AJH 2007; 20:109 –117

REVIEW

Epithelial Sodium Channel

Inhibition in Cardiovascular Disease

A Potential Role for Amiloride

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Justin Teiwes and Robert D. Toto

Amiloride was originally described in 1967 as a potas- ease and protection with mineralocorticoid receptor antag-
sium-sparing diuretic, the mechanism of action of which onists in human trials of heart failure remain unknown.
is to block the epithelial sodium channel (ENaC) within These effects are unexplained by changes in BP, potas-
the distal tubule of the kidney. In addition, higher doses sium, or sodium balance. An additional possibility is that
of amiloride were found to be capable of inhibiting the aldosterone action and mineralocorticoid receptor block-
Na⫹/H⫹ exchangers (NHE) and the Na⫹/Ca2⫹ exchang- ade is conferred by alterations in ENaC activity. Emerging
ers. In time, several amiloride analogs have been synthe- experimental evidence suggests the possibility that sys-
sized to have a marked increase in their specificity to temic or central ENaC inhibition or both may be an
inhibit the ENaC, the NHE or the Na⫹/Ca2⫹ exchangers. alternative to the treatment of hypertension and cardiovas-
Although the NHE inhibitors have received the most re- cular disease states. Clinical trials to evaluate further the
cent attention, large-scale clinical trials using NHE inhib- potential beneficial cardiovascular effects of ENaC block-
itors in ischemic cardiac states have shown them to be ade are needed. This article reviews the case for ENaC
either ineffective or associated with an unacceptable risk inhibition as a potential target for cardiovascular and renal
profile. Aldosterone excess in animal models is known protection in human beings. Am J Hypertens 2007;20:
to cause cardiovascular injury, and blockade of mineralo- 109 –117 © 2007 American Journal of Hypertension, Ltd.
corticoid receptors in human beings with heart disease
improves outcomes. However, the exact mechanisms of Key Words: Hypertension, cardiovascular disease, ep-
aldosterone injury in animal models of hypertensive dis- ithelial sodium channel, amiloride.

he epithelial sodium channel (ENaC) is classically sparing diuretics by blocking the mineralocorticoid re-

T considered a sodium (Na⫹) ion channel residing in

the principal cell of the cortical collecting duct.1
Aldosterone, through activation of the mineralocorticoid
ceptor in the kidney, survival benefit in patients with
heart failure appeared to be independent of natriuretic,
potassium-sparing, or blood pressure (BP)–lowering ef-
receptor (MR), has been shown to increase the expression fects.6 In chronic kidney disease, MRA have been dem-
of the ENaC2 as well as increase the open probability of onstrated to reduce proteinuria in diabetic subjects with
the channel3 with sodium absorption and potassium (K⫹) nephropathy.7–9 Nevertheless, in cardiac and kidney dis-
excretion ensuing. Although the classic role of aldoste- ease, potential benefits of MRA are often offset by their
rone has been associated with modulating Na⫹/K⫹ han- strong potential to cause hyperkalemia in this patient pop-
dling within the kidney,2,3 recent clinical trials have ulation. For example, one study reported an increased rate
called attention to nonrenal effects of aldosterone as a of hospitalization for hyperkalemia and of hyperkalemia-
pathogenic hormone in both cardiac and renal diseases. related deaths in older patients prescribed MRA for con-
For example, two large-scale clinical trials have shown gestive heart failure.10
significant survival benefit by the use of the mineralo- In Liddle syndrome, activating mutations in the ENaC
corticoid receptor antagonists (MRA) spironolactone result in prevention of degradation of the ENaC leading to
and eplerenone in patients with congestive heart fail- excessive Na⫹ absorption, K⫹wasting, and systemic (of-
ure.4,5 Although these medications act as potassium ten severe) hypertension with a high incidence of early

Received January 6, 2006. First decision May 15, 2006. Accepted May Supported by National Institutes of Health grants 5K24DK0281802,
26, 2006. NIH F32 HL80872-01, NIH 5 T32 DK007257-25.
Address correspondence and reprint requests to Robert D. Toto,
From the University of Texas Southwestern Medical Center Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390-8856; e-mail: [Link]@
Dallas, Texas. [Link]

© 2007 by the American Journal of Hypertension, Ltd. 0895-7061/07/$32.00

Published by Elsevier Inc. doi:10.1016/[Link].2006.05.022
110 AMILORIDE IN CARDIOVASCULAR DISEASE AJH–January 2007–VOL. 20, NO. 1

ENaC-specific analogues may represent novel alternatives

NH2
to MRA for treatment of cardiovascular disease in the
N C
future.21–24 The purpose of this article is to examine the
NH2
Cl N C case for using low-dose amiloride (doses associated
O with ENaC-specific blockade) in cardiovascular disease
with an emphasis on its relationship to aldosterone,
when applicable.

