key: cord-0006187-d624q452 authors: Calcutt, Nigel A.; Cooper, Mark E.; Kern, Tim S.; Schmidt, Ann Marie title: Therapies for hyperglycaemia-induced diabetic complications: from animal models to clinical trials date: 2009 journal: Nat Rev Drug Discov DOI: 10.1038/nrd2476 sha: ad09f45cdf18e8813e8bcdcec5dceaae6ee209a0 doc_id: 6187 cord_uid: d624q452 Long-term diabetes increases the likelihood of developing secondary damage to numerous systems, and these complications represent a substantial cause of morbidity and mortality. Establishing the causes of diabetes remains the key step towards eradicating the disease, but the prevention and amelioration of diabetic complications is equally important for the millions of individuals who already have the disease or are likely to develop it before prophylaxis or a cure become routinely available. In this Review, we focus on four common complications of diabetes, discuss the range of pathologies that are precipitated by hyperglycaemia and highlight emerging targets for therapeutic intervention. The increasing prevalence of diabetes, particularly among teenagers 1 , reinforces concerns over the appearance of the complications of long-term diabetes during what should be the most active and productive years of life. Patients with type 1 (insulin-dependent) or type 2 (insulinindependent) diabetes develop secondary complications, the risk of which is related to the duration of diabetes and the degree of glycaemic control 2 . The organs that are susceptible to diabetic complications exhibit insulinindependent glucose uptake and possess the glucosemetabolizing enzyme aldose reductase. Therefore, initial interest focused on hyperglycaemia and the subsequent increased glucose flux that is mediated by aldose reductase and the rest of the polyol pathway as a primary pathogenic insult that initiates diabetic complications 3 . Unfortunately, almost 40 years of clinical investigation have been unable to show the efficacy of aldose reductase inhibitors to the satisfaction of European and North American regulatory bodies. The search for sites of therapeutic intervention has therefore extended to the downstream effects of aldose reductase activity and also to aldose reductase-independent mechanisms of glucose toxicity. Glucose-independent mechanisms of organ damage that arise from other physiological consequences of diabetes, such as impaired insulin and growth factor signalling, hyperlipidaemia and hypertension, could also be instigators of, or contributors to, the aetiology of specific diabetic complications. This mixture of universal and organ-specific mechanisms is reflected in the diverse range of therapeutic approaches that are currently being investigated (FIG. 1) . The four most common complications of diabetes -macrovascular disease, nephropathy, retinopathy and neuropathy -share numerous mechanisms by which hyperglycaemia can disrupt cell and organ function (FIG. 2) , with vascular dysfunction also affecting the kidneys, eyes and nervous system. In this Review, we discuss recent developments in preclinical and clinical research for each of these complications to illustrate therapeutic approaches that target either the full range of diabetic complications or the damage to individual organs. Both type 1 (ReFs 4, 5) and type 2 (ReFs 6-8) diabetes have a largely irreversible and devastating effect on small and large blood vessels, and the consequences of vascular injury, such as hypertension, altered vascular permeability and ischaemia, can also contribute to the other complications of diabetes. Heightened oxidative stress and inflammation are increasingly recognized as being central pathogenic mechanisms and markedly alter patterns of gene expression in the vasculature 9,10 , shifting the balance from antiinflammatory and anti-thrombotic homeostatic set points towards an increased pro-inflammatory and thrombogenic potential. As diabetes is further burdened by failure of vascular repair 11, 12 , complications in many organs cause substantial morbidity and early mortality, with heart attack or stroke being the leading causes of mortality in both type 1 (ReF. 4) and type 2 (ReF. 13) diabetes. Animal models and cardiovascular disease. Assorted animal models have been used to dissect the mechanisms underlying cardiovascular complications of diabetes and to identify possible therapeutic targets. macrovascular complications have been studied using both rodent and larger animal models, such as rabbits, dogs, pigs and non-human primates; each species has its advantages and pitfalls. For example, although diabetes enhances vascular permeability in rodents 14 , advanced atherosclerosis is absent in this model, largely owing to the highly effective lipid-clearance mechanisms that are found in these species 15 . Rodent models that combine hyperglycaemia with dietary modulation or genetic modifications to induce hyperlipidaemia and promote atherosclerosis have therefore been developed. However, a consequence of combined hyperglycaemia and hyperlipidaemia in some of the well-studied mouse models, such as apolipoprotein E-deficient mice with concurrent type 1 diabetes, is that cholesterol levels are higher in diabetic animals compared with non-diabetic animals 16 . This makes it difficult to assess the relative contributions of glucose and/or lipids in driving the acceleration of atherosclerosis. Two particularly creative models have been developed to address this issue. First, to assess the relative roles of hyperglycaemia and dyslipidaemia, mice lacking the low density lipoprotein receptor (lDlR) were crossed with transgenic mice expressing a viral protein under the control of the insulin promoter. when infected with the virus, the mice developed type 1 diabetes. In diabetic animals that were kept on a cholesterol-free diet, atherosclerosis was accelerated compared with non-diabetic animals, suggesting that hyperglycaemia drove accelerated atherosclerosis. However, when the mice were fed diets that were high in cholesterol (which rendered them diabetic), they developed severe hypertriglyceridaemia and advanced atherosclerosis 17 . These studies suggest that diabetes-associated dyslipidaemia accelerated lesion progression. Recent studies using this model have also shown that induction of type 1 diabetes in lDlR-deficient mice promoted plaque disruption, as measured by intraplaque haemorrhage 18 . Interestingly, plaque disruption was associated with the accumulation of monocytes that expressed s100 calcium-binding protein A9, a marker of inflammation. The second model explores the role of the polyol pathway in the vascular complications of diabetes in mice, a species which normally has low levels of aldose reductase, by amplifying aldose reductase expression to physiologically relevant human levels. These aldose reductase-transgenic mice showed enhanced vulnerability to hyperglycaemia-induced atherosclerosis and ischaemia-reperfusion injury 19, 20 . Importantly, hyperglycaemia seemed to be the primary factor driving accelerated atherosclerosis in aldose reductase-transgenic mice that were also deficient in lDlR, as lipid levels were the same in diabetic transgenic and wild-type animals. These and other animal models are helping to dissect the contribution of different pathogenic mechanisms to diabetic vascular damage. The paucity of models for diabetic macrovascular complications has prompted the formation of the Animal models of Diabetes Complications Consortium (AmDCC) to develop and evaluate new models 21 . However, although mouse models are suitable for dissecting and validating the specific role of hyperglycaemia in the pathogenesis of accelerated atherosclerosis in diabetes, they have definite shortcomings. For example, despite developing extensive and highly advanced atherosclerosis and intraplaque haemorrhage, current diabetic mouse models fail to reliably display evidence of thrombosis and overt myocardial infarction. By contrast, hyperglycaemic and hyperlipidaemic pigs and non-human primates with long-standing diabetes can have highly advanced lesions that are more similar to those observed in human subjects and, importantly, that might be vulnerable to thrombosis 22,23 . one key component for accurate modelling may be the duration of diabetes, which may require many months to years to generate overt pathology. This has prompted extensive efforts to identify biomarkers of vascular injury in diabetes. endothelial cell dysfunction, manifested as impaired vasodilatory responses to acetyl choline or reduced blood flow in human subjects, could prove useful in assessing the state of disease and, possibly, the effect of therapeutic intervention on vascular stress 24 . studies that illustrate the potential diagnostic and predictive value of endothelial cell biopsy techniques in diabetes may also hold promise as an easily repeatable means to sample the vasculature and assess its inflammatory and thrombotic potential at any time point 9, 25 . Vascular dysfunction contributes to all diabetic complications, and therapeutic approaches that target