Analysis of ARPKD by Targeted Manipulation of Pkhd1

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DESCRIPTION (provided by applicant): Autosomal Recessive Polycystic Kidney Disease (ARPKD) is a devastating inherited neonatal nephropathy characterized by fusiform collecting duct dilatation and congenital hepatic fibrosis. We recently identified the ARPKD gene, PKHD1, and characterized a rat model of the disease, PCK. The PKHD1 gene is very large (477kb) generating a approximately 16kb mRNA transcribed from 67 exons. The ARPKD protein, fibrocystin, is a large (446kDa) integral membrane protein of unknown function. PKHD1 and the murine ortholog, Pkhd1, are thought to generate multiple splice forms, including possible secreted proteins. Mutation to PKHD1 is associated with a wide range of phenotypes from in utero presentation with greatly enlarged kidneys to hepatic disease only detected in adulthood. Mutation analysis has shown that most patients are compound heterozygotes for PKHD1 mutations and that patients with two truncating changes all have a severe renal phenotype resulting in perinatal death. This proposal is to target murine Pkhd1, to help determine the normal role of the protein and the consequences of various mutations. A targeted removal of exon 2 (Pkhd1 del2) has been engineered and homozygous animals have severe liver and pancreatic disease, but interestingly few renal cysts. Specific Aim 1 will characterize the phenotype and expression in these Pkhd1 del2 homozygotes. The possible influence of the incorporated neo cassette will be tested by floxing out by Cre expression and phenotypic reexamination. If the disease phenotype in the Pkhd1 del2(-neo) homozygotes remains mild further targeted disruption will be designed to generate a more severe renal phenotype: To test the role of the protein after renal and hepatic development, Specific Aim 2 will generate an inducible conditional knockout that will allow Pkhd1 to be mutated in somatic tissue and result in the elimination of fibrocystin in the adult or the neonate. The final set of experiments (Specific Aim 3) will tag the endogenous gene so that the protein can be localized and studied using reliable tag antibodies. Overall these studies should reveal more about the control of expression of Pkhd1, the normal role of fibrocystin and help clarify the mutational mechanism in this complex disorder. These are essential prerequisites before rational therapies can be developed for this disorder.