Endothelial Sodium Channel Research Articles

Activity and function of the endothelial sodium channel is regulated by the effector domain of MARCKS like protein 1 in mouse aortic endothelial cells.

The endothelial sodium channel (EnNaC) plays an important role in regulating vessel stiffness. Here, we investigated the regulation of EnNaC in mouse aortic endothelial cells (mAoEC) by the actin cytoskeleton and lipid raft association protein myristoylated alanine-rich C-kinase substrate like protein 1 (MLP1). We hypothesized that mutation of specific amino acid residues within the effector domain of MLP1 or loss of association between MLP1 and the anionic phospholipid phosphate PIP2 would significantly alter membrane association and EnNaC activity in mAoEC. mAoEC transiently transfected with a mutant MLP1 construct (three serine residues in the effector domain replaced with aspartate residues) showed a significant decrease in EnNaC activity compared to cells transfected with wildtype MLP1. Compared to vehicle treatment, mAoEC treated with the PIP2 synthesis blocker wortmannin showed less colocalization of EnNaC and MLP1. In other experiments, Western blot and densitometric analysis showed a significant decrease in MLP1 and caveloin-1 protein expression in mAoEC treated with wortmannin compared to vehicle. Finally, wortmannin treatment decreased sphingomyelin content and increased membrane fluidity in mAoEC. Taken together, our results suggest constitutive phosphorylation of MLP1 attenuates the function of EnNaC in aortic endothelial cells by a mechanism involving a decrease in association with MLP1 and EnNaC at the membrane, while deletion of PIP2 decreases MARCKS expression and overall membrane fluidity.

Discovery and development of BI 1265162, an ENaC inhibitor for the treatment of cystic fibrosis

Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and development of BI 1265162, the first ENaC inhibitor devoid of the amiloride structural motif that entered clinical trials. The design of BI 1265162 focused on its suitability for inhalation via the Respimat® Soft Mist™ Inhaler and a long duration of action. A convergent and scalable route for the synthesis of BI 1265162 as dihydrogen phosphate salt is presented, that was applied to support clinical trials. A phase 2 study with BI 1265162 did not provide a clear sign of clinical benefit. Whether ENaC inhibition will be able to hold its promise for CF patients remains an open question.

C Type Natriuretic Peptide Receptor Activation Inhibits Sodium Channel Activity in Human Aortic Endothelial Cells by Activating the Diacylglycerol-Protein Kinase C Pathway.

The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA's corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) was augmented after treating hAoEC with cANF compared to vehicle, suggesting EnNaC activity is down-regulated upon NPRC ligand binding in a DAG-PKC dependent manner. The reciprocal cross-talk between NPRC activation and EnNaC inhibition represents a feedback mechanism that presumably is involved in the regulation of endothelial function and aortic stiffness.

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

IntroductionHigh 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 resultsMice 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 αEnNaC−/− and mTOR inhibitor treated groups. Further, depletion of αEnNaC prevented DOCA-salt-induced impairment in EC-dependent vascular relaxation. ConclusionWhile 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.

Increased endothelial sodium channel activity by extracellular vesicles in human aortic endothelial cells: putative role of MLP1 and bioactive lipids

Extracellular vesicles (EVs) contain biological molecules and are secreted by cells into the extracellular milieu. The endothelial sodium channel (EnNaC) plays an important role in modulating endothelial cell stiffness. We hypothesized EVs secreted from human aortic endothelial cells (hAoECs) positively regulate EnNaC in an autocrine-dependent manner. A comprehensive lipidomic analysis using targeted mass spectrometry was performed on multiple preparations of EVs isolated from the conditioned media of hAoECs or complete growth media of these cells. Cultured hAoECs challenged with EVs isolated from the conditioned media of these cells resulted in an increase in EnNaC activity when compared with the same concentration of media-derived EVs or vehicle alone. EVs isolated from the conditioned media of hAoECs but not human fibroblast cells were enriched in MARCKS-like protein 1 (MLP1). The pharmacological inhibition of the negative regulator of MLP1, protein kinase C, in cultured hAoECs resulted in an increase in EV size and release compared with vehicle or pharmacological inhibition of protein kinase D. The MLP1-enriched EVs increased the density of actin filaments in cultured hAoECs compared with EVs isolated from human fibroblast cells lacking MLP1. We quantified 141 lipids from glycerolipids, glycerophospholipids, and sphingolipids in conditioned media EVs that represented twice the number found in control media EVs. The concentrations of sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine were higher in conditioned media EVs. These results provide the first evidence for EnNaC regulation in hAoECs by EVs and provide insight into a possible mechanism involving MLP1, unsaturated lipids, and bioactive lipids.