the vasculature (shown centre in dark pink, where the three areas overlap) show some efficacy against all complications. The kidneys, eyes and nerves could also be targeted by organ-specific approaches. ARI, aldose reductase inhibitor; GAG, glycos aminoglycan; RAGE, receptor for advanced glycation end products; VEGF, vascular endothelial growth factor. Despite the limitations of the various animal models, these studies have shed light on some of the fundamental mechanisms that contribute to accelerated athero sclerosis in diabetes. when testing potential therapeutic targets, smaller animals could be used to probe mechanisms and to carry out early compound screening with a higher throughput, and larger animals could be subsequently used for the final testing of lead compounds and the validation of the mechanisms that were delineated in the smaller rodent species. The key to discovering treatments that target cardiovascular disease in individuals with diabetes lies in identifying the molecular species that promote perturbation of the vessel wall. Extensive epidemiological evidence suggests that glucose is one of the key players in this process 26, 27 . Both the direct and indirect consequences of increased blood glucose levels contribute to the pathogenesis of accelerated cardiovascular disease in diabetes. In the macrovasculature, high levels of glucose probably synergize with superimposed stresses that are common to both diabetes and non-diabetic vascular disease, such as raised levels of serum lipids, hypertension and the sequelae of innate ageing processes, to continuously stress vascular cells. such mechanisms lead not only to primary vascular dysfunction, but also to chronic cycles of stress that injure surrounding cells, which are dependent on intact vascular function. As summarized in FIG. 2, excess glucose directly stimulates activation of the polyol pathway 3 and also the activity of mitochondria 28 , protein kinase C (PKC) 29 and NADPH oxidase 30,31 , which results in the production of reactive oxygen species (Ros). Hyperglycaemia also leads to the formation of advanced glycation end products (AGE), which irrevocably alter the diabetic vasculature, leading to vascular stiffening owing to extensive protein crosslinking 32 . moreover, extracellular AGE also bind and activate the signal transduction receptor RAGE (receptor of AGE). RAGE is a multi-ligand receptor, and its interactions with AGE and non-AGE pro-inflammatory ligands, such as s100-calgranulins and high-mobility group box 1 protein (HmGB1), are potent generators of accelerated vascular inflammation 31,33 . The importance of these pathways has been further suggested by recent studies involving the generation of unique mouse models. For example, the role of RAGE has been examined in both non-diabetic 34 and diabetic 35 mice deficient for the genes that encode apolipoprotein E and RAGE, and these studies have shown that RAGE has a pivotal role in atherosclerosis. Clinical development of therapeutics. The insights gained from experimental models such as those described above have led to clinical trials investigating antagonism of the polyol pathway 36 , PKC isoforms (particularly PKCβ 37,38 ), AGE 39,40 and agents that counter the effect of the enhanced generation of Ros caused by high glucose levels, such as benfotiamine 41 . New small molecule ligands that antagonize RAGE and humanized chimeric soluble RAGE are also being tested in early clinical trials (see the 6-month safety and efficacy study of TTP488 in patients with type 2 diabetes and persistent albuminuria). long-term success in treating the blood vessels of patients with diabetes will probably lie in identifying combinations of these therapeutic targets, as it is likely that at distinct times Organ damage can be triggered by both extracellular and intracellular hyperglycaemia. Increased extracellular glucose leads to non-enzymatic glycosylation of proteins and subsequent formation of advanced glycation end products (AGE) that interact with the receptor for AGE (RAGE) on the plasma membrane and promote the production of reactive oxygen species (ROS). Increased intracellular glucose drives mitochondrial activity, increases the activity of protein kinase C (PKC) and NADPH oxidase and promotes increased flux through the polyol pathway, all of which have many effects on cellular metabolism and phenotype. This figure highlights the consequences of excessive ROS production in the vasculature, where ROS-driven changes in cell phenotype are mediated by a range of signalling pathways and transcription factors. Cells of the kidneys, eyes and nervous system also undergo cell-and organ-specific phenotypic changes as a result of hyperglycaemia-mediated ROS production. ROS production, ROS-unrelated pathogenic consequences of hyperglycaemia and hyperglycaemia-independent mechanisms, such as impaired insulin signalling, are likely to collectively mediate the organ-specific pathologies of diabetic complications. Other pathways that are relevant to diabetic complications but are unrelated to hyperglycaemia per se, such as disruption of the renin-angiotensin system (FIG. 3) , are described in the main text. AP1, activator protein 1; AR, aldose reductase; CCL2, CC-chemokine ligand 2 (also known as MCP1); CDC42, cell division cycle 42; EGR1, early growth response protein 1; ERK, extracellular signal-regulated kinase; ICAM1, intercellular adhesion molecule 1; JAK, Janus-activated kinase; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PI3K, phosphoinositide 3-kinase; RNS, reactive nitrogen species; SDH, sorbitol dehydrogenase; STAT, signal transducer and activator of transcription; VCAM1, vascular cell adhesion molecule 1. A chronic inflammatory response in the wall of large blood vessels during which the vessel wall hardens owing to accumulation of plaques that are formed following oxidation of low density lipoproteins in the blood by reactive oxygen species. Endothelial cell simple squamous epithelial cell that lines the lumen of all blood vessels and is considered an important site of diabetes-induced damage to blood vessels and subsequently other organs. A family of enzymes that, when activated, translocate to the plasma membrane and facilitate phosphorylation of other proteins, thereby activating or deactivating them. Aberrant activation of PKC has been implicated in the pathogenesis of many diabetic complications. A membrane-bound enzyme complex that uses NADPH to catalyse the conversion of oxygen to superoxide and thereby generates reactive oxygen species. Widely studied as a mechanism of cell-mediated bacterial killing, excessive activity of NADPH oxidase isoforms has also been implicated in mechanisms of diabetic complications. A product of the irreversible addition of glucose to proteins or fats. It is produced following a sequence of Amadori, schiff and Maillard reactions, which can change the function of the recipient molecule. The appearance of serum proteins (for example, albumin) in the urine that is associated with early stages of diabetesinduced damage to the filtration system of the kidney. Damage to blood vessels. Usually divided into damage to large (macrovascular disease) and small (microvascular disease) blood vessels. and sites in the vessel wall, many mechanisms synergize to cause diabetic vascular stress. whether suppression of these maladaptive messengers in the vessel wall will also facilitate the recruitment of progenitors and mediate vessel repair is a largely unexplored question. It is plausible that mechanisms which directly injure the vessel wall also disrupt the environmental cues and the flow of repair species and cells to the injured vessel. The longterm safety and tolerability of RAGE antagonism should be more fully investigated, particularly as studies in acute peripheral nerve injury suggest reparative roles for RAGE in inflammatory and axonal element signalling 42, 43 . As new targets and concepts are investigated (TABLe 1) , it is important to note that inflammation and adaptive immune mechanisms that contribute to the pathogenesis of type 1 and type 2 diabetes also play a part in the subsequent macrovascular complications. In this context, strategies that broadly suppress inflammation, such as HmGH-CoA reductase inhibitors (statins) 44 and peroxisome proliferator-activated receptor (PPAR) agonists 45 , have been considered relevant to the treatment of diabetic vascular injury. statins are now considered almost routine in the management of both type 1 and type 2 diabetes, as various studies testing a range of these agents have shown a reduction in cardiovascular morbidity and mortality 46, 47 . The case for PPAR agonists is less clear, despite good efficacy in experimental models of diabetes-associated atherosclerosis 48 . A recent study that evaluated the PPARγ agonist rosiglitazone using intravascular ultrasound suggests a trend towards reduced progression of atherosclerotic events in a cohort of subjects with type 2 diabetes 49 , although this drug has also been associated with increased cardiovascular events 