Accumulation Of Advanced Glycosylation End Products Research Articles

Implication of the MAPK Signalling Pathway in the Pathogenesis of Diabetic Nephropathy

Diabetes has become an emerging public health problem because of its serious complications, and high mortality and morbidity rates. Among the most common microvascular complications of diabetes is diabetic nephropathy (DN), which is a major cause of development of end-stage renal disease worldwide. The aetiopathogenesis of DN is not completely elucidated; however, studies have shown that the components of the MAPK signalling pathway play an essential role in the development and progression of the disease. The MAPK family is mainly composed of three subgroups: extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases (JNK) 1–3, and p38 MAPK, all of which are related to several cellular functions, such as cell death, differentiation, proliferation, motility, survival, stress response, and cell growth. In diabetic kidney disease, the MAPK pathway can be activated by processes resulting from hyperglycaemia (polyol pathway products, oxidative stress, and accumulation of advanced glycosylation end-products) and by angiotensin II, and it is related to several renal pathological processes. This review aims to summarise the role of the MAPK signalling pathway in diabetic nephropathy, as well as to link the biological aspects that contribute to clarify the pathological process behind the disease.

Skeletal Fragility in Type 2 Diabetes Mellitus.

Type 2 diabetes (T2D) is associated with an increased risk of fracture, which has been reported in several epidemiological studies. However, bone mineral density in T2D is increased and underestimates the fracture risk. Common risk factors for fracture do not fully explain the increased fracture risk observed in patients with T2D. We propose that the pathogenesis of increased fracture risk in T2D is due to low bone turnover caused by osteocyte dysfunction resulting in bone microcracks and fractures. Increased levels of sclerostin may mediate the low bone turnover and may be a novel marker of increased fracture risk, although further research is needed. An impaired incretin response in T2D may also affect bone turnover. Accumulation of advanced glycosylation endproducts may also impair bone strength. Concerning antidiabetic medication, the glitazones are detrimental to bone health and associated with increased fracture risk, and the sulphonylureas may increase fracture risk by causing hypoglycemia. So far, the results on the effect of other antidiabetics are ambiguous. No specific guideline for the management of bone disease in T2D is available and current evidence on the effects of antiosteoporotic medication in T2D is sparse. The aim of this review is to collate current evidence of the pathogenesis, detection and treatment of diabetic bone disease.

Osteoporosis and risk of fracture in patients with diabetes: an update

Diabetes mellitus (DM) and osteoporotic fractures are two of the most important causes of mortality and morbidity in older subjects. Recent data report a close association between fragility fracture risk and DM of both type 1 (DM1) and type 2 (DM2). However, DM1 is associated with reduced bone mineral density (BMD), whereas patients with DM2 generally have normal or increased BMD. This apparent paradox may be explained by the fact that, at a given level of BMD, diabetic patients present lower bone quality with respect to non-diabetics, as shown by several studies reporting that diabetes may affect bone tissue by means of various mechanisms, including hyperinsulinemia, deposition of advanced glycosylation endproducts (AGEs) in collagen, reduced serum levels of IGF-1, hypercalciuria, renal failure, microangiopathy and inflammation. In addition, the propensity to fall and several comorbidities may further explain the higher fracture incidence in DM patients with respect to the general population. It is reasonable to expect that close metabolic control of diabetes may improve bone status, although its effect on reduction of fracture risk has not yet been demonstrated. However, metformin has a direct effect on bone tissue by reducing AGE accumulation, whereas insulin acts directly on osteoclast activity, and thiazolidinediones (TZD) may have a negative effect by switching mesenchymal progenitor cells to adipose rather than bone tissue. New prospects include the incretins, a class of antidiabetic drugs which may play a role linking nutrition and bone metabolism. Better knowledge on how diabetes and its treatments influence bone tissue may lie at the basis of effective prevention of bone fracture in diabetic patients. Thus, close glycemic control, adequate intake of calcium and vitamin D, screening for low BMD, and prevention and treatment of diabetic complications are key elements in the management of osteoporosis in both DM1 and DM2. Attention should be paid to treating diabetes with TZD in women with DM2, particularly if elderly. Lastly, patients with osteoporosis and diabetes should be offered the same pharmacological treatments as non-diabetics, although specific trials on the effects of anti-osteoporotic drugs in the diabetic population are lacking.

