r has in mediating cardiovascular aging. the mechanism of the lifespan extension seems to involve preservation of mitochondrial numbers [ , ] . accordingly, inhibition of the classical ras by an ace inhibitor appears effective in attenuating frailty and systemic inflammatory responses in aged mice [ ] . analyses on ace polymorphism [ ] and at r polymorphism [ ] further support the critical negative relationship between angii/at r and healthspan in humans. the angii antagonists are the most frequently used anti-hypertensives. however, only circumstantial information is available regarding the anti-aging actions of angii antagonists in humans [ , ] . while it is challenging, more translational research is encouraged on the ras system in humans, as there are new opportunities to design and evaluate the efficacies of these drugs against human aging or premature aging populations [ ] . angii-induced senescence of vsmcs appears to be mediated through several signaling mechanisms involving nicotinamide adenine dinucleotide phosphate (nadph) oxidase, reactive oxygen species (ros) and mtor ( figure and table ). in contrast, activation of autophagy, cyclic amp (camp)/protein kinase a (pka), sirtuins, nuclear factor erythroid -related factor (nrf ) and klotho counter-regulate angii-induced senescence via several distinct mechanisms ( figure and table ). the following section will aid in explaining the inter-relationship of these signaling events and whether they are required (needed factors) or sufficient (main driver of the alteration) for the phenotypes. signaling mediators of angii-dependent senescence in vsmcs. nox-derived ros production is activated by angii through at r and a small gtpase rac [ , ] . in vsmcs, figure . signaling mediators of angii-dependent senescence in vsmcs. nox-derived ros production is activated by angii through at r and a small gtpase rac [ , ] . in vsmcs, mitochondrial complex i or ii were stimulated by angii causing production of mitochondrial ros (mtros) and subsequent senescent responses [ ] . in addition, mitochondrial respiratory disfunction occurred at complex ix was reported to induce senescence via p [ ] . moreover. major ser/thr kinases involved in senescence (extracellular signal-regulated kinase (erk), p , mtor and s k) are activated via adam -dependent egfr transactivation in vsmcs [ ] . these signaling events are considered major drivers of at r mediated vascular senescence and sasp (table for the references). occurred at complex ix was reported to induce senescence via p [ ] . moreover. major ser/thr kinases involved in senescence (extracellular signal-regulated kinase (erk), p , mtor and s k) are activated via adam -dependent egfr transactivation in vsmcs [ ] . these signaling events are considered major drivers of at r mediated vascular senescence and sasp (table for the references). nox-derived ros nox / [ , ] in vitro nox-derived ros mek/erk, p , akt [ ] in vitro nox-derived ros rac [ ] in vitro mitochondrial ros complex i/ii [ ] in vitro sm α pi k/akt [ ] in vivo p complex iv [ ] in vivo mitochondria fission drp [ ] figure . inhibitory signaling events against angii/at r-mediated senescence in vsmc. various inhibitors that attenuate senescence via antagonizing oxidative stress have been reported. pgc-  deficiency promotes vascular senescence with increased ros and results in reduced expression of sirt [ ] . pgc-  also maintains autophagy and a component of autophagy, autophagy related (atg ) to counter-regulate senescence [ ] . nrf plays a key role in protection of cellular senescence via pka/creb pathway [ ] . soluble klotho, an antiaging hormone, attenuates angii-induced vsmc senescence through nrf induction and ros attenuation [ ] . mitochondrial sirt also protects against mtros [ , ] . in addition, reduction in nad + exaggerates angii-induced vascular senescence [ , ] . inhibitory signaling events against angii/at r-mediated senescence in vsmc. various inhibitors that attenuate senescence via antagonizing oxidative stress have been reported. pgc- α deficiency promotes vascular senescence with increased ros and results in reduced expression of sirt [ ] . pgc- α also maintains autophagy and a component of autophagy, autophagy related (atg ) to counter-regulate senescence [ ] . nrf plays a key role in protection of cellular senescence via pka/creb pathway [ ] . soluble klotho, an antiaging hormone, attenuates angii-induced vsmc senescence through nrf induction and ros attenuation [ ] . mitochondrial sirt also protects against mtros [ , ] . in addition, reduction in nad + exaggerates angii-induced vascular senescence [ , ] . the molecular insight of sips induced by angii had been limited in demonstrating that angii induced sips via at r and p /p induction in vsmcs [ , ] . oxidative stress in the vasculature is considered as a primary mechanism underlying vascular dysfunction and aging. increases in arterial ros leads to elevated expression and activity of nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox), as well as increased endothelial nitric oxide synthase (enos) uncoupling [ ] . in vsmcs, nox is activated by angii via at r leading to superoxide production and subsequent activation of downstream tyrosine and serine/threonine kinases [ , ] . angii also increases superoxide production from mitochondria by increasing activity of the electron transfer chain as well as nox in of vascular cells [ ] . in vsmcs, pharmacological inhibition of mitochondrial complex i or complex ii, or treatment with the mitochondrial ros scavenger significantly reduced superoxide production as well as senescence assessed with sa-βgal staining in vsmcs [ ] . however, nox sirna [ , ] as well as pharmacological inhibitors of serine/threonine kinases (akt, mitogen-activated protein kinase kinase (mek) and p mitogen-activated protein kinase (mapk)) [ ] also attenuated angii-enhanced sa-β gal staining in vsmcs. a small gtpase rac is a contributing component of nox activation complex. inhibition of angii-induced vsmc senescence via rac inhibitor nsc has been interpreted for its ros mitigation [ ] . nrf is a critical transcriptional factor, which mediates antioxidant defense programs upon oxidative stress via binding to antioxidant response elements. it can be also activated by camp/pka responsible creb binding protein. a glucagon-like peptide- analog appears to attenuate angii-induced vsmc senescence via pka-dependent nrf activation [ ] . klotho gene delivery is known to suppress angii-induced nox expression in vsmcs [ ] . soluble klotho further attenuates angii-induced vsmc senescence via nrf induction [ ] . in addition, angii-induced vsmc senescence appears to require inhibition of forkhead box (fox)o transcriptional activity to induce silence information regulator -like (sirt ) via peroxisome proliferator-activated receptor gamma coactivator (pgc)- α (pgc -α) serine phosphorylation. this sirt inhibition reduces anti-oxidative catalase expression leading to vsmc senescence [ ] . in vsmcs, the activation of α nicotinic acetylchoine receptor activation is shown to attenuate angii-induced vsmc senescence by nad + -dependent sirt activation [ ] . taken together, these data support the overall roles of nox and ros in angii-induced senescence in vsmcs. nox and ros in angii signaling and cvds have been studied more than years, albeit not in relation to senescence [ , ] . it seems that the nox/ros dependent mechanism of senescence could be only a minor portion of the ros/nox functions in the angii signaling system and they may be required but not sufficient for vsmc senescence induced by angii. mtor is a master coordinator of cell growth and metabolic adaptation. mtor is a serine/threonine protein kinase that consists of the catalytic subunit of two distinct protein complexes, that are mtor complex (mtorc ) and (mtorc ) [ ] . several lines of evidence support the direct relationship between mtorc and senescence. reduction in mrna translation during mtorc inhibition slows senescence by suppressing the accumulation of proteotoxic and oxidative stress. inhibition of mtorc also slows senescence by increasing autophagy, which helps clear damaged proteins and organelles of mitochondria [ ] . accordingly, contribution of mtor and protective autophagy to angii-induced vsmc senescence has been demonstrated by using mtor inhibitor, rapamycin, and autophagy inhibitors, -methyladenine and bafilomycin a [ ] . the phosphatidylinositol- -kinase (pi k)/akt pathway positively regulates mtor activity. angii-induced vsmc senescence appears to require signal communication between pi k/akt and an actin binding protein, smooth muscle α (sm α). this signal communication leads to akt-dependent phosphorylation and inhibition of mouse double minute homolog (mdm ) which mediates ubiquitination and degradation of p [ ] . in addition, pgc -α appears necessary to maintain autophagy in vsmcs, where angii-induced senescence was enhanced by another autophagy inhibitor, spautin- , or by sirna silencing of autophagy components autophagy related (atg ) or p /sequestosome [ ] . however, conflicting findings have been published regarding whether angii positively or negatively regulates autophagy in vsmcs and whether the vsmc autophagy is protective or detrimental for angii-regulated cvds [ , ] . outside of senescence, angii-induced mtor activation has been implicated in protein synthesis in vsmcs via s kinase activation [ , ] . ribosomal s kinase encoded by s ki is one of the major effectors of mtor . in vsmcs, the pi k/akt/mtor/s k cascade activation via at r is primarily mediated by the epidermal growth factor receptor (egfr) transactivation [ ] . interestingly, s ki deficient mice is protected against age or diet-induced obesity while enhancing insulin sensitivity [ ] , have extended life span and recapitulate the phenotype seen with caloric restriction or pharmacological ampk activation [ ] . a nuclear epigenetic factor zrf has been recently identified as a novel substrate for s kinase, and mediates the s k-dependent senescence program [ ] . these findings thus deserve further investigation for the relationship of the s k cascade to vsmc senescence. angii infusion has been shown to induce sa-βgal in aortas of hyperlipidemic mouse models such as apolipoprotein e (apoe) deficient mice [ ] . angii also caused vascular senescence in a collagen i mutant mouse in which the vascular senescence correlated with aortic stiffness [ ] . similar to findings in a cell culture model, vsmc sirt appears protective against vascular senescence during abdominal aortic aneurysm (aaa) development following angii infusion in apoe deficient mice [ ] . in addition, the aortic senescence induced by angii infusion in ldlr deficient mice was attenuated by concurrent sm α silencing because sm α interacted with and inhibited mdm which control ubiqutination and degradation of p [ ] . the role for p in aaa development and aortic senescence was also confirmed with p deficient mice as well as caloric restriction [ ] . mitochondrial sirt protects against oxidative stress by promoting deacetylation of mitochondrial superoxide dismutase, sod . importance of mitochondrial-derived ros in vascular oxidative stress and senescence has been confirmed with mitochondrial sirt deficient mice and sirt transgenic mice infused with angii [ ] . generation of nad + via nicotinamide phosphoribosyl transferase (nampt) is known to mediate cell vitality. in mice deficient with smooth muscle nampt, angii induced aortic wall degeneration and dissection was exaggerated, which was associated with senescence [ ] . taken together, these studies identify the link between angii and vsmc senescence in vivo, however it is desired to further test pharmacological or genetic senolytic (or senomorphic) treatment in angii-dependent cvd models such as hypertension and aaa. mitochondrial dysfunction including mitochondrial oxidative stress is a hallmark of the aging process, however, recent evidence has emerged that dysfunctional mitochondrial quality control (fission/fusion, biogenesis and mitophagy) also contribute to senescence [ , ] . a small gtpase, dynamin-related protein (drp ) is one such molecule that mediates mitochondrial fission, and excessive drp activity and mitochondrial fission are associated with several models of cvd [ ] . recently, involvement of drp in senescence induction was demonstrated [ , ] . in abdominal aortic smooth muscle cells, angii stimulated drp activity and mitochondrial fission via erk / -dependent drp ser phosphorylation. inhibiting drp activity in mice further attenuated angii-induced aaa development which was associated with reduction of aortic senescence assessed by sa-βgal and p induction [ ] . in endothelial cells, drp also mediates nf-κb activation [ ] . in addition, mitochondrial fission often associates with the turnover of damaged mitochondria through mitophagy, however, the relationship between mitophagy and senescence remains to be explored. interestingly, the concept of a subtype of senescence has been recently proposed as midas (mitochondrial dysfunction-associated senescence) which demonstrates an altered sasp proteome [ ] . therefore, understanding the potential roles of midas sasp elicited by angii over conventional sasp in cardiovascular dysfunction may also be beneficial. the endoplasmic reticulum (er) maintains cellular proteostasis, and is another organelle that is highly involved in aging of the cell. evidence exists that er stress and subsequent unfolded protein response (upr) mediates induction of major senescence markers including sa-βgal. moreover, because the er is responsible for the trafficking and secretion of many proteins, er stress alters the sasp composition while also propagating proteotoxicity and upr signaling to dictate sasp secretions [ ] . in terms of vascular senescent cell development, investigations of the link between mitochondrial and er are only just beginning. in cultured endothelial cells angii/at r-induced senescence appears to involve interaction between drp and er stress [ ] . angii-induced er stress causes protein aggregation in vsmcs [ ] . furthermore, the lamin a variant progerin accelerates aging, and has been shown to induce er stress and upr activation in aortic vsmcs, which overall exacerbates atherosclerosis [ ] . vascular er stress is commonly associated with arterial stiffness [ ] . angii-induced egfr transactivation mediates er stress and subsequent vascular remodeling [ , ] . accordingly, egfr, mitochondrial fission/drp and er stress likely play upstream roles in angii-induced vascular senescence and sasp thus contributing to ras-mediated pathophysiology in cvds (figure ) . . mitochondrial fission and er stress likely play upstream roles in angii-induced vascular senescence and sasp contributing to ras-mediated pathophysiology in cvds. in addition to the molecular mechanisms illustrated in figure , mitochondrial fission and er stress are enhanced under chronic ras activation contributing to vsmc senescence [ ] . it is hypothesized that midas vsmc produce altered sasp, which adversely affects non-senescent vsmc phenotype leading to enhancement of vascular remodeling. such vsmc phenotype has been described as "smooth muscle cell-or arterial-stiffness syndrome" [ , ] . in vivo studies regarding the contribution of senescence to cardiovascular dysfunction and diseases are still limited in certain animal models including mice with angii infusion. in these models, most interventions are not directed towards senescence specifically and show instead only the association between senescence and the phenotype alterations. in mice infused with angii, aortic senescence appears limited whereas the condition is sufficient to induce cardiovascular pathology including hypertension, aortic stiffness, perivascular fibrosis and cardiac hypertrophy. this may mean that senescence is not an essential process for angii-induced pathophysiology or more likely that the mice used may be too young for the experiments to test whether senescence has any relevance in the angii pathology. accordingly, development of better animal models which simulate human premature aging seems essential for testing the effectiveness of senolytics or senomorphics against cvd. it should be noted that in senescence accelerated mice, angii plus cacl -induced aaa appear enhanced [ ] . aging is a nonmodifiable risk factor for cvd. since the lifespan of our population increases, the urgency to study vascular aging as a therapeutic target is evident. accumulation of senescent cells . mitochondrial fission and er stress likely play upstream roles in angii-induced vascular senescence and sasp contributing to ras-mediated pathophysiology in cvds. in addition to the molecular mechanisms illustrated in figure , mitochondrial fission and er stress are enhanced under chronic ras activation contributing to vsmc senescence [ ] . it is hypothesized that midas vsmc produce altered sasp, which adversely affects non-senescent vsmc phenotype leading to enhancement of vascular remodeling. such vsmc phenotype has been described as "smooth muscle cell-or arterialstiffness syndrome" [ , ] . in vivo studies regarding the contribution of senescence to cardiovascular dysfunction and diseases are still limited in certain animal models including mice with angii infusion. in these models, most interventions are not directed towards senescence specifically and show instead only the association between senescence and the phenotype alterations. in mice infused with angii, aortic senescence appears limited whereas the condition is sufficient to induce cardiovascular pathology including hypertension, aortic stiffness, perivascular fibrosis and cardiac hypertrophy. this may mean that senescence is not an essential process for angii-induced pathophysiology or more likely that the mice used may be too young for the experiments to test whether senescence has any relevance in the angii pathology. accordingly, development of better animal models which simulate human premature aging seems essential for testing the effectiveness of senolytics or senomorphics against cvd. it should be noted that in senescence accelerated mice, angii plus cacl -induced aaa appear enhanced [ ] . aging is a nonmodifiable risk factor for cvd. since the lifespan of our population increases, the urgency to study vascular aging as a therapeutic target is evident. accumulation of senescent cells causes production of pathological inflammatory molecules via sasp and negatively affects vascular homeostasis. some anti-senescence therapies suppress the induction of senescent cells not only in vitro but in vivo through various pathways as we discussed. we believe that adapting novel anti-senescent approach into the clinic has potential to effectively prevent or treat cvd. changes in mitochondrial function and er stress appear associated with senescence. therefore, in addition to explore new classes of senolytics and senomorphics, future studies should investigate a novel anti-aging therapy that target multiple organismal phenotypes in senescence. moreover, development of new animal model to simulate cvd linked to human aging in which we can 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creative commons attribution (cc by) license key: cord- -npob n authors: gheblawi, mahmoud; wang, kaiming; viveiros, anissa; nguyen, quynh; zhong, jiu-chang; turner, anthony j.; raizada, mohan k.; grant, maria b.; oudit, gavin y. title: angiotensin-converting enzyme : sars-cov- receptor and regulator of the renin-angiotensin system: celebrating the th anniversary of the discovery of ace date: - - journal: circ res doi: . /circresaha. . sha: doc_id: cord_uid: npob n ace (angiotensin-converting enzyme ) has a multiplicity of physiological roles that revolve around its trivalent function: a negative regulator of the renin-angiotensin system, facilitator of amino acid transport, and the severe acute respiratory syndrome-coronavirus (sars-cov) and sars-cov- receptor. ace is widely expressed, including, in the lungs, cardiovascular system, gut, kidneys, central nervous system, and adipose tissue. ace has recently been identified as the sars-cov- receptor, the infective agent responsible for coronavirus disease , providing a critical link between immunity, inflammation, ace , and cardiovascular disease. although sharing a close evolutionary relationship with sars-cov, the receptor-binding domain of sars-cov- differs in several key amino acid residues, allowing for stronger binding affinity with the human ace receptor, which may account for the greater pathogenicity of sars-cov- . the loss of ace function following binding by sars-cov- is driven by endocytosis and activation of proteolytic cleavage and processing. the ace system is a critical protective pathway against heart failure with reduced and preserved ejection fraction including, myocardial infarction and hypertension, and against lung disease and diabetes mellitus. the control of gut dysbiosis and vascular permeability by ace has emerged as an essential mechanism of pulmonary hypertension and diabetic cardiovascular complications. recombinant ace , gene-delivery of ace , ang – analogs, and mas receptor agonists enhance ace action and serve as potential therapies for disease conditions associated with an activated renin-angiotensin system. rhace (recombinant human ace ) has completed clinical trials and efficiently lowered or increased plasma angiotensin ii and angiotensin - levels, respectively. our review summarizes the progress over the past years, highlighting the critical role of ace as the novel sars-cov- receptor and as the negative regulator of the renin-angiotensin system, together with implications for the coronavirus disease pandemic and associated cardiovascular diseases. k nowledge of the underlying biology and physiology of ace (angiotensin-converting enzyme ) has accumulated over the last years since its discovery and has provided a major stimulus to further our understanding of the renin-angiotensin system (ras). [ ] [ ] [ ] [ ] ace has distinct roles ranging from catalytic activities with various substrates, as functional receptors for severe acute respiratory syndrome (sars) coronaviruses (sars-cov), and as an amino acid transporter. [ ] [ ] [ ] [ ] ace functions as a master regulator of the ras mainly by converting ang (angiotensin) i and ang ii into ang - and ang - , respectively. , both loss-of-function and gain-of-function approaches in experimental models of human diseases have defined a critical role for ace in heart failure (hf), systemic and pulmonary hypertension (ph), myocardial infarction (mi), and diabetic cardiovascular complications. gut dysbiosis and altered gut permeability have emerged as an important mechanism of disease controlled by the ace axis in both vascular and lung diseases, , as well as in diabetes mellitus. clinical and experimental studies support a physiological and pathophysiological role for ace in cardiovascular disease (cvd), and increasing/activating ace may elicit protective effects against hypertension and cvd, although this has yet to be proven clinically. , [ ] [ ] [ ] more recently, ace has garnered widespread interest as the cellular receptor of sars-cov- , the causative virus of the coronavirus disease (covid- ) pandemic, which emerged from wuhan, china, in late . , , ace offers protection in acute lung injury, suggesting that, although it facilitates viral entry at the epithelial surface, the ace /ang - axis can be carefully manipulated to mitigate sars-induced tissue injuries, which represents a potential target for therapeutic intervention. , in experimental models of lung disease, catalytically active ace alleviates pulmonary injury and vascular damage , and prevent ph, decreased lung fibrosis, arterial remodeling, and improved right ventricular performance due to a combination of direct action in the lungs and via the ace -dependent gut-lung axis. , in phase ii clinical trials, administration of ace was shown to reduce systemic inflammation and shifted the ras peptide balance away from ang ii toward ang - . , ongoing global efforts are focused on manipulating the ace /ang - axis to curtail sars-cov- infection while affording maximal protective effects against lung and cardiovascular damage in patients with in this review, we summarize the diverse roles of ace , highlighting its role as the sars-cov- receptor and negative regulator of the ras, and the implications for the covid- pandemic. we also provide a framework for developing novel therapeutic strategies exploring the ace pathway as it relates to cvd and covid- . following the initial and seminal discovery of renin in by tigerstedt and bergman, the ras now encompasses a complex network of enzymes, peptides, and receptors ( figure ). , , , , [ ] [ ] [ ] [ ] [ ] while many metallopeptidases cluster in small inter-related gene families (eg, the neprilysin [nep] family), unusually, no human homolog of the vasoactive zinc-peptidase ace (angiotensin converting enzyme) had been identified at the turn of the century. almost simultaneously, in , independent approaches searching for such ace homologs revealed the existence of a close relative of the ace gene designated aceh or ace . aceh was cloned from a human lymphoma cdna library and the identical ace from a human hf ventricular cdna library, the latter emphasizing a potential role for ace in cardiovascular pathologies. expression of the ace gene was initially established in the heart, kidney, and testis, but subsequent studies have shown a much broader distribution, including the upper airways, lungs, gut, and liver ( figure a ). sequence comparison of ace and ace strongly suggested that ace , like ace, was an integral transmembrane protein (and ectoenzyme) with a transmembrane anchor close to the c-terminus (type i membrane protein). a close evolutionary relationship existed between the ace and ace , genes and it was presumed that the proteins would have similar substrate specificities and involvement in the ras. as it turned out, important differences occur, particularly in the active site regions of the enzymes, such that the enzymes counterbalance rather than reinforce each other's actions. many subsequent studies over the next years have revealed their inter-relationship, respective roles in the ras, and multiple physiological and pathological actions from vasoactive peptide metabolism, importantly including not only ang ii but also apelin, to intestinal amino acid transport affecting innate immunity, to lung function and brain amyloid metabolism (converting aβ to aβ , a substrate for ace). , , another unexpected twist in ace biology was its identification in as the cell-surface receptor for the then newly identified sars-cov that led to > cases of sars and almost deaths, and as the receptor for sars-cov- that is currently devastating many countries worldwide. , the ace gene and basic biochemistry unlike the ace gene, which is located on human chromosome , the kb ace gene is located on chromosome xp and contains exons, most of which resemble exons in the ace gene. whereas somatic ace contains active sites, ace possesses only a single catalytic domain. both ace and ace act as zinc metallopeptidases but of differing substrate specificities defining their distinct and counterbalancing roles in the ras. whereas ace cleaves c-terminal dipeptide residues from susceptible substrates (a peptidyl dipeptidase), ace acts as a simple carboxypeptidase able to hydrolyze ang i, forming ang - and ang ii to ang - ( figure b ). ace does not cleave bradykinin, further distinguishing its specificity from that of ace while it is also insensitive to conventional ace inhibitors. , the c-terminal domain of ace , which has no similarity with ace, is a homolog of a renal protein, collectrin, which regulates the trafficking of amino acid transporters to the cell surface, endowing ace with multiple and distinctive physiological functions. it is the multiplicity of physiological roles that ace plays that has allowed it to be hijacked by sars-cov- as a receptor, resulting in the covid- pandemic. , structural studies have revealed the structures of both the sars-cov and much more recently, the sars-cov- in complex with ace ( figure b ). , in the case of sars-cov- , the major spike glycoprotein (s ) binds to the n-terminal region of ace . the knowledge of the biology and physiology of ace accumulated over the last years since its discovery should provide a major stimulus to understanding some of the key steps in sars-cov- infection and its ultimate prevention. on march , , the world health organization declared the outbreak of sars-cov- a global pandemic, reporting community scale transmissions occurring in every continent outside antarctica. since then, the outbreak has escalated to well over one million cases and caused over deaths worldwide by the start of april . however, before the emergence of sars-cov in , coronaviruses were conventionally viewed as inconsequential pathogens circulating in nature throughout various host and intermediate species that occasionally infected humans causing only mild upper respiratory tract infections and symptoms of the common cold. [ ] [ ] [ ] as such, to better understand the renin was the first component of the ras discovered following the finding that extracts from rabbit kidney produced pressor effects (tigerstedt and bergman, ). constriction of the renal artery was then found to lead to hypertension (htn), thus driving the discovery of hypertensin and angiotonin (and later termed angiotensin; goldblatt et al ; page and helmer ). ang (angiotensin) was subsequently purified, and forms were resolved: ang i and ang ii. therefore, the existence of a converting enzyme was predicted (ace) and subsequently isolated and characterized (skeggs et al severity of global health risks posed by sars-cov- and optimize treatment for infected patients, we must recognize the role of ace in sars-cov- pathogenesis. in addition to respiratory involvement, multiorgan dysfunction occurs in response to sars-cov- infections. [ ] [ ] [ ] while respiratory symptoms are predominant, acute cardiac and kidney injuries, arrhythmias, gut, and liver function abnormalities have all been documented in infected patients, suggesting myocardial, renal, enteric and hepatic damage in covid- . similarly, sars-cov also resulted in systemic manifestations with damages to the heart, gastrointestinal, liver, kidney, and other tissues. , ace as the receptor for sars-cov- sars-cov- differs from the original sars-cov by amino acid substitutions, which translates to differences in five of the six vital amino acids in the receptor-binding domain between the viral spike (s) protein with surface expressed human ace . viral s-proteins are well established as a significant determinant of host tropism and represents a key target for therapeutic and vaccine development. additionally, host cell proteases are important for sars-cov- entry and infection of cells as both s-proteins and ace are proteolytically modified during the process. the binding affinity of sars-cov- with ace seems stronger than sars-cov, with alterations in several amino acid residues allowing for enhanced hydrophobic interactions and salt bridge formations, which may explain the considerably larger global influence of covid- than the initial sars. , moreover, sars-cov- has evolved to utilize a wide array of host proteases including cathepsin l, cathepsin b, trypsin, factor x, elastase, furin, and tmprss (transmembrane protease serine ) for s-protein priming and facilitating cell entry following receptor binding. so far, tmprss and cathepsin l/b mediates s-protein priming of sars-cov- , and camostat mesylate, a serine protease inhibitor combined with cathepsin l/b inhibitor, e- d blocked sars-cov- entry. the entry of both sars-cov and sars-cov- into cells is facilitated by the interaction between viral s-protein with extracellular domains of the transmembrane ace proteins, followed by subsequent downregulation of surface ace expression ( figure ). ace -derived peptides, small molecule inhibitors, ace antibody or single chain antibody fragment against ace . in postmortem autopsy heart tissues from patients who succumbed to sars-cov, heart samples had detectable viral sars-cov genome, which was characterized by increased myocardial fibrosis, inflammation, and reduced myocardial ace expression. these patients also had a much more aggressive illness associated with earlier mortality. additionally, bilateral pleural effusions were frequently observed during autopsy of sars-cov patients, further supporting the evidence of cardiac involvement. individuals with preexisting diabetes mellitus, hypertension, and lung disease are at particular risk of covid- infection , and this is likely due to dysregulated ras that occurs in these conditions. , significance of the sars-cov- infection in the cardiovascular system is reflected through incidences of acute myocardial injury (elevated high sensitivity troponin i levels and/or new ecg/echocardiogram abnormalities), arrhythmias, cardiac arrest, sepsis, septic shock, viral myocarditis, and hf (elevated nt-probnp levels, systolic dysfunction on cardiac magnetic resonance imaging). [ ] [ ] [ ] [ ] further abnormalities from laboratory tests, including elevation in d-dimers reflective of increased thrombosis risk, may lead to acute coronary syndrome, and sustained increased inflammatory cytokines levels throughout the clinical course suggest ongoing systemic and tissue inflammation in patients with covid- . , , gut dysbiosis and a possible link to disease progression in covid- patients ubiquitous expression of ace throughout the luminal surface of the gastrointestinal tract, and most prevalently in enterocytes, may serve as a secondary site for enteric sars-cov- infection (figure a ). leaky gastrointestinal ace -mediated cardiovascular protection is lost following endocytosis of the enzyme along with severe acute respiratory syndrome-coronavirus (sars-cov- ) viral particles. ang ii (angiotensin ii) levels elevate with increased activity of angiotensin receptors (at r) at the cost of ace / ang - driven pathways leading to adverse fibrosis, hypertrophy, increased reactive oxygen species (ros), vasoconstriction, and gut dysbiosis. adam (a disintegrin and metalloproteinase )-mediated proteolytic cleavage of ace is upregulated by endocytosed sars-cov- spike proteins. activation of the at r by elevated ang ii levels also further increases adam activity. adam correspondingly also cleaves its primary substrate releasing soluble tnf-α (tumor necrosis factor-α) into the extracellular region where it has auto-and paracrine functionality. tnf-α activation of its tumor necrosis factor receptor (tnfr) represents a third pathway elevating adam activity. tnf-α along with systemic cytokines released due to sars-cov- infection and in conjunction with comorbidities such as diabetes mellitus and hypertension can lead to a cytokine storm. tmprss indicates transmembrane protease serine . conditions in experimental models of human disease can be ameliorated and worsened with either the gain or loss of ace , respectively. , patients with covid- also suffer from gastrointestinal discomfort and diarrhea, which may arise earlier than respiratory conditions concurrent with the detection of viral rna in feces, as seen with previous coronavirus outbreaks. , , [ ] [ ] [ ] [ ] moreover, common comorbidities of cvd, including diabetes mellitus and obesity, are known to affect the integrity of the gastrointestinal-blood barrier and result in gut dysbiosis, bacteremia, and systemic inflammation ( figure ). development of gastrointestinal leakage and gut dysbiosis have correspondingly been linked to the onset of ph through the gut-lung axis and is closely related to hyperactivation of the ace/ang ii/at r (angiotensin ii type receptor) axis from ace loss. , continued viral production by host enterocytes perpetuates this situation and deteriorates conditions in the gut-lung axis. , evidence supports that sars-cov- infection potentially leads to degeneration of the gut-blood barrier leading to systemic spread of bacteria, endotoxins, and microbial metabolites likely affecting the host's response to covid- infection and cumulating in multisystem dysfunction and septic shock. , , enteric involvement and associated worsening in patient outcomes were documented from the initial sars-cov outbreak in the early s. fecal viral rna was detected in up to % of patients with viral shedding from the gastrointestinal tract associated with a more aggressive clinical course. , in a separate study, sars-cov particles were detected within the cytoplasm and surface microvilli of apical enterocytes in the ileum and colon while in patients with covid- , sars-cov- was detected in feces suggesting fecal-oral transmission. as such, the gastrointestinal tract of sars-cov, and possibly sars-cov- patients, acts as a staging ground for sustained viral replication concurrent with disruption of the enteric ace axis and adverse outcomes. , , , , , in addition to the direct impact of the virus on the microbiome, the predisposing disease states such as diabetes mellitus and pulmonary disease have their own adverse effects on the gut microbiome, , which may be worsened by sars-cov- infection. ang iidependent hypertension in animal models and humans is associated with gut dysbiosis, increased gut leakiness, and gut wall pathology. , , , there is broad support for these observations in pulmonary diseases including ph, copd, and asthma , and in type diabetes mellitus where dysbiosis characterized by decreased microbial richness and diversity, altered representation of bacterial metabolic pathways and modifications in the composition of firmicutes (f) and bacteroidetes (b). [ ] [ ] [ ] ace disruption in biomedical models has shown us that gut dysbiosis is quite prevalent and that this change in microbial profiles can alter systemic pathways exacerbating diabetes mellitus and hypertension. we recently showed that ace deficiency magnifies diabetes mellitus-induced dysbiosis characterized by an increase in peptidoglycan-producing bacteria and loss of gut barrier integrity in ace −/y -akita mice. we also identified a new role of bone marrow cells in the gut. in the ace −/y -akita or akita mice, the disrupted gut barrier was associated with reduced levels of circulating angiogenic cells, hematopoietic cells with reparative function. giving exogenous circulating angiogenic cells from wild-type mice corrected gut barrier dysfunction in ace −/y -akita or akita