Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions

Advanced glycation end products (AGE) include a variety of protein adducts whose accumulation has been implicated in tissue damage associated with diabetic nephropathy (DN). It was recently demonstrated that among AGE, glycoxidation products, whose formation is closely linked to oxidation, such as carboxymethyllysine (CML) and pentosidine, accumulate in expanded mesangial matrix and nodular lesions in DN, in colocalization with malondialdehyde-lysine (MDA-lysine), a lipoxidation product, whereas pyrraline, another AGE structure whose deposition is rather independent from oxidative stress, was not found within diabetic glomeruli. Because CML, pentosidine, and MDA-lysine are all formed under oxidative stress by carbonyl amine chemistry between protein amino group and carbonyl compounds, their colocalization suggests a local oxidative stress and increased protein carbonyl modification in diabetic glomerular lesions. To address this hypothesis, human renal tissues from patients with DN or IgA nephropathy were examined with specific antibodies to characterize most, if not all, carbonyl modifications of proteins by autoxidation products of carbohydrates, lipids, and amino acids: CML (derived from carbohydrates, lipids, and amino acid), pentosidine (derived from carbohydrates), MDA-lysine (derived from lipids), 4-hydroxynonenal-protein adduct (derived from lipids), and acrolein-protein adduct (derived from lipids and amino acid). All of the protein adducts were identified in expanded mesangial matrix and nodular lesions in DN. In IgA nephropathy, another primary glomerular disease leading to end-stage renal failure, despite positive staining for MDA-lysine and 4-hydroxynonenal-protein adduct in the expanded mesangial area, CML, pentosidine, and acrolein-protein adduct immunoreactivities were only faint in glomeruli. These data suggest a broad derangement in nonenzymatic biochemistry in diabetic glomerular lesions, and implicate an increased local oxidative stress and carbonyl modification of proteins in diabetic glomerular tissue damage ("carbonyl stress").     

Restriction of dietary glycotoxins reduces excessive advanced glycation end products in renal failure patients

Advanced glycation endproduct (AGE) levels are elevated in renal failure patients and may contribute to the excessive cardiovascular disease in this population. Diet-derived AGE are major contributors to the total body AGE pool. It was postulated that a reduction in dietary AGE intake might impact on the high circulating AGE levels in renal failure patients. Twenty-six nondiabetic renal failure patients on maintenance peritoneal dialysis were randomized to either a high or a low AGE diet for 4 wk. Three-day dietary records, fasting blood, 24-h urine, and dialysis fluid collections were obtained at baseline and end of study. AGE levels were determined by ELISA for N(epsilon)-carboxymethyl-lysine (CML) and methylglyoxal-derivatives (MG). Eighteen patients completed the study. Low dietary AGE intake decreased serum CML (34%; P < 0.002), serum MG (35%; P < 0.008), CML-LDL (28%; P < 0.011), CML-apoB (25%; P < 0.028), dialysate CML (39%; P < 0.03), and dialysate MG output (40%; P < 0.04). High dietary AGE intake increased serum CML (29%; P < 0.028), serum MG (26%; P < 0.09), CML-LDL (50%; P < 0.011), CML-apoB (67%; P < 0.028), and dialysate CML output (27%; P < 0.01). Serum AGE correlated with BUN (r = 0.6, P < 0.002 for CML; r = 0.4, P < 0.05 for MG), serum creatinine (r = 0.76, P < 0.05 for CML; r = 0.55, P < 0.004 for MG), total protein (r = 0.4, P < 0.05 for CML; r = 0.4, P < 0.05 for MG), albumin (r = 0.4, P < 0.02 for CML; r = 0.4, P < 0.05 for MG), and phosphorus (r = 0.5, P < 0.006 for CML; r = 0.5, P < 0.01 for MG). It is concluded that dietary glycotoxins contribute significantly to the elevated AGE levels in renal failure patients. Moreover, dietary restriction of AGE is an effective and feasible method to reduce excess toxic AGE and possibly cardiovascular associated mortality.     

