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Published in final edited form as: Metabolism. 2022 Feb 17;130:155165. doi: 10.1016/j.metabol.2022.155165

Endothelial sodium channel activation mediates DOCA-Salt-induced endothelial cell and arterial stiffening

Liping Zhang 1,2, Yan Yang 1, Aroor Annayya 3, Jia Guanghong 3, Zhe Sun 1, Alan Parrish 2, Garrett Litherland 1,2, Benjamin Bonnard 4, Frederic Jaisser 4, James R Sowers 1,2,3, Michael A Hill 1,2
PMCID: PMC8977070  NIHMSID: NIHMS1783680  PMID: 35183546

Abstract

Introduction:

High salt intake and aldosterone are both associated with vascular stiffening in humans. However, our preliminary work showed that high dietary salt alone did not increase endothelial cell (EC) or vascular stiffness or endothelial sodium channel (EnNaC) activation in mice, presumably because aldosterone production was significantly suppressed as a result of the high salt diet. We thus hypothesized that high salt consumption along with an exogenous mineralocorticoid would substantially increase EC and vascular stiffness via activation of the EnNaC.

Methods and Results:

Mice were implanted with slow-release DOCA pellets and given salt in their drinking water for 21 days. Mice with either specific deletion of the alpha subunit of EnNaC or treated with a pharmacological inhibitor of mTOR, a downstream signaling molecule involved in mineralocorticoid receptor activation of EnNaC, were studied. DOCA-salt treated control mice had increased blood pressure, EC Na+ transport activity, EC and arterial stiffness, which were attenuated in both the EnNaC−/− and mTOR inhibitor treated groups. Further, depletion of αEnNaC prevented DOCA-salt-induced impairment in EC-dependent vascular relaxation.

Conclusion:

While high salt consumption alone does not cause EC or vascular stiffening, the combination of EC MR activation and high salt causes activation of EnNaC which increases EC and arterial stiffness and impairs vascular relaxation. Underlying mechanisms appear to include mTOR signaling.

Keywords: Mineralocorticoid receptor activation, salt consumption, vascular stiffening, endothelial Na+ channel, DOCA-Salt

Introduction

Previous studies have shown excessive arterial stiffening to be an independent risk factor for cardiovascular disease (CVD) and hypertension, as well as a strong predictor for cardiovascular outcomes in subjects with hypertension [1]. Prior studies have further demonstrated the positive correlation between sodium intake and blood pressure, with excessive dietary salt intake being widely recognized to be a causative factor in the development of hypertension [2]. According to World Health Organization (WHO) guidelines [3] and the American Heart Association (AHA) [4], daily sodium intake for adults should not exceed 2.0 g/day or 2.3 g/day, respectively. Despite this, according to the most recent data released by CDC, from 2013–2014, average U.S. sodium intake is 3.409 g/day [5]. Moreover, based on an extensive meta-analysis, 1.65 million deaths from CVD per year globally were attributed to sodium consumption above a reference level of 2.0 g per day [6].

Sodium homeostasis is regulated by a number of transport mechanisms and ion channels. Relevant to this, the epithelial sodium channel (ENaC) which belongs to the ENaC/degenerin family of ion channels, was first identified in the renal epithelium and classically, ENaC has been demonstrated to be involved in reabsorption of filtered Na+ in the distal nephron, including the aldosterone-sensitive distal nephron and the collecting duct. Activity of renal ENaC has long been implicated in the genesis of hypertension, in particular salt-sensitive hypertension [7, 8]. More recently, the notion that ENaC expression was largely restricted to epithelial tissues was shown to be incorrect when studies demonstrated ENaC gene expression in human vascular endothelial cells [9] and the existence of a functional amiloride-sensitive channel in vascular endothelium [10, 11]. Subsequently, the endothelium sodium channel (EnNaC) has been shown to impact vascular function including flow mediated dilation, nitric oxide-mediated vasorelaxation, and arterial stiffening [12, 13]. Prior studies have shown an association between salt intake and arterial stiffening by showing that high salt consumption acutely exaggerates 2-Kidney, 1-Clip (2K1C) hypertension and promotes aortic stiffness in male C57BL/6J mice [14]. However, whether EnNaC was involved in the underlying mechanism(s) remains unclear. Further, obese, and type 2 diabetic subjects, who typically consume a western diet (WD) that is rich in saturated fat and highly refined carbohydrates, also exhibit salt sensitive related high blood pressure and increased arterial stiffness [15, 16]. Relevant to this, we previously demonstrated that arterial stiffening induced by consumption of a WD is associated with enhanced aldosterone-MR signaling in female mice [17]. We further showed that WD [18] or aldosterone [19] administration-induced arterial stiffening is mediated through EnNaC activation. Thus, in the current study, we aimed to examine the role of EnNaC in a mouse model which exhibits salt-sensitive hypertension and salt-sensitive vascular dysfunction.

