Aminoguanidine inhibits albuminuria, but not the formation of advanced glycation end-products in skin collagen of diabetic rats

Aminoguanidine, an inhibitor of advanced glycation reactions in vitro, inhibits the development of diabetic complications in animal models of diabetes, suggesting that it acts by inhibition of advanced glycation reactions in vivo. However, effects of aminoguanidine on the formation of specific advanced glycation end-products (AGEs) in vivo have not been rigorously examined. Therefore, we studied the effects of aminoguanidine on the formation of pentosidine and N(epsilon)-(carboxymethyl)lysine (CML), measured by analytical chemical methods, in collagen of streptozotocin-diabetic Lewis rats at doses which ameliorated urinary albumin excretion, an index of diabetic nephropathy. At 12 weeks, diabetic animals had fivefold higher blood glucose, threefold higher glycated hemoglobin and fivefold higher collagen glycation, compared to metabolically healthy controls; pentosidine and CML in skin collagen were increased by approximately 30 and 150%, respectively. Administration of aminoguanidine, 50 mg/kg by daily intraperitoneal injection, significantly inhibited the development of albuminuria (approximately 60%, P < 0.01) in diabetic rats, without an effect on blood glucose or glycation of hemoglobin or collagen. Surprisingly, aminoguanidine failed to inhibit the increase in pentosidine and CML in diabetic rat skin collagen. Similar results were obtained in an independent experiment in which aminoguanidine was administered in drinking water at a dose of 0.5 g/l. We conclude that the therapeutic benefits of aminoguanidine on albuminuria may not be the result of inhibition of AGE formation.     

The relative roles of advanced glycation,oxidation and aldose reductase inhibition in the development of experimental diabetic nephropathy in the Sprague-Dawley rat

Summary Advanced glycation is an important pathogenic mechanism in the development of diabetic complications. However, other biochemical processes, such as the polyol pathway or lipid and protein oxidation which can interact with advanced glycation can also yield tissue fluorescence and may also be implicated in the genesis of diabetic microangiopathy. Aminoguanidine is an inhibitor of advanced glycation, but it is not known if all of its effects are mediated by this mechanism. The present study explores the relative contributions of aldose reductase, oxidative stress and advanced glycation on the development of aortic and renal fluorescence and urinary albumin excretion in streptozotocin diabetic rats. The study groups included non-diabetic (control), streptozotocin diabetic rats and diabetic rats receiving aminoguanidine, the anti-oxidants butylated hydroxytoluene and probucol and the aldose reductase inhibitor, ponalrestat. Serial measurements of glycaemic control and urinary albumin excretion were performed every 8 weeks. At 32 weeks, animals were killed, tissues removed and collagen extracted for measurement of fluorescence. Diabetic rats had increased fluorescence in aorta, glomeruli and renal tubules. Aminoguanidine prevented an increase in fluorescence at all three sites suggesting that diabetes-related tissue fluorescence is predominantly due to advanced glycation. Ponalrestat retarded fluorescence in aorta only and butylated hydroxytoluene attenuated fluorescence at the renal sites but not in the aorta. Diabetic rats had increased renal cortical sorbitol levels. Ponalrestat normalized renal cortical sorbitol levels but aminoguanidine did not affect this parameter. The only agent to decrease plasma thiobarbituric acid reactive substances was butylated hydroxytoluene. Diabetic rats developed albuminuria over the 32-week period. This increase in urinary albumin excretion was only attenuated significantly by aminoguanidine therapy, but not by probucol or ponalrestat. The effects of butylated hydroxytoluene on albuminuria were intermediate between aminoguanidine-treated and untreated diabetic rats. The failure of either antioxidants or aldose reductase inhibition to reproduce the renal effects of aminoguanidine suggest that aminoguanidine may act predominantly via inhibition of advanced glycation and not via the alternative biochemical processes evaluated in this study.Abbreviations AGE Advanced glycated end products - AG aminoguanidine - TBARS Thiobarbituric acid reactive substances - BHT butylated hydroxytoluene     

Depletion of nitric oxide synthase-containing neurons in the diabetic retina: reversal by aminoguanidine

