Sodium transporter abundance profiling in kidney: effect of
spironolactone
Jakob
Nielsen1,2,
Tae-Hwan
Kwon2,
Shyama
Masilamani1,
Kathleen
Beutler1,
Henrik
Hager2,
Søren
Nielsen2, and
Mark A.
Knepper1
1 Laboratory of Kidney and Electrolyte Metabolism,
National Heart, Lung and Blood Institute, National Institutes of
Health, Bethesda, Maryland 20892-1603; and
2 The Water and Salt Research Center, University of
Aarhus, DK-8000 Aarhus C, Denmark
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ABSTRACT |
Renal tubule profiling studies were
carried out to investigate the long-term effects of administration of
spironolactone, a mineralocorticoid receptor antagonist, on abundances
of the major Na transporter and Na channel proteins along the rat renal tubule. Oral administration of spironolactone for 7 days to
NaCl-restricted rats did not significantly alter abundances of Na
transporters expressed proximal to the macula densa, while
substantially decreasing the abundances of the thiazide-sensitive Na-Cl
cotransporter (NCC), the
-subunit of the amiloride-sensitive
epithelial Na channel (ENaC), and the 70-kDa form of the
-subunit of
ENaC. A dependency of NCC expression on aldosterone was confirmed by
showing increased NCC expression in response to aldosterone infusion in
adrenalectomized rats. Immunoperoxidase labeling of ENaC in renal
cortex confirmed that dietary NaCl restriction causes a redistribution
of ENaC to the apical domain of connecting tubule cells and showed that high-dose spironolactone administration does not block this apical redistribution. In contrast, spironolactone completely blocked the
increase in
-ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC,
-ENaC, and the 70-kDa form of
-ENaC are regulated via the classical
mineralocorticoid receptor, but the subcellular redistribution of ENaC
in response to dietary NaCl restriction is not prevented by blockade of
the mineralocorticoid receptor.
aldosterone; collecting duct; distal convoluted tubule
 |
INTRODUCTION |
THE MAJOR
CIRCULATING MINERALOCORTICOID in humans and other mammals is
aldosterone. Our laboratory's previous renal tubule NaCl transporter
profiling studies (25, 26) have identified a number of
effects of aldosterone or dietary NaCl restriction along the renal
tubule that are manifested as changes in transporter protein abundance
or distribution within the cell. These effects are the
following: 1) increased abundance of the thiazide-sensitive cotransporter [Na-Cl cotransporter (NCC)]; 2) increased
abundance of the
-subunit of the amiloride-sensitive epithelial Na
channel (ENaC); 3) a partial shift in molecular mass of the
-subunit of ENaC from 85 to 70 kDa, thought to be due to a
physiological proteolytic cleavage of the extracellular loop of
-ENaC; and 4) redistribution of the ENaC complex from a
broad intracellular distribution to the apical region of the collecting
duct principal cell. Both the aldosterone-mediated increase in
NCC (1, 2) and the aldosterone-induced redistribution of
the ENaC complex (22, 23) have also been demonstrated by
others. Although there is strong support for a role for aldosterone in
these responses, recent evidence indicates that aldosterone can bind to
more than one receptor type and may exert effects in the cell by
so-called genomic and nongenomic mechanisms. Genomic mechanisms can
involve activation of the classic mineralocorticoid receptor
[dissociation constant (Kd) for aldosterone,
1.3 nM; see Ref. 4] or glucocorticoid receptors
(Kd for aldosterone, 25-50 nM), both of
which activate gene expression by binding to glucocorticoid regulatory
elements in the 5'-flanking regions of responsive genes
(12). Nongenomic actions of aldosterone are mediated by
binding of aldosterone to plasma membrane-associated steroid receptors
rather than the classic mineralocorticoid receptor (8, 16, 37; and, for
review, see Ref. 11).
One way to discriminate between mineralocorticoid receptor-mediated
responses and mineralocorticoid receptor-independent responses to
aldosterone is to utilize the selective mineralocorticoid receptor blocker spironolactone. Spironolactone acts via a competitive mechanism
and is utilized most often clinically in the treatment of either
primary aldosteronism or clinical states associated with secondary
aldosteronism (e.g., hepatic cirrhosis). Spironolactone and its
congeners are likely to see greater clinical use in the future because
of recent studies demonstrating that their administration reduces the
mortality rate in patients with congestive heart failure (31). Here, we utilize renal tubule NaCl transporter
abundance profiling (21) and immunocytochemistry to assess
the response to long-term spironolactone treatment in rat kidney.
