Molecular Genetics and Pathogenesis of ARPKD

描述(由申请人提供)：这项提案侧重于由PKDH1编码的一种新蛋白的功能分析和表征，PKDH1是导致常染色体隐性遗传性多囊肾病(ARPKD)的基因。最近有几个小组发现了PKHD1(Ward等人，2002年；Onuchic等人，2002年；熊华，2002年)。其中，两个小组独立报道了PKHD1最长的ORF由67个外显子组成，编码4074个氨基酸(AAs)，并分别命名为纤维囊蛋白和多聚蛋白(FPC)，并展示了PKHD1的一系列替代变体。通过定位克隆，本课题组还发现了一种短形式的PKHD1，它的前60个外显子与PKHD1几乎相同(熊华，2002)，我们认为它是PKDH1的一个异构体。使用一组抗体，我们证明了FPC广泛表达于上皮衍生物中，并聚集在碱性小体/睫状亚细胞细胞器(Zhang等人，2004年)。然而，这种大而复杂的蛋白质的生物学功能仍然未知。 为了阐明FPC的功能作用，我们将使用分子、细胞生物学和转基因方法来研究肾和肝脏等受影响组织中肾小管发生和肾小管成熟的细胞生理学。我们还将探讨FPC缺陷导致囊性形成和/或纤维化的机制。此外，我们已经开始建立过表达FPC的小鼠模型。连同我们的其他PKD突变模型，包括空PKD1和-PKD2小鼠，这些模型将提供我们解决几个问题所需的基本工具。这些问题包括：Pkhd1基因产物的缺失(S)是否导致一些器官的显著缺陷，而初级管道对其形态发生至关重要；ARPKD的囊变是否是由于FPC的异常表达；PKHD1是否参与了多囊蛋白途径；如果是，在胚胎发生、器官发生和囊变过程中，FPC和多囊蛋白之间的分子关系；FPC的功能重新表达是否逆转或阻止了FPC缺陷小鼠的囊变表型。通过对这些小鼠及其衍生的细胞系的研究，我们将进一步深入了解FPC在囊性形成的发生和发展中的作用以及对肾上皮细胞的生理调节。我们还将更多地了解FPC与常见致囊途径中其他PKD相关和相关基因的关系。最终，我们希望利用这一知识来建立这种疾病的治疗基础。

DESCRIPTION (provided by applicant): This proposal focuses on the functional analysis and characterization of a novel protein that is encoded by PKDH1, the gene responsible for causing autosomal recessive polycystic kidney disease (ARPKD). Several groups have recently identified PKHD1 (Ward et al., 2002; Onuchic et al., 2002; Xiong H, 2002). Of them, two groups independently reported that the longest ORF of PKHD1 contains 67 exons and encodes 4074 amino acids (AAs), and respectively named them fibrocystin and polyductin (FPC), and also demonstrated a large spectrum of alternative variants for PKHD1. By positional cloning, our group has also identified a short form of PKHD1 in which the first 60 exons are nearly identical to PKHD1 (Xiong H, 2002), and we consider it to be an isoform of PKDH1. Using a panel of antibodies, we demonstrated that FPC is widely expressed in epithelial derivatives and is aggregated at basobodies/ciliary subcellular organelles (Zhang et al., 2004). However, the biologic functions of this large and complex protein remain unknown. To elucidate the functional roles of FPC, 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 an FPC deficiency causes cyst formation and/or fibrosis. Moreover, we have begun to generate mouse models with over-expression of FPC. Together with our other Pkd mutant models, which include null-Pkd1 and -Pkd2 mice, the models will provide the essential tools we need to address several questions. These include whether the absence of Pkhdl gene product(s) causes significant defects in some organs for which the primary duct is essential for their morphogenesis; whether the cystogenesis of ARPKD results from abnormal expression of FPC; whether PKHD1 involves the polycystin pathway; and if so, what the molecular relationship is between FPC and polycystins during embryogenesis, organogenesis, and cystogenesis; whether the functional re-expression of FPC reverse or arrest the cystic phenotypes in FPC-deficient mice. By studying these mice and the cell lines derived from them, we will gain further insight into the role of FPC in the initiation and progression of cyst formation and the regulation of renal epithelial cell physiology. We will also learn more about the relationship between FPC and other PKD-related and -associated genes in the common cystogenic pathway. Ultimately, we hope to use this knowledge to establish a therapeutic basis for this disease.