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The AGE inhibitor pyridoxamine inhibits lipemia and development of vascular and renal disease in Zucker obese rats. Background In previous studies, pyridoxamine (PM) limited the formation of advanced glycation end products (AGEs) and development of nephropathy in streptozotocin-diabetic rats without affecting glycemic control. However, the lipid-lowering effects of PM and the correlation of plasma cholesterol and triglycerides with AGEs in skin collagen suggested that lipids might be an important source of AGEs in the diabetic rat. This study addresses the effects of hyperlipidemia on formation of advanced glycation and lipoxidation end products (AGE/ALEs) and the effects of PM on hyperlipidemia, hypertension, AGE/ALE formation, and development of nephropathy in the nondiabetic, Zucker obese rat. Methods Three groups of Zucker rats were studied: lean (Fa/ fa ), untreated fatty ( fa/fa ), and fa/fa treated with PM (2 g/L drinking water). Blood pressure, plasma lipids and creatinine, and urinary albumin were measured monthly. AGE/ALEs were measured in skin collagen by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Changes in wall thickness of the aorta and renal arterioles were evaluated by light microscopy. Results AGE/ALEs formation was increased two- to threefold in skin collagen of obese versus lean rats. PM inhibited the increases in AGE/ALEs in collagen, and significantly decreased the rise in plasma triglycerides, cholesterol, and creatinine, corrected hypertension and thickening of the vascular wall, and nearly normalized urinary protein and albumin excretion in Zucker fa/fa rats. Conclusion Lipids are an important source of chemical modification of tissue proteins, even in the absence of hyperglycemia. PM inhibited AGE/ALE formation and hyperlipidemia and protected against renal and vascular pathology in a nondiabetic model.

Studies of renal injury III: Lipid-induced nephropathy in type II diabetes Background Nephrotoxicity from elevated circulating lipids occurs in experimental and clinical situations. We tested the hypothesis that lipid-induced nephropathy causes advanced renal failure in rats with type II diabetes and dyslipidemia. Methods First generation (F1) hybrid rats derived from the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) and the LA/NIH-corpulent rat (LA/N-fa) were studied for 41 weeks while being on specific diets. Group 1 (14 rats) ingested 11.5% protein, 47.9% fat, and 40.6% carbohydrate. Group 2 (8 rats) ingested 26.9% protein, 16.7% animal fat, and 56.4% carbohydrate, and group 3 (20 rats) ingested 20.2% protein, 40.4% soy and coconut oil, and 39.4% carbohydrate. Results Hyperglycemia was more severe in rat groups 1 and 2 than in group 3. In contrast, circulating cholesterol and hydroperoxide levels were highest in group 3, intermediate in group 2, and lowest in group 1. Group 3 had severe renal failure secondary to glomerulosclerosis and tubulointerstitial disease, with striking deposition of the lipid peroxidation stress biomarker 4-hydroxynonenal in glomeruli and renal microvessels. Moreover, in group 3, increased arterial wall thickness also connoted vascular injury. In contrast, the glycoxidation stress biomarkers pentosidine and carboxymethyl-lysine were preferentially localized to renal tubules of hyperglycemic rats in groups 1 and 2 and did not segregate with the most severe renal injury. Glomerular and interstitial fibrosis was accompanied by proportional increases in renal transforming growth factor-β1 levels, which were threefold higher in the hypercholesterolemic rats of group 3 than in the hyperglycemic rats of group 1. Conclusions Acquisition of non-nodular glomerular sclerosis and tubulointerstitial disease is dependent on lipoxidation stress in rats with type II diabetes. On the other hand, in the absence of hypercholesterolemia, prolonged glycoxidation stress does not appear to be uniquely nephrotoxic.

