Hypertension represents a major public health concern. To date, each form of inherited hypertension has been linked to defective salt handling in the kidney. This application seeks to explore the mechanisms involved in the regulation of one of the key salt-handling transporters in the kidney, the sodium chloride co- transporter (NCC) located in distal convoluted tubule. Previous studies have reported decreased NCC activity in response to the presence of WNK4 (a kinase associated with a form of genetic hypertension called Gordon's Syndrome) and phorbol esters (chemical analogs of diacylglycerol, a common pathway for hormonal stimulation) but the mechanism by which NCC activity is reduced is unclear. This application will seek to demonstrate that WNK4 and phorbol esters decrease NCC activity by decreasing the amount of NCC expressed at the cell surface, a process that occurs by enhancing the ubiquitination of NCC, thereby promoting degradation of the co-transporter. The experimental model will be a cell model utilizing an immortalized mouse distal convoluted tubule cell line with native NCC activity and intact regulatory pathways. The association between activity, surface expression, and ubiquitination will be established by measuring NCC activity via radiotracer uptake, NCC cell surface expression via biotinylation and immunofluorescence, and ubiquitination via immunoprecipitation and immunoblotting in cells gene-silenced for WNK4 or treated with phorbol esters. The mechanisms of NCC ubiquitination will also be determined, including sites of ubiquitination and required enzymes. This project will help train Dr. Benjamin Ko as a physician-scientist and facilitate his transition to an independent investigator and contributing member of the scientific community. Public Health Relevance Hypertension represents a tremendous public health concern with staggering morbidity and mortality. As inherited hypertension is intimately tied to sodium reabsorption and many of our treatments for hypertension inhibit NCC activity, understanding the regulation of NCC activity will give us insight into the pathogenesis of hypertension and may one day provide further treatment avenues to control hypertension.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (ZDK1-GRB-S (M2))
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Rankin, Tracy L
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University of Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
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Richards, Jacob; Ko, Benjamin; All, Sean et al. (2014) A role for the circadian clock protein Per1 in the regulation of the NaCl co-transporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells. J Biol Chem 289:11791-806
Chavez-Canales, Maria; Arroyo, Juan Pablo; Ko, Benajmin et al. (2013) Insulin increases the functional activity of the renal NaCl cotransporter. J Hypertens 31:303-11
Arroyo, Juan Pablo; Lagnaz, Dagmara; Ronzaud, Caroline et al. (2011) Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway. J Am Soc Nephrol 22:1707-19
Ko, Benjamin; Cooke, Leslie L; Hoover, Robert S (2011) Parathyroid hormone (PTH) regulates the sodium chloride cotransporter via Ras guanyl releasing protein 1 (Ras-GRP1) and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) pathway. Transl Res 158:282-9
Ko, Benjamin; Kamsteeg, Erik-Jan; Cooke, Leslie L et al. (2010) RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter. Am J Physiol Renal Physiol 299:F300-9
Ko, Benjamin; Hoover, Robert S (2009) Molecular physiology of the thiazide-sensitive sodium-chloride cotransporter. Curr Opin Nephrol Hypertens 18:421-7