Effect of renal tubular epithelial cells activated by aristolochic acid on renal interstitial fibroblasts in coculture system

Objective To investigate the effect of human renal tubular epithelial cell line(HK 2) activated by aristolochic acid sodium salt(AA Na)on human renal interstitial fibroblasts (hRIFs) in coculture.Methods (1)HK 2 was incubated with AA Na(20 μg/ml)for 16 h, and then was fully washed. The AA Na activated HK 2 was cocultured with hRIFs for 48 h, with or without neutralizing anti TGF β1 antibody(1 0 or 2 0 μg/ml) in medium, and then the amount of ColⅠin cell layer of hRIFs was determined.(2)Cell proliferation and cytotoxicity were determined by MTT and LDH release assay respectively. Antigen expression of cells was detected by indirect immunofluorescence.TGF β1 and ColⅠconcentrations were measured with ELISA. Results (1)Neither proliferative and cytotoxic effects nor epithelial myofibroblast transdifferentiation were found in HK 2 after AA Na (20 μg/ml) stimulation (P 0.05).(2)TGF β1 secreted by AA Na (20 μg/ml) activated HK 2 was significantly increased [(113.04±11.81)pg/ml at incubation for 48 h vs (86 97±9 33)pg/ml in control,P 0.05]. (3)After hRIFs were cocultured with AA Na(20 μg/ml) activated HK 2 for 48 h, ColⅠsynthesized by hRIFs was significantly enhanced compared with those in control [(217.8±23.4) vs (158.6±15.0) ng/ml,P 0 05]. The synthesis could be partially inhibited by neutralizing anti TGF β1 antibody (1.0 and 2.0μg/ml) [(190 5±18 8) and (186 1±20 5) vs (217 8±23 4) ng/ml, respectively, P 0.05]. No cytotoxic effect on hRIFs was found(P 05). Conclusion HK 2 activated by AA Na can secrete more TGF β1, which in turn acts on hRIFs through cell cell cross talk to enhance their ColⅠproduction.