Epithelial Cell Polarity in Polycystic Kidney Disease

描述(申请人提供)：常染色体显性遗传性多囊肾病(ADPKD)是世界上最常见的遗传性疾病之一。PKD1或PKD2基因突变导致受影响的肾上皮细胞表型改变，包括增殖和凋亡增加，蛋白运输缺陷和跨上皮运输功能的改变。这会导致大的肾囊肿的形成，破坏正常的肾组织，最终导致肾功能衰竭。由于PKD1/2基因产物多囊蛋白-1和多囊蛋白-2的功能目前尚不清楚，因此很难合理设计治疗干预策略。这项研究建议试图弥合我们对PKD1基因突变和最终导致囊肿形成的上皮表型之间存在的知识差距。我们在极化的肾上皮细胞系MDCK中建立了一个体外细胞培养系统，用于四环素调节的多囊蛋白-1结构域的表达，这些结构域被认为是内源蛋白的显性负抑制因子。初步结果表明，这导致了ADPKD中几种典型的囊性上皮细胞表型的诱导，如增殖增加、细胞凋亡和EGF受体的顶端错误定位。位于多囊蛋白-1缺陷下游并负责这些表型的分子机制将被确定，并确定它们之间的相互关系。与显性负性多囊蛋白-1结构域相互作用的蛋白质将被识别出来，以介导这些机制。特别重要的是对细胞内膜运输缺陷的调查，这些缺陷被认为是囊性上皮细胞的几种最终表型的原因。本实验室的初步结果表明，膜转运缺陷ADPKD与SNAR膜融合机制的特异性变化有关。这些陷阱的变化在囊状上皮表型获得中的作用，以及它们是如何由多囊蛋白-1的破坏引起的，将被调查。这些研究将有助于确定多囊蛋白-1的正常功能以及它的干扰所导致的下游影响。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases worldwide. Mutations in the PKD1 or PKD2 genes lead to phenotypic changes in affected renal epithelial cells that include increased proliferation and apoptosis, defects in protein trafficking and changes in transepithelial transport functions. This leads to the formation of large renal cysts, the destruction of the normal renal tissue and finally renal failure. Since the functions of the PKD1/2 gene products, polycystin-1 and polycystin-2, are currently unknown, the rational design of strategies for therapeutic intervention has been difficult. This research proposal seeks to close the gap in our knowledge on what lies between PKD1 gene mutations and the epithelial phenotype that finally leads to cyst formation. We have developed an in vitro cell culture system in the polarized renal epithelial cell line MDCK for the tetracycline-regulated expression of polycystin-1 domains that are proposed to act as dominant-negative inhibitors of the endogenous protein. Preliminary results show that this leads to the induction of several of the typical phenotypes of authentic cystic epithelial cells in ADPKD such as increased proliferation, apoptosis and apical mistargeting of the EGF receptor. The molecular mechanisms that lie downstream of the polycystin-1 defect and are responsible for these phenotypes will be identified and their relationship to each other defined. Proteins that interact with the dominant-negative polycystin-1 domains to mediate these mechanisms will be identified. Of particular importance is the investigation of defects in intracellular membrane trafficking that have been proposed to be responsible for several of the final phenotypes of cystic epithelial cells. Preliminary results from this laboratory suggest that specific changes in the SNARE membrane fusion machinery are involved in membrane trafficking defects ADPKD. The role of these SNARE changes in the acquisition of the cystic epithelial phenotype, and how they are caused by polycystin-1 disruption will be investigated. These studies will help to define the normal function of polycystin-1 and the downstream effects that result from its disruption.