Frontiers in Nephrology: Renal Sodium Handling: The Role of the Epithelial Sodium Channel
Central Role for ENaC in Development of Hypertension
J. Howard Pratt
Department of Medicine, Indiana University School of Medicine, and the Richard L. Roudebush Veterans Administration Medical Center, Indianapolis, Indiana
Address correspondence to: Dr. J. Howard Pratt, Department of Medicine, Indiana University School of Medicine, 541 Clinical Drive, Indianapolis, IN 46202-5111. Phone: 317-274-4347; Fax: 317-274-7700; E-mail: johpratt{at}iupui.edu
Na+ reabsorption by the epithelial Na+ channel (ENaC) in corticalcollecting duct provides the final renal adjustment to Na+ balance,there being no further downstream Na+ transport system. Thisfact coupled with the responsiveness of ENaC to aldosterone,which conveys stimulation inversely proportional to the stateof Na+ balance, places ENaC in a pivotal position to influencethe risk for hypertension. Although several molecular variantsof ENaC subunits have been identified, there has been no consistentdemonstration of an association of any of the variants withhypertension. More compelling is the notion that ENaC activitydoes not fully adjust to an increase in Na+ reabsorption occurringelsewhere in the nephron, there being overstimulation by inappropriatelyelevated aldosterone levels. Additional evidence that the maintenanceof hypertension can be dependent on ENaC is derived from theobserved responses to the treatment of hypertensive individualswith inhibitors of ENaC. Described is a clinical trial in whichblack hypertensive individuals who did not fully respond tomore traditional therapy were given amiloride, spironolactone,a combination of the two drugs, or placebo. Treatment with eitherof the active inhibitors of ENaC resulted in a substantial improvementin BP. In conclusion, evidence to date is supportive of theconcept that an increase in Na+ transport by ENaC may be a commonand requisite component of salt-dependent forms of hypertension.
An increase in Na+ and water retention is required for the developmentof most forms of hypertension as was proposed by Guyton yearsago (1) and to this day has never really been refuted. In instancesin which the mechanism for the hypertension is known, in whichan identified gene mutation defines it, generally speaking anincrease in the function of the epithelial Na+ channel (ENaC)leads to the Na+ retention and elevation in BP (2). The biggerquestion and the issue addressed in the current review is theextent to which ENaC participates in the development of commonforms of hypertension. A strong case can be made for the factthat ENaC is pivotally positioned, both anatomically and physiologically,to convey a dominant influence on the prevalence of hypertension.Studies of hypertension in relation to ENaC and its major regulator,aldosterone, are reviewed here, and a general mechanism fordevelopment of hypertension is formulated. Finally, a clinicaltrial of the effectiveness of ENaC inhibition to lower BP inpatients with hypertension is described.
Common Molecular Variations in ENaC and Risk for Hypertension
The discovery of mutations in - and -subunits of ENaC to explainLiddle syndrome (3,4), a severe form of low-renin hypertension(5), was soon followed by a search for common genetic variantsin ENaC subunits that affect susceptibility in less rare formsof hypertension. Of course variants were identified, and theywere almost universally more common in black individuals, whichfit nicely with the higher prevalence of low-renin hypertensionin black individuals. After a number of years of study by severalinvestigative groups, however, it remains unestablished thata given variant influences the risk for hypertension.
The first molecular variant to show an association with hypertensionwas T594M in the C-terminus of -ENaC (Figure 1) in a study ofblack individuals who lived in London (6) (the variant is rarein white individuals). Frequencies of the variant were foundto be 8.3% in hypertensive individuals and 2.4% in normotensiveindividuals. In addition, plasma renin activity was lower incarriers of T594M. Subsequent studies by others have for themost part failed to replicate the same relationship to hypertension(7,8). In another study, seven molecular variants, all in -ENaC,with overall allele frequencies of 44% in black individualsand 1% in white individuals, showed no association with hypertensionand showed no effect on function using two different in vitrotechniques (7). In our own laboratory, we found a G442V variantin the extracellular loop (Figure 1) in -ENaC (again occurringalmost exclusively in black individuals) significantly associatedwith an index of ENaC activity, the urinary aldosterone/K+ ratio,in normotensive young people, but it did not associate withhypertension in a study of adults (8). We also found that theA663T variant in -ENaC near the PY motif on the carboxyl terminus(Figure 1), the only variant that was more frequent in whitethan in black individuals (30 versus 15%), associated with beingnormotensive as opposed to hypertensive in separate studiesof white and black individuals (8). As an example of how difficultit is to find meaningful associations, Kleyman’s laboratory,using a Xenopus oocyte expression system, found that the amiloride-sensitivecurrent was increased not with the A but with the T allele atposition 663 (9), the opposite of what our clinical studieswould have predicted. Thus, the evidence is still mostly missingthat common variations in ENaC influence the risk for hypertension.The higher frequency of variants in black individuals can beexplained by the greater genetic diversity among black individuals(10).
