Responses in extracellular and intracellular calcium
and magnesium in aldosteronism
ALIYE L. RUNYAN, YAO SUN, SYAMAL K. BHATTACHARYA, ROBERT A. AHOKAS,
VIKRAM S. CHHOKAR, IVAN C. GERLING, and KARL T. WEBER
We hypothesized the hypercalciuria and hypermagnesuria that accompany aldoste-
ronism could be pharmacologically attenuated to prevent shifts in extracellular and
intracellular levels of these divalent cations and the adverse outcomes associated with
them. Accordingly, rats administered aldosterone/salt treatment (ALDOST) were co-
treated with either hydrochlorothiazide (Hctz), to selectively reabsorb urinary Ca2?, or
with Hctz plus spironolactone (Hctz?Spi), where Spi retards the excretion of these
cations in both urine and feces. We monitored urinary excretion and responses in
extracellular and intracellular Ca2?and Mg2?, together with indices of oxi/nitrosative
stress in plasma and ventricular tissue. At 4 weeks ALDOST we found the following: (1)
hypercalciuria was reduced by Hctz and normalized by Hctz?Spi, and this combina-
tion, unlike Hctz alone, also rescued hypermagnesuria; (2) the decrease in plasma-
against a decline in [Mg2?]o; (3) the Ca2?loading of peripheral blood mononuclear
cells and cardiac tissue was not seen with Hctz?Spi; and (4) the induction of oxi/
nitrosative stress, expressed as reduced plasma ?1-antiproteinase activity and activa-
tion of gp91phoxsubunit of NADPH oxidase in inflammatory cells invading intramural
coronary arteries of the right and left ventricles, together with vascular fibrosis, was
completely prevented by Spi cotreatment. In rats with aldosteronism, cotreatment with
Hctz?Spi more effectively (vis-à-vis Hctz alone) protects against adverse iterations in
extracellular and intracellular concentrations of Ca2?and Mg2?, as well as the ap-
pearance of oxi/nitrosative stress to prevent the proinflammatory vascular phenotype.
(J Lab Clin Med 2005;146:76–84)
Abbreviations: ALDO ? aldosteronism; ALDOST ? aldosterone/salt treatment; DOCST ? de-
oxycorticosterone/salt treatment; Hctz ? hydrochlorothiazide; NF?B ? nuclear transcription
factor kappa B; PBMC ? peripheral blood mononuclear cells; PTH ? parathyroid hormone; Spi
matory phenotype. Features include oxi/nitrosative
stress in diverse tissues; inflammatory cells invading
ldosteronism or chronic, inappropriate (rel-
ative to dietary Na?) elevations in plasma
aldosterone (ALDO) results in a proinflam-
the cardiovasculature and fibroblasts that promote an
adverse remodeling of the coronary and systemic
vasculature; and tissue wasting.1–6The involvement
of the heart and systemic organs in aldosteronism is
related to both ALDO and dietary Na?, whereas
From the Divisions of Cardiovascular Diseases and Endocrinology,
Department of Medicine, Departments of Surgery and Obstetrics and
Gynecology, University of Tennessee Health Science Center, Mem-
This work was supported, in part, by National Institutes of Health/
National Heart, Lung, and Blood Institute grant R01-HL73043 (KTW).
Submitted for publication January 28, 2005; accepted for publication
April 10, 2005.
Reprint requests: Karl T. Weber, MD, Division of Cardiovascular
Diseases, University of Tennessee Health Science Center, 920 Madison
Ave., Suite 300, Memphis, TN 38163; e-mail: KTWeber@utmem.edu
0022-2143/$ – see front matter
© 2005 Mosby, Inc. All rights reserved.
