An investigation on nephrotoxicity of Aristolactam I induced epithelial-mesenchymal transition on HK-2 cells

Aristolactam I (AL-I) is the main active ingredient in the Aristolochia plant species, which have been associated with severe nephrotoxicity. In order to investigate the mechanism of AL-I induced renal epithelial-mesenchymal transition (EMT), we established an AL-I induced EMT model in human proximal tubular epithelial cells (HK-2 cells). Biochemical analysis experiment including Morphological examination, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay, and Western blot analysis were performed. The results showed that AL-I accumulates in the cytosol causing cytotoxicity and inhibition of proliferation in a concentration- and time-dependent manner. Morphological examination showed that with the increasing concentration of AL-I, the tendency of HK-2 cells transform form epithelial cell to fibroblast cells was stronger. In the Western blot analysis, the expression of α-Smooth muscle actin (α-SMA) and Transforming Growth Factor β1 (TGF-β1) were significantly up-regulated, the expression of E-cadherin was significantly down-regulated after administrating. The ratio of the expression of P-Smad2/3 and Smad2/3 was significantly up-regulated, suggested that TGF-β/Smad-dependent signaling pathway was activated in this process. With presence of TGF-β receptor inhibitor (LY364947), we found that the expressions of three EMT related proteins (E-cadherin, α-SMA and TGF-β1) were obviously reversed. In conclusion, we acknowledge that AL-I can induce renal EMT process in HK-2 cell, which is triggered by the activation of TGF-β/Smad-dependent signaling pathway.