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。 肾和尿路(CAKUT)。在与CAKUT有关的几个基因突变中有 Notch信号通路基因JAG1和NOTCH2的突变。这些Notch途径基因的突变是 预计会降低Notch信号活性的水平，并与Alagille综合征(ALGS)有关。一 ALGS的成分是肾脏疾病的可变发生率，包括小的多囊、发育不良 肾脏。例如，一项回顾性研究确定，携带JAG1突变的ALGS患者中有40%患有 60%的ALGS患者出现某种形式的肾脏发育不良，伴或不伴肾囊肿 患有肾脏疾病。Notch信号确保的潜在细胞和分子机制 人们对肾脏的正常发育和维持知之甚少。我们知道JAG1可以作为一个 激活NOTCH2的配体，并假定典型的Notch信号通路预防肾脏疾病 与ALGS有关。然而，其他典型的Notch信号通路组件的突变 与ALGS无关。此外，肾脏疾病表现的高度变异性 在ALGS患者中的比例令人费解。在我们的小鼠模型中，多囊肾病的严重程度增加 随着Notch1和Notch2等位基因数量的增加，在发育中的肾上皮细胞中Notch1和Notch2等位基因失活。在这里我们 建议确定肾上皮细胞Notch信号网络(RENSN)并进行完整的Exome 对ALGS患者进行测序以确定RENSN基因除JAG1和/或之外是否还有其他变异 Notch2决定了ALGS中多囊/发育不良肾脏的发生。此外，我们将测试是否 与ALGS相关的Notch2突变改变了肾上皮细胞分裂的方向，初级纤毛 肾上皮细胞Notch靶基因在不同培养肾上皮细胞中的结构和表达我们会 还可以用以下方法确定野生型与ALGS相关的NOTCH2变异体的近端相互作用蛋白 生物识别码。此外，我们将应用三种策略来从分子上干预和防止Notch信号- 肾虚亏虚。这些研究将建立肾脏潜在的细胞和分子机制。 与ALGS相关的疾病和确定在肾脏细胞类型中增加Notch信号的方法 通过确定Notch信号和肾上皮功能之间的分子接口。

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.