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N
NH2 NH2
Amiloride and Its Analogues
Amiloride
FIG. 1. Chemical structure of amiloride.
Amiloride was first described by Cragoe et al26 in 1967
as a potassium-sparing diuretic (Fig. 1). Soon after,
amiloride was found to be a “specific” inhibitor of several
cardiovascular disease.11,12 Inhibition of the ENaC within
ion transporters, including the epithelial sodium channel
the distal nephron by low doses (5 to 10 mg daily) of
amiloride lowers systemic BP in conjunction with natri- (ENaC), the Na⫹/H⫹ exchanger (NHE), and the Na⫹/
uresis, diuresis, and potassium sparing.13 Ca2⫹ exchanger (NCX).25,27 Amiloride is a relatively
Although it is possible that the BP lowering effect of selective inhibitor of the ENaC with an IC50(the concen-
amiloride is purely a reflection of salt/water balance in tration required to reach 50% inhibition of an ion channel)
other hypertensive states, mounting experimental and clin- in the concentration range of 0.1 to 0.5 ␮mol/L.25 Amilo-
ical evidence suggests a role of the ENaC in cardiovascu- ride is a relatively poor inhibitor of the NHE with an IC50
lar disease independent of its effects on renal ion transport. as low as 3 ␮mol/L in the presence of a low external [Na⫹]
In this regard, evidence for the presence of functional but as high as 1 mmol/L in the presence of a high [Na⫹].28
ENaC has been demonstrated in several nonepithelial Amiloride is an even weaker inhibitor of the NCX, with an
cells.14 –18 Moreover, in these nonrenal cell lines, there IC50 of 1 mmol/L.25 As shown in Table 1, the IC50 of
exists a dose–response relationship between aldosterone amiloride dramatically increases for the NHE and NCX in
and ENaC expression, similar to that observed in the concordance with decreased affinity of amiloride for these
renal epithelium.16,18 –20 Finally, in animal models of channels. We use the term “low-dose amiloride” to refer to
hypertension, ⱕ1␮mol/L doses of amiloride have been amiloride at ⱕ1 ␮mol/L, a dose that exhibits a significant
demonstrated to mitigate cardiovascular and renal disease relative inhibitory effect to the ENaC ion transporter as com-
progression despite elevated BP. Moreover, these effects pared with other ion channels. Although most of the studies
occur in the absence of changes in steady-state serum are in vitro, human pharmaocokinetic studies, although
potassium.21–24 limited, indicate an approximate elimination half-life of 6
Amiloride inhibits many sodium transporters; however, to 9 h of amiloride with a prolongation to 21 to 144 h in
in submicromolar (ⱕ1 ␮mol/L) concentrations, it inhibits patients with chronic kidney disease. Amiloride does not
the ENaC much more efficiently than other ion channels.25 appear to be metabolized to any significant extent in
Recent studies suggest that low doses of amiloride and its human beings. Approximately 50% of an oral dose is

Table 1. IC50 (␮mol/L), of amiloride and relative potency of selected analogs for epithelial sodium
channel (ENaC), sodium– hydrogen exchanger (NHE), and sodium calcium exchanger (NCX) as compared
with amiloride

Naⴙ channel Naⴙ/Hⴙ antiporter Naⴙ/Ca2ⴙ exchanger

Substance (ENaC) (NHE1) (NCX)
Amiloride 0.1–0.5 ␮mol/L* 5.3†–50 ␮mol/L‡ 1100 ␮mol/L*
ENaC specific
Benzamil ⫻ 9* ⫻ ⬍0.08* ⫻ 11*
NHE Specific
6-I amiloride ⫻ 0.14* ⫻ 4.6* ⫻ 1*
Dimethylamiloride ⫻ ⬍0.035* ⫻ 20* ⫻ 2*
Cariporide ⫻ 170†–676‡

