Aristolochic acid I-induced apoptosis in LLC-PK1 cells and amelioration ofthe apoptotic damage by calcium antagonist

To examine the effect of different concentrations of aristolochic acid I (A AI) in inducing apoptosis of cultured porcine renal cell line LLC-PK1 and t o investigate the relationship between intracellular free calcium concentra tion ([Ca+ + ] i) and LLC-PK1 apoptosis induced by AAI and the influence of a calcium antagonist, lacidipine on apoptosis and [Ca+ (+) ]i. Methods LLC-PK1 cells were treated in different groups: a. the normal group without treatment; b. the group with AAI alone (0.01 g . L-1, 0.02 g . L-1 , 0.04 g . L-1, 0.08 g . L-1); c. the group with lacidipine alone (10 ng . L-1, 10(2) ng . L-1, 10(3) ng . L-1); d. the group with AAI (0.04 g . L-1) plus lacidipine (10 ng . L-1, 10(2) ng . L-1, 10(3) ng . L-1). Light micros copy, agarose gel electrophoresis, Annexin-V-Flous apoptosis detection kit and flow cytometry using propidium iodide staining to identify or quantify the apoptosis of LLC-PK1 cells. Mean [Ca+ +]i was measured by laser confocu s microscopy using Fluo-3/AM staining. Results A series of morphologic changes that were characteristic of apoptos is, Annexin-V-Flous staining positive apoptotic cells and "DNA ladder" were identified in AAI (0.02 g . L-1 - 0.08 g . L-1) treated LLC-PK1 cells. Qua ntitative analysis of apoptotic cells showed that the percentage of apoptot ic cells in AAI (0.02 g . L-1, 0.04 g . L-1 or 0.08 g . L-1) group was sign ificantly higher than that in normal group (5.3%, 48.5%, 78.7% vs 2.6%, P < 0.001). Mean [Ca+ +]i was significantly higher in cells treated with AAI ( 0.04 g . L-1) than that in normal cells (58.01 +/- 18.89 vs 22.66 +/- 4.78, P < 0.001). In group treated with AAI plus lacidipine (10(2) ng . L-1, 10( 3) ng . L-1), mean [Ca+ (+) ]i was significantly lower than that treated wi th AAI alone (35.47 +/- 12.85, 28.55 +/- 10.16 vs 58.01 rt 18.89, P < 0.001 ). And the percentage of apoptotic cells in group treated with AAI plus lac idipine (10(2) ng . L-1, 10(3) ng . L-1) was also significantly lower than that treated with AAI alone (19.0%, 27.8% vs 34.7%, P < 0.001). Conclusions High concentrations of AAI may induce apoptosis of LLC-PK1 cell s. The mean [Ca+ + ] i in AAl-treated LLC-PK1 cells was increased significa ntly, sugguesting that the increase of [Ca+ + ] i may be related to apoptos is in LLC-PK1 cells. Lacidipine may decrease the raised mean [Ca+ + ] i lev els caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAl-induced apoptotic damage by inhibiting the increase of [Ca + ]i in LLC-PK1 cells.