In the current study, in mice, we used continuous subcutaneous infusion of deoxycorticosterone acetate (DOCA) in combination with NaCl (1%) administered in the drinking water to produce mineralocorticoid receptor (MR) activation in the presence of high salt intake. The rationale for this approach was developed from preliminary studies in wild-type mice where increased salt intake in combination with either a standard chow or a WD led to suppression of endogenous aldosterone production (by approximately 40–50%). Further, in the absence of aldosterone-induced MR activation increased salt intake (in either diet) failed to increase Na+ channel conductance and EC stiffness (see Supplementary Fig. 1 and 2). Similarly, high salt intake did not exacerbate vascular stiffening as measured by pulse wave velocity (PWV) nor attenuate nitric oxide mediated vascular relaxation as assessed by the vasodilatory response to acetylcholine (see Supplementary Fig. 1). Thus, to examine the combined effects of MR activation and increased salt intake (as occurs in salt-sensitive human subjects) the DOCA-salt model was used. We hypothesized that, in the DOCA-Salt hypertensive mouse, increased EC-MR activation results in increased activity of EnNaC, which leads to increased endothelial cell and vascular stiffness.

Materials and Methods

All animal procedures were performed in accordance with guidelines of the National Institutes of Health for the care and use of laboratory animals. All protocols were approved by the Animal Care and Use Committee of the University of Missouri-Columbia.

2.1. Animals

Male and female mice with endothelial cell-specific deletion of the alpha subunit of epithelial sodium channel (αEnNaC−/−) and control littermates (αEnNaC+/+) were generated by crossing αENaCflox/flox mice with transgenic mice expressing Cre recombinase under the control of the Tie2 promoter on a C57BL/6 genetic background, as described previously [13]. The DOCA-Salt mouse model was generated by subcutaneous implantation of slow-release DOCA pellets (50 mg, Innovative Research of America, Sarasota, FL) and the addition of NaCl in the drinking water (1% NaCl with 0.2% potassium chloride) for 21 days. Potassium chloride was added to the drinking water to prevent DOCA-Salt-induced decreases in serum potassium as reported in previous studies [20]. mTOR inhibition was accomplished by daily intraperitoneal injection with the small molecular weight inhibitor, AZD8055, (10mg/kg body weight/day, SelleckChem, Houston, TX). mTOR inhibition was investigated as a downstream signaling molecule involved in mineralocorticoid receptor activation of EnNaC [21, 22]. Overall, there were four experimental groups: αEnNaC knockout mice implanted with DOCA drinking salt water (DOCA-Salt αEnNaC−/−); littermate control mice underwent a sham implantation drinking tap water (Sham αEnNaC+/+); littermate control mice implanted with DOCA drinking salt water (DOCA-Salt αEnNaC+/+); littermate control mice implanted with DOCA drinking salt water and treated with mTOR inhibitor (DOCA-Salt+mTI αEnNaC+/+).

2.2. Tail-cuff Blood Pressure Measurement

Blood pressure was measured using a non-invasive tail-cuff system (CODA-HT2; Kent Scientific, Torrington, CT) immediately prior to initiating treatments and at the end of the study. Mice were acclimatized to physical restraint and the tail-cuff measurement procedures for four consecutive days prior to blood pressure determination. All blood pressure measurements were conducted at the same time of the day (1 to 3PM) and by the same person to limit the influence of the circadian rhythm and reduce handling variability. A minimum of ten blood pressure readings were averaged for each animal to obtain final readings of systolic and diastolic pressures.

2.3. Ultrasound Measurements

As an index of vascular stiffness, PWV was determined in the abdominal aorta using high frequency ultrasound (Vevo 2100, VisualSonics, Toronto, ON, Canada). In vivo ultrasound was performed immediately prior to initiating treatments and at the end of the study using a previously published protocol [23]. As additional indicators of stiffness, aortic distensibility and aortic radial strain were subsequently calculated using Vevo LAB and Vevo Vasc software packages (Visualsonics, Toronto, ON, Canada). To enhance rigor, ultrasound measurements were performed with the operator blinded to the mouse strain or treatment.