50 . By contrast, another PPARγ agonist, pioglitazone, showed modest clinical benefits in a study on subjects with type 2 diabetes 51 . Dual PPARα-PPARγ agonists have been developed as a therapeutic approach to improved glucose and lipid homeostasis in patients with diabetes. However, fenofibrate did not clearly show benefits on cardiovascular outcomes 52 despite a reduction in diabetic microvascular disease 53 , and muraglitazaar was withdrawn because of possible increased cardiovascular events 54 . Although vascular inflammation may be the final common mediator and manifestation of diabetic vascular stress, there is little doubt that inhibiting these harmful pathways at earlier stages of hyperglycaemia is fundamental to preventing the devastating effects of diabetes on blood vessels. The importance of ambient hyperglycaemia per se in macrovascular disease may have to be reconsidered in the context of findings from two recent clinical trials, which explored the effects of tight glycaemic control in subjects with type 2 diabetes and established cardiovascular disease 55, 56 . However, there are potential caveats to these studies. The lack of a positive effect on cardiovascular mortality in these studies could be related to the short duration of the trials (<5 years) or poor tolerance of the demanding and extensive therapeutic regimens by older subjects with type 2 diabetes and its many complications. It is also possible that irreversible vascular changes, such as advanced glycation 57 or 'hyperglycaemic memory' resulting from previous periods of hyperglycaemia 58 , could also contribute, possibly through epigenetic mechanisms such as glucoseinduced histone modifications that alter vascular gene expression [59] [60] [61] . As the effect of diabetes on plasma lipids and blood pressure could also have a substantial role in atherosclerosis, a therapeutic regimen that incorporates antihypertensive and lipid-lowering agents along with drugs that target the consequences of hyperglycaemia may ultimately prove to be the most effective approach, as recently highlighted in the follow-up of a study trialling this approach 62 . Diabetic nephropathy is now the most common cause of end-stage renal failure in the western world 63 . From a clinical perspective, it is characterized by the onset of proteinuria and a subsequent decline in glomerular filtration rate and ultimate progression to uraemia, which is fatal if left untreated 64 . The main clinical associations that frequently precede overt diabetic nephropathy are hyper tension and poor glycaemic control 65 . once nephropathy is established, blood pressure often rises further, but glycaemic control can paradoxically improve as a result of reduced renal insulin clearance 66 . Both glucosedependent pathways that are common to vasculopathy and other complications of diabetes (FIG. 2) , and more organ-specific mechanisms that are linked to systemic and intraglomerular hypertension (FIG. 3) , seem to play important parts in the development and progression of this disease 67 . The drugs currently used to treat diabetic renal disease largely target the hypertensive component. In particular, drugs that interrupt the renin-angiotensin system (RAs), such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor (AT2) antagonists, are currently considered first-line treatments for diabetic nephropathy, and this therapeutic strategy is incorporated into most national and international treatment guidelines 68 . This pharmacological approach has been extended by the recent production of the renin inhibitor aliskerin, which also seems to reduce proteinuria, particularly as part of dual therapy with an AT2 antagonist 69 . The most effective approach to blocking the RAs remains controversial, and various strategies are currently being investigated. with greater understanding of the complexity of the RAs and identification of new components of this pathway, such as ACE2, it has become increasingly evident that there is a complex interaction between the vasoconstrictor and vasodilator arms of the RAs 70 . Indeed, it seems that an alteration in one component, such as ACE2, can influence the renal response to agents such as ACE inhibitors 70 . Furthermore, there is often upregulation of upstream components such as renin with distal interruption of the RAs, which can be suppressed by vitamin D administration 2, 71 . Animal models of diabetic nephropathy. The classical model of streptozotocin-induced type 1 diabetes in rodents is still widely used and has a range of functional and structural changes that are comparable to human diabetic Streptozotocin (sTZ). A glucosaminenitrosurea compound, originally isolated from Streptomyces achromogenes, that enters the pancreatic β-cell by a glucose transporter system and damages DNA by alkylation, leading to cell death and subsequent insulin deficiency. sTZ is used to induce type 1 diabetes in animals and provides the most widely studied models of diabetic complications. Capillary bed of the renal corpuscle from which plasma is extruded and filtered before entering the tubular system of the kidney. The filtering system includes the endothelial cells of the capillary, the glomerular basement membrane, podocytes and associated mesangial cells. Disruption of one or more of these cells and structures can impair blood filtration and contribute to diabetic nephropathy. nephropathy 72 . These include early development of renal hypertrophy, progressive increases in albuminuria and changes to the renal ultrastructure, such as mesangial expansion and glomerular basement membrane thickening. Unfortunately, more advanced renal disease, specifically overt proteinuria, renal impairment and advanced structural lesions, are not prominent in this model 72 . New models, such as the Akita 73 and ovE26 mice 74 , have been developed to address this concern and show more advanced lesions. whether these new mouse strains will ultimately prove to be more useful preclinical models for testing new treatments remains to be determined, and much emphasis is currently being placed on the genetic background of the mice. For example, it has been suggested that manifestations of nephropathy in the Akita mouse model can differ depending on the genetic background of the mice 73 . Mechanisms and interventions derived from animal models. much of the initial research into diabetic nephropathy relied on detailed characterization of the progression of the disease at the functional and ultrastructural levels. These studies identified the phase of microalbuminuria or incipient diabetic nephropathy, characterized by modest increases in urinary excretion of albumin. It was then shown that this phase predicts the subsequent development of overt renal disease and is associated with key structural changes to the renal glomerulus 75 . Recent studies have explored potential molecular and biochemical mechanisms that could be responsible for the progression of renal lesions in diabetes. Initial research focused on the mesangial cells within the glomerulus, but changes in other glomerular cells, including podocytes 76 , and progressive injury to the tubulointerstitium have subsequently been described 77 . Glucose-dependent pathways (FIG. 2) , such as advanced glycation 78 , are clearly important components in the pathogenesis of diabetic renal disease and accompany organ-specific mechanisms involving disruption of vasoactive hormone pathways, such as the RAs 79 . Glucose and AGE-mediated activation of RAGE 78, 80 , key intracellular signalling molecules such as PKC and mitogen-activated protein kinase, and angiotensin II also have a central role in this disease 79 . Furthermore, certain pro-sclerotic growth factors, such as transforming growth factor-β 77,81 and connective tissue growth factor 82 , seem to promote extracellular matrix accumulation, a cardinal structural feature of the kidney in patients with diabetes. It is also becoming increasingly apparent that metabolic and haemodynamic pathways not only interact through common mediators, such as intracellular signalling molecules and growth factors, but can directly interact with each other. For example, angiotensin II can enhance AGE accumulation in the kidney and AGE can directly modulate expression of key components of the RAs 83 . Thus, it seems that metabolic and haemodynamic stimuli, triggered by the diabetic milieu, interact to amplify injury and perpetuate the progression of renal damage in diabetes (FIG. 3) . (TABLe 1) . These trials are focusing primarily on albuminuria as an index of nephropathy, as well as assessing the decline in renal function. Because diabetic nephropathy takes many years to evolve and only occurs in a minority of subjects, these clinical trials have often been difficult to complete. moreover, the Us Food and Drug Administration currently considers renal impairment, and not proteinuria, as the appropriate renal end point of diabetic nephropathy. This requires studies that often involve more than 1,000 patients and a follow-up of up to 5 years 87, 88 . such studies