mice. thus, decreased enteric ace expression from sars-cov- infection may similarly reduce circulating angiogenic cells and compromise the integrity of the endothelium and gut epithelium leading to dysbiosis. further examination is required to validate this link and whether it is a direct or indirect effect of viral infection. link between ace , adam , and inflammation proteolytic cleavage of ace by adam- tnf-α (tumor necrosis factor-α) is a cytokine implicated in chronic inflammation, and its extracellular domain shedding and activation is driven by the membrane-bound protease coined tace (tnf-α-converting enzyme), also known as adam- (a disintegrin and metalloproteinase ). , adam- is a type i transmembrane protein belonging to the adamalysin subfamily of zn-dependent metalloproteases. following the discovery that adam- cleaves tnf-α, the substrate specificity of the enzyme has expanded to include various cytokines and receptors, many of which contribute to initiating and exacerbating inflammation. , importantly, adam- was also found to mediate proteolysis and ectodomain shedding of ace . enhanced ace shedding resulting from ras overactivation, and subsequent adam- upregulation drives pathogenesis in hf, atrial fibrillation, coronary artery disease, and thoracic aortic aneurysm. , , , ang ii-mediated activation of at r triggers a signaling cascade, which culminates in the activation of p mapk (mitogen-activated protein kinase) and adam- phosphorylation by napdh oxidase -induced reactive oxygen species formation. , phosphorylation enhances the catalytic activity of adam- , thus increasing ace shedding, resulting in loss of ace at the membrane and impaired conversion of ang ii (into ang - ), leading to ras-mediated detrimental effects in a positive feedback cycle. , importantly, depletion of ace at the cell surface is a critical pathological outcome of sars-cov- infection. sars-cov- is endocytosed by cells in complex with ace ; thus, the initial detrimental effects of viral infection begins with a loss of ace -mediated tissue protection. adam- activity is upregulated upon binding of sars-cov to ace and facilitates viral entry, while knockdown of adam- by sirna severely attenuated sars-cov cellular entry. the molecular mechanisms of sars-cov and another human coronavirus that only causes mild respiratory symptoms, hnl -cov, were compared. interestingly, although hnl -cov also utilizes ace as a receptor for cellular entry, it does not induce adam- activation and ace ectodomain shedding. , therefore, this study elucidates the unique role of adam- mediated shedding of ace in sars-cov infectivity and may inform the disparity in severity between coronavirus subtypes. furthermore, loss of membrane ace promotes ang ii accumulation, which also activates adam- activity, thus perpetuating membrane shedding of ace , ras overactivation, and inflammation. the modulatory effects on the ang ii/at r and ang - /masr axes make ace a plausible target in preventing and treating chronic inflammation and inflammatory diseases, as highlighted by the recent covid- pandemic. patients with covid- develop pneumonia with acceleration of injury in susceptible patients to multiple organ failure , driven in part by an inflammatory cytokine storm and is a notable cause of death in patients who are critically ill. , when the immune system is activated due to factors such as sars-cov- infection, there is an imbalance of th /treg cell function and overactivation of immune cells, which secrete a large number of proinflammatory cytokines. , , imbalance in the ras system and the loss of ace in patients with covid- are further contributing factors to tissue and systemic inflammation. , lipopolysaccharide-induced acute lung injury decreased expression of ace , precipitated inflammatory injury, and upregulated expression of renin, ang ii, ace, and at receptors. after injection of rhace , lung function and pathological injury improved with attenuation of inflammation. in addition, rhace is beneficial and improves acute lung injury caused by sars-cov, acid inhalation, and sepsis. , , ace knockout (ko) mice showed very severe acute respiratory distress syndrome (ards)/acute lung injury pathology, increased vascular permeability, increased pulmonary edema, neutrophil accumulation, and deterioration of lung function compared with normal wt control mice. , ace deficiency partially rescued the severe phenotype of mice with a single mutation of ace in acute lung injury by further deletion of the ace gene, suggesting that the balance of ace /ace levels is the key to lung injury/lung protection during an inflammatory storm. arbs (at r blockers) induce ace , ang - , and mas expression in line with the reduction of proinflammatory cytokines and induction of il- , an anti-inflammatory cytokine. we showed that ace ko hypertensive mice exhibited enhancement of proinflammatory cytokines, il- β, il , tnf-α, and chemokine (c-c motif) ligand while administration of rhace rescued ang ii-induced t-lymphocyte-mediated inflammation. , blockade of mas receptor by d-ala -ang - (a- ) completely inhibited the ang - mediated anti-inflammatory effects while ave , the agonist of ang - receptors, mimicked the actions of ang - . negative regulator of the ras discovery of ace resulted in a paradigm-changing concept in all aspects of the ras. ace is a monocarboxypeptidase that converts ang i into a nonapeptide, ang - , and ang ii into a heptapeptide, ang - (figure a ). this distinct enzymatic pathway for degradation of ang i and ang ii negatively regulates ras activation and mitigates the deleterious actions mediated by ang ii and at r. this is of particular significance in pathological conditions where the ras is overstimulated. ang - is a biologically active peptide whose vast array of effects are opposite to those attributed to ang ii. [ ] [ ] [ ] [ ] [ ] [ ] [ ] furthermore, ace can antagonize ace-independent formation of ang ii, such as from mast cell chymase. , in , an endogenous orphan receptor, mas receptor (masr), was identified as the ang - receptor, and a , a masr antagonist was shown to inhibit the majority of ang - effects. , - ang - has also shown beneficial biological effects via the at r that result in cardioprotection. [ ] [ ] [ ] [ ] thus, the ace /ang - /masr axis has emerged as a physiological antagonist that counter-regulates the activated ras. , [ ] [ ] [ ] [ ] [ ] the cardioprotective effects of ace taken together can be attributed to ( ) degradation of ang i to ang - , whereby limiting action of ace on its substrate, ( ) reducing ang ii detrimental effects through degradation of the peptide, and ( ) formation of ang - which exercises cardioprotective effects. formation of ang - is an important mechanism of ace mediated protection, as antagonism of ang - using a prevented beneficial effects of rhace in murine model of systolic dysfunction. diminished ace activity results in activation of the ang ii/at r axis, contributing to the increased progression of cvd. elevated ace level and activity result in the formation of ang - and ang - , leading to protection against cvd ( figure a ). the apelin family of peptides act through the apelin receptors mediating protection against cvd. , the x-linked apln gene encodes a amino acids prepro-apelin that is subsequently cleaved by endopeptidases to various bioactive peptides from to amino acids in length. cvd, including hf and hypertension, is characterized by an apelin deficient state in both human myocardium and plasma. [ ] [ ] [ ] apelin ko mice exhibit increased infarct size and systolic dysfunction following coronary ligation and reduced myocardial contractility concomitant with increased susceptibility to hf in pressure-overload models. , reduced myocardial ace mrna and ace protein levels in apelin ko mice, which were rescued by infusion of apelin- , suggest a crucial regulatory role of apelin in ace gene expression. apelin signaling through the apelin receptors specifically increased ace promoter activity leading to an increase in ace mrna and protein. [ ] [ ] [ ] these effects are consistent with the ability of the pyr-apelin- peptide to negatively regulate ang ii-mediated superoxide production, myocardial hypertrophy, dysfunction, and fibrosis and analogs of apelin- preventing abdominal aortic rupture in lowdensity lipoprotein receptor ko models induced by ang ii infusion. however, ace , through its monocarboxypeptidase activity, cleaves and inactivates bioactive apelin peptides apelin- and apelin- through a negative feedback mechanism in the heart and vasculature (figure b) . , due to the short half-life of endogenous apelin peptides in the plasma, synthetic apelin peptide analogs resistant to ace degradation and retaining their binding capability to endogenous apelin receptors elicit protection in the cardiovascular system are being explored as potential new therapies. , ace as a chaperone protein for the amino acid transporter, b at (slc a ) b at is highly expressed in the intestines and kidneys with function in the absorption of neutral amino acids. the ace -b( )at complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ace mediating homo-dimerization. ace has a rasindependent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the gut microbiome. ace is necessary for the expression of the hartnup transporter in the intestine, and the differential functional association of mutant b( )at transporters with ace in the intestine regulates the phenotypic heterogeneity of human hartnup disorder. cardiovascular disease is the leading cause of death worldwide and a major public health concern. heart disease is characterized by the activation of several signaling pathways associated with pathological hypertrophy and maladaptive ventricular remodeling. in the heart, ace is localized to cardiomyocytes, cardiac fibroblasts, epicardial adipose tissue, and the coronary vascular endothelium , , ; ang - /masr is also present on cardiomyocytes, cardiac fibroblasts, and endothelial and vascular smooth muscle cells. , [ ] [ ] [ ] genetic ace deletion resulted in exacerbation of ang ii-mediated cardiorenal fibrosis and oxidative stress in the heart and kidney of hypertensive mice while administration of rhace (recombinant human ace ) remarkably rescued the ang ii-induced hypertension, pathological hypertrophy, oxidant injury, and cardiac dysfunction. , various ace polymorphisms are linked to cvd. post-mi remodeling and coronary artery disease are common causes of hf. , notably, mi increases ace mrna expression in human, mice, and rat hearts, , whereas genetic ace deletion results in worsening of mi-induced cardiac dysfunction, infarct size, mmp (matrix metalloproteinase) /mmp activation, and extracellular matrix disruption. , loss of ace leads to increased neutrophilic infiltration in the infarct and peri-infarct regions, resulting in upregulation of inflammatory cytokines, interferon-γ, il (interleukin)- , and the chemokine, mcp- (monocyte chemoattractant protein- ), as well as increased phosphorylation of erk / (extracellular signal-regulated kinase / ) and jnk / signaling pathways, changes that were blocked with an figure . ace (angiotensin converting enzyme ) role in the renin-angiotensin system peptide cascade and its interaction with the apelinergic peptide system. a, angiotensinogen is processed by renin into ang i (angiotensin i), which is further cleaved by ace or mast cell chymase into ang ii. ang ii can go on to affect the cardiovascular system predominantly through the angiotensin type receptor (at r) or via the angiotensin type receptor (at r). alternatively, ang ii can be degraded by the carboxypeptidase ace or the pcp (prolyl carboxypeptidase) into ang - (angiotensin - ). ang - mediates protective effects throughout tissues which host the mas receptor (masr). ang - can be further formed through the activity of ace on ang i forming ang - which is then cleaved by either ace or nep (neprilysin). b, stimulation of the apelin receptor by apelin peptides leads to cardiovascular protective effects while disrupting ang ii signaling by sequestration of the at r through receptor heterodimerization. apelin is inactivated by ace cleavage of its c-terminal phenylalanine while stimulation of the apelin receptor promotes ace mrna transcription presenting apelin's role as a positive regulator of ace . ros indicates reactive oxygen species. arb ultimately resulting in improvement in myocardial function. in contrast, overexpression of ace and the action of ang - ameliorates mi-induced cardiac remodeling. , importantly, heterozygote loss of ace , as seen in explanted human hearts from patients with dilated cardiomyopathy, was sufficient to increase susceptibility to heart disease. hf with preserved ejection fraction is a proinflammatory state closely linked to obesity-related cardiac and microvascular dysfunction for which there are no approved therapies. , , epicardial adipose tissue is a primary source of inflammatory cytokines that could have detrimental effects on the heart. loss of ace increases macrophage polarization to proinflammatory m -phenotype (alternatively activated, cd c + ) in epicardial adipose tissue from patients with hf with preserved ejection fraction, with decreases in polarization to anti-inflammatory, m -phenotype macrophages, and worsening of hf with preserved ejection fraction in response to diet-induced obesity. importantly, ang - decreased macrophage polarization in epicardial adipose tissue and preserved the cardiac function of obese ace ko mice. , ang - has potent anti-inflammatory effects in adipose tissue of obese type diabetic mice and protects against diabetic cardiomyopathy and nephropathy. [ ] [ ] [ ] the ace /ang - axis also promotes browning of adipose tissue leading to improved metabolic effects and weight loss, which can confer further benefits to the cardiovascular system. , blockade of the deleterious arm of the ras has been the mainstay of the therapeutic management of hypertensive individuals. an increase in ace and the vasoprotective axis of the ras by ace inhibitors and angiotensin receptor blockers (arbs) clearly reinforces this view (see section pharmacological antagonists of the ras and ace expression below). furthermore, increased ace expression protects against hypertension, while ace deficiency exacerbates hypertension. renal ace expression is inversely related to blood pressure in experimental models of hypertension. in the spontaneously hypertensive rat and stroke-prone spontaneously hypertensive rat, renal ace mrna levels are reduced compared with normotensive wistar-kyoto rats. these studies support the essential role of ace in maintaining healthy blood pressure. lentiviral overexpression of ace results in increased expression of antihypertensive components of the ras and attenuates elevated blood pressure. , pretreatment with rhace prevented hypertension induced by ang ii and decreased plasma ang ii while increasing plasma ang - levels. ace and adam were selectively knocked down from all neurons (ac-n), which revealed a reduction of inhibitory inputs to ac-n presympathetic neurons relevant for blood pressure regulation. mice with ace selectively knocked down from sim neurons in mice exhibited a blunted blood pressure elevation and preserved ace activity during the development of salt-sensitive hypertension. the metalloproteinase adam is responsible for mediating ace shedding from the cell membranebound domain, which can be promoted by ang ii, and release of ace as a soluble form in plasma , , impairing brain ace compensatory activity and thus contributing to the development of neurogenic hypertension. genetic ace deficiency is associated with the upregulation of putative mediators of atherogenesis and enhances responsiveness to proinflammatory stimuli suggestive of a key role of ace in suppressing vascular inflammation and atherosclerotic disease. in addition, ace inhibition blocks neuropeptide catestatin-mediated protective effects in the development of atherosclerosis in apoe −/− mice fed a high-fat diet. the counter-regulatory role of the ace /ang - / masr axis of the ras has been well characterized in the progression of diabetic complications, including cardiovascular and kidney disease. , , support for the importance of ace in diabetes mellitus comes from its impact on diabetic complications wherein diabetes mellitus-induced vascular dysfunction is strongly associated with a shift in the ras axis toward the profibrotic, proinflammatory arm of ras with a reduction in the protective arm ( figure ). loss of the protective effects of the ras is related to the regulation of tissue and circulating levels of ang ii and their sequelae in the context of diabetes mellitus. , alterations within the ras are considered pivotal for the development of both diabetic micro and macrovascular complications. , the blockade of the proinflammatory and profibrotic arms of the ras provides significant renoprotection in both experimental models of diabetes mellitus and in patients. while the loss of ace worsens diabetic kidney injury, rhace is therapeutic in an animal model of diabetic nephropathy and experimental alport syndrome. ace inhibitors in t d and angiotensin receptor blockade with losartan and irbesartan in t d retard the progression of nephropathy. in diabetic renal tubules, ace gene expression is decreased by ≈ %, which would reduce ang - formation and allow ang ii accumulation hence directly increasing the expression of tgf-β and ctgf (connective tissue growth factor), leading to tubulointerstitial fibrosis. ras blockade retards renal damage and ace inhibitor therapy, as mentioned above, resulting in a compensatory increase in ace , leading to renoprotection. therefore, support for the loss of ace contributing to vascular complications in diabetes mellitus comes from strong clinical and experimental evidence. retinopathy, the most common complication of diabetes mellitus and one of the leading causes of blindness in working-age adults is linked to activation of oxidative stress, profibrotic, and proinflammatory arm of the ras, which can be effectively curtailed by the ace /ang - axis in experimental models. , increased secretion of proinflammatory cytokines by bone marrow mesenchymal stem cells skews hematopoiesis toward the generation of an increased number of myelo-monocytic cells. target tissues of diabetic complications secrete ccl in response to high glucose-induced stress facilitating the homing of ccr + cells to these regions and promoting the development of vascular complications. [ ] [ ] [ ] [ ] [ ] [ ] in addition to an increase in myeloidosis, diabetics with complications have reduced bone marrow-derived vascular reparative cells and circulating angiogenic cells (cd + cells). levels of ace mrna were also a significant predictor of the presence of microvascular disease in diabetic patients. diabetic individuals who remained free of retinopathy despite > years of poor glycemic control had higher mrna levels for genes of the vasoprotective axis (ace /mas) compared with age, sex, and glycemia-matched diabetics with retinopathy. in dysfunctional cd + cells from diabetic individuals, activation of the protective arm of ras, by exposing the cells to ang - corrected their dysfunction by restoring bioavailable no and reducing reactive oxygen species. ang - gene modification of cd + cells restored the in vivo vasoreparative function of these cells in a mouse retinal ischemia-reperfusion injury model. moreover, intraocular administration of aav-ace or ang - reduced diabetes mellitus-induced retinal vascular leakage and inflammation, thus preventing retinopathy. patients with diabetes mellitus have a dysregulated ras, which may influence their vulnerability to sars-cov- . guan et al examined individuals with confirmed covid . of these individuals, had severe disease, and of this, . % were diabetics. zhang et al studied patients who were hospitalized due to the severity of their covid- infection, of these individuals, % had diabetes mellitus. it is interesting to speculate why diabetics may be more at risk for sars-cov- infection than the general population, and this may be due to the reduced ace levels that are typically observed in the vasculature of diabetic individuals and diabetic animal models. indeed, loss of ace was associated with marked gut dysbiosis, which was further worsened in a model with type diabetes mellitus. lung epithelial cells express high levels of ace , which positively correlates with airway epithelial differentiation. , , involvement of ace in ards, which is triggered by multiple diseases including sars-cov and sars-cov- , has been established in multiple animal models. , ace ko mice exhibit severe pathology of ards. , additional ace deficiency, or treatment with ar r blockers of ace ko mice rescues them from ards implicating the benefit of ace and the critical balance of the protective versus proinflammatory and fibrotic axes of the ras. these findings are consistent with evidence of a beneficial effect of rhace on pulmonary blood flow and oxygenation in a pig model of lipopolysaccharide-induced ards. age-related loss of ace in the lungs correlates with the increased mortality and worsened phenotype in elderly patients with covid- . ace has been implicated in acute lung injury by inducing an imbalance in the ras. evidence includes that in acute lung injury ( ) a decrease in pulmonary ace and an increase in ang ii levels occurs; ( ) supplementation with ace or inhibition of ang ii improves outcomes; and ( ) a lack or decrease of pulmonary ace aggravates viral-induced acute lung injury. ace is also involved in ph and fibrosis. increasing ace activity using rhace reduced bleomycin-induced inflammation and fibrosis, resulting in improved lung function and exercise capacity, and the ace activator, dize, protects animals from ph and fibrosis. moreover, oral feeding of a bioencapsulated form of ace protects and arrests the progression of ph. validation of this protective effect comes from a small human study that showed that ph is characterized by reduced ace activity and supplementation of these individuals with rhace improved pulmonary hemodynamics and reduced oxidative and inflammatory markers. collectively, these studies unequivocally establish the conceptual framework that ace is a central player in normal pulmonary function, and its imbalance leads to pulmonary diseases. pathological neurohormonal activation of the ras drives the development and progression of cvd. current pharmacotherapies aim to achieve multilevel ras inhibition through distinct modes of action. although ace is not the direct cellular target of these therapies, ace gene transcription, translation, and ultimately catalytic activities are modified due to the intricate nature of the ras. blocking the ace/ang ii/at r axis through limiting the formation and actions of ang ii potentiates the effects of ace as the endogenous ras counter-regulator. the arbs consistently increased ace mrna expression, protein levels, and catalytic activities in the heart, kidneys, and thoracic aorta, but the translation to protein levels and activity differs between experimental models and tissues for ace inhibitors (table) . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] combination of lisinopril and losartan treatment in normotensive lewis rats abolished the increase in ace mrna levels observed individually but retained losartan induced rise in ace activity in the heart. moreover, lisinopril in normotensive lewis rats increased ace mrna without affecting ace activity in the heart, but the opposite was observed in the kidneys. , these findings could be attributed to tissue-specific regulation of ace , as higher ace protein levels were reported in the heart, but ace activity was higher in the kidneys of sprague-dawley rats, adding to the complexity of the tissue ras. in type diabetic akita angiotensinogen-transgenic mice, dual ras blockade with perindopril and losartan normalized diseasemediated reduction in kidney ace mrna expression and protein levels. these findings suggest that the accumulation of ang ii in pathological conditions contributes to the modulatory effects of ras blockade on ace . ang ii can regulate ace expression through the at r. healthy hearts and kidneys are characterized by high levels of ace mrna and protein expression, with moderate expression of ace. ras overactivation in cvd increases at r stimulation by ang ii, promoting erk / and p mapk signaling pathways to downregulate ace while upregulating ace expression. activation of p mapk upregulates adam activity though posttranslational phosphorylation of the cytoplasmic domain results in shedding of surface ace in a positive feedback loop and could explain the observed effects of arbs in increasing ace protein levels and activity. , , mechanisms behind the augmentation of ace mrna levels by ace inhibitors and arbs require further characterization. moreover, mineralocorticoid receptor antagonists increased ace mrna expression and activity in samples from patients with chronic hf, wild-type mice, and rats to varying degrees among different tissues but not in the heart of a rat hypertensive disease model (table) . [ ] [ ] [ ] spironolactone, a nonselective mineralocorticoid receptor antagonist, prevented the increase in both ace and at r mrna levels, and the associated increase in at r density from aldosterone signaling in cardiomyocytes. , activation of mineralocorticoid receptors also stimulates overlapping downstream signaling pathways with at r, including the erk / and p mapk pathways mentioned before. , blocking these signaling pathways contributes to the observed effect of mineralocorticoid receptor antagonist on ace gene expression, surface protein levels, and activity. promoting the ace /ang - /mas signaling by rhace or the ang - receptors agonist ave can have salutary therapeutic effects in cvd and lung disease from diverse etiologies. the ang - receptors agonist ave has been shown to exert cardiorenal and pulmonary protective effects, and treatment with rhace improved the symptoms of acute lung injury, cvd, and kidney injury in various preclinical models. , , , maintaining ace levels in patients with or predisposed to common cvd states such as diabetes mellitus, hypertension, and obesity wards off the advancement of these comorbidities in instances where the patient contracts sars-cov- by maintaining a level of ace /ang - / masr negative counter-regulation. rhace functionally sequesters circulating viral particles to prevent s-protein interactions with endogenous ace , while simultaneously regulating the systemic ras may provide therapeutic benefits in covid- and is moving into phase ii clinical trials in europe. a potential limitation of rhace is the restricted penetrance and activity against tissue ras owning to its large molecular size. pharmacological ras blockade agents, arbs, in particular, are capable of modulating both systemic and tissue ras, and simultaneously increasing ace expression and activity in experimental models. the direct implications of ras inhibition in patients with covid- with hypertension remain elusive, and clinical evidence is desperately needed to determine the relative benefits and risks associated with usage of these medications. nonetheless, introducing arbs to patients already infected by sars-cov- may be an effective therapeutic option in addressing the viral-mediated ras imbalance and is currently under investigation in several clinical trials (www.clinicaltrial.gov number nct , nct , and nct ). [ ] [ ] [ ] potential for ace as a therapy is also facilitated by using the probiotic species lactobacillus paracasei (lp), which can be engineered to express recombinant ace indicates angiotensin-converting enzyme; and ras, renin-angiotensin system. proteins. mice treated with the recombinant lp expressing the secreted ace in fusion with the nontoxic subunit b of cholera toxin (acts as a carrier to facilitate transmucosal transport) showed increased ace activities in serum and tissues and reduced diabetic retinopathy. these results provide proof of concept for using bioengineered probiotic species as live vectors for delivery of human ace with enhanced tissue bioavailability for treating diabetic complications but could potentially be repurposed for treating cvd and covid- infection. since the discovery of ace in , tremendous progress has been made in elucidating its biochemical actions and fundamental role in cvd and, more recently, as the sars-cov- receptor. ace is a dominant mechanism for negative regulation of the ras by metabolizing ang ii into the beneficial peptide ang - , and this important biochemical and physiological property is being harnessed as a potential therapy in patients with hf. the activation of the ras axis due to binding of sars-cov- to ace , leading to direct loss of ace and indirectly via proteolytic processing and shedding, partly drives the systemic manifestations of covid- . careful targeting of the ras axes is needed in these patients to optimize their clinical outcomes, including the use of at receptor blockers (arb). role of the ace /angiotensin - axis of the renin-angiotensin system in heart failure a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase a novel angiotensinconverting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - angiotensin converting enzyme : a double-edged sword angiotensin-converting enzyme is a 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coronaviruses: is faecaloral transmission of sars-cov- possible? tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis restructuring of the gut microbiome by intermittent fasting prevents retinopathy and prolongs survival in db/db mice microbiome and diabetes: where are we now? emerging pathogenic links between microbiota and the gut-lung axis chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type receptor antisense intestinal permeability biomarker zonulin is elevated in healthy aging microglial cells impact gut microbiota and gut pathology in angiotensin ii-induced hypertension ace and microbiota: emerging targets for cardiopulmonary disease therapy pulmonary hypertension: pathophysiology beyond the lung churning out safer microbes for drug delivery genetically engineered probiotics lactic acid bacteria for delivery of endogenous or engineered therapeutic molecules a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha adams: focus on the protease domain adam- : the enzyme that does it all cardiomyocyte a disintegrin and metalloproteinase (adam ) is essential in post-myocardial infarction repair by regulating angiogenesis angiotensin ii induced proteolytic cleavage of myocardial ace is mediated by tace/adam- : a positive feedback mechanism in the ras cell-specific functions of adam regulate the progression of thoracic aortic aneurysm detection of soluble angiotensin-converting enzyme in heart failure: insights into the endogenous counter-regulatory pathway of the renin-angiotensin-aldosterone system reactive oxygen species and p mitogen-activated protein kinase mediate tumor necrosis factor α-converting enzyme (tace/adam- ) activation in primary human monocytes direct activation of tace-mediated ectodomain shedding by p map kinase regulates egf receptor-dependent cell proliferation clinical relevance and role of neuronal at receptors in adam -mediated ace shedding in neurogenic hypertension sars coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace induces tnfalpha production and facilitates viral entry human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars trilogy of ace : a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters pulmonary angiotensin-converting enzyme (ace ) and inflammatory lung disease ace exhibits protective effects against lps-induced acute lung injury in mice by inhibiting the lps-tlr pathway angiotensin-converting enzyme 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via akt-dependent pathways angiotensin-( - ) is an endogenous ligand for the g protein-coupled receptor mas angiotensin-( - ) inhibits growth of cardiac myocytes through activation of the mas receptor alteration of cardiac ace /mas expression and cardiac remodelling in rats with aortic constriction adenoviral delivery of angiotensin-( - ) or angiotensin-( - ) inhibits cardiomyocyte hypertrophy via the mas or angiotensin type receptor angiotensin-( - ) attenuates cardiac fibrosis in the stroke-prone spontaneously hypertensive rat via the angiotensin type receptor angiotensin-( - ) regulates cardiac hypertrophy in vivo and in vitro rho kinase inhibition activates the homologous angiotensinconverting enzyme-angiotensin-( - ) axis in experimental hypertension the role of ace in cardiovascular physiology angiotensin-converting enzyme antagonizes angiotensin ii-induced pressor response and nadph oxidase activation in wistar-kyoto rats and spontaneously hypertensive rats angiotensin ii-mediated oxidative stress and inflammation mediate the age-dependent cardiomyopathy in ace null mice cardioprotective effects mediated by angiotensin ii type receptor blockade and enhancing angiotensin - in experimental heart failure in angiotensinconverting enzyme -null mice angiotensin-converting enzyme ii in the heart and the kidney antagonism of angiotensin - prevents the therapeutic effects of recombinant human ace targeting the apelin pathway as a novel therapeutic approach for cardiovascular diseases international union of basic and clinical pharmacology. lxxiv. apelin receptor nomenclature, distribution, pharmacology, and function plasma concentrations of the novel peptide apelin are decreased in patients with chronic heart failure novel role for the potent endogenous inotrope apelin in human cardiac dysfunction reduced circulating apelin in essential hypertension and its association with cardiac dysfunction impaired heart contractility in apelin gene-deficient mice associated with aging and pressure overload loss of apelin exacerbates myocardial infarction adverse remodeling and ischemia-reperfusion injury: therapeutic potential of synthetic apelin analogues apelin is a positive regulator of ace in failing hearts the apelin receptor inhibits the angiotensin ii type receptor via allosteric trans-inhibition apelin is a negative regulator of angiotensin iimediated adverse myocardial remodeling and dysfunction apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs angiotensinconverting enzyme metabolizes and partially inactivates pyr-apelin- and apelin- : physiological effects in the cardiovascular system tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations angiotensin-converting enzyme is a critical determinant of angiotensin ii-induced loss of vascular smooth muscle cells and adverse vascular remodeling ace deficiency worsens epicardial adipose tissue inflammation and cardiac dysfunction in response to diet-induced obesity impairment of in vitro and in vivo heart function in angiotensin-( - ) receptor mas knockout mice angiotensin-( - ) counterregulates angiotensin ii signaling in human endothelial cells angiotensin-( - ) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects from gene to protein-experimental and clinical studies of ace in blood pressure control and arterial hypertension navigating the crossroads of coronary artery disease and heart failure myocardial infarction increases ace expression in rat and humans loss of angiotensin-converting enzyme accelerates maladaptive left ventricular remodeling in response to myocardial infarction angiotensin-converting enzyme inhibits high-mobility group box and attenuates cardiac dysfunction post-myocardial ischemia circulating rather than cardiac angiotensin-( - ) stimulates cardioprotection after myocardial infarction heterozygote loss of ace is sufficient to increase the susceptibility to heart disease epicardial adipose tissue as a metabolic transducer: role in heart failure and coronary artery disease epicardial adipose tissue may mediate deleterious effects of obesity and inflammation on the myocardium ace /ang - axis: a critical regulator of epicardial adipose tissue inflammation and cardiac dysfunction in obesity angiotensin - ameliorates diabetic cardiomyopathy and diastolic dysfunction in db/db mice by reducing lipotoxicity and inflammation angiotensin - mediates renoprotection against diabetic nephropathy by reducing oxidative stress, inflammation, and lipotoxicity angiotensin - stimulates brown adipose tissue and reduces diet-induced obesity ace exerts anti-obesity effect via stimulating brown adipose tissue and induction of browning in white adipose tissue ace gene transfer attenuates hypertension-linked pathophysiological changes in the shr transgenic angiotensin-converting enzyme overexpression in vessels of shrsp rats reduces blood pressure and improves endothelial function targeting the degradation of angiotensin ii with recombinant angiotensin-converting enzyme : prevention of angiotensin ii-dependent hypertension tumor necrosis factor-alpha convertase (adam ) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme- (ace ) brain angiotensinconverting enzyme type shedding contributes to the development of neurogenic hypertension genetic ace deficiency accentuates vascular inflammation and atherosclerosis in the apoe knockout mouse decreased circulating catestatin levels are associated with coronary artery disease: the emerging anti-inflammatory role human recombinant ace reduces the progression of diabetic nephropathy loss of angiotensin-converting enzyme- exacerbates diabetic cardiovascular complications and leads to systolic and vascular dysfunction: a critical role of the angiotensin ii/at receptor axis interaction of diabetes and ace in the pathogenesis of cardiovascular disease in experimental diabetes role of angiotensin-converting enzyme (ace ) in diabetic cardiovascular complications optimizing treatment of hypertension in patients with diabetes loss of angiotensin-converting enzyme- (ace ) accelerates diabetic kidney injury murine recombinant angiotensin-converting enzyme attenuates kidney injury in experimental alport syndrome effects of losartan on renal and cardiovascular outcomes in patients with type diabetes and nephropathy characterization of renal angiotensin-converting enzyme in diabetic nephropathy ace and ang-( - ) confer protection against development of diabetic retinopathy adeno-associated virus overexpression of angiotensin-converting enzyme- reverses diabetic retinopathy in type diabetes in mice diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock long-term type diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model