Molecular Genetics and Pathogenesis of ARPKD

描述（由申请人提供）：该提案重点关注PKDH 1编码的新型蛋白质的功能分析和表征，PKDH 1是导致常染色体隐性遗传性多囊肾病（ARPKD）的基因。几个小组最近已经鉴定了PKHD 1（Ward等人，2002; Onuchic等人，2002; Xiong H，2002）。其中，有两个研究组独立报道了PKHD 1最长的ORF包含67个外显子，编码4074个氨基酸（AA），分别命名为纤维囊蛋白（fibrocystin）和多导管蛋白（polyductin，FPC），并发现了PKHD 1的多种变异体。通过定位克隆，我们的小组还鉴定了PKHD 1的短形式，其中前60个外显子与PKHD 1几乎相同（Xiong H，2002），我们认为它是PKDH 1的同种型。使用一组抗体，我们证明FPC在上皮衍生物中广泛表达，并聚集在基底体/纤毛亚细胞器（Zhang et al.，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解决受影响组织（如肾脏和肝脏）中小管发生和小管成熟的细胞生理学。我们还将讨论FPC缺陷导致囊肿形成和/或纤维化的机制。此外，我们已经开始产生过表达FPC的小鼠模型。与我们的其他Pkd突变模型（包括null-Pkd 1和-Pkd 2小鼠）一起，这些模型将为我们解决几个问题提供必要的工具。这些问题包括：Pkhdl基因产物的缺失是否会导致某些器官的显著缺陷，而初级导管对于这些器官的形态发生是必不可少的; ARPKD的囊形成是否由FPC的异常表达引起; PKHD 1是否涉及多囊蛋白途径;如果是，在胚胎发生、器官发生和囊形成过程中FPC和多囊蛋白之间的分子关系是什么; FPC的功能性重新表达是否逆转或阻止FPC缺陷小鼠中的囊性表型。通过研究这些小鼠及其衍生的细胞系，我们将进一步了解FPC在囊肿形成的启动和进展以及肾上皮细胞生理学调节中的作用。我们还将了解更多关于FPC和其他PKD相关基因和相关基因之间的关系，在共同的囊形成途径。最终，我们希望利用这些知识为这种疾病建立治疗基础。

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.