Relative potency of each analog for the specific transporter is expressed as the fold increase (⫻) or decrease in activity as compared with
parent compound amiloride.
* See Ref. 25.
† See Ref. 31.
‡ See Ref. 32.
AJH–January 2007–VOL. 20, NO. 1 AMILORIDE IN CARDIOVASCULAR DISEASE 111

O H
(CH3)2CH

Cl N C N C NH CH2

NH2
N NH2
N H3CO2S
H2N NH2 O
NH2

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FIG. 2. Chemical structure of benzamil. Substitution of a hydro-
phobic group on terminal nitrogen atom of the guanidino moiety FIG. 4. Chemical structure of cariporide. Replacement of the pyr-
leading to increased specificity toward the epithelial sodium channel azine ring of amiloride by a phenyl, the 6-chloro substituted by a
while markedly lowering potency toward the the Na⫹/H⫹ exchangers. sulfomethyl and the 2-amino deleted markedly increases the spec-
ificity toward the the Na⫹/H⫹ exchangers while completely losing
the inhibitory potency to the Na⫹/Ca2⫹ exchanger and the epithelial
sodium channel.
eliminated unchanged in urine, and up to 40% is elimi-
nated unchanged in the stool by 72 h.29

Amiloride Analogues Amiloride analogues can further be used to increase

selectivity toward the ENaC, NHE, or NCX. Of the
Many analogues of amiloride have been synthesized to numerous analogs synthesized since 1967, the only two
increase the specificity toward one of three ion channels compounds approved for clinical use for treatment of
mentioned above. The specificities of these analogues are hypertension in human beings by the United States Food
reported as the “potency relative to amiloride” and have and Drug Administration that are known to decrease the
been catalogued in detail elsewhere.25 A comparison of activity of the ENaC are amiloride and triamterene. We
the relative potency of some amiloride analogues is pre- will now review the effects of amiloride and its analogues
sented in Table 1. For example, benzamil (Fig. 2), as in experimental and human studies.
compared with amiloride, is ninefold more potent toward
the ENaC30 with a markedly lower relative potency (0.08-
fold) toward the NHE. Conversely, dimethylamiloride The Epithelial Sodium
(Fig. 3), as compared with amiloride, has a dramatically Channel in the Central
lower potency toward the ENaC (⬍0.035-fold) while sub- Nervous System: Cell Studies
stantially increasing its inhibitory effect (12-fold) toward
the NHE.25 Replacement of the pyrazine ring of amiloride Within the principal cell of the kidney, the ENaC is known
by a phenyl, the 6-chloro substituted by a sulfomethyl, and to be a multi-subunit protein consisting of ␣-, ␤- and
the 2-amino deleted or replaced by a methyl group (Fig. 4) ␥-subunits.33 However, a recent study of rat vascular
has led to the benzoylguanidines. These compounds in- smooth muscle cells (VSMC) examined the presence of
clude cariporide and eniporide (see section Translational degenerin/epithelial Na⫹ channel proteins (DEG/ENaC) in
Research of NHE Inhibition in Ischemic Cardiac States) rat cerebral blood vessels and VSMC, and their role as
and have markedly increased specificity toward the NHE1 mechanosensors in mediating the myogenic response in
while completely losing their inhibitory potency to the intact blood vessels.14 The DEG/ENaC cation family is a
NCX and the ENaC.31,32 class of proteins sharing a common topology and sequence
In summary, amiloride at very low doses is highly homology with ENaC and may function as mechanosen-
specific for the ENaC as compared with NHE and NCX. sors in a diverse range of species.15 Using rat cerebral
VSMC, specific ENaC subunit transcripts for ␤-ENaC and
␥-ENaC were identified by reverse transcription–poly-
merase chain reaction (RT-PCR) and the ␣-ENaC subunit
CH3 by nested PCR. Western blot analysis and immunofluo-
CH3 N N NH2 rescence labeling identified both ␤- and ␥-, but not the
␣-ENaC subunits in VSMC from these vessels.14 Effects
of amiloride and benzamil on the myogenic response of
isolated cerebral blood vessels during stepwise increases
N NH2
Cl
N in transmural pressure was also examined. Under control
O conditions, blood vessel diameter decreased in response to
NH2
increasing pressure. However, amiloride (1 ␮mol/L) and
submicromolar doses of benzamil (30 nmol/L), doses
known to inhibit the ENaC, inhibited the myogenic vaso-
FIG. 3. Chemical structure of dimethylamiloride. 5-Amino nitrogen
atom with 2 methyl group substitutes produces one of the most constriction response to increasing perfusion pressure by
potent and specific inhibitors of the Na⫹/H⫹ exchangers. blocking the activity of ENaC proteins.14
112 AMILORIDE IN CARDIOVASCULAR DISEASE AJH–January 2007–VOL. 20, NO. 1