Production of N(epsilon)-(carboxymethyl)lysine is impaired in mice deficient in NADPH oxidase: a role for phagocyte-derived oxidants in the formation of advanced glycation end products during inflammation

Advanced glycation end products (AGEs) derived from glucose are implicated in the pathogenesis of diabetic vascular disease. However, many lines of evidence suggest that other pathways also promote AGE formation. One potential mechanism involves oxidants produced by the NADPH oxidase of neutrophils, monocytes, and macrophages. In vitro studies have demonstrated that glycolaldehyde, a product of serine oxidation, reacts with proteins to form N(epsilon)-(carboxymethyl)lysine (CML), a chemically well-characterized AGE. We used mice deficient in phagocyte NADPH oxidase (gp91-phox(-/-)) to explore the role of oxidants in AGE production in isolated neutrophils and intact animals. Activated neutrophils harvested from wild-type mice generated CML on ribonuclease A (RNase A), a model protein, by a pathway that required L-serine. CML formation by gp91-phox(-/-) neutrophils was impaired, suggesting that oxidants produced by phagocyte NADPH oxidase contribute to the cellular formation of AGEs. To determine whether these observations are physiologically relevant, we used isotope-dilution gas chromatography/mass spectrometry to quantify levels of protein-bound CML in mice suffering from acute peritoneal inflammation. Phagocytes from the gp91-phox(-/-) mice contained much lower levels of CML than those from the wild-type mice. Therefore, oxidants generated by phagocyte NADPH oxidase may play a role in AGE formation in vivo by a glucose-independent pathway.     

Increased levels of N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine in type 1 diabetic patients with impaired renal function: correlation with markers of endothelial dysfunction.

BACKGROUND: Diabetic and non-diabetic patients with renal failure have an increased risk for cardiovascular disease, which may be the result of uraemic toxins, including advanced glycation end-products (AGEs). The aim of the study was to investigate the levels of well-characterized AGEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) in relation to kidney function and to study the relationship of these AGEs to endothelial function and inflammation in type 1 diabetic patients. METHODS: Plasma levels of CML and CEL were measured in 60 type 1 diabetic patients categorized as having normal glomerular filtration rate (GFR) (>80 ml/min, n = 31) or decreased GFR (<80 ml/min, n = 29) as estimated by the Cockcroft-Gault formula. To assess the relationship of these AGEs to endothelial function and inflammation, markers of endothelial function von Willebrand factor (vWf), soluble vascular cellular adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), soluble thrombomodulin (sTM), tissue type-specific plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and C-reactive protein (CRP), a marker of inflammatory activity, were determined by enzyme-linked immunosorbent assays. RESULTS: Plasma levels of CML and CEL were increased in diabetic patients with decreased GFR as compared with patients with normal GFR [CML 4.9 (2-12.6) vs 2.9 (1.7-4.4) micromol/l, P<0.000; and CEL 1.7 (0.9-3.3) vs 1.2 (1.7-4.4) micromol/l, P = 0.004, respectively). Independently of the GFR, the plasma levels of CML and CEL were significantly associated with sVCAM-1, vWf and sTM. CONCLUSIONS: Plasma levels of CML and CEL rise with deterioration of GFR. Furthermore, CML and CEL levels are associated with markers of endothelial activation independently of renal function. This suggests an involvement of these AGEs in the acceleration of cardiovascular complications in patients with renal impairment.     

Glycation free adduct accumulation in renal disease: the new AGE

Glycation adducts formed in the later stages of protein glycation reactions, advanced glycation endproducts (AGEs), are a class of uraemic toxin. Protein glycation was viewed originally as a post-translational modification that accumulated mostly on extracellular proteins. We now know that AGE residues are also formed on short-lived cellular and extracellular proteins. Cellular proteolysis forms AGE free adducts from these proteins, which are released into plasma for urinary excretion. AGE free adducts are also absorbed from food. AGE free adducts are the major molecular form by which AGEs are excreted in urine. They normally have high renal clearance, but this declines markedly in chronic renal failure patients, leading to profound increases in plasma AGE free adducts. Accumulation of plasma AGE free adducts is increased further in end stage renal disease patients on peritoneal dialysis and haemodialysis by increased AGE formation. The impact of AGEs absorbed from food is probably most marked for undialysed patients with mild uraemia. The toxicity of AGEs has been associated with resistance of the extracellular matrix to proteolysis and AGE receptor-mediated responses. AGE free adducts may also contribute to vascular disease in uraemia. They represent an important new age for glycation research in nephrology.     