ccl /mcp- modulation of microglial activation and proliferation ccl induces a pro-inflammatory profile in microglia in vitro role of chemokines in cns health and pathology: a focus on the ccl /ccr and cxcl / cxcr networks critical roles for ccr and mcp- in monocyte mobilization from bone marrow and recruitment to inflammatory sites chronic expression of monocyte chemoattractant protein- in the central nervous system causes delayed encephalopathy and impaired microglial function in mice the role of cc chemokine receptor on microglia activation and blood-borne cell recruitment after transient focal cerebral ischemia in mice activation of the ace / angiotensin-( - )/mas receptor axis enhances the reparative function of dysfunctional diabetic endothelial progenitors decreased glomerular and tubular expression of ace in patients with type diabetes and kidney disease age-and gender-related difference of ace expression in rat lung role of angiotensin-converting enzyme (ace) and ace in a rat model of smoke inhalation induced acute respiratory distress syndrome recombinant angiotensin-converting enzyme improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets diminazene aceturate improves autonomic modulation in pulmonary hypertension upregulation of angiotensin-converting enzyme after myocardial infarction by blockade of angiotensin ii receptors effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme effects of renin-angiotensin system blockade on renal angiotensin-( - ) forming enzymes and receptors angiotensin ii at receptors regulate ace and angiotensin-( - ) expression in the aorta of spontaneously hypertensive rats effect of angiotensin ii blockade on a new congenic model of hypertension derived from transgenic ren- rats enalapril attenuates downregulation of angiotensin-converting enzyme in the late phase of ventricular dysfunction in myocardial infarcted rat localization of ace in the renal vasculature: amplification by angiotensin ii type receptor blockade using telmisartan ace deficiency enhances angiotensin ii-mediated aortic profilin- expression, inflammation and peroxynitrite production organ-specific distribution of ace mrna and correlating peptidase activity in rodents dual ras blockade normalizes angiotensin-converting enzyme- expression and prevents hypertension and tubular apoptosis in akita angiotensinogen-transgenic mice mineralocorticoid receptor blocker increases angiotensin-converting enzyme activity in congestive heart failure patients aldosterone, but not angiotensin ii, reduces angiotensin converting enzyme gene expression levels in cultured neonatal rat cardiomyocytes effects of aldosterone and angiotensin ii receptor blockade on cardiac angiotensinogen and angiotensin-converting enzyme expression in dahl saltsensitive hypertensive rats angiotensin ii up-regulates angiotensin i-converting enzyme (ace), but down-regulates ace via the at -erk/p map kinase pathway aldosterone induces angiotensin-converting-enzyme gene expression in cultured neonatal rat cardiocytes angiotensin at receptor subtype as a cardiac target of aldosterone: role in aldosterone-salt-induced fibrosis aldosterone-induced osteopontin expression in vascular smooth muscle cells involves mr, erk, and p mapk aldosterone increases vegf-a production in human neutrophils through pi k, erk / and p pathways angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target renin-angiotensin-aldosterone system inhibitors in patients with covid- losartan for patients with covid- requiring hospitalization. . www.clinicaltrials.gov identifier: nct losartan for patients with covid- not requiring hospitalization. . www ace inhibitors, angiotensin ii type-i receptor blockers and severity of covid- (covid-ace) expression of human ace in lactobacillus and beneficial effects in diabetic retinopathy in mice we acknowledge patients and their families for participating in our studies. none. key: cord- -che iwu authors: hart, kristen c; donoghue, daniel j title: derivatives of activated h-ras lacking c-terminal lipid modifications retain transforming ability if targeted to the correct subcellular location date: - - journal: oncogene doi: . /sj.onc. sha: doc_id: cord_uid: che iwu to examine the ability of ras to activate signal transduction pathways in the absence of lipid modifications, fusion proteins were constructed that target ras(wt) or activated ras( l) to cellular membranes as integral membrane proteins, using the first transmembrane domain of the e protein of avian infectious bronchitis virus (ibv), which contains a cis-golgi targeting signal. golgi-targeted derivatives of activated ras were completely inactive in transformation assays. however, when examined in focus formation assays, transformation of nih t cells were seen with derivatives of ras( l) containing a mutated e targeting sequence that results in plasma membrane localization. removal of the lipid modification sites in and upstream of the caax motif did not abrogate the transforming activity of plasma membrane-localized ras( l) derivatives, indicating that these lipid modifications are not essential for ras activity, as long as the protein is correctly localized to the plasma membrane. interestingly, the activity of integral membrane versions of ras( l) was strictly dependent on a minimum distance between the transmembrane domain anchor region and the coding sequence of ras. derivatives with only a -amino acid linker were inactive, while linkers of either - or -amino acids were sufficient to restore transforming activity. these results demonstrate that: ( ) activated ras targeted to golgi membranes is unable to cause transformation; ( ) lipid modifications at the c-terminus are not required for the transforming activity of plasma membrane-anchored ras( l) derivatives, and serve primarily a targeting function; ( ) a transmembrane domain can effectively substitute for c-terminal modifications that would normally target ras to the inner surface of the plasma membrane, indicating that ras( l) does not need to reversibly dissociate from the membrane as might be allowed by the normal lipidation; and ( ) in order to function properly, there exists a critical distance that the ras protein must reside from the plasma membrane. introduction ras proteins are critical modulators of signaling pathways involved in cell growth and dierentiation. p ras is a member of a large family of small proteins that act as molecular`switches', cycling between an active gtp-bound state and an inactive gdp-bound state . ras is activated in response to a wide variety of stimuli, including platelet-derived growth factor (pdgf). binding of pdgf to its receptors induces their dimerization and transphosphorylation, creating binding sites for eector molecules (reviewed in hart et al., ) , some of which regulate or activate ras. activation of ras by growth factors recruits raf- to the plasma membrane (stokoe et al., ; leevers et al., ) , leading to raf- activation and subsequent initiation of the mapk cascade. ras also has been shown to activate rac and rho proteins, which control signaling pathways that lead to cytoskeletal modi®cations khosravi-far et al., ; qiu et al., a,b) . activation of this pathway has recently been shown to be crucial for full transformation by ras (prendergast et al., ; khosravi-far et al., ; qiu et al., a,b) . thus, combined activation of the rac/rho and mapk pathways may constitute the entire intracellular response necessary for transformation by activated ras. there are multiple p ras proteins, including n-ras, k-ras, and h-ras (reviewed in santos and nebreda, ; lowy and willumsen, ) , which are localized to the inner surface of the plasma membrane (willingham et al., ; sefton et al., ) through speci®c sequences in the c-terminus (willumsen et al., ) . the mechanism by which ras proteins are targeted to the plasma membrane has been the subject of intense research. since ras is frequently mutated in tumors, interference with ras localization could become an important means of treating some forms of cancer. post-translational modi®cations of ras proteins occur at the c-terminus, which includes a domain known as the`caax' box, where a is an aliphatic amino acid, and x represents any amino acid. processing of this region consists of farnesylation of the conserved cys residue four amino acids from the c-terminus (cys in h-ras and n-ras), cleavage of the last three amino acids of the protein, and carboxymethylation of the new c-terminal cys jackson et al., ; casey et al., ; gutierrez et al., ; clarke et al., ; lowy and willumsen, ; shih and weeks, ; willumsen et al., ; sefton et al., ) . n-ras and hras have cysteines n-terminal of the caax motif, which become palmitoylated after the caax processing (buss and sefton, ) . in the case of k-ras- b, the c-terminus contains a polybasic domain in addition to the caax motif which contributes to plasma membrane localization (hancock et al., (hancock et al., , jackson et al., ) . the palmitoylation reactions are reversible, allowing for potentially dynamic localization of ras proteins (magee et al., ) . although truncation of the c-terminus does not aect the inherent structure or biochemical properties of ras , mutations in these motifs render ras proteins cytosolic and inactive, implying that lipid modi®cations are important for ras function. there is recent evidence suggesting that the lipid modi®cations of ras are also necessary for activation of raf- and b-raf (okada et al., ; kikuchi and williams, ) . however, since these lipid modifications are also responsible for targeting ras to the plasma membrane, it is unclear from these recent reports whether the lipid modi®cations per se are a necessity, or just localization of ras to the plasma membrane. the palmitoylation reactions that occur in the c-terminus of ras proteins have been shown to be reversible (magee et al., ) . there is also some speculation that ras may require reversible association with membranes, and the lipid modi®cations in the cterminus provide the potential for this dynamic localization. however, the experimental methods used to date are unable to conclusively distinguish the precise role of lipid modi®cations in the membrane anchoring and activation of ras. in order to examine these questions, we have used a novel method of targeting ras to the plasma membrane by creating fusion proteins using the transmembrane targeting signal from the e protein of avian ibv placed at the n-terminus (swift and machamer, ) . in its wild-type form, this transmembrane domain is capable of targeting heterologous proteins to cis-golgi membranes. when the point mutation gln ?ile is present, proteins are instead targeted to the plasma membrane. these fusions allow us to address the question of whether ras must be able to reversibly associate with the plasma membrane for biological function as assayed by transformation and gtpase activity. to examine whether c-terminal modi®cations of ras are required for functions other than targeting, we created similar fusion proteins that contain mutations which abolish processing of the caax box. results presented here demonstrate that ras l can transform ®broblasts if targeted to the plasma membrane by an n-terminal transmembrane domain, yet is not active when localized to golgi membranes. we also show that integral membrane versions of ras l require a minimum distance from the membrane in order to activate signal transduction pathways leading to transformation. this work describes the ®rst derivatives of ras l that lack any lipid modi®cations, and demonstrate that cterminal lipid modi®cations are not required for transformation by ras l . to examine the possibility that activated h-ras can transform if targeted to membranes by an n-terminal transmembrane anchor, a variety of fusion proteins were designed. as illustrated in the left side of figure , ras wt and ras l proteins are localized at the inner surface of the plasma membrane by means of lipid modi®cations at the c-terminus. the right side of figure depicts the orientation and design of the transmembrane-anchored ras derivatives. for derivatives containing the e cis-golgi targeting signal, the transmembrane domain anchors the fusion proteins at golgi membranes as type i integral membrane proteins, with the ras-derived portion of the protein still in the cytosol. these derivatives allowed us to examine whether ras could function if localized to any membranous environment, thereby assessing the importance of the interaction between ras and the plasma membrane. fusion proteins were also designed with a mutated e (qi) targeting signal and are expected to be transported to the plasma membrane, with the nterminus outside the cell, and the ras portion of the proteins on the inner surface of the plasma membrane. figure represents all of the fusion proteins designed for this study. the feasibility of targeting ras to the plasma membrane by the n-terminus was previously demonstrated in work by buss et al. ( ) and lacal et al. ( ) , which examined the eect of nterminal myristylation on ras l activity. buss et al. ( ) described a naturally-occurring myristylated derivative of ras, p gag-ras, containing a -amino linker region between the myristylated gly and the ras coding sequence. lacal and colleagues generated nterminally anchored ras derivatives by attaching the amino acid myristylation signal from pp c-src. our derivatives dier in that ras is anchored permanently to membranes as an integral membrane protein. the myristylation targeting signal used in previous studies has the potential to allow reversible association of ras with the plasma membrane. this is an important distinction, which allows us to examine whether ras needs to be able to come o of the membrane in order to function. by exploiting the e localization signal, we were also able to target ras speci®cally to dierent membranes in the cell. we were also able to examine in more detail the minimum distance from the membrane that ras requires for activity, by designing constructs with linker regions of either , or amino acids. additional fusion proteins were designed to examine the general requirement for lipid modi®cations for ras activity. ras derivatives with -or -amino acid linkers figure schematic of ras fusion proteins. all fusion proteins have either the normal c-terminus (amino acids ± ) of ras wt or ras l (cmsckcvls), or contain saax (cmscksvls) or s (smssksvls) mutations, which alter or abolish the normal post-translational processing and lipid modi®cations of the proteins. the n-terminus of each e derivative is oriented either into the lumen of the golgi, or is extracellular if the point mutation q?i is present. the whole of the ras protein is always cytosolic. the transmembrane domain (designated by the shaded box) is either the cis-golgi targeting signal contained in the ®rst transmembrane domain of the ibv e protein, or a mutated version (q ?i) that targets the protein to the plasma membrane. the linker region between the transmembrane domain and the ras protein denoted by the black box is either -, -, or -amino acids in length ras anchored as an integral membrane protein kc hart and dj donoghue were constructed in the context of the unmutated (caax) c-terminus, as well as the saax and s cterminal mutations.`caax' refers to the wild-type cterminal sequences of ras.`saax' indicates a mutation of cys in the caax motif to ser, which abolishes the farnesylation, cleavage, and carboxymethylation reactions.` s' refers to mutation of cys , , and to ser, which in addition destroys the palmitoylation sites . the -amino acid linker derivatives were only made in the context of the saax mutant c-terminus. since mutation of all three c-terminal cys residues results in a ras protein completely unable to be modi®ed by lipid moieties, these mutants allowed us to assay whether these lipid modi®cations are necessary for activity, or just utilized in a membrane targeting capacity. ras derivatives are unable to cause transformation if targeted to golgi membranes when tested in focus forming assays, golgi-targeted derivatives of ras wt with -, -, or -amino acid figure localization and transforming activity of ras derivatives. bars depict the ras wt and ras l constructs tested in this paper. the following abbreviations are used to describe the dierent derivatives.` l' refers to mutation of codon of ras from gln to leu, which oncogenically activates the protein (sekiya et al., ) .`e ' refers to the ®rst transmembrane domain (amino acids ± ) of the e protein of avian ibv, which constitutes a cis-golgi targeting signal that can target heterologous proteins to the golgi (swift and machamer, ) .`e (qi)' indicates a point mutation in the transmembrane domain (gln ?ile) that abrogates its golgi-targeting function, targeting proteins instead to the plasma membrane (swift and machamer, ) . shaded boxes indicate the e or e (qi) transmembrane domain; black boxes indicate the linker region; striped boxes indicate the c-termini of the clones which is either wild-type (caax) or mutant (saax or s). localization was determined by indirect immuno¯uorescence, as described in the legend for figure . transforming activity of the clones was analyzed in a focus forming assay, where nih t cells were transfected with the plasmids encoding the fusion proteins, and examined for foci after approximately ± days. each experiment was repeated at least three times for transforming derivatives, and at least two times for non-transforming constructs. results are expressed as an average of all experiments performed with each given construct. = ± % of ras l ; ++= ± % of ras l ; +++= ± % of ras l ; ++++= ± % of ras l transforming activity linker regions were all inactive. this was expected, since ras wt did not induce focus formation in our assays. the golgi-targeted derivatives of ras l were also unable to cause transformation. this was irrespective of whether the linker region was -, -, or -amino acids in length, or whether the c-terminal post-translational modi®cation signals were intact or not. figure e and i demonstrate localization of e - ras l -saax and e - -ras l -saax, respectively, to a perinuclear region typical of golgi staining. panel k shows the localization of e - -ras l -caax, and figure l shows that this fusion protein colocalizes with mab e (see arrows), which speci®cally reacts with a cis-golgi epitope, and has been used as a marker for the early golgi (hart et al., ; wood et al., ) . this con®rms that the golgi-targeted fusion proteins are localized correctly, and therefore we conclude that ras l cannot initiate transforming signal transduction pathways from golgi membranes, indicating that attachment to any lipid-®lled environment is not sucient for ras to be active. ras l derivatives with -or -amino acid linkers can transform if anchored to the plasma membrane, irrespective of c-terminal lipid modi®cations the plasma membrane-targeted derivatives of ras l with the -amino acid linkers (e (qi)- -ras l -caax, e (qi)- -ras l -saax, and e (qi)- -ras l - s) were all inactive in focus formation assays. however, derivatives with -or -amino acid linker regions were very active in transformation of nih t cells. the results of these assays are summarized in figure , which indicates the focus forming activity of each ras derivative as an average of at least three independent experiments. e (qi)- -ras l -caax, e (qi)- -ras l -saax, e (qi)- -ras l - s and e (qi)- -ras l - focus forming assays of ras derivatives targeted to the plasma membrane as integral membrane proteins. plasmids encoding ras fusion proteins were transfected into nih t cells, and foci were counted after ± days. cells were ®xed with methanol, stained with giemsa, and photographed. ras anchored as an integral membrane protein kc hart and dj donoghue saax exhibited signi®cant transforming activity in comparison with ras l (see figure e ± i). it is of interest to note that mutations in the c-terminus of ras l that abolish the lipidation of the proteins do not aect the ability of activated ras to transform nih t cells. indeed, the derivative with an intact c-terminus was consistently three-to four-fold less ecient at transformation than derivatives with mutated ctermini (see figure ; also compare figure e , f and g). this could re¯ect conformational strains conferred on the protein due to anchoring both termini to the membrane. plasma membrane-targeted derivatives containing -, -, or -amino acid linkers were also examined by immuno¯uorescence. as seen in figure , derivatives that were positive in transformation assays localize to the plasma membrane (f, g, h, and j), and exhibit staining patterns comparable to that of ras l (b). also, derivatives with -amino acid linkers were still targeted to the plasma membrane (d), although unable to transform cells. thus, derivatives of ras l targeted to the plasma membrane via an n-terminal transmembrane domain are able to cause transformation, provided that there is a¯exible linker region of sucient length between the transmembrane anchor and the n-terminus of ras. these results also clearly demonstrate that ras l does not require lipid modi®cations in order to transform cells, and suggests that the c-terminal processing of ras proteins primarily functions in a membraneanchoring capacity. in order to verify that the fusion proteins being expressed were of the correct size, proteins were immunoprecipitated from transiently transfected cells ( figure ) . ras proteins typically run as a doublet on sds ± page gels, one band representing the unprocessed form of ras, the other indicating the mature, processed form of the protein (see arrows) (shih et al., , . the saax and s mutations prevent the c-terminal cleavage step and subsequent lipid mod-i®cations from occurring, leaving only a single band (lanes and , arrows) . the subtleties of these processing steps are dicult to detect in the remaining fusion proteins shown in figure , due to their increased molecular weight. however, it is quite apparent that all fusion proteins do express the additional n-terminal transmembrane domain sequences (lanes ± ), and these targeting domains are not cleaved o during maturation of the proteins. the fusion proteins migrate slower than figure indirect immuno¯uorescence con®rms localization of ras derivatives. nih t cells were transiently transfected with various ras constructs, ®xed, permeabilized, and incubated with rat mab ab- (clone y - ) and¯uorescein-conjugated goat arat secondary antibody. for (l) mab e was used to detect the cis-golgi, and visualized with rhodamine-conjugated goat amouse secondary antibody. (a) mock; (b) ras l -caax; (c) e - -ras wt - s; (d) e (qi)- -ras l - s; (e) e - -ras l -saax; (f) e (qi)- -ras l -caax; (g) e (qi)- -ras l -saax; (h) e (qi)- -ras l - s; (i) e - -ras l -saax; (j) e (qi)- -ras l -saax; (k) e - -ras l -caax -a-ras antibody (¯uorescein); (l) e - -ras l -caax -mab e a-cis-golgi antibody (rhodamine). arrows indicate caax -mab in k and l expected, which may be due to the presence of an nlinked glycosylation site in the extreme n-terminus of the e -derived sequence (machamer et al., ) . the transforming ability of some of the plasma membrane-targeted derivatives of ras l indicates that their function was not adversely aected by addition of an n-terminal transmembrane domain. to analyse whether fusion proteins that were non-transforming were still able to function in some manner, gtpase activity was examined. ras l remains locked in the gtp-bound state longer due to a decreased rate of gtp hydrolysis (temeles et al., ) , despite a -fold increased binding anity for rasgap (krengel et al., ) . if the n-terminal transmembrane domain does not interfere with normal protein function, one would expect to see a higher gtpase activity exhibited by ras wt fusion proteins, as compared to ras l derivatives. using thin-layer chromatography, this dierence in gtpase activities is apparent, as shown by a higher proportion of labeled gdp versus gtp in ras wt samples as compared to ras l samples ( figure , lanes and ) . interestingly, ras l with the s mutation in the cterminus, which renders the protein cytosolic and inactive in transformation, does not aect the gtpase activity (lane ). derivatives of ras wt with the -amino acid linker hydrolyze gtp, irrespective of whether they are targeted to the plasma membrane (lane ) or golgi membranes (lane ). ras l derivatives with transmembrane anchors and -amino acid linkers exhibit impaired gtpase activity as expected (data not shown). finally, the -and -amino acid derivatives of ras wt (lanes and ) or ras l (lanes and ) exhibit the expected gtpase activity pro®les, regardless of the transmembrane domain attached at the n-terminus or the modi®cations present at the c-terminus. these results demonstrate that ras proteins can be targeted to the plasma membrane via a transmembrane anchor without aecting the expected gtpase activity of the proteins. also, we ®nd that c-terminal lipid modifications do not play a role in maintaining the intrinsic gtpase activity of wild-type or activated ras. the experiments described above demonstrate that ras l can activate transforming signal transduction pathways when targeted by the n-terminus to the plasma membrane as an integral membrane protein. this activity was completely independent of the normal post-translational processing that occurs at the c-terminus of ras proteins. since ras l derivatives (e (qi)- -ras l -saax, e (qi)- -ras l - s and e (qi)- -ras l -saax) lacking the c-terminal sequences required for normal processing of ras are still able to cause transformation, it is clear that lipid modi®cations per se are not required for activity of oncogenic ras. this is the ®rst demonstration of nonlipidated transforming derivatives of ras. our data suggests that post-translational modification of h-ras serves primarily to target the protein to the plasma membrane. whether there are other, more subtle functions of these c-terminal lipid modi®cations note that all ras wt derivatives have a higher gtpase activity (lanes , , , and ) compared to ras l derivatives (lanes , , and ), as evidenced by increased amount of labeled gdp versus gtp in ras wt samples. this result is irrespective of localization to golgi or plasma membranes, and regardless of c-terminal modi®cations. lane : ras wt -caax; lane : ras l -caax; lane : ras l - s; lane : e - -ras wt - s; lane : e (qi)- -ras wt -saax; lane : e - -ras wt -saax; lane : e (qi)- -ras l - s; lane : e - -ras wt -saax; lane : e (qi)- -ras l -saax ras anchored as an integral membrane protein kc hart and dj donoghue distinct from their more obvious targeting function remains to be demonstrated. the observation that three of the transmembrane-anchored derivatives, e (qi)- -ras l -saax, e (qi)- -ras l - s, and e (qi)- -ras l -saax, retain transforming activity despite their inability to be lipid-modi®ed strongly implies that downstream signaling molecules such as raf- and the mapk cascade are activated in these cells. this contradicts recent reports that posttranslational modi®cations of ras are required for activation of raf- and b-raf (okada et al., ; kikuchi and williams, ). the precise activation state of eectors downstream from ras, and whether these molecules are activated in a similar fashion by integral membrane versions of ras, is currently under investigation. transforming activity of transmembrane derivatives of ras demonstrates that ras does not require transient or reversible association with the membrane if the lipid modi®cations on ras proteins serve only a targeting function, as indicated by our results, this raises the question of why ras proteins evolved to contain c-terminal lipid modi®cations instead of a more permanent transmembrane anchor. small gtpbinding proteins related to ras are now known to function in a variety of capacities within the cell, such as mediating vesicular transport in the process of endocytosis, sorting and tracking through the secretory pathway, and regulating the structure of the cytoskeleton (reviewed in hall, ; ferro-novick and novick, ; pfeer, ; zerial and stenmark, ) . some of these processes may require the regulatory gtp-binding proteins to be transiently or reversibly associated with lipid bilayers, and only lipid modi®cations would allow this dynamic association to occur. our results suggest, in the case of ras, that a reversible association with the plasma membrane is not required for activation of transforming signal transduction pathways, since integral membrane versions of ras l are transforming. it would be interesting to examine the eects of anchoring other small gtp-binding proteins more permanently to speci®c membranes or compartments using targeting sequences such as the transmembrane domain from the e protein. ras proteins require a minimum spacing from the plasma membrane in order to function transforming activity of ras l derivatives was dependent upon a minimum distance from the plasma membrane mediated by the length of the linker region. altering the distance between ras and the plasma membrane does aect the ability of ras to activate signal transduction pathways, as demonstrated by the inactivity of -amino acid linker derivatives of ras l and activation of these derivatives in transformation assays by addition of -or -amino acid linker regions. the crystal structure of c-h-ras bound to gdp (residues ± ) or gtp (residues ± ) indicates that the extreme c-terminus forms an alpha-helical region that juts out from the globular catalytic region of ras (de vos et al., ; pai et al., ) , suggesting that the catalytic domain is well-removed from the membrane in normal ras proteins. one can imagine that the native c-terminus of ras constitutes a natural`linker' region, not unlike the arti®cial linker regions incorporated into our ras fusion proteins. this would explain the failure of the short amino acid linker at the n-terminus to allow for a functional fusion protein, whereas linkers of -or amino acids, corresponding more closely in length to the c-terminus of normal ras, do result in a transforming ras l derivative. conformational energy analysis performed on the c-terminal amino acids excluded from the crystal structure of the gdp-bound form of ras indicates that this region likely forms a helix ± turn ± helix or helical hairpin' motif, allowing the n-terminus and c-terminus of ras to be in close proximity (brandt-rauf et al., ) . perhaps these two regions of the protein interact in some manner. however, it appears from our results that interactions between the nterminus and lipid modi®cations at the c-terminus are not important for ras function. there could be one or more eectors of ras that bind to a site dierent from the identi®ed eector domain, and access to this site may be dependent upon orientation of the protein with respect to the membrane, or may require binding to sites at both the n-and cterminus of ras proteins. identi®cation of other membrane-associated factors involved in regulation or activation of ras proteins and their eectors will provide clues as to why ras proteins evolved their unique structural elements. clearly the intrinsic gtpase activities of our fusion proteins are not altered when compared to normal versions of ras wt and ras l . gtp binding and hydrolysis are also apparently unaected by mutation of the caax box. thus, it appears that neither the n-nor the c-terminus are important for this function of ras. speci®c amino acids in the eector regions of ras (amino acids ± or switch i, and amino acids ± or switch ii) are involved in association with guanine nucleotides and also in binding to regulators of gtpase and nucleotide exchange activities. since gtpase activity is normal in the fusion proteins, the overall structure of the catalytic regions is not expected to be drastically altered. to our knowledge, this is the ®rst study examining the role of c-terminal lipid modi®cations in regulating gtpase activity of ras. one discrepancy between our results and those of buss et al. ( ) is that anchoring of wild-type ras to the plasma membrane by the e (qi) transmembrane domain does not result in transformation. buss et al. ( ) found signi®cant focus forming ability of wildtype ras after addition of n-terminal myristylation. they speculated that perhaps their protein was binding to inappropriate cellular targets, which may not be promoted by a transmembrane anchor. it is also possible that interaction with nucleotide exchange factors was enhanced in myristylated ras wt derivatives, but this does not occur in e (qi)-ras wt constructs. to further clarify the role of membrane association in regulating the inherent enzymatic activity of ras proteins, it would be interesting to see if there are any subtle eects on the association of integral membrane versions of ras proteins with regulators of gtpase and nucleotide exchange activities. the observation that golgi-targeted derivatives of ras are unable to transform ®broblasts, while perhaps not unexpected, provides some insight into the requirements for signal transduction through the ras pathway. it is clear that some mediators of the transforming pathway initiated by oncogenic ras are only available at the plasma membrane. precisely what these mediators are remains to be elucidated. however, one can conclude that simple juxtaposition of ras to a lipidrich environment is not sucient to allow it to signal. examination of these golgi-targeted ras derivatives and their ability to interact with or activate the normal cytosolic substrates such as raf- will provide future insights into the function of ras. recent evidence demonstrates that other important pathways regulating cytoskeletal structure and organization are also activated by ras. rac regulates membrane ruing in response to growth factor stimulation , while rhoa functions in the regulation of actin stress ®ber and focal adhesion assembly in growth factor-stimulated cells ). it appears that activation of both the mapk and rac/rho pathways is required for full transformation by ras (prendergast et al., ; khosravi-far et al., ; qiu et al., a,b) . in light of this recent evidence, we are currently investigating activation of the rac/rho pathway in response to our integral membrane versions of ras. full-length harvey p ras wt and p ras l cloned into pcdnai at the hindiii-ecori sites were generously provided by j buss. restriction sites in the coding sequences of ras wt and ras l used to make these clones are as follows: bsahi at nt and fspi at nt were used in generation of clones with e or e (qi) transmembrane anchors; a¯iii at nt was utilized to generate mutations in the c-terminus. note that the mutation resulting in the gln?leu at codon occurs at nt , and consists of a a?t transversion. all constructs containing n-terminal transmembrane domains and/or c-terminal mutations described in this paper were constructed using the following strategy. pairs of complementary oligonucleotides were designed and synthesized such that, when annealed, overhangs for restriction sites were formed. more details about the making of the various fusion constructs are available upon request. all synthetic oligonucleotides were gel puri®ed as previously described (xu et al., ) , and all dna sequences derived from oligonucleotides were con®rmed by dideoxy nucleotide sequencing before use. nih t cells were maintained as previously described (hart et al., ) . cells were split at a density of cells per mm plate and transfected the following day using the calcium phosphate precipitation protocol (chen and okayama, ) . cells from each mm plate were split : days later, and scored for foci ± days later. these assays were repeated at least for each construct. nih t cells were split onto mm plates containing glass coverslips and transfected the following day with mg of plasmid dna, as described above. two days after transfection, the cells were ®xed with % paraformaldehyde/pbs and permeabilized with . % triton x- /pbs. the intracellular location of ras fusion proteins was detected with rat monoclonal antiserum ab- (y - ) directed against v-h-ras (oncogene science) and uorescein-conjugated goat a-rat secondary antibody (boehringer mannheim). for double-labeling experiments, cells were ®xed and permeabilized as described above. ras fusion proteins were detected as described above, then the coverslips were treated with monoclonal ab e , which detects the cis-golgi of cells (kindly provided by wj brown and v malhotra) and rhodamine-conjugated goat a-mouse secondary antibody (boehringer mannheim). nih t cells were split onto mm plates and days after transfection, monolayers were washed with tris-saline, incubated min with dme minus cys and met, and then labeled for h with mci each of [ s]-cys and [ s]-met. cells were lysed in ripa ( % triton x- , . m nacl, mm tris-hcl ph . , . % sds, % doc, mg/ml aprotinin), precleared with protein a-sepharose, and incubated with ab- rat monoclonal antibody. immunoprecipitates were collected with protein a-sepharose beads coated with rabbit a-rat igg, washed with ripa, and resuspended in sample buer ( mm tris ph . , % sds, % -mercaptoethanol, % glycerol). proteins were separated by % sds ± page and detected by¯uorography. nih t cells were transfected as described for immunoprecipitation. two days after transfection, monolayers were lysed with lysis buer ( mm tris-hcl ph . , . m nacl, mm mgcl , mm cacl , % triton x- , mg/ml aprotinin) and precleared with protein a-sepharose. one third of the total lysate was then subjected to immunoprecipitation using ab- . immune complexes were collected on protein a-sepharose beads coated with rabbit a-rat igg, washed with lysis buer, with ripa, then incubated for min on ice with ml of . m a-[ p]gtp in ripa. beads were then washed with ripa, with lysis buer, and incubated at c for h in ml of lysis buer. ml of each sample was spotted onto pei-cellulose plates (jt baker, phillipsburg, new jersey) and chromatographed in . m kh po , ph . . abbreviations e , avian coronavirus e glycoprotein; er, endoplasmic reticulum; ibv, infectious bronchitis virus. melanie webster and patricia d'avis for critical reading of the manuscript, and laura castrejon for editorial assistance. kch would like to thank scott robertson, ryan dellinger, and vincent ollendor for advice and support. kch gratefully acknowledges support from the lucille p markey charitable trust. this work was supported by grant rt- from the u.c. tobacco related disease research program and by grant cb- from the american cancer society. proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa proc. natl. acad. sci. usa, proc. natl. acad. sci. usa proc. natl. acad. sci. usa, proc. natl. acad. sci. usa cancer investigation rab gtpases in vesicular transport we thank jan buss for providing the p ras wt and p ras l plasmids that served as the starting point for construction of all clones described in this paper, and for helpful advice on gtpase assays. we thank wj brown and v malhotra for the mab e used for co-localization studies. we also thank april meyer for technical support, ras anchored as an integral membrane protein kc hart and dj donoghue key: cord- -qq h vc authors: fyhrquist, f.; saijonmaa, o. title: renin‐angiotensin system revisited date: - - journal: j intern med doi: . /j. - . . .x sha: doc_id: cord_uid: qq h vc new components and functions of the renin‐angiotensin system (ras) are still being unravelled. the classical ras as it looked in the middle s consisted of circulating renin, acting on angiotensinogen to produce angiotensin i, which in turn was converted into angiotensin ii (ang ii) by angiotensin‐converting enzyme (ace). ang ii, still considered the main effector of ras was believed to act only as a circulating hormone via angiotensin receptors, at and at . since then, an expanded view of ras has gradually emerged. local tissue ras systems have been identified in most organs. recently, evidence for an intracellular ras has been reported. the new expanded view of ras therefore covers both endocrine, paracrine and intracrine functions. other peptides of ras have been shown to have biological actions; angiotensin – heptapeptide (ang iii) has actions similar to those of ang ii. further, the angiotensin – hexapeptide (ang iv) exerts its actions via insulin‐regulated amino peptidase receptors. finally, angiotensin – (ang – ) acts via mas receptors. the discovery of another ace was an important complement to this picture. the recent discovery of renin receptors has made our view of ras unexpectedly complex and multilayered. the importance of ras in cardiovascular disease has been demonstrated by the clinical benefits of ace inhibitors and at receptor blockers. great expectations are now generated by the introduction of renin inhibitors. indeed, ras regulates much more and diverse physiological functions than previously believed. have been shown to have biological actions; angiotensin - heptapeptide (ang iii) has actions similar to those of ang ii. further, the angiotensin - hexapeptide (ang iv) exerts its actions via insulin-regulated amino peptidase receptors. finally, angiotensin - (ang - ) acts via mas receptors. the discovery of another ace was an important complement to this picture. the recent discovery of renin receptors has made our view of ras unexpectedly complex and multilayered. the importance of ras in cardiovascular disease has been demonstrated by the clinical benefits of ace inhibitors and at receptor blockers. great expectations are now generated by the introduction of renin inhibitors. indeed, ras regulates much more and diverse physiological functions than previously believed. keywords: angiotensin, angiotensin-converting enzyme, angiotensin receptor, renin. the classical paper on the discovery of renin by the finnish physiologist robert tigerstedt and his swedish student per bergman in [ ] was based on experiments performed - at the karolinska institute. saline extracts of rabbit kidney were shown to slowly raise the blood pressure (bp) when injected into rabbits. the principle causing this was present in kidney cortex but not in medulla and was destroyed by heating. the authors concluded that the substance was a protein and they named it renin. they speculated that 'renin might in some direct or indirect way be associated with hypertrophy of the heart found in renal disease and high bp'. however, these early results could not be repeated in other laboratories, and it was not until late s when renin was 'rediscovered'. an immense amount of early research on the reninangiotensin system (ras) paved the way for improved understanding of its physiology and pathophysiology. in the early s, the major components of 'classical' circulating ras (fig. ) were identified and there was compelling evidence to indicate important roles for ras in the regulation of fluid balance and bp. at that time, however, there was widespread skepticism review | regarding the role of ras in cardiovascular disease. it was not until the discovery of orally effective angiotensin-converting enzyme (ace) inhibitors, the first of which was captopril [ ] , that the paramount importance of ras in cardiovascular homeostasis and disease was being appreciated. the introduction of losartan, the first orally active and effective angiotensin receptor type blocker [ ] further strengthened this concept. fig. the present view of the expanded renin-angiotensin system. rpr, renin ⁄ prorenin receptor; mas, mas oncogene, receptor for ang - ; at r, angiotensin type receptor; at r, angiotensin type receptor, irap, insulin-regulated aminopeptidase; ang iv receptor ampa, aminopeptidase a; ampm, aminopeptidase m; ace, angiotensin-converting enzyme; ace , angiotensin-converting enzyme ; nep, neutral endopeptidase. angiotensin ii exerts its actions via at and at receptors which in principle, but not invariably, mediate opposite functions. at receptors mediate actions with potentially harmful consequences, if not properly counterbalanced. at receptors are thought to mediate protective actions, the clinical relevance of which has not yet been clearly established. angiotensin ii is a major regulator of fluid and sodium balance and haemodynamics, but also of cellular growth and cardiovascular remodelling. thus, at receptors mediate vasoconstriction, thirst and release of vasopressin and aldosterone, fibrosis, cellular growth and migration. more recently, ang ii has been shown to cause generation of oxidative radicals via at receptors and to be involved in inflammatory processes including atherosclerosis and vascular ageing. at receptors mediate vasodilation, release of nitric oxide (no) and usually inhibition of growth. angiotensin ii infusion caused decrease of plasma adiponectin, an insulin sensitizer, apparently via at receptors in the rat [ ] . suppression of adiponectin may represent a mechanism whereby ang ii causes impaired glucose tolerance. other metabolic actions of ang ii include pro-inflammatory modulation [ ] , increased insulin secretion [ ] , b-cell apoptosis [ ] , reduction of gluconeogenesis and hepatic glucose output [ ] and increased plasma triglycerides [ ] . we decided not to enter the complex field of intracellular at and at receptor signalling and therefore refer the reader to recent reviews [ , ] . angiotensin receptor type is generally reported to mediate effects opposing and counterbalancing those mediated by at receptors (fig. ), e.g. vasodilatation, no release and inhibition of proliferation and growth. however, at receptors may also mediate neurotrophic effects in the central nervous system [ ] . moreover, upregulated at receptors in the peri-ischaemic brain may exert protection against ischaemic damage [ ] . the authors speculate that such a protective effect mediated by at receptors might in part explain superior protection against stroke in patients treated with losartan vs. treatment with atenolol in the life study [ ] . another explanation may be that losartan lowers central bp more effectively than atenolol [ ] . in rat kidney, ang iii but not ang ii was recently reported to induce natriuresis mediated by at receptors [ ] . this natriuresis was augmented by blockade of aminopeptidase n, an enzyme metabolizing ang iii to ang iv ( fig. ) [ ] . the authors speculated that blockers of aminopeptidase n might be developed to treat diseases characterized by sodium and fluid retention such as hypertension and heart failure. in theory, such inhibitors may also exert beneficial actions via reduced tissue levels of ang iv (see below). interestingly, renal interstitial fluid has been shown to contain roughly -fold higher concentrations of ang ii and ang iii than found in the plasma [ ] . effects of unopposed stimulation of at receptors are slightly controversial [ ] . thus, beneficial effects include bradykinin-no vasodilatory effects, natriuretic and antifibrotic effects. potentially detrimental effects are apoptosis, nuclear factor-kappa b (nf-jb) signal transduction and induction of chemokines. despite a plethora of promising experimental results strongly suggesting beneficial actions of at stimulation [ ] the final clinical proof is lacking. though treatment with at receptor blockers (arbs) substantially increase plasma ang ii levels and presumably cause increased stimulation of at receptors, there is no conclusive evidence to prove the clinical relevance of increased at receptor activity. angiotensin ii may be enzymatically generated from ang i by chymase (fig. ) in certain pathological conditions. chymase is stored in macromolecular complex with heparin proteoglycan in secretory granules of mast cells [ ] . to become enzymatically active, complexed chymase must be released from f. fyhrquist & o. saijonmaa | review: renin-angiotensin system mast cell granules, e.g. by vascular damage caused by ballooning or other damage. therefore, chymase is enzymatically inactive in normal vascular tissue and may produce ang ii only in damaged or atherosclerotic arterial walls. it is of note that endogenous serine protease inhibitors present in interstitial fluid [ ] are potent inhibitors of chymase. chymase inhibitors reportedly prevent neointimal lesions following vein grafting or arterial ballooning in dogs, whereas ace inhibitors are ineffective [ ] . however, the effects of chymase inhibitors may depend on other effects of these compounds such as decrease in transforming growth factor-b (tgf-b) generation and stabilization of mast cell granules and not on decreased ang ii formation. in addition, arbs which block ang ii actions irrespective of generating enzyme(s) have not proven superior to ace inhibitors in large clinical trials [ , ] . though animal experimental results with chymase inhibitors are promising [ ] , the possible importance of ang ii generation by chymase is unclear and chymase inhibitors that are safe and useful for human trials have not yet been developed. angiotensin iii has been known since the s to cause vasoconstriction and release of aldosterone. it is generated from ang ii by aminopeptidase a (fig. ). ang iii exerts its actions, in principle similar to those of ang ii, via at and at receptors. while ang ii is considered the main effector of ras, ang iii may be equally or even more important in some actions mediated by at receptors, e.g. release of vasopressin [ ] . systemic infusion of ang ii or ang iii to conscious dogs was recently shown to result in equipotent effects at the same plasma concentration on bp, aldosterone secretion, sodium excretion and plasma renin activity; all effects inhibited by candesartan. however, the metabolic clearance rate of ang iii was five times that of ang ii [ ] . this study indicated that ang ii plays a dominant role as an effector of the 'classical circulating ras'. angiotensin iv may be generated from ang iii by aminopeptidase m (fig. ). this biologically active peptide has caught increasing interest following the discovery and cloning of insulin-regulated amino peptidase receptors (irap), [ , ] , a binding site and a probable receptor (at ) of ang iv. actions of ang iv mediated by irap (fig. ) include renal vasodilation, hypertrophy and activation of nf-jb leading to increased expression of platelet activator inhibitor-i (pai- ), monocyte chemoattractant protein (mcp- ) interleukin- and tumour necrosis factor-a [ , ] . several studies suggest that ang iv has important regulatory functions in cognition, renal metabolism and cardiovascular damage [ , ] . ang iv regulates cell growth in cardiac fibroblasts, endothelial cells and vascular smooth muscle cells. it appears that ang iv is involved in the vascular inflammatory response and could therefore play a role in cardiovascular pathophysiology. angiotensin - heptapeptide was thought for a long time to be devoid of biological actions, in spite of early reports on biological effects [ ] . the importance of ang - was emphasized by the relatively recent discovery of a 'new' ace . this enzyme generates ang - from ang ii. ang - may also be generated from ang i or ang ii by other peptidases. already back in , ang ( - ) was shown to release vasopressin as effectively as ang ii from neurohypophyseal explants [ ] . ang ( - ) was found to have actions opposing those of ang ii (fig. ) , namely vasodilation and antitrophic effects and amplification of vasodilation caused by bradykinin [ ] [ ] [ ] . numerous experiments suggest an important interaction between ang ( - ) and prostaglandin-bradykinin-no systems. ang ( - ) appears to counterbalance several actions of ang ii. ang ( - ) binds to the mas receptor (figs and ) which mediates vasodilating and antiproliferative actions of the heptapeptide. angiotensin-converting enzyme was discovered and cloned rather recently [ , ] . this discovery brought both ace and its main product, ang - into the focus of intense research. ace is a carboxypeptidase which cleaves one residue from ang i to generate angiotensin - and a single residue from ang ii to generate ang - (fig. ) . ace is most abundant in vascular endothelium of kidney, heart, hypothalamus and aortic wall. ace is also found in testis [ ] . regulation of ace expression has not yet been fully clarified [ ] . neither ace inhibitors nor arbs do inhibit ace activity, but they both appear to upregulate ace expression in rat myocardium and renal cortex [ ] . expression of ace in the heart is increased by myocardial infarction [ ] . disruption of the ace gene in mice was reported to result in a severe cardiac contractility defect, increased ang ii plasma levels and upregulation of cardiac hypoxia-induced genes [ ] . the authors concluded that ace is an 'essential regulator of heart function'. genetic ablation of ace in ace null mice completely normalized the cardiac phenotype, which fits the proposed mutually counterbalancing roles for the ace ⁄ ang ii and ace ⁄ ang - arms of ras. the authors interpreted these findings as evidence for ace being an essential regulator of heart function. this interpretation [ ] has recently been challenged by a study also on ace null mice, showing 'no detectable effect on cardiac dimensions or ejection fraction in conscious mice under basal conditions' [ ] . these investigators reported increased pressor sensitivity to ang ii infusion and higher plasma levels and renal concentrations of ang ii during infusion in ace null mice. this study suggested an important role for ace in degrading ang ii and regulation of vascular responses to ang ii. the abundance of ace in kidneys notably in the proximal tubule is of particular note. ace may be critical in regulating the balance between renal effects of ang ii and ang ( - ) and may therefore become a target for future therapeutic approaches [ ] . ace seems to have a protective role in the kidney [ ] . angiotensin-converting enzyme and ang - may play an important role in cardiovascular physiology and pathophysiology, e.g. by modulating or counterbalancing excess activity of the 'classical' ras [ , ] . the expression and activity of ace in heart [ ] and kidney is differently increased by treatment with ace inhibitors. this leads to organ-wise regulated increase of local production of ang - , as demonstrated in rats [ ] [ ] [ ] . this would offer an additional beneficial effect of ace inhibitors, possibly explaining in part why ace inhibitors and arbs review: renin-angiotensin system remain effective despite increased plasma renin activity and angiotensin peptide concentrations [ ] [ ] [ ] . renin receptors were identified and cloned rather recently and shown to be functional [ ] . renin receptors bind both prorenin and renin [ ] . two receptors have been characterized; the m p ⁄ insulin-like growth factor ii receptor, a clearance receptor and the specific renin receptor, which activates intracellular signalling and enhances receptor-bound renin catalytic activity on the cell surface (fig. ) . renin receptors are abundant in heart, brain and placenta, lower levels being found in kidney and liver [ ] . visceral adipose tissue also expresses renin receptor, whereas subcutaneous adipose tissue expressed less renin receptors [ ] . it appears that renin receptors participate in local production of ang ii and may contribute to systemic ang ii levels as well. binding of prorenin to the renin receptor (fig. ) leads to activation of prorenin to active renin, activation of mitogen-activated protein kinases p ⁄ and tgf-b, ultimately increasing contractility, hypertrophy and fibrosis [ , ] . it has been suggested that blocking renin receptors may be a new target for tissue protection. the 'handle and gate' hypothesis of activation of receptor-bound renin offers exciting perspectives [ ] . a pentapeptide reproducing the 'handle' region of the prosegment of prorenin (fig. ) that covers the active site has provided promising results in the treatment of diabetic nephropathy [ ] and in the prevention of hypertensive glomerulopathy in mice [ ] . if local ras systems have been identified in most organs and tissues investigated as recently reviewed [ , ] . they contain all components necessary for the production of ang ii and other angiotensin peptides and their respective receptors, and in addition, renin ⁄ prorenin receptors. the majority of studies indicate that most if not all renin found in local ras systems is derived from renal renin. tissue ras systems exert diverse actions in many organs. in some organs, they operate independently of the 'circulating ras', e.g. the adrenal glands and brain. other local ras systems, e.g. heart and kidney operate in close interaction with the 'circulating' ras. thus, circulating components of ras like renin and agt may be taken up by tissues. circulating ras and local tissue ras are thought to operate in a complementary fashion [ ] , not opposing each other. a proper balance between regulating and counter-regulating factors of tissue ras appears important in maintaining normal physiological functions of many organs. the circulating ras is seen as a regulator of systemic volume and electrolyte balance and of bp homeostasis, whilst 'local' ras systems have local tissue effects involving proliferation, growth, protein synthesis and organ functions, e.g. in kidney, heart, brain, reproductive organs and pancreas [ , ] . some recent discoveries concerning local ras systems may deserve particular interest, namely those of the heart, brain and adipose tissue. thus, ace expression is increased in the heart following myocardial infarction [ ] , in heart failure [ ] and during treatment with ace inhibitors or arbs [ ] . ace is the main generator of ang - from ang ii in the heart, and the amount of ang - is increased in the peri-ischaemic area following myocardial infarction [ ] . brain ras components mediate a large variety of neurobiological activities [ ] which are gradually being understood. for instance, neuronal at receptors mediate ang ii effects on bp, salt and water intake and secretion of vasopressin whereas at receptors mediate, e.g. apoptosis and possibly neural regeneration after neural injury [ ] . in addition to ang ii and ang iii, ang iv and ang - appear to be involved in modulation of brain functions, including learning and memory responses. adipose tissue also contains all components of ras including functional renin receptors co-localizing with renin and may be involved in the regulation of visceral adipose tissue accumulation [ ] . thus, visceral ras may play a role in the pathophysiology of the metabolic syndrome [ ] . adipose tissue was shown to be an important source of both local and circulating agt [ ] and might thereby participate in systemic bp regulation. however, this has not been shown in humans. testicular ace (ca kda), a smaller isoform of ace ( - kda) was recently shown to play a crucial role in fertilization by releasing a glycosylphosphatidylinositol (gpi)-anchored protein from sperm cells. ace knock-out sperm cells showed deficient binding of egg cells [ ] . the impact of this observation awaits further clarification. however, treatment with ace inhibitors is not reported to interfere with male fertility. evidence suggesting the existence a complete, functional intracellular ras within cells has been provided recently [ , ] . intracellular ras is reported to mediate changes in ca + fluxes and activation of genes [ ] . intracellular ang ii reportedly caused cardiac hypertrophy in vivo in mice [ ] . in these experiments, a plasmid construct under the control of an a-myosin promoter caused increased intracellular ang ii and % increase in relative heart weight. the mechanisms by which intracellular ang ii exerts its actions are not fully understood. thus, intracellularly applied arbs can only partly block intracellular ang ii. the role of intracellular ras is presently unclear. surprisingly, ace has been shown to function as a receptor of severe acute respiratory syndrome (sars) coronavirus [ , ] . ace is thought to contribute to pulmonary tissue damage and oedema by generating ang ii. ace is believed to normally counteract these harmful effects, but following attachment of sars virus to ace and replication, ace expression is diminished, less ang - is formed from ang ii and at receptor activation is intensified. in support of this contention, injection of recombinant ace into mice protected these mice from acute lung injury caused by sepsis or acid aspiration [ ] . thus, functioning ace may protect against the potentially lethal lung injury associated with sars [ ] . ageing is associated with alterations of several structural and functional properties of large arteries. increased wall thickness and stiffness, pulse wave velocity and pulse wave augmentation and deterioration of endothelial function are hallmarks of arterial ageing [ ] . these alterations form fertile soil for age-associated cardiovascular disease. conversely, cardiovascular disease causes acceleration of these detrimental changes. several lines of evidence support an important role of ras in arterial ageing as well as in cardiovascular disease. arterial components of the ang ii-signalling cascade increase with ageing [ ] . ang ii signalling via at receptors increases collagen production within the arterial wall, promotes reduced forms of nicotinamideadenine dinucleotide phosphate oxidase activity and enhances migration of vascular smooth muscle cells. increased formation of reactive oxygen species (ros) leads to activation of metalloprotease, less no bioavailability and endothelial dysfunction. formation of ros induced by ang ii may contribute to tissue ageing and age-related cardiovascular disease [ ] . ang ii also causes activation of nf-jb pathway and proinflammatory cytokines [ , ] . thus, judging by mechanistic criteria, ang ii appears to play a central role in many stimuli that govern arterial ageing and its functional responses. a recent study [ ] showed that treatment of male wistar rats with an ace inhibitor (enalapril mg · kg · day) or an arb (losartan mg · kg · day) for months or life-long resulted in prolongation of life span by % (enalapril) or % (losartan) compared with untreated control rats. the difference in life span could not be explained by cardiovascular protection. the authors speculate that prolongation of life span could be explained by reduction by ras inhibitors of ros formation and reduction of oxidative burden. in accordance with the generally acknowledged importance of ras in pathophysiology of cardiovascular disease, many mutations in ras component genes are associated with hypertension and cardiovascular diseases [ ] . the agt gene has been associated with hypertension [ ] , but attempts to predict responses to antihypertensive drugs based on agt polymorphisms have yielded inconsistent results [ ] . variants of the at and the at receptor genes are reportedly associated with hypertension, but they are also inconsistently associated with response to antihypertensive therapy [ ] [ ] [ ] [ ] . in a chinese cohort of hypertensive patients, the association with combined agt and at receptor single nucleotide polymorphisms (snp) haplotypes was modest ( % for systolic, % for diastolic bp reduction with ace inhibitor [ ] . in a prospective study comprising uk men, the at receptor genotype cc was associated with increased cardiovascular risk irrespective of bp [ ] whereas the at ii receptor a allele was associated with increased risk only at high systolic bp (> mm hg). in general, the magnitude of predictive power of ras gene, snps has been rather modest. a possible role for the at receptor in the central nervous system was first suggested by attenuated exploratory behaviour in at receptor-deficient (knockout) mice [ ] [ ] [ ] . in humans, the absence of or mutations in the at receptor gene was shown to be associated with severe x-chromosome linked mental retardation [ ] , showing a link between a ras component and development of cognitive functions. interestingly, a unique mutation of the renin receptor gene was later shown to be present in patients with x-linked mental retardation and epilepsy [ ] . functional analysis revealed that the mutated renin receptor could bind renin and increase renin catalytic activity. 'this finding confirmed the importance of the ras in cognitive processes and indicated a novel specific role for the renin receptor in cognitive functions and brain development' [ ] . considerable widening and deepening of our understanding of ras' physiology and pathopysiology has been achieved by genetical manipulation of experimental animals, e.g. rats [ ] and in mice [ ] . for instance, 'ace ⁄ mice', compound heterozygotes for the ace genes [ ] have no endothelial ace, but are nevertheless capable of maintaining normal physiology. the explanation for this appeared to be a compensatory increase in renal renin production followed by increased ang ii generation by nonendothelial ace, showing the plasticity of ras. this is just one example of fascinating gene manipulation revealing secrets of ras physiology that would otherwise have remained enigmatic. angiotensin-converting enzyme inhibitors and arbs (fig. ) are well established corner stones in the prevention and treatment of hypertension and cardiovascular disease, as demonstrated by numerous clinical trials and world wide clinical practice. according to a recent meta-analysis [ ] , ace inhibitors and arbs have similar bp-dependent effects for the risks of stroke, coronary heart disease and heart failure. for ace inhibitors, but not for arbs, there is evidence for bp-independent effects on the risk of coronary disease events [ ] . the benefits of ace inhibitors or arbs on renal outcomes probably result from bplowering effects [ ] whereas renoprotective benefits in diabetic patients may partly depend on factors beyond bp lowering [ ] . in fact, several studies suggest that both ace inhibitors and arbs offer benefits in addition to those mediated by bp lowering only. several trials have demonstrated a - % reduction of new onset diabetes during treatment with ace inhibitors or arbs [ ] . the mechanism behind this protective effect of ras inhibition is not clear, but it offers a significant advantage for ras inhibitors as we are experiencing a global epidemy of increasing incidence of diabetes. combined blockade of the ras by ace inhibitors and arbs has been shown to provide additional benefits compared with either drug class [ ] [ ] [ ] . however, these expectations were not confirmed by the recently published ontarget study [ ] which compared treatment with telmisartan, ramipril or both drugs combined in a megatrial comprising patients with high cardiovascular risk profile. of particular interest is the use in ontarget of telmisartan, by far, the most prominent activator of peroxisome proliferator activated receptor-c, a mediator of a host of favourable metabolic actions [ , ] . in the ontarget study, mean bp was lower in both the telmisartan group ( . ⁄ . mm hg greater reduction) and the combination therapy group ( . ⁄ . mm hg greater reduction) than in the ramipril group. telmisartan was equivalent to ramipril in terms of primary outcomes in patients with vascular disease or diabetes. the combination of telmisartan and ramipril was associated with more adverse events (hypotension, renal dysfunction) without an increase in benefit. the recent introduction of the first orally effective renin inhibitor, aliskiren, has raised additional interest in new possibilities of almost complete blockade of ras as a tool (fig. ) , perhaps, more effective than earlier, in the prevention and treatment of cardiovascular disease [ , , , ] . early reports on the use of aliskiren are promising, showing at least, an antihypertensive effect of aliskiren potent as those of other antihypertensive drugs [ ] . in particular, the combination of renin inhibitors with ace inhibitors and arbs may offer a solution to the 'renin escape' phenomenon [ ] , which implies that ace inhibitors or arbs may lose part of their effect during long time treatment. many questions are also raised, for instance, what would the consequences be if the 'beneficial' angiotensin peptide, ang - was not generated at all 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harbouring the mouse ren- gene six truisms concerning ace and the renin-angiotensin system deduced from the genetic analysis of mice mice lacking endothelial ace: normal blood pressure with elevated angiotensin ii blood pressure-dependent and independent effects of agents that inhibit the renin-angiotensin system effect of inhibitors of the renin-angiotensin system and other antihypertensive drugs on renal outcomes: systematic review and meta-analysis ace inhibitors and angiotensin receptor antagonists and the incidence of new onset diabetes mellitus: an emerging theme novel therapies blocking the renin-angiotensin-aldosterone system in the management of hypertension and related disorders novel drugs targeting hypertension: renin inhibitors and beyond dual blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers in chronic kidney disease telmisartan, ramipril or both in patients at high risk for vascular events antidiabetic mechanisms of angiotensin-converting enzyme inhibitors and angiotensin ii receptor antagonists: beyond the renin-angiotensin system renin inhibition with aliskiren: where are we now and where are we going? oral renin inhibitors fax: + ; e-mail: frej.fyhrquist@helsinki.fi) no conflict of interest was declared. key: cord- - oa gkrp authors: gemmati, donato; bramanti, barbara; serino, maria luisa; secchiero, paola; zauli, giorgio; tisato, veronica title: covid- and individual genetic susceptibility/receptivity: role of ace /ace genes, immunity, inflammation and coagulation. might the double x-chromosome in females be protective against sars-cov- compared to the single x-chromosome in males? date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: oa gkrp in december , a novel severe acute respiratory syndrome (sars) from a new coronavirus (sars-cov- ) was recognized in the city of wuhan, china. rapidly, it became an epidemic in china and has now spread throughout the world reaching pandemic proportions. high mortality rates characterize sars-cov- disease (covid- ), which mainly affects the elderly, causing unrestrained cytokines-storm and subsequent pulmonary shutdown, also suspected micro thromboembolism events. at the present time, no specific and dedicated treatments, nor approved vaccines, are available, though very promising data come from the use of anti-inflammatory, anti-malaria, and anti-coagulant drugs. in addition, it seems that males are more susceptible to sars-cov- than females, with males % more likely to die from the infection than females. data from the world health organization (who) and chinese scientists show that of all cases about . % of women who contract the virus will die compared with . % of men, and data from hong kong hospitals state that % of male and % of female covid- patients required intensive care or died. on the other hand, the long-term fallout of coronavirus may be worse for women than for men due to social and psychosocial reasons. regardless of sex- or gender-biased data obtained from who and those gathered from sometimes controversial scientific journals, some central points should be considered. firstly, sars-cov- has a strong interaction with the human ace receptor, which plays an essential role in cell entry together with transmembrane serine protease (tmprss ); it is interesting to note that the ace gene lays on the x-chromosome, thus allowing females to be potentially heterozygous and differently assorted compared to men who are definitely hemizygous. secondly, the higher ace expression rate in females, though controversial, might ascribe them the worst prognosis, in contrast with worldwide epidemiological data. finally, several genes involved in inflammation are located on the x-chromosome, which also contains high number of immune-related genes responsible for innate and adaptive immune responses to infection. other genes, out from the ras-pathway, might directly or indirectly impact on the ace /ace balance by influencing its main actors (e.g., abo locus, sry, sox , adam ). unexpectedly, the higher levels of ace or ace /ace rebalancing might improve the outcome of covid- in both sexes by reducing inflammation, thrombosis, and death. moreover, x-heterozygous females might also activate a mosaic advantage and show more pronounced sex-related differences resulting in a sex dimorphism, further favoring them in counteracting the progression of the sars-cov- infection. sars-cov- belongs to the β-coronavirus family and the associated severe acute respiratory syndrome (sars), like the previous sars-cov and middle east respiratory syndrome (mers-cov), it may cause life-threatening diseases [ ] [ ] [ ] [ ] [ ] [ ] . data from past epidemiological studies match with emerging observations, indicating a large, sex-dependent gap in disease infection and outcomes for sars-cov- disease (covid- ) as for previous sars [ , ] . moreover, people aged and over suffer worse outcomes with mortality rates above % [ , ] , and sex differences in incidence and higher fatality rates in males compared to females were also peculiar to earlier sars. conversely, infants and children experience mild symptoms and a better prognosis, without sex differences, and with a mildly elevated proinflammatory cytokines-storm in the early phase of the illness [ , ] . accordingly, thrombosis due to coagulation unbalance or inherited or acquired thrombophilia is a rare event among children [ ] [ ] [ ] [ ] [ ] , and this could in part account for the micro-thromboembolic events or cardiac injury found in the elderly severest cases as well the rarer disseminated intravascular coagulation (dic) [ ] [ ] [ ] , strongly substantiating heparin-based anticoagulant treatments in the selected severe cases [ , , ] . on the other hand, the severity of covid- worsens with advancing age for both sexes, possibly due to a dysregulation of the immune response, a difference in sex-hormones that has become less evident with age between sexes, or a considerable unbalancing in the coagulation/fibrinolytic system and endothelial dysfunction with aging [ ] [ ] [ ] [ ] . accordingly, in a mouse model, gonadectomy did not affect disease outcome in male mice, whilst ovariectomy or estrogen receptor antagonists caused increased mortality in females after sars-cov infection [ ] . this observation also strongly agrees with the fact that females mount stronger innate and adaptive immune responses and are relatively more resistant to virus infections than males. the difference in the copy number of x-linked genes involved in the immune response and the presence of genes dedicated to disease susceptibility in males and females may account for any other possible sex advantage [ ] [ ] [ ] [ ] . as concerns sex hormones, testosterone suppresses innate immune responses, whilst estrogens have immune-suppressive effect at higher levels and immune-stimulant activity at lower levels [ , [ ] [ ] [ ] , with peculiar functions to contrast virus replication in selected tissues such as nasal epithelial cells in humans [ ] . less investigated, is whether differences in the efficiency of the sars-cov- cell-entry might account for at least part of the observed sex-gap. it has been reported that sars-cov- enters human cells using the sars-cov receptor ace and a specific transmembrane serine protease (tmprss ) for the spike (s) protein priming [ ] , an observation that makes the two biomolecules promising therapeutic targets, useful for establishing prevention programs [ ] [ ] [ ] . it has been described how a modest ace expression characterizes the upper human respiratory tract and that this should limit the receptivity of the virus [ , ] . it is controversial whether metallopeptidase domain (adam , also known as tnfα-converting enzyme, tace), involved in the ace ectodomain shedding, by increasing the amount of soluble ace might or might not counteract the virus entry and/or exclusively contribute to ace /ace unbalancing, inflammation and thrombosis [ ] . furthermore, estrogens increase adam and adam expression levels, two putative shedders also responsible for many ectodomain cleavages in atherosclerosis [ ] , suggesting their protective role against cardiovascular events in females, a mechanism potentially accounting for the observed covid- sex-disparity. the efficiency in the ace -receptor/s-protein recognition and interaction is a key determinant for the success of viral infection and receptivity [ ] . recently, the s-protein/ace interface has been elucidated at the atomic level and several crucial amino acid residues are recognized to correctly achieve the homo-dimerization process [ ] . therefore, proper ace functionality is essential for both virus cell entry and local pulmonary homeostasis, and although it has been previously described that polymorphisms in the ace gene do not affect the outcome of sars [ ] , females might have a higher degree of heterodimer assembling than males, which in turn might show different affinity for the sars-cov- spike receptor. similarly, single nucleotide polymorphisms (snps) within the tmprss gene ( q . ) can also have a greater role in the