Summary A close association of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) neurons with the retinal vasculature has been reported and it is proposed that activation of these neurons could be the mechanism by which retinal blood flow and metabolism are linked. Further, advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic retina and aminoguanidine (AG), an inhibitor of advanced glycation, has been shown to attenuate the development of AGE accumulation as well as the progression of experimental diabetic retinopathy. This study examined the effects of short (1 and 3 weeks) and long term (32 weeks) diabetes on nNOS-containing neurons of the retina using NADPH diaphorase (NADPHd) histochemistry. In addition, the effect of aminoguanidine (an inhibitor of advanced glycation and NOS) and N^G-nitro-L-arginine methyl ester (L-NAME) (a non-selective NOS inhibitor) on retinal nNOS-containing neurons was examined in short and long term control and diabetic rats. In a separate study, the effect of 2,3 diamino-phenazine (NN0028) (an inhibitor of advanced glycation, but not NOS) was examined in short term (3 weeks) diabetic rats. The number of NADPHd-positive neurons per retina was reduced after one week of diabetes and remained decreased in long term diabetic rats, an effect not observed in diabetic rats rendered euglycaemic by intensified insulin treatment. Treatment of diabetic animals with aminoguanidine or NN0028 prevented the depletion in the nNOS-containing neuron number, an effect not reproduced by L-NAME. These studies suggest that the action of AG in restoring the number of nNOS-containing retinal neurons is mediated by the inhibition of AGE formation. The depletion of nNOS-containing neurons may contribute to alterations in the autoregulation of blood flow which occurs in diabetes. [Diabetologia (1998) 41: 1419–1425] Received: 23 February 1998 and in final revised form: 4 August 1998     

Advanced glycation end products and their receptors co-localise in rat organs susceptible to diabetic microvascular injury

Summary Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs are increased in experimental diabetes and treatment with the inhibitor of advanced glycation end products, aminoguanidine, has been shown to attenuate the level of these products in tissues undergoing complications. Recently, an AGE-binding protein has been isolated from bovine lung endothelial cells and termed the receptor for advanced glycated end products (RAGE). The present study sought to determine the distribution of AGE and RAGE in tissues susceptible to the long-term complications of diabetes including the kidney, eye, nerve, arteries as well as in a tissue resistant to such complications, the lung. Using polyclonal antisera both AGE and RAGE were found to co-localize in the renal glomerulus. AGE staining was clearly increased with age and was further increased by diabetes. Aminoguanidine treatment reduced AGE accumulation in the kidney. Co-localisation of AGE and RAGE was demonstrated in the inner plexiform layer and the inner limiting membrane of the retina and in nerve bundles from mesenteric arteries. In the aorta, both AGE and RAGE were found in the intima, media and adventitia. Medial staining was increased in diabetes and was reduced by aminoguanidine treatment. A similar pattern was observed for RAGE in the aorta. In the lung, RAGE was found widely distributed throughout the lung whereas the distribution of AGE staining was more limited, primarily localising to macrophages. The co-localisation of AGEs and RAGE in sites of diabetic microvascular injury suggests that this ligand-receptor interaction may represent an important mechanism in the genesis of diabetic complications. [Diabetologia (1997) 40: 619–628] Received: 16 October 1996 and in final revised form: 17 February 1997     

Aminoguanidine treatment reduces the increase in collagen stability of rats with experimental diabetes mellitus

Summary Alterations in the biophysical properties of connective tissues in diabetes mellitus have been attributed to the nonenzymatic glycation of the collagens and the subsequent formation of browning products, cross-linking the proteins. Aminoguanidine may bind to carbonyl groups of these nonenzymatic glycation products and thereby block the process. Rats with streptozotocin-induced diabetes were treated with aminoguanidine, 25mg·kg^–1·day^–1, for 120 days. The aminoguanidine treatment did not counteract the increase in blood glucose concentrations, nor did it prevent the arrest in weight gain of diabetic rats. The increased stability in 7 mol/l urea and increased tensile strength of tail tendons from the diabetic rats, however, were prevented by the aminoguanidine treatment. Aminoguanidine did not reduce the formation of early nonenzymatic glycation products (aldimine and Amadori rearrangement products), whereas the amount of browning products (fluorescent compounds) was reduced in the tail tendon collagen of the diabetic rats. Aminoguanidine treatment of intact rats did not influence these parameters. These findings indicate that the biophysical alterations of collagens induced by experimental diabetes are caused by cross-links derived from the nonenzymatic glycation, and furthermore, that aminoguanidine treatment may prevent the concomitant changes in biophysical properties of connective tissues.     