Differential effects of advanced glycation end-products on renal tubular cell inflammation

Aim: The authors recently showed that advanced glycation end‐products (AGE) in the form of glycated albumin (GA) upregulated renal tubular expression of interleukin (IL)‐8 and soluble intercellular adhesion molecule‐1 (sICAM‐1), but not other important cytokines known to mediate diabetic nephropathy. This implies that other molecules such as the carbonyl intermediates of AGE or other modified protein lysine‐albumin may participate in diabetic tubular injury. Methods: Human proximal tubular epithelial cells (PTEC) were growth‐arrested and exposed to methylglyoxal (MG), MG‐bovine serum albumin (BSA)‐AGE, carboxymethyllysine (CML)‐BSA, AGE‐BSA or BSA with or without prior addition of rosiglitazone that was previously shown to attenuate the pro‐inflammatory effect of GA alone. Results: MG‐BSA‐AGE and AGE‐BSA upregulated tubular expression of connective tissue growth factor (CTGF), transforming growth factor (TGF)‐β, and vascular endothelial growth factor (VEGF), whereas CML‐BSA stimulated expression of IL‐6, CCL‐2, CTGF, TGF‐β and VEGF. These AGE compounds also activated nuclear factor (NF)‐κB and their effects were attenuated by pre‐incubation with anti‐RAGE antibody. MG and BSA did not affect the expression of any of these molecules. Rosiglitazone did not affect the in vitro biological effects of MG, MG‐BSA‐AGE, AGE‐BSA or CML‐BSA on PTEC. Conclusion: AGE exhibit differential inflammatory and fibrotic effects on PTEC via RAGE activation and NF‐κB signal transduction. Rosiglitazone had no effect on these responses. Further investigations on compounds that nullify the downstream effects of these AGE are warranted.     

Connective tissue growth factor plays an important role in advanced glycation end product-induced tubular epithelial-to-mesenchymal transition: implications for diabetic renal disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.     

The effects of valsartan on the accumulation of circulating and renal advanced glycation end products in experimental diabetes

BACKGROUND: Blockade of the RAS with the ACE inhibitor ramipril prevents the accumulation of advanced glycation end products (AGEs) in experimental diabetes. Although AT1 receptor antagonists may inhibit AGE formation in vitro, their effect in normotensive animals with type 1 diabetes has not been established. METHODS: Streptozotocin-induced diabetic and control animals were randomized (N=10/group) to receive the AT1 antagonist valsartan at a dose of 30 mg/kg/day by oral gavage for 24 weeks, or no intervention. Renal and plasma AGE accumulation was correlated with renal functional parameters. RESULTS: Valsartan reduced the albumin excretion rate consistent with its renoprotective effects. Renal and skin collagen accumulation of the non-fluorescent AGE carboxymethyllysine (CML) were increased in animals with diabetes, but normalized following treatment with valsartan. Renal fluorescence and skin collagen pentosidine levels were also increased by diabetes. However, valsartan only provided a modest attenuation of these parameters. In addition, diabetes was associated with increased plasma fluorescence, which was unaffected by AT1 antagonism. CONCLUSION: Renoprotective doses of valsartan are associated with a significant reduction in the accumulation of tissue and plasma CML. These effects were not the same for all AGEs, suggesting combination approaches will be required to optimize renoprotection in diabetes.     

Macrophages in streptozotocin-induced diabetic nephropathy: potential role in renal fibrosis.

BACKGROUND: Renal fibrosis is central to the progression of diabetic nephropathy; however, the mechanisms responsible for fibroblast and matrix accumulation in this disease are only partially understood. Macrophages accumulate in diabetic kidneys, but it is unknown whether macrophages contribute to renal fibrosis. Therefore, we examined whether macrophage accumulation is associated with the progression of renal injury and fibrosis in type 1 diabetic nephropathy and whether macrophages exposed to the diabetic milieu could promote fibroblast proliferation. METHODS: Kidney macrophages, renal injury and fibrosis were analysed in diabetic C57BL/6J mice at 2, 8, 12 and 18 weeks after streptozotocin injection. Isolated rat bone marrow macrophages were stimulated with diabetic rat serum or carboxymethyllysine (CML)-bovine serum albumin (BSA) to determine whether macrophage-conditioned medium could promote the proliferation of rat renal (NRK-49F) fibroblasts. RESULTS: Progressive injury and fibrosis in diabetic nephropathy was associated with increased numbers of kidney macrophages. Macrophage accumulation in diabetic mice correlated with hyperglycaemia (blood glucose, HbA1c levels), renal injury (albuminuria, plasma creatinine), histological damage and renal fibrosis (myofibroblasts, collagen IV). Culture supernatant derived from bone marrow macrophages incubated with diabetic rat serum or CML-BSA induced proliferation of fibroblasts, which was inhibited by pre-treating fibroblasts with interleukin-1 (IL-1) receptor antagonist or the platelet-derived growth factor (PDGF) receptor kinase inhibitor, STI-571. CONCLUSION: Kidney macrophage accumulation is associated with the progression of renal injury and fibrosis in streptozotocin-induced mouse diabetic nephropathy. Elements of the diabetic milieu can stimulate macrophages to promote fibroblast proliferation via IL-1- and PDGF-dependent pathways which may enhance renal fibrosis.     

Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat

BACKGROUND: Nonenzymatic reactions between sugars or lipids and protein and formation of advanced glycation and lipoxidation end products (AGE/ALEs) contribute to the chemical modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is implicated in the development of diabetic complications. In this study, we examined the effect of the AGE/ALE inhibitor pyridoxamine on chemical modification and cross-linking of collagen and development of renal disease in the streptozotocin-diabetic rat. METHODS: Diabetic rats were treated with pyridoxamine; parallel experiments were conducted with aminoguanidine, the prototype AGE inhibitor. Progression of renal disease was evaluated by measurements of albuminuria and plasma creatinine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ALEs in skin collagen by HPLC and GC-MS assays. RESULTS: Pyridoxamine significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia and plasma lactate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and cross-linking of skin collagen increased approximately twofold in diabetic versus control rats after seven months of diabetes. Pyridoxamine caused a significant (25 to 50%) decrease the AGE/ALEs, carboxymethyllysine and carboxyethyllysine, cross-linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. CONCLUSIONS: Pyridoxamine inhibits the progression of renal disease, and decreases hyperlipidemia and apparent redox imbalances in diabetic rats. Pyridoxamine and aminoguanidine had similar effects on parameters measured, supporting a mechanism of action involving AGE/ALE inhibition.     

Advanced glycated end-products (AGE) during haemodialysis treatment: discrepant results with different methodologies reflecting the heterogeneity of AGE compounds.

BACKGROUND: There has been much recent interest in accumulation of advanced glycation end-products (AGE) in uraemic patients. Analysis of AGE has been difficult, because commonly used methodologies, i.e. immunodetection assays or fluorescence measurements, reflect group reactivity and are not specific for chemically defined substances. Some investigators measured individual AGE compounds, e.g. pentosidine, carboxymethyllysine, pyrraline or imidazolone, but a systematic assessment of known compounds using specific HPLC methods in diabetic and non-diabetic end-stage renal disease (ESRD) patients during treatment has not been performed. METHODS: For the present study, the concentrations of early and late products of the Maillard reaction in plasma and ultrafiltrate were monitored during high-flux dialysis sessions in diabetic and non-diabetic patients. AGE were analysed by fluorescence spectroscopy and size exclusion chromatography with fluorescence detection. Specific HPLC methods were used to quantify the Amadori product fructoselysine and the AGE compounds pentosidine and pyrraline in acid or enzymatic hydrolysates. RESULTS: Using size exclusion chromatography, we confirmed a similar fluorescent peak distribution for diabetic and non-diabetic ESRD patients. Main fractions were found at approximately 70, approximately 14 and <2 kDa, confirming results obtained by other authors. In diabetic patients, the fluorescence intensity of the low molecular weight fraction was higher. Uraemic patients differed from controls mainly by the fluorescence of the low molecular weight fraction. The peak spectrum in ultrafiltrates was similar to that in plasma regarding low molecular weight fractions and the 14 kDa peak, but no protein-bound fluorescence was found at 70 kDa. HPLC analysis revealed a significant reduction of plasma pentosidine during high-flux dialysis in non-diabetic (from 9.1+/-5.1 to 8.5+/-4.7 pmol/mg protein; P<0.05) and diabetic patients (from 10.0+/-9.1 to 6.8+/-4.0 pmol/mg protein; P<0.05). In contrast, plasma fructoselysine showed only a non-significant trend to decrease in diabetic (from 3.24+/-0.88 to 3.05+/-0.77 nmol/mg protein) and non-diabetic patients (from 2.69+/-0.52 to 2.56+/-0.50 nmol/mg protein). Pyrraline, a nonfluorescent late AGE product derived from reaction of 3-deoxyglucosone with lysine, could not be detected (detection limit approximately 40 pmol/mg protein). Comparing HPLC and size exclusion analysis, it was found that pentosidine accumulated in the range of low molecular weight substances and was removed by high-flux dialysis. CONCLUSIONS: High-flux dialysis reduces the plasma concentration of fluorescent AGE compounds, i.e. pentosidine, but the Amadori product fructoselysine is not removed, indicating that this compound is protein associated.     