hemodynamic factors, the hypertrophic growth of
cardiomyocytes, and ALDO per se (in the presence
of dietary Na?deprivation) have each been dis-
counted as contributory.7The systemic nature of this
phenotype has recently been underscored by Del-
Cayre and coworkers, who did not find coronary
vascular remodeling in a transgenic rodent having an
overexpression of ALDO synthase in the heart with
accompanying increments in cardiac tissue ALDO.8
Interest in elucidating pathogenic origins and
pathophysiologic expressions of this systemic proin-
flammatory phenotype has turned to rats receiving
1% dietary NaCl and an infusion of ALDO by im-
planted minipump (ALDOST) to increase plasma
levels of this hormone to those found in human
congestive heart failure.9An early immunostimula-
tory state involving peripheral blood mononuclear
cells (PBMC: lymphocytes and monocytes) is seen as
early as week 1 ALDOST10,11and subsequently
eventuates in the invasion of the intramural coronary
and systemic vasculatures at week 4 and be-
yond.4,5,11–13A role for reactive oxygen and nitrogen
species in signal transduction has been suggest-
ed,2,4,5,11,13where the induction of oxi/nitrosative
stress is related to a Ca2?loading of PBMC.10,11,13
The increase in PBMC cytosolic-free Ca2?occurs as
an outcome to events initiated by dietary Na?-de-
pendent enhancement in urinary and fecal excretion
of Ca2?and Mg2?.6,14–16A subsequent decrease in
plasma-ionized concentrations of these divalent cat-
ions provokes the release of parathyroid hormone
(PTH) and resorption of bone.6,14In secondary hy-
perparathyroidism, this calcitropic hormone pro-
motes Ca2?loading of PBMC by receptor-ligand
binding with excessive Ca2?entry occurring through
L-type channels that can be prevented by a Ca2?
channel blocker.13,17–19Dihydropyridine Ca2?chan-
nel antagonists and mibefradil each prevent the ap-
pearance of this vascular remodeling in rats with
chronic mineralocorticoid excess.13,20–22
We used rats with ALDOST to test our hypothesis
that a pharmacologic attenuation of the heightened
excretion of either or both these cations would pre-
vent these consequent pathophysiologic responses.
Accordingly, we monitored urinary Ca2?and Mg2?
excretion, extra- and intracellular concentrations of
these cations in plasma, PBMC, and cardiac tissue at
4 weeks ALDOST, given alone or in combination
with either hydrochlorothiazide (Hctz), to enhance
urinary Ca2?resorption, or Hctz?spironolactone
(Spi), where Spi reduces both urinary and fecal Ca2?
and Mg2?excretion.14Untreated, age- and gender-
matched rats served as controls.
Animal model. Male, 8- to 12-week-old Sprague-Dawley
rats (Harlan, Indianapolis, Ind) were used in this study ap-
proved by the institution’s Animal Care and Use Committee.
There were four groups. Age- and gender-matched, unoper-
ated, untreated rats served as controls. As previously reported,
ALDOST consists of uninephrectomized rats who received
ALDO (0.75 ?g/h) by implanted minipump (Alzet, Cuper-
tino, Calif) together with dietary 1% NaCl/0.4% KCl in
drinking water and standard laboratory chow (Harlan Teklad
2215 Rodent Diet, Madison, Wisc) containing 1.13% Ca2?.
A separate group of animals received ALDOST plus Hctz.
Hctz (50 mg/kg) was mixed into powdered standard chow
with water added. The mixture was placed in a pan, cut into
small squares, and air dried for 24 to 48 hours. We did not
monitor food intake. Yet another group received Hctz plus
Spi (200 mg/kg body weight) in divided doses by twice-daily
gavage. Five rats comprised each experimental and control
group unless otherwise specified.
Experimental protocol. Animals were anesthetized and
killed, and blood and cardiac tissue were harvested at week 4
of each regimen when we monitored the following: 24-hour
urinary Ca2?and Mg2?excretion; plasma-ionized [Ca2?]o
and [Mg2?]o; PBMC cytosolic-free [Ca2?]iand [Mg2?]i;
total Ca2?and Mg2?concentrations in ventricular tissue; and
plasma ?1-antiproteinase (AP) activity. Immunohistochemi-
cal and microscopic examinations of coronal sections of right
and left ventricles was performed (vida infra).
Urinary Ca2?and Mg2?excretion. On the day of the
metabolic study, food was withheld, but water with 1% NaCl
was provided to treated animals. Animals were “bathed” in
distilled water to remove any feces or food that could con-
taminate collected urine. Animals were then placed in a
cleaned, minerally decontaminated and distilled-deionized,
water-rinsed metabolic cage. Urine was collected over 24
hours and kept frozen for Ca2?and Mg2?assay.
Urinary Ca2?and Mg2?concentrations were determined
as reported elsewhere6,14using a Varian model 220 FS dou-
ble-beam fast sequential atomic absorption spectrophotome-
ter (Varian Techtron, Melbourne, Australia). Urinary Ca2?
and Mg2?excretion rates were calculated from the product of
their concentration (micrograms/milliliter) by the 24-hour
urine volume (mL/24 hour) and expressed as ?g/24 hour.
