Advanced glycation end products in clinical nephrology

As a result of oxidative and carbonyl stress, advanced glycation end products (AGEs) are involved in the pathogenesis of severe and frequent diseases and their fatal vascular/cardiovascular complications, i.e. diabetes mellitus and its complications (nephropathy, angiopathy, neuropathy and retinopathy, renal failure and uremic and dialysis-associated complications), atherosclerosis and dialysis-related amyloidosis, neurodegenerative diseases, and rheumatoid arthritis. They are formed via non-enzymatic glycation which is specifically enhanced through the presence of oxidative and carbonyl stress, and their ability to form glycoxidation products in peptide and protein structures finally modulating or inducing biological reactivity. Food can be another source of AGEs; however, high serum AGEs in hemodialysis patients might reflect nutritional status better. Several methods of renal replacement therapy have been studied in connection with the AGE removal, but unfortunately the possibilities are still unsatisfactory even if high flux dialysis, hemofiltration, or hemodiafiltration give better results than conventional low flux dialysis. AGEs are currently being studied in the patients on peritoneal dialysis as their precursors can be formed in the dialysis fluid. AGEs can cause damage to the peritoneum and so a loss of ultrafiltration capacity. Many compounds give promising results in AGE inhibition (inhibition of formation of AGEs, inhibition of their action or degradation of AGEs), are tested for these properties, and eventually undergo clinical studies (e.g. aminoguanidine, OPB-9195, pyridoxamine, antioxidants, N-phenacylthiazolium bromide, antihypertensive drugs, angiotensin-converting enzyme inhibitors and angiotensin II receptor-1 antagonists).     

Angiotensin II receptor antagonists and angiotensin-converting enzyme inhibitors lower in vitro the formation of advanced glycation end products: biochemical mechanisms

The implication of advanced glycation end products (AGE) in the pathogenesis of atherosclerosis and of diabetic and uremic complications has stimulated a search for AGE inhibitors. This study evaluates the AGE inhibitory potential of several well-tolerated hypotensive drugs. Olmesartan, an angiotensin II type 1 receptor (AIIR) antagonist, as well as temocaprilat, an angiotensin-converting enzyme (ACE) inhibitor, unlike nifedipine, a calcium blocker, inhibit in vitro the formation of two AGE, pentosidine and N(epsilon)-carboxymethyllysine (CML), during incubation of nonuremic diabetic, nondiabetic uremic, or diabetic uremic plasma or of BSA fortified with arabinose. This effect is shared by all tested AIIR antagonists and ACE inhibitors. On an equimolar basis, they are more efficient than aminoguanidine or pyridoxamine. Unlike the latter two compounds, they do not trap reactive carbonyl precursors for AGE, but impact on the production of reactive carbonyl precursors for AGE by chelating transition metals and inhibiting various oxidative steps, including carbon-centered and hydroxyl radicals, at both the pre- and post-Amadori steps. Their effect is paralleled by a lowered production of reactive carbonyl precursors. Finally, they do not bind pyridoxal, unlike aminoguanidine. Altogether, this study demonstrates for the first time that widely used hypotensive agents, AIIR antagonists and ACE inhibitors, significantly attenuate AGE production. This study provides a new framework for the assessment of families of AGE-lowering compounds according to their mechanisms of action.     

Advanced glycation and lipidoxidation of the peritoneal membrane: respective roles of serum and peritoneal fluid reactive carbonyl compounds

