NDT Advance Access originally published online on July 19, 2005
Nephrology Dialysis Transplantation 2005 20(10):2215-2225; doi:10.1093/ndt/gfh967
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Cyclosporine A induced epithelial–mesenchymal transition in human renal proximal tubular epithelial cells
Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
Correspondence and offprint requests to: Michael P. Ryan, PhD, Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. Email: michael.p.ryan{at}ucd.ie
Background. Tubulointerstitial fibrosis is a relatively common and sinister complication of cyclosporine A (CsA) therapy that limits its clinical use. CsA may have direct effects on renal tubular epithelial cells by promoting epithelial–mesenchymal transition (EMT). EMT plays an important role in embryonic development and tumourigenesis and has been described in organ remodelling during fibrogenesis. In this study, we investigated the effects of CsA on a human renal cell line as a model system to test the hypothesis that CsA can induce renal EMT.
Methods. Human renal proximal tubular cells were treated with CsA (0.42–42 µm) for periods up to 72 h. Viability was assessed by the Alamar Blue assay. Morphological changes were assessed by phase contrast microscopy. The effects on epithelial adherens molecule, ß-catenin and stress fibre protein, F-actin were analysed by indirect immunofluorescence. Reverse transcription––polymerse chain reaction was performed to measure the mRNA levels of extracellular matrix components. Expression of transforming growth factor-ß was measured by western blotting. Expression and activity of matrix metalloproteinases were measured by gelatin zymography.
Results. CsA induced striking morphological changes in epithelial cells, including changes in cellular morphology, F-actin stress fibre formation, delocalization of the adherens junction protein ß-catenin and increased levels of collagen IV and fibronectin. In addition, CsA-induced EMT was associated with increased TGF-ß1 protein levels and EMT was markedly attenuated in the presence of anti-TGF-ß1 antibody. CsA-induced EMT was also associated with increased expression of connective tissue growth factor (CTGF) suggesting that this molecule may serve as downstream mediator of TGF-ß1 pro-fibrotic activity in this setting.
Conclusions. In aggregate, these data suggest that CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-ß1 and CTGF as mediators of this response. The further delineation of the molecular components of this pro-fibrogenic response may suggest novel strategies through which to prevent CsA-induced tubulo-interstitial fibrosis in vivo.
Keywords: CTGF; cyclosporine; EMT; nephrotoxicity; TGF-ß1
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