Plasma-ionized [Ca2?]oand [Mg2?]o. The plasma con-
centrations of [Ca2?]oand [Mg2?]owere determined by the
direct ion-selective electrode technique using a Nova 8 An-
alyzer (Nova Biomedical, Waltham, Mass) and expressed in
millimoles per liter.
Cytosolic-free [Ca2?]iand [Mg2?]iin peripheral blood
mononuclear cells. Cytosolic-free [Mg2?]i and [Ca2?]i
were measured as previously reported11using a modification
of the ratiometric method and the fluorescent molecular
probes mag-fura-2 and fura-2 (Molecular Probes, Eugene,
Ventricular tissue Ca2?and Mg2?concentrations. As
reported previously,14microdetermination for Mg2?and
Ca2?concentrations in ventricular tissue was carried out in
demoisturized, defatted specimens after complete digestion in
J Lab Clin Med
Volume 146, Number 2Runyan et al
5 mL of 0.75 M Ultrex quality nitric acid (J. T. Baker
Chemical Co., Phillipsburg, Penn) for 15 hours at 68°C. This
procedure extracts more than 99% of Mg2?and Ca2?from
dry, defatted tissue. Tissue Mg2?and Ca2?levels are ex-
pressed as nEq/mg of fat-free dry tissue.
Plasma ?1-antiproteinase activity. As previously report-
ed,10plasma ?1-AP activity was assessed using the ?1AP-410
assay system (Oxis Research, Portland, Ore) and expressed in
micromoles per liter.
NADPH oxidase. In hearts harvested at week 4 ALDOST,
coronal cryostat sections (6 ?m) of right and left ventricular
tissue were prepared, air dried, fixed in 10% buffered forma-
lin for 5 minutes, and washed in phosphate-buffered saline for
10 minutes. Sections were then incubated with primary anti-
body against gp91phoxas previously reported.2
Collagen volume fraction. The appearance of inflamma-
tory cells in the rat heart were detected by hematoxylin-eosin
staining. The interstitial and perivascular distribution of col-
lagen was determined from picrosirius red-stained tissue sec-
tions as previously reported.2
Statistical analysis. Values are presented as mean ?
standard error of the mean. Data were analyzed using
analysis of variance. Significant differences between indi-
vidual means were determined using the post hoc Bonfer-
roni multiple comparisons test. Significance was assigned
to P values less than .05.
Urinary Ca2?and Mg2?excretion. In comparison with
controls, urinary Ca2?excretion at 4 weeks ALDOST
was increased more than fourfold. Cotreatment with
Hctz reduced this hypercalciuria by more than 70%,
whereas the addition of Spi normalized urinary Ca2?
excretion (Fig 1).
Urinary Mg2?excretion was likewise increased four-
fold more than that of controls at 4 weeks ALDOST,
which was not altered by Hctz cotreatment. However,
the addition of Spi significantly attenuated this
Plasma-ionized Ca2?and Mg2?. At 4 weeks AL-
DOST, plasma [Ca2?]owas reduced compared with
controls. This decrease in [Ca2?]o, a stimulus to the
secretion of PTH, was not seen with either Hctz alone
or the combination of Hctz?Spi (Fig 2).
Plasma [Mg2?]owas reduced at 4 weeks ALDOST.
A more marked decrease in plasma [Mg2?]oand addi-
Fig 1. Twenty-four hour urinary Ca2?and Mg2?excretion (u-Ca2?and u-Mg2?, ?g/24 h) (left and right panels,
respectively) for 4 weeks aldosterone/salt treatment (ALDOST) (A), ALDOST ? hydrochlorothiazide (Hctz)
(AH), and ALDOST ? Hctz ? spironolactone (Spi) (AHS). Untreated, age- and gender-matched controls (C).
See text for details.
Fig 2. Plasma-ionized levels (millimoles/liter) of [Ca2?]oand [Mg2?]ofor the three treatment groups and
controls (defined in Fig 1). See text for details.
J Lab Clin Med
Runyan et al
tional stimulus to PTH release were seen with Hctz
cotreatment. This could be the result of increased gas-
trointestinal Mg2?losses. In this study we did not
monitor fecal Mg2?in response to ALDOST?Hctz. In
previous studies the urinary and fecal excretion and its
response to interventions have always been parallel.14
The possibility of a thiazide-sensitive cotransporter or
exchanger, induced by ALDO and upregulated by Hctz
in gut epithelial cells, therefore cannot be discount-
ed.23,24Cotreatment with Spi prevented the decrease in
[Mg2?]oseen with ALDOST and where Spi reduces
urinary and gastrointestinal losses of Mg2?.14
[Ca2?]iand [Mg2?]i. Cytosolic-free [Ca2?]iin PBMCs
was significantly increased at week 4 ALDOST and
with Hctz cotreatment. This Ca2?loading of PBMC
was rescued by Spi cotreatment (Fig 3).
