Cyclosporine Induces Apoptosis of Mouse Terminal Proximal Straight Tubule Cells

Accessible online at: http://BioMedNet.com/karger Dear Sir, Cyclosporine (Cs) is an immunosuppressant which has markedly improved the rate of graft survival of different organ transplants in the last decade. Among its recognized side effects, nephrotoxicity is predominant [1] and often limits the use of Cs. Cs nephrotoxicity encompasses a spectrum of clinical manifestations from precipitous decrease in glomerular filtration rate to chronically progressive scarring. In renal tubules, the major target site of Cs was shown to be the terminal proximal tubule (S3) [2] where isometric vacuolization was reported to occur in response to Cs [3]. In a multicellular organism cell death can occur by two distinct mechanisms, necrosis and apoptosis [4]. Apoptosis is an active form of cell death whereby the cell can be thought of as committing suicide rather than being passively destroyed by the degradative action of proteinases, as in necrosis. Ito et al. [5] have recently demonstrated that apoptosis occurred in a human allografted kidney treated with Cs using an in situ nick end-labeling technique. However, in using this technique it is difficult to discriminate apoptosis and necrosis [6]. To assess the direct effect of Cs on the proximal tubule and to confirm whether Cs induces apoptosis of renal tubules, we determined the effect of Cs on mouse S3 cells derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. As previously reported by us [7], S3 cells were cultured in RITC 80-7 medium (Kyokuto, Tokyo, Japan) containing 5% fetal bovine serum (Gibco, Gaithersburg, Md., USA), 10 Ìg/ml transferrin (Sigma, St. Louis, Mo., USA), 0.08 U/ml insulin (Shimizu, Shizuoka, Japan), 10 ng/ml recombinant human epidermal growth factor (Wakunaga, Hiroshima, Japan), 100 U/ml penicillin and 100 Ìg/ml streptomycin (Gibco). S3 cells retained hydrolase activities associated with the intact proximal tubule including alkaline phosphatase, Á-glutamyltransferase and leucine-aminopeptidase activities, and the levels of these activities were higher than those in LLC-PK1 cells but lower than those in freshly isolated S3 segments [unpubl. observation]. Cs was kindly provided by Sandoz (Basel, Switzerland). The trypan blue exclusion test revealed that Cs induced a time (24 and 48 h)and dose (10–6 and 10–5 M )-dependent decrease in the cell viability of S3 cells (data not shown). As shown in figure 1, agarose gel electrophoresis of DNA extracted from S3 cells treated with vehicle (methanol) revealed no DNA fragments of low molecular weight. In contrast, DNA from S3 cells treated with 10–6 M Cs for 24 h revealed the characteristic ladder pattern resulting from the internucleosomal chromatin cleavage of 180-bp fragments. As shown in figure 2a, S3 cells treated with vehicle retained normal nucleus features with retaining microvilli. In contrast, as shown in figure 2b, margination and condensation of chromatin, characteristic morphological features of apoptosis, were observed in S3 cells treated with 10–6 M Cs for 24 h. Fig. 1. Agarose gel electrophoresis of DNA extracted from S3 cells exposed to cyclosporine (Cs) or vehicle. Lane 1 = vehicle; lane 2 = 10–6 M Cs for 24 h.