Peritoneal Accumulation of Advanced Glycosylation End-Products in Diabetic Rats on Dialysis with Icodextrin

To evaluate and compare the effects of glucose-based solutions to those of icodextrin with respect to peritoneal transport characteristics and formation of advanced glycosylation end-products (AGEs) in the peritoneal membrane in the diabetic rat model of peritoneal dialysis (PD). Thirty-three male Sprague-Dawley rats weighing between 275 - 300 g were divided into 5 groups: group C (n = 6), control rats with catheter but not dialyzed; group D (n = 5), diabetic rats with catheter but not dialyzed; group G (n = 7), diabetic rats dialyzed with standard 2.5% glucose solution for daytime exchanges and 4.25% glucose solution for the overnight exchange; group H (n = 8), diabetic rats dialyzed with standard 2.5% glucose solution for daytime exchanges and 7.5% icodextrin solution for overnight exchanges; group I (n = 7), diabetic rats dialyzed with 7.5% icodextrin solution for all exchanges. Dialysis exchanges were performed three times daily with an instillation volume of 25 mL per exchange for a period of 12 weeks. Tissue sections were stained using a monoclonal anti-AGE antibody. One-hour peritoneal equilibration tests (PET) were performed every 4 weeks for comparison of transport characteristics. The level of immunostaining was lowest in group C and highest in group G. Significant differences were seen between group C and groups G, H, and I (p < 0.001, p = 0.001, and p< 0.05 respectively). Significant differences were also found between group G and groups D and I (p < 0.05 and p < 0.05 respectively). Over time, glucose concentration at the end of an exchange versus concentration at instillation (D/D0 glucose) decreased and dialysate-to-plasma ratio (D/P) of urea increased. Significant differences were found between groups C and H for D/D0 glucose (0.40+/-0.01 vs 0.35+/-0.01, p < 0.05); and between groups C and H for D/P urea (0.87+/-0.03 vs 0.97+/-0.02, p < 0.05). These results suggest that AGE formation is lower with the use of peritoneal dialysis solution containing icodextrin than with glucose-based solutions. We conclude that the use of icodextrin may be helpful in slowing the deterioration of the peritoneal membrane, prolonging its use for dialysis.

Effects of Bicarbonate/Lactate Solution on Peritoneal Advanced Glycosylation End-Product Accumulation

Advanced glycosylation end-products (AGEs) are associated with diabetic complications and peritoneal damage after long-term peritoneal dialysis (PD) with high glucose dialysis solutions. Glucose degradation products (GDPs) derived during heat sterilization of high glucose dialysis solutions are thought to accelerate AGE formation. A new technique of separating glucose from electrolytes has yielded markedly lower GDP levels and permitted the use of dialysis solutions containing the physiologic buffer bicarbonate. Formation of AGEs in vitro with this new solution is significantly lower compared with formation of AGEs with conventional solutions. The purpose of the present study was to investigate the effect of long-term intraperitoneal use of new, neutral dialysis solution (B/L) containing bicarbonate (25 mmol/L) and lactate (15 mmol/L) on peritoneal AGE accumulation and permeability. Normal male Sprague-Dawley rats were used. Twice daily for 12 weeks, 30 mL of new solution (B/L) or conventional solution [Lac (lactate 40 mmol/L)] was injected into the peritoneal cavity of the test rats. As a control, rats that were not injected were kept for 12 weeks in the same manner as the test rats. After 12 weeks, a 2-hour peritoneal equilibration test (PET) was performed in the test rats. After the PET, the parietal peritoneum and liver were obtained for evaluation of peritoneal morphology and for immunohistochemistry for AGE. Intensity of AGE staining was semi-quantitatively graded from 0 to 3. The omentum was also obtained and immediately frozen for analysis of pentosidine content by high-performance liquid chromatography. Compared with findings in the control group, hematoxylin and eosin staining of the parietal peritoneum and liver samples revealed partial denudation of mesothelial cells in the Lac group; denudation was not remarkable in the B/L group. The B/L solution showed significantly less AGE staining in the peritoneal cavity compared to conventional solution. However, B/L solution failed to lower pentosidine levels. Intraperitoneal volume and the ratio of dialysate glucose at 2 hours to dialysate glucose at instillation (D2/D0 glucose) were significantly lower and the ratio of dialysate urea to plasma urea at 2 hours (D2/P2 urea) was significantly higher in the Lac and B/L groups than in the control group. Intraperitoneal volume was significantly higher in the B/L group than in the Lac group; D2/D glucose and D2P2 urea did not differ between the two groups. In conclusion, peritoneal ultrafiltration decreased after long-term PD. The B/L solution showed a small but statistically significant protective effect against decreasing ultrafiltration as compared with Lac solution. The B/L solution attenuated peritoneal AGE accumulation compared with conventional solution, but did not affect peritoneal pentosidine levels. These findings indicate that biochemical kinetics of various AGE peptides are not unique, but multivalent.

