Fluorofenidone suppresses epithelial-mesenchymal transition and the expression of connective tissue growth factor via inhibiting TGF-β/Smads signaling in human proximal tubular epithelial cells

Objectives: The present study was designed to investigate the potential effects and mechanism of fluorofenidone (AKF-PD) on transforming growth factorβ1 (TGF-β1)-induced tubular epithelial-mesenchymal transition (EMT) and the expression of connective tissue growth factor (CTGF) in human proximal tubular epithelial cells. Methods: HK-2 cells were pretreated with AKF-PD, pirfenidone (PFD), Losartan, and SB431542 (an inhibitor of TGF-β type I receptor). The pretreated HK-2 cells were subsequently cotreated with TGF-β1 (5 ng/ml). The morphological changes of HK-2 cells were observed under an inverted microscope. Expression of α-SMA was detected by Western blot and immunofluorescence. The protein expression of ZO-1, fibronectin, CTGF, phosphorylated Smad2 (p-Smad2) and phosphorylated Smad3 (p-Smad3) were evaluated by Western blot. Results: Through down-regulation of p-Smad2 and p-Smad3 proteins, AKF-PD significantly inhibited protein expression of α-SMA, fibronectin, and CTGF. Meanwhile, the depressed ZO-1 expression and morphological changes induced by TGF-β1 were attenuated by AKF-PD. Conclusion: AKF-PD acts as an anti-fibrotic agent through blocking TGF-β/Smads signaling and consequently inhibits TGF-β1-induced EMT and CTGF expression in human proximal tubular epithelial cells.