Role of nephrin in pancreatic beta cell function

描述（由申请人提供）： Nephrin是一种免疫球蛋白样蛋白，在肾小球足细胞中具有重要的结构和信号传导特性。Nephrin的表达最近已被报道在胰腺中，其功能仍然未知。最近的数据表明nephrin在囊泡运输中的作用。我们对胰岛素瘤细胞过度表达nephrin的初步研究表明nephrin在胰岛素分泌中的作用。我们还可以证明nephrin与β细胞中参与胰岛素胞吐的重要分子共定位，即，囊泡相关膜蛋白（VAMP）-2。本申请的目的是了解nephrin在胰腺β细胞的组成性和葡萄糖刺激的胰岛素分泌的调节中的作用。我们推测，在葡萄糖刺激，nephrin有助于胰岛素囊泡颗粒的胞吐。为此，我们将通过shRNAmir技术在胰岛素瘤细胞和整个胰岛中操纵nephrin表达。我们将进一步研究nephrin与VAMP-2在β细胞中的功能和生化相互作用，以及这种相互作用与胰岛素分泌之间的关系。在葡萄糖刺激下，几种GTP酶的活性促进胰岛素分泌：其中，发动蛋白在足细胞和β细胞结构和功能完整性中具有重要作用。因此，我们假设葡萄糖刺激的发动蛋白活性是驱动力，使nephrin招募更多的未成熟的胰岛素囊泡的质膜。本资助提案的短期目标是让申请人了解与糖尿病足细胞功能相关的分子如何也影响胰腺β细胞功能。拟议研究的结果可能允许识别连接糖尿病和糖尿病肾病的共同发病途径，并可能导致开发新的糖尿病治疗方法。该提案将允许申请人通过利用体内β细胞成像、钙电流研究、胰岛素产生和β细胞信号转导的新技术接受β细胞生理学研究的培训。该提案的具体目标和实验设计将支持她作为独立调查员的职业发展。该项目非常符合申请人的长期目标，即追求专注于糖尿病的学术生涯，并旨在开发治疗糖尿病及其并发症的新药。糖尿病研究所的环境是独一无二的，因为它在胰岛细胞生物学和移植领域的领导作用，因为它为申请人提供了与关键教师互动的可能性，这些教师将不断提供智力和技术支持。 公共卫生相关性：拟议的研究是相关的，因为它调查了对肾滤过屏障功能完整性非常重要的分子如何在胰腺的胰岛素分泌细胞中发挥作用。这项研究可能导致：1）确定糖尿病及其主要并发症之一，即糖尿病肾病的共同发病途径; 2）发现治愈糖尿病的新治疗靶点。

DESCRIPTION (provided by applicant): Nephrin is an immunoglobulin-like protein with important structural and signaling properties in the glomerular podocyte. Nephrin expression has been recently reported in the pancreas where its function remains unknown. Recent data suggest a role for nephrin in vesicle trafficking. Our preliminary data with insulinoma cells overexpressing nephrin suggest a role for nephrin in insulin secretion. We could also show that nephrin co-localizes with an important molecule involved in insulin exocytosis in beta cells, i.e., vescicle associated membrane protein (VAMP)-2. The goal of this application is to understand the role of nephrin in the regulation of constitutive and glucose stimulated insulin secretion by pancreatic beta cells. We hypothesize that, upon glucose stimulation, nephrin contributes to insulin vesicle granules' exocytosis. To this aim, we will manipulate nephrin expression by shRNAmir technology in insulinoma cells and whole islets. We will further investigate the functional and biochemical interaction of nephrin with VAMP-2 in beta cells, and the relationship between such interaction and insulin secretion. Upon glucose stimulation, the activity of several GTPases facilitates insulin secretion: among them, dynamin has an important role in both podocytes and beta cell structural and functional integrity. Thus, we hypothesize that glucose stimulated dynamin activity is the driving force that allows nephrin to recruit more immature insulin vesicles to the plasma membrane. The short-term objectives of this grant proposal are to allow the applicant to understand how molecules that are relevant to podocyte function in diabetes may also affect pancreatic beta cell function. The outcome of the proposed research may allow the identification of a common pathogenetic pathway linking diabetes and diabetic nephropathy, and may lead to the development of a new cure for diabetes. This proposal will allow the applicant to receive training for the study of beta cell physiology through the utilization of novel techniques for beta cell imaging in vivo, study of calcium currents, insulin production and beta cell signal transduction. The specific aims and experimental design of the proposal will support the development of her career as an independent investigator. This project fits well into the applicant's long-term goal to pursue an academic career focused on diabetes and aimed at the development of new drugs for the cure of diabetes and its complications. The environment of the Diabetes Research Institute is unique due its leadership role in the field of islet cell biology and transplantation, and because it offers the applicant the possibility to interact with key faculty members that will constantly provide intellectual and technical support. PUBLIC HEALTH RELEVANCE: The proposed research is relevant because it investigates how molecules that are very important for the functional integrity of the renal filtration barrier may play a role in insulin secreting cells of the pancreas. The proposed research may lead to: 1) the identification of a common pathogenetic pathway in diabetes and one of its major complication, i.e. diabetic nephropathy; 2) the discovery of novel therapeutic targets for the cure of diabetes.