BACKGROUND: Advanced glycation of proteins has been incriminated in the progressive alteration of the peritoneal membrane during chronic peritoneal dialysis (PD). Advanced glycation end products (AGEs) result from a modification of proteins by reactive carbonyl compounds (RCOs). RCOs resulting from glucose breakdown are present in commercial PD fluid. They also accumulate in uremic plasma. The present study was undertaken to evaluate the respective contribution of these two sources of RCOs in the genesis of peritoneal AGEs. METHODS: Three major RCOs formed during heat sterilization of PD fluid, that is, glyoxal, methylglyoxal, and 3-deoxyglucosone, and total RCOs were measured in commercial PD fluid and in PD effluent. The generation of pentosidine, used as a surrogate marker for AGEs, during one-week incubations of PD fluid and effluent samples fortified with bovine serum albumin (BSA) was measured by high-performance liquid chromatography. Peritoneal samples were stained with antibodies specific for two AGEs derived from carbohydrate-dependent RCOs, Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, or for two advanced lipoxidation end products (ALEs) derived from lipid-dependent RCOs, malondialdehyde (MDA)-lysine and 4-hydroxynonenal (HNE)-protein adduct. RESULTS: Glyoxal, methylglyoxal, and 3-deoxyglucosone were identified in commercial PD fluid. Their levels in PD effluents decreased with dwell time probably by diffusion into blood circulation. In contrast, the levels of total RCOs were initially low in commercial PD fluid, increased in PD effluent with dwell time probably by diffusion from circulation into the peritoneal cavity, and after 12 hours, reached values observed in uremic serum. The relevance of the rise in total RCOs for AGE formation is demonstrated by a parallel increase in the generation of pentosidine during incubations of PD effluents. In contrast with RCOs present in glucose-rich PD fluid, RCOs diffusing from uremic circulation originate from both carbohydrates and lipids. Their role in the modification of peritoneal proteins is demonstrated by the immunohistochemical study of peritoneal tissue. Two AGEs and two ALEs increase in parallel in the mesothelial layers and in vascular wall of small arteries in the peritoneum. CONCLUSIONS: Protein modification of the peritoneum is determined not only by RCOs originating in PD fluid, but also by RCOs originating from the uremic circulation. The present data might be relevant to current attempts to improve PD fluid toxicity by lowering its glucose content.     

Pyridoxamine improves functional, structural, and biochemical alterations of peritoneal membranes in uremic peritoneal dialysis rats

BACKGROUND: We previously suggested that biochemical alterations of peritoneal membrane associated with long-term peritoneal dialysis might be, at least in part, accounted for by reactive carbonyl compounds overload originating both from uremic circulation and heat sterilization of glucose peritoneal dialysis fluid. In the present study, we utilized a uremic rat model on peritoneal dialysis and evaluated the protective effects of pyridoxamine, a recently developed inhibitor of advanced glycation end product (AGE), on structural, functional, and biochemical alterations of peritoneal membrane. METHODS: Uremic rats were generated by subtotal nephrectomy, some of which were undergone peritoneal dialysis with dialysate and/or given intraperitoneal pyridoxamine. Functional [dialysate/plasma ratio (D/P)(urea, creatinine), D/D(0 glucose)], structural (density of blood vessels in peritoneal membrane tissues), and molecular biochemical [formation of pentosidine, an AGE, by high-performance liquid chromatography (HPLC) assay and expressions of vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2), by semiquantitative polymerase chain reaction (PCR) and/or immunohistochemistry] alterations of peritoneal membrane were assessed. RESULTS: Uremic peritoneal membrane was characterized by an increased functional area of exchange for small solutes between blood and dialysate, vascular proliferation, increased AGE genesis, and up-regulated expressions of angiogenic cytokines. The peritoneal membrane alterations associated with peritoneal dialysis are similar but more severe than those in uremia without peritoneal dialysis. Pyridoxamine given in uremic rats with peritoneal dialysis significantly improved functional and structural alterations. This improvement was accompanied by reduction of AGE accumulation and of angiogenic cytokines expressions. CONCLUSION: Peritoneal carbonyl stress derived from uremia as well as peritoneal dialysis procedure might contribute to the vascular proliferation through induction of bioactive molecules and to an increased functional area, eventually leading to ultrafiltration failure. Pyridoxamine may be beneficial in protection of uremic peritoneal membrane on peritoneal dialysis.     