Spiral Steroids During Pregnancy - It’s All About Potassium

This presentation has two parts. The 1st section describes processes during pregnancy with unknown, underlying biochemistry. The 2nd section shows the role of spiral steroids (SS) in these processes. 1] Fetal nutrition is provided through the placenta. Plasma electrolytes are 145 mM Na+ and 3-5 mM K+. Fetal K+ requirements reach a maximum during the 3rd trimester and must be pumped into cells against the plasma- intracellular gradient. 2] During the 3rd trimester, aldosterone is present in fetal plasma but the signal for endothelial sodium channel (ENaC) synthesis is blocked, leading to fetal Na+ wasting. The mechanism is unknown. 3] After parturition, infants are fed by nursing. Human milk contains 100 mM K+ and 10 mM Na+. Newborn infants are Na+ wasting, despite normal levels of aldosterone. Na+ wasting ends during the 2nd week post-partum without change in serum aldosterone levels. Infant physiology changes from K+ saving to Na+ saving by an unknown mechanism.4] Pre-eclampsia syndrome (proteinuria and hypertension during the 2nd half of pregnancy) affects 3-5 % of pregnant women. These symptoms usually resolve after parturition. 5] Women who have had pre-eclampsia have long-term, excess risk of cardiac and renal disorders.In 2018, we discovered Ionotropin, a SS. SS are phosphocholine esters of steroids with a lactone E-ring, similar to that of spironolactone. SS compounds function as aldosterone antagonists and regulate the NaK-ATPase. SS are involved in each of the 5 steps.1] SS are present in high levels in cord serum and were probably present in fetal plasma throughout gestation. SS stimulate the NaK-ATPase to pump K+ into cells against the gradient, just as does spironolactone.2] SS interfere with aldosterone signaling, just as does amiloride. This leads to increased fetal Na+ wasting, which becomes amniotic fluid.3] SS disappear from the infant circulation during the 1st week after parturition and decrease to adult levels during the 2nd week post-partum. Simultaneously, Na+ wasting ends and growth resumes.4] Women with pre-eclampsia have excess precursors for SS. These would be converted to SS in the fetal-placental unit and, during the 3rd trimester, diffuse into the maternal circulation and could cause pre-eclampsia.5] Ouabain, a plant toxin with a lactone E ring, causes renal and cardiac disorders in rat models. In women with pre-eclampsia, persistent excess SS may cause long-term damage.During gestation, the fetus requires K+ for growth. Our theory is, if (when) a fetus has inadequate K+ (hypokalemia), [a] the mother is signaled to produce SS precursors, [b] the feto-placental unit converts the precursors to SS, [c] fetal SS increase K+ transfer into tissues, and [d] excess SS transfuse back into the maternal circulation and damage maternal organs. We propose that pre-eclampsia is a side effect of fetal efforts to increase supply of K+. There are many possible origins of fetal hypokalemia.

Insulin- and Aldosterone-Mediated Signaling in Endothelial Cells Converge on SGK1 to Promote Endothelial Sodium Channel Activation