Age Accumulation in Peritoneal Membrane and Cavity during Peritoneal Dialysis and Its Effect on Peritoneal Structure and Function

T he conventional dialysis solutions contain a high concentration of glucose to induce sufficient ultra filtration and to maintain fluid balance in uremic patients. During continuous ambulatory peritoneal dialysis (CAPD), the peritoneal cavity is therefore continuously exposed to high glucose for a long time. Histological examination of the peritoneum from peritoneal dialysis patients revealed duplication of submesothelial basement membrane in both diabetic and non diabetic patients and acceleration of preexisting duplication of capillary basement membrane in diabetic patients (1-4). These histologic changes, similar to diabetic complications, are believed to be due to continuous exposure of the peritoneum to an unphysiologic ally high concentration of glucose. Hyperglycemia is associated with the presence or progression of complications in non insulin-dependent diabetes mellitus (NIDDM) (1,5) and insulindependent diabetes mellitus (IDDM) (2). One of the major consequences of prolonged hyperglycemia is accelerated formation of advanced glycosylation endproducts (AGEs) on long-lived structural proteins (3). AGEs are excreted mainly by the kidneys and, therefore, serum level increases as renal function decreases (4). AGE peptide levels in spent dialysate in PD patients is higher than plasma levels (6,7). Peritoneal accumulation of AGEs was reported in PD patients (8,9). Exogenous administration of AGEs induced an increase in vascular permeability in normal animals (10) and the accumulation of AGE induced structural and functional alterations of tis sue proteins (11). Peritoneal accumulation of AGEs, structural and functional alterations of tissue proteins, and increased permeability of the peritoneal vasculature may re

Aminoguanidine has an anti-atherogenic effect in the cholesterol-fed rabbit

Advanced glycosylation endproducts (AGEs) which result from the non-enzymatic interaction of proteins and glucose are implicated in the vasculopathy of diabetes and aging. Since aminoguanidine (A) inhibits the accumulation of AGEs, we explored its effects on the development of atherosclerosis. Male New Zealand white cross rabbits fed a high cholesterol (1%) diet were randomized to control (C) or increasing doses of A treatment (25, 50 and 100 mg/kg A body weight). The animals were sacrificed after 12 weeks. Sudan IV was used to stain the lipid containing plaques of the aortic arch, thoracic and abdominal aorta and the surface area occupied by atheroma was assessed. Increasing doses of A treatment were associated with reduction in plaque formation in the aorta. At a dose of 100 mg/kg A, there was a 30, 49 and 48% reduction in plaque formation in the aortic arch, thoracic and abdominal aorta, respectively. There was a correlation between AGE levels and the degree of atheroma in these cholesterol fed rabbits (control, r=0.75, P<0.01; 100 mg/kg A, r=0.59, P=0.02). These data suggest that advanced glycation may participate in atherogenesis and raise the possibility that inhibitors of advanced glycation may retard this process.

Pathogenesis of diabetic nephropathy, advanced glcation and new therapy

It is well understood that persistent hyperglycemia contributes to diabetic tissue and end-organ damage [1], by promoting the formation of advanced glycosylation endproducts (AGEs) [2, 3]. AGEs form principally from the rearrangement of early glycation products, i.e. Amadori products, which produce a class o f stable moieties that possess distinctive chemical cross-linking and biological properties [2, 31 . It has been generally believed that proteins with half-lives of greater than a few weeks are most susceptible to advanced glycosylation and the highest levels o f AGEs occur on proteins that comprise the long-lived structural components of connective tissue matrix and basement membrane. Of particular interest, however, has been the recent observation that AGEs can also occur on short-lived proteins, on lipid constituents and on nucleic acids. This is most apparent under conditions o£ high AGE accumulation such as diabetes and renal failure, and results from the fact that AGE formation proceeds through a succession of reactive intermediates which can bind indiscriminantly to amino groups present on diverse "bystander" proteins. 1keactive intermediates can also be released from naturally degraded tissue AGE, only to bind again on other substances, if not cleared via the kidney, forming "second" generation AGEs. Cell surface receptors that are specific for the recognition, and degradation of AGE-modified proteins also have been identified on circulating monocytes, lymphocytes, endothelial, renal mesangial cells and other cellular systems to partake in both normal tissue remodeling and tissue damage. In this synoptic review, selected recent studies are highlighted which