Nitric oxide inhibits the formation of advanced glycation end products

BACKGROUND: Advanced glycation end products (AGEs) are elevated in renal failure and have been implicated in the pathogenesis of several uremic complications. Their formation is closely associated with oxidative stress. The recent observation that nitric oxide (NO) has an antioxidant effect led us to examine the possible role of NO in the generation of AGEs. METHODS: We examined the effect of NO donors, 2, 2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) and S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the in vitro formation of pentosidine, which was used as a surrogate marker for AGEs. Bovine serum albumin was incubated under air at 37 degrees C in a medium containing either several AGE precursors or uremic plasma. To elucidate further the mechanism of the NO effect on AGE formation, we examined the generation of free radicals and carbonyls in pentose-driven pentosidine formation. RESULTS: NO donors significantly inhibit the formation of pentosidine in a dose-dependent manner. The effect is abolished by the addition of a NO scavenging agent, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). The inhibitory effect results from NO but not from the NO donor molecule. It is best explained by the ability of NO to scavenge carbon-centered radicals, hydroxyl radical, and carbonyl compounds. CONCLUSIONS: NO inhibits pentosidine formation by scavenging free radicals and by inhibiting carbonyl compound formation. NO might be implicated in the atherogenic and inflammatory effects of AGEs: Reduced NO production and increased oxidative stress associated with atherosclerotic lesions may accelerate AGE formation and, thus, exacerbate endothelial dysfunction and accelerate the development of atherosclerosis in uremia.     

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.     

Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine.

Aminoguanidine-HCl inhibits the formation of advanced glycosylation end products (AGEs) in vitro and in vivo, but the mechanism by which this occurs has not been determined. Aminoguanidine inhibited glucose-derived AGE formation on RNase A by 67-85% at aminoguanidine-glucose molar ratios of 1:5 to 1:50 without affecting the concentration of Amadori products. Fast-atom-bombardment mass spectrometry of RNase peptides incubated with glucose alone or with glucose plus aminoguanidine showed that aminoguanidine inhibited the formation of AGEs without forming an adduct with glycosylated peptide. These data suggest that the primary mechanism of aminoguanidine action is reaction with Amadori-derived fragmentation products in solution. These findings are relevant to the potential clinical use of aminoguanidine in the prevention of diabetic complications.     

Mechanisms for the formation of glycoxidation products in end-stage renal disease

BACKGROUND: Advanced glycation end products (AGEs) accumulate on tissue and plasma proteins in patients with renal failure far in excess of normal aging or diabetes. The aim of these studies was to elucidate the nature of the precursors and the pathways that lead to an accelerated formation of two structurally identified AGEs [pentosidine and Nepsilon(carboxymethyl)lysine (CML)] in the uremic milieu. METHODS: Serum levels of the glycoxidation products, pentosidine and CML, were quantitated by high-performance liquid chromatography in uremic patients treated by dialysis. The formation of early glycation products (as furosine) and late glycoxidation products was modeled in uremic serum and in spent peritoneal dialysate. RESULTS: Clinical factors that affect circulating levels of AGEs included dialysis clearance and dialyzer membrane pore size, but not the presence or absence of diabetes. Both pentosidine and CML form at an accelerated rate in serum from uremic patients. Chelating agents most effectively slow the formation in vitro. In uremic fluids, the primary mechanism of formation of pentosidine is through the Amadori pathway. The primary mechanism of formation of CML is through metal-chelated autoxidation of reducing sugars generating reactive carbonyl precursors. In uremic serum, the presence of an unidentified reactive low molecular weight precursor accelerates the formation of pentosidine. CONCLUSIONS: The formation of the two glycoxidation products, pentosidine and CML, proceeds by different pathways and is enhanced by different precursors in the uremic milieu. The formation of both AGEs is markedly enhanced by metal-catalyzed reactions, evidence for the presence of increased metal-ion mediated oxidant stress in uremia.     

