GSK3, snail, and adhesion molecule regulation by cyclosporine A in renal tubular cells

• Published in: Toxicological sciences 2012-06, Vol.127 (2), p.425-437
• Main Authors: Berzal, Sergio, Alique, Matilde, Ruiz-Ortega, Marta, Egido, Jesús, Ortiz, Alberto, Ramos, Adrián M
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - drug effects; Actin Cytoskeleton - metabolism; Animals; Apoptosis - drug effects; beta Catenin - metabolism; Blotting, Western; Cadherins - metabolism; Calcineurin - metabolism; Calcineurin Inhibitors; Cell Adhesion Molecules - genetics; Cell Adhesion Molecules - metabolism; Cell Line; Claudin-1; Cyclosporine - toxicity; Epithelial-Mesenchymal Transition - drug effects; Glycogen Synthase Kinase 3 - antagonists & inhibitors; Glycogen Synthase Kinase 3 - genetics; Glycogen Synthase Kinase 3 - metabolism; Humans; Immunosuppressive Agents - toxicity; Kidney Tubules, Proximal - drug effects; Kidney Tubules, Proximal - enzymology; Kidney Tubules, Proximal - pathology; Membrane Proteins - metabolism; Mice; Microscopy, Confocal; Phosphoproteins - metabolism; Protein Kinase Inhibitors - pharmacology; Real-Time Polymerase Chain Reaction; RNA Interference; Signal Transduction - drug effects; Snail Family Transcription Factors; Time Factors; Transcription Factors - genetics; Transcription Factors - metabolism; Transforming Growth Factor beta1 - metabolism; Vimentin - metabolism; Zonula Occludens-1 Protein
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/kfs108

Tubular cell injury and fibrosis are key features of calcineurin inhibitor nephrotoxicity, but the molecular processes involved are not fully understood. In cultured murine MCT and human kidney 2 proximal tubular cells, gene expression and protein levels were studied by real-time polymerase chain reaction, Western blot, and confocal microscopy. Protein function was evaluated by pharmacological inhibitors and confirmed by small interfering RNA (siRNA) gene targeting. In renal tubular cells, cytotoxic concentrations of cyclosporine A (CsA) inhibited both gene and protein expression of adherent and tight junction proteins (E-cadherin, ZO-1, claudin-1, and β-catenin) and increased vimentin expression, without involvement of transforming growth factor β1 or caspase activity. CsA upregulated transcriptional repressors (Snail, Slug, and Twist) of the adherent and tight junction proteins were studied. Snail siRNA targeting prevented the downregulation of E-cadherin by CsA. CsA promoted glycogen synthase kinase 3 (GSK3) phosphorylation and increased Snail half-life. The GSK3 inhibitor lithium upregulated Snail and decreased E-cadherin expression in a Snail-dependent manner. Moreover, targeting GSK3 activity by siRNA also upregulated Snail. Furthermore, GSK3 siRNA had a negative impact on CsA-induced upregulation of Snail. Tacrolimus also inhibited GSK3 and mimicked CsA responses in tubular cells. We conclude that calcineurin inhibitors may directly decrease the expression of epithelial adhesion molecules by repressing GSK3 and stabilizing Snail. This offers potential pharmacological targets for prevention of nephrotoxicity.