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Am J Physiol Renal Physiol 283: F923-F933, 2002. First published June 26, 2002; doi:10.1152/ajprenal.00015.2002
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Vol. 283, Issue 5, F923-F933, November 2002

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 alpha -subunit of the amiloride-sensitive epithelial Na channel (ENaC), and the 70-kDa form of the gamma -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 alpha -ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC, alpha -ENaC, and the 70-kDa form of gamma -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 alpha -subunit of the amiloride-sensitive epithelial Na channel (ENaC); 3) a partial shift in molecular mass of the gamma -subunit of ENaC from 85 to 70 kDa, thought to be due to a physiological proteolytic cleavage of the extracellular loop of gamma -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 alpha 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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 alpha -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 gamma -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 gamma -ENaC was unchanged. Furthermore, the mean normalized band density of beta -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), alpha -epithelial Na channel (ENaC) appears as a tight doublet, hypothetically due to differing states of glycosylation. Here, the beta -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 gamma -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

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.

                              
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  		Table 2.   Physiological data for spironolactone-treated rats and vehicle-treated control rats

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.

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.

In the same rats, the opposite kidney was prepared for immunocytochemical labeling of NCC (green) and the alpha 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 alpha 1-subunit (red). Bottom: single labeling for NCC, allowing the exclusively apical NCC labeling to be clearly seen (arrows). Bar = 30 µm.

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 gamma -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 gamma -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.

Representative immunoperoxidase labeling of the gamma -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 gamma -ENaC labeling, similar observations were made with the antibodies to alpha - and beta -ENaC and were confirmed by a blinded observer (not shown). Thus the conclusions appear to apply to trafficking of the alpha beta gamma -ENaC complex.


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  		Fig. 6.   Immunoperoxidase labeling of gamma -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.

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 alpha -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 alpha -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 alpha -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.

An additional experiment was done to address whether spironolactone can fully block the increases in alpha -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 alpha -ENaC abundance (band densities: NaCl restricted, 110 ± 10; NaCl replete, 100 ± 12 normalized densitometry units). Thus the upregulation of alpha -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 alpha -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 alpha -ENaC abundance in the presence of spironolactone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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, alpha -ENaC, and the 70-kDa form of gamma -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, alpha -ENaC, and the 70-kDa form of gamma -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 alpha -ENaC abundance in NaCl-restricted rats (Fig. 7) and completely blocked the ability of dietary NaCl restriction to increase alpha -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 11beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES


                              
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  		Table A1.   Physiological data for spironolactone-treated rats and vehicle-treated rats (see Fig. 2)


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 283(5):F923-F933


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