Background: Abnormal cardiovascular (CV) stiffening precedes overt tissue fibrosis and dysfunction. Although CV stiffening normally occurs with ageing it is accelerated in states of hyperinsulinemia/insulin (INS) resistance including obesity and Type 2 diabetes. While questions remain regarding the underlying cellular mechanisms, heightened activation of endothelial cell (EC) mineralocorticoid receptors (MRs) and INS receptors occur in hyperinsulinemic/INS resistance states. Further, EC sodium (Na+) channel (EnNaC) activation has recently been identified as a key determinant of endothelial dysfunction, CV fibrosis and stiffening in diet-induced obesity and associated INS resistance . As renal epithelial Na+ channels are known to be regulated via sodium glucocorticoid kinase (SGK-1) through INS and MR-dependent mechanisms this study aimed to determine whether vascular EnNaC is similarly regulated. Methods: Lung ECs were isolated from SGK-1 -/- mice and littermate control and maintained under short-term tissue culture conditions. EnNaC activity was determined as Na+ current measured by whole cell patch clamp. Results: The SGK-1 inhibitor, GSK 650394 suppressed amiloride-sensitive Na+ currents in isolated ECs, consistent with an effect on EnNaC activity. Under similar conditions INS caused concentration (0 – 100nM)-dependent stimulation of EnNaC currents. MR activation with aldosterone (24 hrs) caused an increase in EnNaC current which was absent in cells from the SGK-1 -/- mice. INS stimulated EnNaC currents were decreased in cells from SGK-1 -/- mice compared to littermate control.

Role of the vascular endothelial sodium channel activation in the genesis of pathologically increased cardiovascular stiffness.

Cardiovascular (CV) stiffening represents a complex series of events evolving from pathological changes in individual cells of the vasculature and heart which leads to overt tissue fibrosis. While vascular stiffening occurs naturally with ageing it is accelerated in states of insulin (INS) resistance, such as obesity and type 2 diabetes. CV stiffening is clinically manifested as increased arterial pulse wave velocity and myocardial fibrosis-induced diastolic dysfunction. A key question that remains is how are these events mechanistically linked. In this regard, heightened activation of vascular mineralocorticoid receptors (MR) and hyperinsulinaemia occur in obesity and INS resistance states. Further, a downstream mediator of MR and INS receptor activation, the endothelial cell Na+ channel (EnNaC), has recently been identified as a key molecular determinant of endothelial dysfunction and CV fibrosis and stiffening. Increased activity of the EnNaC results in a number of negative consequences including stiffening of the cortical actin cytoskeleton in endothelial cells, impaired endothelial NO release, increased oxidative stress-meditated NO destruction, increased vascular permeability, and stimulation of an inflammatory environment. Such endothelial alterations impact vascular function and stiffening through regulation of vascular tone and stimulation of tissue remodelling including fibrosis. In the case of the heart, obesity and INS resistance are associated with coronary vascular endothelial stiffening and associated reductions in bioavailable NO leading to heart failure with preserved systolic function (HFpEF). After a brief discussion on mechanisms leading to vascular stiffness per se, this review then focuses on recent findings regarding the role of INS and aldosterone to enhance EnNaC activity and associated CV stiffness in obesity/INS resistance states. Finally, we discuss how coronary artery-mediated EnNaC activation may lead to cardiac fibrosis and HFpEF, a condition that is especially pronounced in obese and diabetic females.

Abstract 8: Deletion Of The Alpha Subunit Of EnNaC And MTORC2 Inhibition Attenuate DOCA-salt-induced Endothelial Cell And Vascular Stiffness.

The deoxycortisone acetate (DOCA)/salt model of hypertension and vascular disease replicates a situation of high aldosterone and high salt consumption in humans. We hypothesized that in this model, increased mineralocorticoid receptor (MR) activation would result in increased activity of the endothelial cell sodium channel (EnNaC) leading to increased endothelial cell and vascular stiffness. Female and male mice were implanted with slow release DOCA pellets (50 mg) and given salt in their drinking water (1% NaCl with 0.2% KCl) for 21 days. To probe signaling pathways underlying EnNaC activation induced endothelial cell stiffness we used mice with a specific deletion of the alpha subunit of EnNaC (EnNaC KO) and mice treated with a pharmacological inhibitor of mTORC2 (PP242, 10 mg/kg/day; mTI). Indeed, mTORC2 activation has been shown to increase SGK1 mediated activation of the epithelial Na+ channels in renal epithelial cells (Gleason et al., J. Clin. Invest. 2015). DOCA-salt treated control mice showed an increase in blood pressure and aortic vascular stiffness (ECHO PW analysis) which was attenuated in both the EnNaC KO and mTI-treated groups. Similarly, DOCA-salt administration increased endothelial cell Na + transport activity as measured by whole cell patch clamp. The Na + conductance was blocked by amiloride consistent with a role for the EnNaC ion channel. Further, intrinsic endothelial cell stiffness as measured by AFM was decreased in EnNaC KO and mTI-treated mice compared to control mice given DOCA-salt, alone. Collectively, the data indicate that in the presence of MR activation and high salt consumption, that activation of EnNaC contributes to endothelial cell and vascular stiffness. This appears to involve a contribution of mTOR signaling which is known to stimulate SGK1 mediated increases in the Na + channel at the EC surface and associated increased channel activity. We further conclude that the data are relevant to situations of enhanced aldosterone secretion and MR activation in concert with high salt consumption (ie. salt sensitivitive and resistant hypertension).

