Molecular Determinants of Kidney Podocyte Architecture in Health, Injury, and Recovery

健康、损伤和恢复中肾足细胞结构的分子决定因素

• 批准号: 10522754
• 负责人: Hani Suleiman
• 金额: $ 34.65万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10522754
• 关键词: 3-Dimensional; Actin-Binding Protein; Actins; Actomyosin; Affect; Appearance; Architecture; Area; Biology; Cell Culture Techniques; Cell Shape; Cells; Culture Techniques; Cytoskeleton; Data; Diabetic Nephropathy; Disease; Electron Microscopy; Embryo; Epithelial Cells; Equilibrium; Evolution; Extracellular Matrix; Focal Segmental Glomerulosclerosis; Foot Process; Genes; Genetic Models; Glomerular Capillary; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Homeostasis; Hydrogels; Image; Imaging Techniques; Imaging technology; Injury; Isoactin; Kidney; Kidney Diseases; Kidney Failure; Kidney Glomerulus; Knockout Mice; Labyrinth; Lead; Light; Link; Machine Learning; Mechanics; Membrane; Methods; Microfilaments; Microscopy; Modeling; Molecular; Monomeric GTP-Binding Proteins; Morphology; Mus; Nature; Nonmuscle Myosin Type IIA; Optics; Pathway interactions; Physical condensation; Play; Process; Protein Isoforms; Recovery; Regulation; Renal function; Renal glomerular disease; Resolution; Role; Sarcomeres; Spatial Distribution; Specific qualifier value; Structure; System; Techniques; Testing; Thick; Three-Dimensional Imaging; Tropomyosin; Western Blotting; Work; base; beta Actin; biological systems; biophysical properties; blood filter; blood filtration; cell type; design; gamma Actin; genetic variant; glomerular basement membrane; glomerular filtration; imaging approach; injured; injury recovery; mouse genetics; mouse model; novel; novel therapeutic intervention; podocyte; reconstruction; response to injury; rho; rho GTP-Binding Proteins; slit diaphragm; transcriptome sequencing

Modified Project Summary/Abstract Section The podocytopathies are a group of glomerular diseases that affect the kidney’s ability to filter the blood and often lead to kidney failure. Healthy podocytes cover the glomerular capillaries with thousands of extensions called foot processes that interdigitate with one another and maintain their elaborate cell shape by tightly regulating their actin cytoskeleton. Podocytes respond to insults in a typical fashion by undergoing foot process effacement, a dramatic shift in podocyte morphology and the disappearance of the intricate foot processes, which often associates with the “actin mat”, an actin condensation at the bottom of the effaced areas. We recently used super-resolution imaging to study the podocyte actin cytoskeleton in 3D in both healthy and diseased conditions. We showed that healthy podocyte foot processes contain non-contractile actin cables, while contractile cables are maintained high in the cell bodies. In contrast, injured podocytes appear to have contractile actin cables in effacement areas juxtaposed to the glomerular basement membrane (GBM), indicating a shift in the spatial distribution of actin cables after injury. The overall goal of this proposal is to define the molecular mechanisms that regulate the various types of actin cables in podocytes and the nature of the changes that cause the contractile actin cables in the cell body to shift towards the effaced areas adjacent to the GBM after injury. In Aim 1, we will investigate the roles of the two isoactins, beta and gamma actin, in podocyte pathobiology. Podocytes express high levels of these almost-identical evolutionally-conserved isoactins. While beta actin in non-muscle cells is considered the main isoactin, as evident from the embryonic lethality when inactivated, the role of gamma actin is still elusive. we will use various kidney disease mouse models, including the gamma-actin knockout mouse, to answer some fundamental questions about the role of the two isoactins in podocyte biology. Furthermore, we will utilize a novel technique to study primary podocytes as they spread out of isolated kidney glomeruli onto a substrate-micropatterned hydrogel. This approach will allow us to study the dynamic changes in the actin cytoskeleton in injured podocytes and will shed more light on the fate of the actin mats in effaced podocytes. It will help us identifying the role of Rho small GTPases and its downstream effectors, formins, in the actin mat formation. In Aim 2, we will study the tropomyosin isoform composition in podocytes and their roles in specifying the spatial distribution of different types of actin cables in the kidney podocytes. We hypothesize that changes in tropomyosin composition in injured podocytes causes the ectopic appearance of contractile actin cables in the effaced areas, and this, in turn, is regulated by different formins. Understanding how tropomyosins regulate the various types of actin cables could provide the missing link to podocyte foot process effacement. Our goal is to expand our understanding of the molecular mechanisms that regulate the composition and dynamics of the actin cytoskeleton, a step that will help us in designing novel therapeutic approaches to directly impact podocyte foot process architecture and help cure kidney glomerular diseases.

