Change of autophagy in the transforming growth factor-β1-induced activation of renal fibroblasts in vitro

Abstract

Objective To explore the changes of autophagy in the transforming growth factor (TGF)-β1-induced activation of renal fibroblasts in vitro. Methods (1) NRK-49F cells were cultured with 10 μg/L TGF-β1 for different times (0, 12, 24 h) in vitro. Morphological changes of the cells were observed under inverted microscope. The protein expressions of α-smooth muscle actin (α-SMA) and typeⅠcollagen (ColⅠ) in NRK-49F cells were measured by Western blotting. (2) NRK-49F cells were cultured with 0, 2, 5, 10, 15, 20 μg/L TGF-β1 for 1 hour and with 10 μg/L TGF-β1 for different times (0 min, 7 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h) in vitro. The protein expressions of microtubule-associated protein 1 light chain 3(LC3), p62, total-mammalian target of rapamycin (t-mTor), phospho-mammalian target of rapamycin (p-mTor) and Beclin 1 were detected by Western blotting. (3) NRK-49F cells were cultured with 10 μg/L TGF-β1 for different times (0, 1, 4 h) in vitro after cultured with serum-free medium for 2.5 hours. The protein expressions of LC3 and p62 in NRK-49F cells were measured by Western blotting. Results (1) The morphology of NRK-49F cells changed from stellate to spindle shape after cultured with TGF-β1. The expressions of cell activation markers α-SMA and Col Ⅰ gradually increased as the extend of stimulation time (all P<0.05). (2) TGF-β1 transiently increased the expressions of autophagy proteins p62 (peak value appeared after 4 h) and p-mTor (peak value appeared after 30 min), while decreased Beclin1 expression level (all P<0.05). (3) TGF-β1 decreased the protein expression of LC3-Ⅱ in NRK-49F cells cultured with serum-free medium, whereas increased the protein expression of p62 at the same time (all P<0.05). Conclusions The autophagy activity of renal fibroblasts is inhibited by the TGF-β1-induced cellular activation in vitro, which may contribute to the progression of renal interstitial fibrosis. Key words: Transforming growth factor beta1; Fibrosis; Fibroblasts; Autophagy and its correlated proteins