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Endocrinology Vol. 148, No. 10 4829-4835
Copyright © 2007 by The Endocrine Society

Does Glucocorticoid Receptor Blockade Exacerbate Tissue Damage after Mineralocorticoid/Salt Administration?

Amanda J. Rickard, John W. Funder, James Morgan, Peter J. Fuller and Morag J. Young

Prince Henry’s Institute of Medical Research, Clayton 3168, Australia

Address all correspondence and requests for reprints to: Dr. Morag Young, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton 3168, Australia. E-mail: morag.young{at}princehenrys.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mineralocorticoid receptor (MR) antagonism reverses established inflammation, oxidative stress, and cardiac fibrosis in the mineralocorticoid/salt-treated rat, whereas withdrawal of the mineralocorticoid deoxycorticosterone (DOC) alone does not. Glucocorticoid receptors (GRs) play a central role in regulating inflammatory responses but are also involved in cardiovascular homeostasis. Physiological glucocorticoids bind MR with high affinity, equivalent to that for aldosterone, but are normally prevented from activating MR by pre-receptor metabolism by 11ß-hydroxysteroid dehydrogenase 2. We have previously shown a continuing fibrotic and hypertrophic effect after DOC withdrawal, putatively mediated by activation of glucocorticoid/MR complexes; the present study investigates whether this effect is moderated by antiinflammatory effects mediated via GR. Uninephrectomized rats, drinking 0.9% saline solution, were treated as follows: control; DOC (20 mg/wk) for 4 wk; DOC for 4 wk and no steroid wk 5–8; DOC for 4 wk plus the MR antagonist eplerenone (50 mg/kg·d) wk 5–8; DOC for 4 wk plus the GR antagonist RU486 (2 mg/d) wk 5–8; and DOC for 4 wk plus RU486 and eplerenone for wk 5–8. After steroid withdrawal, mineralocorticoid/salt-induced cardiac hypertrophy is sustained, but not hypertension. Inflammation and fibrosis persist after DOC withdrawal, and GR blockade with RU486 has no effect on these responses. Rats receiving RU486 for wk 5–8 after DOC withdrawal showed marginal blood pressure elevation, whereas eplerenone alone or coadministered with RU486 reversed all DOC/salt-induced circulatory and cardiac pathology. Thus, sustained responses after mineralocorticoid withdrawal appear to be independent of GR signaling, in that blockade of endogenous antiinflammatory effects via GR does not lead to an increase in the severity of responses in the mineralocorticoid/salt-treated rat after steroid withdrawal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASE IS the leading cause of morbidity and mortality in the Western world, and a range of pathways contribute to the development and progression of cardiac fibrosis and heart failure. Mineralocorticoid receptor (MR) activation has been shown to play a central role in cardiac fibrosis. The Randomized Aldactone Evaluation Study (1) and the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (2) have provided convincing clinical evidence for a role of MR activation in heart failure, even when plasma aldosterone levels are normal. The Randomized Aldactone Evaluation Study showed a 30–35% reduction in mortality and 35% in morbidity with low-dose spironolactone in patients with severe heart failure. The Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study reported substantially improved outcomes with eplerenone treatment in patients with systolic dysfunction after myocardial infarction. In both trials the MR antagonist was given in addition to current "standard-of-care" treatment.

In the classical mineralocorticoid/salt model of cardiac fibrosis, administration of aldosterone or deoxycorticosterone (DOC) plus salt to uninephrectomized rats for 8 wk produces hypertension, cardiac hypertrophy, and extensive perivascular and interstitial fibrosis (3, 4), with an early vascular inflammatory response preceding the onset of fibrosis (5, 6, 7, 8). Elevated myocardial and coronary vascular expression of inflammatory markers such as (ED-1) positive macrophages, cyclooxygenase (COX)-2, and osteopontin characterize the inflammatory response after 1-wk mineralocorticoid treatment; more recently the induction of oxidative stress and the associated tissue damage have also been implicated in MR-mediated cardiovascular pathology (9).

