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

修改后的项目摘要/摘要部分 足细胞病变是一组影响肾脏过滤血液能力的肾小球疾病，通常会导致肾功能衰竭。健康的足细胞覆盖着肾小球毛细血管，有数千个被称为足突的延伸，这些足突相互交错，并通过严格调节其肌动蛋白细胞骨架来保持其精细的细胞形状。足细胞以一种典型的方式对侮辱做出反应，经历脚突消失，足细胞形态发生戏剧性变化，错综复杂的足突消失，这通常与“肌动蛋白垫”有关，肌动蛋白凝聚在消失区域的底部。我们最近使用超分辨率成像技术研究了健康和疾病条件下足细胞肌动蛋白细胞骨架的三维结构。我们发现，健康的足细胞足突含有非收缩肌动蛋白电缆，而收缩电缆在细胞体中保持在较高的位置。相反，受损足细胞在与肾小球基底膜(GBM)并列的消失区中似乎有收缩肌动蛋白电缆，这表明损伤后肌动蛋白电缆的空间分布发生了变化。这项建议的总体目标是确定调控足细胞中各种类型的肌动蛋白电缆的分子机制，以及导致细胞体中的收缩肌动蛋白电缆在损伤后向邻近基底膜的消失区域移动的变化的性质。在目标1中，我们将研究两个等肌动蛋白，β和γ肌动蛋白，在足细胞病理生物学中的作用。足细胞表达这些几乎相同的进化保守的同种肌动蛋白的水平很高。尽管非肌肉细胞中的β肌动蛋白被认为是主要的等肌动蛋白，从失活时对胚胎的致死性可以明显看出，伽马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，包括伽马肌动蛋白基因敲除小鼠，来回答关于这两个异肌动蛋白在足细胞生物学中的作用的一些基本问题。此外，我们将利用一种新的技术来研究初级足细胞，当它们从分离的肾小球扩散到底物微图案水凝胶上时。这种方法将使我们能够研究受损足细胞中肌动蛋白细胞骨架的动态变化，并将更多地揭示消失的足细胞中肌动蛋白垫子的命运。这将有助于我们确定Rho小GTP酶及其下游效应因子Forins在肌动蛋白垫形成中的作用。在目标2中，我们将研究足细胞中原肌球蛋白的异构体组成及其在确定不同类型的肌动蛋白缆线在肾足细胞中的空间分布中的作用。我们推测，损伤足细胞中原肌球蛋白组成的变化导致收缩肌动蛋白电缆在消失的区域异位出现，而这反过来又受到不同的福尔马林的调节。了解原肌球蛋白是如何调节各种类型的肌动蛋白电缆的，可以为足细胞足突消失提供缺失的一环。我们的目标是扩大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，这一步骤将有助于我们设计新的治疗方法，直接影响足细胞足突结构，并帮助治愈肾小球疾病。

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.