Project Summary/Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the growth of fluid-filled cysts that disrupt the renal architecture and function, leading to end-stage renal disease (ESRD), dialysis, and renal transplantation. Although ADPKD has a genetic origin, its progression and clinical outcome is highly variable and dependent on agents circulating in blood. Previous work from our laboratory has shown that one of these agents is ouabain. In primary renal epithelial cells obtained from kidneys of patients with ADPKD, ouabain promotes the cystic phenotype by increasing cell proliferation, augmenting cAMP-dependent fluid secretion, and inducing cell dedifferentiation; ouabain also stimulates cyst progression in embryonic renal explants and adult kidneys of ADPKD mouse models. Ouabain exerts these effects via its cell plasma membrane receptor, Na,K-ATPase (NKA), through the activation of signal transduction pathways in the cell. Ouabain also inhibits the exchange of Na+ for K+ across the cell plasma membrane characteristic of NKA ion transport function, which contributes to reduce fluid reabsorption in the renal epithelium and enhance ADPKD cyst progression. While all NKA appears to function as an ion transporter, it is believed that the NKA residing in caveolae is involved in ouabain-induced signal transduction. In caveolae, NKA forms a complex with other proteins in what is known as the “NKA signalosome”; key to this receptor complex is the association of NKA with the kinase Src, which allows NKA to function as a tyrosine kinase-coupled receptor. At present, the mechanisms by which the NKA signalosome functions in ADPKD are unknown. Moreover, it is unclear if ouabain affects both the signaling and ion transport functions of NKA to drive ADPKD cystogenesis. I hypothesize that elimination of NKA signaling capacity, through caveolar depletion or disruption of NKA/Src interaction, will reduce ouabain- induced ADPKD cyst progression. Aim 1 will determine the role of caveolae in ouabain-induced NKA signaling and ADPKD cyst progression. The role of caveolae in ADPKD is currently unknown; I will use a novel mouse model to determine necessity of caveolae for ouabain's effects in ADPKD cyst progression and NKA signaling. Aim 2 will establish NKA/Src interaction as the mechanism driving ouabain effects in ADPKD. The mechanisms involved in the activation of the NKA signalosome are currently unknown. I will create a novel mouse model which interrupts the NKA/Src binding site and determine how the absence of this interaction affects ADPKD cyst progression and NKA signal transduction. Completion of these aims will give a better understanding of how ouabain accelerates ADPKD cyst growth and characterize a novel mechanism of cyst progression which can be targeted to develop future therapeutics for the treatment of ADPKD. The training plan outlined in this fellowship will not only develop my technical research skills, but also ...