Previous studies exploring the effect of MR antagonists in the mineralocorticoid/salt model have focused on their ability to prevent the development of inflammation and cardiac fibrosis (10, 11, 12), rather than upon their potential to reverse established inflammation and fibrosis. In a recent study from our laboratory (13), we explored the potential of eplerenone to reverse established inflammation, oxidative stress, and cardiac fibrosis in the mineralocorticoid/salt model. MR blockade clearly reversed established inflammatory and fibrotic responses, whereas mineralocorticoid withdrawal without eplerenone was followed by a continuing, albeit attenuated, inflammatory and fibrotic response. This continuing response in the withdrawal group has been suggested to reflect continued activation of vascular MR by normal levels of endogenous glucocorticoids in the context of the tissue damage induced by 4 wk of prior DOC/salt administration (14).

MRs bind physiological glucocorticoids (cortisol in humans and corticosterone in rodents) with 10- to 30-fold higher affinity than the glucocorticoid receptor (GR) and with affinity equal to aldosterone. Given that circulating concentrations of corticosterone/cortisol are 3 orders of magnitude greater than those of aldosterone, under normal circumstances, physiological glucocorticoids substantially occupy both 11ß-hydroxysteroid dehydrogenase 2 (11ßHSD2) protected (~90%) and unprotected (~99%) MR (15, 16), presumably in tonic inhibitory mode. Aldosterone specificity for MR in epithelial and vascular smooth muscle cells (VSMCs) is conferred by the nicotine adenine dinucleotide positive (NAD+)-dependent enzyme 11ßHSD2, which converts cortisol and corticosterone to their inactive metabolites cortisone and 11-dehydrocorticosterone; the redox change consequent upon the stoichiometric conversion of nicotine adenine dinucleotide phosphate (NADH) to NAD+ has been suggested to mimic that after generation of reactive oxygen species (ROS) in damaged tissue and be responsible for allowing glucocorticoid activation of MR (14). Thus, we hypothesize that the sustained inflammatory and fibrotic response after DOC withdrawal may be generated by the initial tissue damage and the production of ROS, allowing continuing activation of glucocorticoid-occupied MR complexes.

It is also well established that glucocorticoids have major antiinflammatory actions via GRs and, thus, that the normal activity of this receptor may moderate the inflammatory effects of MR activation. Therefore, we explored whether the suppression of the antiinflammatory effects of GR signaling would exacerbate the continuing inflammatory response seen with DOC withdrawal, after 4 wk of DOC administration, by blocking GR with RU486.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental model
Studies followed the National Health and Medical Research Council Australia Code of Practice for the Care and Use of Animals for Scientific Purposes (1997), and were approved by the Monash University Animal Welfare Committee. Male Sprague Dawley rats (180–200 g) were anesthetized with xylazine hydrochloride (8 mg/kg; Troy Laboratories, New South Wales, Australia) plus ketamine (60 mg/kg; Pfizer Pty. Ltd., New South Wales, Australia). After unilateral nephrectomy, rats were allowed to recover overnight before being randomly assigned to one of six treatment groups: control (CON), no further treatment (n = 8); DOC (20 mg/wk) for 4 wk (DOC4) (n = 7); DOC for 4 wk and no steroid wk 5–8 (DOC4/04) (n = 8); DOC for 4 wk plus the GR antagonist RU486 (2 mg/d) wk 5–8 (DOC4/RU486) (n = 7); DOC for 4 wk plus the MR antagonist eplerenone (50 mg/kg·d) wk 5–8 (DOC4/EPL) (n = 8); and DOC for 4 wk plus RU486 and eplerenone per os for wk 5–8 (DOC4/RU486+EPL) (n = 7). During the study all rats were given 0.9% sodium chloride plus 0.4% potassium chloride solution to drink ad libitum.

Steroid treatment
DOC (Sigma-Aldrich, St. Louis, MO) was administered weekly by sc injection in oil. The progesterone (PR) and GR antagonist RU486 (Mifepristone; Sigma-Aldrich) was administered weekly as sc injection in oil. EPL (Pfizer Pty. Ltd.) was incorporated into the rat chow at a concentration such that each animal received approximately 50 mg/kg·d (13).

