The Study on the Mechanism of Cyclosporine A-induced Nephroptoxicity: The Effects of Ceramide and Phospholipase A2 on Cyclosporine A-induced Apoptosis of Renal Tubular Epithelial Cells / 대한이식학회지

ABSTRACT

PURPOSE: In order to elucidate the mechanisms mediating cyclosporine A (CsA)-induced renal tubular cell injury, we examined the effects of ceramide, second messenger derived from sphingolipid breakdown, and phospholipase A2 (PLA2), enzyme responsible for release of arachidonic acid, on CsA-induced apoptosis of cultured LLC-PK1 renal tubular cell line. METHODS: The apoptosis was evaluated by flow cytometric analysis and DNA gel electrophoresis. The activities of cytosolic (c)- and secretory (s)-PLA2 were measured by ELISA methods and Western blotting of cPLA2 was also investigated. RESULTS: The exposure to CsA (10microgram/mL) significantly increased the percentage of cells displaying annexin-V binding from 5.1+/-2.0% in control to 24.7+/-6.5% (P<0.05), indicating apoptosis. The addition of ceramide (10 micromol/mL) significantly inhibited the increase of apoptosis induced by CsA (24.7+/-6.5% vs. 14.7+/-6.0). The treatment with PLA2 (5 U/mL) also decreased CsA (5microgram/mL)-induced apoptosis (20.4+/-5.7% vs. 13.6+/-5.2, P<0.05). But there was no dose-dependent further protectective effect of ceramide or PLA2. Concerning the changes of PLA2 activities, cPLA2 activity after CsA exposure (10microgram/mL) was increased from 3.92+/-2.01 nmol/min/mL in control to 9.81+/-3.07 (P<0.05), and the addition of ceramide (5 micro mol/mL) significantly inhibited the increase of cPLA2 activity after CsA exposure (4.64+/-1.52). The Western blotting of cPLA2 (110 kD) also showed similar results. Meanwhile there was no significant changes of sPLA2 activitiy which was markedly low. CONCLUSION: The apoptosis of renal tubular epithelial cell following CsA expsoure appears to be mediated by membrane phospholipid breakdown via sphingomyelinase and PLA2. The mechanism(s) mediating the protective effect of ceramide and PLA2 on CsA-induced apoptosis should be further elucidated.