Combination therapy with the advanced glycation end product cross-link breaker, alagebrium, and angiotensin converting enzyme inhibitors in diabetes: synergy or redundancy?

Blockade of advanced glycation end product (AGE) accumulation with alagebrium with concomitant angiotensin converting enzyme inhibition was tested for effects on renal function and on other postulated mediators of diabetic renal disease including the renin-angiotensin system, AGEs, mitochondrial and cytosolic oxidative stress, and intracellular signaling molecules. Sprague Dawley rats were rendered diabetic with streptozocin and followed consecutively for 32 wk with nondiabetic controls. Groups were treated with ramipril (1 mg/kg.d; wk 0-32); alagebrium (10 mg/kg.d; wk 16-32); or a combination of both. Although individual treatments had significant effects on albuminuria, no further improvements were seen with combination therapy. Changes in urinary vascular endothelial growth factor excretion mirrored those seen in albuminuria. Diabetes was associated with suppression of circulating angiotensin II in the context of increased circulating and renal levels of the AGE, carboxymethyllysine. All treatments attenuated circulating but not renal carboxymethyllysine levels. The renal gene expression of AGE receptor 1 and soluble receptor for advanced glycation end products were markedly reduced by diabetes and normalized with alagebrium. Diabetes induced renal mitochondrial oxidative stress, which was reduced with alagebrium. In the cytosol, both therapies were equally effective in reducing reactive oxygen species production. Increases in membranous protein kinase C activity in diabetes were attenuated by all treatments, whereas diabetes-associated increases in nuclear factor-kappaB p65 translocation remained unaltered by any therapy. It is evident that renin-angiotensin system blockade and AGE inhibition have specific effects. However, many of their downstream effects appear to be similar, suggesting that their renoprotective benefits may ultimately involve common pathways and key points of convergence, which could be important targets for new therapies in diabetic nephropathy.     

Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice

We hypothesized that formation of advanced glycation end products (AGEs) associated with diabetes reduces matrix degradation by metalloproteinases (MMPs) and contributes to the impairment of ischemia-induced angiogenesis. Mice were treated or not with streptozotocin (40 mg/kg) and streptozotocin plus aminoguanidine (AGEs formation blocker, 50 mg/kg). After 8 weeks of treatment, hindlimb ischemia was induced by right femoral artery ligature. Plasma AGE levels were strongly elevated in diabetic mice when compared with control mice (579 +/- 21 versus 47 +/- 4 pmol/ml, respectively; P < 0.01). Treatment with aminoguanidine reduced AGE plasma levels when compared with untreated diabetic mice (P < 0.001). After 28 days of ischemia, ischemic/nonischemic leg angiographic score, capillary density, and laser Doppler skin-perfusion ratios were 1.4-, 1.5-, and 1.4-fold decreased in diabetic mice in reference to controls (P < 0.01). Treatment with aminoguanidine completely normalized ischemia-induced angiogenesis in diabetic mice. We next analyzed the role of proteolysis in AGE formation-induced hampered neovascularization process. After 3 days of ischemia, MMP-2 activity and MMP-3 and MMP-13 protein levels were increased in untreated and aminoguanidine-treated diabetic mice when compared with controls (P < 0.05). Despite this activation of the MMP pathway, collagenolysis was decreased in untreated diabetic mice. Conversely, treatment of diabetic mice with aminoguanidine restored collagenolysis toward levels found in control mice. In conclusion, blockade of AGE formation by aminoguanidine normalizes impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation processes that are disturbed as a result of AGE accumulation.     

