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Advanced glycation end products (AGEs) have been linked to the pathogenesis of diabetic nephropathy. Here we tested the effect of AGE-modified bovine serum albumin (AGE-BSA) on differentiated mouse podocytes in culture. Differential display and real-time PCR analyses showed that in addition to neuropilin-1, the entire signaling receptor complex of neuropilin-2, semaphorin-3A, and plexin-A1, was significantly reduced by AGE-BSA as was neuropilin-1 protein. The effect was specific for podocytes compared to isolated mesangial and tubular epithelial cells. Further, AGE-BSA was not toxic to podocytes. Neuropilin-1 expression was decreased in glomeruli of diabetic db/db mice compared to their non-diabetic littermates. Transcripts of both neuropilins were found to be decreased in renal biopsies from patients with diabetic nephropathy compared to transplant donors. Podocyte migration was inhibited by AGE-BSA with similar results found in the absence of AGE-BSA when neuropilin-1 expression was down-regulated by siRNA. In contrast, podocyte migration was stimulated by overexpression of neuropilin-1 even in the presence of AGE-BSA. Our study shows that AGE-BSA inhibited podocyte migration by down-regulating neuropilin-1. The decreased migration could lead to adherence of uncovered areas of the glomerular basement membrane to Bowman's capsule contributing to focal glomerulosclerosis.

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.

The accumulation of advanced glycation end products (AGE) is a key factor in diabetic nephropathy (DN). Pyridoxamine inhibits AGE formation and protects against type I DN. Herein we tested: (1) whether C57BL6 db/db mice as a model of established type II DN resembled patients treated with drugs which inhibit angiotensin II action; (2) whether pyridoxamine was effective as a single therapy; and (3) whether pyridoxamine would add to the benefit of angiotensin-converting enzyme inhibition (ACEi) by enalapril. In first set of experiments mice were treated with ACEi (benazepril) and an angiotensin II receptor blocker (valsartan) combination for 16 weeks after the onset of diabetes. In second group, mice with established DN were treated with pyridoxamine for 8 weeks. In a third set, mice with established DN were treated with pyridoxamine and enalapril combination for 16 weeks. Benazepril and valsartan combination partially prevented the development and progression of DN. Pyridoxamine treatment, as single therapy, decreased the progression of albuminuria and glomerular lesions. The combination of pyridoxamine with enalapril reduced both mortality and the progression of DN. In conclusion, (1) C57 BL6 db/db mice are a model of progressive type II DN; (2) The combination of pyridoxamine with enalapril decreased progression of type 2 DN and overall mortality. Thus, pyridoxamine could be a valuable adjunct to the current treatment of established type II DN.

Advanced glycation end product (AGE)-mediated modification of proteins is enhanced both in the kidneys and aortas of diabetic and uremic patients. However, AGE modification of deoxyribonucleic acid (DNA) has not yet been reported in these patients. We performed immunohistochemistry of kidneys and aortas using a monoclonal antibody against N 2 -carboxyethyl-2′-deoxyguanosine (CEdG), a marker of AGE-linked DNA. A total of 20 kidneys and 20 aortas were obtained by autopsy. The kidney samples consisted of two groups: nondiabetic nonkidney disease (control) and diabetic nephropathy. The aorta samples consisted of four groups: nondiabetic nonkidney disease (control), diabetes, hemodialysis, and diabetic hemodialysis. In the kidneys CEdG was detected predominantly in the nuclei of epithelial cells, mesangial cells, and endothelial cells of the glomeruli, parietal epithelial cells, and tubular cells. The number of CEdG-positive cells in the glomeruli was significantly increased in diabetic nephropathy compared with control. In the aortic walls, CEdG was detected predominantly in the nuclei of macrophages and myofibroblasts. The number of CEdG-positive cells in the aorta was significantly increased in hemodialysis patients and diabetic hemodialysis patients compared with control. The highest number of CEdG-positive cells in the aorta was observed in diabetic hemodialysis patients. In conclusion, AGE-mediated modification of DNA is enhanced in the kidney of diabetic nephropathy and the aorta of uremic atherosclerosis, and may induce a loss of genetic integrity in these diseases.

Background. Advanced glycation end products (AGEs) contribute to diabetic and atherosclerotic end-organ damage, but the mechanisms of AGE-formation and AGE- induced damage are unclear. Glycolaldehyde (GA) is a Maillard-reaction intermediate and can be formed by reaction of L-serine with the myeloperoxidase-system. GA reacts with proteins to form AGEs, such as GA-pyridine, which is specific for protein modification by GA. GA-pyridine accumulates in human atherosclerotic lesions. As atherosclerosis and progressive glomerulosclerosis share many similarities, we hypothesized that GA-pyridine accumulates in renal diseases, especially those with prominent mesangial involvement.Methods. Paraffin-embedded renal biopsies from 55 patients with various renal diseases, as well as control tissue, obtained from the unaffected part of kidneys from 10 patients with renal cell carcinoma were immunohistochemically stained with a monoclonal antibody directed against GA-pyridine and were scored semiquantitatively. Additional sections were scored for mesangial matrix expansion (MME) and focal glomerular sclerosis (FGS).Results. In normal human kidneys, GA-pyridine was mainly localized in tubular epithelial cells, but not in the glomerular mesangium. Significant mesangial GA-pyridine accumulation was found in disorders with mesangial involvement as a common-denominator. In contrast, mesangial GA-pyridine accumulation was less prominent in renal diseases without prominent mesangial involvement. Moreover, mesangial GA-pyridine accumulation was more pronounced in kidneys with higher MME and FGS scores across the different diagnoses.Conclusion. GA-pyridine accumulates in the mesangium in human renal disease, in particular in disorders with mesangial involvement. Further studies should elucidate whether mesangial GA-pyridine plays a role in the progression of glomerular damage.