Systolic blood pressure (SBP)
SBP was measured by tail-cuff plethysmography (ITTC Inc./Life Science, Inc., Woodland Hills, CA) using a procedure adapted from the ITTC Inc./Life Science, Inc., manual. Briefly, rats were acclimated to the measurement procedure twice a week for 3 wk before SBP recording. On the day of measurement, rats were stabilized in the preheated chamber (29 C) for 15 min; SBP was then read over three consecutive manual inflation-deflation cycles. If the SBP readings differed by more than 5 mm Hg, the readings were discarded, rats were allowed to rest, and the procedure was repeated until three consistent readings were obtained.

Tissue collection
Animals were killed by CO2 in air at 8 wk except the DOC4 group, which was killed at 4 wk, and an arterial blood sample and the heart were collected. The apex of the heart was immersion fixed in 4% paraformaldehyde for histological analysis; the midsection was frozen in Optimal Cutting Temperature compound 4583 (Sakura Finetek Inc., Torrance CA) for histological analysis and the apex of the heart snap frozen in liquid nitrogen for quantitative PCR.

Histological analysis
The extent of fibrosis was determined by staining 5-µm heart sections with 0.1% Sirius red (Sigma Diagnostics, St. Louis, MO) in saturated picric acid (AnalaR; BDH, Whatman, Maidstone, UK). Collagen content was then quantified with the Analytical Imaging Station software package (version 4.0 ß 1.5; Imaging Research Inc., St. Catharines, Ontario, Canada), as previously described (13).

Immunohistochemistry
The inflammatory response was characterized by immunohistochemistry as previously described (17, 18), using the following primary antibodies: goat polyclonal COX-2 [1:200 dilution in 1% Tris-buffered saline (TBS); Santa Cruz Biotechnology, Inc., Santa Cruz, CA]; MPIIB101 monoclonal antibody against osteopontin (1:100 dilution 1% TBS; Iowa University Hybridoma Bank, Iowa City, IA); and ED-1, monoclonal antibody against rat monocytes/macrophages (1:200 dilution in 1% TBS; a gift from Professor Peter Tipping, Monash University, Victoria, Australia). Infiltrating ED-1-positive macrophages were quantified by an optical dissector method (19), which provides a value for the average number of macrophages per frame (826,890 µm2) rather than per section; more than 80 ED-1-positive macrophages were counted for each rat to allow accurate statistical between-group comparisons. A semiquantitative grading system (zero to three) was used to measure COX-2 and osteopontin expression in the vessel wall (18). Small, medium, and large vessels were analyzed separately; the pattern of response among the treatment groups was similar for the different-sized vessels, with scores averaged to provide a single score for each heart.

RT-PCR
Total rat heart RNA was prepared with Ultraspec (Fisher Scientific, Pittsburgh, PA). First-strand cDNA synthesis from 500 ng total RNA was performed after DNAase treatment with avian myeloblastosis virus reverse transcriptase (Roche, Indianapolis, IN) and priming with random hexamers. PCR were performed using the primer sets for p22phox, gp91phox, and reduced nicotine adenine dinucleotide phosphate oxidase (NOX)-4 as previously described (13, 18). Expression levels were normalized to those of the 18S ribosomal subunit. To validate the real-time PCR protocol, gene-specific standard curves were generated from one in 10 serial dilutions of previously prepared standards. Standards were diluted as follows: from 10–0.1 pg/µl for 18S and from 500–0.5 fg/µl for all other transcripts. Real-time PCR amplification was performed on a LightCycler (Roche) using SYBR Green reaction mix (Roche) and primers described previously. cDNA samples were diluted 1:20 in water immediately before use for p22phox and 18S; gp91phox and NOX-4 were analyzed undiluted. Relative amounts of mRNA were calculated by normalizing reduced nicotine adenine dinucleotide phosphate [NAD(P)H] oxidase values to 18S rRNA values.

