Roles Of Local Endothelin System On The Development Of Peritoneal Fibrosis

[Objective] To gain a better undstanding of the mechanism and treatment of peritoneal fibrosis, and if local endothelin system play a role in peritoneal fibrosis, we study the level of endothelin-1 (ET-1) in dialysate of continuous ambulatory peritoneal dialysis(CAPD) patients. We also investigate the effect of local endothelin system in cultured human peritoneal mesothelial cells(HPMCs), and a combined ETA/ETB receptor antagonist (PD142893 ) was choosen to further elucidate its mechanism. [Methodology] 1. 26 regular follow-up CAPD patients in our hospital were selected . The expression of ET-1 in plasm and dialysate were detected by radioimmunoassy. Peritoneal equilibration test (PET) were performed to determine their peritoneal transport type and ultrafiltration volume(UF) were recorded. Linear regression, Pearson correlation analysis and oneway analysis of variance (ANOVA) were used to analysis the effect of ET -1 in palsm and dialysate . The following tests were performed on the human peritoneal mesothelial cell line(HMrSV5). Different concentrations of glucose and mannitol were applied in the culture of HMrSV5 cells at different time. The expression of ET-1 were detected by radioimmunoassy and immunocytochemistry. The gene expression of ET-1, ETAR,ETBR and ECE was evaluated by semi-quantitative reverse transcription polymerase chain response(RT-PCR). 2. The changes in cell morphology were observed by phase-contrast microscopy. The cytoplasmic expression of cytokeratins (CK), vimentin were detected by immunocytochemistry. ³H-TdR incorporation and flow cytometry was use to evaluted the cell proliferation. LDH release of the cells were measured to evalute the degree of cell injury.3. The protein synthesis of collagen type IV (ColIV) , fibronectin(Fn) and transforming growth factor betal(TGF-β 1), interleukin-1β (IL-1β)were measured by radioimmunoassy or enzyme-linked immunoadsordent assay(ELISA). The gene expression of ColIV,Fn,matrix metalloproteinase(MMP-2) was evaluated by RT-PCR. To neutralize ET-1effets, we used different concentration of PD142893 to observe the change above. Western blot analysis was performed to determine the level of p38MAPK and phospho- p38MAPK.[Results] 1.The actual level of ET-1 in dialysate was significant higher than the predicted value. The level of ET-1 of plasm(P), dialysate (D) and D/P_ET-1 in different peritoneal transport type had no statistic signigicant difference (P>0.05) .Net ultrafiltration volume (nUF) show negative correlation with the level of ET-1 in dialysate. Normal peritoneal mesothelial cells secreted ET-1 at low level. High glucose stimulated the secretion of ET-1 in a dose-dependent and time-dependent manner. ET-1,ETAR, ETBR, ECEmRNA was found in normal HPMC. High glucose up-regulated the expression of ET-1,ETAR, ETBRmRNA.2. By the treatment of ET-1, the morphology of the HPMC was changed into spindle shapes , the expressions of CK8 and vimentin were still positive. ET-1 stimulated the proliferation of HPMC in a dose-depent manner. And ET-1 promoted the degree of cell injury. 3. ET-1 up-regulated the synthesis of ColIV and expression of ColIV and MMP-2mRNA in HPMC. The expression of ColIV,Fn and MMP-2 up-regulated by high glucose were inhibited by PD142893. ET-1 stimulated the secretion of TGF-β 1,IL—1 β in a dose-dependent manner. 5. High glucose and ET-1 could activate p38MAPK signal transduction pathway, PD142893 could partly inhibit p38MAPK signal transduction activated by high glucose.[Conclusion] 1. Peritoneal ET—1 are probably derived from autocrine or paracrine in the peritoneal cavity. It is possible that the damage of peritoneal ultrafiltration function is correlated with peritoneal ET-1. Normal peritoneal mesothelial cells have endothelin system. It is possible HPMC is one of the source of ET-1 in peritoneal membrane.2. ET-1 induce HPMC contraction , proliferation and cell injury, but not induce the phenotype of HPMC to be transformed. 3. ET-1 up-regulate the synthesis of extracellular matix, which may through TGF-β 1 pathway. And ET-1 promote inflammation , which further increase accumulation of extracellular matix. The effect of ET-1 above may be via p38 MAPK activation.