Effect Of Methylglyoxal And Advanced Glycation End Products On Human Peritoneal Mesothelial Cells

Objective: To study the effects of Glucose degradation products (methylglyoxal, MGO) and AGE-HSA on the angiogenesis, peritoneal fibrosis, and cell injury to clarify the cause of decreased dialysis efficiency during prolonged peritoneal dialysis in vitro. Furthermore, to investigate the effects of simvastatin on the expression of MCP-1 in human peritoneal mesothelial cells.Methods: Cultured human peritoneal mesothelial cells were incubated with MGO (0～320μM), HSA (500μg/mL), AGE-HSA (0～1000μg/mL),NAC (30mM.), SB203580 (20μM), PD98059(50μM), PMA(80～100nm/L), Calphostin C (100nm/L), Simvastatin (0.3μM～3μM), FPP (5μM),and GGPP(10μM). The mRNA and protein expression of the VEGF, MCP-1,and fibronectin were determined by RT-PCR. and ELISA. Cells were marked with Oxidation-susceptible fluroscent probe 2,7-dichorofluoresin diacetate (DCFH) and were assayed by flow cytometry. MAPK phosphorylation was evaluated by Western blot analysis. Then the cell morphologic changes were observed by phase-contrast microscopy. Cell viability was assessed by measuring their ability to metabolize the MTT salt. ~3H-TdR incorporation was use to evaluated the cell proliferation. Results: 1. MGO increased the concentration of ROS in the HPMC with a dose-dependent manner which was accompanied by the activation of p38MAPK. N-acetyl-1-cysteine (NAC) inhibited the MGO-induced p38 MAPK activation. MGO also stimulated the production of VEGF in a dose and time dependent ways; the effects of MGO on HPMC could be" blocked by antioxidant NAC and SB203580 (a specific inhibitor of p38 MAPK).2. AGE-HSA increased the concentration of ROS in the HPMC with a dose-dependent manner which was accompanied by the activation of p38MAPK and P42/44MAPK. NAC inhibited the MGO-induced p38 MAPK or P42/44MAPK activation. AGE-HSA also stimulated the production of VEGF, MCP-1 in a dose and time dependent way; the effects of AGE-HSA on HPMC could be blocked by antioxidant NAC, PD98059 (a specific inhibitor of MEK), and SB203580 (a specific inhibitor of p38 MAPK).3. Simvastatin decreased AGE-HSA-induced MCP-1 synthesis in line with concentration-dependent manner, The effects of simvastatin were arrested in the presence of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, suggesting that the effect of simvastatin on MCP-1 synthesis is mediated through geranylgeranyl-modified intermediates. In contrast, simvastatin did not affect the expression of VEGF.4. Incubation with MGO reduced the proliferation of cells and was paralleled by a gradualloss of cell viability and development of morphologic alterations. In contrast, AGE-HSA did not show any effects on HPMC.5. AGE-HSA up-regulated the expression of fibronectin mRNA and protein dose- and time-dependently. PKC activator phorbol 12-myristate 13-acetate (PMA) induced FN expression, respectively. Depletion of PKC and calphostin C, a PKC inhibitor, effectively prevented both PMA and AGE-HSA-induced expression of the FN. Conclusion: 1.The induction of ROS and MAPK phosphorylation in HPMC by MGO or AGE-HSA, which stimulates the expression of VEGF and MCP-1. It may play an important role in the angiogenesis of peritoneum. 2. Short exposure to MGO, HPMC react with enhanced cytotoxic damage.3. AGE-HSA can directly increase fibronectin expression in HPMC, which may contribute to peritoneal fibrosis, and this is regulated by protein kinase C and ROSAThe simvastatin may have promise for treating or preventing the angiogenesis of peritoneum and leads to ultrafiltration failure.