Mesangial accumulation of GA-pyridine, a novel glycolaldehyde-derived AGE, in human renal disease. Background Advanced glycation end products (AGEs) contribute to diabetic and atherosclerotic end-organ damage, but the mechanisms of AGE-formation and AGE-induced damage are unclear. Glycolaldehyde (GA) is a Maillard-reaction intermediate and can be formed by reaction of L-serine with the myeloperoxidase-system. GA reacts with proteins to form AGEs, such as GA-pyridine, which is specific for protein modification by GA. GA-pyridine accumulates in human atherosclerotic lesions. As atherosclerosis and progressive glomerulosclerosis share many similarities, we hypothesized that GA-pyridine accumulates in renal diseases, especially those with prominent mesangial involvement. Methods Paraffin-embedded renal biopsies from 55 patients with various renal diseases, as well as control tissue, obtained from the unaffected part of kidneys from 10 patients with renal cell carcinoma were immunohistochemically stained with a monoclonal antibody directed against GA-pyridine and were scored semiquantitatively. Additional sections were scored for mesangial matrix expansion (MME) and focal glomerular sclerosis (FGS). Results In normal human kidneys, GA-pyridine was mainly localized in tubular epithelial cells, but not in the glomerular mesangium. Significant mesangial GA-pyridine accumulation was found in disorders with mesangial involvement as a common denominator. In contrast, mesangial GA-pyridine accumulation was less prominent in renal diseases without prominent mesangial involvement. Moreover, mesangial GA-pyridine accumulation was more pronounced in kidneys with higher MME and FGS scores across the different diagnoses. Conclusion GA-pyridine accumulates in the mesangium in human renal disease, in particular in disorders with mesangial involvement. Further studies should elucidate whether mesangial GA-pyridine plays a role in the progression of glomerular damage.

In vitro evidence for immune activating effect of specific AGE structures retained in uremia.BackgroundAdvanced glycation end-products (AGEs) have been identified to be accumulated in blood and tissues of patients with end-stage renal disease (ESRD). AGEs have been shown to modulate immune competent cell activities and in this way they may contribute to the progression of atherosclerosis. All studies in this context have been performed, however, with generated mix of glycation compounds, and not with structures similar to those encountered in uremia. In the present study, the immunologic effect of specific AGE compounds, known to be retained in uremia, has been evaluated.MethodsFour albumin preparations, modified chemically at lysine or arginine residues, respectively, to contain N-ε-carboxymethyllysine (CML albumin), N-ε-carboxyethyllysine (CEL albumin), glyoxal-induced imidazolinones (Arg I albumin) or methylglyoxal-induced imidazolinones (Arg II albumin) were applied. Their effect on chemiluminescence production, CD14 expression, and the DNA synthesis of calcitriol-differentiated HL-60 (monocyte/macrophage phenotype) was studied.ResultsThe phorbol 12-myristate 13-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of CEL albumin (44.1 ± 18.5 vs. 64.7 ± 28.1 counts 103/30 min) (P < 0.05), Arg I albumin (46.4 ± 18.8 vs. 66.1 ± 32.6 counts 103/30 min) (P < 0.05) and CML albumin (41.9 ± 25.5 vs. 60.9 ± 5.5 counts 103/30 min) (P = 0.0625) pointing to an increase in free radical production. The latter AGE compounds also significantly increased the calcitriol-induced CD14 expression on HL-60 cells (1675 ± 796 vs. 2075 ± 1044; 768 ± 143 vs. 890 ± 150; 647 ± 63 vs. 716 ± 69 mean fluorescence intensity) (P < 0.05, respectively) pointing to an increase in expression of the lipopolysaccharide (LPS) receptor. Finally, the DNA synthesis of the calcitriol-differentiated HL-60 cells was enhanced in the presence of Arg I albumin [34.5 ± 4.6 vs. 27.7 ± 9.7% 5-bromo-2′-deoxyuridine (BrdU)-positive cells] (P < 0.05) resulting in an increased cell proliferation.ConclusionGenuine AGE compounds, as they are encountered in the uremic condition, activate leukocyte response, and hence could play a role in uremia related atherogenesis.