修改的项目摘要/摘要部分 足细胞病是一组肾小球疾病，会影响肾脏过滤血液并经常导致肾衰竭的能力。健康的足细胞覆盖了肾小球毛细血管，具有数千个称为脚步过程的扩展，它们相互互相互相互相互相，并通过紧密地调节其肌动蛋白细胞骨架来保持其精致的细胞形状。足细胞通过脚步过程能量，足细胞形态的急剧转移以及复杂的脚部过程的消失，以典型的方式反应侮辱，这通常与“肌动蛋白垫”（肌动蛋白垫子）相关联，肌动蛋白的凝结是侵蚀区域的底部。我们最近使用超分辨率成像研究了健康和解散条件下3D中的足细胞肌动蛋白细胞骨架。我们表明，健康的足细胞脚步过程中包含非收缩肌动蛋白电缆，而收缩电缆在细胞体中保持较高。相比之下，受伤的足细胞似乎在能量区域中有收缩肌动蛋白电缆并置与肾小球基底膜（GBM），表明受伤后肌动蛋白电缆的空间分布发生了变化。该提案的总体目标是定义调节足细胞中各种类型肌动蛋白电缆的分子机制，以及导致细胞体内收缩肌动蛋白电缆的变化的性质，向受伤后的GBM邻近的eSD型区域转移。在AIM 1中，我们将研究两个同肌动物β和伽马肌动蛋白在足细胞病理生物学中的作用。足细胞表达了这些几乎相同的进化同性恋蛋白的高水平。虽然非肌肉细胞中的β肌动蛋白被认为是主要的同肌肌动蛋白，但在灭活时胚胎致死性证明，γ肌动蛋白的作用仍然难以捉摸。我们将使用各种肾脏疾病小鼠模型，包括γ-肌动蛋白敲除小鼠，以回答有关两种同肌蛋白在足细胞生物学中的作用的一些基本问题。此外，我们将利用一种新技术来研究主要的足细胞，因为它们是从孤立的肾脏glomerulli散布到底物 - 麦克罗胶质图水凝胶上的。这种方法将使我们能够研究受伤的足细胞中肌动蛋白细胞骨架的动态变化，并将更多地了解脱落的足细胞中肌动蛋白垫的命运。它将帮助我们确定Rho小GTPases及其下游效应在肌动蛋白垫形成中的作用。在AIM 2中，我们将研究足细胞中的肌球蛋白同工型组成及其在指定肾脏足细胞中不同类型肌动蛋白电缆的空间分布中的作用。我们假设损伤足细胞中横肌球蛋白组成的变化会导致收缩肌动蛋白电缆在有效区域的异位外观，而这反过来又受到不同形式的调节。了解肌动蛋白如何调节各种类型的肌动蛋白电缆可能会提供与足细胞脚步能量的缺失链接。我们的目标是扩展我们对调节肌动蛋白细胞骨架组成和动力学的分子机制的理解，该步骤将有助于我们设计新型的热方法，以直接影响Podocyte脚步过程结构并帮助治疗肾脏肾小球疾病。