Polycystic kidney disease (PKD) is the most common inherited disorder leading to chronic renal failure. Human pedigrees and several mouse models have led to cloning of genes linked to PKD. Characterization of two mice strains, kat and kat2J, both of which develop progressive PKD, has identified Nek1 (Aspergillus NIMA-related kinase 1) as a candidate protein involved in PKD. The mutations found in both kat and kat2J mice result in truncated Nekl without any coiled-coil domain at its carboxyl terminus. To date, little is known about Nekl other than its mRNA expression pattern in mice. In this proposal, Nek1 and its gene product will be characterized at molecular and cellular levels, and the potential role of Nek1 in PKD pathogenesis will be explored. In aim 1, the protein encoded by Nek1 will be characterized for its cell cycle-dependent expression pattern, kinase activity, subcellular localization, and role in kidney development. For this aim, cell and organ culture systems will be used to explore the consequences of wild type and mutant Nek1 expression via tetracycline-regulated and adenoviral vectors. Kidney tissue sections and individual cells will also be examined by immunohistochemistry. A dominant negative Nek1 mutant will be identified and used to examine its effect on kidney development in an organ culture system. In aim 2, a mouse strain in which Nek1 is specifically inactivated by gene targeting will be generated. Using the IRESbeta-geo as selection cassette, the expression of Nek1 will be monitored carefully during kidney development and cystogenesis in the nek1 null mice. Nek1 null kidney cells will be cultured from the mice and used ex vivo to examine the effects of Nek1 and nek1 mutants in a null background. Finally, in aim 3, the interaction between Nek1 and VDAC1, a mitochondrial ion channel potentially involved in apoptotic pathways, will be analyzed by molecular and physiological methods. The significance of the interaction in the pathogenesis of PKD will be explored.