缺氧肾小管上皮细胞Rab7/Caveolin-1/MMP-2轴失调促进肾纤维化

Dysregulation of the Rab7/Caveolin-1/MMP-2 axis in hypoxic tubular epithelial cells promotes renal fibrosis

目的:研究肾纤维化晚期Ras相关GTP结合蛋白7(Ras-related GTP binding protein 7,Rab7)可否通过小窝蛋白-1(Caveolin-1)的介导调节基质金属蛋白酶-2(matrix metalloproteinase-2, MMP-2)的活性,从而对肾纤维化产生影响。方法:通过单侧输尿管结扎(unilateral ureteral obstruction, UUO)法构建C57BL/6小鼠肾纤维化模型,检测肾纤维化组织中缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)、Rab7和Caveolin-1的表达以及MMP-2的活性。利用肾小管上皮细胞株(HK-2)进行体外实验,构建HK-2 Rab7-shRNA细胞,将HK-2和HK-2 Rab7-shRNA细胞分别进行常氧和缺氧培养,并检测各组中HIF-1α、Rab7、Caveolin-1的表达和MMP-2的活性,以及纤维化相关指标I型胶原(Collagen-1)、波形蛋白(Vimentin)和α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)的表达。结果:利用小鼠UUO模型发现,相较正常肾脏组织,肾纤维化组织中Rab7和Caveolin-1的表达均上调,而MMP-2的活性反而下降。进一步体外实验显示,在HK-2细胞中Rab7下调后可通过抑制Caveolin-1的表达进而提高MMP-2的活性,并会使Collagen-1、Vimentin和α-SMA等反映纤维化的指标表达下调。结论:Rab7与MMP-2的活性密切相关;Rab7对MMP-2活性的调节通过Caveolin-1介导;当Rab7表达受抑后,可促进肾纤维化的缓解。

Objective: To study whether Ras-related GTP binding protein 7(Rab7) can regulate the activity of matrix metalloproteinase-2(MMP-2) through the mediation of Caveolin-1 in the advanced stage of renal fibrosis, thereby affecting renal fibrosis. Methods: The renal fibrosis model of C57BL/6 mice was established by unilateral ureteral obstruction(UUO) method, and the expressions of hypoxia inducible factor-1α(HIF-1α), Rab7, Caveolin-1 and the activity of MMP-2 were detected in renal fibrosis tissues.The renal tubular epithelial cell line(HK-2) was used for in vitro experiments. First, HK-2 Rab7-shRNA cells were constructed. HK-2 and HK-2 Rab7-shRNA cells were cultured in normoxia and hypoxia respectively, and HIF-1α, Rab7, Caveolin-1 expression and MMP-2 activity were detected in each group. Then the expression of fibrosis related indicators, such as Collagen-1, Vimentin and α-smooth muscle actin(α-SMA) were also detected. Results: Using the mouse UUO model, it was found that the expression of Rab7 and Caveolin-1 were both upregulated in renal fibrosis tissues compared with normal kidney tissues, while the activity of MMP-2 was decreased. Further in vitro experiments revealed that Rab7 down-regulation in HK-2 cells could increase the activity of MMP-2 in HK-2 supernatant by inhibiting Caveolin-1 expression, and down-regulate the expression of Collagen-1, Vimentin and α-SMA. Conclusion: Rab7 is closely related to MMP-2 activity, and the regulation of MMP-2 activity by Rab7 is mediated by Caveolin-1. When the expression of Rab7 is inhibited, it can promote the remission of renal fibrosis.