general population (rs , rs , rs ) and in a sex-oriented perspective (rs ) hypothesizing that higher expression in males might favor virus membrane fusion, tmprss being an androgen responsive gene [ , ] in line with previous gwas on a(h n ) and a(h n ) influenza [ ] . conversely, estrogen fall in postmenopausal females in turn affects tmprss expression, the gene also being responsive to estrogens [ ] . finally, ace and ace cooperate in the renin-angiotensin system (ras) to balance the local vasoconstrictor/proliferative (ace /ang-ii/at -axis) and vasodilator/antiproliferative (ace /ang - / mas-axis) actions. this results in the protection of organs and blood vessels by anticoagulant, antiinflammatory, anti-proliferation, anti-fibrosis, anti-alveolar epithelial cell apoptosis, and anti-oxidative stress activities antagonizing the ang-ii effects ( figure ). for many ectodomain cleavages in atherosclerosis [ ] , suggesting their protective role against cardiovascular events in females, a mechanism potentially accounting for the observed covid- sex-disparity. the efficiency in the ace -receptor/s-protein recognition and interaction is a key determinant for the success of viral infection and receptivity [ ] . recently, the s-protein/ace interface has been elucidated at the atomic level and several crucial amino acid residues are recognized to correctly achieve the homo-dimerization process [ ] . therefore, proper ace functionality is essential for both virus cell entry and local pulmonary homeostasis, and although it has been previously described that polymorphisms in the ace gene do not affect the outcome of sars [ ] , females might have a higher degree of heterodimer assembling than males, which in turn might show different affinity for the sars-cov- spike receptor. similarly, single nucleotide polymorphisms (snps) within the tmprss gene ( q . ) can also have a greater role in the general population (rs , rs , rs ) and in a sex-oriented perspective (rs ) hypothesizing that higher expression in males might favor virus membrane fusion, tmprss being an androgen responsive gene [ , ] in line with previous gwas on a(h n ) and a(h n ) influenza [ ] . conversely, estrogen fall in postmenopausal females in turn affects tmprss expression, the gene also being responsive to estrogens [ ] . finally, ace and ace cooperate in the renin-angiotensin system (ras) to balance the local vasoconstrictor/proliferative (ace /ang-ii/at -axis) and vasodilator/antiproliferative (ace /ang - /mas-axis) actions. this results in the protection of organs and blood vessels by anticoagulant, anti-inflammatory, anti-proliferation, anti-fibrosis, anti-alveolar epithelial cell apoptosis, and anti-oxidative stress activities antagonizing the ang-ii effects ( figure ). therefore, the coexistence of inherited predispositions or common gene polymorphisms in the ace and ace genes that affect their mutual expression levels might lead to increased capillary permeability, coagulation, fibrosis, and apoptosis in the alveolar cells, accelerating lung damage and pulmonary shut-down triggered/worsened by the sars-cov- infection. therefore, the coexistence of inherited predispositions or common gene polymorphisms in the ace and ace genes that affect their mutual expression levels might lead to increased capillary permeability, coagulation, fibrosis, and apoptosis in the alveolar cells, accelerating lung damage and pulmonary shut-down triggered/worsened by the sars-cov- infection. most likely, it is plausible that a combination of the mechanisms described above might influence the multistep pathogenesis and the age/sex-gaps of such a complex infection and progression, considering also that ace (locus xp . ) and ang-ii receptor type gene (agtr , alias at , locus xq ) are both located on the x-chromosome. essentially, x-linked heterozygous alleles could activate in females a mosaic advantage and a greater sexual dimorphism that might counteract viral infection, local inflammation due to cytokine storms and severe outcomes. in the present work, we will address the hypotheses suggested above and reveal the main underlining genetic mechanisms with the hope that a sex-oriented approach will add new insights for a better understanding of covid- also in the general population. the renin-angiotensin system (ras) acts as a homeostatic regulator of the vascular function, including blood pressure and volume control, starting with renin (ren) mediating the transformation of angiotensinogen (agt) to angiotensin i (ang-i). indirectly, ras is also responsible for local tissue homeostasis by anti-inflammatory, anti-coagulant, anti-proliferation, anti-fibrosis, anti-apoptosis of epithelial cells, and anti-oxidative stress activities, also controlling the local trophic responses to a range of stimuli, viruses included [ , ] . the angiotensin i-converting enzyme (ace ) and the more recently discovered homologue ace [ , ] , are two antagonist enzymes of the ras pathway that act and counterbalance each other [ ] . the main role of ace is the conversion of angiotensin i to angiotensin ii (ang-i > ang-ii), the latter being a powerful peptide causing complex processes such as vasoconstriction, inflammation, fibrosis and proliferation via the at -receptor. conversely, ace firstly converts ang-i to ang - , that is then converted by ace in the vasodilator peptide ang - . moreover, ace directly converts ang-ii to ang - and this latter by acting on mas-receptor exerts organ protection, antagonizing the biological effects of ang-ii [ ] [ ] [ ] . moreover, a high ace /ace ratio protects against endothelial dysfunctions and vascular pathologies, exogenous ace activation promotes antithrombotic activity, and the known antithrombotic properties of captopril (ace inhibitor) and losartan (at -receptor blocker) are attenuated by a selective ang - receptor antagonist [ , ] . ace is a key molecule for the tuning of the ras pathway under both healthy and pathological conditions. it has been recently demonstrated that sars-cov- uses the same receptor (ace ) as sars-cov to enter cells in combination with the action of the serine protease tmprss for s-protein priming [ ] . ace is expressed in several tissues, including endothelium, lung, heart, intestine, and kidney and, as recently demonstrated, on the epithelial cells of oral mucosa and the tongue [ ] , sharing both tissue expression sites and high sequence identity with the homologue ace [ , ] . anomalous tuning of the ace /ace pathway contributes to the development of several complex pathological conditions such as hypertension, atherosclerosis, thrombosis, heart or kidney failure, and severe acute respiratory distress [ ] . in the lung, ace is highly expressed, and ace is mainly clustered in type-ii alveolar cells [ , [ ] [ ] [ ] . during acute respiratory distress syndrome (ards), a local ras unbalance cannot maintain appropriate oxygenation, thus inducing pulmonary edema, inflammation, and hyper-proliferation, establishing in turn severe pulmonary shutdown [ , , ] . the observations from the previous sars epidemic ( - ) that some coronaviruses use heparan sulfate as a receptor entry by acquiring heparan sulfate-binding sites, and that the heparin molecule acts as competitor preventing the binding of the spike protein to the host cell, inhibiting infection rate and mortality, are a valuable rationale to start heparin treatment in selected covid- patients [ ] [ ] [ ] [ ] . this approach might have a double result, to reduce virus entry and avert thrombotic complications or organ dysfunctions [ , , ] . interestingly, in ards animal models, ace knockdown mice experience more severe symptoms [ ] . ace gene deletion causes progressive cardiac fibrosis [ ] , whereas ace deficient mice result in renal injury and glomerulosclerosis [ , ] . these alterations are reversed or ameliorated by treatment with ace-inhibitors or ang-receptors blocker (arbs) [ , , , ] , whether or not they are combined with infusion of a soluble form of recombinant human ace (rhace ) [ ] [ ] [ ] . intuitively, this strategy could also be a possible treatment for covid- patients with a double effect: excessive soluble rhace could competitively bind and neutralize the sars-cov- virus, and rescue the cellular ace activity counteracting unescapably unrestrained ace activity to contrast lung injury. in summary, the ace /ace balance is crucial in contrasting organ dysfunction, so a direct or indirect increase in ace expression together with a modulation of ace activity may be helpful to avoid pulmonary disease progression ( figure ) [ , , [ ] [ ] [ ] [ ] . accordingly, several international societies recommend not to stop treatments with ras pathway antagonists in cardiovascular disease patients [ ] , and a recent editorial comments on several publications discussing the positive effects of ras inhibitors during covid- [ ] . with a double effect: excessive soluble rhace could competitively bind and neutralize the sars-cov- virus, and rescue the cellular ace activity counteracting unescapably unrestrained ace activity to contrast lung injury. in summary, the ace /ace balance is crucial in contrasting organ dysfunction, so a direct or indirect increase in ace expression together with a modulation of ace activity may be helpful to avoid pulmonary disease progression ( figure ) [ , , [ ] [ ] [ ] [ ] . accordingly, several international societies recommend not to stop treatments with ras pathway antagonists in cardiovascular disease patients [ ] , and a recent editorial comments on several publications discussing the positive effects of ras inhibitors during covid- [ ] . finally, other genes directly or indirectly regulate the ras pathway (table ) . firstly, adam by promoting the detaching of ace cell receptor might contribute by downregulating the ace /ang - /mas axis, and in a sex-oriented perspective, sry (y-chromosome) and sox (xchromosome) both by upregulating agt, and downregulating ace , at , and mas. conversely, sry upregulates, whilst sox downregulates, the ren promoter, thus being a potentially detrimental step in limiting the global rate of the ras system that is particularly frail in males [ , ] . finally, other genes directly or indirectly regulate the ras pathway (table ) . firstly, adam by promoting the detaching of ace cell receptor might contribute by downregulating the ace /ang - / mas axis, and in a sex-oriented perspective, sry (y-chromosome) and sox (x-chromosome) both by upregulating agt, and downregulating ace , at , and mas. conversely, sry upregulates, whilst sox downregulates, the ren promoter, thus being a potentially detrimental step in limiting the global rate of the ras system that is particularly frail in males [ , ] . the ace gene maps on chromosome q . , is . kb-long, and comprises exons (genbank, nt ). ncbi records (https://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?locusid= ) include hundreds of intragenic gene variants: most of them are single nucleotide polymorphisms (snps), a minor part of which are located in the coding region, and few of them are missense mutations. interestingly, the gene encodes two isoforms of ace with two different promoters and alternative splicing, and the structure of the ace gene may be the result of a duplication of an ancestral gene [ ] . among the several gene variants, the more investigated is an insertion/deletion (i/d) defined by ncbi [ ] as a sequence of -bp i/d in the alu-sequence of intron in the ace gene and is represented by four individual snps (rs , rs , rs and rs ). it is generally associated with the expression level of ace [ ] , with the d/d genotype having the highest serum/tissue ace levels, the i/d genotype showing intermediate levels, and the i/i genotype the lowest levels [ ] . interestingly, the online mendelian inheritance in man (omim), a catalogue of human genes and genetic disorders particularly focused on gene-phenotype relationships, reports that this gene may be associated with sars progression, assuming that the d-allele can be considered a genetic predisposition affecting the progression from pneumonia to sars, as reported in a study with vietnamese sars patients [ ] . other studies have confirmed these outstanding data [ ] [ ] [ ] , although current conclusions are still controversial [ , , ] . although about % of the ace levels seem to be determined by the ace i/d polymorphism, it is extremely interesting that there is a significant sex-difference in serum ace activity/level, which is lower among females in both healthy and pathological conditions [ , [ ] [ ] [ ] . this evidence, together with the observation that the ace i-allele seems to be overrepresented among females, and that the d-allele (associated with a high level of ace ) seems more prone to express even higher levels among males, suggests a higher chance to have ace /ace imbalance among males during ace receptor suppression, as in the presence of sars-cov- infection [ , ] . interestingly, a quantitative variation in ace levels has been demonstrated to be modulated also by the abo-blood group locus ( q . ). moreover, selected abo polymorphisms (rs , gene promoter, and rs , exon ) influence ace inhibitors treatment response [ ] [ ] [ ] , and might contribute in reducing sars-covs' transmission, in terms of number of infected individuals and epidemic rate reduction. this ascribed to o-blood group a lower risk of infection, hypothesizing that natural anti-a and anti-b antibodies can contribute in protecting against viral diseases at the population level [ ] . the ace gene maps on chromosome xp . , is . kb long, and contains or exons in two isoforms (genbank, nt ), which differ in the presence of an extra exon in the longer isoform at the -end [ ] . the ace and ace catalytic domains share % homology in their amino acid sequence and have a similar exon/intron organization, indicating a common ancestor. among the several gene variants [ ] , particular attention has been paid to the transition g a (rs ) in intron of the ace gene, previously investigated in association studies with hypertension, although some issues remain almost inconclusive [ , ] . unlike autosomal genes (i.e., ace ), x-linked genes (i.e., ace ) cannot show in males any advantageous heterozygous condition in case of mutations or polymorphic at-risk conditions. accordingly, in the presence of a lower activity of the ace gene, as for the one associated with the g-allele [ , ] , male-carriers are certainly hemizygotes unable to compensate with the a-allele counterpart. ace g a is located at the beginning of the intron , theoretically affecting gene expression with alternative splicing mechanisms [ , ] , also having a strong linkage disequilibrium with other snps (rs intron and rs intron ) in the ace gene [ , ] . an interesting recent paper investigated the ace i/d variant in combination with the ace g a transition in hypertensive population. the authors hypothesized that patients characterized by higher ace activity (i.e., d/d-genotype) in conjunction with reduced ace activity (i.e., gg-females or hemizygous g-males) could account for increased susceptibility to hypertension mainly in association with classical cardiovascular risk factors such as old age, dyslipidemia, and diabetes [ ] . noticeably, it emerges that an overly activated ras, exacerbated by genetic predispositions affecting the ace /ace balance and in combination with advanced age and classic acquired cardiovascular risk conditions, might lead to systemic disorders and/or severe local disturbances of the normal tissue homeostasis. overall, this condition can establish a complex multistep mechanism (figure ) leading to organ dysfunction such as in sars-covs where the ace /ace equilibrium is destroyed and incidentally worsened by the suppression of the ace receptor due to sars-cov- binding, and/or adam activity, and/or by the combination of specific ace /ace at risk haplotypes [ , , , , ] . our hypothesis should be demonstrated by dedicated clinical epidemiological studies in genetically selected patients, though it is to be taken into account that the above described ace and ace gene variants do not completely lessen the enzyme levels. rather, they are responsible for a medium-mild reduction with enzyme levels within the low-normal range of the general population. nonetheless, in critical conditions and co-morbidities (e.g., sars-cov- infection), selected genetic variants might favor ras-rebalancing. moreover, among covid- patients, those taking ras-inhibitors may take advantage of the treatment considered an adjuvant drug for ras rebalancing [ , ] . finally, the surface of sars-cov- interacts specifically with ace through its receptor binding domain (rbd) of the s-protein, which is critical to the success of the viral infection. the affinity between ace and the rbd of the sars-cov- is - times higher than that of previous sars-covs, which also explain its higher aggressive performance [ , ] . moreover, the ace peptidase domain (pd), normally deputed to ang-i to ang - cleavage, also makes available a direct binding site for sars-cov- s-proteins. in addition to the pd domain, a neck domain is also crucial for the ace dimerization process and stability. extensive polar interactions have been recognized in the neck domain of ace , and the complex network of polar interactions between amino acid residues ensures a stable and functional dimer assembly. several gene polymorphisms have been listed among those amino acid residues suggested to be crucial for dimer stability in the pd and neck domains of the ace gene [ , ] . theoretically, this might interfere with the ace -sars-cov- interaction, especially in females who have two different ace genes within the two different x-chromosomes, a condition that may give rise to a greater structural variability in females, who potentially assemble "hetero-dimers", particularly in the presence of a skewed x-chromosome inactivation (xci), in comparison to males who necessarily assemble "homo-dimers". obviously, xci is not the same in every female, being potentially advantageous or not in terms of virus cell entry. moreover, a difference in the open or closed conformation of the ace receptor together with the glycosylation rate of some amino acid residues in the pd domain could affect ace -sars-cov- interaction [ , ] , as demonstrated by chloroquine treatment of sars-cov patients, in which chloroquine inhibited virus infection by interfering with the terminal glycosylation of ace receptor [ ] . accordingly, we have previous experience of dimeric/tetrameric molecules in which selected snps seemed to have higher detrimental effects on the molecule structure and activation when combined heterozygous haplotypes were co-inherited in the same carrier rather than in homozygous polymorphic individuals [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sex-disparities have been often observed in response to communicable diseases [ , ] . in viral infections, sex differences in term of intensity, prevalence, severity, and mortality have been reported depending on the etiological agent, as reviewed in [ ] . the general tendency in females is, however, to show a greater humoral and cell-mediated immune response to infection [ ] [ ] [ ] . consequently, females clear infections more quickly and more effectively than males [ , , ] . sex hormones can be partly responsible for this phenomenon: by binding to specific receptors on immune cells, they can trigger their activity (estrogen) or suppress it (testosterone) [ , , ] . however, sex differences may also be due to an imbalanced expression of genes on the x-and y-chromosomes, since immune-related x-linked genes appear to be more activated in female immune cells. the sex chromosomes x and y, which differentiated from ordinary autosomes about million years ago, show great levels of dissimilarities, except in their pseudo-autosomal regions (par and par ) located at the end of both chromosomes [ ] . having two x-chromosomes, females have two different cell types in all their organs with one of the two x-chromosomes being inactivated (xi, while the activate form is xa), a condition that was first described by mary f. lyon in mice [ ] . the xci is due to epigenetic processes that randomly select and permanently silence one of the two x-chromosomes of a female individual. the process occurs at an early phase of embryonic development and is maintained during adulthood. xci is a strategy that aims to balance the x-linked transcriptional dosage between female xx and male xy cells. in general, % of female cells and all cells derived from them have the maternal x-chromosome inactivated, the other % have the paternal x-chromosome inactivated. the knowledge of the complex mechanism of xci and its regulation is summarized in [ ] . xci results in a condition of "cell mosaicism" in a female individual, where a balanced expression of both parental x-linked genes is expected [ ] . this condition may provide females with greater plasticity and adaptability in the response to infectious diseases than males. when it comes to gene expression, females may compensate adverse x-linked mutations by using cells that carry the wild allele on the other x-chromosome [ ] . however, it is clear that xci is incomplete in humans [ ] and that the balance may be disrupted both by the process of skewed xci and by genes escaping silencing [ ] . skewed x-chromosome inactivation appears when one x-chromosome is favored over the other for xci. this may result in - % of cells with the same parental x-chromosome silenced. this predominance of one xi also causes unbalance of the allelic dosage and of x-linked transcription. this loss of mosaicism may lead to a reduced plasticity of the immune system and a loss of immunological memory [ ] . there are several mechanisms underlying skewed xci which appear to be partly age-dependent, showing an increment in older women, at least in the blood, which is also associated with smoking [ ] . with regard to the mechanism of escaping silencing, it seems that about % of x-linked genes can escape inactivation, so that they are expressed by both x-chromosomes in female cells, whereas a further % vary among individuals for their silencing behavior. the level of expression is, however, always lower in the escaped gene [ ] . xci escape signatures vary between individuals, even during growth and aging, as well as between cells in a tissue [ ] . in humans, naïve mature b and t cells were demonstrated to lack xci on some genes (h k me , h a-ubiquitin, h k me and macroh a), which are again partially silenced (h k me and h a-ubiquitin) when lymphocytes are activated [ ] . other immune cells have shown evidence of different xci maintenance in females, as summarized in [ ] . essentially, the results of different studies now support the theory that sexual dimorphism of immune cell is triggered by xci escape mechanisms [ ] . in particular, the distal end of xp and par regions, which contain the highest number of immune-associated genes on the x-chromosome, have a higher chance of escaping xci [ ] . accordingly, in the ace gene maps of the xp-region and in a study carried out without distinguishing between male and female cells [ ] it was demonstrated that ace is abundantly present in human lung epithelia and small intestine enterocytes, while another study [ ] has shown that the expression of the ace receptor is higher on oral mucosa and tongue epithelial cells. a systematic survey of xci that integrated transcriptomes with genomic data [ ] identified ace as a tissue-specific escape gene that showed moderate male-biased expression in lungs, higher male-biased expression in the small intestine, and weak male-biased expression in epstein-barr virus (ebv)-transformed lymphocytes. this finding may be suggestive of lower ace expression in females due to the combination of the two x-linked genes compared to the expression arising from the x-linked and a y homologue in males [ ] . nevertheless, in another recent study [ ] , no sex-mediated differences in expression were found between the sexes. alternatively, the predominant male-biased expression of ace might be explained by increased ace activity in males partially driven by sex hormones [ ] , as it has recently been demonstrated in mice kidneys [ ] . finally, strong results have recently been found in favor of an exceptionally high basal level of ace in asian females than in other ethnic groups and an age-dependent decrease more significant in men than women [ ] . briefly, this study shows an apparent negative correlation between ace quantitative expression and infection susceptibility and severity at population level [ ] . nevertheless, the tissues they have tested are not those of the upper respiratory tract, with the exception of the lung (and blood vessels) which only show a moderately higher ace expression in east asian females. the authors' conclusion that asian women are more protected against covs than men is based on the hypothesis that ace repression induced by sars-cov might be counteracted by high basal levels of ace induced by higher levels of sex hormones (which decrease with age) and reduced by systemic inflammation [ ] . this fascinating theory, which would support the hypothesis of ace /ace unbalance on ards onset in covid- patients, needs to be confirmed in the future when more data will be available. sexual dimorphism in terms of risk, susceptibility, and prognosis has been reported for several inflammatory pathological conditions, including respiratory diseases in which hormonal profile, genetic, and epigenetic factors may play a role [ , , ] . in this scenario, it is relevant that the response to vaccination seems to be improved in females who therefore show a lower risk/vulnerability to several pathogens, while males are more exposed and at higher risk [ ] . furthermore, evidence suggests that males tend to experience the worst prognosis in acute inflammatory settings (e.g., sepsis) [ , ] , while there is a generalized inversion of prognosis between men and women in chronic inflammatory conditions (e.g., asthma) [ , ] . in relation to sars-cov , the accumulation of reports and epidemiological data indeed confirms that, in agreement with other respiratory inflammatory diseases and consistent with previous mers-cov and sars-cov infections, the new coronavirus preferentially affects males than females that show a better prognosis [ ] [ ] [ ] . the onset of inflammatory processes is a key pathological feature of sars-cov- infection. the massive release of inflammatory cytokines and chemokines in covid- patients was first reported by huang and colleagues, showing in particular higher levels of il , il , il , gscf, ip , mcp , mip a, and tnfα in the peripheral blood of intensive care unit (icu) patients compared to non-icu patients [ ] . the impact of the "inflammatory-wave" in covid- suggests that the cytokine storm might be strongly associated with the severity of the disease [ ] . the role of the cytokine storm in sars-cov- infection is attracting great attention as a crucial phase to investigate in order to clarify the disease's pathogenic process (e.g., to elucidate the role of th and th responses) and to identify new therapeutic targets (see the published results and the ongoing clinical trials with tocilizumab, a monoclonal antibody targeting the il- pathway) [ ] [ ] [ ] . sex hormones have been suggested as potential mediators of the reported sexual dimorphism by virtue of their ability to modulate innate and adaptive immunity, as previously reported [ ] and as described above. however, since sex dimorphism observed in several inflammatory diseases, including respiratory pathologies, has also been confirmed in studies dealing with pre-pubertal cohorts [ ] [ ] [ ] , there is growing consensus on the need to consider additional factors/variables that may occur. similarly, there is growing evidence suggesting that the x-chromosome and x-linked genes are the main determinants of the reported sex dimorphism in disease susceptibility and prognosis [ , ] . the x-chromosome carries about , genes [ ] including cytokines/cytokines receptors, toll-like receptor (tlr)-mediated signaling pathway genes, nf-kb and mapk signaling genes, genes involved in apoptosis, genes involved in redox balance, and other immune-modulators such as cd ligand and foxp , as recently reviewed [ ] . with regard to the role of the number of x-chromosomes on inflammation and release of inflammatory cytokines, in a recent work lefèvre and colleagues addressed how the number of x-chromosomes can affect the secretion of inflammatory cytokines after the activation of the tlr signaling pathways [ ] . the authors demonstrated that cytokine production in response to different tlr ligands was improved in males, showing higher inflammatory response than in females and subjects with klinefelter syndrome who carry two x-chromosomes, but are characterized by a hormonal profile more similar to that of males [ ] . the presence of two x-chromosomes carrying genetic variants of x-linked inflammatory genes/receptors undergoing random inactivation may therefore represent an advantage in the acute phase of inflammatory diseases [ , ] . finally, x-linked genes coding for inflammatory mediators/receptors may also escape xci leading to bi-allelic expression [ ] , with strong implications in sex-related differences of inflammatory responses [ ] . interestingly, the x-linked ace gene, in transgender males treated with estrogen therapy and androgen antagonists shows significantly higher expression and increased number of cells expressing ace among testicle cells [ ] . overall, if inflammation is needed to control the invasion/elimination of pathogens, the onset of exacerbated inflammatory responses may be harmful and lead to tissues/organs damage. in this scenario, females result as better "armed" thanks to the presence of a cellular mosaicism that may be more than useful to modulate/balance inflammation [ ] . the lessons learned from different respiratory diseases of viral and non-viral origin, and preclinical research suggest that the number of active x-chromosomes can make a difference in shaping the type, dimension, and lasting of inflammatory responses that, in synergy with sex hormones, may account for the low risk and better prognosis of sars-cov- infection in females, though a multidisciplinary approach for such complex condition is mandatory. according to international disaggregated data, men are faring worse than women in covid- pandemic [ ] . on the other hand, the long-term coronavirus fallout may be worse for women than for men due to social, psychosocial, and occupational reasons as well as environmental factors and differences in lifestyle. biological and behavioral causes directly or indirectly play a role in modifying women's risk such as different comorbidity rates (e.g., hypertension and cardiovascular diseases), smoking and drinking habits, personal hygiene, the number of females among nurses and caregivers both in hospitals and within families, and of course pregnancy and motherhood, just to cite the most relevant. within the several habit-related factors, smoking deserves particular consideration. smoking is a risk for many respiratory diseases and undoubtedly also for covid- [ , ] . the european respiratory society [ ] reported results of different studies concluding that smokers are times more likely to die from covid- [ , ] , but only a few studies definitely advise to stop smoking [ ] and, to our knowledge, no country has taken strong prevention measures. as regards the individual susceptibility of smokers toward respiratory diseases, such as influenza, chronic obstructive pulmonary disease (copd), tuberculosis (tb), or other lung pathological conditions, there are no doubts about a direct association [ ] [ ] [ ] [ ] . accordingly, the european centre for disease prevention and control (ecdc) suggested that smoking may contribute to increasing the number of severe covid- cases [ ] . conversely, it has been speculated that smokers seem less likely to develop covid- , by the hypothesis that smoke exposure might modulate the immune response and the levels of inflammation markers. by attenuating the physiological defense of the immune and inflammatory system, smoking might paradoxically mount a less aggressive cytokine storm [ ] . accordingly, a "nicotinic hypothesis for covid- " has been speculated, suggesting the use of nicotine-patches on coronavirus patients [ ] . overall, no evidence exists that smoking protects covid- patients from developing severe symptoms and recent metanalyses indeed list smoking habits as risk factors [ ] [ ] [ ] . of note, it has been reported that the ace receptor is upregulated in the lungs of smokers or copd patients, including small airway epithelium, brush borders, type-ii alveolar pneumocytes and alveolar macrophages [ , ] . the expression was more evident in patients with copd compared to never-smokers, suggesting that smoking upregulates ace expression and copd further exacerbated it, hypothesizing in them an amplified susceptibility for covid- . environmental and health issues may result associated with covid- and some of them deserve to be mentioned [ ] . first of all, air pollution and poor air quality has been suggested to lead to an increased covid- infection rate probability due to a direct effect of air pollution on humans [ ] . moreover, other studies on flu-like viruses highlighted the potential role of pollen in the atmosphere, that by increasing general immune responses might affect viral spread, speculating that it might also decrease during warm seasons [ ] . hospitals are crucial in covid- pandemic containment and controlling, and for dedicated disinfection techniques for infrastructures personnel and medical equipment are key issues [ ] to avoid front-line healthcare worker infection risk and increased psychological burden, particularly for medical doctors and nurses among which women account for the largest number worldwide [ ] . overall, protective measures for people at such a high risk as hospital workers is of primary importance. efficient personal and protective equipment reduces mortality, and we need to plan strategies to ensure protection also against infections rising from environmental sources. therefore, any useful tool/instrument/product/action should carry a global benefit. finally, considering individual genetic susceptibility or environmental influences on virus infection and/or sars progression, a wide and heterogeneous geographical distribution of absolute numbers and percentages of infected cases is now emerging. different clinical phenotypes and symptoms characterize not only different populations and countries, but also close geographic regions in which opposite situations may coexist. as a paradigmatic example, according to data from "dipartimento protezione civile covid- obviously, other variables also need to be analyzed, first the local containment actions, the number of tests performed and then the geographic locations. in this regard, piacenza is very close to the most critical hot-spot of the infection in italy [region lombardia ( . %); lodi ( . %), codogno area (red zone)] and other additional parameters such as population density may account for local differences in terms of viral spreading (ferrara: habitants/km vs. piacenza habitants/km ). genetic selection pressure by previous epidemics, such as the long-lasting malaria epidemics in the po river area where the city of ferrara is located, cannot be excluded. this is in line with some publications reporting that endemic malaria seems to protect from covid- outbreak and that genetic variations associated with malaria (e.g., ace and abo genes) may play protective roles [ ] [ ] [ ] . in summary, individual susceptibility, mainly driven by gene-environment interactions, has a great role in determining immunity, survival, and treatment responses in different populations. the covid- pandemic by coronavirus sars-cov- , is a severe, complex, and multifactorial disease in which human genetics, due to inherited predispositions, could play a role together with pre-existing comorbidities and acquired risk conditions. unmodifiable factors, such as age and sex, together with modifiable classical cardiovascular risk factors and comorbidities play crucial roles in tuning the fate of sars-cov- infection, worsening prognosis, and mortality rate. in turn, the mechanism of sars-cov- infection, due to ace receptor entry, might be altered by individual genetic or developed susceptibility. an overly activated ras-system, mainly due to local ace /ace unbalancing, might be crucial in determining specific vulnerability/receptivity and in giving indicators of the local balanced inflammation, blood coagulation, and fibrinolysis. these evidences support the use of appropriate anticoagulant and anti-inflammatory therapies properly monitored by specific laboratory markers (as fibrin d-dimer) or with a more refined approach by the dynamic assessing of residual fxiiia levels, as recently suggested during any acute thrombotic event [ , ] . the unexpected reported higher expression of ace in females, the inverse age-dependent ace expression significantly reduced by the presence of diabetes, and the strong repression of ace by inflammatory cytokines, confirm an inverse correlation between ace levels and sars-cov- prognosis. summarizing, this is in contrast with the assumption that "high ace is a culprit in covid- outcome, and on the contrary supports a protective role of high ace expression against sars-cov fatality" [ ] also strengthened by the ace intrinsic anticoagulant properties. interestingly, sars-cov- mediated repression of ace is counteracted by sex hormone inducible mechanisms (though unfortunately sex-hormones decrease with age) and exacerbated by systemic inflammation (which unfortunately increases with age and chronic diseases) [ ] . part of the present findings were already known from previous sars epidemics, but the pandemic proportions of covid- have led researchers and clinicians to fight together more than during previous sars-covs infections. accordingly, some aspects such as the sex gap in favor of the females, particularly for prognosis and survival, have been greatly deepened and investigated through epidemiological studies. different sex and age groups have strongly different susceptibilities to infection and mortality, considering male-sex, old age, and 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renatico, ferrara, italy). the authors declare no conflicts of interest. key: cord- -bqpj ey authors: czick, maureen; shapter, christine; shapter, robert title: covid’s razor: ras imbalance, the common denominator across disparate, unexpected aspects of covid- date: - - journal: diabetes metab syndr obes doi: . /dmso.s sha: doc_id: cord_uid: bqpj ey a modern iteration of occam’s razor posits that “the simplest explanation is usually correct.” coronavirus disease involves widespread organ damage and uneven mortality demographics, deemed unexpected from what was originally thought to be “a straightforward respiratory virus.” the simplest explanation is that both the expected and unexpected aspects of covid- share a common mechanism. silent hypoxia, atypical acute respiratory distress syndrome (ards), stroke, olfactory loss, myocarditis, and increased mortality rates in the elderly, in men, in african-americans, and in patients with obesity, diabetes, and cancer—all bear the fingerprints of the renin-angiotensin system (ras) imbalance, suggesting that ras is the common culprit. this article examines what ras is and how it works, then from that baseline, the article presents the evidence suggesting ras involvement in the disparate manifestations of covid- . understanding the deeper workings of ras helps one make sense of severe covid- . in addition, recognizing the role of ras imbalance suggests potential routes to mitigate covid- severity. medieval english theologian william of occam is widely credited with the eponymous principle known as occam's razor: "entities should not be multiplied without necessity." or, in modern terms, "the simplest explanation is usually correct." in late winter/early spring of , as novel coronavirus case numbers exploded in europe and parts of the united states, prominent newspapers and online newsmedia outlets reported with alarm a broad array of coronavirus disease (covid- ) demographic and clinical outcomes deemed to be surprising and unexpected, even by some of the physicians interviewed. as one washington post headline exclaimed: "once thought a relatively straightforward respiratory virus, covid- is proving to be much more frightening." outcomes that initially jolted both the press and the medical community include cases of severe covid- in young, apparently healthy adults, mortality rates among african-americans and latinos doubled compared to caucasians, , similarly doubled mortality among men compared to women, and a tripled death rate among certain cancer patients. in the uk, government epidemiologic data suggested that blacks there are four times more likely to die of covid- compared to whites, with uk residents of indian/pakistani/bangladeshi ancestry also showing increased mortality rates. journalists warned as covid- patients developed renal failure requiring dialysis, and obesity was linked to higher mortality. subsequent reports revealed occurrences of fatal covid- myocarditis, strokes in young patients, , , "silent hypoxia," a mortality rate among ventilated covid- patients approaching %, and a kawasaki-like syndrome in children. , in the early days of the pandemic, the clear association of pre-existing hypertension with more severe covid- outcomes triggered consternation among physicians, as did cases of the virus presenting with seemingly incongruent hyperglycemia, and loss of taste and smell sensation. the widespread organ involvement and demographic perplexities do at first seem to be a bewildering pathophysiologic maelstrom. but occam's razor posits that the most likely explanation is that a common underlying mechanism unites them all. when viewed through the lens of the renin-angiotensin system (ras), the covid- disarray slips into crystal clear focus. in addition to its better-known circulatory volume/pressure regulatory role, ras is also a cellular-level system, present in the tissues of practically every organ of the body, and mediating a broad array of inflammatory, metabolic, osmotic, growth, and repair functions. the hallmarks of ras imbalance, either pre-existing or covid- -mediated, are a common thread interwoven through wide-ranging covid- symptomatology and disparate sub-population outcomes. the commonality of ras dysregulation does not prove that ras is the sole mediator of covid- pathophysiology; there may be multiple factors that synergize to worsen covid- severity (table ). yet the underlying presence of ras imbalance in so many aspects of covid- does suggest that ras may be the key to understanding and potentially mitigating covid- . only after taking a deeper look at what ras is and how it works, does severe covid- itself begin to make sense. ras was first conceptualized as an exclusively endocrine effector system, activated in response to decreased arterial blood pressure, increased sympathetic nervous system tone, or decreased filtered solute delivery within the kidney tubules as, for instance, during hemorrhage or dehydration. [ ] [ ] [ ] [ ] ras was depicted as a linear sequence of enzymatic activations, with substrates circulating in the bloodstream to reach both their enzymatic activators and their target receptors. renin, released into circulation by renal juxtaglomerular (jg) cells, cleaves hepatically produced angiotensinogen to generate angiotensin i (ai), an intermediate and inactive compound, which circulates to encounter membrane-bound angiotensin converting enzyme (ace) on vascular endothelial cells, primarily within the pulmonary circulation. ace converts ai to angiotensin ii (aii), which transits to the adrenal cortex, triggering the release of aldosterone. aldosterone subsequently stimulates increasing renal reabsorption of sodium and water, thereby augmenting blood volume. this sequence of ras activations has been dubbed the renin/ace/angiotensin/at axis or, more succinctly, the ace axis. aii also binds to angiotensin type- receptors (at ) on vascular smooth muscle cells to mediate vasoconstriction and increase blood pressure. at dispatches a g-protein to activate phospholipase c, which splits the membrane lipid phosphatidyl inositol bisphosphate (pip ) into two products, inositol tri-phosphate (itp) and diacylglycerol (dag), both of which increase intracellular calcium levels. calcium-bound calmodulin next activates myosin light-chain kinase (mlck), which phosphorylates smooth muscle myosin, enabling actin binding, smooth muscle contraction, and vasoconstriction (figure ). circulating pressure/volume regulation within the vascular system is not the only function of ras. after much debate, , there is now widespread agreement that local tissue-level ras systems administer paracrine and autocrine effects in various structures including adipose tissue, the pancreas, vascular walls, and the heart, kidneys, and brain. , evidence suggests that locally produced ras components affect physiology independently of blood pressure. although this premise remains controversial, the original purpose of some ras components may not have involved blood pressure regulation and might even pre-date the evolution of blood vessels; for example, the modern invertebrate fruit fly, which has an open circulatory system and therefore lacks discrete blood vessels, has an ace-like enzyme, and a renin-like enzyme is found in the leech. invertebrates demonstrate the value of a synergistic proinflammatory and procoagulant injury response system. they possess a sole cell-type having both platelet-like and macrophage-like functions which speeds injury response. in open circulatory systems, blood directly contacts cells, so pathogens entering via a breach in the exoskeleton would rapidly begin cellular invasion. in the absence of blood vessels, there is no mechanism to restrict blood flow to the injury site, and this would be fatal without prompt coagulation. with an open circulatory system, there is no time to lose. the evolution of vertebrate closed-circulatory systems interposed the vascular wall as a barrier to shield cells from blood-borne invaders. coagulation and inflammation evolved along separate, specialized cell lines, but the advantages of integration between coagulation and inflammation during injury response were preserved through crosstalk and mutual co-activation. [ ] [ ] [ ] [ ] [ ] vasoconstriction to limit blood flow to injured areas is intertwined with inflammation and coagulation to establish a fully integrated injuryresponse mechanism. the vascular endothelium became the pivotal regulatory interface, with the ace axis mediating all three processes. ras is a critical survival mechanism, yet when dysregulated, it poses a significant danger. under normal conditions, the healthy vascular endothelium promotes vasodilation and inhibits coagulation and inflammation. , however, when injury occurs, aii, together with proinflammatory cytokines from innate immune cells, transforms endothelium into an "activated" state that promotes vasoconstriction, inflammation, and coagulation. , , endothelial activation is intended to be temporary; failure to recover a vasodilatory, anti-inflammatory, and anticoagulant baseline is termed endothelial dysfunction. it results from excessive generation of reactive oxygen species (ros) by overactive aii, with consequent loss of nitric oxide (no) production capacity. (figure ) dysfunctional endothelium, due to ras dysregulation, promotes inflammatory and thrombotic damage in disease states such as diabetes, hypertension, and atherosclerosis. [ ] [ ] [ ] the ace axis has direct growth-promoting effects on cells and cell matrix for post-injury repair. at activation of mitogen-activated protein kinase (map kinase) upregulates growth factors such as vascular endothelial growth factor (vegf), and inflammatory mediators including tumor necrosis factor (tnf) alpha, interleukin- (il ) beta, and nuclear factor kappa b (nf-κb). this upregulated signaling contributes not only to repair but also to inflammation, cancer, and second messenger pathways of the ace axis and bradykinin in vascular wall. to mediate vasoconstriction and increase blood pressure, aii binds to at receptors on vascular smooth muscle cells. at activates plc, which splits the membrane lipid pip into two products, itp and dag, resulting in sr calcium release and increased intracellular calcium levels. calcium binds to calmodulin; together they activate mlck, which phosphorylates smooth muscle myosin. phosphorylated myosin then binds actin, triggering smooth muscle contraction, and vasoconstriction. bk initiates a pathway opposing the vasoconstrictive action of aii. bk binding to the b receptor activates nos to produce no. no diffuses from its site of production in vascular endothelial cells, to neighboring vascular smooth muscle, where it activates a vasodilatory second messenger cascade, culminating in activation of mlcp, which strips phosphate groups from smooth muscle myosin, such that it can no longer bind to actin. nervous system function. acting again via map kinase, at upregulates cytokines, such as transforming growth factor (tgf) beta, that drive fibrotic collagen deposition by myofibroblasts. , fibrosis and hypertrophy can at first be reversible repair responses, but when dysregulated they progressively damage vascular smooth muscle, myocardium, and kidneys. , in addition, some cellular damage may be too extensive for recovery, in which case the ace axis contributes to activating cell death pathways. , , ros derived from at activation can inhibit anti-apoptotic bcl- and activate pro-apoptotic bax pathways, triggering programmed cell death. aii can also impair mitochondrial function and increase protein misfolding, causing pathological protein aggregation. and aii enhances autophagic catabolism of damaged organelles and protein aggregates, necessary for homeostasis, but capable of inciting cell death when dysregulated. it is evident that a system as powerful and far-reaching as ras must be tightly regulated, to prevent potentially lethal misapplication of vasoconstriction, inflammation, and coagulation. the at receptor is generally described as a protective arm of ras, opposing the actions of the at receptor. , the functional roles of at have been challenging to tease out, in part because of low adult at expression levels compared to at , whose effects may overwhelm the more subtle alterations prompted by at . at has extensive expression during fetal development, but comparatively low-level expression in adults in select sites: heart, kidney, adrenal, brain, uterus, pancreas, retina, endothelium, and vascular smooth muscle. aii also binds to at , and in doing so, it counteracts at effects, promoting instead natriuresis, which can slightly lower blood pressure, as well as stimulating antiinflammatory, antithrombotic, and antifibrotic effects. at activates superoxide dismutase (sod), which neutralizes ros and defends against oxidative stress created by at . at knockout mice have increased vascular hypertrophy, indicating that at has protective anti-hypertrophy functions, congruent with at upregulation in the heart during disease states including heart failure, cardiac ischemia, and dilated cardiomyopathy. some of the benefits of angiotensin receptor blockers (arbs), such as reducing vascular and cardiac hypertrophy, appear to be at mediated, by shunting aii away from at . the principal at second messenger pathway involves production of nitric oxide (no), cyclic gmp (cgmp), and bradykinin, all of which promote vasodilation and oppose inflammation. endothelial no upregulates at which then decreases ace activity. cgmp also deactivates rhoa, an at second messenger, thereby antagonizing at -mediated vasoconstriction. ace has a counterbalancing "younger brother" enzyme known as ace . , ace converts aii into angiotensin - (a - ), so named because it possesses only seven of aii's original eight amino acids. a - binds to mas receptors, yielding vasodilatory, anti-inflammatory, and antifibrotic effects. , , in sum, ace depletes aii levels by converting it to a - , which binds to mas receptors, thereby counteracting at actions. [ ] [ ] [ ] through a side-pathway, ace can also convert ai into angiotensin - (a - ), which possesses nine of ai's original ten amino acids. interestingly, ace then converts a - into a - ; therefore, ace plays a role in producing aii, but also in decreasing aii levels by shunting substrates toward a - instead of aii. rather than being a linear cascade of enzymatic reactions, ras is now understood as a web of counterbalancing activations and deactivations , (figure ). the original sars coronavirus (sars-cov or sars), which caused severe acute respiratory syndrome, (also called sars) hitchhiked inside cells using cell-surface ace . the new, closely related coronavirus sars-cov- or sars (which is responsible for covid- ) uses the same method. once inside the host cell, original sars virus dramatically reduces cellular expression of ace . sars virus evidently functions the same way. because ace is critical for regulating the balance between the effects of aii and a - , reduced ace expression leaves the "big brother" enzyme ace, unchecked, enabling unopposed oxidative, inflammatory and procoagulant effects ( figure ). in animal models and human cases of acute respiratory distress syndrome (ards), ace levels are severely decreased, and ace/ace balance is lost. overactive aii/at contributes to severe oxidative and inflammatory lung injury, which in mouse models is blunted by ace . ace inhibitors (aceis) and arbs lessen pulmonary damage in mice injected with the original sars surface spike-protein, , suggesting that unbalanced ace axis activity, after ace is downregulated, is the root of original sars lung pathophysiology. close assessment of covid- pathophysiology suggests the same mechanism. as the following sections show, fingerprints of ras imbalance with ace/aii overactivation and diminished ace /a - are spread across the array of unexpected covid- organ system involvement. subpopulations manifesting higher rates of covid- mortality-including hypertensives, the elderly, the obese, diabetics, men, and african-americans-correlate with preexisting ras imbalance, with ace overactivity and/or ace underactivity priming these patients for more severe covid- outcomes. many hospitalized covid- patients reportedly display silent hypoxia: severe arterial hypoxemia without evident dyspnea, air hunger, or breathlessness. the neural reflex pathways mediating dyspnea have not been fully delineated, but likely involve integration among lung mechanosensory receptors, central and peripheral chemoreceptors, and emotion-regulation centers of the limbic system. , the sensation of dyspnea has been reported to require intact chemosensory function. the carotid bodies (neural crest-derived structures located at the carotid bifurcations), are the peripheral chemoreceptors, regulating the ventilatory response to hypoxia. carotid body glomus cells scan their extensive blood supply for adequacy of oxygenation: , if arterial partial pressure (pao ) falls below approximately mmhg, potassium background leak channels close, halting k + exit and depolarizing the glomus cells. next, calcium influx through voltage-gated channels triggers the release of glomus cell neurotransmitters which initiate firing of action potentials by adjacent afferent terminals of the carotid sinus branch of cranial nerve ix. , the action potentials travel first to the nucleus tractus solitarius (nts), and then to the prebotzinger complex in the medulla, augmenting respiratory drive as an attempt to correct the oxygen shortfall, , ( figure ) and simultaneously invoking the reflex pathways that mediate air hunger. the oxygen sensor which initiates glomus depolarization is still debated, but it may be ros-producing nadph oxidase, which also happens to participate in at receptor signaling. whether or not at and nadph oxidase are involved in hypoxia sensing, ras is definitively activated during the hypoxic response. the carotid body is not only a driver of increased ventilatory effort; it is also a nexus for homeostasis regulation. oxygen perturbations signify severe systemic stress with profound metabolic implications, requiring anti-stress responses including renal compensation to maintain ph, adjustment of cardiovascular functions, and autonomic and endocrine effectors, all of which ras integrates into the ventilatory response. to mediate this integration, components of ras are expressed constitutively in glomus cells, including ace, ace , at , mas, at . , hypoxia increases expression of ace, angiotensinogen, at and at . hypoxia also increases sympathetic tone, which triggers renin release and elevates plasma aii levels. in turn, plasma aii has a stimulating effect on carotid body glomus cells, augmenting intracellular influx of calcium ions, and neurotransmitter release. blood pressure and ventilatory responses crosstalk and reinforce each other. sars coronavirus would be able to enter glomus cells utilizing membrane-bound ace . the subsequent ace downregulation, amid hypoxemic pulmonary infection, would result in significant ace imbalance, leading to carotid body hypoxic-signaling failure via two potential figure the ras web. the ras system is now more appropriately conceptualized as a web of activations and counterbalancing deactivations, rather than its original depiction as a linear cascade. figure routes. first, aii overactivation could drive infected glomus cells into programmed cell death. this would be consistent with experimental aii/at apoptosis induction in rat and human alveolar epithelial cells. [ ] [ ] [ ] alternatively, glomus failure could be akin to cellular dysfunction in heart failure, in which excessive aii triggers cell volume overload and impairment of cardiac electrical function. although aii increases cell volume, a - decreases it. thus, with loss of both ace and a - , aii overactivity would be expected to volume overload glomus cells, impairing depolarization, and blocking neurotransmitter release. either scenario, or both acting synergistically, would result in failure of hypoxic signaling, with an absent dyspnea response despite severe hypoxemia in covid- patients. incidentally, those same two scenarios could also be at work in loss of olfaction and taste, both of these neural in contrast, ace expression is more restricted in the lungs, largely to type ii pneumocytes, although there is evidence of some expression in pulmonary capillary endothelium. ace deactivates aii, by converting it to a - , thereby blunting the pro-inflammatory, pro-fibrotic effects of the ace axis. in covid- , novel coronavirus sars-cov- can enter type ii pneumocytes utilizing ace and then downregulate ace expression, leaving the lungs vulnerable to damaging, unbalanced proinflammatory effects of ace/aii. type ii pneumocytes have multiple protective functions, including producing surfactant, and serving as stem cells to replenish damaged type i pneumocytes (which function in gas exchange). viral impairment of type ii pneumocytes, coupled with aii inflammatory damage to type i pneumocytes, would thus severely damage multiple aspects of alveolar function in covid- , including oxygen exchange and capacity for repair. figure structures requiring depolarization and neurotransmitter release to mediate their sensory functions. both express tissue ras components. , "atypical" ards: ras "mismatched" but why are covid- patients so severely hypoxemic in the first place? critical care anesthesiologists in the thick of the covid- crisis in italy suggested that severe covid- patients may not have classical ards. unlike the situation in ards, lung compliance is preserved in covid- . instead, covid- patients seemingly displayed catastrophic loss of hypoxic pulmonary vasoconstriction (hpv), with resulting ventilation-perfusion (v/q) mismatch hypoxemia. in ards, lung compliance is poor because of decreased alveolar elasticity from noncardiogenic pulmonary edema. , this decreased alveolar elasticity impairs gas entry into alveoli, lowering the alveolar partial pressure (pao ), the top end of the gradient which drives the transfer of oxygen into pulmonary blood. oxygenation in ards patients often improves with ventilation using low tidal volumes and high positive end-expiratory pressure (peep) which increases pao . , but severe covid- patients have not shown a similar benefit with this ventilation strategy. even under normal physiologic conditions, pao is not equal in all alveoli throughout the lungs. because of postural differences in fluid hydrostatic pressures across the lung fields, alveolar expansion is not uniform and some alveoli inevitably have higher pao than others. hpv is a protective physiologic mechanism that optimizes arterial oxygenation by regionally vasoconstricting within the pulmonary vascular bed, thereby diverting pulmonary blood flow away from alveoli with lower pao , and vasodilating in other regions to increase blood flow to alveoli with higher pao . ( figure ) optimal v/q figure cellular mechanism of glomus cell hypoxic signaling. glomus cells, located within the carotid body, are neural crest-derived, and they exhibit voltage-dependent neurotransmitter release upon detection of hypoxemia. , glomus cells detect hypoxemia by an as-yet undefined sensor, which is thought to close k+ channels, halting k+ exit and thereby depolarizing the glomus cell. , , , depolarization leads to opening of voltage-gated ca + channels and neurotransmitter release, which triggers action potential firing in nearby cranial nerve ix. matching yields a pao of approximately mm hg, when breathing air at atmospheric pressure. however, if the hpv response is impaired, blood flows through the lungs in an unregulated fashion, and poorly oxygenated blood with low pao , enters the systemic circulation ( figure ). ras plays a role in hpv that is still not precisely defined but may involve hypoxic activation of nadph oxidase, generating ros that close potassium channels, depolarizing pulmonary vascular smooth muscle, which in turn opens voltage-gated l-type calcium channels, activating the smooth muscle contractile apparatus. , mice lacking cytosolic nadph oxidase had a % decrease in initial phase hpv. aii has been shown to activate pulmonary vasoconstriction in hypoxic humans, and aceis and arbs have been shown to inhibit hpv. , recombinant ace blunts pulmonary vasoconstriction in hypoxic pigs. therefore, the loss of ace due to sars coronavirus together with the resultant ace imbalance would be expected to dysregulate hpv and lead to hypoxemia. mortality rates in ards hover around %. by contrast, a covid- case series of patients in and around new york city reported mortality rates of . % in ventilated patients, essentially doubling the ards rate, and providing further support that covid- is not traditional ards. lung tissue locally expresses almost all components of ras. ace is heavily expressed across the entire alveolar capillary network, and the lungs are the dominant site of circulating aii production. in contrast, ace's antiinflammatory counterpart, ace is more restricted within the lungs, principally to alveolar type ii pneumocytes ( figure ). in addition, in healthy adults, pulmonary expression of anti-inflammatory at is low. with such broad expression of the ace axis, and comparatively restricted expression of the protective arms of ras, the lung is poised for more severe ace imbalance in the event of ace downregulation by the sars virus. without ace , ace actions would be essentially unchecked, with no means to achieve appropriate v/q matching, amidst significant inflammatory destruction of lung tissue. in animal models, mechanical ventilation has been shown to upregulate ace, promoting activation of aii. [ ] [ ] [ ] it may be the case for many severe covid- patients that initiation of mechanical ventilation, and consequent further ace axis upregulation, becomes the final straw, especially when combined with anesthetic/sedating drugs that may further impair hpv. emerging data show a mixed picture of covid- effects on the heart and kidneys. preexisting cardiovascular risk factors are definitively associated with increased covid- severity. in an italian retrospective case series, % of patients admitted to the icu for covid- had preexisting hypertension, and % had cardiovascular disease. among a retrospective cohort of covid- cases hospitalized in wuhan, china, % had hypertension, and % had coronary heart disease. hypertension and atherosclerosis have well-established associations with ace axis dysregulation. [ ] [ ] [ ] [ ] [ ] moreover, ace levels in the heart are reported to be diminished in hypertension and in diabetes-associated cardiovascular disease. thus, it makes sense that these comorbidities would be heavily represented among more severe covid- presentations. studies suggest that myocardial injury worsens covid- prognosis; nevertheless, reported rates of cardiac as well as renal injury are lower than rates of pulmonary damage, suggesting that the heart and kidneys may possess some protection. for example, the wuhan cohort cited that among fatal cases, % had an ards diagnosis, but only % had a myocardial infarction (mi), and % had acute kidney injury (aki) prior to death. among survivors, % had ards while only % had mi or aki. another study reported myocardial injury in % to % of hospitalized covid- patients, and % to % of icu cases. in contrast, the italian icu cohort reported % requiring intubation and mechanical ventilation, indicative of dire pulmonary compromise. kidneys locally express all ace axis components. , additionally, the renal glomeruli filter most circulating ras proteins, which are then reabsorbed by the proximal tubule. combined resorption and local production result in renal aii levels to times higher than in plasma. , dysregulated tissue ras, as occurs in hypertension, results in significant renal inflammatory damage, with progressive destruction of nephrons amid rasmediated remodeling and fibrosis. similarly, the heart has dovepress significant local ras, which mediates inflammatory remodeling in conditions such as hypertension and infarction. however, the counterbalancing anti-inflammatory at and ace arms of ras also have an important role in the heart and kidneys. ace is highly expressed in the heart, in particular in the coronary arteries, and in endothelial cells within the kidneys, achieving significant production of a - . therefore, with higher baseline ace levels, the heart and kidney may have a degree of shielding against the effects of ace loss perpetrated by the sars virus. in addition, cardiac and renal tissue ras often bypass ace, instead using chymase, trypsin, kallikrein, and cathepsins. thus, local ras function in these organs could potentially be less imbalanced by the loss of ace due to the sars virus. additionally, the coronary arteries and kidneys express comparatively higher levels of at during adult life. the heart also has a demonstrated capacity to upregulate at during pathologic states such as ischemia. therefore, the effects of covid- -induced imbalance of ras may be blunted somewhat by aii binding to at . there are also reports of covid- -triggered myocarditis. in one study, myocarditis was deemed likely causative in % of covid- deaths. at has been shown to promote myocarditis in experimental models, secondary to actions on t-cell function. t-cells have an intrinsic ras that regulates their proliferation and migration. at fosters cytoskeletal changes that promote t-cell migration and trigger t-cell production of cytokines and chemokines, thereby increasing t-cell recruitment to regions of active inflammation. by inhibiting at , arbs decrease experimental myocarditis. ras-driven t-cell-mediated effects in the heart, coupled with t-cell-mediated skin effects, which are also well described, , could potentially explain the pediatric kawasaki-like condition linked to sars . besides ischemic and inflammatory tissue destruction, another aspect of cardiac pathology in covid- is the development of arrhythmias and sudden cardiac death. , in addition to the arrhythmia-promoting effects of hypoxemia, arrhythmias in covid- patients bear hallmarks of ras dysregulation. cardiac remodeling by the ace axis is a known driver of arrhythmia events. in the atria, aii promotes fibrosis that facilitates reentry, and aceis and arbs do provide some protection against the recurrence of atrial fibrillation after cardioversion in figure hypoxic pulmonary vasoconstriction (hpv) and ventilation/perfusion (vq) matching. alveolar partial pressure of oxygen (pao ) is the top end of an oxygen gradient, driving transfer of oxygen from the alveolar air spaces into the pulmonary blood. thick red arrows designate a sizable gradient driving oxygen transfer. thin blue arrows designate smaller oxygen gradients, with less potent driving force for oxygen transfer between alveolar air spaces and pulmonary blood. functional hpv regionally vasoconstricts (designated in the figure by small blood vessel tube) within the lungs, to divert pulmonary blood flow away from poorly oxygenated alveoli, which would have a more limited ability to transfer oxygen into the pulmonary blood. such appropriate vq matching ensures that adequately oxygenated blood, with normal arterial partial pressure of oxygen (pao ), is returned to the systemic circulation. when hpv fails, pulmonary flow is instead sent in excess to poorly oxygenated alveoli, such that larger volumes of poorly oxygenated blood, with low pao , are returned to the heart, to then enter the systemic circulation. figure patients with left ventricular dysfunction. aii promotes ventricular arrhythmias by prolonging qrs duration, and by promoting reentry through both fibrosis and slowed ventricular conductance. these factors are likely playing out in covid- pathology, perhaps synergizing with potential arrhythmia-promoting effects of experimental drug treatments, including hydroxychloroquine, which was studied earlier in the pandemic but the use of which has now been curtailed. during the initial united states covid- case escalation, stroke was reported in patients without traditional stroke risk factors. , although unanticipated in the context of a viral respiratory tract infection, stroke risk is known to be elevated in conditions of ras overactivation such as diabetes and hypertension. , ras blockade with aceis and arbs has been shown to reduce stroke rates in high-risk populations. with the downregulation of ace , the coagulation-promoting effects of the ace axis would drive arterial thrombosis. healthy vascular endothelium promotes vasodilation and inhibits coagulation and inflammation, , through expression of natural anticoagulants, including tissue factor pathway inhibitor (tfpi), thrombomodulin (tm), tissue plasminogen activator (tpa), and the anti-inflammatory vasodilator no. produced in endothelial cells by nitric oxide synthase (nos), no diffuses to neighboring vascular smooth muscle to activate guanylate cyclase/cyclic gmp/protein kinase g-mediated vasodilation (figure ). no also has myriad anti-inflammatory and anti-coagulant functions, including inhibiting both platelet aggregation and monocyte adhesion. during injury response, aii inhibits endotheliummediated vasodilation and anti-coagulation. (figure ). at also promotes increased expression of adhesion molecules such as vcam- and e-selectin that recruit inflammatory cells to the injury site, and inflammation synergistically promotes coagulation. , aii prompts endothelial cells to promote coagulation, by new expression of procoagulants-tissue factor (tf) to initiate the blood clotting cascade, , von willebrand factor (vwf) to enhance platelet adhesion, and plasminogen activator inhibitor (pai)- to blunt fibrinolytic degradation of blood clots. , , , activation of these processes would then be the drivers of stroke in covid- -induced ras imbalance, with overactive aii sabotaging the anti-coagulant actions of otherwise healthy endothelium. obesity and diabetes are reported to worsen covid- severity and mortality. in the case series of hospitalized covid- patients, . % had diabetes as a preexisting comorbidity, and . % were obese. in a meta-analysis of published studies that assessed the outcomes among more than patients, diabetes was found to more than double both the risk of severe covid- disease and the risk of mortality. diabetes more than quadrupled the risk of severe ards-like pulmonary outcomes in covid- . a study in new york city reported that patients with body mass index (bmi) equal to or above had more than triple the risk of in-hospital mortality from covid- , compared to leaner patients [or: . ; % ci . - . , p= . ]. multiple factors may be at work in these outcomes. immune function has been shown to be impaired by nutritional deficiencies in obesity, [ ] [ ] [ ] and by poor glycemic control in diabetes. [ ] [ ] [ ] obesity-compromised lung mechanics have been well described. [ ] [ ] [ ] however, both obesity and diabetes also bear the hallmarks of ras dysregulation at baseline. in the developed world, nearly % of diabetes cases are the result of obesity, and the two conditions are mechanistically intertwined via ras overactivation, , - which primes for worse covid- outcomes. adipocytes locally produce a full set of ras components, including angiotensinogen, ace, aii, at . in obesity, this adipose ace axis is highly upregulated, but with diminished counterbalancing ace , this ace imbalance negatively impacts insulin function. the insulin receptor has two principal second messenger cascades. the first, phosphatidyl inositol- kinase (pi k) promotes cell membrane localization of glut transporters that facilitate glucose uptake. pi k also activates nos, which manufactures no to mediate the anti-inflammatory, anti-coagulant effects of insulin. the second cascade, utilizing map kinase, is responsible for the growth-factor-like actions of insulin, including fibrosis, vascular smooth muscle proliferation, and cardiac myocyte hypertrophy. , under normal circumstances, pi k-produced no inhibits the map kinase arm. in ras upregulation, however, superoxide produced by at eliminates no, thus shortcircuiting the pi k function of the insulin receptor, so that glucose uptake fails, hence the connection between obesity and diabetes. this mechanism would also explain the correlation between covid- and hyperglycemia. with ace axis overactivity, insulin receptor function is shunted to map kinase, and as a result of this alteration, insulin receptors and at receptors-which also use map kinase as a second messenger-synergize to promote inflammatory and hypertrophic damage to the cardiovascular system which is a hallmark of diabetes. blocking ras overactivation with aceis or arbs helps restore normal insulin receptor function, improves insulin-mediated glucose uptake, and mitigates vascular damage. as data accumulate showing elevated covid- mortality rates in african-americans, attention has focused on socioeconomic inequalities, including limited access to nutritious food, overcrowded housing conditions, and consequent health-related comorbidities, such as obesity, diabetes, and hypertension. these clearly evident factors undoubtedly play important roles, but less visible factors may also be at work. there is evidence of ras dysregulation that drives hypertension disparities in african-americans. this could also be contributing to the increased severity of covid- among this population. it has been firmly established that african-americans have higher prevalence, earlier onset, and more severe complications of hypertension than caucasians. recent data from the us department of health and human services office of minority health attest that african-americans currently have higher rates of obesity than caucasians. obesity greater than % above ideal body weight doublesto-triples hypertension risk, and obesity significantly increases ace activity. yet obesity alone cannot explain the unequal prevalence of hypertension. according to national health and nutrition examination survey (nhanes) data from to , african-american and caucasian males had almost identical obesity rates during that time frame, but african-americans had % higher rates of hypertension. research has identified critical physiologic distinctions among many african-americans that influence response to hypertension therapies. most african-american hypertensives have low-renin hypertension secondary to either a mutation of aldosterone synthetase (and consequent aldosterone overproduction) or of the epithelial sodium (enac) channels in the kidney, both of which lead to excessive renal sodium resorption. although sodium retention inhibits renin release into the circulation, reduced