Alterations in non-enzymatic biochemistry in uremia: Origin and significance of “carbonyl stress” in long-term uremic complications. Advanced glycation end products (AGEs), formed during Maillard or browning reactions by nonenzymatic glycation and oxidation (glycoxidation) of proteins, have been implicated in the pathogenesis of several diseases, including diabetes and uremia. AGEs, such as pentosidine and carboxymethyllysine, are markedly elevated in both plasma proteins and skin collagen of uremic patients, irrespective of the presence of diabetes. The increased chemical modification of proteins is not limited to AGEs, because increased levels of advanced lipoxidation end products (ALEs), such as malondialdehyde-lysine, are also detected in plasma proteins in uremia. The accumulation of AGEs and ALEs in uremic plasma proteins is not correlated with increased blood glucose or triglycerides, nor is it determined by a decreased removal of chemically modified proteins by glomerular filtration. It more likely results from increased plasma concentrations of small, reactive carbonyl precursors of AGEs and ALEs, such as glyoxal, methylglyoxal, 3-deoxyglucosone, dehydroascorbate, and malondialdehyde. Thus, uremia may be described as a state of carbonyl overload or “carbonyl stress” resulting from either increased oxidation of carbohydrates and lipids (oxidative stress) or inadequate detoxification or inactivation of reactive carbonyl compounds derived from both carbohydrates and lipids by oxidative and nonoxidative chemistry. Carbonyl stress in uremia may contribute to the long-term complications associated with chronic renal failure and dialysis, such as dialysis-related amyloidosis and accelerated atherosclerosis. The increased levels of AGEs and ALEs in uremic blood and tissue proteins suggest a broad derangement in the nonenzymatic biochemistry of both carbohydrates and lipids.

Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: Is there oxidative stress in uremia?BackgroundAdvanced glycation end products (AGEs), formed by non-enzymatic glycation and oxidation (glycoxidation) reactions, have been implicated in the pathogenesis of several diseases, including normoglycemic uremia. AGE research in uremia has focused on the accumulation of carbohydrate-derived adducts generated by the Maillard reaction. Recent studies, however, have demonstrated that one AGE, the glycoxidation product carboxymethyllysine (CML), could be derived not only from carbohydrates but also from oxidation of polyunsaturated fatty acids in vitro, raising the possibility that both carbohydrate and lipid autoxidation might be increased in uremia.MethodsTo address this hypothesis, we applied gas chromatography-mass spectrometry and high performance liquid chromatography to measure protein adducts formed in uremic plasma by reactions between carbonyl compounds and protein amino groups: pentosidine derived from carbohydrate-derived carbonyls, malondialdehyde (MDA)-lysine derived from lipid-derived carbonyls, and CML originating possibly from both sources.ResultsAll three adducts were elevated in uremic plasma. Plasma CML levels were mainly (>95%) albumin bound. Their levels were not correlated with fructoselysine levels and were similar in diabetic and non-diabetic patients on hemodialysis, indicating that their increase was not driven by glucose. Pentosidine and MDA-lysine were also increased in plasma to the same extent in diabetic and non-diabetic hemodialysis patients. Statistical analysis indicated that plasma levels of CML correlated weakly (P < 0.05) with those of pentosidine and MDA-lysine, but that pentosidine and MDA-lysine varied independently (P > 0.5).ConclusionsThese data suggest that the increased levels of AGEs in blood, and probably in tissues, reported in uremia implicate a broad derangement in non-enzymatic biochemistry involving alterations in autoxidation of both carbohydrates and lipids.

The serum concentration of the advanced glycation end-product Nε-(carboxymethyl)lysine is increased in uremia. Advanced glycation end products (AGEs) such as pentosidine and Nε-(carboxymethyl)lysine (CML) have been traditionally quantified by HPLC or gas chromatography—mass spectrometry (GC/MS). Enzyme-linked immunosorbent assays (ELISA) have been introduced as a convenient alternative to simplify the detection and measurement of AGEs in proteins and tissues, but some of these studies are limited by the lack of information on the structure of the epitopes recognized by antibodies to AGE-proteins. In this work we demonstrate that an antibody used in a previous study, reporting increased levels of AGEs in patients with diabetes or on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD), recognizes CML as its major epitope. We also show that there is a significant correlation between the concentration of AGEs in serum measured by ELISA and a GC/MS assay for CML in serum proteins. Both analyses yielded comparable results, with patients on CAPD and HD having about threefold higher AGE- or CML-concentrations in their serum. Our data suggest that ELISA assays for CML should be useful for the clinical measurement of AGEs in serum proteins.