2.4. Atomic Force Microscopy

Aortic endothelial cell (EC) stiffness was measured in en face aortic segments using indentation atomic force microscopy (AFM) according to a previously published protocol [19]. In brief, a 2×2 mm segment of the thoracic aorta explant was obtained from mice and opened longitudinally to allow access to the endothelial surface. Repeated cycles of nano-indentation and retraction cycles on the cell surface were interrogated using an AFM cantilever (MLCT, Bruker-nano, Goleta, CA). AFM measurements were performed with the operator blinded to the mouse strain or treatment.

2.5. Wire Myography

Vascular relaxation was determined using myography as previously described [19]. Briefly, a 2 mm segment of thoracic aorta was dissected and immediately placed in ice-cold physiological salt solution (PSS, pH ~7.4). The vessel ring was then mounted under isometric conditions in a wire myograph (Danish Myo Technologies). Aortic segments were pre-constricted with U46619 (20 nM) for 20 min before assessment of vasodilation responses to acetylcholine (Ach, 10−9 – 10−4 M),) and sodium nitroprusside (SNP, 10−9 to 10−4 M). Myography studies were performed with the operator blinded to the mouse strain or treatment.

2.6. Whole Cell Patch Clamp

Fresh lung tissue was harvested from mice and dissociated using a lung dissociation kit (Miltenyi Biotec Inc., Auburn, CA, USA). Lung endothelial cells (ECs) were isolated using two step magnetic bead separation (CD45 and CD31 antibody-conjugated microbeads; Miltenyi Biotec Inc., Auburn, CA, USA). ECs were seeded on gelatin-coated glass chips and cultured for 5 to 8 days at 37°C, 5% CO2 incubator. Glass chips were subsequently transferred to a recording chamber and whole cell Na+ currents were measured from holding potential of 0 mV to potentials ranging from −80 mV to +80 mV using an EPC-10 amplifier (Heka) and Patchmaster software. Patch pipette solution contained (in mM) 40 KCl, 100 K-gluconate, 1 CaCl2, 0.1 EGTA, 4 Na2ATP, 10 Glucose, 10 HEPES, 2 GTP-Na2 (pH 7.2 with KOH). Bath solution contains 120 NaCl, 4.5 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, 10 HEPES (pH 7.2 with NaOH). As in previous studies [19, 24], ECs were treated with amiloride (1 μM) to determine the relative proportion of amiloride-sensitive Na+ currents and as a functional measure of the contribution of EnNaC to total current. Igor Pro software was used for data analysis. Patch clamp measurements were performed with the operator blinded to the mouse strain or treatment.

2.7. Histology and Immunohistochemistry

A 2 mm segment of thoracic aorta was dissected and fixed by immersion in 4% paraformaldehyde, paraffin-embedded, and transversely processed as 5μm-thick sections. Sections were coded for unbiased examination. Sections were stained with hematoxylin-eosin reagent for general histological examination. To evaluate fibrosis, additional sections were stained with picro-sirus-red. Further sections were incubated with antibodies to 3-nitrotyrosine (3-NT; Millipore, Billerica, MA) overnight and then incubated with appropriate secondary antibodies (details in Supplementary Table 2). Sections were examined under an Olympus IX51 microscope and images were taken using cellSense Dimension software with an automatic exposure time and white balance on all slides. The areas (fibrosis) and the intensities (3-NT) of staining were quantified using Image J software (NIH, Bethesda, MD). Histological determinations were performed with the operator blinded to the mouse strain or treatment.

2.9. Statistical analysis

Data are shown as mean ±SEM. Differences in outcomes were determined using one-way ANOVA together with Dunnett’s multiple comparisons analysis and paired-t test after passing normality testing. When data did not conform to a Gaussian distribution, the Brown-Forsythe and Welch ANOVA was used. A value of P<0.05 was considered statistically significant. Figures were generated and statistical analyses were performed using GraphPad Prism 8.0 software.

