Phosphorylation of the podocyte cytoskeleton in diabetic nephropathy

糖尿病肾病足细胞细胞骨架的磷酸化

基本信息

批准号：
10456149
负责人：
Di Feng
金额：
$ 13.13万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-23 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456149
关键词：
Actinin Actins Address Affect Animal Model Animals Award Biomedical Engineering Cell model Chronic Kidney Failure Collaborations Complications of Diabetes Mellitus Crosslinker Culture Media Cytoskeleton Data Defect Diabetes Mellitus Diabetic Nephropathy Disease Event Exposure to Filtration Focal Segmental Glomerulosclerosis Fostering Functional disorder Funding Future Genetic Glucose Goals Health Care Costs Human Hyperglycemia In Vitro Investigation Kidney Kidney Diseases Knowledge Lead Link Mass Spectrum Analysis Mechanical Stress Mechanics Mediating Mediator of activation protein Mentored Research Scientist Development Award Microfluidics Mission Modeling Morbidity - disease rate Mus Mutation National Institute of Diabetes and Digestive and Kidney Diseases Nature Pathogenesis Pathway interactions Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Population Postdoctoral Fellow Public Health Rattus Research Role Sampling Serine Stress Stretching Structure Techniques Technology Testing Tissues United States National Institutes of Health Work alpha Actinin blood filter cost diabetic in vivo in vivo Model innovation insight mortality mutant novel podocyte shear stress

项目摘要

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.

足细胞功能障碍是糖尿病性肾病发病机理的早期关键事件。足细胞依靠他们细胞骨架以保持其结构和功能，同时面对内部恒定的机械应力肾小球。 A-肌动蛋白4（ACTN4）是肌动蛋白细胞骨架的必不可少的交联链； ACTN4铅中的突变人类肾脏疾病。最近的证据已经确定了ACTN4在Serial处的一个重要的辐射事件（s）159。在磷酸化细胞和动物模型中，ACTN4的这种磷酸化与机械应力下的足细胞脆弱性。此外，S159处的野生型（WT）ACTN4的磷酸化为由高葡萄糖刺激并与细胞骨架进化相关，类似于相关的进化与引起疾病的突变体ACTN4。该R03应用程序进步的长期目标是了解细胞骨架在糖尿病性肾病基础的足细胞功能障碍中的作用。总体目标当前的建议是阐明高葡萄糖导致ACTN4磷酸化的途径足细胞脆弱性的潜在介体。中心假设是ACTN4的磷酸化增加由高葡萄糖刺激，并促进足细胞易受机械应力的脆弱性。理由项目是找到导致足细胞脆弱性的新途径可以填补与知识相关的关键空白为了实现此应用程序的总体目标，以下两个将追求具体目标。 AIM 1将定义ACTN4和糖尿病的磷酸化之间的关联体内肾病。野生型（WT）对照和糖尿病性肾病肾脏组织将从小鼠中获得老鼠和人类。靶向质谱法将用于量化所有的ACTN4磷酸化 AIM 2将在体外确定高葡萄糖介导的ACTN4磷酸化的影响。微流体将使用携带WT ACTN4或人足细胞的人口足细胞观察肾小球携带不可磷酸的S159A ACTN4。这些chip片将暴露于培养基含有高葡萄糖，同时受到机械拉伸和剪切应力的影响。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。拟议的研究很重要，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）在糖尿病性肾病的体内模型中进行了更新，并且（2）在机械压力下导致足细胞脆弱性的途径将为进一步的理由提供强有力的理由研究ACTN4作为糖尿病肾病发作和进展的基础机制的磷酸化。来自该R03的数据将支持R01，以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涉及的足细胞功能障碍提供新的机械见解糖尿病性肾病，以及减轻足细胞功能障碍的新型治疗方法的潜在靶标。