modification and cross-linking of tissue proteins with age. Accelerated formation of AGE/ALEs during hyperglycemia is such as fructoselysine. The Amadori product is a precurimplicated in the development of diabetic complications. In sor to AGEs, which are a more permanent, irreversible this study, we examined the effect of the AGE/ALE inhibitor modification of proteins. According to the AGE hypothpyridoxamine on chemical modification and cross-linking of esis [1, 2], the increased rate of chemical modification collagen and development of renal disease in the streptozotocin-diabetic rat. and cross-linking of tissue proteins during hyperglycemia Methods. Diabetic rats were treated with pyridoxamine; par- compromises the structure and function of proteins and allel experiments were conducted with aminoguanidine, the contributes to the development of chronic complications prototype AGE inhibitor. Progression of renal disease was in diabetes. The accumulation of AGEs in collagen, a evaluated by measurements of albuminuria and plasma creatilong-lived protein in the extracellular matrix, is thought nine concentration. Plasma triglycerides, cholesterol, lactate and pyruvate were measured by enzymatic assays, and AGE/ to effect changes in elasticity, ionic charge, thickness and ALEs in skin collagen by HPLC and GC-MS assays. turnover of basement membrane components, setting Results. Pyridoxamine significantly inhibited the increase in the stage for development of diabetic renal, retinal and albuminuria, plasma creatinine, hyperlipidemia and plasma lac- vascular disease. tate/pyruvate ratio in diabetic rats, without an effect on blood glucose or glycated hemoglobin. AGE/ALEs, fluorescence and Reactive carbonyl and dicarbonyl compounds derived cross-linking of skin collagen increased approximately twofold from sugars are intermediates in the formation of AGEs, in diabetic versus control rats after seven months of diabetes. and oxygen, redox active transition metal ions and reacPyridoxamine caused a significant (25 to 50%) decrease the tive oxygen species are recognized as catalysts of AGE AGE/ALEs, carboxymethyllysine and carboxyethyllysine, crossformation [3]. The precise chemical origin of specific linking and fluorescence in skin collagen of diabetic rats, but did not affect pentosidine. AGEs is not always clear. Some AGEs, such as pentosiConclusions. Pyridoxamine inhibits the progression of renal dine, are clearly derived from carbohydrates, but may disease, and decreases hyperlipidemia and apparent redox im- be formed from glucose, ascorbate, 3-deoxyglucosone balances in diabetic rats. Pyridoxamine and aminoguanidine or other sugars in vivo [4]. The major AGEs, including had similar effects on parameters measured, supporting a the lysine adducts N e

Advanced glycation end products (AGEs) such as pentosidine and N epsilon-(carboxymethyl)lysine (CML) have been traditionally quantified by HPLC or gas chromatography--mass spectrometry (GC/MS). Enzyme-linked immunosorbent assays (ELISA) have been introduced as a convenient alternative to simplify the detection and measurement of AGEs in proteins and tissues, but some of these studies are limited by the lack of information on the structure of the epitopes recognized by antibodies to AGE-proteins. In this work we demonstrate that an antibody used in a previous study, reporting increased levels of AGEs in patients with diabetes or on continuous ambulatory peritoneal dialysis (CAPD) and hemodialysis (HD), recognizes CML as its major epitope. We also show that there is a significant correlation between the concentration of AGEs in serum measured by ELISA and a GC/MS assay for CML in serum proteins. Both analyses yielded comparable results, with patients on CAPD and HD having about threefold higher AGE- or CML-concentrations in their serum. Our data suggest that ELISA assays for CML should be useful for the clinical measurement of AGEs in serum proteins.