Experimental study on epithelial-mesenchymal-transition of peritoneal mesothelial cells induced by indoxyl sulfate

Abstract

Objective To induce epithelial mesenchymal transition (EMT) of immortalized human peritoneal mesothelial cell line (HMrSV5) with indoxyl sulfate (IS) and investigate the role of transforming growth factor (TGF-β1) . Methods HMrSV5 cells were randomly divided into 3 groups: the control group: with 4.25﹪peritoneal dialysate fluid in culture medium; IS groups: with IS at final concentrations oft 250μmol/L, 500μmol/L, and 1 000μmol/L and 4.25﹪peritoneal dialysate fluid; TGF-β1 inhibitor group: with 2μmol/L LY364947, IS and 4.25﹪peritoneal dialysate fluid. At 0 h, 4 h, 12 h, 24 h, 48 h, and 72 h, the morphological changes were observed by immunofluorescence method, and alpha smooth muscle actin (α-SMA) expression was evaluated with Western Blot. At 48 h, supernatant was collected to determinate fibronectin and laminin5 by ELISA. Results After IS induction, cells change from typical cobblestone-like shape to long fusiform shape, which was prevented but TGF-β1 inhibitor. Western Blot showed that: compared with control group the expression level ofα-SMA increased significantly in the IS treatment group. The level ofα-SMA was highest at 48 h with 1 000μmol/L IS (39.929±2.610vs0.996±0.001;P< 0.05) , which was decrease to 7.418±3.075 in the presence of TGF-β1 inhibitor (P< 0.05) .Compared with control group, IS increased the secretion of FN (1.368±0.040vs1.094±0.036, P< 0.05) and laminin 5 (1.031±0.085vs0.860±0.063,P< 0.05) , which were decreased by TGF-βinhibitor (0.925±0.15 and 0.795±0.426 respectively,P< 0.05) . Conclusion EMT of PMC could be induced by IS via TGF-β1 signaling pathway and is potential target for the treatment of peritoneal fibrosis. Key words: Peritoneum; Stromal cells; Sulfuric acids; Indophenol; Epithelial-mesenchymal-transition; Transforming growth factor beta