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METHODS |
Animal protocol 1 (effect of spironolactone in NaCl-restricted
rats).
Male Sprague-Dawley rats (n = 12, 220 g; Taconic
Farms, Germantown, NY) were maintained in metabolic cages on a low-NaCl
gel diet. The diet was prepared by combining commercially available synthetic rat chow containing no added NaCl (0.0041% NaCl wt/wt; formula 53140000, Ziegler, Gardner, PA) with deionized water (25 ml/15
g rat chow) and agar (0.5%) for gelation. No NaCl was added to the
base diet for experimental rats, so these rats received 0.01 mmol · 200 body
wt
1 · day
1 of Na. All
rats were given the equivalent of 15 g · 200 g
body wt
1 · day
1 of
the synthetic chow and 25 ml · 200 g body
wt
1 · day
1 of water.
Spironolactone (S-3378, Sigma, St. Louis, MO) was dissolved in olive
oil (500 mg spironolactone/ml olive oil) and added to the food mixture
before gelation in an amount sufficient to give rats 0.35 mg · g
body
1 · day
1. An
equal amount of olive oil was given to the control low-NaCl rats. The
rats received a half dose of spironolactone or olive oil for the first
3 days and the full dose for the last 4 days. All rats were
euthanized at the same time and kidneys were prepared for
immunoblotting (see Semiquantitative
immunoblotting). Trunk blood was collected at the time of
decapitation for the measurement by radioimmunoassay of aldosterone
concentration (Coat-a-Count, Diagnostic Products, Los Angeles, CA), Na,
and creatinine concentrations (Monarch 2000 autoanalyzer,
Instrumentation Laboratories, Lexington, MA). Urinary samples
were analyzed for Na, K, urea, and creatinine concentrations (Monarch
2000 autoanalyzer).
Animal protocol 2 (effect of spironolactone on moderately
NaCl-restricted rats).
Male Munich-Wistar rats (n = 11, 225 g;
Møllegaard Breeding Center, Skensved, Denmark) were maintained as
above with ration feeding of a gel diet. These rats were less severely
NaCl restricted than in protocol 1 (0.32 meq
Na · 200 g body
wt
1 · day
1) by using
another synthetic low-Na powdered food (Altromin 1321, Chr. Petersen,
Ringsted, Denmark) with added deionized water (30 ml/10 g of food) and
agar. All animals received the equivalent of 10 g
food · 200 g body
wt
1 · day
1.
Spironolactone was mixed with the food and no olive oil vehicle was
used. The dose of spironolactone was 0.1 mg · g
body wt
1 · day
1 for 7 days. Control rats received the low-NaCl gel diet but no spironolactone. The left kidney was fixed by perfusion, as described in
Immunocytochemistry, under halothane anesthesia (Halocarbon Laboratories). Blood was collected from the inferior vena cava. Urine
and serum was analyzed for protocol 1. The unfixed
right kidney was removed from each rat and frozen in liquid nitrogen for later preparation of whole kidney homogenates for immunoblotting (see Semiquantitative immunoblotting).
Animal protocol 3 (NaCl-restricted vs. NaCl-replete rats).
In experiments addressing the effect of spironolactone administration
on ENaC trafficking, male Munich-Wistar rats were maintained on the
moderately NaCl-restricted diet described above (protocol 2)
or an NaCl-replete diet for 7 days. The NaCl-replete diet was the same
as the NaCl-restricted diet except that a supplemental amount of NaCl
was included in the gel diet to give the rats 2.0 meq of
Na · 200 g body
wt
1 · day
1. The
kidneys were fixed as described in
Immunocytochemistry.
Animal protocol 4 (response to dietary NaCl restriction in
spironolactone-treated rats).
Male Sprague-Dawley rats (n = 12, 220 g, Taconic
Farms) were maintained in metabolic cages and given spironolactone as
described in protocol 1 in either an NaCl-restricted gel
diet (see protocol 1 for dietary formulation) or an
NaCl-replete formulation of the same diet. For the latter, enough NaCl
was added to allow the rats to receive 2.0 meq · 200 g body
wt
1 · day
1 of Na.
After 7 days, all rats were euthanized and the left kidneys were
prepared for immunoblotting.
Animal protocol 5 (response to aldosterone administration in
adrenalectomized rats).