The in vitro biocompatibility performance of a 25mmol/L bicarbonate/10mmol/L lactate-buffered peritoneal dialysis fluid. Background. The biocompatibility profile of a new peritoneal dialysis (PD) solution (Physioneal 35) was determined using a selection of in vitro assay systems. Physioneal 35 is buffered by a combination of 25mmol/L bicarbonate and 10mmol/L lactate, thereby providing a solution with a total of 35mmol/L of alkali to complement the currently available 25mmol/L bicarbonate and 15mmol/L lactate combination solution, Physioneal 40. In addition, the new solution contains a calcium concentration of 1.75mmol/L rather than 1.25mmol/L present in Physioneal 40. Physioneal 35 and 40 are manufactured in double chamber bag systems that permit separation of glucose from the buffer during sterilization. When the two chambers are mixed just before patient use, the resulting solution has a neutral pH and reduced glucose degradation content. Physioneal 35 was evaluated for its cytotoxicity potential using a murine fibroblast assay, its acute effect on human neutrophil and human peritoneal mesothelial cell function, and its in vitro potential to form advanced glycation end products (AGE). The biocompatibility characteristics of this new formulation were compared with that of a conventional, lactate-based solution and to that of its parent formulation, Physioneal 40. Methods Proliferation of murine fibroblasts was determined after exposure to dialysis fluids for 72hours. Cell viability was assayed by the ability to take up neutral red dye. Human neutrophils were exposed for 15 minutes to dialysis fluids, and their ATP content and phorbol 12-myristate 13-acetate (PMA) stimulated chemiluminescence response was determined as a measure of viability and respiratory burst activity, respectively. Cellular interleukin (IL)-1β–driven IL-8 synthesis by human mesothelial cells following acute exposure to dialysis fluids was also assessed. Advanced glycation end product formation in the dialysis fluids was measured after 5 and 20days of incubation with human serum albumin (HSA) as the model protein. Results In all assays employed, the biocompatibility profile of Physioneal 35 was similar to that of the Physioneal 40 parent formulation. Physioneal 35 showed a significant improvement in biocompatibility performance compared to a pH neutralized conventional lactate-buffered peritoneal dialysis solution in the murine fibroblast assay. In the acute exposure assays, human neutrophil viability and respiratory burst were significantly improved compared with the acidic, conventional solution; however, no statistically significant improvement were seen in mesothelial cells. AGE formation, which is thought to be an important mechanism by which glucose and glucose degradation products cause structural and functional changes of the peritoneal membrane, was significantly lower in Physioneal 35 compared with the conventional dialysis solution. Conclusion The biocompatibility profile of Physioneal 35 was similar to that of the original Physioneal 40 bicarbonate/ lactate-buffered dialysis solution, confirming that differences in both buffer content and calcium concentration do not affect biocompatibility performance. Both bicarbonate/lactate formulations (Physioneal 35 and Physioneal 40) were more biocompatible than a conventional lactate-buffered dialysis solution in this in vitro biocompatibility assessment.

AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression.BackgroundExcess advanced glycation end-products (AGEs) are formed during renal failure, and AGE formation also may be connected with the high glucose concentration of peritoneal dialysis (PD) fluids. To determine the effect of human peritoneal mesothelial cell (HPMC) exposure to glycated proteins, we studied the HPMC receptor of AGE expression (RAGE), and analyzed the results of AGE–RAGE interaction on adhesion molecule expression and leukocyte binding.MethodsRAGE was detected by FACS analysis, and RAGE mRNA by reverse transcription–polymerase chain reaction (RT–PCR). Vascular and intercellular cell adhesion molecule (VCAM-1 and ICAM-1) expression was measured by a known radiometric technique under basal conditions, after the addition of an AGE-specific compound, Nε-carboxylmethyllysine (CML–albumin). Leukocyte adhesion on HPMC was analyzed by videomicroscopy after HPMC stimulation.ResultsRAGE protein was detected on HPMC, and RAGE mRNA was evidenced by RT-PCR. VCAM-1 expression was stimulated by CML-albumin (P < 0.01), while ICAM-1 was unchanged. By blocking the AGE–RAGE interaction, anti-RAGE antibodies or recombinant RAGE inhibited the increase in VCAM-1 expression. CML–albumin stimulation potentiated leukocyte adhesion to HPMC (P < 0.001). This effect was prevented by the incubation of leukocytes with recombinant VCAM-1 (P < 0.001).ConclusionsAGE binding to RAGE stimulated mesothelial cell activity, and resulted in the overexpression of VCAM-1, a structure for leukocyte adhesion. The AGE–RAGE interaction resulted in HPMC activation, which may promote local inflammation, and thus is implicated in the peritoneal injury found in long-term PD patients.

Fluvastatin suppresses oxidative stress and fibrosis in the interstitium of mouse kidneys with unilateral ureteral obstruction. Background Recently, we demonstrated increased oxidative stress in the interstitium of ureteral obstructed kidneys based on the increased expression of heme oxygenase-1 and immunohistochemical detection of advanced glycation end products (AGE) in the interstitium. Antioxidant therapy may have a therapeutic potential toward interstitial fibrosis of unilateral ureteral obstruction (UUO) kidneys. Fluvastatin is an HMG-CoA reductase inhibitor and has been demonstrated to have an antioxidant activity in vitro. Methods The effects of fluvastatin on UUO kidneys from the viewpoints of antioxidant action in vivo and antifibrosis action were studied. To investigate the antioxidant action and its therapeutic efficacy of fluvastatin in UUO kidneys, AGE accumulation and fibrosis in the obstructed kidneys was compared among vehicle-, pravastatin-, or fluvastatin-treated (10 or 40 mg/kg/day) groups. Results Tubulointerstitial fibrosis was significantly attenuated in fluvastatin-treated animals. Fluvastatin significantly suppressed the degree of immunostaining of AGE in UUO kidneys. Conclusions These results provide evidence for the antioxidant action of fluvastatin in vivo. The decreased interstitial fibrosis along with a decreased oxidative stress marker in the interstitial lesion strongly suggests the existence of a causal relationship between them. Fluvastatin may have therapeutic value in slowing or preventing interstitial fibrosis in progressive renal disease.

β 2 -Microglobulin increases the expression of vascular cell adhesion molecule on human synovial fibroblasts. Background β 2 -Microglobulin (β 2 m) amyloidosis is a destructive articular disease that affects patients on dialysis. The disease presentation is similar to other forms of arthritis in which adhesion molecules are felt to be pathogenic. Therefore, we hypothesized that β 2 m directly increases the expression of vascular cell adhesion molecule-1 (VCAM-1) by synovial fibroblasts. We also examined the effect of alteration of β 2 m by advanced glycation end products on this cellular response. Methods Human synovial fibroblasts were isolated and incubated with β 2 m with and without alteration with advanced glycation end products. VCAM-1 expression was determined by immunohistochemistry, flow cytometry, and Western blot and Northern blot analyses. Results β 2 m increased the protein expression of VCAM-1 by synovial fibroblasts in a dose-dependent manner. β 2 m altered with advanced glycation end products had no effect. However, all forms of β 2 m increased VCAM-1 mRNA. β 2 m also increased the adhesion of peripheral blood mononuclear cells to synovial fibroblasts. Conclusion β 2 m directly increases the expression of VCAM-1 by synovial fibroblasts, indicating that synovial fibroblasts may play a key role in the pathogenesis of β 2 m amyloidosis.

