Wang Yanying, Zhang Zhongwen, Shen Hong, Lu Yan, Li Huanrong, Ren Xiaoming, Wu Guojuan
Laboratory of Pharmacology of Chinese Veterinary Medicine, Department of Animal Science and Technology, Beijing University of Agriculture, Beijing, China.
J Recept Signal Transduct Res. 2008;28(4):413-28. doi: 10.1080/10799890802176741.
A progressive tubulointerstitial nephropathy is mainly induced by aristolochic acid I (AAI), but a comprehensive understanding of this process is still missing. By using mouse primary renal tubular epithelial cells (RTECs) cultured in vitro and combining with two AAI treatment types (dose-response studies and time-response studies), we sought to investigate the nephrotoxicity of AAI further. Following our molecular and pharmacological studies, we found that high doses of AAI could lead to the death of RTECs within a short time, but low doses in a long duration only induce the epithelial cells to transform into myofibroblasts (MFs). This was also immediately identified by the increased expression of vimentin and de novo expression of alpha-smooth muscle actin (alpha-SMA) with the loss of cytokeratin 18 (CK18) by semiquantitative reverse transcriptase-PCR (RT-PCR) and immunofluorescence staining. The transcriptional level of transforming growth factor-beta1 (TGF-beta1) in the group treated with AAI significantly increased twice as much as the control. Smad2 mRNA level in the group with 50 ng/mL AAI declined by 23.4% at 24 hr, then increased by 180.0% at 36 hr; it was also evidently increased (217.4%) after being treated with 30 ng/mL AAI for 24 hr. Meanwhile, Smad7 mRNA level was down-regulated by AAI in dose- and time-dependence. Furthermore, by cotransfecting in mouse primary RTECs, the transcriptional level of Smad7 promoter-luciferase reporter gene was significantly down-regulated by AAI (300 ng/mL), and the expression of myofibroblast-specific markers induced by AAI was also suppressed by the specific antagonist of TGF-beta1 receptors (SB-431542). Collectively, the present results suggest that AAI may induce cytotoxicity through its conductive epithelial to mesenchymal transition, and TGF-beta1/Smad7 signaling can stimulate renal tubulointerstitial fibrosis induced by AAI.
进行性肾小管间质性肾病主要由马兜铃酸I（AAI）诱导，但对这一过程仍缺乏全面了解。通过使用体外培养的小鼠原代肾小管上皮细胞（RTECs），并结合两种AAI处理类型（剂量反应研究和时间反应研究），我们试图进一步研究AAI的肾毒性。经过分子和药理学研究，我们发现高剂量的AAI可在短时间内导致RTECs死亡，但低剂量长时间作用仅诱导上皮细胞转化为肌成纤维细胞（MFs）。通过半定量逆转录聚合酶链反应（RT-PCR）和免疫荧光染色检测波形蛋白表达增加、α-平滑肌肌动蛋白（α-SMA）从头表达以及细胞角蛋白18（CK18）丢失，也立即证实了这一点。AAI处理组中转化生长因子-β1（TGF-β1）的转录水平显著升高，是对照组的两倍。50 ng/mL AAI组中，Smad2 mRNA水平在24小时时下降23.4%，然后在36小时时升高180.0%；用30 ng/mL AAI处理24小时后也明显升高（217.4%）。同时，Smad7 mRNA水平在剂量和时间依赖性上被AAI下调。此外，通过在小鼠原代RTECs中共转染，AAI（300 ng/mL）显著下调了Smad7启动子-荧光素酶报告基因的转录水平，并且TGF-β1受体特异性拮抗剂（SB-431542）也抑制了AAI诱导的肌成纤维细胞特异性标志物的表达。总的来说，目前的结果表明，AAI可能通过其诱导的上皮-间充质转化诱导细胞毒性，并且TGF-β1/Smad7信号通路可刺激AAI诱导的肾小管间质纤维化。
