Renal NKCC2 Is Dual Regulated by the Synergy of 20-HETE and High-Fat Diet in CYP4F2 Transgenic Mice

Abstract

<b><i>Introduction:</i></b> 20-Hydroxyeicosatetraenoic acid (20-HETE) is the metabolite of cytochrome P450, which modulates blood pressure by inhibiting renal sodium transport. However, the molecular mechanisms underlying the role of 20-HETE in the development of obesity-related hypertension remain unclear, necessitating this study. <b><i>Methods:</i></b> Cytochrome P450 4F2 (<i>CYP4F2</i>) transgenic mice fed high-fat diet (HFD) were used as research animal models. The expression of renal ion transport molecules targeted by 20-HETE was evaluated by real-time PCR and Western blot (WB). The regulatory effect of 20-HETE and HFD on renal Na⁺-K⁺-2Cl⁻ cotransporter, isoform 2 (NKCC2) was explored by immunoprecipitation, WB, and luciferase assay. <b><i>Results:</i></b> A 2-week HFD feeding dramatically decreased protein abundance but increased renal <i>NKCC2</i> mRNA expression in <i>CYP4F2</i> transgenic mice. The decrease in NKCC2 protein was demonstrated to be due to ubiquitination induced by the synergy between 20-HETE and HFD. The increased PPAR-γ protein in <i>CYP4F2</i> transgenic mice fed HFD and the activation of rosiglitazone on the luciferase reporter construct of the <i>NKCC2</i> promoter demonstrated that the increase in <i>NKCC2</i> mRNA in <i>CYP4F2</i> transgenic mice fed HFD was a consequence of elevated PPAR-γ protein induced by the synergy between 20-HETE and HFD. <b><i>Conclusions:</i></b> Our data demonstrated that the synergy between 20-HETE and HFD could decrease NKCC2 protein via posttranslational ubiquitination, which was thought to be the main mechanism underlying the short-term effect in response to HFD and might be responsible for the adaptive modulation of renal NKCC2 to resist sodium retention. Moreover, the increased <i>NKCC2</i> mRNA expression via PPAR-γ-induced transcriptional regulation was thought to be the main mechanism underlying the long-term effect in response to HFD and plays a pivotal role in the development of obesity-related hypertension.