Aminoguanidine reduces regional albumin clearance but not urinary albumin excretion in streptozotocin-diabetic rats

Summary Advanced glycation end-product-formation is thought to play a role in the development of diabetic angiopathy. By altering the structure of different extracellular matrix components advanced glycation end-products might affect vascular and glomerular permeability. In this study we investigated the effect of treatment with an inhibitor of advanced glycation end-product-formation, aminoguanidine, on vascular permeability and the development of albuminuria in streptozotocin-induced diabetic rats. Male Wistar Rp rats were randomized into a control group, a diabetic group, and an aminoguanidine-treated diabetic group. After 8 weeks, 24-h urine collections were taken and rats were implanted with an arterial and a venous catheter. Mean arterial blood pressure was determined by intra-arterial measurement. Regional albumin clearances were assessed in the eye, ileum, lung, skeletal muscle and skin using an isotope technique. Mean arterial pressure in the diabetic group was significantly lower in the control and aminoguanidine-treated groups (p<0.02). Regional albumin clearances were significantly increased in all tissues of diabetic rats compared to control rats (p<0.05). Aminoguanidine treatment of diabetic rats resulted in a significant decrease of regional albumin clearance in all tissues except the lung (p<0.05, lung p=0.07). The development of albuminuria in diabetic rats however, was not affected by aminoguanidine.     

晚期糖基化终末产物对糖尿病大鼠主动脉结构和功能的影响

目的探讨晚期糖基化终末产物(AGEs)对主动脉血管结构和功能影响及氨基胍的保护作用。方法将80只雄性SD大鼠随机分为4组:空白对照组(A组),糖尿病组(B组),糖尿病氨基胍干预组(C组)和空白氨基胍干预组(D组),每组20只。药物应用12周观察主动脉功能和结构变化。结果与A组和D组比较,B组、C组主动脉环对去甲肾上腺素引起的收缩反应明显增强,对乙酰胆碱引起的舒张反应则均明显减弱,C组反应强度较B组明显减轻(P0.05,P0.01)。主动脉壁均有AGEs沉积,B组、C组较明显,C组较B组明显减轻(P0.01);超微结构观察显示,A组、D组主动脉内膜大致正常,B组、C组内膜破坏明显,与动脉环结果呈一致性趋势。结论糖尿病大鼠主动脉AGEs含量增多,损伤了主动脉的结构和功能,氨基胍能够减少AGEs的沉积,并对主动脉结构和功能有保护作用。     

Aminoguanidine inhibits semicarbazide-sensitive amine oxidase activity: implications for advanced glycation and diabetic complications

Summary Aminoguanidine, a nucleophilic hydrazine, has been shown to be capable of blocking the formation of advanced glycation end products. It reduces the development of atherosclerotic plaques and prevents experimental diabetic nephropathy. We have found that aminoguanidine is also quite potent at inhibiting semicarbazide-sensitive amine oxidase (SSAO) both in vitro and in vivo. The inhibition is irreversible. This enzyme catalyses the deamination of methylamine and aminoacetone, which leads to the production of cytotoxic formaldehyde and methylglyoxal, respectively. Serum SSAO activity was reported to be increased in diabetic patients and positively correlated with the amount of plasma glycated haemoglobin. Increased SSAO has also been demonstrated in diabetic animal models. Urinary excretion of methylamine is substantially increased in the rats following acute or chronic treatment with aminoguanidine. Urinary methylamine levels were substantially increased in streptozotocin (STZ)-induced diabetic rats following administration of aminoguanidine. The non-hydrazine SSAO inhibitor (E)-2-(4-fluoro-phenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been shown to reduce urinary excretion of lactate dehydrogenase (an indicator of nephropathy) in STZ-induced diabetic rats. Formaldehyde not only induces protein crosslinking, but also enhances the advanced glycation of proteins in vitro. The results support the hypothesis that increased SSAO-mediated deamination may be involved in structural modification of proteins and contribute to advanced glycation in diabetes. The clinical implications for the use of aminoguanidine to prevent glycoxidation have been discussed. [Diabetologia (1997) 40: 1243–1250] Received: 8 May 1997 and in revised form: 24 June 1997     

Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications.

