Diabetes Care

Volume 22 Supplement 2
Improving Prognosis in Type 1 Diabetes
Proceedings from an Official Satellite Symposium
of the 16th International Diabetes Federation Congress

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Advanced Glycosylated End Products and Hyperglycemia in the Pathogenesis of Diabetic Complications

Eli A. Friedman, MD

Protein alteration resulting from a nonenzymatic reaction between ambient glucose and primary amino groups on proteins to form glycated residues called Amadori products is termed the Maillard reaction. By dehydration and fragmentation reactions, Amadori products are transformed to stable covalent adducts called advanced glycosylation end products (AGEs). In diabetes, accelerated synthesis and tissue deposition of AGEs is proposed as a contributing mechanism in the pathogenesis of clinical complications. Uremia in diabetes is associated with both a high serum level of AGEs and accelerated macro- and microvasculopathy. Diabetic uremic patients accumulate advanced glycosylated end products in "toxic" amounts that are not decreased to normal by hemodialysis or peritoneal dialysis but fall sharply to within the normal range within 8 h of restoration of half-normal glomerular filtration by renal transplantation. It follows that the higher mortality of hemodialysis-treated diabetic patients compared with those given a renal transplant may relate, in part, to persistent AGE toxicity. Pharmacologic prevention of AGE formation is an attractive means of preempting diabetic microvascular complications because it bypasses the necessity of having to attain euglycemia, an often unattainable goal. Pimagidine (aminoguanidine) interferes with nonenzymatic glycosylation and reduces measured AGE levels leading to its investigation as a potential treatment. The mechanism by which pimagidine prevents renal, eye, nerve, and other microvascular complications in animal models of diabetes is under investigation. Separate multicenter clinical trials of pimagidine in type 1 and type 2 diabetes, where proteinuria is attributable to diabetic nephropathy, are in progress. The effect of treatment on the amount of proteinuria, progression of renal insufficiency, and the course of retinopathy will be monitored.

Diabetes Care 22 (Suppl. 2):B65–B71, 1999

Attention to the pathogenesis of tissue and organ damage in diabetic patients has increased in proportion with their growing acceptance for treatment of renal failure. End-stage renal disease (ESRD) registries in the U.S., Japan, and most nations in industrialized Europe show that diabetes, in 1997, was the leading cause of renal failure worldwide. Mauer and Chavers (1) aptly predicted over a decade ago that "diabetes is the most important cause of ESRD in the Western world." Furthermore, in every European and North American registry of renal failure patients, both the incidence and prevalence of diabetic patients has risen continuously over the past 10 years. According to the 1997 report of the United States Renal Data System, of 257,266 U.S. patients receiving either dialytic therapy or a kidney transplant in 1995, 80,667 had diabetes (2)—a prevalence rate of 31.4%. Moreover, of 71,875 new (incident) cases of ESRD in 1995, 28,740 (40%) had diabetes (Fig. 1).

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Figure 1Diabetes is the disease of greatest prevalence and incidence in ESRD registries throughout the industrialized world. According to the most recent report from the U.S. Renal Data System (USRDS), diabetes accounted for 40% of new ESRD patients in 1995.

HYPERGLYCEMIA RESPONSIBLE FOR VASCULAR COMPLICATIONS— How diabetes induces perturbed micro- and macrovascular function and organ injury is far from clear (Fig. 2). Hyperglycemia is increasingly linked to the pathogenesis of nephropathy, retinopathy, and atherosclerosis in individuals with long-duration diabetes (3). Vascular endothelial cells in those with diabetes exhibit multiple cellular and molecular abnormalities that presage vascular damage (4). Mechanisms proposed to explain the metabolic pathway between a high ambient glucose concentration and end organ damage in diabetes include (5) 1) activation of the aldose-reductase pathway, leading to toxic accumulation of sorbitol in nerves; 2) accelerated nonenzymatic glycosylation with deposition of advanced glycosylated end products (AGEs) (6); and 3) activation of isoform(s) of protein kinase C (PKC) in vascular tissue, initiating a cascade of events culminating in diabetic complications (7).