are difficult to carry out in terms of recruitment and are expensive. It is hoped that surrogate markers such as albuminuria may be accepted for approval by regulatory agencies to register drugs as renoprotective in diabetes 89 , although the finding that some subjects with diabetes develop renal impairment in the absence of albuminuria 90 suggests that serial assessment of urinary albumin excretion may also not be ideal. many research groups are trying to identify alternative biomarkers of renoprotection, and much of this work focuses on urine proteomics 91 . This research is at an early phase, and a biomarker has not yet been established. Ultimately, the goal is to develop more robust strategies for monitoring diabetic nephropathy and in particular to generate a more reliable approach to identifying truly renoprotective regimens that not only decrease albuminuria but also reduce renal morphological injury and preserve renal function. Diabetes is the leading cause of new cases of blindness among adults aged 20-74 years 92 . It is characterized by a range of retinal lesions and abnormalities that indicate vascular damage (capillary microaneurysms, capillary degeneration, increased vascular permeability and new vessel formation) and death or dysfunction of the neural retina ('cotton wool spots', alterations in retinal electrophysiology, loss of colour or hue discrimination). Clinically, It is notable that many of the metabolic disorders of the kidney reflect those occurring in other organs that are compromised by diabetes (FIG. 2) , whereas aspects of the haemodynamic component may be more specific to nephropathy. Therapeutic approaches that intervene in the core pathogenic mechanisms that are common to all diabetic complications may therefore require combination with organ-specific therapies for maximal benefit. AGE, advanced glycation end product; CTGF, connective tissue growth factor; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; RAGE, receptor of AGE, TGFβ, transforming growth factor-β; VEGF, vascular endothelial growth factor. Microaneurysm small, focal points of damage to a blood vessel (usually capillary) wall, leading to pressure-induced swelling that may cause the vessel to rupture and allow leakage of blood (haemorrhage) into the surrounding environment. Growth of new blood vessels. Can be beneficial when blood vessels have been damaged, but can cause diabetic complications such as proliferative retinopathy if uncontrolled or inappropriate. Oedema swelling of the extracelluar tissue space by influx of fluid in response to the accumulation of osmotically active molecules. Oedema has a protective role following tissue injury but can also cause damage if it is prolonged or develops inappropriately. A non-neuronal cell of the nervous system, including schwann cells (in peripheral nerve), astrocytes and oligodendrocytes (in brain and spinal cord) that regulates the environment surrounding neurons. smaller microglial cells are scavengers of cellular debris that can accumulate in the nervous system, particularly following damage. diabetic retinopathy has been separated into non-proliferative and proliferative disease stages. only the late stages of the retinopathy, especially neovascularization and retinal oedema, have adverse effects on vision 93 , but these disorders seem to be dependent on changes that develop in the earlier stages of the disease. New vessels growing out of the retina into the normally avascular vitreous fluid are particularly threatening for vision owing to excessive leakage from these new vessels (pre-retinal haemorrhage) or to the development of a fibrovascular membrane 93 . Available evidence suggests that occlusion or degeneration of retinal capillaries is strongly associated with the development and progression of diabetic retinopathy 94 , presumably by contributing to the development of ischaemia and the subsequent release of hypoxia-inducible vasoproliferative factors. Accumulation of fluid within the retina also contributes to the visual impairment in diabetes, in part by distorting the retinal architecture 93 . Diabetic retinopathy takes many years to develop, and almost all patients with type 1 (ReF. 95) and type 2 (ReF. 96) diabetes exhibit some lesions after 20 years of disease. Nevertheless, only a fraction of these patients will progress to having visual impairments. There does not seem to be a difference in the clinical picture of diabetic retinopathy between patients with type 1 and type 2 diabetes. Animal models of retinopathy. Numerous species, including monkeys, dogs, cats, pigs, hamsters, rats and mice, have been used as models for the study of diabetic retinopathy 97 . All mammalian species studied to date develop at least the early stages of retinopathy, including degeneration of retinal capillaries. Development of retinopathy in many of these models occurs more rapidly than in humans, with early lesions developing within months to years of the onset of diabetes. The severity of retinal disease increases with duration of diabetes, but remains mild compared with that seen in many patients with diabetes. This is due in part to the limited lifespan of laboratory animals. with the exception of occasional contrary claims, diabetes alone has not been found to cause pre-retinal (intravitreal) neovascularization in any animal model 98 . Again, this is probably due in part to insufficient severity of capillary degeneration and other lesions during the limited time over which these models have been studied. No data have yet been generated to suggest that the current animal models are inappropriate reflections of human diabetic retinopathy because they are mechanistically inadequate. Instead, the development and appearance of early retinopathy is similar in many diverse animal models of diabetes, in which the differences in the severity of retinopathy are related to the differences in the severity of hyperglycaemia. Investigators who are interested in studying or inhibiting retinal neovascularization in vivo have turned to non-diabetic models in which retinal neovasculari zation occurs after branch vein occlusion, oxygen-induced retinopathy 99 or overexpression of growth factors such as vascular endothelial growth factor (vEGF) 100, 101 or insulinlike growth factor 102 in the eye. Because degeneration or occlusion of retinal capillaries in diabetes has been closely linked to eventual retinal neovascularization in humans, inhibition of diabetes-induced capillary degeneration is also used to evaluate therapeutic interventions 103 . Retinal neurons, particularly the retinal ganglion cells that connect the eye to the brain, have also been reported to degenerate in diabetic rats 104, 105 and some strains of diabetic mice 106 . Diabetes-induced retinal neuron loss seems to begin before death of retinal vascular cells occurs but is not dramatic in these models, again perhaps owing to the short duration of the study. Despite this progressive neuronal loss, animal models have not yet been found to show evidence of visual impairment or blindness because of diabetes. There remain important gaps in our understanding of the pathogenesis of retinal neovascularization in diabetes, the role of neural dysfunction and neurodegeneration in loss of vision and in the translation of efforts to inhibit retinal capillary degeneration in animal studies into the prevention of vision loss in patients with diabetes. many cell types, both within and outside the retina, are likely to contribute to the development of diabetic retinopathy, and it is becoming apparent that the interrelationship between the vascular, neural, myeloid and glial cells needs further study in both health and disease. ongoing efforts to develop genetically modified animals may offer new ways to investigate the pathogenesis of diabetic retinopathy and identify new therapeutic targets. Although it is widely accepted that diabetic retin opathy is caused by poor glycaemic control, the progression of retinopathy does not reverse, or even immediately halt, when better glycaemic control is achieved 26,107 . The observation that diabetic-like retinopathy develops following a long-term increase in galactose levels in the blood, even in the absence of diabetes, provides strong evidence that hyperglycaemia per se, as opposed to insulin deficiency or alterations in lipid profile that are characteristic of poor diabetic control, is sufficient to initiate the development of aspects of retinopathy in mice 108 and rats 109, 110 . However, disorders that commonly parallel hyperglycaemia in patients with diabetes, such as altered blood pressure and dyslipidaemia, have been found to influence the rate of progression of retinopathy 27,111 . In recent years, there has been a great increase in the number of potential therapies that are reported to inhibit the diabetes-induced degeneration of retinal capillaries and neurons as well as the increase in retinal vascular permeability in animals. These range from pan-complication therapies, such as aldose reductase inhibitors, to tissue-specific