activation of the circulating ras does not prevent hypertension. however, in mouse experiments, renin gene deletion solely within the kidneys did confer protection against developing aii-mediated hypertension, suggesting that tissue ras is more critical than circulating ras for blood pressure regulation-and for the development of hypertensive nephropathy. in chronic kidney disease, renal tissue ras was found to be elevated, independent of circulating ras levels. african-american hypertensives have increased incidence of ras-mediated organ damage, congruent with overactive tissue ras despite low circulating renin. in keeping with this, african-americans taking aceis and arbs have comparatively poor blood pressure decrease, yet have significant protection against hypertensive end-organ damage. several gene polymorphisms have been discovered to increase ras activity. for example, increased hypertension prevalence among african-americans has been linked to the − t polymorphism of at . best studied, however, is the ace gene i/d polymorphism, distinguished by insertion (i) or deletion (d) of a nearly base pair dna segment. the dd genotype doubles ace activity, in both circulation and tissue, compared to the ii homozygous genotype; heterozygous ace id is intermediate between the two. in a population-based study, the ace d polymorphism was slightly more common with african ancestry than with european or japanese ancestry. african-american hypertensives in particular have been shown to have higher prevalence of the d allele. , ` it is important to stress, however, that although those of african descent may be more likely to carry the d allele, d is found among many ethnicities. for instance, it was found to occur at increased frequency among caucasian australians who suffered myocardial infarction compared to the general healthy population. when ace d polymorphism occurs, it has often been linked to adverse medical conditions, including increased susceptibility to ards. in children, the d allele was associated with worse outcomes in focal glomerulosclerosis and single ventricle congenital heart disease. ace d allele correlates with ventricular hypertrophy in endurance athletes. additionally, the rotterdam study showed an association of d allele with early mortality. despite current potentially negative consequences, ethnobiological studies have hypothesized an evolutionary survival benefit during ancient times for ras upregulation polymorphisms such as ace d allele. in tropical areas with sodium scarcity, these polymorphisms promoted sodium and water retention, and maintenance of blood pressure. , however, because of the current abundance of dietary sodium, modern descendants face increased damaging consequences from those same polymorphisms. , other ethnicities with reportedly increased covid- mortality, including latinos and south asians, are less well represented in the hypertension literature than african-americans. there is, nonetheless, some evidence for ras upregulation. low-renin hypertension is common in caribbean hispanics. the ace-upregulating d allele has also been found with increased frequency among south indians with hypertension. these observations suggest again that ras upregulation portends the worse covid- outcomes seen in some populations. in a retrospective case series of consecutive patients requiring icu level care for covid- in milan, italy, % were male. researchers from beijing retrospectively analyzed mortality data from the earliest -plus reported cases of covid- in wuhan, china. they reported that, while men and women had similar covid- prevalence, men were nearly . times more likely to die of covid- . simultaneous retrospective re-analysis of mortality data collected in showed that the original sars coronavirus also skewed toward higher mortality in men, in keeping with prior published reports. although gender-specific differences in immune response may play a role in unequal outcomes of both sars and covid- , there are definitive differences between the genders in the ras system and, in keeping with occam's razor, these differences likely contribute to worse male outcomes. sex hormones regulate the expression and function of ras components. males generally have higher levels of ras than premenopausal females, perhaps explaining why male hypertensive rats show a greater blood pressure decrease with aceis. estrogen downregulates the expression of the at gene , and suppresses both ros production in vascular smooth muscle and the enzymatic activity of ace. estrogen also promotes the production of no, the counterweight to aii in the endothelium. estrogen also positively impacts the protective arms of ras, including increasing ace levels in rats. female mice display higher renal at expression than male counterparts, an effect that dissipates if ovaries are removed, and estrogen upregulates at levels in the mouse kidney. both the at and ace genes are found on the x chromosome, , so it is reasonable to conclude that women would regulate and express these genes differently than men. in keeping with this, women have been reported to have higher levels of protective a - than men, both in plasma and in urine, suggesting higher levels of ace activity both in the circulation and at the renal tissue level. these factors likely synergize to confer comparative covid- protection in females, especially pre-menopausally. when assessing the elevated death rates among cancer patients who develop covid- , immune impairment evoked by therapies and nutritional depletion cannot be ignored as contributory factors. however, ras dysregulation has also been shown to be associated with cancer. , increased ace and at expression have been reported in patients with metastatic ovarian cancer and prostate cancer, compared to those with benign neoplasms. at is also increased in squamous cell carcinoma, and in % of estrogen-receptor-positive breast cancers. the cellular growth-promoting aspects of ras are brought into play by tumor cells to facilitate tumor expansion and invasion. aii promotes differentiation of tumorassociated macrophages (tams) from immature progenitors. tams are abundant in the stroma surrounding the tumor, and they release factors that modulate tumor progression and metastatic migration. tams also inhibit the body's anti-tumor immune mechanisms. tams express at , whose effect profile is proangiogenic, augmenting levels dovepress of vascular endothelial growth factor (vegf), and vegf type receptor. at facilitates epithelial-to-mesenchymal transition, which is crucial to metastatic progression. knockout mice lacking the murine version of at have decreased tumor growth compared to normal counterparts. arbs blunt tumor size in animal models, and they also decrease vegf levels, metastatic burden, and tumor vascularization. similarly, aceis have been reported to inhibit tumor growth, angiogenesis, and metastasis. among gastric cancer patients taking either aceis or arbs, prolonged survival has been noted. also, fiveyear recurrence-free survival was significantly higher among patients with non-invasive bladder cancer who were receiving ras-blocking drugs. a small retrospective study of advanced non-small-cell lung cancer estimated a -month prolongation in survival with ras blockers. human studies of ras blockade to limit cancer progression may be confounded by the invisible effects of ras polymorphisms, which are only rarely assessed in trial subjects. in the prospective rotterdam study, however, ace genotyping was performed and revealed that the high ace-activity dd genotype had the most significant benefit in cancer risk reduction with ras blockade. the ace dd genotype has been associated with a higher risk of cancer, while the ace ii homozygote pools with a lower risk. , these findings are congruent with ras overactivation portending poorer covid- prognosis. from the outset of the covid- epidemic, elderly patients have accounted heavily among fatalities. a retrospective comparison of covid- data from italy and china revealed a large increase in case fatality rates with increasing age in both population groups. the italian case fatality rate was % in patients aged - , . % in those aged - , . % in those aged - , and . % in persons above years of age. among the chinese patients, case fatality rate was . % in those - , . % in the - age group, % in those - , and . % above age . elderly people often have nutritional deficiencies that can impair immune function. increasing age is also associated with impairment of mucociliary clearance, which contributes to increased susceptibility to respiratory infections in the elderly, because mucociliary clearance is the primary barrier defense mechanism of the lungs. the elderly account for a higher percentage of patients having the various co-morbidities associated with poor covid- prognosis, such as hypertension, diabetes, cardiovascular disease, and cancer. however, even healthy aging also leads to ras imbalance, with elevated tissue ras components, and lower levels of counterbalancing protective arms of ras. it is well described that circulating aii levels typically decline in the elderly. , importantly, however, tissue levels of aii, ace, at , and angiotensinogen increase with aging in the heart, kidney, and vasculature. , data from african-american hypertensives, including those treated with aceis and arbs, revealed that tissue ras has a more significant deleterious effect on organ function than circulating ras. , it is reasonable to expect that tissue ras levels, rather than circulating levels, would also be the more important variable in elderly individuals, coinciding with the higher incidence of the ras dysregulationassociated comorbidities in aged adults, such as hypertension and diabetes, which portend worse covid- outcomes. at the same time, there is evidence of lower levels of the protective counterbalancing arms of ras, leaving the elderly comparatively more vulnerable to the damaging effects of the ace axis. animal experiments showed that the aortic vasodilatory response to a - is lost in elderly female rats, but restored by exogenous estrogen replacement. in rat lungs of both genders, ace levels have been found to decrease with aging , , this loss of ace was more pronounced in elderly male rats than in similarly aged females, , , which is in keeping with the worse prognosis reported for men compared to women with covid- . it has been posited that patients with baseline ace deficiency would be particularly vulnerable to severe outcomes because of unchecked ace activity with sars virus downregulating ace expression. in elderly covid- patients, the higher basal tissue ace axis components and lower counterweight ace levels even at the start of infection would disadvantage these patients for poor outcome. the covid- outbreak in the west brought unexpectedly severe cases affecting younger adults. the explanation for this situation remains undefined and is probably multifactorial. lifestyle factors may impair immune defense against covid- in younger people. for instance, dehydration which may occur during strenuous exercise, impairs mucociliary clearance, as does aerobic exercise in polluted urban environments. rising obesity rates may play a role, as well as genetic factors, including high ras activity polymorphisms in the general population. [ ] [ ] [ ] acquired, non-genetic ras dysregulation may also be playing a role here, due to the resurgence of inhaled nicotine and its effects upon ras expression. data reported on patients with confirmed covid- from two hospitals in china showed that the odds ratio of dying among current smokers, compared to nonsmokers, was . ( % ci: . ). the impact of e-cigarettes ("vaping") has not yet been reported in the context of covid- . but experimental evidence has been accumulating, showing that e-cigarettes may be even more deleterious than traditional cigarettes, so it is reasonable to suppose that vaping will also influence covid- outcomes. smoking is known to inhibit mucociliary clearance, an essential defense feature against airway pathogens. recent experimental evidence suggests that high nicotine levels in vaping may be even more harmful to this critical defense against inhaled pathogens. , in a sheep model, high-nicotine-containing e-cigarettes caused a % reduction in experimentally modelled mucociliary clearance. moreover, nicotine also adversely impacts ras levels, which would be deleterious during covid- infection. nicotine has been shown to decrease levels of mas receptor and a - , while increasing ace, aii, and at . additionally, nicotine decreased both ace and at expression in several cell types, including cardiac fibroblasts. the rising use of e-cigarette products among young americans has alarmed the medical community and public health officials. cdc data showed that . % of us adults -more than million people-used e-cigarettes in . a gallup poll reported that vaping had increased to more than % of us adults; in addition, % of respondents reported smoking traditional cigarettes. the national youth tobacco survey revealed that million teenagers vaped in , up from . million in . this constitutes a sizable portion of the younger us population who would be at increased risk during the current pandemic, given the evidence that baseline ace deficiency predisposes to poorer covid- outcomes. with covid- deaths now surpassing , worldwide, and further increases becoming evident in some areas as economies reopen-let alone the possibility of a second wave of infections surging late in the yeardeveloping a therapeutic means to mitigate the severity of covid- has taken on utmost urgency. with the evidence of ace axis overactivation at the root of varied organ system pathology in covid- , the most obvious recourse to attempt to blunt covid- progression and mortality would seem to be aceis and arbs. these drugs are widely available, inexpensive, and they target the very system that is unbalanced in covid- . yet the use of these drugs in covid- is not clear cut. early in the covid- pandemic, concerns were raised that aceis and arbs might predispose to increased covid- severity. in covid- , the role of ace appears paradoxical. ace facilitates entry of sars virus into cells, but at the same time, ace expression in alveolar cells protects against experimentally induced lung injury. , , after viral entry into alveolar cells, the spike protein of the sars-cov- virus downregulates further expression of ace , thereby reducing the protective effect of ace on lung tissue. in animal models, aceis and arbs elevate the expression of ace in the heart. , during fetal kidney development, at exerts negative feedback to downregulate ace levels. it has been suggested that aceis and arbs upregulate ace levels in cardiac and diabetic patients, , and therefore could potentially accelerate sars entry. , yet these drug classes have shown clear cardiovascular and renal protective effects, , which might prove beneficial in blunting covid- organ damage. in the midst of this "double-edged sword" debate, the european society of cardiology and the american college of cardiology recommended that, without clear clinical evidence, already-prescribed aceis and arbs should not be discontinued. a case series of patients from wuhan, china found that aceis and arbs were not associated with increased covid- case severity or mortality. an italian case-control study comparing records of covid- positive patients to more than , registered recipients of lombardy's regional health service came to the same conclusion. an observational study of medicare advantage health records, conducted by yale university in partnership with united health group insurance corporation, announced that aceis-but not arbs-might even be protective against more severe covid- outcomes, since acei use correlated with a lower rate of covid-related hospitalization in persons over years of age in this study. multiple ongoing prospective trials are attempting to assess whether differences in covid- outcomes result from maintaining versus stopping aceis and arbs (eg, study nct in the who international clinical trials registry platform). readers may access the database of international clinical studies at http://www.who.int.ictrp). independent of a potential impact on virus entry into cells, these two drug classes have been shown in human studies and animal models to lower the risk and severity of numerous problems that covid- precipitates. among their many benefits, aceis are lung protective in ards, and aceis and arbs reduce stroke rates. arbs reduce cardiovascular oxidative stress, reverse endothelial dysfunction, and decrease inflammatory and oxidative renal damage in patients with diseases associated with ras dysregulation, such as hypertension and diabetes. , arbs have mitigated the severity of experimental myocarditis and have been shown to upregulate at in vascular endothelial cells. moreover, at blockade may shunt existing aii to activate at , yielding antiinflammatory effects. , therefore, aceis and arbs might hold the potential to mitigate covid- organ damage. but clarity may come only once the drugs have been assessed as individual agents, rather than as aceis and arbs en masse. although there are definitive class effects of aceis and arbs, such as blood pressure lowering, the drugs themselves each have unique pharmacological characteristics. evaluating them en masse may wash out otherwise important distinctions. for example, not all aceis have equivalent tissue penetration, due to differences in lipophilicity. in a rat model, fosinopril had better cardiac entry than captopril, lisinopril, and ramipril. similarly, quinapril had higher tissue penetration and better prevention of left ventricular hypertrophy than enalapril. in spontaneously hypertensive rats, moexipril more potently inhibited lung, aortic, and cardiac ace than enalapril. and among arbs, the unique structure of eprosartan seems to confer high renal affinity. in addition, some aceis, such as captopril and perindopril, cross the blood brain barrier (bbb); others, such as enalapril and imidapril, do not. this distinction could potentially hold importance, given that neurons express ace , and some severe covid- patients have manifested cns dysfunction, including encephalitis and a neurocognitive, dementia-like syndrome. parenthetically, these reports are consistent with accumulating evidence that ras is also involved in the amyloid pathology of alzheimer's dementia. among the arbs, only telmisartan has been shown to cross the bbb, thereby reinforcing the importance of carefully evaluating arbs individually, rather than as whole drug classes, in covid- studies. there may be potential pitfalls deploying aceis and arbs against covid- . arbs have two broad categories of action: irbesartan, candesartan, and telmisartan are noncompetitive inhibitors of at , while losartan, valsartan, and eprosartan are competitive blockers. , high concentrations of aii have been shown to overcome competitive blockade in vitro, possibly negatively impacting the therapeutic potential of competitive at blockers in covid- . additionally, many aceis are prodrugs, dependent on hepatic metabolism for activation, but there is evidence that, in some cases, the prodrugs may inhibit the function of the activated counterpart. lisinopril, by contrast to other class members, is orally formulated as the active drug. in the arb class, losartan and candesartan are prodrugs, but activated candesartan has times higher affinity for at than activated losartan. such pharmacological differences may impact the outcome of these therapies in covid- . at least some aceis are dialyzable, but arbs are not, and this may introduce additional difficulties using these agents in covid- patients with renal failure. because ras blockade inhibits hpv, , it is not known whether aceis and arbs would worsen hypoxia in covid- patients, or alternatively, push v/q matching back toward normal. because aceis, but not arbs, impact the metabolism of the vasodilator bradykinin (bk), the distinction between the two drug classes has the potential to differentially impact the course of treatment in covid- patients. bradykinin (bk) is the major vasoactive peptide in the kallikrein-kinin system, [ ] [ ] [ ] interfacing with two principal receptors: , constitutively expressed b , and inflammation-inducible b . in the lungs, bk receptors are situated on the endothelial cells of alveolar capillaries, with b upregulated in patients with covid- . to inactive fragments; thus, aceis may prolong the action of bk. [ ] [ ] [ ] [ ] [ ] bk dysregulation can cause angioedema. [ ] [ ] [ ] b receptors inhibit the function of adherens junctions between vascular endothelial cells, leading to plasma fluid extravasation into the interstitium (figure ) . clinical data indicate that this occurs as a side effect in about one or two patients per people taking an acei, , but the reason why only such a small percentage of patients are affected remains unknown. [ ] [ ] [ ] the concept that angioedema-like changes occur in the lungs of patients with covid- is supported by both indirect (radiologic and clinical observation) evidence , and by more direct evidence at autopsies. [ ] [ ] [ ] other than producing aii and inactivating bradykinin, ace also contributes to the production of a - , the counterbalance to aii. so, the interruption of these additional effects by aceis may potentially impact outcomes in covid- in ways that the arbs do not. as of this time, a small number of prospective clinical trials have begun enrolling patients to assess the potential of individual acei and arb drugs as a therapy to lessen covid- severity. among them, a study at the university of california san diego is evaluating the acei ramipril (nct ). trials at the university of kansas medical center (nct ) and the university of minnesota (nct ) are assessing the arb losartan, and telmisartan is being assessed at universidad de buenos aires (nct ). many other agents remain to be separately evaluated. aside from aceis and arbs to downregulate the ace axis, there are also potential agents to augment the activity of the protective arms of ras. recombinant ace has shown benefit in animal models of ards, so it may prove beneficial in covid- treatment. apeiron biologics has announced that its recombinant ace , called apn , will enter phase ii trials in covid- patients. in animal models, an at agonist called c has proven effective at countering damaging effects of ras. for example, c decreased collagen deposition fibrosis after mi and deceased arterial stiffness. in addition, c blocked the rise in aii that accompanies a high sodium diet in obese zucker rats and blunted progression of glomerulosclerosis. c also decreased renal at protein levels as measured by western blot. a human trial of c in pulmonary fibrosis has been conducted in europe (the european union registry number of this trial is eudract - - ). this agent could potentially be studied in covid- patients. in animal studies, the vitamin d receptor inhibits ras activity, especially at the tissue level. in human studies, vitamin d supplementation did not significantly lower blood pressure, although these studies were small and had few african-american participants, who are at elevated risk from covid- and who typically exhibit lower vitamin d levels than caucasians. perhaps vitamin d could have a positive impact on covid- course if given to high-risk patients. no outcome in medicine is ever guaranteed, but the evidence of ras dysregulation in covid- supports attempting to mitigate covid- outcomes by modulating ras. such an approach would not be a shot in the dark, but rather would be guided by decades of experience modifying ras in other diseases. trials of rasmodulating therapies in covid- could be tailored to patients with high-ras profiles, and would likely need initiation of medication early in the symptomatic course, before the tipping point of severe organ damage has occurred. unfortunately, outpatient therapy opens doors to patient noncompliance, but enrollment of patients early in a hospitalization course may also be beneficial. despite potential pitfalls, the urgency to combat covid- requires tapping the full armamentarium of ras modifier agents for possible efficacy. a recent model predicts that even with continued social distancing, covid- fatalities could triple in the us alone by year's end. human beings are now the ones with no time to lose. cha ae a mysterious blood-clotting complication is killing coronavirus patients belluck p days on a ventilator: one covid patient's fight to breathe again virus is twice as deadly for black and latino people than whites in nyc reyes-velarde a younger blacks and latinos are dying of covid- at higher rates in california the coronavirus is killing men at twice the rate of women mcginley l patients with certain cancers are nearly three times as likely to die of covid- , study says black people four times more likely to die of covid- in the uk compared to white people. cnn international but doctors are finding its damage in kidneys, hearts and elsewhere rabin rc obesity linked to severe coronavirus disease, especially 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from other united states cities covid- autopsies apeiron biologics initiates phase ii clinical trials of apn . news release estimating the infection fatality rate among symptomatic covid- cases in the united states angiotensin converting enzyme: a review on expression profile and its association with human disorders with special focus on sars-cov- infection the control of breathing in clinical practice carotid body oxygen sensing original research, review, case reports, hypothesis formation, expert opinion and commentaries are all considered for publication. the manuscript management system is completely online and includes a very quick and fair peer-review system the authors would like to thank ms melinda judson and ms cassandra graybeal brown for assistance in digitizing the manuscript figures from the authors' hand-sketched originals. the authors report no conflicts of interest in this work. key: cord- -yh ndtgm authors: mohammed el tabaa, manar; mohammed el tabaa, maram title: targeting neprilysin (nep) pathways: a potential new hope to defeat covid- ghost date: - - journal: biochem pharmacol doi: . /j.bcp. . sha: doc_id: cord_uid: yh ndtgm covid- is an ongoing viral pandemic disease that is caused by sars-cov , inducing severe pneumonia in humans. however, several classes of repurposed drugs have been recommended, no specific vaccines or effective therapeutic interventions for covid- are developed till now. viral dependence on ace- , as entry receptors, drove the researchers into ras impact on covid- pathogenesis. several evidences have pointed at neprilysin (nep) as one of pulmonary ras components. considering the protective effect of nep against pulmonary inflammatory reactions and fibrosis, it is suggested to direct the future efforts towards its potential role in covid- pathophysiology. thus, the review aimed to shed light on the potential beneficial effects of nep pathways as a novel target for covid- therapy by summarizing its possible molecular mechanisms. additional experimental and clinical studies explaining more the relationships between nep and covid- will greatly benefit in designing the future treatment approaches. coronavirus disease (covid- ) is an infectious viral disease that is caused by a newly discovered coronavirus, namely severe acute respiratory syndrome coronavirus- (sars-cov- ) [ ] . it is a virus belonging to order nidovirales of family coronaviridae, which are singlestranded rna viruses [ ] and broadly distributed in both humans and other mammals [ ] . majority of the people infected with novel coronavirus ( -ncov) may recover without requiring special treatment [ ] , except the elderly people and those with some medical problems such as hypertension, diabetes [ ] and chronic respiratory disease [ ] . such patients are more liable to be infected with covid- and might develop a fatal pneumonia with clinical presentation greatly resembling that caused by the previous beta-coronavirus sars-cov appeared in ; severe acute respiratory syndrome (sars) [ ] . in such cohort, patients firstly complain of fever, dry cough, and at late stage, may suffer from dyspnea that makes them in a desperate need of intensive care unit (icu) admission and oxygen therapy [ ] . however, the time between icu admission and ards development is recorded to be as short as days [ ] , being the reason for the high mortality rate associated with covid- at this stage [ ] . till present, no covid- -specific vaccines or medications are available, although some national medical authorities recommend testing the efficacy of antiviral medication in especially sever clinical trials [ ] . in addition, several medical researchers suggest starting the supportive and symptomatic treatment till the new medications are developed [ , ] . early evidences have shown that the pathogenicity for covid- may be enhanced with increased the body susceptibility to induce a phenomenon called cytokine storm, in which the immune system gears up by overreacting and producing more inflammatory mediators to fight the infection [ ] . as a result, a severe inflammatory reaction is produced accompanied with significant damage, including organ failure [ , ] . receptors [ ] ; disrupting, of course, the normal physiological function of ace- /ang ( - )/masr axis of the renin-angiotensin system (ras) pathway [ ] . therefore, researchers suggested that the use of angiotensin converting enzyme inhibitors (aceis) and/or angiotensin receptor blockers (arbs), may show a positive trend towards the severe inflammatory reactions and endothelial dysfunction caused by stimulating the function of ace/ang ii/at- axis and thereby, towards the bad pulmonary effects associated with the covid- infection [ , ] . however, no clinical trial, till this time, has been proved its efficacy in preventing or even reducing the covid- severity [ , ] . pursuing clinical trials with the absence of whole data describing the structure of covid- virus and its related mechanisms may lead to unsatisfied outcomes. as well, understanding such data will greatly help in determining the most appropriate effective treatment protocols. consequently, the first step to prevent and slow down the disease progression should involve the deduction of proper pathophysiology of that novel viral disease. identifying ace- as a viral entry receptor will emphasize on the important role of classical ras pathway in covid- pathophysiology, however, neither the link between ace- and other ras components nor their exact roles in the covid- pathogenesis have been neatly studied till now. one ras component, namely neprilysin (nep), has been established to potentiate the physiological beneficial role ascribed to the ace- /ang ( - )/masr axis; by an alternative pathway [ ] . nowadays, nep has emerged as a pharmaceutical target for many drugs; especially those used in treating cardiovascular diseases and alzheimer's disease (ad) [ , ] . considering the respiratory system, nep was reported to play a vital role in protecting lungs from inflammation and fibrosis [ ] [ ] [ ] . a long time ago, mentioned that rats infected with common respiratory tract pathogens, such as parainfluenza virus type- , rat coronavirus, and mycoplasma pulmonis showed low nep activity; resulting in an increase in their susceptibility to inflammatory responses [ ] . however, the precise nep's role against life-threatening lung injuries has not been investigated yet. consequently, this current review aims to examine the cellular signaling pathways involving nep in covid- pathogenesis and to summarize the evidences supporting the potential beneficial effects of nep as a novel target for therapy. all previous and recent studies agreed with the fact that the surface of all human pathogenic coronaviruses is covered with crown like projections called spike (s) glycoproteins; giving the viruses their name [ ] . in addition to s protein, another three main structural proteins have been recognized in sars-cov- , namely envelope (e), nucleocapsid (n) and membrane (m) proteins [ ] , figure ( ) . specifically, s proteins are very important for sars-cov- infection to get started [ ] . they possess two functional subunits by which the virus can enter into its target host cells involving; s subunit, which enables it to bind with receptors on the host cell surface and s subunit, that allows the virus to fuse into the cellular membrane [ ] . unlike other coronaviruses, several experimental analyses proved that sars-cov- does not use the common known viral entry receptors, such as aminopeptidase n (apn) [ ] and dipeptidyl peptidase (dpp ) [ ] , but, instead, can utilize ace- receptor [ ] . additionally, it was revealed that transmembrane protease serine (tmprss ) is very critical for the host cell entry of sars-cov- [ ] and its plasma membrane fusion [ ] resembling sars-cov [ ] . firstly, the virus starts its destructive journey with binding of s subunit to ace- enzyme receptor on the cell membrane surface, which in turn activates tmprss . activation of tmprss is followed by cleavage of both virus's s subunits from its s subunits and so of the ace- receptors. secondly, activated tmprss can also act on the s subunit, activating it through prompting an irreversible conformational change that will facilitate the virus-cell fusion [ , ] . within this view, understanding the precise involvement ace- , as a member of pulmonary ras pathway, in covid- pathophysiology may open new therapeutic possibilities for management. for years, ras was depicted as a hormonal circulating system involved in fluid and electrolyte balance, systemic vascular resistance and blood pressure regulation [ , ] . however, a wealth of data showed that lung epithelial cells, fibroblasts and alveolar macrophages could also express the major constituents of the ras [ , ] supporting the existence of a "local" pulmonary ras that can be differentiated from the "systemic" circulating ras [ ] . as regards "local" ras in the lung, it has been reported to involve multiple cellular mechanisms affecting the vascular permeability, fibroblast activity and alveolar epithelial cells [ , ] . by the time, it is well recognized that activating pulmonary ras can influence the pathogenesis of lung injury in different lung diseases such as pulmonary hypertension, acute respiratory distress syndrome (ards), asthma and pulmonary fibrosis [ ] . as a consequence, pulmonary ras has been described to participate in disease process or play a protective role against tissue injury [ ] and thus, modulating ras in the lung can successfully play a good role in the treatment of inflammatory lung disease [ , ] . the functional steps of ras was firstly initiated by hepatic synthesis of a plasma globulin called renin substrate (or angiotensinogen) which could be enzymatically converted through renin secreted by juxtaglomerular cells of the kidney, into the biologically inactive decapeptide, namely angiotensin (ang) i [ ] . subsequently, ras exhibits two main axes based on two distinct enzymes responsible for cleavage of ang i into angiotensin (ang) ii or ang - [ ] , first axis involves generation of ang ii as the main effector via the angiotensin converting enzyme (ace) and named the classical vasopressor axis ace/ ang ii/ ang ii type receptor (at ) [ ] . for the second axis, angiotensin converting enzyme ii (ace- ) was identified as a depressor for ras activation through producing ang - [ ] . henceforth, this axis ace- / ang - / mas- has become an important area of scientific research interest [ ] . the ace/ang ii/at- axis has been well documented to drive a set of deleterious reactions in the lung through generating ang ii, the principal effector of this classical axis, involved in ras mediated vasoconstriction, sodium retention, fluid overload, inflammation and fibrosis [ , ] . in addition, ace has been reported to evoke bradykinin and substance p degradation, two local pro-inflammatory and protussive peptides which can stimulate cough reflex and nitric oxide (no) release [ , ] . specifically, pulmonary vascular inflammation contributes to a phenomenon called ace "shedding," in which endothelial surface-bound ace is released into the interstitium resulting in significant increase in the level of ang ii [ ] , which explains the detection of higher ang ii level in covid- patients than normal [ ] . several studies declared that ang ii could act through two distinct g protein-coupled receptors (gpcr) subtypes, angiotensin ii receptor type (at r) and type (at r), that were found to be expressed in human lung tissue; proving the local generation of ang ii in lung [ , ] . the majority of actions evolved by ang ii could be mediated by at receptor via enhancing many complex intracellular signaling pathways including mapk/erk, plcb/ip /diacylglycerol, tyrosine kinases, and nf-kb [ ] . activating at receptor had been shown to further stimulate monocytes, macrophages and vascular smooth muscle cells to produce the proinflammatory cytokines such as tnf-α and il- [ ] [ ] [ ] . on the contrary, at receptor was documented to have a number of counterregulatory interactions against lung tissue injury via inhibiting inflammation, and fibrosis [ , ] . in addition to the known ang ii effects on promoting vasoconstriction, and pro-inflammatory cytokine release, there is also an increasing evidence that ang ii could induce vascular endothelial dysfunction [ , ] , promoting the activation and aggregation of platelets and thereby, pro-thrombotic milieu [ ] . ang ii could be associated with noticeable rise in the plasma level of endothelin- (et- ) [ ] , which is a peptide secreted mainly from vascular endothelial cells (ecs), and basically works through the endothelin type a receptor (eta) in vascular smooth muscle cells and the endothelin type b receptor (etb) in ecs [ ] . consequently, et- is described as the most potent bronchoconstrictor [ ] and vasoconstrictor in the airways [ ] . practically, et- acts as a key player of angiotensin ii-induced endothelial dysfunction and platelets activation via inducing il- release [ , ] , which was documented to be correlated directly with the extent of endothelial dysfunction [ ] . endothelial dysfunction is generally characterized by an imbalance between endotheliumdependent relaxing and contracting factors, attributed to reduced production/availability of nitric oxide (no); namely endothelium-derived