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：
10287650
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 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10287650
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肾病发病机制中的一个早期关键事件，其依赖于足细胞。细胞骨架维持其结构和功能，同时面临内部持续的机械应力肾小球肌动蛋白 4 (ACTN4) 是肌动蛋白细胞骨架的重要交联剂；最近的证据已经确定了 ACTN4 丝氨酸的一个重要磷酸化事件。 (S) 159. 在拟磷细胞和动物模型中，ACTN4 的磷酸化与此外，野生型 (WT) ACTN4 在 S159 处磷酸化。受高葡萄糖刺激并与细胞骨架紊乱相关，类似于相关紊乱 R03应用推进的长期目标是了解致病突变ACTN4。细胞骨架在糖尿病肾病足细胞功能障碍中的作用。目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调节因素核心假设是 ACTN4 磷酸化增加。受到高葡萄糖的刺激并促进足细胞对机械应力的脆弱性。该项目的目的是寻找导致足细胞脆弱性的新途径可以填补相关知识的关键空白为了达到本申请的总体目的，以下两点阐述了糖尿病肾病的发病机制。将追求具体目标目标 1 将确定 ACTN4 磷酸化与糖尿病之间的关联。野生型（WT）对照和糖尿病肾病肾组织将从小鼠获得，靶向质谱法将用于量化所有大鼠和人类的 ACTN4 磷酸化。目标 2 将确定高葡萄糖介导的 ACTN4 磷酸化的体外影响。肾小球芯片将接种携带 WT ACTN4 的人足细胞或人足细胞携带不可磷酸化的 S159A ACTN4 的这些肾小球芯片将暴露于培养基中。含有高葡萄糖，同时受到机械拉伸和剪切应力。创新，因为它采用最新、最先进的技术来研究细胞骨架的磷酸化拟议的研究意义重大，因为它将定义一条新的途径，通过该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) 该通路在糖尿病肾病体内模型中上调，并且 (2) 该通路途径有助于足细胞在机械应力下的脆弱性，这将为进一步研究提供强有力的理由研究 ACTN4 磷酸化作为糖尿病肾病发病和进展的机制。来自 R03 的数据将支持 R01 识别调节磷酸化的激酶和/或磷酸酶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，也是减轻足细胞功能障碍的新疗法的潜在目标。