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Mineralocorticoid Action and Sodium-Hydrogen Exchange: Studies in Experimental Cardiac Fibrosis

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

Address all correspondence and requests for reprints to: Morag Young, Endocrine Genetics, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: morag.young{at}med.monash.edu.au.

	Abstract

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There is increasing evidence that the trigger for cardiac fibrosis in response to mineralocorticoid/salt administration is coronary vasculitis and that effects can be seen within days of deoxycorticosterone acetate (DOCA) administration. Furthermore, rapid, nongenomic mineralocorticoid effects on the sodium-hydrogen exchanger (NHE-1) in vascular smooth muscle cells have recently been described. That this mechanism may act as an inflammatory or profibrotic signal was tested by comparing the specific NHE-1 antagonist cariporide and the mineralocorticoid receptor antagonist K canrenoate in the rat model of mineralocorticoid/salt perivascular fibrosis over 8 d of DOCA/salt administration. Interstitial collagen, inflammatory cell infiltration, and inflammatory markers were determined. DOCA elevated blood pressure above control, cariporide +DOCA, or K canrenoate +DOCA rats, without cardiac hypertrophy. At 8 d interstitial collagen was significantly elevated in the DOCA-alone group, with levels in cariporide- and K canrenoate-treated rats not different from control. Expression of osteopontin, cyclooxygenase-2, and ED-1 were elevated by DOCA treatment, blocked by potassium canrenoate, and (for ED-1 and osteopontin) partially reduced by cariporide. These results suggest mineralocorticoid/salt-induced cardiac fibrosis may involve coronary vascular smooth muscle cell NHE-1 activity as a possible contributor to the cascade of transcriptional events that produce the characteristic coronary vasculitis seen with excess mineralocorticoid and salt.

	Introduction

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OVER THE PAST decade, following pioneering work from Weber’s laboratory (1), there have been a series of studies on the direct cardiovascular actions of aldosterone in general, and in particular on its ability to produce cardiac hypertrophy and fibrosis in rats fed a high-salt diet (reviewed in Refs.2, 3, 4). These cardiac effects are not seen in animals given aldosterone on a low-salt diet (1) and are clearly mediated via mineralocorticoid receptors (MRs) in that they have been shown to be blocked by spironolactone, potassium canrenoate, and eplerenone.

The time course for these pathophysiologic effects of mineralocorticoids on the heart and coronary vessels has been studied in a number of laboratories. In studies from both Weber’s (5) and Robert’s (6) groups, changes in collagen synthesis could not be seen before 2–3 wk and gradually evolved thereafter. In subsequent 4-wk studies (7), earlier changes were noted under the same experimental conditions of aldosterone/salt administration to uninephrectomized animals, with significant changes in coronary and perivascular inflammatory markers, such as ED-1, osteopontin, and cyclooxygenase (COX)-2, at the first time point measured (1 wk). In studies on deoxycorticosterone acetate (DOCA)/salt hypertension (8), inflammatory cell infiltration was seen at 4–8 d; whether this more rapid time course reflects the high bolus dose of DOCA, compared with the infusion of aldosterone or other methodological differences, remains to be explored.

In all of the above studies, it was assumed that the mechanisms of action of aldosterone on blood cells, blood vessels, and cardiomyocytes were at the genomic level, in that the effects were MR mediated and occurred over a relatively extended (days/weeks) time course. Recently, however, it has become clear that at least some of the acute, nongenomic effects of aldosterone are also almost certainly MR mediated, from studies from Marusic’s laboratory (9) on the rapid effect of aldosterone on vascular smooth muscle cell (VSMC) sodium-hydrogen exchange (NHE), a protein kinase C (PKC)-dependent phenomenon previously demonstrated by Wehling (10) and colleagues (11). In these studies, 1–10 nM aldosterone was shown to raise intracellular pH to a maximum within 5 min, an effect that was neither blocked by spironolactone nor mimicked by cortisol and consistent with an effect via a putative membrane-located, non-MR aldosterone receptor. When, however, carbenoxolone was used to block 11ß-hydroxysteroid dehydrogenase activity in VSMCs, cortisol could be shown to mimic aldosterone, consistent with its effect on epithelial MR under similar circumstances. Second, the water-soluble MR antagonist RU28318, in contrast with spironolactone, completely blocked the effect of aldosterone or cortisol, again consistent with a MR-mediated rapid, nongenomic action of aldosterone. The reason spironolactone is ineffective acutely remains to be explored.

On this background, then, we determined to explore a possible role for effects of NHE-1 blockade in the uninephrectomized, 0.9% saline-drinking rat model. On the basis of our previous studies, we compared the effect of the MR antagonist potassium canrenoate with the NHE-1 blocker cariporide on the cardiac responses to a standard 20-mg dose of DOCA 8 d previously. The results of the study suggest that some, but not all, of the responses to mineralocorticoid/salt administration may represent effects of NHE-1 blockade, possibly nongenomic, which may thus play a role in triggering some of the secondary transcriptional changes responsible for the tissue damage seen.