approaches, such as anti-vEGF agents 103 . likewise, the range of therapies that have been found to inhibit retinal neovascularization in non-diabetes animal models is expanding and includes both those with potential efficacy against many diabetic complications, such as PKCβ inhibitors, and those that specifically inhibit mechanisms of retinopathy, such as blocking vEGF signalling 112 . A large group of diabetes-induced biochemical and physiological abnormalities in the retina, which were previously regarded as being independent of each other, are now known to associate in the context Demyelination Retraction of the myelin sheath from around a neuron that is caused by damage to, or death of, myelin-forming schwann cells (in the peripheral nervous system) or oligodendrocytes (in the central nervous system). Demyelination is a prominent feature of diabetic neuropathy but has been difficult to model in rodent models of diabetes. A small unmyelinated sensory neuron of the peripheral nervous system, the peripheral terminal of which projects above the dermis of the skin into the epidermis and allows sensation of heat pain and other stimuli. Also referred to as an intra-epidermal nerve fibre. of inflammation 103, 113 . The non-proliferative stages of diabetic retinopathy include altered vascular permeability and function, vascular degeneration, and neural dysfunction and degeneration. The interplay between these abnormalities is an ongoing area of research, and it is conceivable that individual therapies might not have comparable effects on each of these different areas. Numerous clinical studies over the past several decades have provided strong evidence that surrogate markers that quantify the severity of retinopathy, such as the Early Treatment of Diabetic Retinopathy study (ETDRs) retinopathy grading scale 114,115 , can predict progression of the retinopathy and can indicate inhibition of disease progression. Randomized, multi-centre clinical trials have similarly provided valuable data on the ability of treatments, such as those providing improved glycaemic control, to inhibit the development and/or progression of retinopathy. Intensive treatment to normalize blood glucose levels resulted in a 63% reduction in retinopathy progression and a significant inhibition of other microvascular complications 26,116 in patients with diabetes. Furthermore, laser photocoagulation reduced the risk of severe visual loss by more than 50% in the eyes of patients with high-risk characteristics 117 . on the basis of positive preclinical studies 118 , a clinical study recently assessed the role of the angiotensin II antagonist candesartan in both type 1 and type 2 diabetes with or without early diabetic retinopathy 119, 120 . overall, this agent had a positive effect, with reduced progression and in some groups regression of retinopathy as measured by the ETDRs scale. There is also increasing evidence of benefits from intravitreal injections of steroids such as triamcinolone, often in conjunction with laser photocoagulation on vision-threatening retinopathy, including diffuse macular oedema 121, 122 . However, there are risks associated with intraocular steroid therapy, including increased intraocular pressure and enhanced cataract formation. several other clinical trials have not produced the outcomes that were hoped for, probably owing in part to insufficient study duration, slow development or progression of retinopathy, insufficient inhibition of the therapeutic target or a lack of involvement of the therapeutic target in the pathogenesis of the retinopathy [123] [124] [125] [126] . The development of non-invasive methods to assess the vascular and neural components of the eye is offering new opportunities to investigate the pathogenesis of retinopathy and to document responses to therapeutic intervention. Directed therapies against abnormalities that are believed to play key parts in the later stages of diabetic retinopathy, including inhibitors of PKC 127, 128 or vEGF 129 , are providing therapeutic tools to inhibit clinically important aspects of retinopathy in patients with diabetes (TABLe 1) . However, it is important to recognize that there is a disconnection between what is studied in animal models using high resolution microscopy and isolated tissue preparations, and what can currently be measured in patients with diabetes using colour fundus photographs, fluorescein angiograms and retinal function tests. New techniques, such as optical coherence tomography 130 and magnetic resonance imaging 131,132 , may be useful for both animal and clinical investigations and will hopefully lead to more interaction between preclinical and clinical scientists. Diabetic neuropathy affects the somatic and autonomic divisions of the peripheral nervous system, and there is a growing appreciation that the spinal cord 133 and higher central nervous system (CNs) 134 may also be damaged. over half of all patients with diabetes develop some form of neuropathy 135 , resulting in sensory loss, pain and autonomic dysfunction. These manifestations of neuropathy, along with their contribution to impaired wound healing and cardiovascular and erectile dysfunction, can severely reduce a patient's quality of life. As with the other complications discussed above, duration of diabetes and lack of adequate glycaemic control are important risk factors for neuropathy in both type 1 and type 2 diabetes 26, 116 . No therapy for diabetic degenerative neuropathy, other than maintenance of normoglycaemia, is approved by regulatory bodies in Europe and the United states, and current pain management strategies are not consistently effective and do not target the causes of diabetes-induced pain. The development of new therapies has been hindered by an incomplete understanding of the aetiological mechanisms involved, which is largely a reflection of the lack of a suitable animal model. Animal models of neuropathy. most studies have been carried out in rodent models of type 1 or type 2 diabetes, in which hyperglycaemia is induced by genetic, pharmacological or dietary manipulations. short-term rodent models of diabetes quickly develop a slowing of nerve conduction but generally lack overt demyelination and fibre loss in nerve trunks, which are prominent features of clinical diabetic neuropathy 136 . longer durations of diabetes may produce discernable pathology in nerve trunks of some rodent models 137, 138 , and the retraction of small sensory fibre terminals in the skin of short-term rodent models of diabetes 139,140 offers a measurement of nerve pathology that can be used in drug screening assays. The current rodent models of diabetic neuropathy may best reflect initial biochemical and functional disorders that precede degenerative neuropathy and could be useful for understanding early pathogenic events. However, there remains a prominent knowledge gap regarding the mechanisms of demyelination and neurodegeneration, such that a leap of faith has been required when translating any therapy that targets degenerative neuropathy from preclinical studies to clinical trials. The lack of overt degenerative neuropathy in most rodent models of diabetes may be due to their short lifespan and physically shorter axons. larger, long-lived diabetic animals have therefore also been used as alternative models. Diabetic dogs develop a slowing of nerve conduction and corneal hyposensitivity after years of hyperglycaemia 141, 142 , but degenerative neuropathy is minimal 143 Inappropriate oxidation of proteins, lipids and DNA that may change the function of these molecules, caused by excessive production of reactive oxygen species beyond the local capacity to remove them. A term generally used to encompass any molecule that supports neuronal growth and repair or prevents neuronal death. (FIG. 4) that replicates many features of the human condition 145, 146 . The extent to which the growing population of domestic cats with diabetes can be used to test potential therapies for degenerative neuropathy and act as a bridge between traditional preclinical models and clinical trials is currently being evaluated. Prevention, or preferably reversal, of the slowing of large-fibre conduction in diabetic rodents has historically been the preclinical 'gold standard' for establishing the therapeutic potential of drugs targeting degenerative diabetic neuropathy. over 100 compounds have been reported to prevent or reverse the slowing of large-fibre conduction, and the successful agents seem to target diverse pathogenic mechanisms. However, these have yet to translate to a successful therapy for diabetic neuro pathy. whether this reflects a complex and interconnected pathogenesis of diabetic neuropathy or the unsuitability of conduction defects in diabetic rodents for predicting efficacy in humans remains controversial. A more cautious approach that involves showing efficacy against both functional and structural indices of neuropathy may be warranted. many of the potential therapies that have been investigated to treat diabetic peripheral neuropathy