Results

3.1. Characteristics of animal groups

No obvious sex differences were observed in BP, ultrasound, wire myography, patch clamp or histology and immunohistochemistry measurements. Therefore, female and male data have been combined. Plasma sodium levels were significantly increased in DOCA-Salt αEnNaC+/+ mice compared to all other groups, while plasma potassium levels were similar across groups (Supplementary Table 1). Of note, there were no significant differences in fasting (4 hrs) glucose, fasting insulin,or homeostatic model assessment-insulin resistance (HOMA-IR) among the groups (Supplementary Table 1). Food intake was similar in all groups, while all DOCA-Salt treated groups exhibited polydipsia, particularly within the first week of treatment (Supplementary Fig. 3). These observations are consistent with previous reports [25, 26] supporting the validity of the model.

After the 21-day treatment period, DOCA-Salt administration significantly increased blood pressure in αEnNaC+/+ mice, which was prevented by αEnNaC deletion or mTOR inhibition (Fig. 1A–B). Picrosirius red staining of aortic sections, an indicator of fibrosis, was increased in all groups of DOCA-salt treated mice (Fig. 2A and C). 3NT staining, an indicator of oxidative stress, tended to be increased in aorta segments from mice treated with DOCA-Salt, alone (Fig. 2B and C).

Figure 1: DOCA-Salt-induced increases in blood pressure were prevented by αEnNaC deletion or mTOR Inhibition.

Figure 1:

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(A) Systolic blood pressure measured before and at the end of each treatment. (B) Mean blood pressure measured before and at the end of each treatment. n=8–15 mice per treatment group. * P<0.05, compared to Sham αEnNaC+/+; # p<0.05, compared to DOCA-Salt αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis. N.S. indicates no significant differences.

Figure 2: DOCA-Salt-induced aortic fibrosis.

Figure 2:

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(A) Group data showing the calculated area of Picrosirius Red (PSR) staining in cross sections of aorta. n=7–12 mice per treatment group. * p<0.05, compared to Sham αEnNaC+/+ using Brown-Forsythe and Welch ANOVA. (B) Group data showing the intensity of 3-nitrotyrosine (3-NT) staining in cross sections of aorta. n=6–11 mice per treatment group. (C) Representative images for PSR and 3-NT staining together with the regions of interest (ROI) used for quantification. In the 3-NT images lumen of the aorta are indicated and arrows point towards areas of positive endothelial staining.

3.2. DOCA-Salt-induced increases in arterial stiffness were prevented by either αEnNaC deletion or mTOR inhibition

To evaluate arterial stiffness, high frequency ultrasound measurements were performed on all experimental groups immediately before and at the end of the respective treatments. Increased aortic PWV, decreased aortic distensibility and radial strain were observed in DOCA-Salt αEnNaC+/+ mice compared to pre-treatment levels. There were no significant differences in these parameters when comparing pre to post treatment values in the other experimental groups.

3.3. DOCA-Salt-induced increases in aortic intimal cellular stiffness were prevented by either αEnNaC deletion or mTOR inhibition.

We further evaluated stiffness of the endothelium by en face indentation AFM using freshly prepared aortic explants. The data show that endothelial stiffness was increased in DOCA-Salt treated αEnNaC+/+ mice when compared to sham αEnNaC+/+ mice (Fig. 3D). In contrast, the DOCA-Salt induced increase in aortic endothelium stiffness was prevented by either αEnNaC deletion or mTOR inhibition (Fig. 3D).

Figure 3: αEnNaC deletion or mTOR inhibition prevented DOCA-Salt-induced increase in arterial and endothelium stiffness.

Figure 3:

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(A) Aortic pulse wave velocity measured in vivo. (B) Aortic distensibility measured in vivo. (C) Aortic radial strain measured in vivo. (D) In Situ aortic endothelium stiffness measured by atomic force microscopy. n=9–12 mice per treatment group for ultrasound measurements. n=7–11 mice per treatment group for AFM measurements. ‡ P<0.05, using paired t-test between before and after respective treatments; * P<0.05, compared to Sham αEnNaC+/+; # p<0.05, compared to DOCA-Salt αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis.