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Figure 2—A: A simplistic view of the association between hyperglycemia and kidney injury used until specific intermediary metabolites and kinins were identified. B: Multiple steps, several still obscure, take place as the impact of high ambient glucose levels are transmitted to end organs. C: Variables linked to the progression of renal damage in association with hyperglycemia are shown, though neither the start point nor the end point of their interaction has been defined.

PROTEIN KINASE C— Indicting a central role for PKC is attractive because PKC activity is increased in renal glomeruli, retina, aorta, and heart of diabetic animals, probably due to increased synthesis de novo of diacylglycerol, a major endogenous activator of PKC (8). Substantive evidence that PKC is involved in diabetic complications has been gleaned from experiments in induced diabetic rats: 1) PKC is activated in glomeruli isolated from diabetic rats (9); 2) activation of PKC by either intravitreal injection of the PKC agonist phorbol 12,13-dibutyrate (10) or by exposure of granulation tissue to the PKC agonist 12-O-tetradecanolylphorbol-3-acetate in normal rats reproduces the vascular abnormalities induced by high glucose levels in diabetes (11); and 3) mesangial cells cultured in high (27.8 mmol/l) concentration of glucose for 5 days increase PKC and mitogen-activated protein kinase activity in their membrane fraction, supporting the hypothesis that hyperglycemia induces abnormalities in the glomerular mesangium (12). Additionally, in glomeruli isolated from streptozotocin-induced diabetic rats, PKC promotes elevated levels of mRNA encoding matrix components, and matrix synthesis is
accelerated (13).

Support for PKC action in the genesis of diabetic complications is also derived from experiments in which an orally effective inhibitor of PKC-2 ameliorated vascular dysfunction in streptozotocin-induced diabetic rats (14). LY333531, the macrocyclic bis(indolyl)maleimidestructure (LY333531), selectively inhibits PKC-—normalizing renal functional perturbations in diabetic rats—but has no effect on renal function in normal rats. After oral treatment with LY333531 (1.0 and 10 mg/kg) for 8 weeks, glomerular filtration rate—which had increased from 3.0 ± 0.2 ml/min in nondiabetic rats to 4.6 ± 0.4 ml/min in diabetic rats—returned to normal. At the same time, the urinary albumin excretion rate was reduced from 11.7 ± 0.5 to 4.9 ± 1.6 mg/day (normal 1.6 ± 0.5 mg/day). Whether these early trials of PKC inhibition in diabetes will have clinical application is speculative. It is encouraging to note, however, that an effective treatment strategy was documented without a requirement for euglycemia.

ALDOSE REDUCTASE— Labeled "the longest running controversy among researchers and clinicians studying this disease" (15), the role of sorbitol in diabetic complications has been and is continuously debated. In induced diabetic rats, detection of excess sorbitol in cataracts (16) stimulated the polyol/sorbitol hypothesis that high ambient glucose levels damage cells by increasing intracellular osmolality and decreasing myo-inositol levels, thereby altering Na+/K+ ATPase activity (nerves) or shifting redox potential in cells. Sorbitol production in diabetic rats is markedly enhanced by hyperglycemia, which leads to its accumulation and injury to cells (17). Significantly higher levels of erythrocyte aldose reductase have been noted in type 2 patients with either proliferative or nonproliferative retinopathy than in type 2 patients without retinopathy, despite equivalent mean HbA1c and blood pressure levels. Detracting from a central role for aldose reductase, however, is the observation that erythrocyte aldose reductase activity in type 2 patients does not correlate with age, duration of diabetes, fasting blood glucose, or HbA1c (18). Several large clinical trials of drugs that block aldose reductase, including spirohydantoins (sorbinil), carboxylic acid derivatives (tolrestat and ponalrestat), and flavonoids, have been disappointing. Although some positive results have been reported, the benefits have been of no clinical moment. Presently, tolrestat is the only aldose reductase inhibitor being tested in the U.S., in a multicenter trial for diabetic nephropathy, neuropathy, and retinopathy.