Immunohistochemical detection of advanced glycosylation end-products in the peritoneum and its possible pathophysiological role in CAPD

It has recently been suggested that advanced glycosylation end-products (AGEs) are formed in the peritoneum in patients on CAPD. However, the exact location of AGE accumulation, the relation with the duration of CAPD and its pathophysiological role in CAPD remain unclear. If the peritoneum is glycosylated, it could bring about altered peritoneal function. Therefore, the aim of this study is to clarify the localization of AGEs in the peritoneum in accordance with the duration of CAPD and to examine its relation to the peritoneal permeability. Fifteen non-diabetic patients were divided into three groups (each 5 patients) on the basis of the mean duration (D) of CAPD (Group 1, D = 0 month; Group II, D = 34 months; Group III, D = 84 months). The AGE staining by monoclonal anti-AGE antibody in the peritoneum and the four-hour peritoneal equilibration test (PET) were compared among these groups. AGE was absent or found only weakly in Group I. However, in groups II and III, AGE was moderately or strongly positive especially in the vascular walls and it was dominant in group III. PET revealed that peritoneal permeability for glucose, creatinine, beta2-microglobulin and albumin was increased in Group II as compared to Group I, and it was further increased in Group III. The results of this study indicate that AGEs become dominantly accumulated in the vascular wall in accordance with the prolongation of CAPD treatment, and this might play some roles for the increased permeability of the peritoneal membrane in CAPD.

Advanced glycosylation end-products and heat shock proteins accumulate in the basophilic degeneration of the myocardium and the corpora amylacea of the glia.

Using monospecific antibody for the advanced glycosylation end-products (AGEP), it was revealed that the AGEP localized in the basophilic degeneration of the myocardium and the corpora amylacea of the glia. The stability of the proteins that constitute those degenerative deposits suggests that they would be ideal substrates for non-enzymatic glycation, a process that occurs over a long time under a high glucose content, and ultimately results in the formation of the AGEP. Such deposits also exhibited evidence of stress reactions: the accumulation of HSP72, heme oxygenase-1 and ubiquitin. As recent studies have shown that AGEP-modified proteins aggregate and that they generate reactive oxygen intermediates, the accumulation of such heat shock proteins may reflect the oxidative stress concomitant with AGEP accumulation, and thereby promote their cellular dysfunction. Hereby, it is proposed that the age-related increase in the AGEP, that is, a fundamental aging process, is involved in the formation of the basophilic degeneration in the myocardium and the corpora amylacea of the glia.

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.

Slowing of peripheral motor nerve conduction was ameliorated by aminoguanidine in streptozocin-induced diabetic rats

The aims of this study were to investigate the effect of aminoguanidine (AG) on slowing of motor nerve conduction velocity (MNCV) of the sciatic nerve in streptozocin-induced diabetic rats and to assess its mechanism of action. The MNCV of the sciatic nerve was measured electrophysiologically in diabetic rats treated with and without AG for 16 weeks. To elucidate the action of AG, morphological lesion and abnormality of polyol pathway metabolism in the nerve were examined and tissue levels of advanced glycosylation end-products (AGE) were determined as an indicator of AGE accumulation in tissue. Diabetic rats were treated with AG at three doses of 10, 25 and 50 mg/kg for 16 weeks. Myelinated fiber morphometry and nerve Na+,K(-)-ATPase activity were determined. The AGE levels in renal cortex were measured by a specific ELISA. Aminoguanidine dose-dependently ameliorated slowing of MNCV 16 weeks after the treatment without changing body weight or blood glucose levels. No difference in myelinated fiber morphometry or Na+,K(+)-ATPase activity with or without AG treatment was detected in diabetic rats. Diabetes increased the AGE level in the renal cortex by six times compared to non-diabetic rats, and AG reduced the rise in the AGE level by 40%. The MNCV was inversely correlated with the AGE levels. We conclude that improvement of conduction slowing by AG in experimental diabetes may be through decreasing the AGE level in the peripheral tissues. Aminoguanidine may have a therapeutic potential in controlling diabetic peripheral neuropathy.

