Guo Jia, Yang Lili, Qiao Yingjin, Liu Zhangsuo
Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China.
Mol Med Rep. 2016 Nov;14(5):4091-4098. doi: 10.3892/mmr.2016.5786. Epub 2016 Sep 26.
Epithelial-mesenchymal transition (EMT) is important for diabetic nephropathy (DN). Podocytes are specialized epithelial cells, which form a major component of the glomerular filtration barrier. Podocyte damage has been suggested to be the primary mechanism behind the albuminuria associated with DN. The present study aimed to determine the function of glycogen synthase kinase (GSK)‑3β in EMT and barrier dysfunction of mouse podocytes exposed to high glucose (HG) conditions. Matured and differentiated podocytes were treated with normal glucose (NG), HG or NG + mannitol. Podocytes were also transfected with a small interfering RNA (siRNA) against GSK‑3β or a scrambled siRNA, or were treated with lithium chloride (LiCl), a GSK‑3β inhibitor, under NG or HG conditions. The expression levels of the epithelial cell markers, nephrin and podocin, and the myofibroblast cell markers, α‑smooth muscle actin (SMA) and fibronectin, in podocytes by western blot analysis and immunofluorescence staining, respectively. The monolayer barrier function was assessed by albumin inflow. The phosphorylation and activity levels of GSK‑3β were also quantified. It was observed that HG promotes EMT in podocytes, due to the increased levels of podocin and nephrin expression and the reduced α‑SMA and fibronectin expression levels. HG also induced barrier dysfunction and increased the expression level of total GSK‑3β, Try216‑phosphorylated‑GSK‑3β and the GSK‑3β activity in podocytes. Transfection of GSK‑3β siRNA or treatment with LiCl reversed the HG‑induced EMT and barrier dysfunction in podocytes. In conclusion, the present study determined that GSK‑3β is required for EMT and barrier dysfunction in podocytes under HG conditions; therefore, GSK‑3β may be a novel target for the treatment of DN.
上皮-间质转化(EMT)在糖尿病肾病(DN)中起重要作用。足细胞是特殊的上皮细胞,构成肾小球滤过屏障的主要成分。足细胞损伤被认为是DN相关蛋白尿的主要机制。本研究旨在确定糖原合酶激酶(GSK)-3β在暴露于高糖(HG)条件下的小鼠足细胞EMT和屏障功能障碍中的作用。将成熟分化的足细胞用正常葡萄糖(NG)、HG或NG + 甘露醇处理。在NG或HG条件下,足细胞还转染针对GSK-3β的小干扰RNA(siRNA)或乱序siRNA,或用GSK-3β抑制剂氯化锂(LiCl)处理。分别通过蛋白质印迹分析和免疫荧光染色检测足细胞中上皮细胞标志物nephrin和podocin以及肌成纤维细胞标志物α-平滑肌肌动蛋白(SMA)和纤连蛋白的表达水平。通过白蛋白流入评估单层屏障功能。还对GSK-3β的磷酸化和活性水平进行了定量。观察到HG促进足细胞的EMT,这是由于podocin和nephrin表达水平增加以及α-SMA和纤连蛋白表达水平降低。HG还诱导屏障功能障碍,并增加足细胞中总GSK-3β、Try216磷酸化GSK-3β的表达水平和GSK-3β活性。转染GSK-3β siRNA或用LiCl处理可逆转HG诱导的足细胞EMT和屏障功能障碍。总之,本研究确定GSK-3β是HG条件下足细胞EMT和屏障功能障碍所必需的;因此,GSK-3β可能是治疗DN的新靶点。