Uremic toxins and peritoneal dialysis. Uremic toxicity is related in part to the accumulation of toxic substances, the nature of which has only partly been characterized. Because of the use of a highly permeable membrane and better preservation of the residual renal function, it could be anticipated that some of these uremic toxins are more efficiently cleared across the peritoneal membrane, and that the plasma and tissue levels of these compounds are lower than in hemodialysis patients. This article analyzes the generation and removal of several uremic toxins in peritoneal dialysis patients. The following uremic toxins are discussed: β2-microglobulin, advanced glycation end products, advanced oxidation protein products, granulocyte inhibitory proteins, p-Cresol, and hyperhomocysteinemia. Some recent studies are reviewed suggesting that uremic toxins are involved in the progression of renal failure and are at least partially removed by peritoneal dialysis. We conclude that, although the plasma levels of some of these compounds are lower in peritoneal dialysis versus hemodialysis patients, it does not mean that the peritoneal dialysis patient is “better” protected against the numerous disturbances caused by these toxins.

Mass spectrometric monitoring of albumin in uremia.BackgroundAdvanced glycation end products (AGEs) are a novel class of uremic toxins. In plasma, they are present in proteins and also in low molecular mass peptides. AGE-modified peptides are thought to bind and modify plasma proteins. Monitoring of the consequent increase in molecular mass of serum albumin may be used in surveillance of the clinical management of uremia.MethodsWe investigated molecular mass changes of human serum albumin (HSA) glycated by glucose and methylglyoxal in vitro and of subjects with moderate renal impairment, end-stage renal disease (ESRD), ESRD on hemodialysis, and normal healthy controls by matrix-assisted laser desorption ionization mass spectrometry.ResultsFatty acid-free HSA had a molecular mass of 66,446 ± 114 D. Mean (±SD) molecular mass increases were HSA minimally glycated by glucose 399 ± 88 D (N = 5, P < 0.001), HSA highly glycated by glucose 6780 ± 122 D (N = 5, P < 0.001), HSA minimally glycated by methylglyoxal 73 ± 121 D (N = 5, P > 0.05), and HSA without fatty acid removal 535 ± 90 D (N = 5, P < 0.001). For HSA of human subjects, mean (± SD) molecular mass increases were normal healthy controls 243 ± 97 D (N = 5), moderate renal impairment 350 ± 83 D (P > 0.05 with respect to controls, N = 5), ESRD 498 ± 128 (P < 0.02 with respect to controls, N = 3), and ESRD on hemodialysis 438 ± 85 D (P < 0.02 with respect to controls, N = 5). The mean molecular mass of albumin of all groups was increased significantly with respect to that of fatty acid free albumin (P < 0.001).ConclusionsOnly ESRD was associated with a significant increase in the molecular mass of HSA in vivo. Since this mass increase was very low and much lower than reported for AGE-modified peptides, it may reflect AGE formation on HSA by α-oxoaldehydes that accumulate in uremia, rather than modification of albumin by AGE-modified peptides. The molecular mass of HSA in vivo was indicative of a minimal and not a high extent of glycation.

Apoptosis and extracellular matrix-cell interactions in kidney disease. Extracellular matrix (ECM)-cell interactions have major effects on phenotypic features such as cell growth, differentiation, and gene expression. Apoptosis is an active form of cell death that is crucial for maintaining an appropriate number of cells as well as tissue organization. Recent reports have implied that ECM can influence survival and apoptosis of several cell lineages including glomerular mesangial cells (MC). Numerous glomerular diseases are associated with the expansion of the mesangial ECM, which may eventually produce glomerular scarring. Glomerular cell apoptosis is associated with the deletion of glomerular cells and the accumulation of ECM in the progression of glomerulosclerosis in rat remnant kidney model induced by 5/6 nephrectomy. Our recent study indicated that basement membrane matrix (a model for normal ECM components) prevented cultured MC from undergoing apoptosis after serum deprivation, thus promoting their survival, compared with type I collagen matrix (a model for abnormal ECM components). Inhibition of matrix-derived signals by antisense oligonucleotides against beta1 integrin increased MC apoptosis. Data suggest that the survival and death of MC are regulated by the surrounding ECM through integrin molecules. The mechanism of regulation of MC apoptosis by ECM requires further in vivo study to gain new insight into the treatment of glomerular diseases as well as the pathophysiology of the mesangium. Diabetic nephropathy is characterized by the abnormal ECM accumulation and the phenotypic change of MC. Some speculations on the possible involvement of apoptosis in diabetic nephropathy are also discussed.

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 N ϵ (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.