In this protocol, the effect of aldosterone administration on renal NCC
abundance was investigated in glucocorticoid-replaced adrenalectomized
rats. Control rats were male Munich-Wistar rats (n = 13, 220-230 g, Møllegaard Breeding Center, Skensved, Denmark), which were adrenalectomized and implanted with osmotic minipumps containing dexamethasone (delivering 0.012 µg · g body
wt
1 · day
1) for 10 days (n = 7). Aldosterone-treated rats were the same as
the controls except for administration of aldosterone (delivering 0.02 µg · g
1 · day
1)
for 10 days (n = 6) in addition to dexamethasone. The
doses were chosen on the basis of previous studies (24, 33,
34). The dose of dexamethasone used has been reported to
increase plasma dexamethasone concentration to 21 nM, which is two to
four times the dissociation constant for the glucocorticoid receptor
(5-10 nM) (33).
Surgery to remove adrenal glands and implant osmotic minipumps was
carried out at the same time under halothane anesthesia. Both adrenal
glands were removed through bilateral flank incisions. Osmotic
minipumps were implanted subcutaneously. For implantation, minipumps
(2002, Alzet, Palo Alto, CA) were filled with D-aldosterone (A6628, Sigma) or dexamethasone (D1756, Sigma) dissolved in DMSO and
diluted with sterile isotonic saline. The pumps were equilibrated with
normal saline for 4 h before insertion.
The rats were maintained in metabolic cages with a fixed amount of
daily water (35 ml · rat
1 · day
1)
and food (15 g · rat
1 · day
1;
Altromin 1324, Chr. Petersen) intake. The NaCl intake was 1.3 meq
Na · rat
1 · day
1.
After 10 days of hormone replacement, all rats were anesthetized under
halothane inhalation and left kidneys were rapidly removed and
processed for semiquantitative immunoblotting. Right kidneys were
perfusion fixed as described in
Immunocytochemistry.
Semiquantitative immunoblotting.
Kidneys were homogenized intact and prepared for immunoblotting as
described previously (19, 35). Equal loading was confirmed by staining identically loaded gels with Coomassie blue dye as described previously (35). Incubation of blots with
primary antibodies and peroxidase-conjugated secondary antibodies
(31458 or 31434, Pierce) was followed by band visualization with an
enhanced chemiluminescence substrate (VC110 LumiGLO for Western
Blotting, Kirkegaard and Perry) before exposure to X-ray film (Kodak
165-1579). The band densities were quantitated by laser densitometry
(PDS1-P90, Molecular Dynamics). The densitometry values were normalized
to control to facilitate comparisons, defining the mean for the control group as 100%.
Immunocytochemistry.
A perfusion needle was inserted into the abdominal aorta of
halothane-anesthetized rats, and the vena cava was cut to establish an
outlet. Blood was flushed from the kidneys with cold PBS (pH 7.4) for
15 s before switching to cold 4% paraformaldehyde in 0.1 M
cacodylate buffer (pH 7.4) for 3 min. The left kidney was removed and
the midregion was sectioned into 2- to 3-mm transverse sections and
postfixed for 1 h, followed by 3 × 10-min washes with 0.1 M
cacodylate buffer (pH 7.4). The tissue was dehydrated in graded ethanol
and left overnight in xylene. The tissue was embedded in paraffin and
2-µm sections were cut on a microtome (Leica Microsystems, Herlev,
Denmark). NCC and ENaC subunits were localized by using indirect
immunoperoxidase labeling or immunofluorescence as previously described
(17).
Antibodies.
Affinity-purified rabbit polyclonal antibodies to the following renal
NaCl transporters were utilized: the type 3 Na/H exchanger of the
proximal tubule (13), Na-K-2Cl cotransporter of the thick ascending limb (19), thiazide-sensitive cotransporter NCC
of the distal convoluted tubule (DCT) (20), and three
subunits of ENaC (25). The antisera were affinity purified
against the immunizing peptides as previously described (19,
20). Specificity of the antibodies has been demonstrated by
showing unique peptide-ablatable bands on immunoblots and a unique
distribution of labeling by immunocytochemistry. In addition, a mouse
monoclonal antibody recognizing the
1-subunit of
Na-K-ATPase was used.
Presentation of data and statistical analyses.
Quantitative data are presented as means ± SE. Statistical
comparisons were accomplished by unpaired t-test (when
variances were the same) or by Mann-Whitney rank-sum test (when
variances were significantly different between groups). P
values <0.05 were considered statistically significant.
 |
RESULTS |
Profiling the effects of spironolactone on Na transporter protein
abundances in kidney.
