The thiazide-sensitive NaCl cotransporter (NCC) mediates salt reabsorption in the distal nephron (DN) of the kidney and is a key determinant of the blood pressure set point. With-No-Lysine (WNK) kinases regulate NCC membrane trafficking, phosphorylation status, and activity. One member of this family, WNK1, is expressed in the distal nephron as two major classes of isoforms with opposing functions. Long isoforms of WNK1 that possess intact serine-threonine kinase activity (L-WNK1) stimulate NCC. In contrast, short kidney-specific WNK1 isoforms that lack a kinase domain (KS-WNK1) inhibit NCC by antagonizing L-WNK1. Prior work indicates that the balance of these kinase-active and -defective products controls NCC activity, effectively functioning as an isoform switch. The upstream mechanisms regulating WNK1 isoform balance, however, remain obscure. Our preliminary data suggest that aldosterone is a physiologically relevant stimulus that signals through the WNK1 switch to activate NCC-mediated salt reabsorption in the kidney. This proposal is driven by three novel observations: (1) First, in DN cell lines, aldosterone increases total WNK1 protein expression, but has a stronger effect on kinase active L-WNK1 than kinase defective KS-WNK1; this triggers downstream signaling events that increase NCC plasma membrane abundance and phosphorylation. (2) Second, WNK1 isoforms enriched at the protein level in the DN contain PY motifs- sequences which bind to Nedd4-2, an E3 ubiquitin ligase whose activity is suppressed by aldosterone. (3) Third, although KS-WNK1 transcript levels are high in the DN, it is an inherently unstable protein, and comparative studies with L-WNK1 indicate striking differences in steady state expression and protein turnover. Based on these findings, we hypothesize that aldosterone increases the total protein abundance of WNK1 isoforms in the distal nephron via Nedd4-2 inhibition, adjusting their ratio to favor increased L-WNK1 activity and NCC activation. To critically test this model, we propose to answer three questions about the regulation of WNK1 isoforms that remain incompletely addressed. First, how does the inhibition of Nedd4-2 by aldosterone regulate WNK1 protein expression and NCC activation? Second, why do L-WNK1 and KS-WNK1 exhibit different protein turnover rates? Third, how does aldosterone affect the WNK1 isoform switch in vivo? Answering these questions should provide novel insights into the molecular basis of aldosterone action, NCC regulation, and blood pressure homeostasis. Completion of the proposed aims will therefore improve our understanding of the pathogenesis of essential hypertension and highlight new strategies for its treatment.

Public Health Relevance

High blood pressure places one third of the U.S. population at risk for end stage kidney disease, stroke, and death from heart disease. The kidney regulates blood pressure by controlling the rate of urinary salt excretion. This proposal seeks to understand how a blood pressure regulatory hormone, aldosterone, fine-tunes salt handling in the kidney by adjusting the balance of two molecules, L-WNK1 and KS-WNK1. By understanding how this balance is achieved, the studies will provide insights into the regulation of blood pressure in health and disease while identifying new strategies for treatment.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Special Emphasis Panel (KMBD)
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Ketchum, Christian J
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University of Pittsburgh
Internal Medicine/Medicine
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