Glycolaldehyde, a reactive intermediate for advanced glycation end products, plays an important role in the generation of an active ligand for the macrophage scavenger receptor

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs) that are recognized by AGE receptors. Glyoxal, glycolaldehyde (GA), and methylglyoxal are potential intermediates for the formation of AGE structures such as Nomega-(carboxymethyl)lysine (CML). We evaluated the contribution of these aldehydes to the formation of AGE structure(s), particularly the structure important for the receptor-mediated endocytic uptake of AGE proteins by macrophages. GA-modified bovine serum albumin (BSA), methylglyoxal-modified BSA (MG-BSA), and glyoxal-modified BSA (GO-BSA) were prepared, and their physicochemical, immunological, and biologic properties were compared with those of glucose-derived AGE-BSA. CML contents were high in GO-BSA and low in GA-modified BSA (GA-BSA) but did not exist in MG-BSA. The fluorescence patterns of GA-BSA and MG-BSA were similar to those of glucose-derived AGE-BSA but were weak in GO-BSA. Immunochemically, the antibody against non-CML structures of glucose-derived AGE-BSA reacted strongly with GA-BSA and weakly with GO-BSA but did not react with MG-BSA. The negative charge of these ligands increased to a similar extent. However, GA-BSA, but not MG-BSA or GO-BSA, underwent receptor-mediated endocytosis by the macrophage-derived cell line RAW 264.7, which was effectively inhibited by glucose-derived AGE-BSA, acetylated LDL, and oxidized LDL, which are well-known ligands for the macrophage type I and type II class A scavenger receptors (MSR-A). The endocytic uptake of GA-BSA by mouse peritoneal macrophages was also significant, but that by peritoneal macrophages from MSR-A-deficient mice was markedly reduced. Our results suggest that GA serves as an important intermediate for the generation of AGE structure(s) responsible for recognition by MSR-A.     

Effect of dwell time on carbonyl stress using icodextrin and amino acid peritoneal dialysis fluids

BACKGROUND: Deterioration of the peritoneal membrane limits the technical survival of peritoneal dialysis (PD). Advanced glycation of the membrane has been incriminated in this evolution. Advanced glycation end products (AGEs) develop under the influence of glucose and of its degradation products, mainly reactive carbonyl compounds (RCOs) such as glyoxal (GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG). The present study was undertaken to evaluate the impact of recently developed glucose-free PD fluids on AGE generation. METHODS: Recently developed glucose-free PD fluids containing either icodextrin or amino acids were investigated. GO, MGO, and 3-DG [high-performance liquid chromatography (HPLC)] and total RCOs (spectrophotometry) were measured in fresh solutions and in effluents after various dwell duration. The AGE formation potential of PD fluids and effluents was assessed by incubation at 37 degrees C, for one week, with bovine serum albumin and by the eventual measurement of pentosidine (HPLC) and Nepsilon-carboxymethyllysine (CML; gas chromatography/mass spectrometry). RESULTS: GO, MGO, and 3-DG (P < 0. 001) as well as total RCOs levels (P < 0.01) were significantly lower in icodextrin and amino acid PD fluid than in commercial, heat-sterilized, 1.36% glucose PD fluid. Pentosidine and CML generation were also significantly lower (P < 0.001) in icodextrin and amino acid PD fluid than in conventional 1.36% glucose PD fluid. The levels of total RCOs, however, increased in icodextrin and amino acid PD fluid effluents with dwell time. AGE formation potential rose accordingly, as demonstrated by a parallel increase in the generation of pentosidine and CML during incubation of PD effluents. CONCLUSION: The present data demonstrate lower RCO contents and AGE formation potential in fresh icodextrin and amino acid PD fluids than in fresh heat-sterilized glucose PD fluids. However, this difference decreases progressively during dwell time, mainly as a result of the influx of total RCOs.     

