Candidate: Gustavo Ares, Ph.D., is a Research-Scientist Instructor training in the integrative renal physiology related to hypertension at Henry Ford Health system. In this revised application, Dr. Ares aims to determine whether a high salt diet stimulates the E3 ubiquitin-ligase FBXL13-NKCC2 interaction, enhancing NKCC2 degradation, favoring NaCl excretion thereby preventing an increase in blood pressure. Dr. Ares' immediate goal is to acquire the research training and professional skills necessary to transition to an independent extramurally funded investigator. His long-term goal is to establish his own research program with a focus on identifying novel targets of loops diuretics and pharmacological interventions to treat hypertension. Dr. Ares' Career Development Plan consists of improving his: 1) research skills; 2) Networking and collaborations; 3) professional development through attendance of presentations at weekly journal clubs, seminars, course and national scientific meetings; 4) mentoring skills; 5) writing manuscripts and grants. Dr. Ares' progress will be assessed thru bi-weekly to monthly meetings with each member of the mentoring team. Environment: Dr. Ares and his mentor, have assembled a strong team of co-mentors and advisors to guide him through the research project. Primary mentor: Dr. Pablo Ortiz a NIH-funded scientist with strong records of successful in renal physiology and hypertension. Secondary mentor: Dr. Jeffery L. Garvin is a professor of physiology at CWRU Cleveland, with extensive experience in regulation of salt and water transport along the renal nephron. Secondary mentor: Dr. Peter Kaiser, professor and Chair of Biological Chemistry at UC Irvine, has extensive experience in the ubiquitin-proteasome system, and the E3-ubiquitin ligases with special interest in the Skp1- Cullin-FBox family complex. The supportive research team includes T. Pavlov, Ph.D. and Mariela Mendez, Ph.D. working in renal physiology; Pamela Harding Ph.D., N-E. Rhaleb, Ph.D., Suresh Palaniyandi, Ph.D. working in cardiovascular physiology/pathophysiology. Research: Hypertension is a highly prevalent condition involving the kidney's inability to excrete excess salt. Abnormally enhanced NaCl reabsorption thru the apical Na+/K+/2Cl- cotransporter (NKCC2) by the thick ascending limb of the loop of Henle (TAL) is associated with salt-sensitive hypertension in humans. NKCC2 inhibitors has many side effects, therefore they are not used. This project study the role of a novel E3 ubiquitin ligase FBXL13 on NaCl reabsorption and blood pressure regulation under normal or high salt diet. In Aim I, we study the effect of high salt on NKCC2 ubiquitination, surface phosphor (Thr96-101 and Ser126) and total NKCC2 expression and activity. In Aim II, we will study the FBXL13-NKCC2 interaction. In Aim III We study the role of FBXL13 on NaCl reabsorption and blood pressure regulation. This application will advance our knowledge which may lead to new strategies for the treatment of hypertension and promote the development of novel and specific loop diuretics.

Project Narrative/Relevance Hypertension is a growing epidemic condition that afflicts a third of the US population. We have identified the E3 ubiquitin ligase adaptor FBXL13 that interacts with the Na/K/2Cl co-transporter (NKCC2) which is associated to hypertension, but the function of this E3-ubiquitin ligase remains unclear. This proposal will elucidate the effect of high salt diet on the mechanism by which the E3 ubiquitin ligase adaptor FBXL13 regulates NKCC2- dependent NaCl reabsorption which, may lead to new strategies for the treatment of hypertension and promote the development of specific loop diuretics.