have targeted mechanisms such as vascular dysfunction 147 , oxidative stress 148 and RAGE signalling 149 , all of which are common to all the diabetic complications (FIGs 1,2) . Evidence that impaired neurotrophic support contributes to diabetic peripheral and autonomic neuropathy 150,151 suggests a more organ-specific pathogenic mechanism (FIG. 1) , although some aspects of neurotrophic insufficiency are clearly downstream consequences of hyperglycaemia 152, 153 . several neuroactive proteins and polypeptides have been investigated as replacement therapies to counteract impaired neurotrophic support 150 , but difficulties with delivery routes and systemic effects have impeded clinical development. small molecules that promote endogenous production of neurotrophic factors have also shown some success in diabetic rodents [154] [155] [156] . most recently, gene therapy to facilitate endogenous production of nerve growth factor 157, 158 , hepatocye growth factor 159 , neurotrophin 3 (ReF. 160) or vEGF 161, 162 has been explored. Advancement of this approach must clearly incorporate the goal of delivering localized and regulated production of such factors. Impaired signalling through insulin receptors, whether driven by insulin deficiency or resistance, is also emer ging as a possible primary pathogenic mechanism that contributes to diabetes-induced damage to the nervous system. Peripheral nerves have insulin receptors 163 , and insulin treatment regimes that do not modulate circulating glucose levels can protect against neurochemical 164 , behavioural 165 and functional 166, 167 indices of neuropathy in diabetic rodents. This suggests that insulin acts as a support factor for peripheral nerves. As well as impeding neurotrophic support, reduced insulin signalling can also activate neurotoxic mechanisms. This is becoming increasingly apparent in the CNs, where deficient insulin signalling has been linked to accumulation of amyloid-β and hyperphosphorylation of the neuronal protein tau in models of Alzheimer's disease (ReF. 168) . Amyloid-β and hyperphosphorylated tau also accumulate in the brain of diabetic rats 169 and mice 170, 171 , in which there is evidence of synaptic and neuronal loss. There is continuing debate regarding the extent to which patients with type 1 and type 2 diabetes are prone to developing cognitive impairments and manifestations of dementia 172, 173 . Nevertheless, the apparent convergence of pathogenic mechanisms for these two common diseases of ageing on defective insulin signalling is attracting considerable interest 174 and may reveal new targets for therapeutic intervention. Therapeutic strategies to treat the pain associated with diabetes have traditionally drawn on experience from other neuropathic pain states rather than targeting diabetes-specific mechanisms; an example of this is the widespread use of tricyclic antidepressants 175 . The anticonvulsants gabapentin and pregabalin 176, 177 and the antidepressant duloxetine 178 have been used for alleviating diabetes-induced neuropathic pain, albeit with caveats concerning their efficacy, side effects, cost effectiveness 179 and a lack of effect on the underlying degenerative neuropathy. Although these treatments have emerged from broad anti-neuropathic pain programmes, preclinical studies suggest that a wider therapeutic window may exist in diabetes because of the increased expression of the presumed targets 180, 181 . Reports that diabetes increases the expression of other potential targets, such as peripheral κ-opioid receptors 182 and spinal cyclooxygenase 2 (ReF. 183) , may provide additional avenues for drug development. Clinical development of therapeutics. many agents have entered clinical trials to treat diabetic neuropathy. Twenty five years of failure to translate the mechanistic and therapeutic findings in animal models to a clinically effective treatment for diabetic degenerative neuropathy has prompted sharp divides in the research community. A distal sensory branch of the sciatic nerve trunk that passes close to the dermis at the ankle and can therefore be biopsied. An imaging technique that allows sensory neurons in the cornea of the eye to be viewed without tissue incision or invasion. specifically, some researchers see the animal models as being inadequate tools for discovering effective therapeutics and others think that the clinical trials have been poorly designed and executed or have relied on inappropriate or unreliable end points; both views probably have some merit. Approaches currently under investigation range from refinement of aldose reductase inhibition and other interventions that may have pan-organ efficacy to the neuronspecific targeting of neurotrophic support mechanisms (TABLe 1) . Previous clinical trials have largely relied on the slowing of nerve conduction and on sensory testing as quantifiable predictors of progressive degenerative neuropathy 184, 185 . sural nerve biopsies have also been used as a direct measure of nerve pathology 186, but the technique is invasive, difficult to quantify and does not allow multiple measurements. The continued failure to show an acceptable clinical efficacy of therapies that were developed from preclinical screening against slowing of nerve conduction in diabetic rodents could be due to mechanistic differences between species or the lack of reliable measures of early neuropathy. one advance that may assist drug development studies is the emerging use of skin biopsies as a minimally invasive measure of small fibre distal degenerative neuropathy 187, 188 . Epidermal fibre loss can be detected even before the onset of clinically overt diabetes 189 and is associated with functional indices of sensory loss 190 . Although the technique has currently been restricted to the evaluation of small sensory fibres and not of myelinated sensory, motor or autonomic fibres that are also affected by diabetes, it offers the advantage of providing a direct measurement of fibre loss that may ultimately result in loss of thermal sensation. Repeated biopsies can be carried out and allow monitoring of the progression of neuropathy and the effects of drug treatment. The use of corneal confocal microscopy to view changes in sensory nerve fibres in the eyes of patients with diabetes is also currently being evaluated against other measures of early neuropathy 191 . Corneal confocal microscopy offers the additional advantage of being entirely non-invasive, which allows many sequential measurements. It also facilitates local and topical drug delivery and can be used to detect nerve regeneration 192 . skin biopsy and corneal microscopy may therefore prove useful additions to future clinical trials of drugs that target diabetic neuropathy. It is likely that refinement of pancreatic transplantation and other approaches to maintaining long-term regulation of insulin and glucose levels, such as the artificial pancreas, will ultimately reduce the incidence and severity of diabetic complications. However, initial periods of poorly controlled diabetes can have a protracted negative effect on the subsequent protection that is afforded by improved glycaemic control 193 , and numerous patients have, or are in the process of developing, diabetic complications. These caveats provide sufficient concern to support efforts directed at understanding the pathogenic mechanisms that cause diabetic complications and at developing therapeutic interventions. The effects of diabetes on the organ systems described above emphasize that the main complications share numerous glucose-driven pathologic mechanisms (FIGs 1,2) . Targeting glucose-mediated vascular dysfunction with approaches such as inhibition of RAGE signalling, aldose reductase activity and oxidative stress is a particularly appealing therapeutic approach as it offers potential efficacy against multiple complications. moreover, some of these targets may also have additional organ-specific effects that are independent of their impact on vascular function. For example, aldose reductase also localizes in schwann cells of peripheral nerves and in cells of the kidneys and eyes 194, 195 , whereas RAGE expression is induced or increased by diabetes in each of these organs independently of its expression in blood vessels 78, 196, 197 . some treatments therefore have the potential to disrupt both universal and organ-specific pathogenic mechanisms. The limitations of current rodent models of diabetic complications, which tend to show early metabolic and functional disorders but lack marked structural pathology, are a cause for concern. The use of these models is driven in part by the desire of researchers to accelerate the onset of complications in inexpensive species that are amenable to genetic manipulation. Although current rodent models are useful for identifying the initial pathogenesis of diabetic complications and for studying the effect of potential prophylactic and early interventional therapies, there is persistent uncertainly as to whether investigators are studying mechanisms and drugs that are