Phosphorylation of the podocyte cytoskeleton in diabetic nephropathy

足细胞功能障碍是糖尿病肾病发病机制中的早期关键事件。足细胞依靠它们的 细胞骨架，以维持其结构和功能，同时面对内部恒定的机械应力 肾小球。α-肌动蛋白4(ACTN4)是肌动蛋白细胞骨架的重要交联剂；ACTN4基因突变 人类的肾脏疾病。最近的证据已经确定了ACTN4丝氨酸的一个重要的磷酸化事件 (S)159.在仿磷细胞和动物模型中，ACTN4的这种磷酸化与 足细胞在机械应力下的脆弱性。此外，野生型(WT)ACTN4在S159位的磷酸化是 受高糖刺激并与细胞骨架紊乱相关，类似于相关的紊乱 致病突变株ACTN4。这个R03应用程序推进的长期目标是理解 细胞骨架在糖尿病肾病基础足细胞功能障碍中的作用。《公约》的总体目标 目前的建议是阐明高糖导致ACTN4作为一种 足细胞脆弱性的潜在调停者。中心假说是ACTN4的磷酸化增加 在高糖刺激下，足细胞容易受到机械应力的影响。这样做的理由是 项目是找到导致足细胞漏洞的新途径，可以填补相关知识的关键空白 对糖尿病肾病的发病机制有一定的影响。要实现此应用程序的总体目标，请使用以下两个 将追求具体的目标。目标1将确定ACTN4的磷酸化与糖尿病之间的联系 活体肾病。野生型(WT)对照和糖尿病肾病肾组织将从小鼠身上获得， 老鼠，还有人类。靶向质谱学将被用来量化所有 样本。目的2体外研究高糖对ACTN4磷酸化的影响。微流控 芯片上的肾小球将种植携带WT ACTN4的人足细胞或人足细胞 携带非磷酸化的S159A ACTN4。这些芯片上的肾小球将暴露在培养介质中 在机械拉伸和剪切应力作用下含有高葡萄糖的。拟议的研究是 具有创新性，因为它采用了最新、最先进的技术来研究细胞骨架的磷酸化 和足细胞的脆弱性。这项拟议的研究具有重要意义，因为它将定义一种新的途径，通过 高糖通过足细胞骨架的磷酸化介导足细胞功能障碍。 证明(1)这一途径在糖尿病肾病的体内模型中上调，以及(2)这 在机械应力下导致足细胞脆弱性的途径将为进一步 研究ACTN4的磷酸化作为糖尿病肾病发生和发展的机制。 来自R03的数据将支持R01来识别调节磷酸化的激酶和/或磷酸酶 ACTN4。这项未来的工作不仅将为足细胞功能障碍提供新的机制见解， 糖尿病肾病，但也是缓解足细胞功能障碍的新疗法的潜在靶点。

Podocyte dysfunction is an early, key event in the pathogenesis of diabetic nephropathy. Podocytes rely on their cytoskeleton to maintain their structure and function while facing constant mechanical stress inside the glomerulus. a-Actinin 4 (ACTN4) is an essential crosslinker of the actin cytoskeleton; mutations in ACTN4 lead to human kidney disease. Recent evidence has identified an important phosphorylation event in ACTN4 at serine (S) 159. In phosphomimetic cellular and animal models, this phosphorylation of ACTN4 is associated with podocyte vulnerability under mechanical stress. Moreover, phosphorylation of wild type (WT) ACTN4 at S159 is stimulated by high glucose and associated with cytoskeletal derangements, similar to derangements associated with disease-causing mutant ACTN4. The long-term goal that this R03 application advances is to understand the cytoskeleton’s role in podocyte dysfunction underlying diabetic nephropathy. The overall objective of the current proposal is to elucidate the pathway by which high glucose leads to phosphorylation of ACTN4 as a potential mediator of podocyte vulnerability. The central hypothesis is that increased phosphorylation of ACTN4 is stimulated by high glucose and fosters podocyte vulnerability to mechanical stress. The rationale for this project is that finding new pathways leading to podocyte vulnerability could fill critical gaps in knowledge related to the pathogenesis of diabetic nephropathy. To attain the overall objective of this application, the following two specific aims will be pursued. Aim 1 will define the association between phosphorylation of ACTN4 and diabetic nephropathy in vivo. Wild type (WT) control and diabetic nephropathy kidney tissue will be obtained from mice, rats, and humans. Targeted mass spectrometry will be used to quantify ACTN4 phosphorylation across all samples. Aim 2 will determine the impact of high glucose-mediated ACTN4 phosphorylation in vitro. Microfluidic glomeruli-on-chips will be seeded with either human podocytes carrying WT ACTN4 or human podocytes carrying nonphosphorylatable S159A ACTN4. These glomeruli-on-chips will be exposed to culture media containing high glucose while subjected to mechanical stretch and shear stress. The proposed research is innovative since it employs the latest, state-of-the art techniques to study phosphorylation of the cytoskeleton and podocyte vulnerability. The proposed research is significant because it will define a novel pathway by which high glucose mediates podocyte dysfunction through phosphorylation of the podocyte cytoskeleton. Demonstrating that (1) this pathway is upregulated in in vivo models of diabetic nephropathy and that (2) this pathway contributes to podocyte vulnerability under mechanical stress will provide strong justification to further study phosphorylation of ACTN4 as a mechanism underlying the onset and progression of diabetic nephropathy. Data from this R03 will support an R01 to identify the kinases and/or phosphatases that regulate phosphorylation of ACTN4. This future work will not only provide new mechanistic insights into podocyte dysfunction involved in diabetic nephropathy, but also potential targets for novel treatments that mitigate podocyte dysfunction.