Figure 1. The locations along the subunits of representative molecular variants that have shown significant associations with BP (-A663T, -T594M) or an index of epithelial Na+ channel (ENaC) activity (-G442V). Allele frequencies are in brackets; B, black; W, white. Illustration by Josh Gramling—Gramling Medical Illustration.
Several explanations can be proposed for why the associationstudies of ENaC variants are so often inconclusive. To beginwith, a modification that increases the activity of ENaC wouldnot result in Na+ retention if at the same time there were anappropriate downward adjustment in aldosterone secretion. Thus,a variant that affected function in vitro would in general failto affect the in vivo expression, unless of course aldosteronesecretion does not adjust appropriately (below). There obviouslyare many pressor and depressor influences that exist apart fromENaC that make it additionally difficult ever to detect associationsusing the phenotype hypertension.
The inconclusiveness of association studies, however, does notdiminish the unlikelihood that all ENaC are equal. Its complexityand the multiplicity of factors that regulate it would seeminglylend ENaC vulnerable to genetic modification. Future studiesare more likely to succeed in showing significant associationsif the gene’s net influence can be studied in contrastto the effect of an isolated variation. This can be accomplishedthrough use of a series of single-nucleotide polymorphisms inlinkage disequilibrium and with construction of haplotypes thatare representative of the entire gene. The influence of thecombined variation of a given ENaC subunit or of a regulatoryprotein, for example Nedd4-2, can be better appreciated.
Adaptations of ENaC to Increases in Na+ Reabsorption
The key to whether a salt-dependent form of hypertension ensuesor not may depend on the fidelity of the adjustments that takeplace at the level of ENaC. An example of where ENaC adjuststo increased Na+ reabsorption (resulting in there being no increasein BP) and evidence of when it may not adjust (with increasedrisk for hypertension) are presented.
Increased Na+ Reabsorption in Proximal Nephron Accompanied by an Appropriate Decrease in ENaC Activity: Studies in Normotensive Black and White Individuals
We tested the hypothesis that black individuals, a group athigh risk for developing hypertension, have a greater levelof ENaC activity when compared with white individuals, a groupat lower risk. Participants were from a cohort of young people(mean age 17 yr), all of whom were normotensive (but inclusiveof prehypertensives); thus, we avoided the confounding influencesassociated with hypertension and in particular its treatment.Black individuals of the cohort had consistently demonstratedlower aldosterone levels than the white individuals (Figure 2)(11), suggesting to us that greater Na+ retention was suppressingthe renin-angiotensin-aldosterone axis in black individuals.Black and white individuals were treated for 1 wk with a 5-mg/ddose of amiloride. The BP response was used to gauge ENaC activity(12). Measurements of BP were made after an overnight hospitalizationin the General Clinical Research Center so as to minimize differencesbetween the conditions at baseline and the posttreatment period.The results indeed were surprising to us. In response to amiloride,systolic and diastolic BP decreased in white but not in blackindividuals, with the differences between groups significantfor both systolic (P = 0.034) and diastolic (P = 0.010) BP.Black individuals seemed to have less ENaC activity to inhibit.In a cross-sectional sampling of participants from the samecohort, the urinary excretion of K+ was significantly lowerin black than in white individuals (3.2 ± 0.1 versus3.8 ± 0.1 mmol/mmol creatinine; P < 0.0001), and theserum K+ concentration was significantly higher in black thanin white individuals (4.35 ± 0.05 versus 4.21 ±0.03 [SE] mmol/L; P = 0.012), both findings indicating lessK+ secretion in black individuals and consistent with less ENaCactivity.
Figure 2. Plasma aldosterone concentrations in white and black school-aged children (pmol/L, mean ± SD). The difference between groups was significant at P < 0.001. Illustration by Josh Gramling—Gramling Medical Illustration.
We concluded that the lower aldosterone levels in black individualswas the primary event and that ENaC activity was secondarilyreduced—the opposite of what we had conjectured previously.We deduced from the findings that increased Na+ reabsorptionwas occurring in another nephron region that led to suppressedaldosterone secretion and in turn a reciprocal decrement inNa+ reabsorption by ENaC. Na+ transport by ENaC adjusted appropriatelyto the increase in Na+ reabsorption because the BP in blackand white individuals were similar.