Functional studies indicate that neural mechanisms act- the first, endothelial cell volume response to a 20-min
ing through the MR and the ENaC are crucial to the exposure to aldosterone (0.1 ␮mol/L) was assessed by
development of sympathoexcitation and hypertension in atomic force microscopy, a measure of cell volume and
experimental models of salt-sensitive hypertension.34 –36 volume shift (cytosol to nucleus). Aldosterone produced
Amin et al demonstrated the ENaC and MR expression at a maximal cell volume increase of 18% at 10 min and
the level of mRNA and protein levels in the cardiovascular nuclear swelling at 5 min, followed by nuclear shrink-
regulatory centers of the brain of Wistar rats by RT-PCR age 15 min later. Spironolactone (1 ␮mol/L) and amilo-
and immunohistochemistry, respectively.37 Both mRNA ride (1 ␮mol/L) prevented these shifts in cytosol and

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and protein for ␣-, ␤-, and ␥-ENaC subunits were found to nuclear volume. In contrast, the NHE blocker, cariporide,
be expressed in rat brain. In the cells of the hippocampus, was ineffective despite using a dose 10-fold higher (10
hypothalamus, and cerebral cortex, the ENaC subunits and ␮mol/L).19 In the second study, endothelial cell volume
the MR were found to follow a membranous and perinu- response was assessed over a period of 72 h in endothelial
clear distribution, respectively. cells exposed to a 10 nmol/L concentration of aldosterone.
At this concentration, aldosterone produced an 18% in-
The Epithelial Sodium Channel crease in cell volume during this time. Aldosterone-in-
duced swelling was prevented by spironolactone (100
in the Vasculature: Cell Studies nmol/L). When amiloride (1 ␮mol/L) was added to endo-
The MR and the ENaC have been demonstrated at the thelial cells in the presence of aldosterone, a dramatic
level of mRNA and at the protein level within fibro- reduction in cell volume ensued. In contrast, cells that
blasts,17 VSMC,14,38 and endothelial cells.16,18 Kornel were deprived of aldosterone failed to respond to amilo-
et al showed that VSMC from the aorta of rabbits treated ride.20 The authors concluded that aldosterone responsive
with physiologic doses of aldosterone (5 nmol/L) for 7 to ENaC was in part responsible for these observed changes
10 days were found to have significant increases in Na⫹ in cell and nuclear volume. These data support the view
influx. Furthermore, Na⫹ influx was almost completely that aldosterone exerts important physiologic/patho-
inhibited by amiloride in doses known to be relatively physiologic effects on the cardiovascular system and that
specific for ENaC (1.5 ␮mol/L), but not by either the low-dose amiloride may antagonize the effects of adverse
NHE-specific amiloride analogue ethylisopropyl-amilo- cardiovascular effects of aldosterone.40,41 However, al-
ride or the NCX-specific dichlorobenzamil. In a separate though cellular studies can be helpful in discerning certain
study, rabbits were treated with aldosterone (2 mg/day) for mechanistic data, one can only begin to assess potentially
4 weeks. Subsequently, VSMC were isolated from the clinically relevant efficacy by using animal models.
aorta and quantification of Na⫹ channels was performed
using ENaC specific [3H] amiloride binding. Aldosterone-
Systemic EnaC Inhibition: Animal Studies
treated animals had double the number of VSMC Na⫹
channels, suggesting a possible relationship between aldo- Campbell et al examined myocardial fibrosis in a high-
sterone and ENaC within VSMC.38 salt/aldosterone state. Uninephrectomized Sprague-Daw-
Golestaneh et al examined whether similar findings ley rats were placed on 1% NaCl drinking solution and
occur in the endothelial cells obtained from human um- given one of the following for 8 weeks: 1) aldosterone
bilical cords. After double-labeled immunofluorescence 0.75 ␮g/h subcutaneously; 2) amiloride 1 mg/kg/day sub-
recorded by confocal microscopy, both the MR and the cutaneously; 3) aldosterone ⫹ amiloride subcutaneously;
ENaC were found to be co-localized in endothelial or 4) vehicle. Aldosterone increased BP significantly, an
cells. Immunocytochemical localization showed that the effect that was attenuated by amiloride. Microscopic scar-
intracellular MR was labeled primarily as a cytoplasmic ring, a reparative fibrosis indicative of myocyte loss, was
protein with perinuclear preference, whereas the mem- apparent in animals treated with aldosterone. However,
brane-bound ENaC was revealed as diffuse grains in a when amiloride was given simultaneously with aldoste-
perinuclear pattern within the cytoplasm.16 Both of these rone, the microscopic scarring of both the left and right
studies examined the relationship of the ENaC to the pres- ventricles was completely prevented. The finding of scar-
ence of aldosterone at levels of 10 ␮mol/L and 1 nmol/L, ring to the nonhypertensive right ventricle suggests that
respectively. Aldosterone was found to increase significantly the benefits of amiloride were not from BP-lowering ef-
levels of the ENaC by RT-PCR and immunocytochemical fects alone. The authors postulated that myocyte necrosis
localization in the respective studies. These data show that in hyperaldosteronism is likely a result of enhanced po-
aldosterone-mediated signaling in endothelial cells is com- tassium excretion that can be prevented by amiloride.21
parable to that in cells of the epithelial origin.39 Although this remains a possibility, measurements of uri-
Two physiologic studies have been performed by Ober- nary Na⫹/K⫹handling and serum K⫹ were not performed.
leithner et al on human umbilical endothelial cells in the Furthermore, subsequent studies suggest other possible
presence and absence of aldosterone and low-dose amilo- mechanisms of benefit (see later here).
ride (1 ␮mol/L) to provide indirect evidence for the pres- Mirkovic et al studied the attenuation of cardiac fibrosis
ence of both the MR and the ENaC in endothelial cells. In with amiloride in another high mineralocorticoid hor-
AJH–January 2007–VOL. 20, NO. 1 AMILORIDE IN CARDIOVASCULAR DISEASE 113

