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

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.