Disorders of transepithelial ion transport underlie clinical disorders of extracellular volume, blood pressure, andelectrolytes, but molecular mechanisms of transepithelial ion transport are difficult to directly examine in themammalian nephron. The applicants' long-term goal is to better understand epithelial ion transportmechanisms relevant to human kidney function, in sufficient molecular detail to define new therapeuticstrategies. The overall objective of this application is to identify regulators of a kinase cascade, consisting ofWNK (With No Lysine) and SPAK/OSR1 (Ste20-related proline alanine rich kinase/oxidative stress response)kinases, that plays an essential role in sodium and potassium homeostasis through the regulation of renaltransepithelial ion transport. The application builds on three recent findings: Cl- directly binds to the WNKkinase domain to inhibit autophosphorylation and activation; the scaffold protein Mo25 (Mouse protein25/Cab39) enhances the activity of SPAK/OSR1; and low potassium diet activates WNK-SPAK/OSR1signaling. The central hypothesis is that transepithelial ion flux is directly regulated by transported ions (Cl- andK+) through modulation of WNK-SPAK/OSR1 signaling, while Mo25 provides additional regulatory control. Therationale is that better understanding of these molecular mechanisms will allow the design of noveltherapeutics with fewer off-target effects. Guided by strong preliminary data, the central hypothesis will betested by pursuing three specific aims: 1) Determine the roles of Cl- and K+ in the regulation of WNK isoformsin transepithelial ion transport; 2) Determine the role of Mo25 in WNK signaling in a transporting epithelium;and 3) Probe tubule physiology using newly developed chemical WNK inhibitors. The approach is innovativeby bridging fundamental molecular insights gained from biophysical studies, with the functional physiologicalroles of those molecular mechanisms, using newly developed platforms and tools to probe questions oftransporting epithelium biology. Assays have been established, and demonstrated feasible in the investigators'hands, to examine regulation of Drosophila and mammalian WNKs by Cl- and K+ in vitro and in the fly renaltubule, and to measure intracellular Cl- in live tubules, with temporal resolution; and to measure transepithelialion flux in genetically modified, or pharmacologically treated, tubules. The proposed research is significant,because it is expected to advance understanding of molecular mechanisms of WNK-SPAK/OSR1 regulation ina transporting renal epithelium. The studies will determine: 1) how quickly changes in intracellular Cl- changeWNK activity; 2) whether WNKs act as K+ sensors; and 3) the role of Mo25 in transepithelial ion transport. Inaddition, these studies will further develop recently identified pharmacological WNK inhibitors, which will be auseful tool for further probing the biology of WNK-SPAK/OSR1 signaling in Drosophila and mammaliansystems, and potentially serve as the basis for future development of therapeutic compounds for the treatmentof volume overload, hypertension and hyperkalemia.
Inadequate excretion of sodium and potassium by the kidney results in volume overload; high blood pressure;and high blood potassium levels; conditions which are especially common in patients with chronic kidneydisease; and are associated with increased morbidity and mortality. The proposed research will examineunique mechanisms of regulation of a kinase pathway that regulates kidney sodium and potassium excretion.Ultimately; this is expected to lead to the development of drugs targeting this kinase pathway; with beneficialeffects on volume overload; high blood pressure and high potassium levels; and decreased risk of side effects.