mone state by using the DOCA-salt hypertensive rat.22 In selective NHE inhibitor amiloride analog)25 at a dose of
Wistar rats given 1% NaCl drinking solution and deoxy- 0.7 mg/kg/day subcutaneously and values in control rats.
corticosterone acetate (DOCA) 25 mg subcutaneous every Dimethylamiloride-treated rats survived until an average
4 days, collagen deposition was found to be significantly of 14.7 weeks of age, which was significantly (P ⬍ .005)
increased in the interstitium at 2 weeks with further in- longer than control rats (0% survival at 12.7 weeks).
creased scarring of the left ventricle after 4 weeks. Amilo- However, benzamil treated SHRSP rats survived, on av-
ride at 1 mg/kg/day subcutaneously was found to reverse erage, until 16.1 weeks of age, which was significantly
the initial increases in collagen deposition and prevent (P ⬍ .05) longer than the dimethylamiloride-treated rats.

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any further increases. These benefits occurred without Blood pressure was severely elevated in all rats, with no
any significant change to SBP. Because previous studies differences between groups throughout the study. Further-
using angiotensin-converting enzyme (ACE) inhibitors, more, benzamil delayed the onset of proteinuria compared
angiotensin receptor blockers, and MRA demonstrated with dimethylamiloride. Acute administration of benzamil
that cardiac fibrosis can be reversed in the absence of (1 mg/kg) or dimethylamiloride (1 mg/kg) did not change
significant BP lowering or decreasing cardiac hypertro- plasma sodium or body weight. A slight, but significant,
phy,42 this study adds amiloride to the list of agents that elevation occurred in the plasma potassium at 4 h (but not
seem to prevent or mitigate cardiac fibrosis in experimen- at 24 h) in both groups (unexpectedly in the dimethyl-
tal hypertension animal models. The authors postulate that amiloride group) compared with control. However, the
prevention of scarring by amiloride may be related to dose administered was larger than that used in the survival
maintenance of myocardial potassium concentration. How- analysis and was administered as a bolus, rather than a
ever, they did not measure blood or tissue levels of amilo- continuous infusion throughout the day. Although reasons
ride, nor did they determine the intracellular potassium why saline-drinking SHRSP do not exhibit a hyperkalemic
concentration or the effects on potassium transport. In fact, response to benzamil are not clear, the low-potassium diet,
their study showed no significant difference in plasma increased sodium intake, or a genetic defect in potassium
potassium between the DOCA-salt and the subsequent transport in these rats (or all of these factors) may be
DOCA-salt ⫹ amiloride treated rats. responsible.
Sepehrdad et al performed survival analysis with the
administration of amiloride in the saline-drinking, stroke-
prone spontaneously hypertensive rats (SHRSP). First, the EnaC Inhibition of the Central
authors demonstrated that acute administration of escalat-
Nervous System: Animal Studies
ing doses of amiloride (1 to 30 mg/kg/day) did not alter
urine output, urinary Na⫹/K⫹ ratio, or body weight. Fur- Several experiments suggest a mechanism of benefit by
thermore, elevated mean arterial pressure in SHRSP was central nervous system ENaC inhibition as demonstrated
affected only at the highest dose of amiloride, namely by the intracerebroventricular infusion of benzamil. Go-
