Molecular Genetics and Pathogenesis of ARPKD

描述（由申请人提供）：该提案侧重于通过产生具有PKDH1靶向突变的小鼠系以及产生针对该基因产物的多克隆和单克隆抗体来表征由PKDH1编码的新蛋白，PKDH1是导致常染色体隐性多囊肾病（ARPKD）的基因。最近，我们发现了一个名为PKHD1-T的新基因，其表达主要在肾脏、胰腺和肝脏中检测到8。该基因编码一种含有3396个氨基酸的蛋白质，被命名为紧蛋白。当我们验证这个基因为PKHD1时，两个小组报告了他们自己的PKHD1鉴定，他们显示这个基因编码一种新的蛋白质，他们分别称之为纤维囊蛋白或多ductin，编码4074个氨基酸。纤维囊蛋白和多导蛋白与紧蛋白具有几乎相同的氨基酸序列（c端除外），表明它们是由同一基因编码的。为了了解PKHD1的功能作用，我们将使用分子、细胞生物学和转基因方法来解决肾和肝等受影响组织中小管形成和小管成熟的细胞生理学问题。我们还将探讨PKHD1缺失导致囊肿形成和/或纤维化的机制。针对这种新蛋白的抗体的产生将为揭示PKHD1的细胞和亚细胞定位以及评估其在肾脏和肝脏发育过程中的生物学特征提供有力的工具。此外，通过对人类PKHD1的小鼠同源物进行基因诱变，并在该基因中插入可检测的报告基因，建立小鼠模型，将加强对其基因产物的表征和对ARPKD分子发病机制的解剖。我们将利用我们已经或即将生成的具有Pkhdl、Pkdl和Pkd2靶向突变的小鼠模型，以及已经自发产生并通过实验创建的现有小鼠arpkd样模型来剖析这些基因产物之间的遗传关系。综上所述，这些研究结果将显著促进我们对多囊肾病的发病机制和囊肿形成和生长的分子机制的理解。

DESCRIPTION (provided by applicant): This proposal focuses on the characterization of a novel protein that is encoded by PKDH1, the gene responsible for causing autosomal recessive polycystic kidney disease (ARPKD), by the generation of mouse lines with a targeted mutation in PKDH1 and by the production of poly- and monoclonal antibodies against the gene product. Recently, we identified a novel gene, named PKHDl-tentative (PKHD1-T), whose expression can mainly be detected in the kidney, pancreas, and liver 8. This gene encodes a 3396-amino-acid protein, which was named tigmin. While we were validating this gene as PKHD1, two groups reported their own identification of PKHD1 9,10 They showed this gene to encode a novel protein they called fibrocystin or polyductin, respectively, which encoded 4074 amino acids. Both fibrocystin and polyductin share an almost identical amino acid sequence with tigmin (except at the C-terminus), indicating that they are encoded by the same gene. To understand the functional roles of PKHD1, we will use molecular, cell biological, and transgenic approaches to address the cellular physiology of tubulogenesis and tubular maturation in affected tissues such as the kidney and liver. We will also address the mechanisms by which the deficiency of PKHD1 causes cyst formation and/or fibrosis. The generation of antibodies against this novel protein will provide a powerful tool for revealing the cellular and subcellular localization of PKHD1 and assessing its biological features during kidney and liver development. In addition, the establishment of mouse models by genetically mutagenizing the murine homologue of human PKHD1 and by inserting into the gene the cDNAs for detectable reporters will enhance the characterization of its gene product and the dissection of the molecular pathogenesis of ARPKD. We will take advantage of mouse models with targeted mutations in Pkhdl, Pkdl, and Pkd2, which were or will be generated by us, as well as of existing mouse ARPKD-like models that have risen spontaneously and been created experimentally to dissect the genetic relationship between these gene products. Taken together, the results of these studies will significantly advance our understanding of the pathogenesis of polycystic kidney diseases and the molecular mechanism of cyst formation and growth.