Piwkowska Agnieszka, Rogacka Dorota, Kasztan Małgorzata, Angielski Stefan, Jankowski Maciej
Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdańsk, Poland.
Biochim Biophys Acta. 2013 Jun;1832(6):791-804. doi: 10.1016/j.bbadis.2013.02.011. Epub 2013 Feb 21.
The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.
肾小球滤过屏障对白蛋白通透性增加是糖尿病微血管病变的一个众所周知的特征，也是血管并发症的一个不良预后因素。然而，其潜在机制尚未完全明确。我们最近证明，超氧阴离子的产生会增加Iα型蛋白激酶G（PKGIα）亚基的二聚化，导致足细胞功能障碍。在此，我们研究了高胰岛素浓度是否参与糖尿病肾小球滤过屏障的PKGI依赖性高通透性。我们通过测量Wistar大鼠、肥胖和瘦型Zucker大鼠分离肾小球中肾小球毛细血管对白蛋白的通透性以及培养的大鼠足细胞中的跨膜白蛋白通量，评估胰岛素诱导的肾小球通透性变化。使用蛋白质印迹法和免疫荧光法在足细胞中证实了PKGIα及其上游蛋白的表达。胰岛素（300nM，5分钟）增加了Wistar大鼠中NAD(P)H依赖性肾小球白蛋白通透性以及培养足细胞中PKGI依赖性跨膜白蛋白通量。在非还原条件下，足细胞暴露于胰岛素会增加PKGIα蛋白间二硫键的形成，改变PKG靶蛋白MYPT1和MLC的磷酸化，并破坏肌动蛋白细胞骨架。用NOX2和NOX4小干扰RNA证实了NADPH氧化酶（NOX）在胰岛素诱导的活性氧（ROS）生成以及胰岛素引起的足细胞白蛋白通透性增加中的作用。高胰岛素血症的Zucker肥胖大鼠的肾小球白蛋白通透性增加，分离的肾小球显示PKGIα和NOX4表达增加。综上所述，这些数据表明胰岛素通过涉及NAD(P)H依赖性超氧阴离子生成的PKGI依赖性机制增加肾小球屏障白蛋白通透性。这些发现揭示了胰岛素在糖尿病肾小球肾病病理生理学中的作用。