AGEs— Throughout life, reducing sugars such as glucose react nonenzymatically and reversibly with free amino groups in proteins to form small amounts of stable Amadori products through Schiff base adducts. During aging, spontaneous further irreversible modification of proteins by glucose results in the formation of a series of compounds termed AGEs, a heterogeneous family of biologically and chemically reactive compounds with cross-linking properties. In diabetes, this continuous process of protein modification is magnified by high ambient glucose concentrations (19).

The extent of protein binding by Amadori products is proportional to the degree and duration of hyperglycemia (20). Amadori products undergo dehydration and rearrangements to produce highly reactive carbonyl compounds, including 3-deoxyglucosane (3-deoxy-D-erythro-hexos-2-ulose [3-DG]) (21). 3-DG in turn reacts with free amino groups, leading to cross-linking and the so-called browning of proteins as AGEs accumulate in the Maillard reaction. Candidate active AGE compounds include N-(carboxymethyl)-L-lysine (CML) (22), pyrraline, pentosidine (23), and cross-links (24). Of late, Niwa et al. (25) demonstrated that several imidazolones, the reaction products of the guanidino group of arginine with 3-DG, are common epitopes of AGE-modified proteins produced in vitro and are significantly increased in erythrocytes of diabetic patients. Through immunologic histopathological techniques, imidazolone has been noted in nodular lesions and expanded mesangial matrix of glomeruli and renal arteries in diabetic nephropathy (25). CML, an oxidative product of glycated proteins, accumulates in arteries, atherosclerotic plaques, foam cells, and serum proteins in diabetic patients, which suggests that it may be an endogenous marker of oxidative tissue damage and glomerulopathy in diabetic individuals (26).

AGEs bind to specific receptors on endothelial cells (27). After cellular attachment, AGEs have been shown to increase vascular permeability, procoagulant activity, adhesion molecule expression, and monocyte influx, actions that may contribute to vascular injury (28).

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Figure 3ESRD death rates from 1993 to 1995. Excess mortality in ESRD attributable to diabetes is evident in this comparison of treatment by dialysis and renal transplantation in diabetic and nondiabetic patients. According to the most recent report from the U.S. Renal Data System (USRDS), diabetic ESRD patients suffer higher mortality, whether treated by dialysis or a kidney transplant. Note that the superior survival in transplant recipients compared with those diabetic patients treated by dialysis may reflect both selection bias in assigning those with less comorbidity to a transplant as well as the impact of AGE toxicity on dialysis patients. The dialysis groups are composed of both peritoneal- and hemodialysis-treated patients.

AGE peptides are normally excreted by the kidney; their plasma concentration is inversely proportional to the glomerular filtration rate. As diabetic patients develop renal insufficiency, there is a progressive and marked increase in plasma and tissue levels of AGEs. Vlassara's group (29) recently demonstrated that renal excretion of orally absorbed AGEs is suppressed in individuals with diabetic nephropathy, a perturbation that may contribute to progressive elevation of plasma AGE levels as residual renal function declines. Neither peritoneal dialysis nor hemodialysis decrease the high level of AGEs noted in uremic diabetic patients to normal. Within days of restoration of half-normal glomerular filtration by transplantation of a single kidney, AGE levels fall sharply to within the normal range. Based on the foregoing, the markedly greater mortality of diabetic ESRD patients treated by dialysis when compared with diabetic recipients of renal allografts (Figs. 3 and 4) has been attributed—at least in part—to the toxicity of AGEs.