Figure 1 shows semiquantitative
immunoblots for each of the major apical Na transporters expressed
along the renal tubule in experiments in which NaCl-restricted rats
were treated with either spironolactone or vehicle (protocol
1). Densitometric quantification is given in
Table 1. The band densities for the two
major Na transporters expressed in pre-macula densa segments, the type 3 Na/H exchanger and the Na-K-2Cl cotransporter, were not significantly changed. However, the mean normalized band density for NCC, the thiazide-sensitive NaCl transporter of the DCT, was markedly decreased by spironolactone administration (Table 1). Similarly, the mean normalized band density for
-ENaC was decreased by nearly 50% in
the spironolactone-treated rats vs. the vehicle-treated rats. In
addition, there was a significant increase in the abundance of the
85-kDa form of
-ENaC in the spironolactone-treated rats and a
corresponding decrease in the 70-kDa form. However, the sum of the
densities of the two bands for
-ENaC was unchanged. Furthermore, the
mean normalized band density of
-ENaC was not significantly changed.

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Fig. 1.
Immunoblots assessing Na transporter abundances in whole
kidney homogenates from vehicle-treated rats on a low-Na diet and
spironolactone-treated rats on the same low-Na diet. Each lane was
loaded with a sample from a different rat. Preliminary 12%
SDS-polyacrylamide gels were run and stained with Coomassie blue dye to
confirm equality of loading in each lane. Band densities were assessed
by laser densitometry. Note that, as seen before (25),
-epithelial Na channel (ENaC) appears as a tight doublet,
hypothetically due to differing states of glycosylation. Here, the
-ENaC blot was moderately overexposed to reveal peptide-ablatable
ladderlike ancillary bands of slightly higher molecular mass,
hypothetically due to differing levels of glycosylation or
ubiquitination. Similarly, the -ENaC blot was moderately overexposed
to reveal both 85- and 70-kDa bands as well as a weak peptide-ablatable
band just above the 85-kDa band, presumably due to an unknown
posttranslational modification. NCC, Na-Cl cotransporter;
NHE3, type 3 Na/H exchanger. *P < 0.05, significant
change in band density.
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Table 1.
Densitometric analysis of immunoblots for major Na transporters in
whole kidney of NaCl-restricted rats receiving spironolactone or
vehicle
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Table 2 shows physiological data from the
same rats. These data are compatible with the view that the
spironolactone-treated rats manifested a moderate degree of
extracellular fluid volume contraction (increased serum aldosterone
concentration, increased serum urea concentration, increased serum NaCl
concentration, increased serum osmolality, decreased urinary volume,
increased urinary osmolalities). This volume contraction presumably was due to Na losses early in the course of spironolactone treatment, although urinary Na excretion was not measurably increased at the time
the animals were euthanized for analysis of the kidneys. This finding
indicates that compensatory mechanisms, likely dependent on the
extracellular fluid volume contraction, allowed Na balance to be
reestablished despite continued spironolactone administration. In
addition, spironolactone administration was associated with positive K
balance (decreased urinary K excretion) and moderate metabolic acidosis
(decreased serum total CO2 concentration). There was no
significant difference in creatinine clearance between the
spironolactone-treated group and controls. We conclude from the data in
Table 2 that the administered spironolactone was efficacious in
blocking mineralocorticoid receptors at a renal tubule level.
Effect of spironolactone on subcellular distribution of NCC in DCT.
Figure 2 shows immunoperoxidase
labeling for NCC in DCT cells in sections of kidney tissue from a
different set of spironolactone-treated rats and control rats
(right and left, respectively; protocol 2). Microscope settings and labeling conditions were the
same for both groups. The labeling intensity in the
spironolactone-treated rats was markedly decreased. Labeling was only
seen in the most apical region of the DCT cells in both groups, with no
sign of subcellular redistribution. Thus the NCC protein abundance was reduced in response to spironolactone treatment. To confirm this response in these animals, we carried out immunoblotting for NCC in the
contralateral kidney from the same rats (immunoblot not shown). The
mean normalized NCC band density in the spironolactone-treated rats was
46 ± 4 normalized densitometry units vs. 100 ± 10 normalized densitometry units in control rats (P < 0.05).

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Fig. 2.
High-power images of immunoperoxidase labeling of NCC in the distal
convoluted tubule (DCT) of rat renal cortex. Left: images
from two different control rats (C-1 and C-2) on a low-Na diet.
Right: image from two different spironolactone-treated rats
(S-1 and S-2) on the same low-Na diet. Arrows, decrease in intensity of
apical labeling in DCTs of spironolactone-treated rats. Bar = 15 µm.