30 mg/kg/day. In a survival analysis of 8.5 week-old mez-Sanchez et al found that intracerebroventricular in-
SHRSP rats given amiloride (1 mg/kg/day) along with jection of benzamil (0.3 to 1 ␮g/h) significantly attenuated
1% NaCl drinking solution, exhibited no decrease in SBP the increase in salt-induced hypertension in the inbred
nor change in urine Na⫹/K⫹ excretion or urine output as Dahl salt-sensitive (SS/jr) rat, whereas the subcutaneous
compared with untreated rats. All of the control SHRSP infusion of benzamil (0.5 ␮g/h) did not.34 Nishimura et al
died by 16.4 weeks, whereas 75% of the amiloride-treated showed that the continuous intracerebroventricular infu-
SHRSP were alive at the end of the 20-week study period. sion of benzamil (1 or 10 nmol/kg/day) for 7 days atten-
All of the amiloride-treated SHRSP rats alive at the study uated salt-induced hypertension in both DOCA-salt
end showed no signs of stroke, whereas all control rats hypertensive and SHRSP rats but not in renovascular
displayed neurologic signs of stroke before death. More- hypertensive rats.35 Huang et al examined the effects of
over, despite prolonging survival by an average of 6 weeks, intracerebroventricular infusion of benzamil in cardiovas-
amiloride delayed the onset of proteinuria and improved cular disease. Wistar rats received intracerebroventricular
brain and kidney histologic scores compared with con- infusions of benzamil (4 ␮g/kg/h) or its vehicle, or spi-
trols.23 This study indicated that amiloride is similar to ronolactone (400 ng/kg/h) or its vehicle, 3 days after
ACE inhibitors,43 angiotensin subtype-1 antagonists,44 having their coronary artery ligated. Blockade of brain
and MRA45,46 for markedly reducing stroke, proteinuria, ENaC or MR similarly prevented sympathetic hyperactiv-
and vascular injury, in the absence of BP lowering, in the ity and improved cardiac function as compared with ve-
saline-drinking SHRSP model. hicle.36 However, although benefit of ENaC inhibition is
To determine whether inhibition of ENaC or NHE seen with direct infusion into the central nervous system,
plays a more significant role in improving survival in none of these studies examined the degree to which ben-
saline-drinking SHRSP rats, Sepehrdad performed a sec- zamil or amiloride crosses the blood– brain barrier, and
ond survival study.24 Benzamil (an ENaC specific amilo- therefore it is unclear whether the systemic use of ENaC
ride analog)25 administered at a dose of 0.7 mg/kg/day inhibitors can derive their benefit from their effects in the
subcutaneously was compared with dimethylamiloride (a central nervous system.
114 AMILORIDE IN CARDIOVASCULAR DISEASE AJH–January 2007–VOL. 20, NO. 1

In summary, the ENaC appears to be present and func- strategy. However, at doses used to inhibit NHE1 in clin-
tional in nonrenal epithelial cells of the cardiovascular and ical trials of ischemic heart disease, the risks and adverse
central nervous system including endothelial, smooth mus- events of these agents appear to outweigh the potential
cle, and fibroblast cells. Amiloride antagonizes or prevents benefits.
actions of aldosterone in these cells and in cardiovascular
and renal tissues in animals with salt-dependent forms of
Amiloride in
hypertension. These studies give rise to the notion that
Low-Renin Forms of Hypertension
ENaC inhibition may be a potential new target in the