Endothelial sodium channel activation promotes cardiac stiffness and diastolic dysfunction in Western diet fed female mice

ObjectiveObesity is associated with myocardial fibrosis and impaired diastolic relaxation, abnormalities that are especially prevalent in women. Normal coronary vascular endothelial function is integral in mediating diastolic relaxation, and recent work suggests increased activation of the endothelial cell (EC) mineralocorticoid receptor (ECMR) is associated with impaired diastolic relaxation. As the endothelial Na+ channel (EnNaC) is a downstream target of the ECMR, we sought to determine whether EC-specific deletion of the critical alpha subunit, αEnNaC, would prevent diet induced-impairment of diastolic relaxation in female mice. Methods and materialsFemale αEnNaC KO mice and littermate controls were fed a Western diet (WD) high in fat (46%), fructose corn syrup (17.5%) and sucrose (17.5%) for 12–16 weeks. Measurements were conducted for in vivo cardiac function, in vitro cardiomyocyte stiffness and EnNaC activity in primary cultured ECs. Additional biochemical studies examined indicators of oxidative stress, including aspects of antioxidant Nrf2 signaling, in cardiac tissue. ResultsDeletion of αEnNaC in female mice fed a WD significantly attenuated WD mediated impairment in diastolic relaxation. Improved cardiac relaxation was accompanied by decreased EnNaC-mediated Na+ currents in ECs and reduced myocardial oxidative stress. Further, deletion of αEnNaC prevented WD-mediated increases in isolated cardiomyocyte stiffness. ConclusionCollectively, these findings support the notion that WD feeding in female mice promotes activation of EnNaC in the vasculature leading to increased cardiomyocyte stiffness and diastolic dysfunction.

Endothelial sodium channel (EnNaC) activation contributes to mineralocorticoid receptormediated increases in coronary artery and cardiac fibrosis/stiffness leading to diastolic dysfunction in obesity

BackgroundWhile obesity, induced by an industrialized or Western diet (WD), has been shown to impact structural and functional properties of both coronary arteries and myocardium, the exact mechanisms by which these interact to cause diastolic dysfunction (DD) are uncertain. We hypothesized that the mineralocorticoid receptor (MR) activation in coronary vascular endothelial cells (ECs) impairs endothelial function via EC Na+ channel, EnNaC, activation. Further, we posited that such coronary dysfunction initiates increased myocyte stiffness and fibrosis ultimately leading to DD. The objective was to demonstrate that WD‐induced DD is dependent on EnNaC activation.MethodsTo cause MR activation, aldosterone was administered to female or male mice (C57BL6) by osmotic minipump for 3 wks. Further, female wild‐type and EC MR deficient mice (2–3 mths of age) or low aldosterone implanted as subcutaneous pellet were fed a WD, high in saturated fat and fructose (16 wks). In an additional experiment, female wild‐type and EnNaC α subunit deficient mice (3–4 mths of age) were fed the WD (12–16 wks). Vascular function was assessed in isolated coronary arteries by pressure myography, cardiac and perivascular fibrosis was evaluated by picosirius red (PSR) staining and diastolic function was assessed by echocardiography. Using in vitro cellular approaches, intrinsic cardiomyocyte stiffness was measured by atomic force microscopy (AFM) and EnNaC activity (Na+ current) determined by whole cell patch clamp.ResultsAldosterone infusion caused impaired endothelium‐dependent relaxation and coronary vascular fibrosis, which were prevented by the MR antagonist, spironolactone (1 mg/kg/day). WD feeding was associated with DD, but not systolic dysfunction, that was prevented by either spironolactone or EC‐specific MR deletion. Furthermore, DD was accompanied by increased vascular EnNaC currents and increased stiffness of both ECs and cardiomyocytes, which were attenuated by EC MR deletion. Finally, WD‐induced increases in activation of coronary EnNaC, EC stiffness and cardiomyocyte stiffness, together with DD, were suppressed in EnNaC α deficient mice.ConclusionsCollectively, these results demonstrate that MR activation (aldosterone infusion and diet‐induced) elicits coronary vascular dysfunction associated with tissue and cellular cardiac changes and DD. Further, activation of EnNaC, a known target of MR signaling, promotes coronary EC‐dependent cardiomyocyte stiffness, cardiac fibrosis and impairs DD. Therapeutic targeting to prevent EnNaC activation may be a viable approach for the management of obesity‐associated diastolic dysfunction.