	Materials and Methods

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Protocols for animal use in this study were approved by the Animal Ethics Committee of the Baker Medical Research Institute. Male Sprague Dawley rats (190–210 g starting weight) were uninephrectomized on d 0 under anesthesia with xylazil (8 mg/kg, Troy Laboratories, Smithfield, NSW, Australia) and ketamine (60 mg/kg, Park-Davis, Auckland, New Zealand), with carprofen (5 mg/kg, Pfizer, New York, NY) for postoperative analgesia. Rats were then maintained on chow ad libitum and 0.9% NaCl solution to drink and allocated into four treatment groups of eight animals as follows: 1) control, receiving vehicle (ethanol, corn oil) sc on study d 2 (CON, n = 8); 2) DOCA (n = 8; Sigma, St. Louis, MO; 20 mg in corn oil sc on study d 2; 3) DOCA plus potassium canrenoate 20 mg/kg·d (KCAN, n = 8; Pharmacia, Skokie, IL); and 4) DOCA plus cariporide 100 mg/kg·d (CAR, n = 8; Aventis, Heidelberg, Germany). For groups 3 and 4, the concentration of cariporide or potassium canrenoate in the drinking fluid was adjusted daily on the basis of body weight and volume consumed.

Rats were acclimated to the blood pressure-measuring device for 1–2 wk before surgery, with measurements made every 3 d over the course of the study. Animals were killed on study d 10; kidneys and hearts were excised, weighed, fixed in buffered paraformaldehyde for 12–16 h, and then rinsed and stored overnight in PBS before paraffin embedding.

Histological analysis
Tissue blocks were sectioned in the midcoronal plane at 5-µm thickness onto glass slides and myocardial collagen levels determined by picrosirius red staining. Immunohistochemistry for inflammatory cytokines used antibodies against COX-2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), against osteopontin from the University of Iowa Hybridoma Bank (Iowa City, IA), and against ED-1 from Dr. P. Tipping (Monash University, Clayton, Australia). Sections were dewaxed by 2 x 5-min washes in xylene, and rehydrated through graded alcohol by 3 min washes with a final wash in water. They were then blocked in 0.3% H₂O₂ in Tris-buffered saline (TBS), and washed 3 x 5 min in TBS. For osteopontin and ED-1, a standard citrate boiling antigen retrieval procedure was used, but for COX-2 no retrieval was necessary. Sections were then washed 3 x 5 min in TBS, the primary antibody added (COX-2 1:100; osteopontin 1:10; ED-1 1:400), and sections incubated overnight at 4 C. The following day they were rinsed (3 x 5 min TBS) and the appropriate secondary antibody added at a dilution 1:200 in TBS for 1 h, followed by 3 x 5 min washes in TBS. Preincubated avidin biotin complex and filtered diaminobenzidine were applied and nuclei counterstained with hematoxylin before mounting in Depex (BDH, Poole, UK). Collagen content and level of inflammatory marker expression are expressed as percent area per field analyzed; for each marker 10–12 fields were analyzed in each individual rat, the average of which was used for between-group comparisons by one-way ANOVA. Small- and medium-sized coronary arteries were scored on a scale of 0–3 for marker expression in vessel wall.

	Results

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Figure 1, A–H, shows the range of responses measured to 8 d DOCA, and the effects on such responses of concurrent administration of potassium canrenoate or cariporide. Figure 1A shows that blood pressure in response to DOCA rose on average 12 mm Hg, which was significantly attenuated by both potassium canrenoate and cariporide, to levels not significantly different from control. Figure 1B shows data for heart weight as a percentage of body weight and Fig. 1C kidney weight similarly. In both cases mean values for DOCA are fractionally higher than control, but in neither case did DOCA significantly alter organ weight after 8 d in this study, nor were any effects of potassium canrenoate or cariporide seen.

View larger version (19K): [in this window] [in a new window]   FIG. 1. A, Final systolic blood pressure (SBP) after 8 d of DOCA treatment is shown. DOCA treatment for 8 d produced a small but significant increase over control, whereas no changes were seen with any other treatment. B and C, Data for cardiac and renal hypertrophy, respectively, are shown. In both cases there was a tendency for an increase with DOCA treatment, but this did not reach significance; no change was seen with CAR+DOCA or KCAN+DOCA. D–G, Results for ED-1, osteopontin, and COX-2. DOCA produced about a 3-fold increase in vascular (D) and interstitial (E) ED-1 levels and about a 10-fold elevation of osteopontin (F) and COX-2 (G). Levels of inflammatory markers were significantly reduced by KCAN to values not significantly different from control for ED-1 and osteopontin and to a level midway between control and DOCA for COX-2, significantly different from both. CAR also reduced marker expression in the case of ED-1 and osteopontin to levels significantly different from both control and DOCA but had no significant effect on COX-2 staining. H, Collagen content is shown. DOCA significantly increased collagen deposition; both KCAN and CAR produced levels not significantly different from control, with CAR+DOCA not different from DOCA alone (0.05 < P < 0.10). *, Significantly different from control at the P < 0.05 level; , significantly different from DOCA at the P < 0.05 level.

