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，同时面临不断的机械应力内 肾小球α-辅肌动蛋白4（ACTN 4）是肌动蛋白细胞骨架的必需交联剂; ACTN 4的突变导致肌动蛋白细胞骨架的改变。 to human人类kidney肾脏disease疾病.最近的证据已经确定了一个重要的磷酸化事件，在ACTN 4的丝氨酸 (S)159.在拟磷酸化细胞和动物模型中，这种ACTN 4的磷酸化与 足细胞在机械应力下的脆弱性。此外，野生型（WT）ACTN 4在S159处的磷酸化是 受高糖刺激，并与细胞骨架紊乱相关，类似于 致病突变型ACTN 4此R 03应用程序推进的长期目标是了解 细胞骨架在糖尿病肾病足细胞功能障碍中的作用。的总体目标 目前的建议是阐明高葡萄糖导致ACTN 4磷酸化的途径， 足细胞脆弱性的潜在介质。核心假设是ACTN 4磷酸化水平的增加 高葡萄糖刺激并促进足细胞对机械应力的脆弱性。这样做的理由 该项目是，寻找导致足细胞脆弱性的新途径可以填补知识相关的关键空白 糖尿病肾病的发病机制。为了实现本申请的总体目标， 将追求具体目标。目的1将明确ACTN 4磷酸化与糖尿病的关系。 体内肾病。野生型（WT）对照和糖尿病肾病肾组织将从小鼠获得， 老鼠和人类。靶向质谱法将用于定量所有受试者中的ACTN 4磷酸化。 样品目的2将确定体外高糖介导的ACTN 4磷酸化的影响。微流体 将用携带WT ACTN 4的人足细胞或人足细胞接种芯片上的肾小球 携带不可磷酸化的S159 A ACTN 4。这些芯片上的肾小球将暴露于培养基中 含有高葡萄糖，同时受到机械拉伸和剪切应力。拟议的研究是 创新，因为它采用了最新的，最先进的技术来研究细胞骨架的磷酸化 和足细胞脆弱性。这项拟议中的研究意义重大，因为它将定义一种新的途径， 高葡萄糖通过足细胞细胞骨架的磷酸化介导足细胞功能障碍。 证明（1）该途径在糖尿病肾病体内模型中上调，并且（2）该途径 通路有助于足细胞在机械应力下的脆弱性，这将为进一步研究足细胞在机械应力下的脆弱性提供强有力的理由。 研究ACTN 4磷酸化作为糖尿病肾病发生和发展的潜在机制。 来自R 03的数据将支持R 01鉴定调节磷酸化的激酶和/或磷酸酶 ACTN4这项未来的工作不仅将提供新的机制的见解足细胞功能障碍参与 糖尿病肾病，而且是减轻足细胞功能障碍的新型治疗的潜在靶点。

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.