The importance of the epithelial sodium channel in determining salt sensitivity in people of African origin.

Hypertension is highly prevalent in Black Africans and has been found to be associated with worse blood pressure (BP) control and more cardiovascular disease. Black Africans are more salt sensitive with low renin and aldosterone levels. This can be explained in part by variants in the epithelial sodium channel (ENaC) causing an increase in channel activity resulting in sodium and water retention. These variants in the ENaC are increased in the Black African populations presumably due to selective pressure for sodium retention in traditionally low-salt diets. Furthermore, increased endothelial sodium channel activity contributes to the risk of vascular stiffness, which may also result in more difficult to control hypertension. Patients with increased activity of the ENaC are more likely to respond to amiloride (a selective sodium channel antagonist), which has implications for the management of severe and resistant hypertension in Black Africans. A large-scale controlled trial on the use of amiloride compared to usual care is warranted in Blacks with severe or resistant hypertension.

Abstract P3019: Endothelial Epithelial Sodium Channel is a Novel Mediator of Early Macrovascular and Microvascular Stiffening That Precedes Kidney Injury in Diet-Induced Obesity

Consumption of a Western Diet (WD) high in saturated fat and refined carbohydrates causes obesity and leads to insulin resistance and associated vascular stiffening in females earlier than males. This diet-induced obesity is characterized by enhanced activation of vascular mineralocorticoid receptors (MRs). Our recent work suggests that enhanced MR signaling in female mice prompts increased expression and translocation of the α-subunit of the epithelial sodium channel to the endothelial cell (EC) surface (EnNaC). However, the influence of EnNaC on endothelial function in the kidney remains unknown. Therefore, we hypothesized that WD feeding would induce kidney microvessel dysfunction and kidney injury through inflammatory pathways. Using a model of cell specific deletion of the α-subunit of EnNaC, female KO mice and littermate controls were fed a diet high in fat (46%) and fructose (17.5%) for 12 weeks. There were increases in indices of weight gain and fat mass in WD fed mice along with increased measures of central aortic stiffness, renal arteriolar remodeling, perivascular fibrosis and aortic and kidney endothelial stiffening in the absence of increases in blood pressure or proteinuria. The central aortic stiffness, renal arteriolar remodeling, perivascular fibrosis and aortic and kidney endothelial stiffening occurred in temporal relation to reductions in bioavailable nitric oxide. All of these abnormalities were attenuated in the EnNaC α-subunit knockout (endothelial stiffness, WD, 9.2 ± 0.65 kPA; WD-KO, 5.38 ± 0.75kPA). Our observations suggest, that central aortic and kidney vessel stiffening along with arterial wall remodeling occur in conjunction to renal injury.