3.4. DOCA-Salt-induced increases in EC Na+ currents were prevented by αEnNaC deletion or mTOR inhibition

The impact of DOCA-Salt treatment on endothelial Na+ currents was determined using whole cell patch clamp on isolated lung ECs. After 21 days of DOCA-Salt treatment, a significant increase in inward Na+ current was observed in ECs isolated from DOCA-Salt αEnNaC+/+ mice compared to those of sham αEnNaC+/+ mice (Fig. 4A–C). In contrast, EC Na+ currents were attenuated in DOCA-Salt αEnNaC−/− mice compared to similarly treated αEnNaC+/+ mice. Moreover, the increase in EC Na+ currents in DOCA-Salt treated αEnNaC+/+ mice was prevented by treatment with the mTOR inhibitor (Fig. 4A–C). As shown in previous studies [19, 27] and Supplementary Figure 4, amiloride (1 μM) inhibited inward currents to a similar exent (> 75%) in all groups, consistent with a major contribution of EnNaC to total whole cell currents that was proportionally similar despite treatment (Supplementary Fig. 4). Taken together, these data suggest that MR/mTOR-mediated EnNaC activation is required for DOCA-Salt enhancement of inward EC sodium current.

Figure 4: DOCA-Salt-induced increases in EC Na+ currents were prevented by αEnNaC deletion or mTOR Inhibition.

Figure 4:

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(A) Na+ current tracings. (B) Peak inward Na+ currents measured at −80 mV. n=8–15 mice per treatment group, and 6–9 cells per mouse. * P<0.05, compared to Sham αEnNaC+/+; # p<0.05, compared to DOCA-Salt αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis.

3.5. DOCA-Salt-induced impairment of aortic relaxation was attenuated in EnNaC−/− mice or by mTOR inhibition.

To evaluate vascular function, we examined aortic relaxation using isometric wire myography. DOCA-Salt treatment impaired both endothelium-dependent relaxation to acetylcholine (Ach) and endothelium-independent relaxation as assessed by sodium nitroprusside (SNP) (Fig. 5A–D). DOCA-Salt treatment significantly attenuated the efficacy of the Ach-induced vasorelaxation both in terms of normalized (% maximum relaxation; Fig. 5A) and absolute (mN; Fig. 5C) responses. Both αEnNaC−/− and mTOR inhibitor treated mice showed an attenuation of the DOCA-Salt-induced impairment in endothelium-dependent aortic relaxation (Fig. 5A and C). Concentration-dependent relaxation to the endothelial independent vasodilator, SNP, was similar in all DOCA-Salt treatment groups (Fig. 5B). As with the response to Ach, absolute relaxtion (Fig. 5D) was decreased in the DOCA-Salt EnNaC+/+ group (Fig. 5D). These data are consistent with aldosterone/MR-mediated EnNaC activation mediating a relatively specific impairment in nitric oxide-mediated vasorelaxation in this model, as previously observed with WD feeding [17, 18, 28].

Figure 5: Deletion of αEnNaC attenuated DOCA-Salt-induced impairments in endothelium-dependent aortic relaxation.

Figure 5:

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(A) Concentration dependent relaxation of isolated aortic rings in response to the endothelial-dependent agent, acetylcholine (Ach). Data are normalized as percent maximal response. (B) Concentration dependent relaxation of isolated aortic rings in response to the endothelial-independent agent, sodium nitroprusside (SNP). Data are normalized as percent maximal response. (C) Maximal relaxation to Ach in absolute terms (mN). (D) Maximal relaxation to SNP in absolute terms (mN). n= 4–8 mice per treatment group. Black * p<0.05, all the other groups compared to Sham αEnNaC+/+; red * p<0.05, DOCA-Salt αEnNaC+/+ compared to Sham αEnNaC+/+; black # p<0.05, all the other group compared to DOCA-Salt αEnNaC+/+; Orange # p<0.05, DOCA-Salt αEnNaC−/− compared to DOCA-Salt αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis.

Discussion

Excessive dietary salt intake has been widely shown to increase blood pressure in human subjects. However, as salt-related changes in blood pressure have shown significant variability among individuals in the general population, subpopulations showing significant blood pressure elevation with increasing salt intake have been referred to as exhibiting salt-sensitivity [2]. Under physiological conditions a high level of dietary sodium would be expected to suppress the renin-angiotensin-aldosterone system (RAAS) and decrease aldosterone levels, contributing to reduced renal sodium reabsorption. However, such suppression has been reported to be attenuated in salt-sensitive individuals, potentially leading to renal damage and salt-sensitive hypertension [29]. Indeed, studies have shown that a high salt diet completely suppresses plasma aldosterone in Dahl salt-resistant rats while increasing plasma aldosterone levels in Dahl salt-sensitive rats [30]. Further, obese populations who manifest high plasma aldosterone levels are often salt sensitive [31]. In concert with these observations prior studies have shown that diet-induced obesity is accompanied by elevated plasma aldosterone levels [31, 32], increases in EnNaC activity and EC stiffness, which were prevented in EnNaC−/− mice fed a WD [18]. Accordingly, findings in the current investigation are consistent with these previous observations suggesting that both MR activation as well as high salt are needed to increase EC and vascular stiffness and elevate blood pressure (ie. salt sensitivity).