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Figure 4Survival during treatment for ESRD is compared in diabetic patients treated by cadaver donor kidney transplant, live donor kidney transplant, or dialysis. In the most recent report from the U.S. Renal Data System (USRDS), survival of peritoneal dialysis and hemodialysis patients is grouped together. AGEs retained by dialysis patients may be toxic, contributing to death from vascular complications of accelerated atherosclerosis. This hypothesis is being tested in a clinical trial of aminoguanidine, an AGE blocking agent, conducted in patients undergoing maintenance hemodialysis.

INFERENCES OF AGE TOXICITY DRAWN FROM ANIMAL MODELS OF DIABETES— A number of observations in diabetic animals (mainly in the rat) are compatible with a pathogenetic role for AGEs in microvascular disease. Vascular dysfunction resembling that seen in diabetes is observed after administration of AGEs to nondiabetic rabbits and rats (30). The administration of AGE-modified albumin to nondiabetic rats for 4 weeks causes glomerular hypertrophy and increased extracellular matrix production in association with activation of the genes for collagen, laminin, and transforming growth factor- (31).

Similar changes are noted when AGEs alone are given to achieve plasma concentrations equivalent to those seen in diabetic animals (32). After 5 months, the renal AGE content in AGE-treated rats was 50% above that in controls, while the plasma concentration was 2.8 times greater than that of controls. AGE-treated rats had a 50% expansion in glomerular volume, basement membrane widening, and increased mesangial matrix, which indicates significant glomerulosclerosis compared with untreated controls.

AGEs probably contribute to the rapidly progressive atherosclerosis that develops in patients with diabetes and renal insufficiency. AGEs promote an influx of mononuclear cells and stimulate cell proliferation (33,34). Utilizing AGE-specific enzyme-linked immunosorbent assay techniques, AGEs are identified in vascular-wall, lipoprotein, and lipid constituents of atherosclerotic lesions (35). Within the atherosclerotic lesion, collagen-linked AGEs bind plasma proteins, interact with macrophage receptors to induce cytokine and growth factor release, and quench nitric oxide activity (36). There is also evidence that they modify LDL, making it less susceptible to clearance by LDL receptors. In one study, for example, LDL modified in vitro by AGE peptides (at the concentration present in azotemic diabetic patients) markedly impaired LDL clearance when injected into transgenic mice expressing the human LDL receptor.

Study of coronary arteries obtained from patients with type 2 diabetes using immunohistochemical analysis detected high levels of AGE reactivity within atherosclerotic plaques stained with anti-AGE antibodies (37). This observation is consistent with a link between hyperglycemia, hyperlipidemia, and atherosclerosis in diabetes. AGE formation also contributes to development of diabetic complications by changing the structure and function of the extracellular matrix in the glomerular mesangium and elsewhere. In type IV collagen from basement membrane, for example, AGE formation decreases binding of the noncollagenous NC1 domain to the helix-rich domain, thereby interfering with the lateral association of these molecules into a normal lattice structure (38). In addition, alterations by AGEs of type I collagen, a substance also found in the glomerular mesangium, expand molecular packing. These alterations to the integrity of collagen adversely affect biological functions important to normal vascular tissue integrity, such as reaction to endothelium-derived relaxing factor (nitric oxide) and antiproliferative
factors (39).

A central effector pathway for AGE action may be the consequence of impaired nitric oxide synthesis and action. In rodents, AGEs impair a variety of important nitric oxide–mediated processes, including neurotransmission, wound healing (40), blood flow in small vessels, and decreased cell proliferation (41). It follows that the toxicity of AGEs may be mediated in part by their interference with the actions of nitric oxide (42).

