key: cord-322654-6nccarjn authors: Luzes, Rafael; Muzi-Filho, Humberto; Pereira-Acácio, Amaury; Crisóstomo, Thuany; Vieyra, Adalberto title: Angiotensin-(3–4) modulates angiotensin converting enzyme 2 (ACE2) downregulation in proximal tubule cells due to overweight and undernutrition: implications regarding the severity of renal lesions in Covid-19 infection date: 2020-06-29 journal: bioRxiv DOI: 10.1101/2020.06.29.178293 sha: doc_id: 322654 cord_uid: 6nccarjn The renal lesions – including severe acute kidney injury – are severe outcomes in SARS-CoV-2 infections. There are no reports regarding the influence of the nutritional status on the severity and progress of these lesions. Ageing is also an important risk factor. In the present communication we compare the influence of overweight and undernutrition in the levels of renal angiotensin converting enzymes 1 and 2. Since the renin-angiotensin-aldosterone system (RAAS) has been implicated in the progress of kidney failure during Covid-19, we also investigated the influence of Angiotensin-(3–4) (Ang-(3–4)) the shortest angiotensin-derived peptide, which is considered the physiological antagonist of several angiotensin II effects. We found that both overweight and undernutrition downregulate the levels of angiotensin converting enzyme 2 (ACE2) without influence on the levels of ACE1 in kidney rats. Administration of Ang-(3–4) recovers the control levels of ACE2 in overweight but not in undernourished rats. We conclude that chronic and opposite nutritional conditions play a central role in the pathophysiology of renal Covid-19 lesions, and that the role of RAAS is also different in overweight and undernutrition. with high levels of inflammatory factors [19] that can stimulate the renin-angiotensin-79 aldosterone system (RAAS) [20, 21] . The experiments were conducted during the 80 development of 2 projects in our laboratory during the last year, and we believe that the 81 resultsif presented together to allow comparisonscould shed some light on the 82 mechanisms underlying the progression and the severity of renal lesions due to Covid-83 Male Wistar rats were fed: (i) with the RBD from weaning ( Figure 1A ,B) represents, per se, an increase in the risk of kidney proximal 101 tubules damageand therefore of AKIduring SARS-CoV-2 infection in 102 normonourished rats, and this could also be applicable to human kidneys, as in the 103 lungs [12] . Figure 1A ,C shows that ACE levels remained unmodified over that relatively 104 long period of life and, therefore, this could contribute to the ACE/ACE2 imbalance that 105 would worsen kidney injuries, as is the case with lung and heart [27] , especially in the 106 presence of comorbidities or modifications in the renal local RAAS. 107 In the next figures our results show that modifications in the nutritional status 108 profoundly modified ACE2 abundance in the renal cortex corticis, which is also 109 modulated in a different manner by antagonizing RAS in cases of overweight or 110 undernutrition. Figure 2A ,B shows that the abundance of ACE2 decreased 30% in the 111 HL (overweight) rats whose body mass was ~20% higher than that of the CTR group. product of a pathway that involves progressively shorter Ang II-derived peptides, as we 120 demonstrated a decade ago [29] . The mechanism underlying the effect of the peptide 121 could be an action as "allosteric enhancer" that induces a second binding site in AT2R 122 with a very high affinity for Ang II [30], possibly involved in the modulation of ACE2 123 formation. Another possibility would be a beneficial formation of heterodimers involving 124 these modified AT2R and Mas receptors, and that are able to act on the ACE2, as in 125 the case of blood pressure [31] . That Ang-(3-4)-mediated upregulation of renal ACE2 126 occurred in overweight rats, but not in the CTR group, is indicative that a "pro-127 hypertensive tissular microenvironment" (high Ang II) develops in animals fed with a 128 diet rich in lipids, causing downregulation of ACE2 (Figure 2A The influence of chronic undernutrition on renal ACE2 levels present some 132 similarity, but also a huge difference, compared with overweight rats (Figure 3) . The 133 similarity is in the downregulation of the enzyme level by RBD alone (~40%) (Figure 134 3A,B), i.e. a value that did not differ from the overweight-induced downregulation seen 135 5 in Figure 2A ,B (~35%). Again, it may be that activation of the Ang II/AT1R axis now in a 136 "pro-hypertensive tissular microenvironment" induced by the multideficient diet 137 contributes for the notable