Laboratory of Molecular and Cellular Nephrology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Wita Stwosza 63, 80-308 Gdańsk, Poland.
Faculty of Chemistry, Department of Molecular Biotechnology, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
J Biochem. 2020 Dec 26;168(6):575-588. doi: 10.1093/jb/mvaa059.
Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes.
足细胞是肾小球滤过屏障的主要组成部分，通过其收缩特性调节肾小球对白蛋白的通透性。胰岛素和高糖（HG）依赖性激活蛋白激酶 G 型 Iα（PKGIα）均可导致肌动蛋白细胞骨架重排和足细胞破坏。血管扩张刺激磷蛋白（VASP）是 PKGIα 的底物，参与肌动蛋白细胞骨架动力学的调节。我们研究了 PKGIα/VASP 途径在调节足细胞白蛋白通透性中的作用。我们评估了培养的大鼠足细胞单层中高胰岛素和/或 HG 诱导的跨上皮白蛋白通量的变化。通过 Western blot 和免疫荧光证实了 PKGIα 和下游蛋白的表达。我们证明胰岛素和 HG 通过 PKGIα 依赖性调节 VASP 磷酸化状态来改变足细胞的收缩装置，增加 VASP 与 PKGIα 的共定位，并改变这些蛋白在足细胞中的亚细胞定位。此外，VASP 参与了胰岛素和 HG 依赖性肌动蛋白细胞骨架的动态重塑，从而在高胰岛素血症和高血糖条件下增加了足细胞对白蛋白的通透性。这些结果表明，在培养的大鼠足细胞中，胰岛素和 HG 依赖性白蛋白通透性的调节是由 PKGIα/VASP 途径介导的。这种分子机制可能解释了糖尿病中的足细胞病变和白蛋白尿。
