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

Objective To examine the effect of different concentrations of aristolochic acid I (AAI) in inducing apoptosis of cultured porcine renal cell line LLC-PK1 and to investigate the relationship between intracellular free calcium concentration (［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 microscopy, 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 confocus microscopy using Fluo-3/AM staining. Results A series of morphologic changes that were characteristic of apoptosis, 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.Quantitative analysis of apoptotic cells showed that the percentage of apoptotic cells in AAI (0.02 g·L(-1) , 0.04 g·L(-1) or 0.08 g·L[-1]) group was significantly 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 (102 ng·L(-1) , 103 ng·L(-1) ), mean ［Ca++］i was significantly lower than that treated with AAI alone (35.47±12.85, 28.55±10.16 vs 58.01±18.89, P<0.001).And the percentage of apoptotic cells in group treated with AAI plus lacidipine ](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 cells.The mean ［Ca++］i in AAI-treated LLC-PK1 cells was increased significantly, sugguesting that the increase of ［Ca++］i may be related to apoptosis in LLC-PK1 cells.Lacidipine may decrease the raised mean ［Ca++］i levels caused by AAI and the percentage of apoptotic cells, and lacidipine may ameliorate AAI-induced apoptotic damage by inhibiting the increase of ［Ca++］i in LLC- PK1 cells.     

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