Renal proximal tubular metabolism of protein-linked pentosidine,an advanced glycation end product

BACKGROUND: Pentosidine, an advanced glycation end product, accumulates in plasma proteins of uremic patients. Its fate is, however, yet to be fully understood. METHODS: Three cell lines, JTC-12 (proximal tubular cells), MDCK (distal tubular cells), and BALB3T3 (nonrenal cells), were cultured in a double chamber system and were exposed to uremic serum, and the contents of protein-linked pentosidine derived from uremic sera were determined in each medium by HPLC assay. The presence of pentosidine in the cytoplasm of these cells was assessed by immunoperoxidase staining. RESULTS: When the apical cell membrane was exposed to uremic serum (fortified in the upper chamber), the contents of protein-linked pentosidine in the upper medium decreased by up to 30% after 24- and 48-hour incubations of JTC-12 cells but not of other cells. On the other hand, the contents of protein-linked pentosidine in the lower medium did not change. By contrast, exposure of the basolateral cell membrane of the three cell lines to uremic serum (fortified in the lower chamber) did not change the contents of protein-linked pentosidine both in the upper and lower medium after a 24-hour incubation. Pentosidine was detected immunohistochemically in the cytoplasm of JTC-12 cells, but not of BALB3T3 and MDCK cells, the apical membranes of which were exposed to uremic sera for 8 h. The immunoreaction disappeared 48 h after exposure. Pentosidine was not detected in the cytoplasm of JTC-12 cells, the basolateral membranes of which were exposed to uremic sera. The relevance of the in vitro results to humans was demonstrated by immunohistochemical studies in normal human kidney tissues showing that pentosidine was identified in the proximal renal tubules. CONCLUSION: These results suggest that the proximal tubular cells play a role in the disposal of plasma pentosidine.     

Oxidative stress, advanced glycation end product, and coronary artery calcification in hemodialysis patients

Coronary artery calcification is an index of the severity of atherosclerotic vascular disease, and may predict future adverse cardiovascular events in uremic patients undergoing hemodialysis (HD). HD patients are exposed to oxidative stress, and show high plasma levels of advanced glycation end products (AGEs). The association between oxidative stress, AGEs, established cardiovascular risk factors, and coronary artery calcification score (CACS) was studied in 225 HD patients (123 male, 102 female patients). CACS was measured by using multi-detector row computed tomography. Age, systolic blood pressure, calcium, calcium x phosphate, malondialdehyde, lipid peroxides, and pentosidine were significantly and positively correlated with CACS. Duration on HD tended to be positively correlated with CACS. From the independent variables included in the forward stepwise multiple linear regression analysis, only age, systolic blood pressure, lipid peroxides, calcium, and pentosidine were independently associated with CACS. The odds ratios for past history of coronary artery disease and the presence of diabetes mellitus for high CACS (> or =100) were 6.25 (95% confidence interval; 1.83-21.4) and 2.03 (95% confidence interval; 1.02-4.05), respectively. The plasma pentosidine was significantly and positively correlated with indoxyl sulfate. In conclusion, in addition to such traditional cardiovascular risk factors as past history, diabetes mellitus, aging, systolic blood pressure and calcium overload, oxidative stress (lipid peroxides), and AGE (pentosidine) are associated with extensive coronary artery calcification in HD patients. Lipid peroxidation and glycoxidation may be involved in the pathogenesis of coronary artery calcification.     

Reduced systemic advanced glycation end products in children receiving peritoneal dialysis with low glucose degradation product content.

