Autosomal dominant polycystic kidney disease is caused primarily by mutations in two genes, Pkd1 and Pkd2, which function together as a calcium channel complex. Subcellular localization studies suggest that the polycystin2 protein may act in the apical cell membrane associated with cilia or in internal cell membranes as a calcium release channel. However, it is currently not known where polycystin2 channel activity acts in epithelia or which cellular signaling systems may impinge on polycystin2 function. Disruption of zebrafish polycystin2 gene expression using antisense morpholino oligos results in rapid kidney cyst development, randomized organ laterality, hydrocephalus, and body axis curvature. These defects are rescued by co-injection of the human pkd2 mRNA.
In Aim 1. we propose using the zebrafish as an in vivo system for structure function analysis of polycystin2. We hypothesize that specific amino acid motifs in polycystin2 target it to its cellular site of action. By disrupting these either in the endogenous zebrafish polycystin2 or in rescuing human polycystin2 mRNAs we will test whether subcellular localization affects the cellular function of polycystin2.
In Aim 2. we hypothesize that polycystin 2, acting as a calcium channel, mediates the effects of physical forces on the epithelium. We will measure calcium responses in isolated kidney tubules that lack polycystin2 function or that express altered pkd2 alleles. Finally we propose that mutations in polycytin2 interacting proteins or downstream mediators may effect signaling or function of polycystin2.
In Aim 3. we outline a plan to identify cellular components that interact with polycystin2 by screening for dominant suppressors of the zebrafish polycystin2 phenotype. This proposal exploits unique advantages of the zebrafish as a model organism to more fully explore the function of polycystin2 in vivo and further our understanding of the cellular mechanisms of autosomal dominant polycystic kidney disease.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Special Emphasis Panel (ZDK1-GRB-6 (J1))
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Rasooly, Rebekah S
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Massachusetts General Hospital
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