pertinent to overt pathological damage in humans. Another recurring theme is the difficulty in designing viable clinical trials and identifying which end points should be used as indicators of therapeutic efficacy. It is clear that the regulatory bodies are challenged by conflicting requirements to establish consistent end points which can be used in trials that may take many years to complete, while needing to have the flexibility to adopt new surrogate biomarkers as they are identified and validated by preclinical and clinical studies. The fact that diabetes disrupts such a diverse range of highly specialized organs also presents substantial challenges for drug development. The example of vEGF, for which inhibitors are being developed to treat diabetic retinopathy 129 and increased expression provoked to treat neuropathy 162 , illustrates the complexities that have to be considered when addressing individual complications within the context of a disease that impairs so many systems. Nevertheless, new animal models, creative drug delivery systems and improvements in clinical trial design and biomarkers will hopefully combine to accelerate the development of therapies for diabetic complications and improve approaches that are already being investigated. A single drug that is effective against all diabetic complications may not be a realistic goal. Therapeutic strategies that incorporate interventions targeting the glucose-mediated pathogenic axis outlined in FIG. 2, in combination with those that address organ-specific and glucose-independent mechanisms, may provide the most successful approach to drug development in this area. Diabetes mellitus in children and adolescents Lessons from UK prospective diabetes study The sorbitol pathway and the complications of diabetes An early attempt to outline a unifying hypothesis for diabetic complications, based on exaggerated glucose metabolism by aldose reductase Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus United Kingdom Prospective Diabetes Study 17: a 9-year update of a randomized, controlled trial on the effect of improved metabolic control on complications in non-insulindependent diabetes mellitus 10-year follow-up of intensive glucose control in type 2 diabetes Long-term follow-up after tight control of blood pressure in type 2 diabetes Chronic vascular inflammation in patients with type 2 diabetes: endothelial biopsy and RT-PCR analysis Microcirculation and oxidative stress Number and function of endothelial progenitor cells as a marker of severity for diabetic vasculopathy Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures Secular decline in mortality from coronary heart disease in adults with diabetes mellitus: cohort study Alteration of the blood-retinal barrier in experimental diabetes mellitus Age-induced hypercholesterolemia in the rat relates to reduced elimination but not increased intestinal absorption of cholesterol Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions Type 1 diabetes promotes disruption of advanced atherosclerotic lesions in LDL receptor-deficient mice Central role for aldose reductase pathway in myocardial ischemic injury Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice Recipes for creating animal models of diabetic cardiovascular disease Nonhuman primate models of atherosclerosis: potential for the study of diabetes mellitus and hyperinsulinemia A description of vascular damage in diabetic pigs that closely resembles the condition in humans and offers an animal model for late stages of diabetes Benfotiamine prevents macro-and microvascular endothelial dysfunction and oxidative stress following a meal rich in advanced glycation end products in individuals with type 2 diabetes Antagonism of RAGE suppresses peripheral nerve regeneration RAGE modulates peripheral nerve regeneration via recruitment of both inflammatory and axonal outgrowth pathways Statins for diabetic cardiovascular complications Effects of PPAR gamma agonists on cardiovascular function in obese, nondiabetic patients Heart Protection Study of cholesterollowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial Efficacy of cholesterol-lowering therapy in 18,686 people with diabetes in 14 randomised trials of statins: a meta-analysis Rosiglitazone attenuates atherosclerosis in a model of insulin insufficiency independent of its metabolic effects Assessment on the Prevention of Progression by Rosiglitazone on Atherosclerosis in diabetes patients with Cardiovascular History (APPROACH): study design and baseline characteristics Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitAzone Clinical Trial In macroVascular Events): a randomised controlled trial Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial Effect of fenofibrate on the need for laser treatment for diabetic retinopathy (FIELD study): a randomised controlled trial Effect of muraglitazar on death and major adverse cardiovascular events in patients with type 2 diabetes mellitus Effects of intensive glucose lowering in type 2 diabetes Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes Clinical review: the role of advanced glycation end products in progression and complications of diabetes UKPDS and the legacy effect Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia An epigenetic clue to diabetic vascular disease This study introduces evidence that short periods of hyperglycaemia can have long-term epigenetic effects by altering histone structure and function, providing a plausible mechanism for cellular metabolic memory of hyperglycaemia Effect of a multifactorial intervention on mortality in type 2 diabetes Projecting the number of patients with end-stage renal disease in the United States to the year 2015 The stages in diabetic renal disease. With emphasis on the stage of incipient diabetic nephropathy UK Prospective Diabetes Study (UKPDS). X. Urinary albumin excretion over 3 years in diet-treated type 2, (non-insulin-dependent) diabetic patients, and association with hypertension, hyperglycaemia and hypertriglyceridaemia Diabetic management in patients with renal failure Pathogenesis, prevention, and treatment of diabetic nephropathy Nephropathy in diabetes Aliskiren combined with losartan in type 2 diabetes and nephropathy ACE2 deficiency modifies renoprotection afforded by ACE inhibition in experimental diabetes Combination therapy with AT1 blocker and vitamin D analog markedly ameliorates diabetic nephropathy: blockade of compensatory renin increase Mouse models of diabetic nephropathy Impact of genetic background on nephropathy in diabetic mice Development of late-stage diabetic nephropathy in OVE26 diabetic mice Structural-functional relationships in diabetic nephropathy Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE) RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy Interaction of metabolic and haemodynamic factors in mediating experimental diabetic nephropathy Long-term renal effects of a neutralizing RAGE antibody in obese type 2 diabetic mice Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-β antibody in db/db diabetic mice Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease These studies in rats show that AGE formation and disruption of the RAS may not be entirely independent mediators of diabetic nephropathy and that interaction of these two pathogenic mechanisms may occur Role of advanced glycation end products in diabetic nephropathy The effect of ruboxistaurin on nephropathy in type 2 diabetes Mechanisms of kidney fibrosis and the role of antifibrotic therapies Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes The future of clinical trials in chronic renal disease: outcome of an NIH/FDA/Physician Specialist Conference. Evaluation of Clinical Trial Endpoints in Chronic Renal Disease Study Group Nonalbuminuric renal insufficiency in type 2 diabetes Prediction of diabetic nephropathy using urine proteomic profiling 10 years prior to development of nephropathy National diabetes fact sheet: general information and national estimates on diabetes in the United States (Department of Health and Human Services Diabetic retinopathy Patterns of ischemia in diabetic retinopathy The prevalence of diabetic retinopathy among adult type 1 diabetic persons in the United States The prevalence of diabetic retinopathy among adults in the United States Retinopathy in animal models of diabetes Retinal and Choroidal Angiogenesis Animal models of oxygeninduced retinopathy Inducible expression of vascular endothelial growth factor in adult mice causes severe proliferative retinopathy and retinal detachment Characterisation of a model for retinal neovascularisation. VEGF model characterisation Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin Vascular and neural changes in the rat optic nerve following induction of diabetes with streptozotocin The Ins2Akita mouse as a model of early retinal complications in diabetes Progression of incipient diabetic retinopathy during good glycemic control A mouse model of diabetic retinopathy Experimental galactosemia produces diabetic-like retinopathy Diabetic-like