This NIH mentored Career Development Award proposal describes a five year training program for the candidate, a physician scientist with the long-term goal of becoming an independent academic investigator with a research focus on epithelial ion transport related to kidney physiology and diseases. The candidate proposes to build on a background in basic research developed during undergraduate, Ph.D., and fellowship studies, and in particular previous experience studying Drosophila melanogaster, by developing new scientific skills in physiology and biochemistry. These will be applied to the immediate goal of understanding the molecular mechanisms of regulation of SLC12 cation-chloride cotransporters by the WNK and SPAK/OSR1 kinases, which play essential roles in epithelial ion transport in the kidney. The candidate will develop these skills with the support of a primary mentor, a co-mentor, and advisory committee members with extensive experience in fields related to the candidate's proposed field of study. The candidate and her advisors are located at the University of Texas Southwestern Medical Center, a leading academic medical center with the substantial physical and intellectual resources necessary for the career development of young investigators and the performance of cutting-edge research. In addition to the intensive immersion in research in the laboratory, the candidate will take advantage of the numerous research and career development seminars and courses available at UT Southwestern to further develop her career. Project Description: Epithelial ion transport underlies essential kidney functions, such as the regulation of extracellular volume and blood pressure and acid-base regulation. The SLC12 family of cation-chloride cotransporters, which includes sodium-chloride, sodium-potassium-two-chloride, and potassium-chloride cotransporters (NCC, NKCC, and KCC), plays a key role in renal epithelial ion transport. Loss-of-function mutations in human NCC and NKCC2 result in hypotension due to renal sodium wasting, and these transporters are the targets of thiazide and loop diuretics, respectively. Knockout of the KCC4 gene in mice results in distal renal tubular acidosis. Regulation of these cotransporters remains incompletely understood, but recent evidence suggests that the WNK and SPAK/OSR1 kinases are involved. Human WNK gain-of-function mutations lead to hypertension and hyperkalemia, and loss-of-function mutations in WNK and SPAK lead to renal salt wasting and hypotension in the mouse. Data from in vitro, cell culture, and Xenopus oocyte models suggest that WNKs phosphorylate and activate SPAK and OSR1, which then phosphorylate and activate NCC and NKCCs, and oocyte data also suggest regulation of KCCs by WNKs. However, many open questions remain. The immediate goal of this project is to use the fruitfly Drosophila melanogaster to better understand the molecular mechanisms of SLC12 regulation by WNKs and SPAK/OSR1. The sophisticated genetics of the fly, its rapid life cycle, and the well-characterized physiology of its renal tubule allows for efficient, yet detailed, study of the molecular mechanisms of epithelial ion transport in vivo. Moreover, the predominant single gene representation of most mammalian gene families decreases combinatorial complexity and gene compensation as encountered in mammalian models. Preliminary and published data suggest a role for SLC12 cotransporters in the fly renal tubule.
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 mechanisms by which WNK and Fray regulate NKCC versus KCC. This will be tested by assaying tubule physiology, including measurement of transepithelial potential and potassium flux, rates of urine secretion, and lethality of adult flies on high-potassium food, as well as in vitro and cell culture assays of protein-protein interactions and kinase activity. The differences between the four mammalian WNK isoforms will be explored by their introduction into flies lacking endogenous WNK. Finally, the importance of specific phospho-serines and phospho-threonines on the in vivo functioning of fly and mammalian NKCC and KCC will be tested by assaying tubule physiology in flies expressing mutant transporters. Insights gained from these studies can in future be directly tested in mouse models, with the long-term goal of better understanding mammalian renal physiology and human disorders such as hypertension and distal renal tubular acidosis.
SLC12 cotransporters are important in the kidney's ability to regulate blood pressure and acid-base balance in the body. How these cotransporters are regulated is not well understood. This project seeks to better understand the molecular mechanisms by which these cotransporters work and how they are regulated, to improve understanding and treatment of high blood pressure and disorders of acid-base balance.
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