Advanced glycosylation end products (AGEs) have been implicated in many of the complications of diabetes and normal aging. Markedly elevated vascular tissue and circulating AGEs were linked recently to the accelerated vasculopathy of end-stage diabetic renal disease. To determine the pathogenic role of AGEs in vivo, AGE-modified albumin was administered to healthy nondiabetic rats and rabbits alone or in combination with the AGE-crosslink inhibitor aminoguanidine. Within 2-4 weeks of AGE treatment, the AGE content of aortic tissue samples rose to six times the amount found in controls (P < 0.001). Cotreatment with aminoguanidine limited tissue AGE accumulation to levels two times that of control. AGE administration was associated with a significant increase in vascular permeability, as assessed by 125I label tracer methods. This alteration was absent in animals that received aminoguanidine in addition to AGE. Significant mononuclear cell migratory activity was observed in subendothelial and periarteriolar spaces in various tissues from AGE-treated rats compared to normal cellularity noted in tissues from animals treated with aminoguanidine. Blood pressure studies of AGE-treated rats and rabbits revealed markedly defective vasodilatory responses to acetylcholine and nitroglycerin compared to controls (P < 0.001), consistent with marked NO. inactivation; aminoguanidine treatment significantly prevented this defect. These in vivo data demonstrate directly that AGEs, independent of metabolic or genetic factors, can induce complex vascular alterations resembling those seen in diabetes or aging. AGE administration represents an animal model system for the study of diabetic and aging complications as well as for assessing the efficacy of newly emerging therapies aimed at inhibiting advanced glycosylation.     

Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine.

Human aging is impacted severely by cardiovascular disease and significantly but less overtly by renal dysfunction. Advanced glycation endproducts (AGEs) have been linked to tissue damage in diabetes and aging, and the AGE inhibitor aminoguanidine (AG) has been shown to inhibit renal and vascular pathology in diabetic animals. In the present study, the effects of AG on aging-related renal and vascular changes and AGE accumulation were studied in nondiabetic female Sprague-Dawley (S-D) and Fischer 344 (F344) rats treated with AG (0.1% in drinking water) for 18 mo. Significant increases in the AGE content in aged cardiac (P < 0.05), aortic (P < 0.005), and renal (P < 0.05) tissues were prevented by AG treatment (P < 0.05 for each tissue). A marked age-linked vasodilatory impairment in response to acetylcholine and nitroglycerine was prevented by AG treatment (P < 0.005), as was an age-related cardiac hypertrophy evident in both strains (P < 0.05). While creatinine clearance was unaffected by aging in these studies, the AGE/ creatinine clearance ratio declined 3-fold in old rats vs. young rats (S-D, P < 0.05; F344, P < 0.01), while it declined significantly less in AG-treated old rats (P < 0.05). In S-D but not in F344 rats, a significant (P < 0.05) age-linked 24% nephron loss was completely prevented by AG treatment, and glomerular sclerosis was markedly suppressed (P < 0.01). Age-related albuminuria and proteinuria were markedly inhibited by AG in both strains (S-D, P < 0.01; F344, P < 0.01). These data suggest that early interference with AGE accumulation by AG treatment may impart significant protection against the progressive cardiovascular and renal decline afflicting the last decades of life.     

Secondary intervention with aminoguanidine retards the progression of diabetic retinopathy in the rat model

