Characterization of a novel protein required for glomerular podocyte function

描述(申请人提供)：足细胞是肾脏的特殊细胞，参与过滤血液中的废物。然而，在各种肾病综合征中，这些细胞受损，其足突脱离肾小球基底膜(GBM)。这种脱落被认为是蛋白尿等疾病症状的主要原因，在许多情况下最终会导致肾衰竭。因此，了解足细胞如何形成并保持其足突与基底膜的附着是一个重要的研究领域。这项提议中的实验旨在表征参与维持这些附着的分子，并可能导致各种肾脏疾病的新治疗靶点。这些目标直接关系到NIH/NIDDK通过新的研究改善人类健康的使命，在这种情况下，特别是与肾功能和疾病有关的研究。足细胞-基底膜附着因子(PGAF)是一种新的足细胞足突与基底膜附着所必需的蛋白质。它是Wilms的肿瘤抑制因子-1(WT1)的下游靶点，并与半胱氨酸蛋白酶Calain的小亚基Capnsi结合。由于活性钙蛋白酶促进细胞粘连的脱离，目前的假说是PGAF抑制足细胞中的钙蛋白酶活性，以防止足突脱离。这项建议的目的是使用体外和体内技术相结合的方法来研究PGAF和钙调蛋白在维持足细胞与基底膜附着中的作用。体内研究将在斑马鱼和老鼠模型的组合上进行。由于斑马鱼胚胎肾脏为研究足细胞功能提供了一个简单、快速和易于操作的系统，因此我们将在该系统中进行PGAF和Calain功能的初步体内分析。基于吗啡的基因敲除和mRNA过度表达等技术将被用来扰乱胚胎的基因功能。对足细胞的电子显微镜分析和肾小球滤过能力的评估将用于分析由此产生的突变胚胎。此外，将使用类似的技术产生和评估PGAF功能丧失的小鼠。体外分析将包括蛋白质相互作用的生化特征和PGAF在培养的小鼠足细胞中的下调/过表达。与公共健康相关：肾脏通过使用称为足细胞的特殊细胞来过滤血液中的废物。然而，这些细胞可能会因糖尿病或各种肾脏疾病等疾病而受损。我们的研究将检查足细胞是如何形成和维持的，这将有助于我们了解疾病期间它们发生了什么，并可能导致新的治疗方法。

DESCRIPTION (provided by applicant): Podocytes are specialized cells of the kidney involved in filtering waste products from the blood. However, in various nephrotic syndromes, these cells become compromised and their foot processes detach from the underlying glomerular basement membrane (GBM). This detachment is thought to be the major cause of disease symptoms such as proteinuria and will eventually lead to kidney failure in many cases. Understanding how the podocytes form and retain their foot process attachments to the GBM is therefore an important area of research. The experiments in this proposal are designed to characterize molecules involved in maintaining these attachments, and may lead to novel therapeutic targets for various kidney diseases. These goals directly relate to the mission of the NIH/NIDDK to improve human health through novel research, in this case specifically related to renal function and disease. Podocyte-GBM attachment factor (PGAF) is a novel protein required for the attachment of podocyte foot processes to the GBM. It is a downstream target of Wilms' tumor suppressor-1 (WT1) and binds capnsi, the small subunit of the cysteine protease calpain. As active calpains promote detachment of cellular adhesions, the current hypothesis is that PGAF inhibits calpain activity in podocytes to prevent foot process detachment. The aims of this proposal are to use a combination of in vitro and in vivo techniques to investigate the role of PGAF and calpains in the maintenance of podocyte attachments to the GBM. In vivo studies will be conducted on a combination of zebrafish and mouse models. As the embryonic zebrafish kidney provides a simple, rapid and easily manipulatable system in which to study podocyte function, our primary in vivo analysis of PGAF and calpain function will be carried out in this system. Techniques including morpholino-based gene knockdown and mRNA over-expression will be used to perturb gene function in embryos. Electron microscopy analysis of podocytes and assessment of glomerular filtration ability will be used to analyze the resulting mutant embryos. In addition, PGAF loss-of-function mice will be generated and assessed using similar techniques. In vitro analysis will involve biochemical characterization of protein interactions and knockdown/overexpression of PGAF in cultured murine podocyte cells. Relevance to public health: The kidney filters waste from the blood by using specialized cells called podocytes. However, these cells can become damaged by conditions such as diabetes or various kidney diseases. Our research will examine how podocytes form and are maintained, which will facilitate our understanding of what happens to them during disease, and potentially lead to new therapies.