Molecular and cellular basis of the renal diseases associated with Alagille Syndrome

项目摘要 5岁以下儿童终末期肾病的主要原因是先天性肾功能异常。 肾脏和泌尿道（CAKUT）。在与CAKUT有关的几种基因突变中， Notch信号通路基因JAG 1和NOTCH 2的突变。这些Notch途径基因的突变是 预测降低Notch信号传导活性的水平，并且与Alagille综合征（ALGS）相关。一 ALGS的组成部分是肾脏疾病的可变发生率，包括小的多囊性、发育不良的 肾脏例如，一项回顾性研究确定，40%携带JAG 1突变的ALGS患者 在60%的ALGS患者中发生某种形式的肾脏疾病，伴有或不伴有囊肿的发育不良肾脏 患有肾病Notch信号传导确保 对正常的肾脏发育和维持知之甚少。我们知道JAG 1可以作为 配体激活NOTCH 2，并假设经典Notch信号通路可预防肾脏疾病 与ALGS有关。然而，在其他经典Notch信号传导途径组分中的突变具有 与ALGS无关。此外，肾脏疾病表现的高度变异性 在ALGS患者中是令人费解的。在我们的小鼠模型中， 随着Notch 1和Notch 2等位基因在发育中的肾上皮中失活数量的增加。这里我们 建议确定肾上皮Notch信号网络（RENSN）并进行全外显子组 对ALGS患者进行测序以确定除了JAG 1和/或 NOTCH 2决定ALGS中多囊性/发育不良肾脏的发生。此外，我们还将测试 与ALGS相关的NOTCH 2突变改变了肾上皮细胞分裂、初级纤毛 不同肾上皮细胞培养物中肾上皮Notch靶基因的结构和表达。我们将 还使用确定野生型与ALGS相关NOTCH 2变体的近端相互作用蛋白 生物识别此外，我们将应用三种策略来分子干预和阻止Notch信号传导， 肾功能缺陷这些研究将建立肾损害的细胞和分子机制 与ALGS相关的疾病和鉴定在肾细胞类型中特异性增加Notch信号传导的方法 通过确定Notch信号传导和肾上皮功能之间的分子界面。

Project Summary The leading cause of end stage renal disease among children under 5 years is congenital abnormalities of the kidney and urinary tract (CAKUT). Among the several genetic mutations that have been linked to CAKUT are mutations in Notch signaling pathway genes, JAG1 and NOTCH2. Mutations in these Notch pathway genes are predicted to reduce the level of Notch signaling activity, and are associated with Alagille Syndrome (ALGS). One component of ALGS is the variable occurrence of kidney disease including that of small multicystic, dysplastic kidneys. For instance, a retrospective study determined that 40% of ALGS patients with JAG1 mutations had some form of kidney disease with dysplastic kidneys with or without cysts occurring in 60% of ALGS patients with kidney disease. The underlying cellular and molecular mechanisms by which Notch signaling ensures normal kidney development and maintenance are poorly understood. We know that JAG1 can function as a ligand to activate NOTCH2, and assume the canonical Notch signaling pathway prevents kidney disease associated with ALGS. However, mutations in the other canonical Notch signaling pathway components have not been associated with ALGS. Additionally, the high degree of variability in the manifestation of kidney disease among ALGS patients is puzzling. In our mouse models the severity of multi-cystic kidney disease increases with increasing number of Notch1 and Notch2 alleles inactivated in the developing renal epithelium. Here we propose to determine the Renal Epithelial Notch Signaling Network (RENSN) and perform whole exome sequencing of ALGS patients to determine if additional variations in RENSN genes in addition to JAG1 and/or NOTCH2 determine the occurrence of multicystic/dysplastic kidneys in ALGS. Additionally, we will test if NOTCH2 mutations associated with ALGS alter the orientation of renal epithelial cell division, primary cilia structure and expression of renal epithelial Notch target genes in different renal epithelial cell cultures. We will also determine the proximal interacting proteins of wild type versus ALGS associated NOTCH2 variants using BioID. Additionally, we will apply three strategies to molecularly intervene and prevent the Notch-signaling- deficient kidney defects. These studies will establish the underlying cellular and molecular mechanisms of kidney disease associated with ALGS and identify methods to increase Notch signaling specifically in kidney cell types by determining the molecular interface between Notch signaling and renal epithelial functions.