Summary Primary prevention with aminoguanidine — an inhibitor of advanced glycation end product (AGE) formation — has been successfully employed to prevent diabetic retinopathy in the rat. However, it is unknown whether inhibition of AGE formation is still effective in a secondary intervention strategy. The present study addresses this question by comparing secondary intervention with aminoguanidine with syngeneic islet transplantation in the rat model. After 6 months of diabetes, one group was treated with aminoguanidine (50 mg/100 ml drinking water; D-AG) while another group received syngeneic transplantation of collagenase-ficoll isolated islets by intraportal injection (Tx). After an additional 4 months, both groups were compared to a normal (NC 10) and diabetic (DC 10) control group. Retinal autofluorescence was increased 2.5-fold after 6 months and increased 3.7-fold after 10 months of diabetes (p<0.001). Aminoguanidine and islet Tx retarded the further accumulation of autofluorescence equally (p<0.001 vs DC 10), although the values were higher than those observed in DC at 6 months (p<0.001). Diabetes was associated with a 2.7-fold increase in acellular capillaries after 6 months and a 4.1-fold increase after 10 months. Treatment with aminoguanidine or islet Tx reduced but did not completely attenuate the progression of vascular occlusion (p<0.001 vs DC 10; D-AG vs DC 6, p<0.05; Tx vs DC 6, p<0.01). Both treatments reduced endothelial proliferation (22.4% after 10 months; p<0.001) and completely arrested pericyte dropout (40% after 10 months; p<0.001).These data demonstrate that aminoguanidine is as effective as islet transplantation in retarding the progression of diabetic retinopathy in a secondary prevention setting.Abbreviations Tx Islet transplantation - AGE advanced glycation end product - iNOS inducible form of nitric oxide synthase     

Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats.

High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur in diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosis of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were administered daily to healthy rats alone or in combination with the AGE inhibitor aminoguanidine. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), significant periodic acid/Schiff reagent-positive deposits, basement membrane widening, and mesangial extracellular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with significant loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and functional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. The effects are AGE-specific, as they are ameliorated by a pharmacological AGE inhibitor, aminoguanidine.     

Renoprotective effects of a novel inhibitor of advanced glycation

Aims/hypothesis. ALT-946, an inhibitor of advanced glycation with a minimal inhibitory effect on nitric oxide synthase, was compared with aminoguanidine in experimental diabetic nephropathy.¶Methods. In vitro and in vivo assays were used to assess the ability of ALT-946 to inhibit AGE-protein cross-link formation. Diabetic animals were randomly allocated into groups receiving aminoguanidine for 32 weeks, ALT-946 or vehicle (untreated). As a delayed intervention protocol, an additional diabetic group was treated with ALT-946 from week 16 to week 32 of the study. Non-diabetic rats were studied concurrently. Systolic blood pressure, body weight, plasma glucose, glycated haemoglobin and urinary albumin excretion were measured serially. Accumulation of advanced-glycation end products in the kidney was assessed by immunohistochemistry.¶Results. The ALT-946 inhibitor was more potent than aminoguanidine in inhibiting AGE-protein cross-linking both in vitro and in vivo. Increased albuminuria observed in diabetic rats was attenuated in all three treatment groups. We found no difference in body weight, blood pressure or glycaemic control with any of the treatments. The untreated diabetic group had a twofold increase in glomerular staining for advanced-glycation end products compared with the diabetic groups which received treatment.¶Conclusion/interpretation. ALT-946 is a potent inhibitor of advanced renal glycation end-product accumulation and reproduces the renoprotective effects of aminoguanidine. Therefore, ALT-946 should be considered as a treatment for preventing or retarding diabetic nephropathy. [Diabetologia (2001) 44: 108–114]     

Potential benefit of inhibitors of advanced glycation end products in the progression of type II diabetes: A study with aminoguanidine in C57/BLKsJ diabetic mice

Prolonged hyperglycemia in type II diabetic patients is linked both with diabetic complications and with further impairment of glucose homeostasis, possibly due to glucose toxicity of the β cell. While the connection between the accumulation of extracellular advanced glycation end products (AGEs) and the development of complications is well established, it has only recently been suggested that intracellular glycation may be equally adverse and could be involved in the pathogenesis of glucose toxicity in vitro. Aminoguanidine is a recognized inhibitor of the formation of both extracellular and intracellular AGEs. In this study, we show that the development of diabetes, measured by increased water intake and concomitant midday blood glucose levels in type II genetically diabetic mice, is reduced by treatment with aminoguanidine at a dosage of 500 mg/kg/d for 12 weeks in the diet. In addition, at the end of the study, aminoguanidine reduced the decline in serum and pancreatic insulin levels and the degree of pancreatic islet morphological degeneration, all of which are associated with pancreatic insufficiency following prolonged hyperglycemia in this animal model. These results suggest that AGEs may be involved in the aggravation of type II diabetes in vivo and aminoguanidine may be beneficial in its treatment.     

