Change of autophagy in the transforming growth factor-β1-induced activation of renal fibroblasts in vitro / 中华肾脏病杂志

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 type Ⅰ collagen (Col Ⅰ) 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 Beclinl expression level (all P ＜ 0.05).(3) TGF-β1 decreased the protein expression of LC3-Ⅱ in NRK-49F cells cultured with serumfree 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.