Enhanced plasma levels of advanced glycation end products (AGE) and pro-inflammatory cytokines in children/adolescents with chronic renal insufficiency and after renal replacement therapy by dialysis and transplantation--are they inter-related?

AIMS: Advanced glycation end products (AGEs) are formed by non-enzymatic glycation or glycoxidation. After their interaction with specific receptors, they may induce expression of various proinflammatory cytokines. AGEs were shown to accumulate with advanced age, in diabetes mellitus and, in particular, in patients with chronic renal failure. In contrast to numerous investigations in adults, there are no data on plasma levels of AGEs in children with chronic renal insufficiency (CRI) and after renal replacement therapy. To elucidate the specific role of renal impairment for the formation of AGEs, these data become especially interesting by exclusion of the age-dependent modulatory effects occurring in adults. Therefore, we investigated the concentrations of fluorescent (FL) AGEs, carboxymethyllysine (CML) and markers of inflammation (CRP, IL-6, TNF-alpha) in children/adolescents with chronic renal insufficiency (CRI) and on renal replacement therapy with maintenance dialysis (D) or renal transplantation (TX). PATIENTS: Eleven CRI patients on conservative treatment (CRI, age: mean 12.6, median 12.80, SD 5.8 +/- 1.7 years, serum creatinine: 205.7, 157.5, 58.0 micromol/l, respectively), 10 patients on D (13.6, 13.0, 5.4 years, and 698.2, 633.8, 296.1 micromol/l, respectively) and 9 patients after TX (15.9, 16.0, 3.4 years, and 115.9, 128.0, 35.1 micromol/l, respectively) were included. METHODS: Plasma levels of CML, TNF-alpha, and IL-6 were determined by ELISA, FL-AGEs spectrofluorimetrically (lambda(ex)/lambda(em): 370/440 nm). RESULTS: FL-AGEs and CML levels were increased in all 3 groups with the highest levels in the D patients, a successful renal transplantation did not lead to normalization of plasma AGEs. The mean CRP and IL-6 concentrations were marginally elevated, and no significance among groups was revealed. TNF-alpha was noticeably elevated in all groups, with the highest values in CRI and TX patients, while in the dialysis patients the rise was less pronounced. Stepwise multiple regression did not reveal any correlation between AGEs and proinflammatory parameters, even after exclusion of the TX group from analysis. CONCLUSIONS: In children with CRI and on maintenance dialysis therapy, plasma AGE levels are markedly increased. After renal transplantation, AGE levels decrease without normalization. Proinflammatory parameters (except for TNF-alpha) are only mildly to moderately elevated. No association between AGE levels and data characterizing a proinflammatory state was revealed.     

Serum levels of advanced glycosylation end products in diabetic nephropathy

BACKGROUND/AIMS: Advanced glycosylation end products (AGEs) are important pathogenetic factors underlying diabetic complications. Recently, a highly reliable new enzyme-linked immunosorbent assay for these metabolites has been established. We used the assay to correlate AGEs in serum with renal function categories and histopathology in patients with diabetic nephropathy. METHODS: Type 2 diabetic patients (n = 71) and healthy control subjects (n = 35) were studied. The diabetic subjects were divided into two groups: normal renal function and chronic renal failure not requiring dialysis. In renal biopsy specimens from 22 diabetic subjects with a normal renal function, the severity of glomerular lesions was assessed morphometrically in terms of the ratio of the area of periodic acid-Schiff stained mesangium to total glomerular area. RESULTS: The serum AGE concentrations were higher in both undialyzed diabetic groups, especially in those with renal failure, than in the controls. The serum AGE concentrations increased with the severity of glomerular lesions (morphometric ratio under 15%, 2.85 +/- 0.73 mU/ml; 15-24%, 4.01 +/- 0.71 mU/ml; 25% or more, 5.12 +/- 0.64 mU/ml). CONCLUSIONS: The serum AGE levels measured by the new enzyme-linked immunosorbent assay reflected the severity of glomerulopathy, and, therefore, it may be a clinically useful tool for assessing diabetic renal complications.     

