Protective effect of bone morphogenetic protein 7 on TGF-β1 induced epithelial-mesenchymal transition of podocyte

Abstract:

Objective：To observe the protective effect of bone morphogenetic protein 7 on TGF-β1 induced epithelial-mesenchymal transition of podocyte. Methods：The pcDNA 3.1rh BMP-7 plasmid was constructed and identified. Human podocytes were cultured in vitro，with different concentrations（10，20，50，100，200 μg/mL） of recombinant BMP-7 stimulating podocytes for 24，48，72 h and were collected in the cell respectively. The death rates of cells were detected by flow cytometry，and the optimal stimulation concen－trations and time points of recombinant BMP-7 were selected. The experiment was divided into normal control group，TGF beta 1 stimulation group（TGF-β1） and plasmid transfection group（PEGF-BMP）. PEGF-BMP group was transfected podocyte with 100 μg/mL recombinant BMP-7 pretreatment after 48 hours；the human podocytes of TGF-β1 and PEGF-BMP groups were stimulated respectively with 5 ng/mL TGF-β1. Alterations of specific markers in cultured podocytes were observed by immunofluorescence；and 72 hours after treatment，the cells of different groups were harvested，the mRNA expressions of Podocin，SMA，VIM in podocyte were detected by RT-PCR and the protein expressions of Podocin，SMA，VIM in podocyte were detected by Western blot. Results：①pcDNA 3.1rh BMP-7 plasmid was constructed successfully and stably expressed BMP-7 was transfected in human podocytes. The expression level of BMP-7 protein in PEGF-BMP group（0.487±0.012） was significantly higher than that in normal control group（0.251±0.012） and TGF beta induction group（0.151±0.015）（P<0.001）. ②Real-time PCR analysis showed that mRNA expression of podocin was significantly decreased in TGF beta 1 group（0.285±0.013） than in normal control group （1.057±0.090）（P<0.001），while mRNA expression of podocyte was increased in PEGF-BMP-7 group（0.693±0.077） than in TGF beta 1 group（P=0.003）；the mRNA expression of α-SMA，VIM were significantly increased in TGF beta 1 group（1.257±0.039，1.114±0.097） and PEGF-BMP-7 group（1.028±0.093，0.821±0.059） than in normal control group（0.357±0.089，0.403±0.020）（P＜0.001），but mRNA expression of α-SMA，VIM were decreased in PEGF-BMP group than in TGF beta 1 group（P=0.011，P=0.002）. Western blot showed that protein expression of podocin was significantly decreased in TGF beta 1 group（32.923±5.301） than in normal control group（101.807±9.208）（P<0.001），while protein expression of podocyte was increased in PEGF-BMP-7 group（90.507±7.810） than in TGF beta 1 group（P＜0.001）；the protein expression of α-SMA，VIM were significantly increased in TGF beta 1 group（116.120±8.300，124.016±9.702） and PEGF-BMP group（93.832±10.602，100.801±6.801） than in normal control group（35.730±4.892，43.801±2.600）（P＜0.001），but protein expressions of α-SMA，VIM were decreased in PEGF-BMP-7 group than in TGF beta 1 group（P=0.017，P=0.007）. Conclusion：The expression of α-SMA and VIM in human podocyte induced by TGF beta 1 was in－creased，podocin expression decreased，while BMP-7 inhibited this effect and showed the protective effect on podocyte.