Statistics
All data sets were analyzed by one-way ANOVA (SPSS statistical software package, version 11.5; SPSS, Inc., Chicago, IL), and Tukey’s comparisons test was applied to identify significant effects between groups; differences were considered significant at P ≤ 0.05. All data are reported as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SBP
SBP measurements were recorded at 4 and 8 wk (Fig. 1Go). DOC treatment for 4 wk significantly increased SBP in all groups at this time point (P ≤ 0.05 vs. CON; Fig. 1AGo). As shown in Fig. 1BGo, at 8 wk the increase in SBP induced by DOC treatment over wk 1–4 was reversed by steroid withdrawal (DOC4/04; SBP at 4 wk, 150 ± 11 mm Hg vs. SBP at 8 wk, 130 ± 9 mm Hg; P ≤ 0.05). RU486 treatment over wk 5–8 after steroid withdrawal (DOC4/RU486) sustained the DOC-induced increase in SBP at wk 4 (143 ± 4 mm Hg vs. SBP at wk 8, 129 ± 5 mm Hg; P ≤ 0.05 vs. both CON and DOC4/EPL). Eplerenone administered alone (DOC4/EPL) or with RU486 for wk 5–8 (DOC4/RU486/EPL) gave values similar to CON and significantly less than DOC4/RU846 (P ≤ 0.05) but produced no further decrease in SBP above steroid withdrawal alone (DOC404). Of note, whereas DOC4/RU486 sustained SBP to a level significantly above CON, the addition of eplerenone (DOC4/RU486/EPL) restored SBP to a level not different to CON.


Figure 1
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  		FIG. 1. SBP determined by tail-cuff plethysmography. Treatment groups are as follows: CON, DOC4, DOC4/04, DOC4/RU486, DOC4/EPL, and DOC4/RU486+EPL. Values are mean ± SEM for CON and DOC404, n = 8; and for DOC4, DOC4/RU486, DOC4/RU486+EPL, n = 7. A, SBP at 4 wk. DOC treatment for 4 wk increased SBP compared with CON; this increase was significantly greater in DOC4 than DOC4/04. *, P ≤ 0.05 vs. CON. **, P ≤ 0.05 vs. CON and DOC4/04. B, SBP at 8 wk. RU486 treatment wk 5–8 (DOC4/RU486) sustained the DOC-induced increase in SBP compared with all other groups. *, P ≤ 0.05, other groups.

 
Index of cardiac hypertrophy and fibrosis
An index of cardiac hypertrophy was determined by dividing the wet heart weight by end body weight. As shown previously (4), DOC4 produced an increase in cardiac weight (DOC4 vs. CON; P ≤ 0.01; Fig. 2AGo), which persisted after steroid withdrawal (DOC4/04; P ≤ 0.05 vs. CON), but not after eplerenone treatment for wk 5–8, alone or with RU486 (DOC4/EPL and DOC4/RU/EPL, respectively). As previously shown in our laboratory, DOC4 induced a fibrotic response (DOC4 vs. CON; P ≤ 0.01; Fig. 2BGo) that was sustained despite DOC withdrawal (DOC4/04 vs. CON; P ≤ 0.05), whereas tissue fibrosis returned to CON levels in rats treated with eplerenone for wk 5–8 (DOC4/EPL; P ≤ 0.05 vs. DOC4). RU486 treatment alone for wk 5–8 did not enhance the sustained fibrotic response after DOC withdrawal. In contrast, treatment with DOC and RU486 plus eplerenone (DOC4/RU486+EPL) significantly reduced fibrosis to a level that remained not different to CON or DOC4/EPL (P ≤ 0.05; Fig. 2BGo).


Figure 2
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  		FIG. 2. Index of cardiac hypertrophy and fibrosis. Treatments are the same as for Fig. 1Go. A, Index of cardiac hypertrophy. DOC4 significantly increased cardiac hypertrophy, and this increase was sustained in DOC4/04 compared with DOC4/EPL. *, P ≤ 0.05 vs. DOC4/EPL and DOC4/RU486+EPL. **, P ≤ 0.05 vs. CON, DOC4/RU486, DOC4/EPL and DOC4/RU486+EPL. B, Cardiac fibrosis DOC4 significantly increased cardiac collagen content compared with CON, DOC4/EPL, and DOC4/RU486+EPL. DOC4/04 and DOC4/RU486 sustained the DOC-induced increase in collagen compared with CON and DOC4/EPL. *, P ≤ 0.05 vs. CON and DOC4/EPL. **, P ≤ 0.05 vs. CON, DOC4/EPL, and DOC4/RU486+EPL. {dagger}, P ≤ 0.05 vs. DOC4; NS vs. CON and DOC4/EPL.

