Chronic hypoxia induced abnormal autophagy of tubular epithelial cells through activation of HIF-1α/microRNA-96 signaling pathway invovled in renal interstitial fibrosis
Objective:To study the mechanism of chronic hypoxia induced renal fibrosis.Methodology:Hypoxia induced renal tubular epithelial cells,the expression of HIF-1α,autophagy key molecules LC3Ⅰ/Ⅱ and P62 were measured by western blot,and microRNA-96(miR-96)was measured by PCR.HIF-1α siRNA and pcDNA-HIF-1α were transfected into renal tubular depithelial cells lines(HK-2)induced by hypoxia,and PCR was used to measure the expression of miR-96,and explore whether HIF-1α up-regulation affected the expression of miR-96.miR-96 mimic was transfected into HK-2 cells induced by normoxia,and western blot was used to measure the expression of fibrotic factor α-smooth muscle actin(α-SMA),Collagen4A1(COL4A1)and autophagy key molecules LC3Ⅰ/Ⅱ and P62.Mice were injected intraperitoneally with the viral vector of miR-96 inhibitor.The degree of renal fibrosis and the expression of fibrosis factor COL4A1 was detected by Masson staining.Results:Compared with normoxia group,hypoxia group showed significant increase of HIF-1α and miR-96(P＜0.05),and increase of autophagy key molecule LC3Ⅰ/Ⅱ and down-regulation of P62(P＜0.05).HIF-1α siRNA and pcDNA-HIF-1α were transfected into HK-2 induced by hypoxia,found that HIF-1α can promote the expression of miR-96.The up-regulation of miR-96 significantly increased the expression of fibrotic factor α-SMA and COL4A1 in normoxia condition,and increase of autophagy key molecule LC3Ⅰ/Ⅱ and down-regulation of P62(P＜0.05).The down-regulation of miR-96 significantly restrain the expression of LC3Ⅰ/Ⅱ and regain expression of P62(P＜0.05).Mice injected with the viral vector of miR-96 inhibitor intraperitoneally can inhibit the degree of renal fibrosis and the expression of the fibrosis factor COL4A1 in the(Unilateral Ureteral Obstruction,UUO)model compared with mice injected with the control virus.Conclusion:HIF-1α/miR-96 signaling pathway induced abnormal autophagy of tubule epithelial cells,which caused the increase of pro-fibrotic factors and promotes renal fibrosis.
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