The collecting duct principal cell (PC) is responsible for the final adjustment of renal Na and water excretion. Derangements in PC Na reabsorption are associated with most forms of monogenic hypertension, while altered PC water transport accompanies disorders of water metabolism. PC Na and water transport are subject to the influences of multiple autocrine, paracrine and endocrine factors. A key mechanism for mediating many of these effects involves signaling through adenylyl cyclase (AC)-derived cAMP. The PC expresses AC3, 4 and 6, raising the fundamental question as to which biologic effects individual AC isoforms exert in the PC. We have engineered mice with PC-specific disruption of AC3, 4 or 6, and have found exciting new evidence for unique roles of the PC AC isoforms, AC3 and 6. Our findings suggest that AC6 regulates PC Na and water transport, while AC3 selectively affects PC water transport. Based on these novel findings, together with the currently increasing interest in designing inhibitors specific for individual AC isoforms as therapeutic modalities, this proposal will define the role of the specific AC isoforms, AC3 and 6 in the regulation of PC Na and water transport. In addition, cAMP in the PC is not only important in regulating Na and water transport, but it can be of primary importance in disease. In particular, disordered cAMP production and actions in autosomal dominant polycystic kidney disease play a fundamental role in cyst development and expansion. In preliminary studies, we have obtained exciting new data suggesting that targeting AC6 is markedly protective against the development of cystic kidney disease in a mouse model. Consequently, this proposal will define the role of specific AC isoforms in the PC in the pathogenesis of polycystic kidney disease. The major hypotheses of this proposal are: 1) AC3 and 6 in the PC exert distinctive effects on PC Na and water transport in health; and 2) AC3 and 6 in the PC differentially modulate PC development into cysts in polycystic kidney disease. The proposal has three specific aims: 1) Aim 1 will determine the physiologic role of PC AC isoforms (AC3 and 6) in renal Na and water handling. Each AC isoform will be selectively targeted in mouse PC in vivo. Intact animals and acutely isolated collecting ducts will be used to assess the role of the individual AC isoforms by loss of function in the regulation of blood pressure and Na and water excretion. 2) Aim 2 will determine the biochemistry of PC AC isoforms (AC3 and 6) related to control of cAMP levels and cAMP effects on Na and water transport. The mechanisms by which individual AC isoform-derived cAMP is modulated by vasopressin and exerts its biologic effects will be studied using mice with PC-specific knockout of AC3, 6 or both isoforms. In particular, these studies will define how cAMP derived from each AC isoform in the PC is metabolized by phosphodiesterases and which signaling molecules (protein kinase A and/or exchange protein directly activated by cAMP) are directly activated by each AC isoform in transducing vasopressin actions in the PC. 3) Aim 3 will determine the pathophysiologic effects of all PC AC isoforms (AC3 and 6) in polycystic kidney disease. A mouse model of polycystin-1 deficiency will be employed and the effect of simultaneously disrupting individual AC isoforms together with the Pkd1 gene on renal structure and function will be assessed. These studies will define which AC isoform(s) is/are important in the pathogenesis of polycystic kidney disease and will begin to identify the specific signaling pathways that individual AC isoforms modulate in polycystic kidney disease.