通过共培养观察被马兜铃酸活化后的肾小管上皮细胞对肾间质成纤维细胞的作用

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

目的通过共培养观察被马兜铃酸钠盐(AA-Na)活化后的肾小管上皮细胞株(HK-2)对肾间质成纤维细胞(hRIFs)的作用。方法用AA-Na刺激HK-2,16h后用此活化的HK-2与hRIFs共培养(培养液中加或不加抗TGF-β1抗体),48h后检测hRIFs细胞层中Ⅰ型胶原(ColⅠ)含量。结果(1)AA-Na(20μg/ml)对HK-2无刺激增殖、转分化及细胞毒作用。(2)用此浓度AA-Na刺激HK-216h,能使HK-2分泌TGF-β1显著增加(P<0.05)。(3)用此活化后的HK-2与hRIFs共培养48h,能使后者合成ColⅠ显著增多(P<0.05);而抗TGF-β1抗体(1.0或2.0μg/ml)能部分抑制此反应(P<0.05)。结论被AA-Na刺激活化的HK-2能分泌TGF-β1促进hRIFs合成ColⅠ。

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.