 
Markers of inflammation
The inflammatory response was characterized by the level of expression of COX-2 and osteopontin in the vessel wall and by the number of infiltrating ED-1-positive monocytes/macrophages detected in the cardiac interstitium (Fig. 3Go). Consistent with our previous studies (13, 18), DOC4 increased COX-2 and osteopontin expression in the vessel wall and the number of infiltrating ED-1 positive macrophages (DOC4 vs. CON; P ≤ 0.05; Fig. 3Go, A–C, respectively). Furthermore, this response persisted after steroid withdrawal (DOC4/04 vs. CON; P ≤ 0.05; Fig. 3Go, A–C, respectively). Interestingly, RU486 treatment wk 5–8 did not affect the level of expression of the inflammatory markers after steroid withdrawal (DOC4/RU486 vs. CON; P ≤ 0.05; Fig. 3Go, A–C, respectively), whereas eplerenone alone or with RU486 reversed the DOC-induced increase in COX-2, osteopontin, and macrophage expression to CON levels.


Figure 3
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  		FIG. 3. Markers of inflammation. Treatments are the same as for Fig. 1Go. COX-2 (A), osteopontin (B), and ED-1 positive macrophage (C) expression. Expression levels of all three inflammatory markers were increased by DOC4, and this increase persisted in DOC4/04 and DOC4/RU486 compared with CON, DOC4/EPL, and DOC4/RU486+EPL. *, P ≤ 0.05 vs. CON, DOC4/EPL, and DOC4/RU486+EPL.

 
Oxidative stress
To determine the role of the oxidative stress in this model, quantitative RT-PCR was used to measure mRNA levels of subunits of the NAD(P)H oxidase system (NOX4, p22phox, and gp91phox; Fig. 4Go, A–C). As shown previously (13) DOC4 increased mRNA levels of the NOX-4 subunits, an increase which persisted after steroid withdrawal (Fig. 4AGo). Furthermore, the DOC-induced increase was reversed by eplerenone in the presence of RU486 treatment (DOC4/RU486+EPL; P ≤ 0.05 vs. DOC4, DOC404). No further increase in these markers was seen with the addition of RU486 post-steroid withdrawal. Gp91phox expression was increased in the DOC04 group in comparison with CON and DOC4/RU486+EPL (Fig. 4Go; P ≤ 0.05), whereas in terms of p22phox and mRNA expression (Fig. 4BGo), although trends consistent with previous studies (7) are apparent, no significant differences between treatment groups were observed.


Figure 4
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  		FIG. 4. NAD(P)H oxidase subunit mRNA expression relative to 18S rRNA. Treatments are the same as for Fig. 1Go. p22phox (A), gp91phox (B), and NOX-4 (C) mRNA expression. mRNA levels in whole heart were not significantly altered by the different treatment conditions in terms of p22phox and gp91phox, whereas NOX-4 mRNA expression was increased significantly by DOC4, and this increased was sustained in DOC4/04. *, P ≤ 0.05 vs. DOC4/EPL. **, P ≤ 0.05 vs. CON and DOC4/RU486+EPL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Administration of mineralocorticoid/salt to uninephrectomized rats for 4 or 8 wk produces hypertension, vascular inflammation, cardiac hypertrophy, and fibrosis, a process that can be blocked by the concurrent administration of eplerenone (13, 18). More recently, mineralocorticoid withdrawal in an established mineralocorticoid/salt model has not reversed the sustained inflammatory and fibrotic response (13, 18); we have interpreted these data as a role for corticosterone-occupied MR in sustaining inflammatory and oxidative responses (16), consistent with studies demonstrating MR activation after vascular damage subsequent to coronary angioplasty (32). In the present study, we examined whether inflammation and oxidative stress due to corticosterone-activated MR might be limited by antiinflammatory actions of the GR. This possibility appears not to be the case on the basis of the results of the current study.