ACTION OF AMINOGUANIDINE— Pharmacological prevention of AGE formation is an attractive means of preventing diabetic microvascular complications because it bypasses the usually difficult goal of achieving euglycemia. Brownlee et al. (43) selected aminoguanidine because its structure is similar to -hydrazinohistidine, a compound known to reduce diabetes-induced vascular leakage. Over the past decade, a substantive literature has documented the efficacy of aminoguanidine in blocking or slowing progression of major diabetes-related organ damage in the induced diabetic rat and other models. Aminoguanidine is beneficial to the retina, kidney, nerve, and isolated arteries in streptozotocin-induced diabetes in the rat. In a key study in the retina, for example, aminoguanidine reduced the number of acellular capillaries (which is increased 19-fold in diabetes) by 80%. Aminoguanidine also blocks retinal capillary closure, the principal pathophysiological abnormality underlying diabetic retinopathy (44). Treatment with aminoguanidine completely prevents arteriolar deposition of periodic acid– Schiff–positive material and microthrombus formation after 26 weeks of induced diabetes in spontaneous hypertensive rats (45). Cataracts are prevented in rats 90 days after the rats are made "moderately diabetic" (<350 mg/dl plasma glucose); lens-soluble and -insoluble AGE fractions were inhibited by 56 and 75%, respectively, by treatment with 25 mg/kg body weight of aminoguanidine starting from the day of streptozotocin injection (46).

Experimental diabetic glomerulopathy is virtually preempted by continuous treatment with aminoguanidine starting at the time of induction of hyperglycemia. Major renal abnormalities minimized or blocked entirely by aminoguanidine include proteinuria, mesangial matrix expansion, matrix gene expression, and basement membrane thickness (47). Treatment with aminoguanidine also diminishes the deposition of AGEs in glomeruli and tubules, a benefit directly related to duration of therapy (48). AGE accumulation (measured by tissue fluorescence) in glomeruli and renal tubules in rats 32 weeks after induction of diabetes is prevented by aminoguanidine. By contrast, ponalrestat, an aldose reductase inhibitor, does not block AGE accumulation. Demonstration by Cohen et al. (49) of a similar benefit by administration of antibodies directed against glycated albumin is further evidence for a pathogenetic role of AGEs.

Experimental diabetic neuropathy in the rat is attenuated by aminoguanidine, as evidenced by increased motor nerve conduction velocity and diminished neural accumulation of AGEs (38). Abnormally slowed sciatic nerve conduction velocity improves after treatment at doses of 10–50 mg/kg for 16 weeks (50). In the diabetic rat, the "stiff myocardium" that is a main component of diabetic cardiomyopathy (51) is prevented by aminoguanidine in a dose of 7.35 mmol · kg–1 · dl–1 for 4 months, as indicated by both decreased myocardial AGE accumulation and increased left ventricular end-diastolic compliance.

AMINOGUANIDINE ACTION— Aminoguanidine prevents formation of reactive AGEs and their subsequent cross-linking with albumin, leading to a reduction in AGE levels (52). It has been proposed that aminoguanidine may act as a glucose competitor for the same protein-to-protein bond that becomes the link for the formation and tissue accumulation of irreversible and highly reactive AGEs (53). Another action of aminoguanidine may be a vascular protective effect. In one study, for example, aminoguanidine and methylguanidine normalized vascular albumin permeability in diabetic rats, even though only aminoguanidine inhibited AGE formation (54). One possible explanation is that both guanidines inhibit nitric oxide synthase, sustaining the inference that nitric oxide contributes to vascular dysfunction in diabetes (55). Supporting this view is the observation that treatment of streptozotocin-induced diabetic rats with aminoguanidine for 2 months at a dose of 1 g · kg–1 · day–1 prevented diabetes-induced 24% impairment in maximal endothelium-dependent relaxation to acetylcholine for phenylephrine precontracted aortas (56).

Studies in rodents suggest that AGEs exert their toxicity by impairing nitric oxide–mediated vital processes, including neurotransmission, wound healing, and blood flow in small vessels (57). Thus, AGEs, by blocking the synthesis of nitric oxide, almost certainly interfere with maintenance of normal physiological processes such as autoregulation of blood flow. The myriad actions of nitric oxide pertinent to nephrologists have been recently reviewed (58).