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Urinary and plasma measurements for these animals are available as
supplementary data (see Table A1 in the APPENDIX).
Effect of aldosterone infusion on NCC abundance in kidneys of
adrenalectomized rats.
To further address the role of the mineralocorticoid receptor in
regulation of NCC abundance, we carried out studies in adrenalectomized rats to test the effect of aldosterone infusion. The possibility of
indirect effects due to binding to the glucocorticoid receptor was
eliminated by infusing all adrenalectomized rats (both control and
experimental) with dexamethasone. The rate of dexamethasone infusion
used has been reported to increase plasma dexamethasone levels to two-
to fourfold above the dissociation constant for the glucocorticoid
receptor and to maintain glomerular filtration rate at normal levels
(see METHODS). Figure 3 shows
an NCC immunoblot for whole-kidney homogenates from these rats. In
dexamethasone-treated adrenalectomized rats, aldosterone infusion
strongly increased the abundance of NCC in whole kidney homogenates
(mean normalized band density of NCC: aldosterone infused, 748 ± 88 normalized densitometry units; control, 100 ± 29 normalized
densitometry units; P < 0.05).

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Fig. 3.
Immunoblot comparing NCC abundances in whole kidney
homogenates from control animals (Dexamethasone; dexamethasone-replaced
adrenalectomized rats) and rats receiving aldosterone
(Dexamethasone+Aldosterone; aldosterone-treated, dexamethasone-replaced
adrenalectomized rats). All rats were euthanized for analysis of
kidneys after 10 days of steroid treatment. Each lane was loaded with a
sample from a different rat. Preliminary 12% SDS-polyacrylamide gels
were run and stained with Coomassie blue dye to confirm equality of
loading in each lane.
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In the same rats, the opposite kidney was prepared for
immunocytochemical labeling of NCC (green) and the
1-subunit of the Na-K-ATPase (red), shown in Fig.
4. Representative images of control adrenalectomized rats treated only with dexamethasone (left)
and images from rats treated with both dexamethasone and aldosterone (right) are shown. Microscope settings and labeling
conditions were identical for all images. There is a marked increase in
NCC labeling (green) in the most apical region of the DCT cells after aldosterone administration. This confirms the increased NCC
abundance observed on immunoblots. There is no sign of subcellular
redistribution of NCC in response to aldosterone. There was strong
basolateral labeling with the anti-Na-K-ATPase antibody in both control
and aldosterone-treated rats. Note that Na-K-ATPase protein is much more abundant in the DCT than in surrounding cortical structures, as
previously observed (14).

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Fig. 4.
Confocal immunofluorescence
images of NCC (green) in the DCT in control rats (Dexamethasone;
adrenalectomized and dexamethasone-replaced without aldosterone
infusion; left) vs. experimental rats
(Dexamethasone+Aldosterone; adrenalectomized and dexamethasone-replaced
plus aldosterone infusion; right). Top: double
labeling of NCC (green) and Na-K-ATPase 1-subunit (red).
Bottom: single labeling for NCC, allowing the exclusively
apical NCC labeling to be clearly seen (arrows). Bar = 30 µm.
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Effect of spironolactone on subcellular redistribution of ENaC in
response to low-NaCl diet.
ENaC has been shown to redistribute to the apical cell domain in
collecting duct principal cells and connecting tubule cells in response
to dietary NaCl restriction or aldosterone administration (22,
23, 25). We investigated here whether the effect of dietary NaCl
restriction on ENaC redistribution is blocked by high-dose
spironolactone in rats (protocols 2 and 3). Representative immunoperoxidase labeling for the
-subunit of ENaC in the
superficial cortex of three control rats receiving the NaCl-replete
diet and three rats on the NaCl-restricted diet is shown in Fig.
5 (left and right,
respectively). The rats receiving the NaCl-replete diet showed labeling
dispersed throughout the cytoplasm, whereas the NaCl-restricted diet is
associated with labeling limited to the apical region of the cells
(arrows).

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Fig. 5.
Immunoperoxidase labeling of -ENaC in cortical tissue sections
from rats on Na-replete (left, C-1- C-3) and
Na-deficient diet (right, LS-1-LS-3). No rats received
spironolactone. Notice the difference in cellular localization of
labeling between groups. Rats on Na-replete diet only show dispersed
intracellular labeling, whereas rats on Na-deficient diet show
predominant labeling of the apical cell domain (arrows). Bar = 15 µm.
