Effect of forkhead transcription factor O1 on high glucose-induced epithelial-mesenchymal transition in mice podocytes and its mechanism

Abstract

Objective To study the role and molecular mechanism of forkhead transcription factor O1(FoxO1)on epithelial-mesenchymal transition(EMT)in mice podocytes cultured under high glucose conditions through inhibiting transforming growth factor-β1(TGF-β1)/Smad3 signaling. Methods The podocytes were transfected with lentiviral vectors expressing constitutively active FoxO1(LV-CA-FoxO1), the lentiviral vectors expressing FoxO1 short hairpin RNA(LV-FoxO1-shRNA), and lentiviral vectors expressing empty vectors(LV-NC). The podocytes were divided into five groups of normal glucose(5.6 mmol/L, group A), high glucose(25 mmol/L, group B), high glucose plus LV-CA-FoxO1(group C), high glucose plus LV-FoxO1-shRNA(group D), and high glucose plus empty lentiviral vectors(group E). After treatment with different periods of time(24, 48, and 72 h), the mRNA levels of podocytes were measured through realtime PCR in each group, including FoxO1, TGF-β1, Smad3, nephrin, and desmin. The protein expressions of FoxO1, p-FoxO1, TGF-β1, Smad3, p-Smad3, nephrin, and desmin were assessed by Western blot. The distribution of FoxO1 in podocytes was detected by immunofluorescence. In addition, the TGF-β1 secretion in cell culture supernatants was analyzed by ELISA. Results Compared with group A, there was no significant difference in either mRNA or protein levels of FoxO1 in group B(both P>0.05), whereas the p-FoxO1/FoxO1 ratio markedly increased(P<0.05). The expressions of TGF-β1 and Smad3, as well as the ratio of p-Smad3/ Smad3 all increased(all P<0.05). The nephrin mRNA and protein levels decreased, with increased desmin levels in a time-dependent manner(all P<0.05). Compared with group B, the levels of FoxO1 mRNA and protein increased in group C but decreased in group D(all P<0.05). There was a significant decrease of p-FoxO1/FoxO1 ratio in group C(P<0.05). The expressions of TGF-β1, p-Smad3, and desmin decreased in group C but increased in group D, with the nephrin mRNA and protein levels elevated in group C, but reduced in group D(all P<0.05). Conclusion FoxO1 plays a crucial role in down-regulating the activation of TGF-β1 pathway and thereby ameliorating EMT of podocytes. (Chin J Endocrinol Metab, 2016, 32: 767-772) Key words: Forkhead transcription factors O1; Diabetic nephropathy; Podocyte; Epithelial-mesenchymal transition; Transforming growth factor-β1