Influence Of Osteopontin Silencing On Cyclosporine A-Induced Epithelial To Mesenchymal Transition In Rat Renal Tubular Epithelial Cells

ObjectiveTo study the effect of Osteopontin silencing on Cyclosporine A (CsA)-Induced Epithelial-to-Mesenchymal Transition in Rat Renal Tubular Epithelial Cells, and investigate the mechanism of its antifibrotic effect.MethodsCultured NRK52E cells were divided into four groups:(A) the control group, cells were treated without any medicine; (B) the cells treated with CsA (4.2μmol/L) for 72h; (C) the cells were transfected with OPN shRNA expression vector for 72h, then treated with CsA(4.2μmol/L) for 72h; (D) the cells were transfected with control shRNA expression vector for 72h, then treated with CsA(4.2μmol/L) for 72h; Morphological changes of the cell in different groups were assessed by phase-contrast microscopy. The immunofluorescence technology was adopted to detect the expression of E-cadherin,α-SMA,OPN in each group. Additionally, Western blot was performed to assessed the protein expression of E-cadherin;α-SMA and OPN.ResultsThe GFP-conjugated OPNshRNA expression vector was successfully transfected into NRK52E cells, we found the green fluorescence signal was the strongest at 72h which indicated the transfection efficiency arrived at a peak at 72h, we also found the expression of OPN protein was markedly decreased in the NRK52E cells transfected with OPN shRNA expression vector. Decreased OPN by OPN shRNA expression vector can markedly ameliorate the morphological changes of NRK52E cells stimulated by CsA; The immunofluorescence staining revealed the expression of E-cadherin was markedly decreased in NRK52E cells stimulated with CsA for 72 comparing with the control group, while the expression ofα-SMA and OPN were significantly higher in CsA group than the control group. In addition, the expression of E-cadherin in the third group was higher than the CsA group, while the expression ofα-SMA; OPN in the third group was lower than the CsA group. In Western blot analyses, the protein level of E-cadherin in CsA group was dramatically lower than that of the control group(P<0.05), while the protein level ofα-SMA; OPN in CsA group were significantly higher than those of the control group(P<0.05); Simultaneously, the protein level of E-cadherin in the third group was markedly higher than that of the CsA group(P<0.05), the protein level ofα-SMA;OPN in the third group were significantly lower than those of the CsA group(P<0.05).ConclusionOPN plays a important role in the tubulointerstitial fibrosis, OPN silencing can ameliorate the Cyclosporine A-Induced Epithelial to Mesenchymal Transition in NRK52E cells.