Cystic Dilatation of Nephrons in Transgenic inv Mice

转基因小鼠肾单位囊性扩张

• 批准号: 6869238
• 负责人: CARRIE L PHILLIPS
• 金额: $ 7.58万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6869238
• 关键词: MDCK cell; RNA interference; cadherins; clinical research; computer program /software; confocal scanning microscopy; deoxyribonucleotides; embryo /fetus tissue /cell culture; epithelium; fluorescence microscopy; functional /structural genomics; gene interaction; gene mutation; immunoprecipitation; nephrogenesis; pathologic process; polycystic kidney; protein binding; protein protein interaction; protein structure function; renal tubule; western blottings

DESCRIPTION (provided by applicant): Congenital and inherited polycystic renal disorders are among the most frequent primary diagnoses in children with chronic kidney disease and are responsible for 15% of renal transplants. Inherited polycystic kidney diseases (PKD) of childhood include nephronophthisis, autosomal recessive polycystic kidney disease and medullary cystic kidney disease. Nephronophthisis is the most common cause of end stage renal disease in children and young adults and the infantile form (NPHP2) has recently been linked to mutations in the inv gene. The function of inversin, the inv gene product, is not known. We developed antibodies to inversin that localized to plasma membranes, nuclei, perinuclear cytoplasm and primary cilia, and co-immunoprecipitated with proteins involved in junctional complexes, axis development and PKD, i.e. N-cadherin and beta-catenin. We found fusiform cysts in proximal tubules and collecting ducts of inv/inv mice. Cilia are implicated in the formation of renal cysts and many PKD-associated proteins have been localized to cilia, including polaris, cystin and polycystins-1 and -2. We propose to define the function of inversin by determining its interaction with other PKD-associated proteins. Our long-term objective is to determine how defective proteins in cilia or plasma membranes lead to PKD so that therapeutic modalities may be developed. Our immediate objective is to establish the molecular and cellular basis of PKD by investigating the functional relationship of inversin with known PKD-associated proteins including the polycystins, cadherins and catenins. We hypothesize that inversin interacts with the polycystins, cadherins and catenins in normal development and maturation of renal epithelium. This interaction allows renal epithelial cells to progress through developmental checkpoints by integrating junctional complexes with cilia. Our general strategy is to study the potential role of inversin in the multi-protein polycystin/PKD complex by applying techniques of functional genomics to established culture model systems. We propose one overall specific aim with two components. First, we will determine the structural and functional significance of inversin in renal development by blocking in vitro translation of inversin in cultured renal epithelial cells and transfilter embryonic kidney cultures. Second, we will identify crucial binding partners of inversin in these cultured systems by blocking in vitro translation and assaying for changes in PKD-associated proteins.

描述（由申请人提供）： 先天性和遗传性多囊肾疾病是慢性肾病儿童最常见的原发性诊断之一，占肾移植的 15%。儿童遗传性多囊肾病（PKD）包括肾痨、常染色体隐性遗传性多囊肾病和髓样囊性肾病。肾结核是儿童和年轻人终末期肾病的最常见原因，最近发现婴儿型肾结核 (NPHP2) 与 inv 基因突变有关。 inv 基因产物 inversin 的功能尚不清楚。我们开发了定位于质膜、细胞核、核周细胞质和初级纤毛的反转蛋白抗体，并与参与连接复合物、轴发育和 PKD 的蛋白质（即 N-钙粘蛋白和 β-连环蛋白）进行共免疫沉淀。我们在 inv/inv 小鼠的近曲小管和集合管中发现梭形囊肿。纤毛与肾囊肿的形成有关，许多 PKD 相关蛋白已定位于纤毛，包括 Polaris、胱氨酸和多囊蛋白-1 和 -2。我们建议通过确定反转蛋白与其他 PKD 相关蛋白的相互作用来定义其功能。我们的长期目标是确定纤毛或质膜中的缺陷蛋白如何导致 PKD，以便开发治疗方式。我们的直接目标是通过研究反转蛋白与已知 PKD 相关蛋白（包括多囊蛋白、钙粘蛋白和连环蛋白）的功能关系来建立 PKD 的分子和细胞基础。我们假设反转蛋白在肾上皮的正常发育和成熟过程中与多囊蛋白、钙粘蛋白和连环蛋白相互作用。这种相互作用使肾上皮细胞通过将连接复合物与纤毛整合来通过发育检查点。我们的总体策略是通过将功能基因组学技术应用于已建立的培养模型系统来研究反转蛋白在多蛋白多囊蛋白/PKD复合物中的潜在作用。我们提出一个包含两个组成部分的总体具体目标。首先，我们将通过在培养的肾上皮细胞和转滤胚胎肾培养物中阻断反相蛋白的体外翻译来确定反相蛋白在肾脏发育中的结构和功能意义。其次，我们将通过阻断体外翻译和检测 PKD 相关蛋白的变化来确定这些培养系统中反相蛋白的关键结合伴侣。