Abstract 009: Endothelial Epithelial Sodium Channel Promotes Intrinsic Cardiomyocyte Stiffness and Diastolic Dysfunction in Response to a Diet Induced Obesity in Female Mice

Obesity is associated with a metabolic cardiomyopathy characterized by diastolic dysfunction with preserved systolic function. Although premenopausal non-obese women show relative protection against development of metabolic cardiomyopathy and cardiovascular disease compared to men, this relationship is lost in obese women. Diastolic dysfunction in such cardiomyopathy is caused not only by extrinsic extracellular remodeling and interstitial fibrosis but also through alterations in intrinsic stiffness of cardiomyocytes. One mechanism by which intrinsic cardiomyocyte stiffness is increased in obesity is decreased coronary endothelial cell production of nitric oxide. We have described a clinically relevant diet induced obesity murine model that exhibits increased vascular stiffness associated with cardiac dysfunction. In this model, female mice have high plasma aldosterone levels and increased mineralocorticoid receptor (MR) expression in both the vasculature and heart. One of the mechanisms by which MR activation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in ECs (EnNaC). In this study, we tested the hypothesis that specific deletion of EnNaC ameliorates increases in endothelial stiffness and decreases diastolic dysfunction occurring during diet induced obesity (DIO) in female mice. To produce cell specific deletion of the EnNaC gene,”floxed” EnNaC mice were serially crossed with Tie 2-Cre transgene mice. This resulted in marked suppression of EnNaC expression in ECs. Female KO mice and littermate controls were fed a diet high in fat (46%) and fructose (17.5%) for 12 to 16 weeks. Compared to mice fed a control diet (CD), whole cell sodium currents in pulmonary ECs and aortic EC stiffness were significantly increased in DIO mice and this was prevented in EnNaC KO mice with DIO. Moreover, deletion of EnNaC also prevented DIO increases in stiffness of isolated cardiomyocytes (WD, 7.43 ± 1.21 kPA, WD-KO, 3.47 ± 0.14 kPA) as well as normalizing impaired diastolic relaxation. Collectively, these findings support the notion that DIO promotes coronary ECMR mediated activation of EnNaC and associated increases in intrinsic cardiomyocyte stiffness and diastolic dysfunction.

The Absence of Endothelial Sodium Channel α (αENaC) Reduces Renal Ischemia/Reperfusion Injury.

The epithelial sodium channel (ENaC) has a key role in modulating endothelial cell stiffness and this in turn regulates nitric oxide (NO) synthesis. The physiological relevance of endothelial ENaC in pathological conditions where reduced NO bioavailability plays an essential role remains largely unexplored. Renal ischemia/reperfusion (IR) injury is characterized by vasoconstriction and sustained decrease in renal perfusion that is partially explained by a reduction in NO bioavailability. Therefore, we aimed to explore if an endothelial ENaC deficiency has an impact on the severity of renal injury induced by IR. Male mice with a specific endothelial sodium channel α (αENaC) subunit gene inactivation in the endothelium (endo-αENaCKO) and control littermates were subjected to bilateral renal ischemia of 22 min and were studied after 24 h of reperfusion. In control littermates, renal ischemia induced an increase in plasma creatinine and urea, augmented the kidney injury molecule-1 (Kim-1) and neutrophil gelatinase associated lipocalin-2 (NGAL) mRNA levels, and produced severe tubular injury. The absence of endothelial αENaC expression prevented renal tubular injury and renal dysfunction. Moreover, endo-αENaCKO mice recovered faster from renal hypoxia after the ischemia episode as compared to littermates. In human endothelial cells, pharmacological ENaC inhibition promoted endothelial nitric oxide synthase (eNOS) coupling and activation. Altogether, these data suggest an important role for endothelial αENaC in kidney IR injury through improving eNOS activation and kidney perfusion, thus, preventing ischemic injury.