GR blockade
RU486 treatment after mineralocorticoid withdrawal neither enhanced nor attenuated the sustained inflammation, the increase in cardiac hypertrophy index, or fibrosis in this model, in contrast with MR blockade by eplerenone. Given the pivotal role of GR signaling in moderating inflammatory responses, these findings provide evidence for the key pathophysiological role for inappropriate MR activation in the development of cardiovascular inflammation and cardiac fibrosis, independent of GR signaling. In the vessel wall, glucocorticoids are not only potent participants in terms of the inflammatory response but can also directly alter arterial function and structure. GRs are expressed in all cell types found in the vessel wall, and have increased vascular reactivity, endothelial-nitric-oxide-synthase (eNOS) expression, and endothelial cell permeability, consistent with their CON of volume homeostasis via nonrenal mechanisms (19). In the present study, DOC-induced hypertension was reversed by steroid withdrawal alone, whereas the increase persisted with RU486 administration; in previous studies, administration of corticosterone or RU486 alone for 8 wk neither increased nor lowered SBP (4). The action of RU486 in modulating vascular reactivity and, thus, regulating blood pressure has not been widely investigated. Previously, in a model of ACTH-induced hypertension, high doses of RU486 (70 mg/kg every 3 d) were found to partially reduce increased SBP (20); in a model of cortisol-induced hypertension, RU486 had no effect on hypertension (21). RU486 acts as both a GR and PR antagonist, and recent studies have suggested that PR signaling may play a role in mediating inflammation in the vasculature (22); in addition, it has been shown that PR has vasorelaxant effects on the endothelium (23). Although RU486 was administered at a dose (2 mg/d) sufficient to block GR signaling (24, 25, 26), those needed to block PR receptors in this model have not been determined. Thus, the findings of the present study suggest that reducing activity at the GR by RU486 administration has little or no effect on the sustained pathology in the model of mineralocorticoid withdrawal.

MR blockade and steroid withdrawal
Eplerenone has successfully reversed MR-mediated cardiac pathology, even in the face of continuing DOC and salt (13). In the present study, eplerenone administered from wk 5 is sufficient to reverse the cardiac pathology, despite suppressed antiinflammatory GR signaling. The increase in cardiac hypertrophy index induced by DOC4 is partially sustained after steroid withdrawal, whereas DOC-mediated hypertension decreases to CON levels (Fig. 2BGo). These findings highlight the specific cardiotropic effects of MR activation in the heart, which have been clearly demonstrated previously in vivo (18, 27) and in vitro (28). Given that SBP was reduced by steroid withdrawal, hypertrophy at 4 wk in this model does not appear to be an irreversible structural response to the increased hemodynamic load (Fig. 2AGo) (27).

Steroid withdrawal in this model of MR-mediated vascular damage is followed by a sustained fibrotic and inflammatory response (13). Changes in oxidative responses are difficult to demonstrate after 4-wk DOC, although the present values are consistent with those shown previously (7). DOC treatment for 4 wk (DOC4) or steroid withdrawal (DOC4/04) showed no change in the mean mRNA levels of NAD(P)H oxidase system subunits. The current study did not include an 8-wk treatment time point that, in previous studies, showed that values for mRNA expression of these subunits continue to increase with ongoing treatment (13, 18). There is increasing experimental evidence to show that NAD(P)H oxidase-generated ROS is important in the establishment and progression of inflammation and fibrosis. The present findings are supported by studies using antioxidants or mice in which various components of the NAD(P)H oxidase system have been rendered inactive (29). Moreover, given that such interventions specifically prevent cardiac fibrosis, but not hypertrophy, it is highly likely that this reflects the presence of distinct cellular signaling pathways (30).

Redox regulation of glucocorticoid-occupied MR
Although the mechanism responsible for the persisting pathology after DOC withdrawal remains to be fully elucidated, we hypothesize that the sustained response is produced by activation of normally quiescent corticosterone-occupied MR in the presence of tissue injury (16). One mechanism that may alter the activation state of glucocorticoid-occupied MR is the redox state of the cell. Pre-receptor metabolism of active corticosterone into inactive 11-dehydrocorticosterone by 11ßHSD2 stoichiometrically reduces NAD+ to NADH. Thus, when 11ßHSD2 is operational, high-intracellular levels of NADH are generated and glucocorticoid-occupied-MR complexes held inactive. In contrast, when 11ßHSD2 activity is blocked by the 11ßHSD2 inhibitor carbenoxolone, intracellular levels of NADH decrease, and glucocorticoid-occupied MR complexes are activated (8, 31), suggesting a mechanism whereby these complexes in epithelial tissues and vessel wall may be activated (16). Intracellular levels of NADH can be similarly reduced by the production of ROS after tissue damage and may explain the protection afforded by selective MR blockade after acute coronary angioplasty in pigs (32). The study showed that administration of the MR antagonist eplerenone preserved the luminal diameter, which supports the aforementioned hypothesis in that the animals were not given exogenous aldosterone or salt; eplerenone appears to be blocking activated cortisol-occupied MR complexes in the vessel wall activated by tissue damage-induced ROS. The precise mechanism whereby redox state leads to activation of corticosterone/cortisol-occupied MR complexes is unclear, although previous studies have demonstrated redox-dependent transcriptional changes in other systems (33, 34). In support of such a hypothesis is the demonstration that the oxidation state of the heme iron-bound steroid receptor homolog E75 in Drosophila determines whether E75 can interact with its heterodimer partner DHR3 (35).