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Representative immunoperoxidase labeling of the
-ENaC subunit
in the superficial cortex of three spironolactone-treated rats on the
NaCl-replete diet and three spironolactone-treated rats on the
NaCl-restricted diet is shown in Fig. 6
(left and right, respectively). Interestingly,
the distribution of ENaC was no different from what was observed in the
rats that did not receive spironolactone. The spironolactone-treated
rats on the NaCl-replete diet showed disperse cytoplasmic labeling,
whereas the spironolactone-treated rats on the NaCl-restricted diet
showed labeling only in the apical cell domain (arrows). A
statistically significant redistribution was confirmed by blinded
examination of sections from all eight rats
studied.1 Thus there was no
evidence of an effect of the spironolactone treatment on the cellular
redistribution of ENaC in response to dietary NaCl restriction. The
labeling conditions and microscope settings were the same for all
images. Although the images shown in Figs. 5 and 6 present only
-ENaC labeling, similar observations were made with the antibodies
to
- and
-ENaC and were confirmed by a blinded observer (not
shown). Thus the conclusions appear to apply to trafficking of the


-ENaC complex.

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Fig. 6.
Immunoperoxidase labeling of -ENaC in cortical tissue sections
from spironolactone-treated rats on an Na-replete (left,
C-4-C-6) and Na-deficient diet (right, LS-4-LS-6).
Rats receiving the Na-replete diet and spironolactone show dispersed
intracellular labeling, whereas rats on the Na-deficient diet with
spironolactone show labeling limited to the apical cell domain
(arrows). This finding indicates that the ENaC trafficking induced by
dietary NaCl restriction is insensitive to mineralocorticoid receptor
blockade. Bar = 15 µm.
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To demonstrate that the lack of effect of spironolactone on cellular
redistribution of ENaC was not due to ineffective mineralocorticoid receptor blockade, immunoblotting was carried out using the
contralateral kidney from the same rats. An immunoblot of
-ENaC
comparing the control rats on the NaCl-restricted diet vs. the
spironolactone-treated rats on the NaCl-restricted diet showed a
significantly decreased band density for
-ENaC (Fig.
7). Mean normalized band density for
spironolactone-treated rats on the NaCl-restricted diet was 67 ± 5 vs. 100 ± 11 normalized densitometry units in untreated rats on
the NaCl-restricted diet (P < 0.05). Furthermore, the plasma K concentrations were substantially higher in
spironolactone-treated rats. Untreated rats on the NaCl-replete and
NaCl-restricted diets had plasma K concentrations of 4.9 ± 0.1 and 5.0 ± 0.2 mmol/l, respectively; spironolactone-treated rats
on the NaCl-replete and NaCl-restricted diets had plasma K
concentrations of 5.8 ± 0.1 and 6.0 ± 0.2 mmol/l,
respectively. These observations confirm the efficacy of the drug at
the dose given to block renal mineralocorticoid receptors.

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Fig. 7.
Immunoblot comparing protein abundance of the -ENaC
subunit in whole kidney homogenates from control rats on a low-NaCl
diet without spironolactone treatment and experimental rats on low-NaCl
with spironolactone treatment. These kidneys were from the same rats
used for immunocytochemistry (Figs. 5 and 6). Band densities were
assessed by laser densitometry. *P < 0.05, significantly different mean band density between groups.
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An additional experiment was done to address whether
spironolactone can fully block the increases in
-ENaC protein
abundance in response to dietary NaCl restriction (protocol
4). Spironolactone-treated rats were placed on a normal NaCl
intake (NaCl replete, 2.0 meq · 200 g body
wt
1 · day
1) or a
severely reduced NaCl intake (NaCl restricted, 0.01 meq · 200 g body
wt
1 · day
1), and
whole kidneys were processed for immunoblotting (Fig.
8). As shown, high-dose spironolactone
blocked the expected increase in
-ENaC abundance (band densities:
NaCl restricted, 110 ± 10; NaCl replete, 100 ± 12 normalized densitometry units). Thus the upregulation of
-ENaC
protein abundance in response to NaCl restriction is virtually totally
dependent on the mineralocorticoid receptor.

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Fig. 8.
Immunoblot showing effect of dietary NaCl
restriction on -ENaC expression in spironolactone-treated rats. Each
lane was loaded with a sample from a different rat. Preliminary 12%
SDS-polyacrylamide gels were run and stained with Coomassie blue dye to
confirm equality of loading in each lane. Dietary NaCl restriction had
no significant effect on -ENaC abundance in the presence of
spironolactone.
