Aims: The nephrotoxic side effects of cyclosporine A (CsA) are partly due to induction of the epithelial-to-mesenchymal transition (EMT), possibly through upregulation of connective tissue growth factor (CTGF). Bone morphogenetic protein (BMP-7) has been reported to protect against and reverse renal injury via its renotropic and antifibrotic effects. We therefore designed a method to investigate the mechanism by which BMP-7 inhibits CSA-induced EMT in cultured human renal proximal tubular epithelial (HK-2) cells and whether BMP-7 can antagonize CsA-induced profibrogenic effects.
Methods: Cultured HK-2 cells were treated with CsA or a combination of CsA and BMP-7 for 72 h. Morphological changes were assessed by phase-contrast microscopy. The expression of alpha-smooth muscle actin (alpha-SMA), E-cadherin, collagen type I and CTGF was analyzed by immunofluorescence, RT-PCR, and Western blot. Additionally, the effect of CTGF silencing on CsA-mediated gene expression was assessed.
Results: CsA-induced EMT was associated with decreased expression of E-cadherin, increased expression of alpha-SMA, collagen type I and CTGF, and loss of epithelial morphology. BMP-7 inhibited these effects in a dose-dependent manner. Using siRNA, CTGF knock-down also attenuated EMT-associated phenotypic changes induced by CsA.
Conclusion: These data suggest that BMP-7 can block CsA-induced EMT by downregulating the expression of CTGF, a downstream mediator of EMT.