Characterization of a novel protein required for glomerular podocyte function

描述（由申请人提供）：足细胞是肾脏的特化细胞，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，它们的足突与肾小球基底膜（GBM）分离。这种脱离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附着在GBM上是一个重要的研究领域。本提案中的实验旨在表征参与维持这些附着物的分子，并可能导致各种肾脏疾病的新治疗靶点。这些目标与NIH/NIDDK的使命直接相关，即通过新颖的研究改善人类健康，在这种情况下，特别是与肾功能和疾病相关的研究。足细胞-GBM附着因子（PGAF）是足细胞足突附着于GBM所需的一种新型蛋白。它是Wilms' tumor supprestor -1 （WT1）的下游靶点，并结合半胱氨酸蛋白酶calpain的小亚基capnsi。由于活性钙蛋白酶促进细胞粘附的脱离，目前的假设是PGAF抑制足细胞中的钙蛋白酶活性以防止足突脱离。本提案的目的是使用体外和体内技术的结合来研究PGAF和钙蛋白酶在维持足细胞附着于GBM中的作用。体内研究将在斑马鱼和小鼠模型的组合上进行。由于胚胎斑马鱼肾脏提供了一个简单、快速和易于操作的系统来研究足细胞功能，我们的PGAF和calpain功能的初步体内分析将在这个系统中进行。包括基于morpholino的基因敲除和mRNA过表达在内的技术将用于干扰胚胎中的基因功能。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，PGAF功能丧失小鼠将使用类似的技术产生和评估。体外分析将涉及培养小鼠足细胞中蛋白相互作用和PGAF敲低/过表达的生化表征。与公众健康相关：肾脏通过一种叫做足细胞的特殊细胞来过滤血液中的废物。然而，这些细胞会因糖尿病或各种肾脏疾病而受损。我们的研究将检查足细胞是如何形成和维持的，这将有助于我们理解在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.