HYPERTENSION is considered the leading cause of “loss of health” worldwide, involving the kidney’s inability to excrete excess salt. During high salt intake the kidneys excrete the extra salt load by increasing the blood pressure, phenomenon known as the pressure-natriuresis response. On the other hand, salt-sensitive hypertension is a sustained increase in blood pressure caused by an acute salt intake, which is prevalent in 50% of African-Americans and in 30% of Caucasians. Enhanced salt retention by the thick ascending limb of Henle’s loop (TAL) involving the Na+/K+/2Cl- cotransporter (NKCC2) has been described in patients and genetic animal models of salt-sensitive hypertension. However, the molecular mechanism for this defect is not fully understood. Ubiquitination is a post-translational modification that regulates expression of channels and transporters. Recently a novel E3 ubiquitin ligase adaptor F-Box leucine-rich domain 13 (FBXL13) was identified as novel locus for blood pressure regulation in humans. We found that FBXL13 recognizes and interacts with NKCC2. We found that the high salt-induced increase in NKCC2 ubiquitination is blunted in FBXL13-KO mice. Global FBXL13-KO mice show exacerbated total NKCC2 expression. However, the global FBXL13-KO mice show high levels of ubiquitinated NKCC2, indicating that other E3-ubiquitin ligases or adaptors mediates the ubiquitination of NKCC2. Moreover, global FBXL13-KO mice are not salt sensitive, nor they develop hypertension. Therefore, this R03 project aims to discover other E3-ubiquitin ligases that mediates NKCC2 ubiquitination and play a role on NaCl reabsorption and blood pressure regulation under normal or high salt diet. In Aim I, we hypothesize that high salt diet stimulates the 48-linked poli-ubiquitination of NKCC2 via multiple E3-ubiquitin ligases. This proposal is significant to human health because in most hypertensive patients and in animal models of hypertension, the natriuretic effect of nitric oxi