Activation of MR by corticosterone
As discussed previously, MRs in mineralocorticoid responsive tissues are protected from inappropriate activation by the activity of 11ßHSD2 and are, thus, primarily responsive to aldosterone. It is well accepted that inactivation of 11ßHSD2 in epithelial cells (e.g. kidney, salivary gland) and VSMCs allows endogenous corticosterone/cortisol to act as MR agonists (7, 31). Where 11ßHSD2 is not present (cardiac myocytes, hippocampus), MR acts primarily as a cortisol or corticosterone receptor, normally in tonic inhibitory mode (36). Thus, tissue responses to cortisol/corticosterone and aldosterone differ. In VSMCs, MRs are a normal physiological target for mineralocorticoids, consistent with the return of blood pressure to CON levels after 4-wk DOC withdrawal. However, in cardiomyocytes, MRs are normally not aldosterone target tissues but can be activated by aldosterone, by experimentally overexpressing 11ßHSD2 (36), or by corticosterone in the presence of ROS. The latter is again consistent with the maintained hypertrophy and fibrosis despite mineralocorticoid withdrawal.

Conclusions
In the present study, we have shown: 1) that reducing activity at the GR does not enhance or attenuate the inflammatory and fibrotic response in the model of mineralocorticoid withdrawal; and 2) thus, the difference between mineralocorticoid treatment for 8 wk and steroid withdrawal cannot be defined by the counteracting antiinflammatory actions via GR signaling. These data highlight the role of MR in promoting an inflammatory response and suggest that signaling via the MR is controlled by cellular context, i.e. redox state of the cell.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia. A.J.R. is supported by a Monash University Graduate Scholarship.

Disclosure Statement: A.J.R. and J.M. have nothing to declare. J.W.F. has received consulting fees from Merck, Pfizer, Sankyo, Lilly, Schering-Plough, Wyeth, Exelisis, and CBio. P.J.F. has received consulting fees from Merck and lecture fees from Novartis. M.J.Y., P.J.F., and J.W.F. have been the recipients of a previous research grant from Pfizer, and M.J.Y. and J.W.F. from Merck. The present study does not relate to these activities.

First Published Online July 19, 2007

Abbreviations: CON, Control; COX, cyclooxygenase; DOC, deoxycorticosterone; DOC4, deoxycorticosterone (20 mg/wk) for 4 wk; DOC4/04, deoxycorticosterone for 4 wk and no steroid wk 5–8; DOC4/EPL, deoxycorticosterone for 4 wk plus the mineralocorticoid receptor antagonist eplerenone (50 mg/kg·d) wk 5–8; DOC4/RU486, deoxycorticosterone for 4 wk plus the glucocorticoid receptor antagonist RU486 (2 mg/d) wk 5–8; DOC4/RU486+EPL, deoxycorticosterone for 4 wk plus RU486 and eplerenone for wk 5–8; GR, glucocorticoid receptor; 11ßHSD2, 11ß-hydroxysteroid dehydrogenase 2; MR, mineralocorticoid receptor; NAD, nicotine adenine dinucleotide; NADH, NAD phosphate; NAD(P)H, reduced NAD phosphate; NOX, reduced nicotine adenine dinucleotide phosphate oxidase; PR, progesterone; ROS, reactive oxygen species; SBP, systolic blood pressure; TBS, Tris-buffered saline; VSMC, vascular smooth muscle cell.

Received February 13, 2007.

Accepted for publication July 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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