Article

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.

Download full-text

Full-text

Available from: Tara Mcmorrow, Jun 20, 2014
0 Followers
 · 
141 Views
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: End Stage Renal Disease (ESRD) is an ever increasing problem worldwide. However the mechanisms underlying disease progression are not fully elucidated. This work addressed nephrotoxicity induced by the immunosuppressive agents' cyclosporine A (CsA) and sirolimus (SRL). Nephrotoxicity is the major limiting factor in long term use of CsA. SRL causes less nephrotoxicity than CsA. Therefore investigations into the differential effects of these agents may identify potential mechanisms of nephrotoxicity and means to prevent ESRD induced by therapeutic drugs. Using ELISA, Western blotting, quantitative PCR and a reporter gene assay we detailed the differential effects of CsA and SRL in human renal mesangial cells. CsA treatment increased profibrotic TGF-β1 secretion in human mesangial cells whereas SRL did not, indicating a role for TGF-β in CsA toxicity. However we observed a synergistic nephrotoxic effect when CsA and SRL were co-administered. These synergistic alterations may have been due to an increase in CTGF which was not evident when the immunosuppressive drugs were used alone. The CsA/SRL combination therapy significantly enhanced Smad signalling and altered the extracellular matrix regulator matrix metalloproteinase 9 (MMP-9). Inhibition of the ERK 1/2 pathway, attenuated these CsA/SRL induced alterations indicating a potentially significant role for this pathway.
    Journal of Transplantation 01/2012; 2012:980910. DOI:10.1155/2012/980910
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tubular epithelial cells play a central role in the pathogenesis of chronic nephropathies. Previous toxicogenomic studies have demonstrated that cyclosporine- (CsA-) induced epithelial phenotypic changes (EPCs) are reminiscent of an incomplete epithelial to mesenchymal transition (EMT) in a TGF-β-independent manner. Furthermore, we identified endoplasmic reticulum (ER) stress as a potential mechanism that may participate in the modulation of tubular cell plasticity during CsA exposure. Because c-jun-N-terminal kinase (JNK), which is activated during ER stress, is implicated in kidney fibrogenesis, we undertook the current study to identify the role of JNK signaling in EPCs induced by CsA. In primary cultures of human renal epithelial cells, CsA activates JNK signaling, and the treatment with a JNK inhibitor reduces the occurrence of cell shape changes, E-cadherin downregulation, cell migration, and Snail-1 expression. Our results suggest that CsA activates JNK signaling, which, in turn, may participate in the morphological alterations through the regulation of Snail-1 expression.
    Journal of Transplantation 01/2012; 2012:348604. DOI:10.1155/2012/348604
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The increasing prevalence of BK virus (BKV)-associated diseases in immunosuppressed patients has prompted an investigation of the immune response to BKV, especially the role of cytokines in regulating viral replication. We examined the effect of TGF-beta, a cytokine that is stimulated by certain immunosuppressive therapies, on BKV gene expression during lytic infection of renal proximal tubule epithelial cells. Viral gene expression, and specifically the activity of the BKV early promoter, is regulated by TGF-beta in a strain-dependent manner. Promoter activity is upregulated in the presence of TGF-beta for the TU strain of BKV, and not for the Dik, Dunlop, or Proto-2 strains. Using site-directed mutagenesis, we have identified a small segment of the TU promoter that is required for stimulation in response to TGF-beta. These results demonstrate that BKV strains can respond differently to cytokine treatment and suggest that TGF-beta may play a role in the reactivation of BKV.
    Virology 07/2008; 378(1):6-12. DOI:10.1016/j.virol.2008.05.009 · 3.28 Impact Factor