Abstract 097: Interaction of miRNA, Endothelial Mineralocorticoid Receptor and Epithelial Sodium Channel in Endothelium Stiffness and Aortic Dysfunction

Excessive endothelial cell (EC) mineralocorticoid receptor (ECMR) and epithelial sodium channel activation in endothelium (EnNaC) increase oxidative stress and inflammation with associated cardiovascular abnormalities. Previous studies implicate elevations in circulating aldosterone (Aldo) in obesity enhance ECMR/EnNaC signaling that contribute to the development of vascular stiffness, in part, by reducing endothelial nitric oxide (NO) synthase (eNOS) activity and NO production. However, the specific role of ECMR/EnNaC signaling and its molecular mechanisms have not been explored. We hypothesized interactions between ECMR, dysregulation of miRNA expression, and EnNaC contribute to obese-/Aldo-induced endothelium dysfunction and aortic stiffness. Six week-old ECMR -/- and wild type littermate mice were fed a mouse chow or Western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. The EnNaC alpha subunit KO (EnNaC -/- ) and EnNaC +/+ female mice were administrated Aldo (250 μg/kg/day) via osmotic minipumps for 3 weeks beginning at 25-28 weeks of age. RNA sequencing showed that in ECMR +/+ mice WD induced increased miR-7a, miR-34, and miR-6973a and reduced miR-99, miR-486a, miR-6904, miR-6916, miR-6240 that potentially target EnNaC expression. For EnNaC in vascular function, EnNaC -/- significantly reduced inward sodium currents by patch clamp in isolated mouse ECs. Meanwhile, EnNaC -/- significantly inhibited Aldo-induced endothelium stiffness and endothelium dependent relaxation observed in EnNaC +/+ . Further, chronic Aldo infusion induced aortic endoplasmic reticulum stress, reduced eNOS activation, endothelium permeability, expression of pro-inflammatory cytokines IL1 and IL6, oxidative stress, and aortic collagen 1 deposition and these abnormalities were attenuated in EnNaC -/- mice. These data indicate that interaction of endothelial specific ECMR/miRNA/EnNaC mediates vascular endothelium dysfunction and prompts aortic stiffness.

Arterial Stiffness in Hypertension: an Update.

To update and review novel insights into the mechanisms, measurements, and therapeutic approaches to arterial stiffness. Arterial (e.g., vascular) stiffness has been shown over time to prognosticate for cardiovascular and kidney outcomes. In this context, there has been increased interest behind the mechanisms that drive arterial stiffness beyond aging and interest in how to apply newer technologies in measurement of arterial stiffness. Pulse wave velocity has been the gold standard for measurement but industry has been innovating to improve measurement with use of single-point PWV as well as pharmacologic approaches with anti-hypertensives and oral hypoglycemic agents. Emerging data on the role of the mineralocorticoid receptor, the endothelial sodium channel (EnNaC), and uric acid in arterial stiffness are promising a number of potential therapies. Newer techniques of measuring PWV for arterial stiffness and novel therapies may soon lead to better outcomes from hypertension complications.

The Deletion of Endothelial Sodium Channel α (αENaC) Impairs Endothelium-Dependent Vasodilation and Endothelial Barrier Integrity in Endotoxemia in Vivo.

The role of epithelial sodium channel (ENaC) activity in the regulation of endothelial function is not clear. Here, we analyze the role of ENaC in the regulation of endothelium-dependent vasodilation and endothelial permeability in vivo in mice with conditional αENaC subunit gene inactivation in the endothelium (endo-αENaCKO mice) using unique MRI-based analysis of acetylcholine-, flow-mediated dilation and vascular permeability. Mice were challenged or not with lipopolysaccharide (LPS, from Salmonella typhosa, 10 mg/kg, i.p.). In addition, changes in vascular permeability in ex vivo organs were analyzed by Evans Blue assay, while changes in vascular permeability in perfused mesenteric artery were determined by a FITC-dextran-based assay. In basal conditions, Ach-induced response was completely lost, flow-induced vasodilation was inhibited approximately by half but endothelial permeability was not changed in endo-αENaCKO vs. control mice. In LPS-treated mice, both Ach- and flow-induced vasodilation was more severely impaired in endo-αENaCKO vs. control mice. There was also a dramatic increase in permeability in lungs, brain and isolated vessels as evidenced by in vivo and ex vivo analysis in endotoxemic endo-αENaCKO vs. control mice. The impaired endothelial function in endotoxemia in endo-αENaCKO was associated with a decrease of lectin and CD31 endothelial staining in the lung as compared with control mice. In conclusion, the activity of endothelial ENaC in vivo contributes to endothelial-dependent vasodilation in the physiological conditions and the preservation of endothelial barrier integrity in endotoxemia.