Renal connective tissue growth factor induction in experimental diabetes is prevented by aminoguanidine

The aim of this study was to determine whether aminoguanidine (AG), an inhibitor of advanced glycation, prevents expression of the profibrotic cytokine, connective tissue growth factor (CTGF), as well as accumulation of the previously reported CTGF-dependent matrix protein, fibronectin, in a model of experimental diabetic nephropathy. Diabetic animals were randomly allocated into groups receiving 32 wk of AG or vehicle. Diabetic rats showed increases in CTGF mRNA and protein expression as well as in advanced glycation end-product (AGE) and fibronectin immunostaining, compared with nondiabetic rats. In the diabetic kidney, the increase in CTGF gene and protein expression as well as expression of the extracellular matrix protein fibronectin were prevented by AG. To further explore the relationship between AGEs and mesangial CTGF and fibronectin production, cultured human mesangial cells were exposed in vitro to soluble AGE-BSA and carboxymethyl lysine-BSA, and this led to induction of both CTGF and fibronectin. On the basis of our in vitro findings in mesangial cells linking AGEs to CTGF expression, the known prosclerotic effects of CTGF, and the ability of AG to attenuate mesangial expansion, it is postulated that the antifibrotic effects of AG in this animal model may be partially mediated by CTGF.     

Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease.

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGE-modified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extracellular matrix alpha 1(IV) collagen, laminin B1, and transforming growth factor beta 1 mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.     

橙皮苷对STZ糖尿病大鼠肾脏功能和形态的影响

观察橙皮苷对链脲佐菌素导致的糖尿病大鼠肾脏功能和形态的影响,并与氨基胍进行比较。结果表明:（1）两治疗组大鼠尿蛋白排泄量显著低于对照组P<0.05）;（2）两治疗组肾组织AGEs和LPO含量显著低于对照组（P<0.01,P<0.05）:（3）治疗组肾小球系膜增生和基底膜增厚明显减轻。提示橙皮苷在抑制蛋白非酶糖基化、预防糖尿病肾脏并发症方面具有与氨基胍相似的作用。     

Superior renoprotective effects of combination therapy with ACE and AGE inhibition in the diabetic spontaneously hypertensive rat

Aims/hypothesis Diabetic renal disease has been postulated to progress as a result of an interaction between metabolic and haemodynamic pathways. Our aim was to assess the functional, structural, molecular and cellular aspects of renal disease in an experimental model of diabetes with associated hypertension.Method Streptozotocin-induced diabetic spontaneously hypertensive rats were randomised to no treatment, the ACE inhibitor, perindopril (2 mg/l), the AGE formation inhibitor, aminoguanidine (1 g/l) and a combination of both agents and were followed for 32 weeks.Results Diabetes was associated with a considerable increase in albumin excretion rate. Both aminoguanidine and perindopril retarded the increase in albuminuria, which was completely abrogated by combination therapy. Glomerulosclerosis and tubulointerstitial damage was reduced by both monotherapies with further renoprotection afforded by combination therapy in both cases. Combination therapy was also associated with a superior restoration in diabetes-induced nephrin protein depletion compared to either monotherapy. TGF1 expression as assessed by in situ hybridisation was increased in the diabetic rats and reduced by perindopril and aminoguanidine.Conclusion/interpretation These findings indicate that in the context of diabetes-related renal injury, blocking both the renin-angiotensin and advanced glycation pathways offers superior renoprotection and could be considered as a therapeutic strategy in the prevention and retardation of progressive-diabetic renal injury.Abbreviations AG Aminoguanidine - AG/PER aminoguanidine/perindopril - GS glomerulosclerosis - PER perindopril - RAS renin-angiotensin system - SBP systolic blood pressure - SHR spontaneously hypertensive rat - STZ streptozotocin - TGF1 transforming growth factor beta-1 - TIA tubulointerstitial area