Effect of advanced glycation end products on endotoxin-induced TNF-alpha, IL-1beta and IL-8 in human peripheral blood mononuclear cells

BACKGROUND/AIMS: Advanced glycated end products (AGE) are endogenous proteins that have formed covalent complexes with sugars by a nonenzymatic process. Being proinflammatory molecules, AGE are thought to contribute to chronic systemic and local inflammatory processes associated with pathological changes in various diseases. In patients with end-stage renal disease, AGE are believed to play a role in the progression of atherosclerosis and worsening of renal failure. In patients receiving hemodialysis, AGE are thought to contribute to the inflammatory components of the therapy, particularly in diabetic patients. METHODS: In the present study, AGE were produced using 5% human serum albumin (HSA) and 50% glucose, both used for intravenous infusion into humans and both released after strict control for endotoxin content. The presence of AGE formed by HSA and glucose was confirmed using 2 independent assays. The inflammatory properties of these AGE were assessed using synthesis and release of the proinflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF) and IL-8, a chemokine. RESULTS: Alone, AGE did not induce these cytokines from peripheral blood mononuclear cells (PBMC) obtained from 14 healthy human donors. However, in the presence of 1 or 10 ng/ml of endotoxin, AGE augmented the production of IL-1 and TNF above that induced by endotoxin alone. Although the amount of augmentation of LPS-induced cytokines by AGE varied between the blood donors, the response was consistently observed and reached statistical significance. The augmentation of cytokine production was confirmed using AGE prepared with different lots of HSA and glucose. CONCLUSION: These results demonstrate that in the strict absence ofendotoxins, AGE are formed that do not stimulate cytokine production from PBMC of healthy donors, however, AGE significantly augment the synthesis and release of proinflammatory cytokine in the presence of low concentrations of endotoxins. The data suggest that renal replacement therapies should consider the role of microbial products in potentiating the biological consequences of naturally formed AGE and their potential to contribute to systemic and local inflammation in renal replacement therapies. Therefore, although the formation of AGE is unavoidable, excluding microbial products during renal replacement therapy should reduce the pathological consequences of AGE.     

Accumulation of free adduct glycation, oxidation, and nitration products follows acute loss of renal function

Glycation, oxidation, and nitration of endogenous proteins occur spontaneously and these modifications are also present in foods. Increased levels of these chemical changes are associated with chronic renal failure; however, little is known about acute kidney failure. We measured these modifications of plasma protein and related free adducts in plasma following bilateral nephrectomy and bilateral ureteral obstruction. Advanced glycation end-product (AGE) residues of plasma protein were increased 3 h post-surgery, and thereafter slowly decreased in all groups, reflecting changes in plasma protein synthesis and transcapillary flow post-surgery. Ureteral ligation increased oxidation and nitration adduct residues. There were, however, marked increases in AGE, dityrosine, or 3-nitrotyrosine free adducts in both nephrectomized and ureter-ligated rats compared to rats that had undergone sham operations. There were lower modified adduct concentrations in the ureter-ligated compared to the nephrectomized rats, reflecting residual glomerular filtration and tubular removal. There was no increase in glycated, oxidized, and nitrated proteins. Glyoxal and methylglyoxal were also increased in both renal failure models. Our study shows that the acute loss of renal function and urinary excretion leads to the accumulation of AGE, oxidation, and nitration free adducts in the plasma.     

Accelerated nephropathy in diabetic apolipoprotein e-knockout mouse: role of advanced glycation end products

