Immunohistochemical 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 epsilon-(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.

Role of advanced glycation end products and growth factors in peritoneal dysfunction in CAPD patients.

High levels of glucose degradation products in peritoneal dialysis fluids are believed to cause excess accumulation of advanced glycation end products (AGEs) in the peritoneum during continuous ambulatory peritoneal dialysis (CAPD) treatment, resulting in functional and structural changes in the peritoneal membrane of CAPD patients. In this study, we investigated whether AGEs, the receptor for AGE (RAGE), and growth factors are involved in deteriorating ultrafiltration (UF) capacity of the peritoneal membrane in patients on CAPD therapy. Immunohistochemical staining showed that ODI-GLC19, a novel monoclonal anti-AGE antibody, was localized exclusively in peritoneal cells, in contrast to imidazolone, localized mostly in peritoneal degenerative collagen. Numbers of ODI-GLC19- and RAGE-positive cells in the peritoneum were increased significantly in CAPD patients, even before a decrease in UF capacity, compared with patients with nonrenal disease. Cells positive for ODI-GLC19 were identified as myofibroblasts and RAGE-positive cells and partly as CD68-positive macrophages in the peritoneum. The peritoneal membrane was thickened significantly in CAPD patients, especially patients with low UF. The number of blood vessels was increased significantly in CAPD patients with low UF. Transforming growth factor-beta1, macrophage colony-stimulating factor, and vascular endothelial growth factor were recognized in the peritoneum of CAPD patients, especially those with low UF, where imidazolone was deposited. Focal hepatocyte growth factor expression was noted in the peritoneum of patients with low UF in moderate intensity, specifically in the area without severe structural changes. In conclusion, progressive accumulation of AGEs in the peritoneum may promote peritoneal expression of various growth factors and subsequently deteriorate UF capacity in CAPD patients.

An inhibitor of advanced glycation end product formation reduces N epsilon-(carboxymethyl)lysine accumulation in glomeruli of diabetic rats.

BACKGROUND: An inhibitor of advanced glycation, OPB-9195, retards the progression of nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of non-insulin-dependent diabetes mellitus. The aim of this study is to evaluate histologically the role of N(epsilon)-(carboxymethyl)lysine (CML) in the development of diabetic nephropathy and investigate whether inhibition of CML accumulation by OPB-9195 is associated directly with the prevention of glomerular lesions in OLETF rats. METHODS: Kidneys of OLETF and Long-Evans Tokushima Otsuka rats were obtained at ages 7, 20, 50, and 68 weeks after collecting their blood and urine samples. OPB-9195 had been administered to the rats from age 24 weeks to the end of the experiments. CML in kidneys was detected by using a monoclonal antibody against CML according to an indirect immunofluorescence technique. CML-positive glomerular area was measured using NIH Image software (Research Services Branch of NIMH, Bethesda, MD). Hyalinized and/or sclerotic areas in glomeruli and mesangial and glomerular volume were measured using a point-counting technique. RESULTS: CML-positive area in glomeruli correlated closely not only with urinary albumin excretion (r = 0.912; P = 0.001), but also with volumes of mesangium and hyalinized and/or sclerotic lesions (r = 0.859; P = 0.0019 and r = 0.833; P = 0.0027, respectively). Treatment with OPB-9195 reduced CML-positive area and prevented the increase in mesangial volume, with no significant change in glomerular volume at age 68 weeks. The volume of hyalinized and/or sclerotic lesions also decreased by treatment with OPB-9195 in three of four rats at age 68 weeks. CONCLUSION: CML is a major advanced glycation end product contributing to the development of diabetic nephropathy, and inhibition of its accumulation by OPB-9195 results in amelioration of glomerular lesions in OLETF rats.