Synergistical effect of 20-HETE and high salt on NKCC2 protein and blood pressure via ubiquitin–proteasome pathway

Abstract

We previously generated a cytochrome P450 4F2 (CYP4F2) transgenic mouse model and demonstrated that overexpressed CYP4F2 and overproduced 20-HETE in the kidneys contribute to the increase of blood pressure in the CYP4F2 transgenic mice with normal salt intake. We currently expect to elucidate a potential mechanism of salt-related hypertension whereby diverse levels of 20-HETE interact with dietary salt on Na(+)-K(+)-2Cl(-) cotransporter, isoform 2 (NKCC2) in the kidneys of the transgenic and wild-type mice with high salt intake. High salt intake reduced about 85 % abundance of renal NKCC2 protein in the transgenic mice and about 24 % in the wild-type mice by Western blot. Furthermore, we first found that NKCC2 was ubiquitinated and interacted with Nedd4-2 by immunoprecipitation in the transgenic mice with high salt intake. In addition, inhibition of 20-HETE synthesis or proteasome activity reversed the reduction of NKCC2 expression induced by 20-HETE and high salt intake. These results suggest that 20-HETE and high salt intake synergistically decrease the expression of NKCC2 protein via Nedd4-2-mediated ubiquitin-proteasome pathway, and thereby modulate natriuresis and blood pressure. We propose that diverse levels of 20-HETE have diverse effects on blood pressure in different salt concentrations.

OS9 Protein Interacts with Na-K-2Cl Co-transporter (NKCC2) and Targets Its Immature Form for the Endoplasmic Reticulum-associated Degradation Pathway

“…This is of particular interest because NKCC2 protein expression is down-regulated by high salt intake (4) and in the aged kidney (7). Consequently, an increase in OS9 expression in response to ER stress could contribute to the down-regulation of NKCC2 expression via the ubiquitin-proteasome pathway under these conditions (4,7).…”

“…Moreover, although distinct cellular mechanisms may account for NKCC2 loss of function in BS1 disease, our preliminary data in the laboratory revealed that ER-associated protein degradation is the most common mechanism underpinning BS1. 4 Consequently, understanding the involvement of this process in BS1 is essential to provide novel therapeutic strategies for the treatment of BS1. Indeed, the identification and selective modulation of ERAD components specific to NKCC2 and its disease causing mutants might help to define a strategy to release fractions of misfolded mutant proteins from the ER by preventing their interactions with the quality control components to rescue their surface expression and restore their functional activity.…”

“…Each bar represents the mean Ϯ S.E. rates of cell pH recovery (dpH i /dt in pH units/min) from NH 4 ϩ -induced alkaline load of three independent experiments. #, p Ͻ 0.03 versus NKCC2 alone (n ϭ 3).…”

“…The initial rate of intracellular pH recovery (dpH i /dt) was measured over the first 20 s of recordings as reported earlier (36,38). The dpH i caused by NH 4 Cl addition was used to calculate the cell buffer capacity, which was not different between the studied groups (data not shown). Hence, the Na-K-2Cl cotransporter transport activity is expressed as dpH i /dt.…”

“…Briefly, cells were first bathed at 37°C in a CO 2 -free Hepes/Tris-buffered medium to measure baseline pH i . 20 mM NH 4 Cl was then added to the medium, causing a very rapid initial cellular alkalinization followed by a pH i recovery. The initial rate of intracellular pH recovery (dpH i /dt) was measured over the first 20 s of recordings as reported earlier (36,38).…”