[Mg2?]iin PBMCs was significantly reduced at week
4 ALDOST and with ALDOST?Hctz. With the addi-
tion of Spi to the ALDOST?Hctz regimen, cytosolic-
free [Mg2?]iwas greater than ALDOST alone or
Ventricular tissue Ca2?and Mg2?. Total Ca2?concen-
tration of ventricular tissue at 4 weeks ALDOST was
significantly increased compared with controls (Fig 4).
This intra- and extracellular Ca2?loading of myocar-
dial tissue was not seen with either Hctz or Hctz?Spi
cotreatments. This may relate to a cardioprotective ef-
fect of this thiazide mediated by its reducing L-type
Ca2?current and opening mitochondrial ATP-sensitive
K?channels in cardiomyocytes.25,26
Total Mg2?concentration of myocardial tissue was
significantly reduced at 4 weeks ALDOST. It was also
reduced, although not significantly, with Hctz cotreat-
ment. The addition of Spi, however, prevented this
decline in tissue Mg2?.
controls, ?1-AP activity was significantly reduced at 4
weeks ALDOST and with ALDOST?Hctz. A decrease
in this systemic marker and inverse correlate of oxi/
nitrosative stress was not seen with the addition of Spi
Activation of NADPH oxidase. Inflammatory cells in-
vaded the perivascular space of intramural coronary arter-
ies of the right and left ventricles at 4 weeks ALDOST
(Fig 6). As noted previously2these are ED1? macro-
phages and CD4? lymphocytes. Evidence of gp91phox
activation in these cells was found by immunohistochem-
activity. Compared with
(PBMCs) for the three treatment groups and controls (defined in Fig 1). See text for details.
Cytosolic-free [Ca2?]iand [Mg2?]ilevels (millimoles/liter) in peripheral blood mononuclear cells
Fig 4. Total Ca2?and Mg2?levels (nEq/mg fat-free dry tissue) for ventricular tissue for the three treatment
groups and controls (defined in Fig 1). See text for details.
J Lab Clin Med
Volume 146, Number 2Runyan et al
istry. The number of these cells and their expression of
gp91phoxwere attenuated by Hctz cotreatment and com-
pletely prevented by the addition of Spi.
Collagen volume fraction. The proinflammatory cor-
onary vascular phenotype seen with ALDOST gives
way to perivascular and interstitial fibrosis of involved
vessels, which we previously showed to be related to
myofibroblasts and their upregulated mRNA expres-
sion of type I collagen, a major component of fibrous
tissue (Fig 7).1,2The importance of cell–cell signaling
involving inflammatory cells and myofibroblasts is ev-
idenced by a reduction in fibrous tissue formation sur-
rounding intramural coronary arteries and within the
interstitial space with the attenuation of the inflamma-
tory cell response seen with Hctz cotreatment and that
did not appear with Spi cotreatment that prevented the
inflammatory cell response.
Our study led to several major findings. First were
responses in extracellular Ca2?and Mg2?. The hypercal-
ciuria seen with ALDOST was attenuated by Hctz. This
served to prevent a decrease in plasma-ionized Ca2?.