BACKGROUND: Glucose degradation products (GDP) in peritoneal dialysis (PD) solutions are toxic to the peritoneal membrane and promote the formation of advanced glycation end products (AGE), which contribute to accelerated atherosclerosis and amyloidosis. Double chamber PD solutions have a markedly reduced GDP content. METHODS: We analysed GDP and AGE kinetics in 21 children (7 months to 18 years) on automated PD in a prospective multicentre trial with randomized administration of single chamber, high-GDP and double-chamber, low-GDP dialysis solution for 12 weeks each. Total AGE fluorescence, carboxymethyllysine (CML, ELISA) and 3-deoxyglucosone (3-DG, HPLC) were measured in plasma and PD effluent during a 4 h peritoneal equilibration test. Plasma AGE profiles were assessed by size selective gel permeation chromatography and compared with 23 healthy controls. RESULTS: Initial effluent 3-DG concentrations were 140+/-55 and 25+/-4 micromol/l with high- and low-GDP PD fluid, respectively and declined to 53+/-32 and 7+/-2 micromol/l within 4 h dwell time (P<0.001). The ex vivo AGE generating capacity was three times higher with the high-GDP solution and decreased significantly with dwell time. Plasma AGE levels were 1.8-7.4-fold above those of healthy controls; the elevation was most marked for the small molecular fraction (<2 kDa). Plasma AGE and CML levels were significantly higher after 12 weeks exposure to high-GDP solution (20991+/-4145 AU and 1505+/-617 ng/ml) than following treatment with low-GDP fluid (17518+/-4676 AU and 1151+/-438 ng/ml; both P<0.05). Four hour AGE clearance was higher with low-GDP solution (0.74+/-0.3 vs 0.44+/-0.15 ml/min*1.73 m2, P<0.01). CONCLUSION: GDP are rapidly absorbed from the peritoneal cavity. Administration of PD solutions with low-GDP content reduces plasma AGE levels and may thus improve the cardiovascular risk profile of dialysed children.     

Age-related increase in an advanced glycation end product in penile tissue

Summary Nonenzymatic glycosylation (glycation) of proteins, often referred to as the Maillard reaction, has been proposed to play a role in age and diabetes-related processes by forming protein and DNA adducts and cross-links. These cross-links may contribute to erectile dysfunction by scavenging nitric oxide, which is needed for erection. As the basis for a possible role of the advanced Maillard reaction in age-related erectile dysfunction, we investigated the presence of the specific advanced glycation endproduct (AGE) pentosidine in penile corpus cavernosum tissue and penile tunica albuginea tissue as a function of age. A total of 23 penile tissue specimens were obtained at autopsy, from which 19 samples of tunica albuginea and 21 samples of corpus cavernosum were derived. In addition, 13 penile corporal and tunical specimens were procured at the time of insertion of a penile prosthesis, from which 12 tunica albugineal specimens and 10 samples of corpus cavernosum were derived. Collagen was extracted withacetic acid and pepsin digestion, and the final insoluble collagen product was acid-hydrolyzed with 6 N HCL for 24 h at 110°C. Pentosidine was quantified by high-performance liquid chromatography using a reverse-phase column. The level of pentosidine (expressed in picomoles per milligram of insoluble collagen) was found to increase with age in cadaver as well as living penile corporal and tunical albugineal tissues. Best-fit analysis revealed an exponential increase in both types of cadaver penile tissue, with regression equations of y=15.29×10^(9.9e–3x), R ²=0.79, being obtained in the tunica and y=13.2×10^(7.63e–3x), R ²=0.56, in the corpora. These correspond to 6- and 4-fold increases in pentosidine levels from puberty to the age of 100 years (P<0.05), respectively. Mean pentosidine levels were higher in the tunica than in the corpora. Comparison of pentosidine levels in the tunica versus the corpora revealed a weakly linear correlation (y=24.88+1.08x, R ²=0.32). Levels in the tunical and corporal specimens from the living human specimens fell with the predicted confidence intervals of the cadaveric tissue. Tunical specimens from patients who underwent repair or revision of a previously inserted penile prosthesis had very low levels of pentosidine. The exponential age-related increase in pentosidine observed in both types of penile tissue suggests and impairment of collagen turnover, which could be related to the advanced glycation reaction in aging. It is not known whether pentosidine itself is directly associated with erectile dysfunction, but its formation is usually accompanied by extensive tissue modification. Formation of advanced Maillard reaction products, which is greatly accelerated in aging, diabetes, and uremia, could contribute to erectile dysfunction in these syndromes.     