relaxing factor (edrf) [ , ] . no acts as a potent endogenous vasodilator that could prevent platelet aggregation and leukocyte adhesion to ecs [ , ] . since il- would inactivate endothelial nitric oxide synthase (enos), it could disrupt no production [ ] , decreasing its level and inducing a state of oxidative stress that may lead to ang ii-induced impairment in endothelial responses [ ] postulating impaired endothelium functions as a principal factor in the pathogenesis of heart failure, hypertension and diabetes, it will be expected to classify the patients of such diseases as high risk groups for covid- development [ ] [ ] [ ] . furthermore, platelets activation and aggregation may be noticed as a result of inflammatory reactions aggravated by endothelial dysfunction, leading to imbalance between coagulation and fibrinolysis [ ] . it is clearly evident that continuing release of il- can enhance hepatic thrombopoietin (tpo) mrna expression resulting in thrombopoiesis stimulation and increase in circulatory platelets' numbers, known as (inflammatory thrombocytosis). within this context, et- can also mediate the synthesis of platelet activating factor (paf), a potent phospholipid mediator of platelet activation and aggregation, that may activate platelets to stick together and aggregate. as a result, a platelet plug is formed as an initiator for blood clotting and intravascular thrombus formation [ , ] , which is considered as a starting point for developing stroke [ ] . even though the high incidence of inflammatory thrombocytosis in covid- patients, the laboratory results of severe cases showed the opposite; suffering from thrombocytopenia [ ] .the possible explanation is the depletion of both platelets and megakaryocytes as a result of multiple blood clots formed at the injured site, leading to less platelet production with more consumption as the disease severity increases [ ] . interestingly, endothelial dysfunction and platelet activation can successively together worsen the severity of covid- infection. as known, both et- and activated platelets, associated with endothelial dysfunction, [ ] could promote leukocytes rolling, adherence to endothelium, activation and migration into the inflammatory sites, sharing in enhancement of leukocytes recruitment [ , ] . in addition, endothelial dysfunction can drive the fibrotic consequences following sars-cov- infection, developing pulmonary fibrosis as a result of releasing transforming growth factor-β (tgf-β ), the main fibrogenic cytokines implicated in pulmonary fibrosis, which could be induced by et- action [ , ] . taken into consideration the numerous harmful effects possibly induced by ang ii during covid- pathogenesis, we found that most novel studies aim to use the anti-hypertensive drugs which act either by inhibiting the ace activity or by blocking at receptor, suggesting that action may mitigate the disease severity in covid- patients. on the other side, it has been proposed that enhancing the counter regulatory axis composed by ace- /ang ( - )/masr axis may be the most helpful. ace- /ang ( - )/masr axis, the depressor arm of ras, was identified to mitigate the deleterious actions mediated by ace/ ang ii/ at [ ] . within this axis, ace- competes with ace by hydrolyzing ang i into the nonapeptide angiotensin (ang - ) which is further cleaved by the action of ace into heptapeptide angiotensin (ang - ), thus decreasing the amount of ang i available for ang ii generation by ace [ , ] . to the same extent, ace- could also hydrolyze ang ii converting it into ang ( - ) [ , ] . until the year , ace has emerged as the key enzyme in the pulmonary ras, but this was challenged by the discovery of its homologue; ace- [ ] which was found out to be broadly expressed in almost all types of lung cells, within the vascular endothelial and smooth muscle cells, types i and ii alveolar epithelial cells and bronchial epithelial cells [ , ] . ace- could negatively regulate the ras in the lung through reducing ang ii/at receptor signaling and activating the counterregulatory ang ( - )/ mas receptor pathway [ ] . that finding was compatible with the animal studies showed that the use of ras inhibitors could effectively relieve the symptoms of acute severe pneumonia and respiratory failure [ ] . consequently, the increased ace- was addressed as a target for protection from predisposition to inflammatory lung diseases such as, acute respiratory distress syndrome (ards) [ , ] . concerning covid- pathogenesis, binding of sars-cov- to ace- resulted in exhaustion of ace- , breaking the balance of the ras system within the lung and then, exacerbation of pulmonary inflammatory reactions [ , ] . being ace- receptors widely expressed on vascular endothelial cells (ec) within the lungs [ ] , sars-cov- can exploit them to induce and diffuse inflammatory cascades within endothelial cells disrupting their function; evidenced by the existence of viral elements and inflammatory cells within endothelial cells [ ] . thus, distributing ace- , as the main functional receptor for sars-cov- , within human tissues will be the determinator for spreading of viral infection within the lung and other organs [ , ] . both alveolar and bronchial membranes were reported to highly express ace- , which may explain the higher tendency of sars-cov- virus to harmfully affect lower airways than the upper ones [ , ] . however, recent reports indicate that few covid- patients may also develop some signs and symptoms of upper respiratory tract infection (e.g. rhinorrhea, sneezing, or sore throat) [ , , ] . furthermore, it was reported that ace- is also expressed in many epithelial cells of other organs than lung including kidney, blood vessels, intestine [ ] and brain [ ] . the fact which may explain the existence of some extra-pulmonary co-morbidities as myocardial dysfunction [ ] and acute kidney injury (aki) [ ] , gastrointestinal manifestations (diarrhea, vomiting or abdominal pain) [ ] , neurologic manifestations (altered mental status or seizures) [ , ] . therefore, increased ace- may be useless by promoting viral entry into lung cells, potentiating its devastating effect and enhancing mortality [ ] . consistent with these findings, suggesting ace- activators such as, diminazene aceturate (dize) [ ] , -[ -( carbamimidoyl phenyl) imino hydrazinyl] benzene carboximidamide and xanthenone (xnt) [ ] for counteracting covid- pathogenesis will be in vain. as a consequence, in order to diminish sars-cov- entry and its subsequent lung injury, pulmonary ace- activity should be reduced. however, at the same time, reduced ace- activity may contribute to worsening of the lung inflammation, by unopposed angiotensin ii accumulation [ ] and forcing the ras to continuously increase the expression of cytokines [ ] . simultaneously, a previous data indicated that the decrease in the ace- activity in a rat model of ards was paralleled by low amounts of ang-( - ) [ ] , which has been reported to play a beneficial role against pulmonary inflammation and fibrosis [ , ] . in this context, another study stated that treatment with protease-resistant, a cyclic form of ang ( - ) (cang - ), could restore the ace- activity; attenuated the inflammatory response; decreased lung injury and improved lung function [ , ] . that confirms the positive relationship between ang - and preserving the depressor role of ace- /ang ( - )/masr axis in pulmonary ras. hence, these data will attract the attention to the pivotal role of ang ( - ) in covid- pathophysiology and therapy. ang ( - ) is a biologically active metabolite of the ras that had become a peptide of interest in the last decade, because of its effective role in activating a number of crucial events for the homeostasis of normal physiological functions [ ] . it was reported that ang ( - ) could exert various effects, which are greatly in opposition to those of at- receptor activation such as vasodilator, anti-inflammatory, anti-hypertrophy, anti-proliferative, anti-fibrosis and antioxidant effects [ , ] . clinical and epidemiological studies have revealed the existence of a powerful link between the vasodilator effect of ang - and its higher plasma levels in females; making them less liability to hypertension than males [ , ] . several mechanisms have been attributed to ang - in lowering blood pressure which can be explained as follows (i) triggering enos to stimulate the release of no, which could play a critical role in promoting the relaxation of blood vessels and inhibiting the platelet aggregation [ , ] (ii) inducing natriuresis/diuresis [ ] , and (iii) activating peroxisome proliferator activator receptors (ppars), which in turn supports the availability of no [ ] . interestingly, ang - was documented to directly blunt the activation of pro-inflammatory signaling pathways induced by the ang ii-associated phosphorylation of mapks and nf-kb signaling [ , ] , suggesting also the anti-hypertrophic effects of ang - through normalization of mapks activity [ ] . moreover, ang ( - ), by counteracting the ang ii effects, could also preserve the endothelial function through increasing nitric oxide bioavailability and inhibiting oxidative stress [ ] . taken together, ang - could also reduce lung injury by suppressing the expression of fibrogenic molecules such as tgf-β [ ] , which acts as a key mediator involved in pulmonary fibrosis [ ] . moreover, several researches have pointed out the antioxidant role of ang - [ , ] that might be established by; (i) limiting the activation of nadph oxidase, which is a membranebound enzyme complex involved in triggering ros production through generating superoxide radicals [ ] , and/or (ii) normalizing the expression of antioxidant enzymes such as catalase and heme oxygenase- (ho- ) [ ] , as well as the nuclear factor erythroid -related factor (nrf ), that acts as an emerging regulator of cellular resistance to oxidants [ ] . other observation and experimental evidence suggested that endogenous aceis effects of ang ( - ) can be mediated by its unique membrane bound g protein-coupled receptor known as mas (masr) [ ] , which was revealed to be found in the thin areas of the bronchial epithelium and smooth muscle [ ] . masr could direct the ang - biological responses via the masr/camp/protein kinase a (pka) signaling [ ] , which was previously validated by administrating ave (a nonpeptide mimetic of ang - as a specific ligand (agonist) for masr [ ] . subsequently, attention should be drawn to specifically trigger the ang ( - ) as a potential therapeutic agent able to mitigate the lung injury in patients with covid- infection, without increasing ace- activity. there are different formulations of ang - are being developed e.g. ave- [ ] , hpβcd/ang - [ ] , cgen- [ ] , norleu -a [ ] and cyclic ang - [ ] and used to demonstrate its therapeutic potential in numerous animal models of human diseases, including hypertension, heart failure, stroke, diabetes mellitus, atherosclerosis, renal disease and pulmonary arterial hypertension [ , , ] . however, therapeutic attempts and clinical trials are still underway because of ang ( - ) rapid in vivo degradation by ace [ ] . on the other hand, previous studies emphasized that ang ( - ) could induce vasodilatation in rats of mas-deficient vessels [ ] and in rats pretreated with a (masr blocker) [ ] . the former observed data suggested that ang ( - ) might also interact with an additional specific receptor other than masr to elicit vasodilatation [ ] . indeed, ang ( - ) have been shown to stimulate also the bradykinin (bks) pathway via preventing the bk hydrolysis [ ] . bks are one of the formed kinins that can play significant roles in regulating tissue injury, inflammatory responses and vascular permeability [ ] . they have a little direct impact on the activation and recruitment of inflammatory cells, but they could work indirectly through stimulating the airway epithelial cells and lung fibroblasts through producing a wide array of cytokines, including il- , il- , il- , granulocyte colony stimulating factor (g-csf), gm-csf and macrophage chemoattractant protein- (mcp- ) [ ] [ ] [ ] [ ] . bk action was mediated by g-coupled receptors, namely bk receptor (br) [ ] that amazingly found to be interacted with the mas receptors in order to regulate the vasodilator effect of the ang ( - ) [ ] . at the same time, it was found that the reduction in ace- mrna expression within the lungs of stz diabetic rats was linked with an increase in circulating ang ii, but without any significant change in the production of ang ( - ) [ , ] , ensuring that pulmonary ace- was not the only enzyme responsible for ang ( - ) production, but there might be another enzyme contributing to its synthesis. by the time, the classical view of ras has been further expanded and become well established than previously conceived. as a consequence, it was discovered that there was another alternative pathway by which ang ( - ) is produced, instead of that based on ace- . that way was disclosed to degrade ang i directly into ang ( - ) by another ras member, called nep. nep (neutral endopeptidase or neprilysin, previously known as cd ) is a member of transmembrane zink-metalloendopeptidase that particularly highly expressed in both kidney and lung [ ] [ ] [ ] . nep was also found in a number of other tissues, as epithelia of breast, prostate, stomach and in the central nervous system [ ] [ ] [ ] . nep had been also shown to be present in a soluble circulating form (cnep) within several body fluids including urine, cerebrospinal fluid and plasma [ ] . although nep can discriminately hydrolyze a broad spectrum of physiologically relevant substrates, it was found to possess an obvious substrate specificity. classically, nep exhibits a size-related specificity that enables it to hydrolyze only peptides with a small molecular weight generally at or below , dalton [ ] . as well, nep has been also described to show a distinction between substrates being cleaved 'in vitro' and that being cleaved 'in vivo'. initially, nep was reported to specifically cleave more than peptides 'in vivo' including natriuretic peptides (nps) (atrial natriuretic peptide (anp), c-type natriuretic peptide (cnp), and b-type natriuretic peptide (bnp)), bks, neuropeptides (substance p, enkephalins), gastrin, chemotactic peptide formyl-met-leu-phe (fmlp), versus peptides 'in vitro' such as il-lβ, oxytocin, gastrin-releasing peptide (grp), et- , ang i and ii……etc [ ] [ ] [ ] . by time, more substrates had been proposed with varying levels of 'in vitro' and/or 'in vivo' proof of cleavage. among that, grp, et- , ang i which have been proved to be additionally metabolized 'in vivo' by nep [ , , ] , emerging the evidence that nep could play an important role in many physiological and pathological conditions [ ] , table for cvs patients, nps were known to be of therapeutic importance in lowering blood volume by inducing natriuresis, in which excess sodium can be excreted in urine with accompanying water by the renal tubules [ ] . previous clinitcal trials proved that administering synthetic nps might be associated with some limitations especially on the long run [ , ] , that pushed them to depend on using neprilysin inhibitors (nepi) such as (thiorphan or candoxatrilat) to prolong and potentiate the beneficial effects of vasoactive/nps via inhibiting endogenous nps degradation [ ] . nepi have been used for decades to treat acute diarrhea [ ] . however, they are now developed and emerged as a pharmaceutical target for different cvs diseases. nepi have been used mainly in people with congestive heart failure and a reduced left ventricular ejection fraction (lvef) [ ] . however, using nepi (e.g. candoxatril) solely for hypertension treatment was ineffective, because nepi could inhibit ang ii degradation, increasing its associated simultaneous detrimental effects. therefore, combining nepi either with acei (e.g. omapatrilat) or arbs (e.g. lcz ) become a necessity to overcome this limitation [ , ] . another critical aspect for the pharmacological role of nepi was achieved in treating diabetes via inhibiting the breakdown of some substrates known to modulate glucose metabolism, such as incretin glucagon-like peptide- (glp- ), nps and bks [ ] . within the brain, nep had also proved to hold a great beneficial role in the neurological disorders such as alzheimer's disease (ad) on both in vitro and in vivo studies [ ] [ ] [ ] . multiple lines of evidence highlighted the presence of many amyloid plaques in the form of βamyloid (aβ) peptide in the brains of ad patients with dementia [ ] . it was worthy mentioned that nep was one of the major aβ peptide-degrading enzymes in the brain, whose expression was documented to be lower in the brain of ad patients [ ] supporting the role that nep can play in the prevention and treatment of ad. in addition to the above mentioned, nep seems to play a protective role in the pathogenesis of lung injury since significant decrease in the nep enzymatic activity in the lung of mice with ali was associated with inactivation of the tachykinins degradation pathway and consequently, reducing uncontrolled inflammation in ali/ards and in other neurogenic respiratory diseases [ , , ] . of both respiratory bronchioles and alveoli [ , ] , resulting in excessive production of grp [ ] . grp was known to be one of the bombesin-like peptides that can be expressed and released by pnecs into the surrounding airway parenchyma in response to various stimuli like hypoxia or irritation [ , ] to regulate the neutrophil chemotaxis and macrophage infiltration within the lung tissue [ , ] . grp could act via stimulating gastrin-releasing peptide receptor (grpr) at the surface of macrophages, which in turn, would enhance the release of early inflammatory mediators contributing to the recruitment of neutrophils [ , ] . more neutrophil infiltration within the lung is usually associated with high tendency for lung tissue damage [ , ] , since they would be involved in breakdown of basement membrane integrity within the bronchiolar/alveolar architecture and thereby, diminution of pulmonary function. moreover, during the deleterious inflammatory reactions, as in pneumonia, neutrophil lifespan is prolonged to generate more superoxide radicals resulting in damage of the surrounding normal tissue [ ] . interestingly, nep can play a vital role during lung inflammation through its catabolic effect on (grp) [ ] in addition to its presence on the plasma membrane of neutrophils modulating their chemotactic responsiveness via cleaving the chemotactic peptide formyl-met-leu-phe (fmlp) which resembles an effective chemotactic agonist in response to grp [ , ] . thus, breaking both grb and fmlp peptide by nep will cut the way for recruiting more neutrophils into site of injury and consequently, grab the reins. on the other hand, once neutrophils being activated at inflammatory sites, they could secrete high concentration of several serine proteases into the extracellular environment to degrade host pathogens, recruit more cytokines and stimulate further tissue damage [ , ] . cathepsin g was reported to be one of the neutrophil-derived serine proteases that is abundantly found in the azurophil granules and known to degrade both angiotensinogen and ang i into ang ii [ , ] . so far, nep can additionally take a part in decreasing the pro-inflammatory, oxidative and profibrotic effects of ang ii by minimizing the release of cathepsin g and consequently, its action on ang i [ ] [ ] [ ] . as nep was reported to have more catalytic activity than ace- in cleaving ang i into ang ( - ), it could also effectively enhance the protective activities associated with ang ( - ) in the lung [ ] . as well, nep could not affect lung ang ( - ) metabolism because it was involved in the metabolism of ang ( - ) within tissues other than pulmonary tissues, as renal cortex. on the other hand, ace was recorded to be the major enzyme responsible for ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) metabolism in the pulmonary membranes by hydrolyzing it to ang ( - ), and then, into ang ( ) ( ) ( ) by the action of aminopeptidases [ ] . besides, nep shows higher activity than ace towards bks degradation, resulting in inhibition of the bradykinin-induced inflammatory cells influx [ , ] . despite multiple data confirming the expected role of nep in relieving the pulmonary inflammatory response, its effect on reducing exacerbation of acute severe pneumonia in covid patients has not been highlighted yet. hence, the question now, may the reviewed actions of nep pathways be sufficient to impose a new effective strategy for covid- management as compared to the suggested repurposed drugs? guidelines only provide supportive care. however, many drugs repurposed based on host response in order to defeat covid- , table [ ]. recently, there is an empirical use of anti-inflammation therapy in critical patients of covid- presented with severe complications in order to prevent further injury and suppress cytokine storm manifestations as ards and other organs damage till even death. main antiinflammatory medications include, non steroidal anti-inflammatory drugs (nsaids), corticosteroids, chloroquine and statins [ ] . numerous observational data support a strong association between use of nsaids in management of lower respiratory tract infections and higher incidence of complications including fulminating pneumonia, pleural effusions and dissemination of infection [ ] . nsaids may also induce nephrotoxicity among susceptible covid- patient groups and is exacerbated by fever and dehydration [ ] . consequently, some studies recommend avoiding use of nsaids e.g. ibuprofen and diclofenac as the first choice for fever control and pain symptoms in covid- infection and using paracetamol instead [ , ] . previous study demonstrated that corticosteroids-treated asthmatic patients showed enhanced nep expression in their airway epithelium as compared to nonsteroid-treated ones. this fits the hypothesis that the anti-inflammatory effect of corticosteroids within the airways may be partially mediated by upregulating nep [ ] . despite of possible benefits gained from the anti-inflammatory effect of corticosteroids, they will be associated with serious impairment in the immune system of severe covid- cases [ ] . corticosteroids may delay the viral elimination and increase susceptibility for the secondary infection resulting in deterioration of the disease especially with immune system impairment [ ] . other side effects associated with corticosteroid treatment include hyperglycemia, central obesity and hypertension which represent an obstacle against their use in people at higher risk for covid- especially diabetic, cardiac and hypertensive patients [ ] . auyeung et al., reported that there is no survival benefit for treatment of sars patients with corticosteroids [ ] . another study on corticosteroid therapy of mers patients has revealed no mortality difference with delayed clearance of mers-cov from lower respiratory tract [ ] . moreover, there is no evidence from any clinical trials supporting its administration for covid- [ ] . in order to overcome the immune suppression induced by corticosteroids, a variety of other therapies have been developed to act directly as specific anti-cytokines (e.g. anakinra or tocilizumab) [ ] or anti-inflammatory cytokines (e.g. il- or il- ) [ ] without targeting the immune system and have proven to be effective in treating several syndromes that triggered by cytokine storm [ ] . according to previous studies, chloroquine (cq) and its less toxic metabolite, hydroxychloroquine (hcq) possess anti-inflammatory and immunomodulatory benefits by affecting cell signaling in viral infections. cq/hcq also exhibit a wide variety of antiviral reactions against several viruses including members of the flaviviruses, retroviruses, and coronaviruses [ ] . cq and hcq can prevent the attachment of viral particles to their cell surface receptor, modulate ph in order to inhibit ph-dependent steps of viral replication or interfere with posttranslational modifications (ptms) of viral proteins [ , ] . an enough pre-clinical evidence considering cq effectiveness for treatment of covid- showed reduction in the pneumonia exacerbation, improving lung imaging findings and high rate of virus nucleic acid test negativity. accordingly, cq phosphate in guidelines (version ) for treatment of covid- has been recommended with oral administration twice daily at a dose of mg ( mg for chloroquine) for adults and no more than days [ ] . however, there is a narrow margin between cq/hcq therapeutic and toxic dose. its poisoning has been favored and life-threatening in patients with cardiac disorders [ ] . it is also contraindicated for people with retinopathy, elevated liver enzymes, heart rhythm disorders (as qt prolongation) or allergy to cq/hcq [ ] . further, efficiency and safety of cq/hcq for covid- is still unclear and needs a confirmation depending on more preclinical and clinical trials [ ] . statins are the widely used cholesterol lowering drugs, that were also reported to improve endothelial functions via lipid-independent mechanisms; mediated by their anti-inflammatory and anti-oxidant properties as well as their ability to restore vascular no bioavailability [ ] . because of their immunomodulatory effect, they have also proven to be beneficial as adjuvant therapy in patients with different auto-immune inflammatory conditions (e.g. systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis) [ ] . as a consequence, some hospitals included statins in the treatment protocol of covid- . although statin therapy is usually well tolerated, their use in covid- patients may increase the incidence and severity of myopathies and acute kidney injury [ ] . moreover, statin drugs may increase il- levels which can promote severe pneumonia, deteriorating sars-cov- -induced ards and mortality, especially in elderly patients who are more likely to use these drugs [ ] . sex differences in health outcomes following covid- may be attributed to sex-dependent production of steroid hormones. estrogen has been reported to attenuate inflammation which might protect women compared to men [ ] . estrogen signaling are also known to downregulate mcp- expression and promote adaptive t cell response by stimulating neutrophil recruitment [ ] . it has been also reported that serms, like toremifene, exhibits potential effects in blocking various viral infections as sarscov and merscov virus [ ] through interacting with and destabilizing the virus membrane glycoprotein resulting in inhibition of its replication [ ] . wang et al., suggests that treatment with estrogens and estrogen-related compounds as estradiol (e ) could suppress the expression of tmprss in the lung resulting in decreased mortality to sars-cov infection [ ] . an estrogen receptors/ras interaction has been demonstrated in several studies. e was detected to drive ras to increase ang-( - ) production through estrogen receptor (er α) mediated stimulation of ace and ace mrna expression and activity. a sex difference has been revealed in the expression of ras components [ ] . there is a controversy about the role of ras blockers including, ace inhibitors and/or angiotensin ii type receptor blockers (arbs) in treating covid- and their exact roles still remain unclear [ , , ] . their use has been suggested to be of a value through increasing ace- that may have a protective effect against virus-induced lung injury by preserving ace- in competition with sars-cov- entry into the cells [ ] . another mechanism by which ace- can prevent lung damage and attenuate the pulmonary fibrosis, will be via enhancing the ace- /ang-( - )/mas axis to increase the production of ang ( - ), which in turn can counteract the activity of the ace/angii/at r axis [ ] . on the other hand, it was known that ace inhibitors could block the breakdown of bradykinin increasing its level and then, promoting its associated inflammatory reactions resulting in more deterioration in the health state [ ] . additionally, it has been expected that patients receiving arbs may show upregulation in the membrane bound ace- facilitating the coronavirus entry and worsen then its course [ ] . the suggested explanation may be attributed to the increase in angiotensin ii level that probably pushes it to act as an increased substrate loaded on the ace enzyme, resulting in shifting a part of ang ii to be converted by the action of ace into ang ( - ) , that may be associated with ace- upregulation [ ] . other agents acting on the ras, such as beta-blockers and direct renin inhibitors (dri) to lower angi formation and thereby, angii and ang ( - ). however, till now, no one discussed their impact on the severity and prognosis of covid- [ ] . on the contrary, lacking ang ( - ) will be of negative effect on lung health. so, there is an urgent need to suppose a pathway that can ensure the increase of ang ( - ) level without upregulating ace- . that effect may be attained by keeping ace activity to enhance the pulmonary metabolism of ang ( - ) and at the same time, shifting the ras system away from ace/ang ii/at- axis to avoid its associated inflammatory and oxidative activities. we suggest that nep may achieve this complicated equation. based on previous literature that addressed several beneficial and protective effects displayed by nep during lung injury, we postulate that increasing nep activity may mitigate covid- pathogenesis. lung of covid- patients showed pneumocyte hyperplasia with inflammatory cellular infiltration [ ] , confirming the release of excessive grp into the surrounding airway parenchyma as a result of pnecs hyperplasia [ , , , ] . considering the documented links between high grp level and both neutrophil chemotaxis and infiltration as well as reduction in food and water intake [ ] , it is not surprising to detect high neutrophil count [ ] and anorexia in severe covid- patients [ ] . thus, we expect that grp is the first spark in initiating neutrophils recruitment as well as cytokine storm, which are the main pillars in covid- pathophysiology. our suggested hypothesis herein depends on two main aspects, figure ( nep may abrogate grp-induced neutrophil chemotaxis via cleaving grp and degrading fmlp peptide that can modulate the chemotactic responsiveness of neutrophils. on the other aspect, nep may withstand the potent cytokine storm, which was prescribed to be one of causes for lung damage progression and death in covid- patients. we suggest that nep can diminish the release of inflammatory cytokines that may increase sensitivity of target cells for further stimulation by sara-co- virus [ , ] . thus, nep can improve lung histopathology and enhance tissue survival through two mechanisms: firstly, interfering with ang ii formation via preventing the proteolytic cleavage of angiotensinogen and ang i into ang ii by neutrophil-derived cathepsin g [ ] [ ] [ ] that is expected to be released continuously in response to uncontrolled neutrophil activation associated with covid- patients and via regenerating the synthesis of endogenous ang ( - ), expected to be minimized because of ace- exhaustion by sara-co- virus [ ] . ang ( - ) by itself may protect against pulmonary fibrosis through reducing tgf-β expression [ ] . secondly, breaking bks and thereby, inhibiting its role in activation and recruitment of the inflammatory cells. however, recorded findings suggest that lung damage caused by covid- is induced by an alternative mechanism rather than hyperinfammatory injury. it likely seems that endothelial activation and associated pulmonary intravascular coagulopathy are the contributing factors in covid- pathogenesis [ ] . within this context, nep may exert a critical role in suppressing et- that mediates angiotensin ii-induced endothelial and platelets dysfunction. yet, nep may minimize the chance for ang ii formation, which was reported to be a potent stimulator of et- in endothelial cells [ ] . even, nep can additionally degrades et- , preventing its associated inflammatory injury and eventual fibrotic cascade in the lung [ , ] . furthermore, we speculate that nep may be helpful in dealing with individuals at high risk groups for covid- that exhibit many obstacles in their management. nep may regulate blood pressure in cardiovascular and hypertensive patients indirectly via decreasing both blood and tissue levels of ang ii by: (i) increasing ang i substrate availability a result of inhibiting cathepsin g-mediated neutrophil release, and (ii) augmenting the rate of ang i conversion into ang ( ) ( ) ( ) ( ) ( ) ( ) ( ) , that previously reported to exert a stimulatory effect on anp secretion via masr [ ] . for diabetic patients, we demonstrate that nep may regulate pancreatic ras flux to improve glycemic conditions. in response to nep-mediated degradation 'in vitro', nep may evoke the insulin secretory ability of ang ( - ) via hydrolyzing it into the biologically active ang ( ) ( ) dipeptide in pancreatic islets [ , ] . numerous 'in vivo' and 'in vitro' experiments have been made to increase nep expression. one study speculated that dexamethasone could enhance nep expression in airway epithelial cells via promoting its transcription and synthesis [ ] . another prior finding demonstrated that valproic acid could reduce plaque formation and improve learning deficits via up-regulating nep in app/ps transgenic mice [ ] . recently, serotonin and its derivatives were explored to ameliorate symptoms of ad induced in mouse via enhancing nep up-regulation [ ] . several lines of evidence identified that hormones such as androgens [ ] and estrogen [ ] could also positively regulate nep expression, suggesting that decline in levels of androgen or estrogen associated with aging would accompany by decrease in nep synthesis, which may be an important factor for increasing the risk of covid- infection among elderly. on the other hand, several findings investigated the up-regulating effect of some natural products on nep expression such as apigenin, luteolin, and curcumin, (-)-epigallocatechin- -gallate as well as resveratrol [ ] [ ] [ ] . in china, naoerkang (nek), a traditional chinese herbal medicine, improved the ability of learning and memory in rats model of ad by increasing nep expression in their hippocampal tissues [ ] . however, some scientists aimed to produce recombinant nep (r nep) instead, as park et al., who prepared recombinant soluble nep from insect cells to be intracerebrally injected into ad mice [ ] . therefore, we finally suggest that therapeutic strategies aimed to increase nep expression and/or activity may be of great benefit for prevention and treatment of covid- . despite the high widespread of covid- contagion worldwide, there is no specific efficient treatment that has been proved till now. several pharmacological interventions for covid- have been suggested targeting the host's immune response. following sars-cov- exposure, there is an overproduction of grp within lung tissue resulting in increased release of inflammatory cytokines such as il- β, il- , tnf-α, vegf, gm-csf and mcp- . such cytokines are widely known to enhance neutrophil infiltration which, in turn, induce lung inflammation and respiratory distress as reported in covid- infected patients. as well, cytokines release is also presumed to develop sars-cov- -associated pulmonary fibrosis. novel findings define covid- as one of the pulmonary diseases associated with endothelial and platelets dysfunction. now, it is definitely known that viral invasion can be mediated by one of the ras signaling system components, namely ace- . hence, covid- occurrence and progression can be attributed to imbalance in the pulmonary ras signaling resulting from sars-cov- -induced ace- drain. interestingly, the decrease in ace- activity will be accompanied with a decrease in the generation of ang - which was known to be the light side of ras. regarding the data emphasized on the protective role of ang ( - ) in lung injury, it may be recommended as a covid- therapy, but because of its short half-life, ang ( - ) exhibits a limitation for its use. since nep is a more potent alternative way than ace- for producing ang - , it is suggested to assess the possible beneficial role of nep in covidinduced lung injury. few studies have discussed nep-mediated protective pathways in experimental models of lung injury and fibrosis, however its actual role as a lung protective therapy has not been yet recognized. nep has been involved in degradation of many peptides that may be incorporated in covid- pathophysiology. so, we expect that nep can effectively interfere with the chemotactic responsiveness and recruitment of neutrophils by degrading both fmlp peptide and gpr, respectively. furthermore, we suggest that nep can minimize cytokine storm associated with sars-cov- invasion through inhibiting ang ii formation by neutrophil-derived cathepsin g and directing ang i for generating ang ( - ) which can in turn suppress tgf-β expression and its fibrogenic action, protecting against fibrosis. degrading both bks and et- by nep may be associated with low il- levels, which will be beneficial for stabilizing endothelium and restoring its function. in addition to its catabolic properties, we postulate that nep may possess an advantage for covid- high risk patients through modulating blood pressure and glucose homeostasis. practically, numerous invivo and in-vitro experimental manipulations were made to upregulate nep expression either by using drugs (dexamethasone and valproic acid), hormones (androgens and estrogen) or natural substances (apigenin, luteolin, curcumin and (-)-epigallocatechin- -gallate). however, others directed their efforts towards preparing the recombinant nep (r nep). because most pre-clinical and clinical studies within the medical field are interested in studying nep inhibitors, there is a little data concerning use of nep as a therapeutic agent. consequently, its associated adverse effects have not yet been studied well. finally, we hope our hypothesis will be somewhat enough to direct a future work towards the therapeutic role of nep in modulating covid- pandemic and to target the subsequent therapies for enhancing nep activity in covid- patients. the authors declare no conflict of interest. the authors and their institutions are the only responsible for the financial support and the content of this work in the submitted manuscript. all other authors have no conflict of interests to disclose. 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