Hyperlipidemia not only may be relevant to cardiovascular disease in diabetes but may also play a role in the development and progression of diabetic nephropathy. Furthermore, there is increasing evidence that advanced glycation end products (AGE) play an important role in diabetic renal disease. The objectives of this study were first to characterize renal injury in diabetic apolipoprotein E knockout (apo E-KO) mice and second to explore the role of AGE in the development and progression of renal disease in this model. Diabetes was induced by injection of streptozotocin in 6-wk-old apo E-KO mice. Diabetic animals received no treatment or treatment with the inhibitor of AGE formation aminoguanidine (1 g/kg per d) or the cross-link breaker [4,5-dimethyl-3-(2-oxo2-phenylethyl)-thiazolium chloride] ALT-711, which cleaves preformed AGE (20 mg/kg per d) for 20 wk. Nondiabetic apo E-KO mice as well as nondiabetic and diabetic C57BL/6 mice served as controls. Compared with nondiabetic apo E-KO mice, induction of diabetes in apo E-KO mice resulted in accelerated renal injury characterized by albuminuria and glomerular and tubulointerstitial injury. These abnormalities were associated with increased expression of collagen type I and type IV and transforming growth factor-beta1 (TGF-beta1), increased alpha-smooth muscle actin immunostaining and macrophage infiltration, and increased serum and renal AGE. The two treatments, which attenuated renal AGE accumulation in a disparate manner, were associated with less albuminuria, structural injury, macrophage infiltration, TGF-beta1, and collagen expression. The accelerated renal injury that was observed in diabetic apo E-KO mice was attenuated by approaches that inhibit renal AGE accumulation.     

Effect of diabetes and aging on carboxymethyllysine levels in human urine

Carboxymethyllysine (CML) has been identified as a modified amino acid that accumulates with age in human lens proteins and collagen. CML may be formed by oxidation of fructoselysine (FL), the Amadori adduct formed on nonenzymatic glycosylation of lysine residues in protein, or by reaction of ascorbate with protein under autoxidizing conditions. We proposed that measurements of tissue and urinary CML may be useful as indices of oxidative stress or damage to proteins in vivo. To determine the extent to which oxidation of nonenzymatically glycosylated proteins contributes to urinary CML, we measured the urinary concentrations of FL and CML in diabetic (n = 26) and control (n = 28) patients. The urinary concentration of FL correlated strongly with HbA1 measurements and was significantly higher in diabetic compared with control samples (9.2 +/- 6.5 and 4.0 +/- 2.8 micrograms/mg creatinine, respectively; P less than 0.0001). There was also a strong correlation between the concentrations of CML and FL in both diabetic and control urine (r = 0.67, P less than 0.0001) but only a weakly significant increase in the CML concentration in diabetic compared with control urine (1.2 +/- 0.5 and 1.0 +/- 0.3 micrograms/mg creatinine, respectively; P = 0.05). The molar ratio of CML to FL was significantly lower in diabetic compared with control patients (0.25 +/- 0.12 and 0.43 +/- 0.16, respectively; P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma levels of advanced glycation end products during haemodialysis, haemodiafiltration and haemofiltration: potential importance of dialysate quality.

BACKGROUND: Advanced glycation end products (AGEs) accumulate in patients with end-stage renal disease (ESRD). The aim of this study was to investigate the potential influence of different modalities of renal replacement therapies on plasma AGE levels. METHODS: The removal of AGEs by high-flux haemodialysis (HD) using standard and ultrapure dialysis fluid (SDF and UDF), by haemodiafiltration (HDF) and by haemofiltration (HF) was studied by fluorescence spectroscopy and by a carboxymethyllysine (CML)-specific ELISA. In addition, molecular weight distribution of fluorescent AGE products in serum of several patients was analysed by gel filtration. RESULTS: The highest AGE-typical fluorescence was found in the serum of patients on HD using SDF (114,667+/-18,967 arbitrary units (AU)), followed by patients on HDF (86,912+/-24,411 AU, P<0.005), by patients on HD using UDF (74,953+/-21,152 AU, P<0.0001) and by patients on HF (74 039+/-17 027 AU, P<0.0001). Similar results were found for serum CML levels with the highest values in HD patients on SDF (1609+/-504 ng/ml), followed by patients on HF (1354+/-614 ng/ml, P<0.001), then by HD patients on UDF (1310+/-403 ng/ml, P<0.001) and by patients on HDF (1132+/-338 ng/ml, P<0.001). The removal rate of AGEs, as evaluated by the determination of the pre-/post-dialysis AGE differences, was comparable across all groups. CONCLUSION: These findings suggest that factors other than removal are responsible for the lower pre-dialysis AGE levels found in patients on convective dialysis as well as on HD with UDF. A role of water quality is assumed. This is corroborated by the finding that the high molecular weight AGE-fraction is preferentially lowered in comparison with patients on HD with SDF, as analysed by gel filtration chromatography. These findings could be best explained by a less severe oxidative stress (i.e. resulting in decreased AGE generation) with HF and HDF, as well as with ultrapure HD.