Epithelial Cell Polarity in Polycystic Kidney Disease

描述（由申请人提供）：常染色体显性遗传性多囊肾病（ADPKD）是全球最常见的遗传性疾病之一。PKD 1或PKD 2基因突变导致受影响肾上皮细胞的表型变化，包括增殖和凋亡增加、蛋白质运输缺陷和跨上皮转运功能变化。肾囊肿是一种慢性肾功能衰竭的常见病，也是一种慢性肾功能衰竭的常见病。由于PKD 1/2基因产物多囊蛋白-1和多囊蛋白-2的功能目前尚不清楚，因此难以合理设计治疗干预策略。这项研究旨在缩小我们对PKD 1基因突变和最终导致囊肿形成的上皮表型之间的知识差距。我们已经开发了一种体外细胞培养系统中的极化肾上皮细胞系MDCK四环素调节表达的多囊蛋白-1域，建议作为显性负抑制剂的内源性蛋白质。初步结果表明，这导致ADPKD中真实囊性上皮细胞的几种典型表型的诱导，例如增殖增加、细胞凋亡和EGF受体的顶端错误定位。位于下游的多囊蛋白-1缺陷，并负责这些表型的分子机制将被确定和它们之间的相互关系的定义。将鉴定与显性负性多囊蛋白-1结构域相互作用以介导这些机制的蛋白质。特别重要的是调查的缺陷细胞内膜贩运已被提出负责几个最终的表型的囊性上皮细胞。该实验室的初步结果表明，SNARE膜融合机制的特定变化与ADPKD膜运输缺陷有关。这些SNARE变化在获得囊性上皮表型中的作用，以及它们是如何由多囊蛋白-1破坏引起的，将进行研究。这些研究将有助于确定多囊蛋白-1的正常功能及其破坏所产生的下游效应。

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.