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.
Nephrology Dialysis Transplantation (Impact Factor: 3.49). 11/2005; 20(10):2215-25. DOI: 10.1093/ndt/gfh967
Source: PubMed

ABSTRACT 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.
Human renal proximal tubular cells were treated with CsA (0.42-42 microm) 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, beta-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-beta was measured by western blotting. Expression and activity of matrix metalloproteinases were measured by gelatin zymography.
CsA induced striking morphological changes in epithelial cells, including changes in cellular morphology, F-actin stress fibre formation, delocalization of the adherens junction protein beta-catenin and increased levels of collagen IV and fibronectin. In addition, CsA-induced EMT was associated with increased TGF-beta1 protein levels and EMT was markedly attenuated in the presence of anti-TGF-beta1 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-beta1 pro-fibrotic activity in this setting.
In aggregate, these data suggest that CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-beta1 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.

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Available from: Tara Mcmorrow, Jun 20, 2014
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    • "The SDS- PAGE procedure used was that of Laemmli [19]. Expression levels of renal proteins following CsA treatment was determined by Western blot and has been described previously [15] [20] [21]. Proteins of interest were detected using the following antibodies according to the manufacturer's protocol (rabbit anti-ERK 1/2, Cell Signalling Technology, 9211S and 9211). "
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    Journal of Transplantation 01/2012; 2012:980910. DOI:10.1155/2012/980910
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    • "Transcriptomic analysis of tubular cell response to CsA has shown that CsA induces EMT in vitro in a transforming growth factor β-(TGF-β-) dependent manner [10]. In a previous study, we have shown that CsA promotes TGF-β-in dependent epithelial phenotypic changes (EPCs). "
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    Journal of Transplantation 01/2012; 2012:348604. DOI:10.1155/2012/348604
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    • "Our studies of cytokine-mediated regulation of BKV gene expression and replication are targeted at understanding the process of viral reactivation in immunosuppressed transplant patients. Previously, it has been shown that common immunosuppressive therapies used in renal transplantation patients, such as cyclosporine and tacrolimus, result in upregulation of TGF-β expression in various cell types, including kidney epithelial cells (Khanna et al., 1999a; Khanna et al., 1999b; McMorrow et al., 2005; Shihab et al., 1996). Therefore, we hypothesized that TGF-β-mediated upregulation of the TU strain of BKV would result in an enhanced ability to reactivate in renal transplant recipients and, consequently, an increase in virulence of this particular strain. "
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