Endothelial cell adhesion molecule and PMNL response to inflammatory stimuli and AGE-modified fibronectin. Background Atherosclerotic vascular disease is the leading cause of death in patients with diabetes mellitus and end-stage renal disease. Advanced glycation end products (AGEs) are strongly suggested to be involved in the pathogenesis of atherosclerosis in these patients who also frequently experience infectious complications. We hypothesized that the interaction of AGEs and inflammatory mediators contributes to the up-regulation of endothelial cell activation. Methods We investigated the effect of advanced glycated fibronectin in the presence or absence of inflammatory stimuli on the endothelial cell surface and mRNA expression of cell adhesion molecules. Furthermore, the influence of advanced glycated fibronectin on the transendothelial migration pattern of polymorphonuclear cells was analyzed. Results Exposure to advanced glycated fibronectin together with inflammatory stimuli such as interleukin (IL)-1α, tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) led to a significant increase in the surface expression of the cell adhesion molecules E-selectin, ICAM-1, VCAM-1 and PECAM-1 on endothelial cells. Soluble AGEs in combination with advanced glycated fibronectin significantly enhanced the endothelial cell surface expression of ICAM-1, VCAM-1 and PECAM-1, whereas this was not the case for E-selectin. At the transcriptional level short-time exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators resulted in an increased expression of E-selectin, ICAM-1 and VCAM-1 mRNA levels, whereas PECAM-1 repeatedly showed a significant decrease of gene transcript levels. An increase of mRNA levels was also observed for E-selectin, ICAM-1, VCAM-1 and PECAM-1 following incubation with a combination of advanced glycated fibronectin and soluble advanced glycation end-products. Furthermore, polymorphonuclear cells responded with a sevenfold increase in transendothelial migration following exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators. Conclusions These results suggest that the combination of matrix glycation and inflammation up-regulates the activation of the endothelial cell adhesion cascade, a mechanism that might contribute to the increased burden of atherosclerotic morbidity and mortality in patients suffering from diabetes mellitus or chronic renal failure.

Free pentosidine and neopterin as markers of progression rate in diabetic nephropathy. Background Patients with diabetic nephropathy experience a progressive and usually inexorable decline in renal function. The presence of the structurally defined advanced glycation end product (AGE) pentosidine on tissue and circulating proteins has been correlated with the severity of diabetic complications. Methods To delineate a role for this AGE in the progression of diabetic nephropathy, glycohemoglobin and free and protein-bound pentosidine were measured in baseline stored serum and urine from a subgroup of patients with diabetes mellitus and proteinuria originally followed by the Collaborative Study Group Trial. To delineate a potential role for an immune-activation response to AGEs, the inflammatory markers, interleukin-6 (IL-6), C-reactive protein (CRP), and the monocyte activation marker neopterin were also measured at baseline. The patients chosen represented 67 subjects whose creatinine levels had "doubled" over the course of the study whether or not they later were treated with captopril, and 67 paired "non-doublers." Results Baseline disease activity, as manifested by glycohemoglobin, serum creatinine and degree of proteinuria was equal in the two groups, as was protein-bound pentosidine and the immune-markers IL-6 and CRP. At baseline the "doublers" as compared to the "non-doublers" had elevated serum levels of free pentosidine and neopterin. Baseline increases in these two parameters were also associated with an increased rate of "doubling" of serum creatinine by the proportional hazards method. Conclusion Differences in individual responsiveness to AGEs, as manifested by either the production of free pentosidine or its release from a protein-bound form, and by evidence of monocyte/macrophage activation, are associated with progression of diabetic nephropathy.

β2-microglobulin modified with advanced glycation end products modulates collagen synthesis by human fibroblasts. β2-microglobulin amyloidosis (Aβ 2 m) is a serious complication for patients undergoing long-term dialysis. β2-microglobulin modified with advanced glycation end products (β 2 m-AGE) is a major component of the amyloid in Aβ 2 m. It is not completely understood whether β 2 m-AGE plays an active role in the pathogenesis of Aβ 2 m, or if its presence is a secondary event of the disease. β2-microglobulin amyloid is mainly located in tendon and osteo-articular structures that are rich in collagen, and local fibroblasts constitute the principal cell population in the synthesis and metabolism of collagen. Recent identification of AGE binding proteins on human fibroblasts lead to the hypothesis that the fibroblast may be a target for the biological action of β 2 m-AGE. The present study demonstrated that two human fibroblast cell lines exhibited a decrease in procollagen type I mRNA and type I collagen synthesis after exposure to β 2 m-AGE for 72 hours. Similar results were observed using AGE-modified albumin. Antibody against the RAGE, the receptor for AGE, attenuated this decrease in synthesis, indicating that the response was partially mediated by RAGE. In addition, antibody against epidermal growth factor (EGF) attenuated the decrease in type I procollagen mRNA and type I collagen induced by β 2 m-AGE, suggesting that EGF acts as an intermediate factor. These findings support the hypothesis that β 2 m-AGE actively participates in connective tissue and bone remodeling via a pathway involving fibroblast RAGE, and at least one interposed mediator, the growth factor EGF.

Nonenzymatic glycation of type IV collagen and matrix metalloproteinase susceptibility. The glomerular basement membrane (GBM) is damaged in diabetes through complex mechanisms that are not fully understood. Prominent among them is nonenzymatic protein glycation leading to the formation of so-called advanced glycation end products (AGEs). We examined the effects of in vitro glycation of intact collagen type IV in bovine lens capsule (LBM) and kidney glomerular (GBM) basement membranes on their susceptibility to matrix metalloproteinases, using stromelysin 1 (MMP-3) and gelatinase B (MMP-9). Sites of cleavage of unmodified LBM collagen were located in the triple helical region. In vitro glycation by glucose severely inhibited the release of soluble collagen cleavage peptides by MMP-3 and MMP-9. The distribution of AGEs within the three domains of collagen IV (7S, triple helical, and non-collagenous NC1) were compared for LBM glycation using AGE fluorescence, pentosidine quantitation, and immunoreactivity towards anti-AGE antibodies that recognize the AGE carboxymethyllysine (CML). Marked asymmetry was observed, with the flexible triple helical domain having the most pentosidine and fluorescent AGEs but the least CML. The in vivo relevance of these findings is supported by preliminary studies of AGE distribution in renal basement membrane (RBM) collagen IV domains from human kidneys of two insulin-dependent diabetics and one normal subject. Pentosidine and fluorescent AGE distributions of diabetic RBM were similar to LBM, but the CML AGE in diabetic kidney was less in the triple helical domain than in NCI. Our results support the hypothesis that nonenzymatic glycation of collagen IV contributes to the thickening of basement membranes, a hallmark of diabetic nephropathy.