In a preliminary investigation we evaluated the impact of a high salt consumption added either to a normal chow or a WD to determine if high salt intake exacerbated WD-induced increases in EnNaC activity, EC or vascular stiffness. However, the addition of high salt did not increase EnNaC activity (Supplementary Fig. 2A–B), EC or arterial stiffness (Supplementary Fig. 1A–D) in either study group. We attribute this finding to the fact that plasma aldosterone levels were substantially suppressed by high salt feeding. This notion is consistent with prior in vitro observations that both salt and MR activation were necessary for increased EC stiffness [33]. Consistent with our conclusions it was previously observed that feeding Sprague-Dawley (SD) male rats a high salt diet (8%, 3 weeks) leads to a significant increase in blood pressure and a decrease in plasma aldosterone levels together with a reduction in EnNaC activity and expression due to feedback inhibition, which suggested an important role for EnNaC activation in allowing the vasculature to adapt to high salt conditions [34]. Further, another study showed incubation of ECs in an aldosterone-free high-sodium (150 mM, 72h) medium significantly enhanced αEnNaC abundance and stiffened the EC cortex compared to low-sodium conditions, which were prevented by addition of the aldosterone synthase blocker FAD286, suggesting local aldosterone synthesis and MR-dependent signaling which operates in a feedforward manner [35]. In contrast, this group later showed that a high salt diet (16 mg NaCl per gram of body weight, 14 days) significantly increased EC αEnNaC abundance at the plasma membrane and enhanced cortical stiffness in 129/SvEv wildtype and aldosterone synthase (Cyp11b2)-deficient mice [36]. These data suggest that high salt could conceivably increase EnNaC activity and EC stiffening by both aldosterone-dependent and independent effects. While the current studies were not designed to examine the impact of high salt in the absence of aldosterone/MR activation, some of the contrasting findings in such studies may relate to differences in species/strains as well as quantity and duration of salt treatment.

In the current investigation we utilized a DOCA-Salt mouse model, which exhibits an increased mineralocorticoid levels [25], and thus replicates a situation of high salt consumption in salt sensitive humans who do not manifest appropriate suppression of aldosterone production. Our data showed that DOCA-Salt administration increased blood pressure and arterial stiffening as indicated by PWV, aortic distensibility and aortic radial strain, which were attenuated by αEnNaC deletion. Moreover, DOCA-Salt-induced EC stiffening as measured in aortic explants by AFM was also decreased in αEnNaC knockout mice compared to control mice receiving DOCA-Salt, alone. Taken together, these data suggest that DOCA-Salt-induced arterial stiffening is mediated via a pathway involving MR activation of EnNaC.

Previous studies have shown that mammalian target of rapamycin (mTOR) activation is essential for the phosphorylation/activation of SGK-1 and downstream activation of ENaC. Specifically, mTORC2, but not mTORC1, is required for Na+ transport in kidney epithelial cells [37]. In vivo studies have further shown that mTORC2 regulates renal tubule sodium uptake through SGK1-dependent modulation of ENaC activity in C57BL/6 mice [21]. In the current study, treatment of mice with a pharmacological inhibitor of mTOR prevented DOCA-Salt-induced increases in blood pressure, ENaC currents and endothelial and aortic stiffness. This is consistent with the notion that inhibition of mTOR/SGK-1 signaling blunts the salt sensitivity arising from increased MR activation. Taken together, these observations provide further support that DOCA-Salt-induced EC and arterial stiffening is mediated via mTOR/SGK-1-mediated regulation of EnNaC.