Resistance to glomerular injury in the diabetic biobreeding rat.

This study was designed to determine whether the diabetic BioBreeding rat develops significant renal injury following long-term moderate to severe hyperglycaemia. Diabetic and control rats were followed from the onset of diabetes (2-4 months) to 18 months of age. Frank proteinuria and/or albuminuria were always absent. Glomerular filtration rate, measured by inulin clearance (ml min-1 (100 g body weight)-1), was significantly higher in diabetic rats than in controls at 10, 12 and 18 months of age. Advanced glycosylation end-product cross-links assessed by percentage solubility of tail tendon collagen were moderately increased in diabetic compared with control animals. Urinary excretion of advanced glycosylation end-products in unfractionated urine and in urine fractionated for low molecular mass peptides (< 10 kDa) was 11-fold greater in the diabetic rats than in the control group. Urinary excretion of nitric oxide metabolites (nmol NO2- and NO3- (24 h)-1) were significantly (P < 0.05) greater in diabetic rats than in controls after 8 months of age. Mild histopathology resembling human diabetic nephropathy, including increased mesangial volume and glomerular basement membrane thickness, was detected at 18 months of age. The findings of hyperfiltration and mild glomerular morphological changes in diabetic BioBreeding rats are similar to the abnormalities seen in stage 2 human diabetic nephropathy. We hypothesize that two factors which may contribute to the resistance or tolerance to renal injury in the BioBreeding diabetic rat are increased nitric oxide production and the decreased accumulation of advanced glycosylation end-products.

Aminoguanidine treatment increases elasticity and decreases fluid filtration of large arteries from diabetic rats.

The accumulation of advanced glycosylation end-products (AGEs) on collagen and the subsequent stiffening of this matrix protein in diabetes has been described many years ago. Structural modification of collagen in the arterial wall might have important effects on arterial elasticity. Aminoguanidine is known to decrease the formation of AGEs. In this study we evaluated the effects of aminoguanidine treatment on different parameters reflecting arterial wall elasticity in diabetic rats. We demonstrated that treatment of diabetic rats with aminoguanidine resulted in a significant increase in carotid static compliance (+39%, P < 0.01 under control conditions, and +27%, P < 0.01 after abolition of vascular tone by KCN), and a decrease in characteristic aortic input impedance (-40%, P < 0.01). The arterial pulse pressure in aminoguanidine-treated rats was decreased (-15%, P < 0.05) and the pulsatile component of left ventricular power output was relatively diminished (-35%, P < 0.05). In addition, we observed a lower fluid filtration across the carotid wall. These results indicate an increased vascular elasticity, an improved left ventricular-arterial coupling, and a decreased vascular permeability in diabetic rats after aminoguanidine treatment, suggesting that AGE-accumulation on collagen negatively affects arterial wall properties in experimental diabetes.

Mesangial cells in diabetes

The glomerular mesangium is the site of prominent structural lesions in diabetic nephropathy, including excess deposition of extracellular matrix in a focal, nodular pattern. Increased single-nephron blood flow and filtration are early signs of glomerular involvement in diabetes, and may initiate or contribute to mesangial damage. Vasodilatation results from arteriolar insensitivity to vasoconstrictors. In turn, this may reflect primary metabolic defects of glomerular smooth muscle, including the afferent arteriole and mesangial cells themselves. Parallel alterations in the glomerular basement membrane and related structures, such as mesangial matrix, are likely to result from glycosylation of intrinsic proteins, or accumulation of advanced glycosylation end-products. Structural and haemodynamic changes account for (micro)-albuminuria, with additional possible overloading of the measangium. Mesangial proliferative changes eventually ensue, with excess matrix deposition and progressive fibrosis. Recent evidence that long-standing hyperglycaemia modifies mesangial metabolism, sensitivity to vasoconstrictors and matrix biosynthesis in vitro is reviewed here, in the light of its potential implications for experimental and human diabetic nephropathy.