Relationship between advanced glycoxidation end products, inflammatory markers/acute-phase reactants, and some autoantibodies in chronic hemodialysis patients. Uremia and dialysis treatment are associated with uncorrected oxidative and carbonyl stress and microinflammation. Elevation of both oxidative/carbonyl stress end products (advanced oxidation protein products (AOPP), advanced glycation end products (AGEs), and advanced lipoperoxidation end products (ALEs), autoantibodies against modified biological structures, and acute-phase reactants (e.g., C-reactive protein [CRP], fibrinogen) seems to take part in the development of various complications, among them accelerated atherosclerosis. These pathogenic mechanisms are supposed to act synergically; nevertheless, oxidative stress shows a closer relationship to inflammation and acute-phase reaction than advanced glycation. Its end product, AOPP, could, thus, represent a biochemical marker of specific importance.

Glycated and carboxy-methylated proteins do not directly activate human vascular smooth muscle cells.BackgroundAdvanced glycation end products (AGEs) accumulate in patients with diabetes, particularly at sites of vascular damage and within atherosclerotic lesions, but whether they have direct actions on vascular smooth muscle cells (VSMCs) is controversial.MethodsAGEs were constructed and characterized by protein content, level of modification, fluorescence, and molecular size. Human VSMCs were derived from different vascular beds. Glucose consumption, de novo protein synthesis, and proteoglycan biosynthesis were measured using a colorimetric assay and metabolic radiolabeling. Receptor for AGEs (RAGE) expression was assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.ResultsTreatment with AGEs under low or high glucose conditions showed no change in cellular glucose consumption or in cellular protein synthesis under low glucose conditions. Treatment of VSMCs with Nε-(carboxymethyl)lysine in the presence of low glucose increased [35S]-sulfate incorporation into secreted proteoglycans by 72% (P < 0.001) and 67% (P < 0.001); however, the control proteins also increased [35S]-sulfate incorporation into proteoglycans by 56% (P < 0.01), with similar effects observed under high glucose conditions. Human VSMCs showed no difference in response to glycated and non-glycated protein. Protein and gene expression of RAGE in VSMC was approximately 50-fold lower compared to HMEC-1 and U937 cells, consistent with the immunohistochemical staining of RAGE in vivo.ConclusionVSMCs show very low levels of RAGE expression; thus, activation of VSMCs by AGEs does not occur. In diabetes, RAGE expression in VSM may increase to the extent that it becomes activated by AGEs in a manner that would contribute to the process of atherosclerosis.

Myeloperoxidase in kidney disease. In glomerular and tubulointerstitial disease, polymorphonuclear- and monocyte-derived reactive oxygen species may contribute to oxidative modification of proteins, lipids, and nucleic acids. In part, the processes instigated by reactive oxygen species parallel events that lead to the development of atherosclerosis. Myeloperoxidase (MPO), a heme protein and catalyst for (lipo)protein oxidation is present in these mononuclear cells. The ability of MPO to generate hypochlorous acid/hypochlorite (HOCl/OCl − ) from hydrogen peroxide in the presence of chloride ions is a unique and defining activity for this enzyme. The MPO-hydrogen peroxide-chloride system leads to a variety of chlorinated protein and lipid adducts that in turn may cause dysfunction of cells in different compartments of the kidney. The aim of this article is to cover and interpret some experimental and clinical aspects in glomerular and tubulointerstitial diseases in which the MPO-hydrogen peroxide-chloride system has been considered an important pathophysiologic factor in the progression but also the attenuation of experimental renal disease. The colocalization of MPO and HOCl-modified proteins in glomerular peripheral basement membranes and podocytes in human membranous glomerulonephritis, the presence of HOCl-modified proteins in mononuclear cells of the interstitium and in damaged human tubular epithelia, the inflammation induced and exacerbated by MPO antibody complexes in necrotizing glomerulonephritis, and the presence of HOCl-modified epitopes in urine following hyperlipidemia-induced renal damage in rodents suggest that MPO is an important pathogenic factor in glomerular and tubulointerstitial diseases. Specifically, the interaction of MPO with nitric oxide metabolism adds to the complexity of actions of oxidants and may help to explain bimodal partly detrimental partly beneficial effects of the MPO-hydrogen peroxide-chloride system in redox-modulated renal diseases.