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treatment of cardiovascular disease. The antihypertensive properties of amiloride have long
been known53; however, an emerging role for amiloride in
Clinical Studies low-renin forms of hypertension has recently been exam-
ined. Eide et al evaluated 90 patients referred for the
Translational Research of NHE1
treatment of resistant hypertension; these patients re-
Inhibition in Ischemic Cardiac States
mained hypertensive despite treatment with at least three
The NHE1 inhibitor, cariporide, has been found in rabbits drugs (including a diuretic agent).54 Of these referred
to reduce dramatically the size of myocardial infarction by patients, 60 (67%) were found to have a low-renin form of
50%, when a dose of 0.3 mg/kg is given intravenously hypertension as defined as plasma renin activity (PRA)
15 min before a 30-min coronary artery occlusion with 3 h ⬍0.5 nmol/L/h. Of these 60 patients, 38 went on to par-
of reperfusion.47 Although animal models have shown a ticipate in a prospective, randomized, placebo-controlled,
protective benefit of NHE1 inhibition in coronary artery– double-blind, cross-over study. Patients were placed on a
occlusive states,47,48 when the NHE1 inhibitors cariporide fixed combination of amiloride 2.5 mg and hydrochlo-
and eniporide were used in large-scale, double-blind, ran- rothiazide (HCTZ) 25 mg or placebo. After 1 week, if the
domized clinical trials, they were found to be either inef- study pill was tolerated and BP was clinically insuffi-
fective or potentially dangerous. The Guard During ciently reduced, the study dosage was doubled (which was
Ischemia Against Necrosis (GUARDIAN) trial examined done in 26 of the 38 patients). Patients were then followed
the use of intravenous cariporide at doses of 20 mg, 80 mg, for 2 weeks more with a washout period of 1 to 2 months.
and 120 mg versus placebo in 2918 patients requiring The amiloride/HCTZ combination was found to reduce the
urgent coronary artery bypass graft to evaluate the effects mean SBP and DBP by 31/15 mm Hg at 3 weeks as
on all-cause mortality or myocardial infarction at 36 days. compared with placebo. A nonsignificant increase in mean
Although there were no significant adverse effects with serum potassium from 4.0 mmol/L to 4.1 mmol/L oc-
cariporide, the primary end point of all-cause mortality or curred. However, although the increase in serum potas-
myocardial infarction (MI) by day 36 was not different sium was minimal, 17 of the 38 patients met criteria of
between groups, although a nonsignificant trend was seen primary aldosteronism with a mean urine aldosterone ex-
at the highest dose of cariporide.49,50 The sodium– hydro- cretion of 68 ⫾ 14 nmol/24 h.
gen exchange inhibition to prevent coronary events in Saha et al examined the use of amiloride in 98 African
acute cardiac conditions (EXPEDITION) trial was per- American patients who remained hypertensive despite the
formed in a much larger number of patients (n ⫽ 5770), use of HCTZ or lasix with amlodipine and who met
and used a higher dose of cariporide (180 mg), to evaluate criteria for a low-renin form of hypertension (defined as
whether cariporide reduces all-cause mortality or myocar- PRA ⱕ0.56 ng/L/sec).55 The study was a randomized,
dial infarction in patients before coronary artery bypass placebo-controlled, double-blind, parallel-group trial with
graft surgery as compared with placebo. In this study, a primary endpoint of reduction in SBP/DBP over a
cariporide significantly decreased the incidence of myo- 9-week period. All patients remained on their HCTZ or
cardial infarction; however, an increase in all-cause mor- lasix and then randomized to 1 of 4 treatment groups:
tality and cerebrovascular events was observed in the amiloride 10 mg, spironolactone 25 mg, combination of
cariporide group. The authors concluded that the imbal- amiloride 10 mg/spironolactone 25 mg, or placebo. Perti-
ance in the safety profile of cariporide outweighs the nent exclusion criteria included use of ACE inhibitors or
benefits of reduction in MI.51 In the Evaluating the Safety angiotensin receptor blockers and a serum creatinine ⬎1.5
and Cardioprotective Effects of Eniporide in Acute Myo- mmol/L in men and ⬎1.4 mmol/L in women. As com-
cardial Infarction (ESCAMI) trial, the use of another pared with placebo, spironolactone significantly reduced
NHE1 inhibitor, eniporide, was investigated in 1411 pa- SBP and DBP (7.3 ⫾ 2.3 mm Hg and 3.3 ⫾ 1.4 mm Hg);
tients with acute myocardial infarction. There was no however, use of amiloride led to a greater reduction in
difference in the infarct size between groups, although a SBP and DBP (12.2 ⫾ 2.2 mm Hg and 4.8 1.3 mm Hg).
similar trend toward excess death and stroke, as seen in There was no synergism in BP reduction with a combina-
EXPEDITION, was observed in patients receiving enipo- tion of amiloride/spironolactone as compared with amilo-
ride.49,52 ride alone. Use of 10 mg of amiloride raised the serum
In summary, in vitro and in vivo studies support the potassium by only 0.35 ⫾ 0.06 mmol/L, and no patients in
potential for inhibitors of NHE1 as a cardioprotective the amiloride arm had a serum potassium ⬎5.0 mmol/L.
AJH–January 2007–VOL. 20, NO. 1 AMILORIDE IN CARDIOVASCULAR DISEASE 115

