Background

Dietary sodium and potassium affect the fluctuation in blood pressure (BP) and renal function. Corin, with its enzymatic activity to convert pro-atrial natriuretic peptide (pro-ANP) to biologically active ANP, regulates BP, cardiac, and renal functions. We investigated whether corin expression responds to a high-salt (HS) diet to regulate salt and water balance.

Methods

Forty-two volunteers followed 3 sequential diets for 7 days each: a low-salt (LS) diet (3.0 g/day NaCl), a HS diet (18.0 g/day NaCl), followed by an HS diet with K+ supplementation (HS + K+) (18.0 g/day NaCl and 4.5 g/day KCl).

Results

Corin level was higher with the HS diet than the LS and HS + K+ diets and was positively correlated with systolic BP (SBP) and 24-hour urinary Na+ and microalbumin (U-mALB) excretion. In rodents, serum and renal levels of corin were transiently increased with the HS diet and were decreased if the HS diet was continued for up to 7 days. HS loading increased SBP, 24-hour urinary Na+, U-mALB excretion, and the expression of proprotein convertase subtilisin/kexin-6 (PCSK6), a corin activator. Knockdown of PCSK6 or corin in high salt-treated M1-cortical collecting duct (M1-CCD) cells increased the expression of aquaporin 2 (AQP2) and β-epithelial Na+ channel (β-ENaC).

Conclusions

Short-term HS may induce the PCSK6-corin-ANP-AQP2/β-ENaC pathway in the kidney. Enhanced serum corin level in humans and rodents is positively correlated with HS-induced SBP and 24-hour urinary Na+ and U-mALB excretion, which suggests that corin is involved in the salt-water balance in response to HS intake.

Clinical trials registration

Public Trials Registry Number NCT02915315.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects cholesterol homeostasis by targeting hepatic LDL receptor (LDLR) for lysosomal degradation. Clinically, PCSK9 inhibitors effectively reduce LDL-cholesterol (LDL-C) levels and the incidence of cardiovascular events. Because microRNAs (miRs) are integral regulators of cholesterol homeostasis, we investigated the involvement of miR-483 in regulating LDL-C metabolism. Using in silico analysis, we predicted that miR-483-5p targets the 3'-UTR of PCSK9 mRNA. In HepG2 cells, miR-483-5p targeted the PCSK9 3'-UTR, leading to decreased PCSK9 protein and mRNA expression, increased LDLR expression, and enhanced LDL-C uptake. In hyperlipidemic mice and humans, serum levels of total cholesterol and LDL-C were inversely correlated with miR-483-5p levels. In mice, hepatic miR-483 overexpression increased LDLR levels by targeting Pcsk9, with a significant reduction in plasma total cholesterol and LDL-C levels. Mechanistically, the cholesterol-lowering effect of miR-483-5p was significant in mice receiving AAV8 PCSK9-3'-UTR but not Ldlr-knockout mice or mice receiving AAV8 PCSK9-3'-UTR (ΔBS) with the miR-483-5p targeting site deleted. Thus, exogenously administered miR-483 or similarly optimized compounds have potential to ameliorate hypercholesterolemia.

Background

Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II, a potent vasoconstrictor, to angiotensin-(1-7) and is also a membrane protein that enables coronavirus disease 2019 (COVID-19) infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of posttranslational modification of ACE2 in vascular endothelial cells is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein MDM2 (murine double minute 2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the posttranslational modification of ACE2 through its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH.

Methods

Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH.

Results

Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH.

Conclusions

Maladapted posttranslational modification (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.

Rationale

Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive.

Objectives

We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH.

Methods

Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout (ACE2^-/-) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK-p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension.

Measurements and main results

Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (angiotensin) 1-7 and endothelial nitric oxide synthase-derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2-knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell-specific deletion of AMPKα2. Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension.

Conclusions

Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.