Estrogen receptor alpha mediated activation of the endothelial epithelial sodium channel: role in the genesis of arterial stiffness

Premature arterial stiffening is often seen in obesity and type 2 diabetes (T2D). Although both men and women are affected, T2D women are at greater risk. As augmented arterial stiffness is an independent predictor of cardiovascular disease, the increased susceptibility of women to arterial stiffening may explain their higher overall risk. In healthy females, estrogen signaling prevents stiffening, but these effects are blunted in obesity where signaling via estrogen receptor alpha (ERα) appears deleterious. We have previously reported that deletion of ERα in endothelial cells (EC) results in decreased arterial stiffening in conditions of over‐nutrition and insulin resistance. Importantly, the EC epithelial sodium channel (ENaC) has been hypothesized as a central mediator in the genesis of endothelial dysfunction and arterial stiffness, and estrogen has been shown to activate ENaC activation. Here we examine the preferential activation of the EC ENaC in females under conditions of insulin resistance and the role of estrogen signaling, specifically via ERα, on the activation of the EC ENaC. In the present investigation we report that cultured aortic EC from female mice exhibited a significant increase in amiloride‐sensitive sodium currents (ENaC currents) assessed by patch‐clamp after treatment with 100 nM 17beta‐estradiol (E2) for 24 hours when compared to control. In contrast, there was no significant effect of E2 treatment on ENaC currents in EC from male mice. Further, we found that C57BL6/J female mice fed for 16 weeks with a diet high in fat and high in fructose (WD; TestDiet 58Y1; calories: 46% fat, 17.5% sucrose, 17.5% high fructose corn syrup; 19% protein) exhibit augmented aortic endothelial surface stiffness assessed via atomic force microscopy when compared with male mice. Moreover, primary lung ECs from female mice fed WD exhibited elevated E2‐stimulated ENaC currents when compared with control fed female mice and with male mice. In addition, in primary ECs isolated from female EC‐specific ERα knockout mice there was a significant abrogation of the E2‐stimulated amiloride sensitive sodium currents without significant changes in aldosterone (10 uM) induced sodium currents. Our results show that in conditions of over‐nutrition, there is enhanced EC ENaC activation and highlights the important role of estrogen, via ERa signaling, as a modulator of the expression and activity of the EC ENaC. Future studies will determine the direct impact of ERα‐mediated activation of ENaC in the genesis of arterial stiffness in obesity and T2D.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 120: Endothelial Specific Sodium Channel Activation in Endothelium Dysfunction and Vascular Stiffness in Obese Female Mice

Excessive activation of endothelial cell (EC) mineralocorticoid receptor (ECMR) signaling induces EC epithelial sodium channel (EnNaC) activity to promote cardiovascular stiffness. Our previous study has demonstrated that activated ECMR signaling prompts expression and translocation of EnNaC to the EC surface inducing fibrosis, inflammation, and macrophage infiltration in the vasculature of female mice fed a western diet (WD). As ECMR KO also prevented these abnormalities, we posit that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC-specific EnNaC activation would mediate endothelium dysfunction, vascular stiffness, and impair flow-mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO and wild-type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC, significantly inhibited inward Na+ currents and EnNaC activity in the cultured endothelial cells. Amiloride treatment significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. In addition, amiloride improved flow mediated dilation in mesenteric arteries and endothelium-dependent relaxation in response to acetylcholine (10 -9 -10 -4 mol/L). Furthermore, amiloride prevented WD-induced increases in coronary endothelium permeability that were associated with decreased expression of claudin-5 and occluding. This also resulted in reduction of total macrophage recruitment (CD11b) and M1 polarization (CD11c). Importantly, genetic knock-out EnNaC KO also prevented aldosterone-induced endothelium stiffening and impairment of endothelium-dependent relaxation. These data indicate that EC specific EnNaC activation decreases bioavailable NO, increases vascular endothelium dysfunction, and prompts vascular stiffening in obese female mice.