However, the mean baseline serum potassium in the amilo- ride to lowering BP via its effects on salt/water balance.
ride arm was 3.6 mmol/L. Moreover, the mean plasma Outside of its antihypertensive properties in low-renin
aldosterone was 17.1 ng/dL and the mean aldosterone/ forms of hypertension, if ENaC inhibition is proved to be
PRA ratio was ⬎15, which can be suggestive of primary of therapeutic benefit in cardiovascular disease states as
hyperaldosteronism. Nonetheless, these studies suggest well, wide-scale use could result in rates of hospitalization
the use of amiloride as a viable option of treatment in for hyperkalemia similar to those recently observed with
hypertensive patients in low-renin states who fail to reach MRA.10 A possible solution can again be found by exam-
BP goals with standard therapy. ining the pharmacokinetics of amiloride. The average

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amount of amiloride excreted unchanged in the urine of
Amiloride in Cardiovascular Disease healthy volunteers is 49%, but rises to as much as 100% in
patients with hepatitis.29 Although one explanation for the
To date, there is one published study examining the use of increased urinary excretion in hepatitis patients would be
low-dose amiloride in relation to cardiovascular disease. from decreased biotranformation, another possibility
Farquharson and Struthers investigated whether increasing would be decreased biliary excretion. Therefore, as at least
plasma potassium by the oral administration of 5 mg/day 50% of the route of elimination for amiloride is through
of amiloride for 1 month reduces endothelial dysfunction. the kidney, and any benefits of systemic blockade of ENaC
This was assessed by forearm blood flow to the brachial could be masked by the inability to reach the required
artery via forearm venous occlusion plethysmography in systemic concentrations (0.5 to 1.0 ␮mol/L). Because of
response to acetylcholine in patients with New York Heart the potential for hyperkalemia with amiloride and other
Association class II to III chronic heart failure. In a pre- potassium sparing diuretics, we believe that further studies
vious study using this technique, the authors demonstrated with amiloride are warranted, including those in popula-
that spironolactone at 50 mg/day for 1 month was found to tions of patients who may be susceptible to hyperkalemia,
improve endothelial dysfunction to a significant extent.56 such as those with diabetes and those with chronic kidney
However in this study, amiloride did not decrease endo- disease.59,60 The use of additional amiloride analogues
thelial dysfunction, despite the increase in serum potas- that have increased ENaC selectivity (eg, benzamil) do not
sium by 0.4 mmol/L.57 This study was performed by the have approval by the Food and Drug Administration for
authors to show that the benefits of spironolactone to use in human beings and would require additional human
improve endothelial dysfunction are not mediated through studies regarding the route of elimination and effects on
its effect on serum potassium. However, as noted above, serum K⫹ balance. Thus, modification of existing ana-
several animal studies suggest the benefits of survival and logues to a hepatic clearance route could open new doors
improved histopathologic scores with low-dose amiloride in examining the possibility of systemic benefits of ENaC
are not related to effects on serum potassium. The question inhibition.
then arises: if low-dose amiloride does have cardioprotec-
tive properties, how could this study produce a negative
result? A possible answer lies with a closer look at the
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