Effect of a novel adsorbent on cytokine responsiveness to uremic plasma. Background Middle molecules such as β 2 -microglobulin (β 2 M) and advanced glycation end products (AGE)–modified proteins contribute to inflammation in uremia. The BetaSorb™ column is a new adsorptive device, which contains copolymeric beads, suitable for removal of β 2 M and other middle molecules. We assessed the effect of this column on the bioreactivity of uremic plasma, as measured by cytokine responsiveness. Methods Uremic plasma was perfused in vitro through the column (10 mL/min) and samples were collected after 10 to 30 passes. Endotoxin-stimulated tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) production by THP-1–derived monocytes was measured following brief exposure to uremic plasma. β 2 M levels were measured. The contribution of AGE-modified proteins to the bioreactivity of uremic plasma was explored. Results TNF-α and IL-10 production markedly decreased after 30 passes (629 ± 78 vs. 144 ± 62 pg/mL; 207 ± 25 vs. 117 ± 23 pg/mL; P = 0.04). The column removed β 2 M efficiently with a marked decline in plasma levels by 99% after 30 passes. Neutralization of AGE receptor (RAGE) resulted in a further reduction in the bioreactivity of uremic plasma. This was observed with nonperfused, as well as perfused, uremic plasma, suggesting that AGE-modified proteins were biologically active and still present after perfusion. Conclusion The sorbent beads removed uremic solute(s) that prime monocytes to enhanced cytokine production. Removal of β 2 M was efficient, and of native and AGE-modified middle molecules likely.

Glucose degradation products in PD fluids: Do they disappear from the peritoneal cavity and enter the systemic circulation? Background Glucose degradation products (GDP) are generated in peritoneal dialysis (PD) fluid during heat sterilization and storage. They are thought to adversely affect the peritoneal membrane. The fate of GDP within the peritoneal cavity has not been well characterized. Methods A clinical study was designed to determine ( 1 ) whether during the dwell in the peritoneal cavity GDP concentration decreases in the PD fluid as assessed by ex vivo formation of AGE; ( 2 ) whether exposure to GDP-containing PD fluids increases plasma fluorescence (as an index of plasma AGE concentration) as well as plasma carboxymethyllysine (CML) concentration; and ( 3 ) whether exposure to GDP-containing PD fluids adversely affects glycoprotein CA 125 concentration. A two-group crossover design was adopted comprising two consecutive observation periods of eight weeks each. Stable PD patients were exposed in random order either to conventional PD fluid (heat sterilized at pH 5.5) and subsequently to PD test fluid (or the 2 fluids in reverse order). The PD test fluid was sterilized using a multicompartment bag system separating highly concentrated glucose at pH 3 from the buffer solution. Conventional and test fluids differed with respect to concentrations of GDP, that is, 3-deoxyglucosone (118 vs. 12.3 μmol/L), methylglyoxal (5.3 μmol/L vs. below detection threshold), 3, 4-dideoxyglucosone-3-ene (10 μmol/L vs. below detection threshold) and acetaldehyde (226 vs. Results The following results were obtained. First, methylglyoxal disappeared completely as early as two hours after intraperitoneal instillation of conventional PD fluid. Second, when spent conventional dialysate was recovered after a two hour and particularly an eight hour dwell and subsequently incubated ex vivo with 40mg of human serum albumin, there was a continuous decrease of AGE-forming capacity, that is, less generation of fluorescence (AGE) and pyrraline (non-fluorescent Amadori product), and an increase of advanced oxidation protein products (AOPP) in the spent dialysate. Third, plasma fluorescence (exc. 350/em. 430 nm) as an index of circulating AGE compounds as well as plasma CML concentrations were significantly higher in the conventional PD fluid period versus low GDP PD fluid period. Fourth, CA 125 concentrations in spent dialysate were higher during the low GDP PD fluid period compared to the conventional PD fluid period. Conclusion Conventional PD fluid undergoes modifications during intraperitoneal dwell with a loss of AGE forming capacity, suggesting breakdown, precipitation or resorption of GDP in vivo. This is accompanied by an increase in plasma AGE compounds.

Renal catabolism of advanced glycation end products: The fate of pentosidine. Advanced glycation end products (AGEs) generated through the Maillard reaction significantly alter protein characteristics. Their accumulation has been incriminated in tissue injury associated with aging, diabetes, and renal failure. However, little is known about their clearance from the body. The present study delineates the catabolic pathway of a well-defined AGE product, pentosidine. Synthesized pentosidine given intravenously in rats with normal renal function was rapidly eliminated from the circulation through glomerular filtration, but was undetectable in the urine by chemical analysis. Immunohistochemistry with anti-pentosidine antibody disclosed that pentosidine accumulated transiently in the proximal renal tubule one hour after its administration, but had disappeared from the kidney at 24 hours. After an intravenous load of radiolabeled pentosidine, radioactivity peaked in the kidney at one hour and subsequently decreased, whereas it rose progressively in the urine. Over 80% of the radioactivity was recovered in the 72-hour collected urine. However, only 20% of urine radioactivity was associated with intact pentosidine chemically or immunochemically. In gentamicin-treated rats with tubular dysfunction, up to 30% of the pentosidine load was recovered as intact pentosidine in the urine. The present study suggests that free pentosidine (and possibly other AGEs) is filtered by renal glomeruli, reabsorbed in the proximal tubule where it is degraded or modified, and eventually excreted in the urine. Kidney thus plays a key role in pentosidine disposal.

