High salt-dependent regulation of the Na+/K+/2Cl- co-transporter (NKCC2) ubiquitination by E3-ubiquitin ligases in Thick Ascending Limb

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 oxide is decreased, yet the mechanisms involved in the development of hypertension
are poorly understood. Although we already have drugs (loop diuretics) that effectively block NKCC2 activity,
they are not used to treat hypertension due to their side effects and offside targets combined with the lose
potency over time. This proposal will explore the mechanism and signaling cascade by which high-salt diet
stimulates NKCC2 ubiquitination and will characterize a new E3-ubiquitin ligases that may play a critical role in
salt-sensitive hypertension. This application will focus on specific interactions between E3-ubiquitin ligases and
adaptors with NKCC2. Moreover, this proposal will increase the knowledge on post translational mechanism that
regulates blood pressure. The proposal also will help us to understand a post-translational mechanism that we
know little about and how it regulates NKCC2 expression, which would be crucial for the development of new
strategies for the treatment of hypertension in special salt-sensitive hypertension, which could lead to the
development of novel and specific loop diuretics.

Grant Number: 5R03DK140261-02
NIH Institute/Center: NIH
Principal Investigator: Gustavo Ares-Sarmiento