Luo Peng-Li, Wang Yan-Jun, Yang Yan-Yan, Yang Jia-Jia
Department of Nephrology, Hospital of Qinghai University, Xining, China.
Braz J Med Biol Res. 2018;51(7):e6201. doi: 10.1590/1414-431x20186201. Epub 2018 May 17.
This study aimed to investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia conditions. The expression level of HIF-2α and tight junction proteins (occludin and ZO-1) in rGENCs were examined following 5% oxygen density exposure at different treatment times. HIF-2α lentivirus transfection was used to knockdown HIF-2α expression. Cells were divided into four groups: 1) control group (rGENCs were cultured under normal oxygen conditions), 2) hypoxia group (rGENCs were cultured under hypoxic conditions), 3) negative control group (rGENCs were infected with HIF-2α lentivirus negative control vectors and cultured under hypoxic conditions), and 4) Len group (rGENCs were transfected with HIF-2α lentivirus and cultured under hypoxic conditions). The hypoxia, negative control, and Len groups were kept in a hypoxic chamber (5% O2, 5% CO2, and 90% N2) for 24 h and the total content of occludin and ZO-1, and the permeability of rGENCs were assessed. With increasing hypoxia time, the expression of HIF-2α gradually increased, while the expression of occludin decreased, with a significant difference between groups. ZO-1 expression gradually decreased under hypoxia conditions, but the difference between the 24 and 48 h groups was not significant. The permeability of cells increased following 24-h exposure to hypoxia compared to the control group (P<0.01). The knockdown of HIF-2α expression significantly increased occludin and ZO-1 content compared with hypoxia and negative control groups (P<0.01), while permeability was reduced (P<0.01). Hypoxia increased HIF-2α content, inducing permeability of rGENCs through the reduced expression of occludin and ZO-1.
本研究旨在探讨缺氧诱导因子-2α（HIF-2α）在缺氧条件下对大鼠肾小球内皮细胞（rGENCs）紧密连接蛋白表达及通透性改变中的作用。在不同处理时间进行5%氧浓度暴露后，检测rGENCs中HIF-2α和紧密连接蛋白（闭合蛋白和紧密连接蛋白1）的表达水平。采用HIF-2α慢病毒转染来敲低HIF-2α表达。细胞分为四组：1）对照组（rGENCs在正常氧条件下培养），2）缺氧组（rGENCs在缺氧条件下培养），3）阴性对照组（rGENCs感染HIF-2α慢病毒阴性对照载体并在缺氧条件下培养），4）慢病毒组（rGENCs用HIF-2α慢病毒转染并在缺氧条件下培养）。将缺氧组、阴性对照组和慢病毒组置于缺氧箱（5% O₂、5% CO₂和90% N₂）中24小时，评估闭合蛋白和紧密连接蛋白1的总含量以及rGENCs的通透性。随着缺氧时间增加，HIF-2α表达逐渐升高，而闭合蛋白表达降低，组间差异显著。缺氧条件下紧密连接蛋白1表达逐渐降低，但24小时和48小时组间差异不显著。与对照组相比，缺氧暴露24小时后细胞通透性增加（P<0.01）。与缺氧组和阴性对照组相比，敲低HIF-2α表达显著增加了闭合蛋白和紧密连接蛋白1的含量（P<0.01），同时通透性降低（P<0.01）。缺氧增加了HIF-2α含量，通过降低闭合蛋白和紧密连接蛋白1的表达诱导rGENCs的通透性。