A limitation of the current study is the inability to determine the temporal and causal relationship between blood pressure and arterial stiffening in our experimental models as our data showed that DOCA-Salt hypertensive mouse manifested increased arterial stiffness, while deletion of αEnNaC or mTOR inhibition decreased both blood pressure and arterial stiffening. Data from human subjects have, however, shown that consumption of a high salt diet can lead to EC dysfunction independent of changes in blood pressure [38]. Studies have also indicated the causality between arterial stiffening and hypertension is complex due to many confounding factors (e.g., aging, diet, concurrent disease, etc.) [39]. It is suggested that arterial stiffening is a precursor for hypertension while on the other hand arterial stiffness increases in hypertension due to increases in distension pressure [40]. Thus, whether arterial stiffness acts as a cause or a consequence of hypertension is still unclear. Further, clinical studies and various animal models have shown a consistent temporal sequence of arterial stiffening preceding hypertension [39]. However, whether increased arterial stiffness alone can lead to hypertension requires further exploration.

In addition, the comparatively short duration (21 days) of our study likely lessened the development of marked fibrosis consistent with changes in vascular stiffness being an early event. Importantly, vascular stiffening results from changes to both the active and passive properties of blood vessels [41] and thus changes in vascular reactivity were observed in the present study (Fig. 5A). Nevertheless, longer during studies may assist in further delineating underlying mechanisms. On the other hand, previous studies [42, 43] have suggested a link between EnNaC expression and mechanical properties of the endothelium, specifically that increased EnNaC activity negatively regulates nitric oxide (NO) production. A deficiency in NO might be expected to contribute to overt vascular stiffening through multiple mechanisms possibly through VSM tone and altered interactions between VSMC and matrix proteins [27, 44, 45]. Other studies [46, 47] have previously proposed that endothelial stiffening is EnNaC-mediated by a mechanism involving the interaction of the C-terminus of the α subunit with F-actin in the membrane cortical cytoskeleton. Further investigation is required to determine whether mTOR or EnNaC mediate eNOS activation and remodeling of the cytoskeleton by triggering the polymerization of G-actin to F-actin under the high aldosterone and high salt conditions of the current study.

In the current study, we combined the data for both male and female mice since we did not observe any obvious sex differences in DOCA-Salt effects on blood pressure, EC or arterial stiffening. Of note, large population studies have shown that salt sensitivity is clinically more evident in women than in men. In contrast to clinical reports, most animal studies of salt-sensitive hypertension show an opposing sex discrepancy, in which the males were more susceptible to blood pressure changes in response to excess dietary salt [48–50]. However, one study using Balb/C mice demonstrated a high salt diet to cause salt-sensitive hypertension in females but not in the male mice due to lack of suppression of aldosterone production [48], which is more analogous to the human situation. Additional experiments are needed to explore the sexual dimorphism on the DOCA-Salt model using age-matched and blood pressure normalized animals. A further limitation of this study is the lack of specifically targeting EC SGK-1, particularly as SGK-1 has been implicated in ENaC regulation in multiple tissues [37, 51–53]. Future studies, therefore, require the development of an EC-specific SGK-1 knockout model, which will allow more insight into the exact role of SGK-1 in aortic and vascular cell stiffening.

In regard to methodological/technical limitations, we note that EnNaC activity can be modulated via regulation of channel conductance, open probability, number of channels on the cell surface by regulation of gene/protein synthesis, trafficking to the membrane or ubiquitination which lead to internalization and degradation [27]. The increase in the Na+ current could be due to either of the possibilities listed above or a combination of them. While the exact mechanism cannot be defined based on the whole cell patch clamp, future experiments are required to determine single channel conductance and open probability using single channel patch clamp and plasma membrane localization of EnNaC using approaches such as cell biotinylation. Furthermore, we are aware of the limitations of using AFM nanoindentation on en face aortic explants to assess endothelium stiffness in that the mechanical characteristics of the underlying vascular smooth muscle cells and extracellular matrix may also contribute to the magnitude of stiffness. Similarly, interactions between elements of the vessel wall likely impact EC stiffness (for example through modulation of cellular adhesion) [44, 45]. Despite this, the nanoindentation approach occurs to a depth of approximately 130 – 160 nm which would be expected to limit measurement to properties of the endothelial cell cortical cytoskeleton.

In summary, our data suggest that in the presence of both MR activation and high salt consumption activation of EnNaC contributes to EC and arterial stiffening. This appears to involve mTOR signaling which is known to stimulate SGK-1-mediated increases in Na+ channel at EC surface and associated increases in channel activity [37]. We further conclude that the data are relevant to situations of enhanced aldosterone secretion and MR activation in concert with high salt consumption, as seen with salt-sensitive hypertension and resistant hypertension.

