This NIH mentored Career Development Award proposal describes continuation of a five year training programfor the candidate, a physician scientist with the long-term goal of becoming an independent academicinvestigator with a research focus on epithelial ion transport related to kidney physiology and diseases. Thecandidate proposes to build on a background in basic research developed during undergraduate, Ph.D., andfellowship studies, and in particular previous experience studying Drosophila melanogaster, by developing newscientific skills in physiology and biochemistry. These will be applied to the immediate goal of understanding themolecular mechanisms of regulation of SLC12 cation-chloride cotransporters by the WNK and SPAK/OSR1kinases, which play essential roles in epithelial ion transport in the kidney. The candidate will develop these skillswith the support of a primary mentor with extensive experience in fields related to the candidate?s proposed fieldof study. The candidate and her advisor are located at the University of Utah School of Medicine, a leadingacademic medical center with the substantial physical and intellectual resources necessary for the careerdevelopment of young investigators and the performance of cutting-edge research. In addition to the intensiveimmersion in research in the laboratory, the candidate will take advantage of the numerous research and careerdevelopment seminars and courses available at University of Utah to further develop her career.Project Description: Epithelial ion transport underlies essential kidney functions, such as the regulation ofextracellular volume and blood pressure and acid-base regulation. The SLC12 family of cation-chloridecotransporters, which includes sodium-chloride, sodium-potassium-two-chloride, and potassium-chloridecotransporters (NCC, NKCC, and KCC), plays a key role in renal epithelial ion transport. Loss-of-functionmutations in human NCC and NKCC2 result in hypotension due to renal sodium wasting, and these transportersare the targets of thiazide and loop diuretics, respectively. Knockout of the KCC4 gene in mice results in distalrenal tubular acidosis. Regulation of these cotransporters remains incompletely understood, but recent evidencesuggests that the WNK and SPAK/OSR1 kinases are involved. Human WNK gain-of-function mutations lead tohypertension and hyperkalemia, and loss-of-function mutations in WNK and SPAK lead to renal salt wasting andhypotension in the mouse. Data from in vitro, cell culture, and Xenopus oocyte models suggest that WNKsphosphorylate and activate SPAK and OSR1, which then phosphorylate and activate NCC and NKCCs, andoocyte data also suggest regulation of KCCs by WNKs. However, many open questions remain. The immediategoal of this project is to use the fruitfly Drosophila melanogaster to better understand the molecular mechanismsof SLC12 regulation by WNKs and SPAK/OSR1. The sophisticated genetics of the fly, its rapid life cycle, andthe well-characterized physiology of its renal tubule allows for efficient, yet detailed, study of the molecularmechanisms of epithelial ion transport in vivo. Moreover, the predominant single gene representation of mostmammalian gene families decreases combinatorial complexity and gene compensation as encountered inmammalian models. Preliminary and published data suggest a role for SLC12 cotransporters in the fly renaltubule.
The aims of this proposal are to assess whether WNK and Fray (the Drosophila SPAK/OSR1 homolog)regulate epithelial ion transport; if they do so by regulation of NKCC and/or KCC; and the detailed mechanismsby which WNK and Fray regulate NKCC versus KCC. This will be tested by assaying tubule physiology, includingmeasurement of transepithelial potential and potassium flux, rates of urine secretion, and lethality of adult flieson high-potassium food, as well as in vitro and cell culture assays of protein-protein interactions and kinaseactivity. The differences between the four mammalian WNK isoforms will be explored by their introduction intoflies lacking endogenous WNK. Finally, the importance of specific phospho-serines and phospho-threonines onthe in vivo functioning of fly and mammalian NKCC and KCC will be tested by assaying tubule physiology in fliesexpressing mutant transporters. Insights gained from these studies can in future be directly tested in mousemodels, with the long-term goal of better understanding mammalian renal physiology and human disorders suchas hypertension and distal renal tubular acidosis.
SLC12 cotransporters are important in the kidney's ability to regulate blood pressure and acid-base balance inthe body. How these cotransporters are regulated is not well understood. This project seeks to betterunderstand the molecular mechanisms by which these cotransporters work and how they are regulated; toimprove understanding and treatment of high blood pressure and disorders of acid-base balance.
Showing the most recent 10 out of 15 publications