However, Hctz did not disrupt the accompanying hyper-
magnesuria, and accordingly plasma-ionized Mg2?was
well-known sites of high-density ALDO receptor binding,
Spi disrupts urinary and gastrointestinal losses of these
divalent cations.6,14Microgram quantities of Ca2?and
Mg2?are found in urine, whereas milligram quantities of
these cations are present in feces.14Here, we found the
combination of Hctz?Spi to normalize urinary Ca2?ex-
cretion while attenuating Mg2?losses; therefore, plasma-
ionized Ca2?and Mg2?homeostasis were preserved with
The importance of reducing the excretion of both
Ca2?and Mg2?resides in preventing a decrease in
their plasma-ionized levels, each of which are an im-
portant determinant of the parathyroid glands’ release
of PTH.27,28We and others have found elevated plasma
PTH levels in rats with either ALDOST or deoxycor-
ticosterone/salt treatment (DOCST).14,29Plasma PTH
is persistently elevated at weeks 1 to 4 ALDOST.14
Hyperparathyroidism has been reported in patients with
primary aldosteronism30,31and in patients with conges-
tive heart failure,32–34in whom secondary aldosteron-
ism is expected. In patients with symptomatic heart
failure the long-term usage of furosemide, a loop di-
uretic that enhances urinary Ca2?and Mg2?excretion,
must also be taken into consideration. PTH, a calci-
tropic hormone, seeks to maintain extracellular ho-
meostasis of these cations. Toward this end, one of its
important actions is to promote bone mineral resorp-
tion. We have found a marked reduction in bone
mineral density for both tibia and femur at weeks 4
to 6 ALDOST, which is accompanied by a reduction
in bone strength to flexor stress.6,14Bone loss in
ALDOST can be prevented by Spi, Hctz, and the
combination of Hctz?Spi.14,35Osteopenia and os-
teoporosis have been reported in patients with ad-
vanced heart failure who are awaiting cardiac trans-
plantation, in whom furosemide treatment may also
lead to bone loss.32–34Furosemide-related bone loss
in rats with ALDOST has recently been reported,
which could be rescued by Spi cotreatment.36
Our second finding relates to responses in intracellu-
lar concentrations of Ca2?and Mg2?that occur with
ALDOST. In PBMC, cytosolic-free [Ca2?]iis in-
creased, whereas [Mg2?]ilevels are reduced at 4 weeks
ALDOST. PTH promotes a Ca2?loading of PBMC
through L-type channels, which can be inhibited by a
Ca2?channel blocker.17–19We previously showed that
amlodipine prevents Ca2?loading of PBMC in rats
with aldosteronism.13In addition to Ca2?loading of
cells, PTH may reduce Ca2?efflux.37Cotreatment with
Hctz did not prevent Ca2?loading of these cells, which
is likely in keeping with its failure to prevent the
decline in plasma-ionized [Mg2?]oand expected in-
crease in circulating PTH.14On the other hand, in
preventing the decrease in [Ca2?]oand [Mg2?]o, Spi
cotreatment prevented PBMC Ca2?loading. Spi alone
as cotreatment during ALDOST attenuates urinary and
fecal wasting of Ca2?and Mg2?to prevent a decrease
in [Ca2?]oand [Mg2?]oand an increase in [Ca2?]iof
PBMC.14Parathyroidectomy or a Ca2?-supplemented
diet (to attenuate hyperparathyroidism) each has been
shown to reduce intracellular Ca2?loading in rats with
We also found the total Ca2?concentration of ven-
tricular tissue, including intra- and extracellular com-
partments, to be increased at 4 weeks ALDOST. We
Fig 5. Plasma ?1-antiproteinase activity (?1-AP, ?mol/L) for the
three treatment groups and controls (defined in Fig 1). See text for
J Lab Clin Med
Runyan et al
have not identified the specific cell(s) contributing to
this response, but would suspect that cardiomyocytes
are involved. Voltage-gated Ca2?channels and intra-
cellular Ca2?release channels are in greater abundance
in excitable versus nonexcitable, nonmyocyte cells.
Several signals are candidates for promoting Ca2?
loading during ALDOST. ALDO upregulates the Ca2?
current in cardiomyocytes, and the number of cardio-
myocyte Ca2?channels are increased with chronic
mineralocorticoid excess.39–41Antagonism of the min-
eralocorticoid receptor prevents electrical remodeling
of isolated cardiomyocytes, and this includes the L-type
Ca2?channel current.42In addition, cultured adult rat
cardiomyocyte [Ca2?]iincreases in response to incuba-
tion with PTH and is a PTH receptor-mediated activa-
tion of L-type channels with augmented Ca2?entry and
mobilization of Ca2?from sarcoplasmic reticulum.43,44
Future studies will address the importance of ALDO
Fig 6. Immunohistochemical detection of cardiac gp91phoxexpression. In the normal rat heart, gp91phoxwas
negatively labeled (A). In ALDOST rats, gp91phoxwas expressed in inflammatory cells appearing in the
perivascular space of intramural coronary arteries (B). Cotreatment with Hctz (C) or Spi (D) attenuated or
prevented, respectively, inflammatory cell infiltration and gp91phoxexpression in perivascular space (?400).
surrounding intramyocardial coronary arteries (perivascular) and within the interstitial space for the three
treatment groups and controls. See text for details.
Morphometric determination of collagen volume fraction (CVF, %) is presented for fibrous tissue
J Lab Clin Med
Volume 146, Number 2 Runyan et al
versus PTH in Ca2?loading of myocardial tissue and
the involvement of cardiomyocytes.