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").     

Anti-hypertensive agents inhibit in vivo the formation of advanced glycation end products and improve renal damage in a type 2 diabetic nephropathy rat model

Prevention or retardation of diabetic nephropathy (DN) includes anti-hypertensive treatment with angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor blockers (ARB) on the premises that these drugs have an added protective effect beyond their influence on BP. The present study used a strain of spontaneously hypertensive/NIH-corpulent rats [SHR/NDmc-cp (fat/fat)] as a model of type II DN to unravel the renoprotective effects of anti-hypertensive drugs. Olmesartan (1 or 5 mg/kg per d), an ARB, and hydralazine (5mg/kg per d), an anti-hypertensive drug without effect on the renin-angiotensin system (RAS), were given for 20 wk. BP, renal function, glucose and insulin levels, and proteinuria were monitored. Glomerular lesions and kidney pentosidine content were assessed at the end of the study. Olmesartan (1 and 5 mg) significantly reduced BP and kidney pentosidine content and improved histologic renal damage and proteinuria. The changes were dose-dependent. The effect of hydralazine (5 mg) was similar to that of olmesartan (1 mg) but reached statistical significance only for kidney pentosidine content. The similarity of both drugs' effects on kidney damage and proteinuria suggest that renoprotection does not hinge on manipulation of RAS in these rats. By contrast, the inhibition of renal pentosidine formation assessed both by immunohistochemistry and HPLC suggests a critical role of advanced glycation end product (AGE) formation together with hypertension in the genesis of diabetic nephropathy. This view is supported by the correlation found between renal pentosidine content and proteinuria. The unsuspected AGE-lowering effect of hydralazine was further confirmed in vitro and elucidated; it is due to both reactive carbonyl compounds trapping and modifications of the oxidative metabolism. It is concluded that AGE inhibition should be included in the therapeutic strategy of DN.     

Effect of the peritoneal dialysis prescription on pentosidine in children

Enhanced formation of advanced glycation end products (AGEs) by peritoneal dialysate containing high dextrose concentrations has been implicated as a source of peritoneal membrane toxicity and loss of viability in patients treated with peritoneal dialysis (PD). The goal of this project was to elucidate the relationship between the structurally defined AGE pentosidine accumulation on peritoneal and plasma proteins and peritoneal membrane function, and to identify clinical factors leading to alterations in these parameters. The study comprised 27 pediatric patients (14 continuous ambulatory PD, 13 chronic cycling PD) on PD for a mean duration of 37.0+/-22.8 months (range 1-120 months) and with a mean age of 13.3+/-4.4 years (range 2.4-20 years). The pentosidine contents of plasma and peritoneal proteins were significantly lower in patients with residual renal function than in patients who were anuric (plasma pentosidine 11.2+/-8.8 vs. 24.1+/-16.6, P=0.02, respectively, peritoneal pentosidine 14.9+/-11.9 vs. 31.1+/-3.7, P=0.01, respectively). There was no effect of treatment modality on plasma pentosidine (18.1+/-11.2, 18.8+/-19.3, CAPD vs. CCPD, P>0.05) or peritoneal pentosidine content (24.1+/-14.1, 24.9+/-19.6, CAPD vs. CCPD, P>0.05). There was no evidence that increased levels of pentosidine on peritoneal proteins reflect or affect peritoneal membrane function in these patients. Furthermore, there was no effect of peritonitis on the pentosidine content of peritoneal proteins or peritoneal function as measured by peritoneal equilibration test. In conclusion, PD represents a well-tolerated therapy in children with no evidence that current practice causes changes in peritoneal membrane function, or in the peritoneal clearance of plasma or peritoneal proteins rich in pentosidine.