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 |
DISCUSSION |
In this paper, an NaCl transporter abundance profiling approach (a
form of targeted proteomics; see Ref. 21) has been applied to an analysis of the effects of administration of the
mineralocorticoid receptor antagonist spironolactone on Na transporter
abundances along the renal tubule. Spironolactone is used clinically as
a K-sparing diuretic and for treatment of primary and secondary hyperaldosteronism. Recent studies demonstrating efficacy in reducing mortality in patients with severe congestive heart failure
(31) predict greater clinical use in the future.
Therefore, studies profiling spironolactone's effects on kidney NaCl
transporter expression are timely. It is clear from these results that
the major effects of long-term spironolactone administration were in
the post-macula densa segments of the renal tubule, i.e., the DCT,
connnecting tubule, and collecting duct. Specifically, spironolactone significantly decreased the renal abundance of NCC,
-ENaC, and the
70-kDa form of
-ENaC. The responses are the reverse of those identified in previous studies examining responses to dietary NaCl
restriction and aldosterone administration (25).
Therefore, the results are consistent with the conclusion that the
effects of aldosterone or dietary NaCl restriction in increasing the
abundance of NCC,
-ENaC, and the 70-kDa form of
-ENaC are
mediated, at least in part, by the mineralocorticoid receptor.
Spironolactone does not block apical ENaC redistribution.
The most surprising observation in the present study was that
spironolactone did not block the ability of dietary NaCl restriction to
trigger a redistribution of ENaC to the apical cell domain of
connecting tubule cells (Figs. 5 and 6). Two previous studies (22, 25) provided persuasive evidence for such a
redistribution in collecting duct and connecting tubule in response to
dietary NaCl restriction. The apical redistribution of ENaC is
hypothetically involved in the action of aldosterone to increase
amiloride-sensitive transport of Na across the connecting tubule
epithelium. Additional studies demonstrated that aldosterone could
produce a similar redistribution tied to the induction of expression of
the serum- and glucocorticoid-regulated kinase (sgk) (3, 6, 23,
28, 32). Although sgk abundance in the distal nephron is
strongly regulated by aldosterone (6, 28, 32), its
activity is also regulated by the peptide hormones vasopressin and
insulin and perhaps other factors through phosphorylation of the sgk
protein (10, 30). Thus a failure of the mineralocorticoid
receptor blocker to block apical ENaC redistribution does not rule out a role for sgk in the redistribution in response to dietary NaCl restriction, but rather it raises the possibility that dietary NaCl
restriction activates sgk by alternative mechanisms. It is also
conceivable that the apical redistribution of ENaC in response to
dietary NaCl restriction is dependent on aldosterone but is mediated by
nonclassic (nongenomic) aldosterone receptors that are not blocked by
spironolactone. These nonclassic receptors are high-affinity
membrane-associated aldosterone receptors thought to trigger changes in
the s messengers inositol trisphosphate, diacylglycerol, cAMP, and
intracellular calcium and to activate a variety of downstream kinases
(11). These receptors are activated in the physiological
range of circulating aldosterone concentrations. In contrast, the
plasma aldosterone levels realized in response to dietary NaCl
restriction (3-8 nM in this study) are unlikely to be high
enough to activate glucocorticoid receptors, which have a
Kd for aldosterone in the range 25-50 nM.
An additional possibility is that the ENaC redistribution in response
to dietary NaCl restriction could be unrelated to changes in
circulating hormone concentrations but could instead be mediated
directly by physical factors, such as altered intracellular Na
concentration (15).
The spironolactone dose administered to the rats was >10-fold higher
than what has been reported to block 95% of the mineralocorticoid receptors in vivo (9). Furthermore, the efficacy of the
drug in these experiments was documented both by the measurements of serum K concentration and by ancillary immunoblotting. In particular, high-dose spironolactone not only failed to block apical ENaC redistribution but also strongly decreased
-ENaC abundance in NaCl-restricted rats (Fig. 7) and completely blocked the ability of
dietary NaCl restriction to increase
-ENaC abundance in separate experiments (Fig. 8). Thus the failure of spironolactone to prevent the
cellular redistribution of ENaC in response to dietary NaCl restriction
was apparently not due to failure of the drug to block the
mineralocorticoid receptor.
Mineralocorticoid regulation of NCC abundance.
