Experimental Study On Epithelial-mesenchymal-transition Of Peritoneal Mesothelial Cells Induced By Indoxyl Sulfate

Peritoneal dialysis is a form of renal replacement alternative to the hemodialysis.During this treatment,the peritoneal membrane acts as a permeable barrier for exchange of solutes and water.Continual exposure to dialysis solutions,as well as episodes of peritonitis and hemoperitoneum,can injury to the peritoneal membrane,which undergoes progressive fibrosis,eventually leading to discontinuation of the peritoneal dialysis.Among the different events controlling this pathological process,epithelial to mesenchymal transition of mesothelial cells plays a main role in the induction of fibrosis and in subsequent functional deterioration of the peritoneal membrane.Studies have shown that,IS play a role in EMT-related fibrosis of organs,including heart and blood vessels,kidney,and osteoblastand.However,the role of IS in peritoneal mesothelial cells EMT and peritoneal fibrosis is unclear.Our study will investigate the mechanisms of IS in EMT of HPMCs.[Objective]To stimulate with indoxyl sulfate(Indoxyl sulfate,IS)into human peritoneal mesothelial cells immortalized cell line(HMrSV5),and then to study if epithelial mesenchymal transition(EMT)could be induced and investigate the role of transforming growth factor(TGF-?)played in it.[Methods]Cell culture in vitro,with different concentrations of IS(250umol/L,500umol/L,1000 umol/L)+4.25% peritoneal dialysis fluid of different coculture time(0h,4h,12 h,24h,48 h,72h),the control group with the culture medium +4.25% peritoneal dialysis solu tion for peritoneal dialysis fluid group;2umol/L LY364947+ 4.25% peritoneal dialys is fluid for TGF-?R inhibitor group.The morphological changes of cells were observed by immunofluorescence method,and then use the Western Blot method of alpha smooth muscle actin(a-SMA)expression,finally select the transdifferentiation process where the peak time point,determination the expression of two kinds of matrix protein about FN and laminin5.in the supernatant by ELISA method.[Results](1)Cells after IS stimulated,cells from the typical cobblestone,become thin andlong,fusiform shape change,while the expression of a-SMA on IS treatmentgroup increased significantly compared with the control group,the differencewas statistically significant(P<0.01);the expression of TGF-?R inhibitor groupdecreased,the difference was statistically significant(P<0.01);(2)ISstimulated peritoneal mesothelial cells after 48 h,upregulation of matrix proteinFN,laminin5 expression,the difference was statistically significant(P<0.05),TGF-? inhibitor group was decreased,the difference was statisticallysignificant(P<0.05).[Conclusion](1)IS can upregulate the expression of ?-SMA,FN and LN5 in peritonealmesothelial cells,while the TGF-?R inhibitor has an inhibitory effect on theexpression of ?-SMA,FN,and LN5;(2)IS can induce EMT in peritoneal mesothelial cells,and TGF-? may play animportant role.