retinopathy in rats prevented with an aldose reductase inhibitor The RAGE axis in early diabetic retinopathy Ocular neovascularization: basic mechanisms and therapeutic advances A description of the role of inflammatory and immunological molecules in the development of early and late stages of diabetic retinopathy, and of potential therapeutic approaches to inhibit these processes Grading diabetic retinopathy from stereoscopic color fundus photographs -an extension of the modified Airlie House classification Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes Laser treatment in diabetic retinopathy The renin-angiotensin system influences ocular endothelial cell proliferation in diabetes: transgenic and interventional studies Effect of candesartan on prevention (DIRECT-Prevent 1) and progression (DIRECT-Protect 1) of retinopathy in type 1 diabetes: randomised, placebo-controlled trials Effect of candesartan on progression and regression of retinopathy in type 2 diabetes (DIRECT-Protect 2): a randomised placebo-controlled trial Efficacy and safety of multiple intravitreal triamcinolone injections for refractory diabetic macular oedema Triple therapy of vitrectomy, intravitreal triamcinolone, and macular laser photocoagulation for intractable diabetic macular edema A randomized trial of sorbinil, an aldose reductase inhibitor, in diabetic retinopathy. Sorbinil Retinopathy Trial Research Group Effects of aspirin treatment on diabetic retinopathy. ETDRS report number 8. Early Treatment Diabetic Retinopathy Study Research Group The effect of ruboxistaurin on visual loss in patients with moderately severe to very severe nonproliferative diabetic retinopathy: initial results of the Protein Kinase C β Inhibitor Diabetic Retinopathy Study (PKC-DRS) multicenter randomized clinical trial Effect of ruboxistaurin in patients with diabetic macular edema: thirty-month results of the randomized PKC-DMES clinical trial Effect of ruboxistaurin on visual loss in patients with diabetic retinopathy Effect of ruboxistaurin on the visual acuity decline associated with long-standing diabetic macular edema Vascular endothelial growth factor and the potential therapeutic use of pegaptanib (macugen) in diabetic retinopathy Three-dimensional optical coherence tomography of proliferative diabetic retinopathy Impaired apparent ion demand in experimental diabetic retinopathy: correction by lipoic acid Early supernormal retinal oxygenation response in patients with diabetes Early involvement of the spinal cord in diabetic peripheral neuropathy Microvascular disease in type 1 diabetes alters brain activation: a functional magnetic resonance imaging study Diabetes mellitus and its degenerative complications: a prospective study of 4,400 patients observed between 1947 and 1973 (author's transl Peripheral nerve structure and function in experimental diabetes Distal degenerative sensory neuropathy in a long term type 2 diabetes rat model Degeneration of the Golgi and neuronal loss in dorsal root ganglia in diabetic BioBreeding/Worcester rats Erythropoietin both protects from and reverses experimental diabetic neuropathy Noninvasive monitoring of diabetes-induced cutaneous nerve fiber loss and hypoalgesia in Thy1-YFP transgenic mice Nerve conduction and aldose reductase inhibition during 5 years of diabetes or galactosaemia in dogs Corneal sensitivity in dogs with diabetes mellitus Nerve pathology in the type 1 diabetic dog: effects of treatment with sulindac Differential hypertrophy and atrophy among all types of cutaneous innervation in the glabrous skin of the monkey hand during aging and naturally occurring type 2 diabetes Endoneurial microvascular pathology in feline diabetic neuropathy This paper documents the first species to show nerve pathology during diabetes that replicates most features of human diabetic neuropathy Vascular factors and metabolic interactions in the pathogenesis of diabetic neuropathy Diabetic neuropathy and oxidative stress RAGE, diabetes, and the nervous system Growth factors as therapeutics for diabetic neuropathy Glucose neurotoxicity Aldose reductase inhibition increases CNTF-like bioactivity and protein in sciatic nerves from galactose-fed and normal rats Neurotrophin-3-induced production of nerve growth factor is suppressed in Schwann cells exposed to high glucose: involvement of the polyol pathway Protection of sensory function in diabetic rats by neotrofin A vitamin D(3) derivative (CB1093) induces nerve growth factor and prevents neurotrophic deficits in streptozotocin-diabetic rats Orally active neurotrophinenhancing agent protects against dysfunctions of the peripheral nerves in hyperglycemic animals Herpes simplex-mediated gene transfer of nerve growth factor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse HSV-1-mediated NGF delivery delays nociceptive deficits in a genetic model of diabetic neuropathy Nonviral gene transfer of human hepatocyte growth factor improves streptozotocininduced diabetic neuropathy in rats Continuous delivery of neurotrophin 3 by gene therapy has a neuroprotective effect in experimental models of diabetic and acrylamide neuropathies HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy Gene transfer of an engineered transcription factor promoting expression of VEGF-A protects against experimental diabetic neuropathy Insulin receptor in rat peripheral nerve: its localization and alternatively spliced isoforms Insulin prevents depolarization of the mitochondrial inner membrane in sensory neurons of type 1 diabetic rats in the presence of sustained hyperglycemia The effect of low-dose insulin on mechanical sensitivity and allodynia in type I diabetes neuropathy Direct insulin signaling of neurons reverses diabetic neuropathy Insulin deficiency rather than hyperglycemia accounts for impaired neurotrophic responses and nerve fiber regeneration in type 1 diabetic neuropathy The role of insulin and neurotrophic factor signaling in brain aging and Alzheimer's disease Alzheimer-like changes in rat models of spontaneous diabetes The pathological interaction between diabetes and presymptomatic Alzheimer's disease Defective insulin signaling pathway and increased GSK-3 activity in the brain of diabetic mice: parallels with Alzheimer's disease and correction by insulin Long-term effect of diabetes and its treatment on cognitive function Diabetes and other vascular risk factors for dementia: which factor matters most? A systematic review Common pathological processes in Alzheimer disease and type 2 diabetes: a review EFNS guidelines on pharmacological treatment of neuropathic pain Gabapentin dosing for neuropathic pain: evidence from randomized, placebocontrolled clinical trials Efficacy of pregabalin in neuropathic pain evaluated in a 12-week, randomised, doubleblind, multicentre, placebo-controlled trial of flexible-and fixed-dose regimens A double-blind, randomized multicenter trial comparing duloxetine with placebo in the management of diabetic peripheral neuropathic pain Switching from pathogenetic treatment with α-lipoic acid to gabapentin and other analgesics in painful diabetic neuropathy: a real-world study in outpatients Changes of sodium channel expression in experimental painful diabetic neuropathy Injury type-specific calcium channel α 2 δ 1 subunit up-regulation in rat neuropathic pain models correlates with antiallodynic effects of gabapentin Dynorphin A, kappa opioid receptors and the antinociceptive efficacy of asimadoline in streptozotocin-induced diabetic rats Pathogenesis of spinally mediated hyperalgesia in diabetes Value of repeated measures of nerve conduction and quantitative sensory testing in a diabetic neuropathy trial Long-term effects of ranirestat (AS-3201) on peripheral nerve function in patients with diabetic sensorimotor polyneuropathy Effect of aldose reductase inhibition on nerve conduction and morphometry in diabetic neuropathy EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy Skin biopsy as a diagnostic tool in peripheral neuropathy Epidermal nerve innervation in impaired glucose tolerance and diabetes-associated neuropathy Intraepidermal nerve fibers are indicators of small-fiber neuropathy in both diabetic and nondiabetic patients Surrogate markers of small fiber damage in human diabetic neuropathy This study provides evidence that both skin biopsies and corneal confocal microscopy can detect early evidence of nerve damage in diabetic patients Corneal confocal microscopy detects early nerve regeneration after pancreas transplantation in patients with type 1 diabetes Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus Immuno histochemical localization of aldose reductase. II. Rat eye and kidney Immunohistochemical localization of aldose reductase. I. Enzyme purification and antibody preparationlocalization in peripheral nerve, artery, and testis Upregulation of RAGE and its ligands in proliferative retinal disease Receptor for advanced glycation end products (RAGEs) and experimental diabetic neuropathy The authors declare competing financial interests: see web version for details.