	Discussion

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NHE-1 has previously been shown to be expressed in VSMCs, cardiomyocytes, and cardiac fibroblasts, all of which cell types are potentially involved in the processes of vascular inflammation, cardiac hypertrophy, and cardiac fibrosis seen in the experimental model of mineralocorticoid/salt hypertension. In cardiac cells, attention has focused on its role in mediating inotropic responses to stretch (12) and neurohumoral factors such as angiotensin (13), endothelin (14), and -adrenergic stimulation (15). In addition, several studies have demonstrated protection by NHE-1 blockade in myocardial ischemia (16) and postinfarction (16, 17), although whether the site of action is on cardiomyocytes, blood vessels, or both is not well established. NHE-1 expression has similarly been shown in cardiac fibroblasts (18), as has its regulation by mitogenic factors in such cells at the transcriptional level (19).

In VSMCs there is not only ample evidence for NHE-1 expression but also for its regulation by aldosterone (9, 20, 21). As previously noted, Wehling (20) showed aldosterone to stimulate NHE-1 activity in VSMCs, and thus elevate intracellular pH, by a rapid, nongenomic, PKC-dependent mechanism. Subsequently Alzamora et al. (9) extended these studies, providing evidence that this rapid nongenomic effect is mediated via a classical, 11ß-hydroxysteroid dehydrogenase-protected MR. Simultaneously Ebata et al. (21) reported both nongenomic and genomic actions of aldosterone on VSMCs to increase both NHE-1 activity and NHE-1 mRNA levels. This complex response makes effects observed over 8 d, as in the present series, difficult to partition between genomic and nongenomic.

The present studies were undertaken to determine whether the previously described vascular inflammatory response to mineralocorticoid/salt administration might be mediated or modulated by effects on sodium-hydrogen exchange via NHE-1. In terms of specificity, cariporide has almost two orders of magnitude lower IC₅₀ for NHE-1 than for NHE-2 and over three orders of magnitude lower than for NHE-3 (16, 22). Although the present studies primarily address the coronary vascular effects of NHE-1 blockade, cariporide clearly blocks NHE-1 activity in other organs: The extent to which central or renal NHE-1 blockade is involved in the effect seen on blood pressure, for example, remains moot. On the other hand, the findings for the inflammatory markers ED-1, COX-2, and osteopontin can be reasonably ascribed to an effect on the vessel wall and on the processes involved in establishing an inflammatory response to MR activation.

By these criteria, the putative primary events are modestly (ED-1, osteopontin) or not at all (COX-2) antagonized by NHE-1 blockade, whereas what has often been presumed to be a secondary event (collagen deposition) in response to DOCA/salt is equivalently blocked by potassium canrenoate and cariporide. Our interpretation of these findings is as follows. First, in circumstances in which MR blockade reduces inflammatory markers to levels not different from control, their expression may be modulated by the intracellular pH of the cells involved. This is consistent with the reported action of aldosterone to increase NHE-1 expression in VSMCs (21), presumably as part of the totality of the VSMC response to MR activation. In contrast to ED-1 and osteopontin, the COX-2 response to DOCA/salt is substantially lowered by potassium canrenoate but to levels clearly well above control.

The mechanisms underlying this differential sensitivity to MR blockade are unclear. One possible explanation is of a range of downstream sensitivity to MR activation, potentially distinguishing acute nongenomic effects via PKC (10, 23), protein-protein interactions involving MR and activator protein-1 or nuclear factor B (24), and classical transcriptional effects via pentadecamer hormone response element. Although this difference may indicate a useful direction for exploring a subcellular portfolio of responses to MR activation, elevation of COX-2 may be a bystander effect rather than a necessary link in the chain of causation of the inflammatory response. This is also suggested by the finding that COX-2 blockade (unlike MR blockade) does not block the tissue damage and vascular inflammation seen with mineralocorticoid/salt excess (Rocha, R., personal communication).

Finally, the single tissue response measured wherein cariporide and potassium canrenoate appear equivalently able to block the DOCA/salt effect is that of collagen deposition. Although this may reflect one end of the spectrum of VSMC response seen for osteopontin/ED-1/COX-2, it may also reflect involvement of cardiomyocytes/cardiac fibroblasts as well as VSMCs in perivascular and interstitial collagen accumulation. If this is the case, it would appear that, of the responses to inappropriate MR activation, those primarily driven by the vessel wall may be modulated rather than directly mediated by NHE-1 activity; in contrast, NHE-1 activity would appear crucial in mounting a putatively downstream response, that of fibrogenesis. This tentative conclusion might therefore serve as a point of departure for further studies on coronary inflammation and cardiac fibrosis in response to inappropriate MR activation, particularly in the way in which such activation may involve differential responsiveness in terms of intracellular pH and cellular redox state.

	Footnotes

 
Abbreviations: COX, Cyclooxygenase; DOCA, deoxycorticosterone acetate; MR, mineralocorticoid receptor; NHE, sodium-hydrogen exchange; PKC, protein kinase C; TBS, Tris-buffered saline; VSMC, vascular smooth muscle cell.

Received January 8, 2003.

Accepted for publication April 14, 2003.

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