The rapidity of aminoguanidine correction of nerve conduction velocity and nerve blood flow deficits in streptozotocin-induced diabetic rats suggests a neurovascular mechanism involving improved action of nitric oxide. Such quick action is consistent with inhibition of free radical production by autoxidative glycosylation or glycoxidation.

CLINICAL TRIALS OF AMINOGUANIDINE IN DIABETES— Concurrent multicenter clinical trials are in progress to evaluate the effect of aminoguanidine (Pimagidine) on the course of diabetic nephropathy after onset of clinical proteinuria in type 1 and type 2 diabetes. Adults with documented diabetes, fixed proteinuria >500 mg/day, and a plasma creatinine concentration >1.0 mg/dl (88 µmol/l) in women and >1.3 mg/dl (115 µmol/l) in men have been randomly assigned to treatment with aminoguanidine or placebo for 4 years. The effect of treatment on the amount of proteinuria, progression of renal insufficiency, and the course of retinopathy will be monitored. An additional multicenter clinical trial of aminoguanidine in diabetic patients undergoing maintenance hemodialysis was initiated in mid-1996. The hypothesis under evaluation is that the higher mortality of diabetic patients on dialysis (both peritoneal and hemodialysis) as contrasted with survival of diabetic patients after a kidney transplant reflects, in part, toxicity of AGEs, the level of which falls to near-normal after a successful allograft is in situ.

OTHER INNOVATIVE STRATEGIES TO REDUCE AGE TOXICITY— Authenticating the case for seminal participation of AGEs in the genesis of diabetic complications stimulated an intense quest for methods and techniques to minimize AGE deposition. Although aminoguanidine inhibits initial stages of glycation in the presence of high concentrations of glucose, it shows minimal inhibition of post-Amadori AGE formation in RNase and bovine serum albumin. In this system, pyridoxamine and thiamine pyrophosphate are effective post-Amadori inhibitors that decrease the quantity of AGEs formed (59). This observation appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential.

Other novel inhibitors of advanced glycation have salutary effects on the course of diabetic glomerulopathy in the rat. OPPB-9195, a thiazolidine derivative, lowered serum levels of AGEs and attenuated AGE deposition in glomeruli of the Otsuka-Long-Evans-Tokushima-Fatty rat, a model of type 2 diabetes (60). Another direction of AGE prevention being explored is being followed by Shoda et al. (61), who found that tenilsetam, an antidementia drug, retarded both glucose and fructose-induced polymerization of lysozyme, restoring the enzymatic digestibility of collagen by preventing AGE formation. It may also prove possible to cleave established AGE cross-links; AGEs irreversibly bind to macromolecules through covalently cross-linked proximate amino groups. A prototypic AGE cross-link "breaker," N-phenacylthiazolium bromide, separates AGE cross-links in vivo in induced diabetic rats, suggesting an alternate means of interrupting the diabetes-induced hyperglycemia to tissue injury sequence (62).

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From the Department of Medicine, State University of New York, Health Science Center at Brooklyn, Brooklyn, New York.

Address correspondence and reprint requests to Eli A. Friedman, Department of Medicine, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Ave., Brooklyn, NY 11203. E-mail: elifriedmn@aol.com.

Received for publication 27 May 1998 and accepted in revised form 20 August 1998.

E.A.F. is a paid consultant of the Alteon Corporation.

Abbreviations: 3-DG, 3-deoxy-D-erythro-hexos-2-ulose; AGE, advanced glycosylated end product; CML, N-(carboxymethyl)-L-lysine; ESRD, end-stage renal disease; PKC, protein kinase C.

This article is based on a presentation at a satellite symposium of the 16th International Diabetes Federation Congress. The symposium and the publication of this article were made possible by educational grants from Hoechst Marion Roussel AG.

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