decrease in ACE2, and, since there was no modification in 138 ACE ( Figure 3A,C) , to the important ACE/ACE2 imbalance in proximal tubules that 139 might be crucial in the kidney, as seems to be the case in the lung [27] . On this 140 account, we have strong evidence for the existence of a "pro-hypertensive tissular AT2R acts in parallel with AT1R in promoting tissular damage in kidney tissue (recently 147 reviewed in [27] ). There is a possibility of an adjuvant and independent AT2R-148 associated pathway that, being altered by an amino acid imbalance [33] due to 149 undernutrition, downregulates ACE2 synthesis and levels. A second possibility is the 150 formation of heterodimers between AT1R and abnormal AT2R, which could be 151 modulated by Ang II and Ang-(3-4), as in the case of spontaneously hypertensive rats 152 [34]. As noted above, these rats are of a juvenile age. Therefore, undernutrition could 153 favor severe kidney lesions in young people affected by Covid-19, an issue that has not 154 been explored until the present. World Health Organization. Novel Coronavirus -China European Centre for Disease Prevention and Control. COVID-19 situation update 180 worldwide Severe COVID-19: A review of recent progress with 183 a look toward the future Possible cause of inflammatory storm and 186 septic shock in patients diagnosed with (COVID-19) Multiple organ dysfunction in SARS-189 CoV-2: MODS-CoV-2. Expert Rev Respir Med (2020) Online ahead of print Should COVID-19 concern nephrologists? Why 192 and to what extent? The emerging impasse of angiotensin blockade Covid-19: Risk factors for severe disease and 195 death SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a 199 clinically proven protease inhibitor Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein Glomerular localization 205 and expression of angiotensin-converting enzyme 2 and angiotensin-converting 206 enzyme: Implications for albuminuria in diabetes The contralateral kidney presents with impaired 210 mitochondrial functions and disrupted redox homeostasis after 14 days of unilateral 211 ureteral obstruction in mice The pivotal link between ACE2 214 deficiency and SARS-CoV-2 infection Age-and gender-related difference of 217 ACE2 expression in rat lung Age-associated changes in the vascular renin-angiotensin system in 221 mice COVID-19 and endocrine diseases. A 224 statement from the Two modes of Na extrusion in cells from guinea pig 227 kidney cortex slices A regional basic diet from 229 northeast Brazil as a dietary model of experimental malnutrition Components of the AIN-93 diets as improvements in the AIN-76A diet Perivascular adipose tissue as a 234 relevant fat depot for cardiovascular risk in obesity Obesity-hypertension: An ongoing pandemic Obesity-induced 239 hypertension: interaction of neurohumoral and renal mechanisms I-converting enzyme inhibitory peptides in an alkaline protease hydrolyzate derived 243 from sardine muscle Antihypertensive effects of peptide in 246 sake and its by-products on spontaneously hypertensive rats the shortest angiotensin II-derived peptide, opening new vistas on the renin-250 angiotensin system? Comparison between calcium transport and adenosine triphosphatase 254 activity in membrane vesicles derived from rabbit kidney proximal tubules Comparing rat's to human's age: How old is my rat in people years? A hypothesis for pathobiology and treatment of COVID-19: The 259 centrality of ACE1/ACE2 imbalance is an endogenous ligand for the G protein-coupled receptor MAS A scrutiny of the biochemical pathways from Ang II to Ang-(3-4) in renal 268 basolateral membranes Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on 272 renal Ca 2+ -ATPase through a cAMP/PKA pathway Dimerization of AT2 and Mas Receptors in Control of Blood 275 Altered 279 signaling pathways linked to angiotensin II underpin the upregulation of renal Na + -280 ATPase in chronically undernourished rats Effects of ingestion of 283 disproportionate amounts of amino acids inhibits renal Na + -ATPase in hypertensive rats through a mechanism that involves 287 dissociation of ANG II receptors, heterodimers, and PKA administration as indicated on the abscissae. Statistical differences were estimated 326 using one-way ANOVA followed by Bonferroni's test for the selected pairs indicated 327 within the panels. Significant differences were set at P<0.05. In A, the representative 328 immunoblottings were derived from the same gel, and were cut to remove information 329 irrelevant to the work described in this letter. Loading controls were run on the same 330 blot. 331