Abstract: Purpose After the cultured human renal tubular epithelial cells (HK-2 cells) were stimulated with aristolochic acid-1 (AA-I) for a period of 12-48 hours, whether removal of AA-1 in culture media can lessen the proliferation inhibition effect of AA-1. Methods HK-2 cells were cultured and stimulated with AA-1 for 12, 24 or 48 hours, washed with Hank抯 solution for 3 times, and then cultured in the medium free of AA-1 for 24 hours. Proliferation of the living cells was measured by the trypan blue method, and cell cycle was assessed by flow cytometry. Results The living cell number decreased by 8%, 25% and 57%, after the HK-2 cells were stimulated with AA-1(10μg/ml) for 12, 24 and 48 hours, respectively. AA-1 stimulated HK-2 cells showed higher proportion of G2/M stage, which increased to 1.28 and 3.8 times of the normal controls in those stimulated for 24 and 48 hours, respectively.Removal of AA-1 in the media did not improve the above changes. Of the HK-2 cells stimulated with AA-1 for 12, 24 and 48 hours and then removed the media AA-1, cell number deceased further by 45%, 32% and 30%, respectively. Of the HK-2 cells stimulated with AA-1 for 24 hours and then removed the media AA-1, the proportion of G2/M stage increased further by 2.91 times, equal to the value of the cells stimulated with AA-1 for 48 hours. Conclusion AA-1 (10μg/ml) inhibits the proliferation of HK-2 cells considerably, manifested by the decrease of living cell number and the blockage of cell cycle at G2/M stage. Removal of the extracellular AA-1 can not reverse the AA-1 induced cell proliferation inhibition, indicating that the intracellular accumulated AA-1 and its metabolites may be the important factors for the sustained injury of the cells.

Key words: Renal tubular epithelial cell, Cell proliferation, Cell cycle