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

修改项目摘要/摘要部分 足细胞病是一组肾小球疾病，影响肾脏的过滤血液的能力，往往导致肾衰竭。健康的足细胞覆盖着肾小球毛细血管，有数千个称为足突的延伸部分，这些延伸部分相互交错，并通过严格调节肌动蛋白细胞骨架来维持其精致的细胞形状。足细胞以典型的方式通过经历足突消失（足细胞形态的急剧转变和复杂足突的消失）来响应损伤，所述足突消失通常与“肌动蛋白垫（actin mat）”相关，所述肌动蛋白垫是在消失区域底部的肌动蛋白凝聚物。我们最近使用超分辨率成像来研究健康和患病条件下的3D足细胞肌动蛋白细胞骨架。我们发现，健康的足细胞足突包含非收缩性肌动蛋白电缆，而收缩性电缆保持在细胞体的高度。与此相反，受伤的足细胞似乎有收缩性肌动蛋白电缆在擦除区并列的肾小球基底膜（GBM），表明在损伤后的肌动蛋白电缆的空间分布的转变。该提案的总体目标是确定调节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性肌动蛋白电缆在损伤后向邻近GBM的擦除区域转移的变化的性质。在目的1中，我们将研究两种同工肌动蛋白，β和γ肌动蛋白，在足细胞病理生物学的作用。足细胞表达高水平的这些几乎相同的进化保守的isoactins。虽然β肌动蛋白在非肌肉细胞中被认为是主要的异肌动蛋白，从胚胎死亡时失活，γ肌动蛋白的作用仍然是难以捉摸的。我们将使用各种肾脏疾病小鼠模型，包括γ-肌动蛋白基因敲除小鼠，来回答关于两种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原代足细胞，因为它们分散到一个基板上的微图案化的水凝胶分离的肾小球。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，并将更多地揭示肌动蛋白垫在消失足细胞中的命运。这将有助于我们确定Rho小GTP酶及其下游效应物，formins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的原肌球蛋白亚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。我们推测，在受伤的足细胞原肌球蛋白组成的变化，导致异位外观的收缩肌动蛋白电缆在抹去的地区，这反过来又是由不同的formins。了解原肌球蛋白如何调节各种类型的肌动蛋白电缆可以提供缺失的环节足细胞足突消失。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.