"Normal" Aldosterone Levels Lead to an Inappropriate Level of ENaC Activity
Unlike what we observed in young people without hypertension,ENaC may not adjust to an increase in Na+ reabsorption in adultswith the same facility. In a study of normotensive individualsfrom the Framingham Offspring Study cohort (n = 1688; mean age55 yr), Vasan et al. (13) found a significant relationship betweennormal aldosterone levels and the BP 4 yr later. Specifically,there was a 16% increase in risk for an increase in BP and a17% increase in risk for actually becoming hypertensive witheach quartile increment in serum aldosterone level. The authorssuggested that the regulation of aldosterone secretion developedalong the lines that improved survival during an early ancestralperiod, when there was a scarcity of salt and water. There wasno similarly compelling need for a full downward adjustmentin aldosterone secretion to accommodate the higher intakes ofNa+ that typify modern-day diets (Figure 3). Thus, many people,some more than others, may be exposed to an excess of aldosteronewith the potential for an inappropriately increased reabsorptionof Na+ by ENaC.
Figure 3. Aldosterone excretion rates in the Yanomamo Indians (21), who consumed an extremely low Na+ diet, probably typical of the early ancestral period, when regulation of aldosterone secretion evolved to optimize the conservation of Na+ for purposes of survival (Salt-Scarce World), and in individuals who consume a diet replete with Na+ (Today). There presumably was no similar survival advantage for aldosterone secretion to decrease to where there would be an avoidance of an excess of Na+ retention. Under low-salt conditions, excretion of aldosterone matches the amount required for Na+ and water homeostasis. Today, with diets containing an abundance of salt, aldosterone excretion may exceed what is required, leading to an inappropriately elevated level of ENaC activity. The "required" aldosterone excretion rate when diets are replete with Na+ was arbitrarily set at 3 µg/d. Illustration by Josh Gramling—Gramling Medical Illustration.
ENaC’s Pivotal Position Lends Itself to a "Two-Hit Model" for Development of Hypertension
If there is increased Na+ reabsorption in a proximal regionof the nephron, then a downstream site must respond with anequivalent in magnitude reduction in Na+ reabsorption if normalNa+ and volume homeostasis are to be maintained. A principaldownstream site is ENaC, with aldosterone delivering the levelof stimulation to ENaC in keeping with the prevailing stateof Na+ balance. If ENaC fails to decrease reciprocally its activityand because there is no additional downstream site to adjustto the increase in Na+ reabsorption by ENaC, then net Na+ retentionwill take place. Thus, a scenario can be proposed, one requiring"two hits": An increase in Na+ reabsorption at a nephron siteproximal to ENaC or possibly at ENaC itself (hit 1) togetherwith a failure on the part of aldosterone secretion to decreasefully (hit 2), leaving ENaC operating at an inappropriatelyincreased level. The orchestration of the kidney’s functionsof course is more complex and fluid than such a simple schemeimplies (Figure 4). For example, the pressure natriuresis thatdevelops in response to the increase in BP would work towardrestoring Na+ homeostasis followed by normalization of BP. Whethera hypertensive state ensues would also depend on the pressure-natriureticresponsiveness of the kidney and thus the possible requisitefor a third hit. Nonetheless, ENaC, because of its distal locationin the nephron and because of its regulation being driven byaldosterone, would seem to be at the center of the developmentof commonly encountered forms of hypertension. Proof of theconcept will rest with demonstration of a combination of molecularor genetic variations acting together to increase risk for hypertension.That such a pairing of genes can affect BP was suggested inan association study in which individuals with a HindIII restrictionsite on the Y-chromosome were at increased risk for hypertensionif they were also carriers of a variant in the aldosterone synthasegene (14).
Figure 4. An ENaC-mediated mechanism for the development of hypertension. In the normotensive state, there is no increase in Na+ reabsorption (example 1), or there is an increase in Na+ reabsorption that is accompanied by an appropriate downward adjustment in aldosterone secretion (example 2). An increase in BP occurs when there is an increase in Na+ reabsorption, with incomplete suppression of aldosterone secretion leading to incomplete suppression of ENaC activity (example 3). Because of the pivotal position of ENaC within the nephron, there being no downstream Na+ reabsorptive site to adjust reciprocally to the increase in Na+ reabsorption by ENaC, an increase in BP can ensue. Illustration by Josh Gramling—Gramling Medical Illustration.
To test further whether ENaC is integral to the pathophysiologyof hypertension, one can remove its influence through targeteddrug therapy. We had early anecdotal evidence that small dosesof spironolactone, an aldosterone receptor antagonist, normalizedBP when added to the existing antihypertensive therapy of blackindividuals with suppressed plasma renin activity (15). A studyof larger numbers of subjects by Calhoun et al. (16) showedthat regardless of ethnicity, spironolactone was effective inotherwise treatment-resistant hypertensive individuals. We recentlyreported on a prospective, randomized, double-blind, placebo-controlledclinical trial of ENaC inhibition in hypertension (17). Enrollmentwas limited to black individuals because of their known greaterpredilection to retain Na+ (18).
