The Study on the Mechanism of Cyclosporine A Induced Apoptosis in Renal Tubular Epithelial Cells

Kim, Soon Il; Choi, Kyu Hun; Chong, Deug Lim; Kim, Kyung Sup

Abstract

A major limiting factor in the use of cyclosporine A (CsA) is nephrotoxicity, but the mechanisms of nephrotoxicity are not fully understood. In order to elucidate the pathogenesis of CsA tubulotoxicity, we examined mechanisms (DNA synthesis, necrosis and apoptosis) of cellular injury induced by CsA in cultured LLC-PK1 renal tubular cell line. The possible role of Fas antigen in the mediation of CsA-induced cell death and the hypothesis that CsA-mediated injury activates the glucose transporter GLUT1, a stress response gene in renal tubular cells were also investigated. CsA treatment for 24 hours in LLC-PK1 cells showed significantly decreased 3H-thymidine uptake in a dose dependent manner (0.1 microgram/ml to 1 mg/ml), indicating that DNA damage is a sensitive indicator of CsA induced nephrotoxicity. A dose of 10 microgramml CsA caused a significant increase in LDH release (M+/-S.D., 11.0+/-3.0% vs 27.0+/-9.8, p<0.05). On flow cytometric analysis, 9.9 4.2% of control cells appeared in a region of decreased forward light scatter and increased side scatter, respectively. Both indices representing characteristics of apoptotic cell death. Compared to control, treatment with 10 ng/ml of CsA for 24 hours significantly increased the proportion of cells in apoptotic region to 38.9 13.5%. This finding was supported by electrophoretic analysis of the DNA extracted from CsA-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. CsA (0.1 microgram/ml) treatment for 24 hours was seen to cause a significant elevation in the expression of the 45 kD Fas protein by Western blot analysis. In addition, the exposure to CsA was also associated with an increase of GLUT1 protein levels up to 2.2 fold (mean) on Western blot analysis. In conclusion, CsA is directly toxic to tubular cells with inhibiting DNA synthesis and inducing cell death in the form of necrosis or apoptosis. Fas antigen-ligand system and glucose transporter GLUT1 may play roles in mediating CsA induced tubular cytotoxicity.