Supplementary Material

1

Supplementary Table 1: Characteristics of mice. Values are shown as Mean ± SEM. n = 8–15 mice per treatment group. * p<0.05 compared to Sham αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Table 2: Chemicals and Reagents Used.

2

Supplementary Figure 1: High salt did not exaggerate western diet-induced increases in arterial stiffness, endothelium stiffness or aortic relaxation (A) Aortic pulse wave velocity measured in vivo. (B) In situ endothelium stiffness measured ex vivo using atomic force microscopy. (C) Response of isolated aortic rings to endothelium-dependent dilator, acetylcholine and (D) endothelium-independent dilator, sodium nitroprusside. ‡ p<0.05, CD compared to WD; $ p <0.05, CD+HS compared to WD+HS using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Figure 2: High salt suppress inward sodium currents. Peak inward Na+ currents measured at −80 mV. * p<0.05, compared to CD; # p<0.05, compared to WD using one way ANOVA with Dunnett’s multiple comparison analysis..

Supplementary Figure 3: Characteristics of mice. (A) Food intake, and (B) water intake were monitored from day 0 to 18 post treatments. n=8–15 mice per treatment group. * p<0.05, all the other groups compared to Sham αEnNaC+/+using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Figure 4: DOCA-Salt induced increases in sodium current are amiloride-sensitive. Peak inward Na+ currents were measured at −80 mV. * p<0.05, paired t-test in the presence and absence of amiloride (1 μM).

Highlights:

  • In mice, high salt consumption alone does not cause endothelial cell or vascular stiffening.

  • A combination of endothelial cell mineralocorticoid receptor activation and high dietary salt causes activation of the sodium channel EnNaC

  • EnNaC activation increases endothelial cell and arterial stiffness and impairs vascular relaxation.

  • Underlying mechanisms appear to include mTOR signaling.

Acknowledgements:

Appreciation is extended to the Dalton Cardiovascular Research Center Small Animal Phenotyping Core for the provision of facilities for ultrasound imaging and measurement of blood pressure.

Footnotes

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Declaration of Competing Interest

No relevant disclosures exist for this work.

Credit Author Statement

Michael A. Hill and James R. Sowers conceived and planned the experiments, supervise the experiment and manuscript writing; Liping Zhang gave treatments to and monitored animal groups, performed blood pressure measurement, tissues collection, statistical analysis, graph making, and wrote the initial manuscript; Yan Yang performed patch clamp experiment; Aroor Annayya performed atomic force microscopy experiment, Jia Guanghong performed wire myography experiment; Zhe Sun performed ultrasound experiments, Alan Parrish and Garrett Litherland performed immunohistochemistry experiment; Benjamin Bonnard and Frederic Jaisser provide the animal source.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplementary Table 1: Characteristics of mice. Values are shown as Mean ± SEM. n = 8–15 mice per treatment group. * p<0.05 compared to Sham αEnNaC+/+ using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Table 2: Chemicals and Reagents Used.

NIHMS1783680-supplement-1.pdf (1.6MB, pdf)
2

Supplementary Figure 1: High salt did not exaggerate western diet-induced increases in arterial stiffness, endothelium stiffness or aortic relaxation (A) Aortic pulse wave velocity measured in vivo. (B) In situ endothelium stiffness measured ex vivo using atomic force microscopy. (C) Response of isolated aortic rings to endothelium-dependent dilator, acetylcholine and (D) endothelium-independent dilator, sodium nitroprusside. ‡ p<0.05, CD compared to WD; $ p <0.05, CD+HS compared to WD+HS using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Figure 2: High salt suppress inward sodium currents. Peak inward Na+ currents measured at −80 mV. * p<0.05, compared to CD; # p<0.05, compared to WD using one way ANOVA with Dunnett’s multiple comparison analysis..

Supplementary Figure 3: Characteristics of mice. (A) Food intake, and (B) water intake were monitored from day 0 to 18 post treatments. n=8–15 mice per treatment group. * p<0.05, all the other groups compared to Sham αEnNaC+/+using one way ANOVA with Dunnett’s multiple comparison analysis.

Supplementary Figure 4: DOCA-Salt induced increases in sodium current are amiloride-sensitive. Peak inward Na+ currents were measured at −80 mV. * p<0.05, paired t-test in the presence and absence of amiloride (1 μM).

NIHMS1783680-supplement-2.xlsx (11.7KB, xlsx)

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