A considerable amount of evidence supports the conclusion that
the DCT is a target for regulation by aldosterone. Early micropuncture studies showed increased tubule fluid-to-plasma concentration ratios of
Na in the entire accessible distal tubule in adrenalectomized rats
(18). The ratio was decreased to control levels by
aldosterone administration throughout the accessible distal tubule
including the earliest portions, which undoubtedly included the DCT.
Administration of aldosterone and dexamethasone has been shown to
increase [3H]metolazone binding in membrane fractions, a
measure of NCC abundance (7). Furthermore, in vivo
microperfusion studies have shown that aldosterone increases
thiazide-sensitive NaCl transport in the DCT (36).
Recently, we showed by immunoblotting that elevated plasma aldosterone
concentration is associated with increased renal cortical NCC
abundance, regardless of whether plasma aldosterone was increased
by dietary NaCl restriction or aldosterone infusion (20). Finally, in this paper, we showed in
dexamethasone-replaced adrenalectomized rats that aldosterone
infusions markedly increase NCC abundance in renal cortex (Figs.
3 and 4). Collectively, we view these findings as strong evidence for
an important role for aldosterone in regulation of NCC.
The data in this paper establish that aldosterone's effects in
increasing NCC abundance are mediated by the mineralocorticoid receptor. The finding that spironolactone administration decreases NCC
protein abundance in kidney points to a role of the classic mineralocorticoid receptor in the regulation of NCC abundance. A
similar conclusion was also drawn in a recent paper demonstrating that
an increase in renal NCC abundance brought about by chronic furosemide
administration is blocked by spironolactone administration (1).
Although the results presented in this paper support the view
that the mineralocorticoid receptor plays an important role in the
regulation of NCC abundance, the interpretation of the results is
complicated by the fact that in rodents, NCC is expressed in two
dissimilar subsegments of the DCT called DCT1 and DCT2 (29). Although the mineralocorticoid receptor and the
glucocorticoid-metabolizing enzyme 11
-hydroxysteroid dehydrogenase
type 2 are strongly expressed in the DCT2 segment, these proteins are
expressed at much lower levels in the DCT1 segment (5).
Although not specifically investigated in this paper, it appears
possible that the mineralocorticoid-mediated regulation of NCC
abundance may occur predominantly in the late portion of the DCT, that
is, in the DCT2 subsegment. Further studies using immunomorphometric
and microdissection techniques will be required to investigate this possibility.
Despite the evidence supporting the view that NCC is a target for
regulation by mineralocorticoids, existing evidence indicates that this
effect must be indirect, i.e., unrelated to NCC gene transcription.
Specifically, there has been a consistent failure to detect changes in
NCC mRNA levels in response to dietary NaCl restriction (27,
38) or aldosterone administration (2). Indeed, we
have recently demonstrated with simultaneous NCC mRNA and protein
measurements that the increase in NCC protein evoked by dietary NaCl
restriction is not associated with a measurable change in NCC mRNA
(26).
 |
APPENDIX |
 |
ACKNOWLEDGEMENTS |
The authors thank Inger Merete Paulsen for technical assistance at
Aarhus University and Dr. Christian A. Combs, manager of the National
Heart, Lung, and Blood Institute (NHLBI) Light Microscopy Imaging
Facility at the National Institutes of Health.
 |
FOOTNOTES |
This study was funded by the Intramural Budget of the NHLBI
(Z01-HL-01282-KE to M. A. Knepper). S. Masilamani was supported by
an NHLBI Career Transition Award (K22-HL66994). Studies at Aarhus
University were supported by the Danish Medical Research Council, the
Karen Elise Jensen Foundation, the Commission of the European Union
(EU-TMR Program and K.A. 3.1.2 Program), and Dongguk University. The
Water and Salt Research Center, Aarhus University, is supported by The
Danish National Research Foundation (Danmarks Grundforskningsfond).
1
Analysis was achieved by a blinded observer who
examined ENaC labeling in connecting tubules from the superficial
cortex in all spironolactone-treated rats and all control rats. The
observer ranked the sections with regard to distribution of ENaC in the labeled cells, giving the highest rank to those with the most apically
oriented ENaC labeling. Statistical significance was tested with the
Mann-Whitney rank-sum test.
Address for reprint requests and other correspondence:
M. A. Knepper, National Institutes of Health, Rm. 6N260,
Bldg. 10, 10 Center Dr. MSC 1603, Bethesda, MD 20892-1603 (E-mail:
knep{at}helix.nih.gov).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 26, 2002;10.1152/ajprenal.00015.2002
Received 10 January 2002; accepted in final form 23 June 2002.
 |
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