Toward better dialysis compatibility: Advances in the biochemistry and pathophysiology of the peritoneal membranes. Peritoneal dialysis (PD) has modified our concept of the peritoneal membrane, which is now a topic of active research. Peritoneal solute transport progressively increases with time on PD, enhances the dissipation of the osmotic gradient and, eventually, reduces ultrafiltration capacity. The causes of peritoneal membrane failure remain elusive. Recurrent episodes of peritonitis are not a prerequisite for the development of ultrafiltration failure. Functionally, the changes of the failing peritoneal membrane are best described as an increased functional area of exchange for small solutes between blood and dialysate. Histologically, these events are associated with vascular proliferation and structural changes of pre-existing vessels. Gathered evidence, including information on the composition of peritoneal cavity fluids and its dependence on the uremic environment, have cast a new light on the molecular mechanisms of decline in peritoneal membrane function. Chronic uremia per se modifies the peritoneal membrane and increases the functional area of exchange for small solutes. Biochemical alterations in the peritoneum inherent to uremia might be, at least in part, accounted for by severe reactive carbonyl compounds overload originating both from uremic circulation and PD fluid (“peritoneal carbonyl stress”). The molecular events associated with long-term PD are similar but more severe than those present in chronic uremia without PD, including modifications of nitric oxide synthase (NOS) and angiogenic growth factors expression, and advanced glycation and lipoxidation of the peritoneal proteins. This review focuses on reactive carbonyls and their association with a number of molecular changes observed in peritoneal tissues. This hypothetical approach will require further testing. Nevertheless, the insights gained on the peritoneal membrane offer a new paradigm to assess the effect of uremic toxins on serosal membranes. Furthermore, the progresses made in the dissection of the molecular events leading to peritoneal membrane failure open new avenues to develop safe, more biocompatible peritoneal dialysis technologies.

AGEs activate mesangial TGF-β–Smad signaling via an angiotensin II type I receptor interaction. Background The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-β (TGF-β)–Smad signaling in cultured rat mesangial cells. Methods The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H 2 DCFDA. Ang II was measured by radioimmunoassay. TGF-β released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27 Kip1 (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-β–inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2′-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [ 3 H]leucine incorporation. Results AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N -acetylcysteine (NAC). AGE-induced TGF-β overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT 1 R) antagonist. Both candesartan and neutralizing antibody against TGF-β completely prevented AGEs-induced Smad2 phosphorylation and TGF-β–inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-β. Conclusion The present study suggests that AGE-RAGE–mediated ROS generation activates TGF-β–Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

Increased erythrocyte 3-DG and AGEs in diabetic hemodialysis patients: Role of the polyol pathway. Background 3-Deoxyglucosone (3-DG) accumulating in uremic serum plays an important role in the formation of advanced glycation end products (AGEs). To determine if 3-DG is involved in the formation of intracellular AGEs, we measured the erythrocyte levels of 3-DG and AGEs such as imidazolone and Ne-carboxymethyllysine (CML) in hemodialysis (HD) patients with diabetes. Further, to determine if the polyol pathway is involved in the formation of erythrocyte 3-DG and AGEs, an aldose reductase inhibitor (ARI) was administered to these patients. Methods The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were measured in ten diabetic HD patients before and after treatment with ARI (epalrestat) for eight weeks, and were compared with those in eleven healthy subjects. 3-DG was incubated in vitro with hemoglobin for two weeks to determine if imidazolone and CML are formed by reacting 3-DG with hemoglobin. Results The erythrocyte levels of sorbitol, 3-DG, imidazolone, and CML were significantly elevated in diabetic HD patients as compared with healthy subjects. The erythrocyte levels of 3-DG significantly decreased after HD, but sorbitol, imidazolone or CML did not. The administration of ARI significantly decreased the erythrocyte levels of sorbitol, 3-DG and imidazolone, and tended to decrease the CML level. Imidazolone was rapidly produced in vitro by incubating 3-DG with hemoglobin, and CML was also produced, but less markedly as compared with imidazolone. Conclusion The erythrocyte levels of 3-DG and AGEs are elevated in diabetic HD patients. The administration of ARI reduces the erythrocyte levels of 3-DG and AGEs, especially imidazolone, as well as sorbitol. Thus, 3-DG and AGEs, especially imidazolone, in the erythrocytes are produced mainly via the polyol pathway. ARI may prevent diabetic and uremic complications associated with AGEs.

Imunohistochemical detection of an AGE, a ligand for macrophage receptor, in peritoneum of CAPD patients. Background In patients on long-term continuous ambulatory peritoneal dialysis (CAPD), ultrafiltration (UF) capacity of peritoneal membrane may be impaired due to accumulation of advanced glycation end products (AGEs). This study aimed to elucidate the characteristics of a novel anti-AGE antibody, ODI-GLC19, and to demonstrate AGE accumulation in the peritoneum of CAPD patients using the antibody. Methods A monoclonal anti-AGE antibody (ODI-GLC19) was prepared by immunizing female balb/c mice using D-glucose-modified keyhole limpet hemocyanin. The characteristics of ODI-GLC19 were determined by enzyme-linked immunosorbent assay and receptor binding inhibition assay. Immunohistochemistry using ODI-GLC19 was performed to detect AGE in peritoneal tissues obtained from patients with nonrenal disease, and CAPD patients with normal and low UF. Results ODI-GLC19 reacted with glycolaldehyde-modified BSA (GA-BSA) and glucose-modified BSA (GLC-BSA), but not with imidazolone or N γ -(carboxymethyl)lysine. GA-BSA and GLC-BSA strongly bound to cultured macrophages. Time-dependent recognition of newly formed GA-BSA by ODI-GLC19 was similar to that by macrophages. The binding of GA-BSA to macrophages was inhibited by ODI-GLC19 in a dose-dependent manner. Immunohistochemical studies revealed that ODI-GLC19-positive AGE was exclusively detected in peritoneal cells including macrophages, and its staining intensity was more prominent in the peritoneum of CAPD patients, especially with low UF, than in patients with nonrenal disease. Conclusions A novel monoclonal anti-AGE antibody, ODI-GLC19, recognizes a ligand for an AGE receptor on macrophages. Incorporation of AGE into peritoneal cells including macrophages may be involved in progressive peritoneal dysfunction in CAPD patients.