TGF-beta1/Smad7 signaling stimulates renal tubulointerstitial fibrosis induced by AAI.

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.