PTH reduces [Mg2?]iin PBMC, erythrocytes, and
renal epithelial cells.45,46In our present and previous
studies,10,11,13we found reduced levels of cytosolic-
free [Mg2?]iin PBMC at 4 weeks ALDOST. Lympho-
cyte [Mg2?]iis reduced in patients with primary aldo-
steronism.47Additional mechanisms that may account
for the decrease in PBMC [Mg2?]iinclude the observed
hypermagnesuria that accompanies ALDOST. We
found that a Mg2?-supplemented diet will prevent the
decrease in PBMC [Mg2?]i.13Finally, an enhanced
Mg2?efflux through a putative Na?/Mg2?exchanger
cannot be discounted.47
Our third major finding relates to the presence of
oxidative stress in blood and cells invading the myo-
cardium of the right and left ventricles at 4 weeks
ALDOST, which was respectively identified by a de-
crease in plasma ?1-AP activity, an inverse correlate of
oxi/nitrosative stress, and activation of the gp91phox
subunit of NADPH oxidase at sites of the proinflam-
matory coronary vascular phenotype. Unlike Spi co-
treatment, Hctz did not prevent the decrease in ?1-AP
activity, which we interpret to be in keeping with its
failure to prevent a decrease in [Mg2?]oand Ca2?
loading of PBMC. We previously found treatment with
an antioxidant to prevent Ca2?loading of PBMC and
the appearance of this phenotype.2,13This included its
prevention of the activation of a redox-sensitive nuclear
transcription factor (NF)?B and upregulated mRNA
expression of a proinflammatory mediator cascade reg-
ulated by NF?B, which includes intercellular adhesion
molecule-1 and monocyte chemoattractant protein-1.
Antioxidants also prevented the appearance of 3-nitro-
tyrosine, a stable tyrosine residue formed in response to
nitrosylation by short-lived peroxynitrite, a reactive
nitrogen species, in these invading cells. In the present
study, cotreatment with Hctz was partially protective in
preventing the invasion of the coronary vasculature by
inflammatory cells, whereas Spi abrogated this re-
sponse. In attenuating the inflammatory cell response
and thereby cell–cell signaling with fibroblasts respon-
sible for the expression and deposition of fibrillar col-
lagen, these interventions reduced or prevented the
appearance of the fibrogenic phenotype, respectively,
which presents as a perivascular/interstitial fibrosis.
Our study has several limitations. Although we did
not measure plasma PTH levels in this study, we pre-
viously found PTH to be persistently elevated in rats
receiving ALDOST for 1 to 4 weeks.14We did not do
so here because the limited blood volume available
from these rats was used to analyze the multiple pa-
rameters presented. However, we have strong biochem-
ical evidence of hyperparathyroidism in the present
study. This included (1) reduced plasma-ionized Ca2?
and Mg2?, each of which are major determinants of
PTH release; (2) elevated [Ca2?]iand reduced [Mg2?]i
in PBMC, each of which is a response to elevated
circulating PTH levels; and (3) elevated total Ca2?
concentration of ventricular tissue. We also have doc-
umented a marked reduction in bone mineral density to
accompany ALDOST, which we consider biologic ev-
idence of hyperparathyroidism.6,14Because we have
previously reported on the response to ALDOST?Spi
cotreatment alone,11,14we did not repeat this interven-
In summary, findings of the present study underscore
the importance of urinary losses of Ca2?and Mg2?that
occur with aldosteronism and the accompanying de-
cline in plasma-ionized levels of these divalent cations
and paradoxic Ca2?loading of PBMC and cardiac
tissue with subsequent induction of oxi/nitrosative
stress. The consequences of this chemical remodeling
of these immune cells favor cell activation, expressed
in their transcriptome as a proinflammatory geno-
type.10,11The resultant immunostimulatory state leads
to coronary vascular invasion. This proinflammatory
vascular phenotype, the Ca2?overload of ventricular
tissue, or the two in combination could favor arrhyth-
mogenesis.48–52Hctz rescues urinary Ca2?but not
Mg2?losses and therefore is only partially protective
against the proinflammatory phenotype. Hctz?Spi, on
the other hand, provides a more complete rescue of
these losses and thereby is protective of intracellular
Ca2?loading, oxi/nitrosative stress, and vascular re-
modeling. In addressing the pharmacologic manage-
ment of symptomatic heart failure, in which aldoste-
ronism is expected, the impact of any given agent on
Ca2?and Mg2?homeostasis should be taken into con-
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