Study Design and Procedures
The study used a two-by-two factorial design with four treatmentgroups: Amiloride (10 mg/d), a direct inhibitor of ENaC; spironolactone(25 mg/d); the combination of both drugs; and placebo. Patients(n = 98) were between 18 and 75 yr of age (mean age 46 yr),with a systolic BP >140 and/or a diastolic BP >90 mmHgwhile receiving hydrochlorothiazide (minimum dose of 25 mg)or furosemide (minimum dose of 40 mg) or equivalent doses ofsimilar diuretics and amlodipine 5 or 10 mg or equivalent dosesof a similar calcium channel blocker. Other drugs could be used(e.g., blockers), but triamterene, angiotensin-converting enzymeinhibitors, and/or angiotensin receptor blockers were discontinuedfor 1 mo before the study was started because of the added riskfor hyperkalemia. Exclusion criteria were an elevated plasmarenin activity and evidence of a secondary form of hypertension.BP measurements were made at baseline and at weeks 1, 3, 5,7, and 9 of active treatment. The study was designed with theprimary end points being the changes from baseline in systolicand diastolic BP.
Outcomes
The reductions in systolic and diastolic BP (mmHg; mean ±SEM) in responses to the additional treatment were, respectively,9.8 ± 1.6 and 3.4 ± 1.0 for amiloride and 4.6± 1.6 and 1.8 ± 1.0 for spironolactone. Amiloridesignificantly reduced both systolic and diastolic BP (P <0.001). Spironolactone produced a significant reduction in systolicBP (P = 0.006) and a marginally significant reduction in diastolicBP (P = 0.07). Figure 5 shows the adjusted mean changes frombaseline in BP. The largest drop in BP occurred at 3 wk, butit continued to decrease until week 7, at which point valuesstabilized for each of the treatment groups. The amiloride-spironolactonecombination group showed the greatest reduction in BP followedby amiloride alone and spironolactone alone. We observed, however,no significant interaction of amiloride with spironolactone.
Figure 5. Changes from baseline in systolic and diastolic BP. The decrease in systolic BP was significant for all of the treatment groups when compared with placebo: P < 0.001 for amiloride, P < 0.010 for spironolactone, and P < 0.001 for the combination of amiloride and spironolactone. The decrease in diastolic BP was significant for the amiloride-treated group (P = 0.003) and for the combination of amiloride and spironolactone (P = 0.002) when compared with placebo. Illustration by Josh Gramling—Gramling Medical Illustration.
The BP-lowering response was greater for amiloride than forspironolactone, but a response similar to that seen with amiloridemight have occurred had a larger dose of spironolactone beenused. The superior response to amiloride suggests that it wasindeed ENaC inhibition that led to the reduction in BP ratherthan another aldosterone response site for sodium reabsorption(19,20). The results of targeting ENaC for inhibition are consistentwith a significant role for ENaC in the maintenance if not thedevelopment of common forms of hypertension.
Evidence has been presented that taken together places ENaCin a central position for creating the Na+ retention that isnecessary to achieve a state of hypertension. It would seema mistake in considering mechanisms for hypertension to thinkthat a given Na+ reabsorptive site acts alone—the evidenceis compelling for consideration of ENaC as the additional requisiteparticipant.
Acknowledgments
This study was supported by National Institutes of Health grantsRO1-HL6730, RO1-HL-3579, and MO1-RR00750.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
- and 
-subunits of ENaC to explain Liddle syndrome (3,4), a severe form of low-renin hypertension (5), was soon followed by a search for common genetic variants in ENaC subunits that affect susceptibility in less rare forms of hypertension. Of course variants were identified, and they were almost universally more common in black individuals, which fit nicely with the higher prevalence of low-renin hypertension in black individuals. After a number of years of study by several investigative groups, however, it remains unestablished that a given variant influences the risk for hypertension. 
-ENaC near the PY motif on the carboxyl terminus (Figure 1), the only variant that was more frequent in white than in black individuals (30 versus 15%), associated with being normotensive as opposed to hypertensive in separate studies of white and black individuals (8). As an example of how difficult it is to find meaningful associations, Kleyman’s laboratory, using a Xenopus oocyte expression system, found that the amiloride-sensitive current was increased not with the A but with the T allele at position 663 (9), the opposite of what our clinical studies would have predicted. Thus, the evidence is still mostly missing that common variations in ENaC influence the risk for hypertension. The higher frequency of variants in black individuals can be explained by the greater genetic diversity among black individuals (10). 
