Nedd4-2 upregulation is associated with ACE2 ubiquitination in hypertension.

Abstract

ACE2 is a critical component of the compensatory renin-angiotensin system that is downregulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establish causal relationships, clinical relevance and test a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension. Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST) and capillary Western were used to assess the role of ACE2 ubiquitination in neurogenic hypertension.Ubiquitination was first validated as leading to ACE2 downregulation and Nedd4-2 identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to Ang-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and reduction of hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST. Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of a tonic inhibitory tone to the PVN, and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination leading to the development of neurogenic hypertension and highlight potential novel therapeutic strategies. While ACE2 conversion of Ang-II to Ang-(1-7) is supposed to limit the overactivity of the renin-angiotensin system (RAS), the enzyme is downregulated during the development of hypertension. As antihypertensive RAS blockers on the market only provide limited control of BP among hypertensive patients, understanding the mechanisms responsible for this blunted compensation provides new possible targets for the treatment of hypertension. In this study we show that Nedd4-2 upregulation is associated with ACE2 ubiquitination while prevention of this post-translational modification prevents the development of hypertension. Accordingly, targeting of ACE2 ubiquitination provides a new treatment strategy to reduce hypertension.