Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

ISSN: 1524-4563 Copyright © 2009 American Heart Association. All rights reserved. Print ISSN: 0194-911X. Online

72514Hypertension is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX

DOI: 10.1161/HYPERTENSIONAHA.109.131110 published online Jul 27, 2009; HypertensionMorag J. Young

Amanda J. Rickard, James Morgan, Greg Tesch, John W. Funder, Peter J. Fuller and Pressure

Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Deletion of Mineralocorticoid Receptors From Macrophages Protects Against

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Deletion of Mineralocorticoid Receptors From MacrophagesProtects Against Deoxycorticosterone/Salt-Induced Cardiac

Fibrosis and Increased Blood PressureAmanda J. Rickard, James Morgan, Greg Tesch, John W. Funder, Peter J. Fuller, Morag J. Young

Abstract—Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling.Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/mac-rophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macro-phages, their role in regulating macrophage function to date has not been investigated. We, thus, used theCre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages withthe lysozyme M promoter used to drive Cre expression (MRflox/flox/LysMCre/� mice). Male mice from each genotype(MRflox/flox or wild-type and MRflox/flox/LysMCre/� mice) were uninephrectomized, given 0.9% NaCl solution to drink,and treated for 8 days or 8 weeks with either vehicle (n�10) or deoxycorticosterone (n�10). Equivalent tissuemacrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogenactivator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MRflox/flox/LysMCre/� mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced inMRflox/flox/LysMCre/� mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophagerecruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similarfor each genotype; in contrast, MRflox/flox/LysMCre/� mice showed no increase in cardiac fibrosis or blood pressure, aswas seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptorsignaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocor-ticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulationof cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model. (Hypertension. 2009;54:00-00.)

Key Words: macrophages � monocytes � mineralocorticoid receptor � cardiac fibrosis � inflammation� tissue remodeling

The clinical use of mineralocorticoid receptor (MR) an-tagonists added to the current standard of care reduces

morbidity and mortality in patients with congestive heartfailure1,2 and reduces blood pressure and proteinuria asmonotherapy in essential hypertension.3 Although the precisemechanism for this protection remains to be determined,considerable insights have been obtained from experimentalmodels of mineralocorticoid/salt-mediated cardiac fibro-sis4–6; hypertension, cardiac hypertrophy, and fibrosis are keyresponses to the administration of aldosterone or deoxycorti-costerone (DOC) concurrently with a high salt intake for 8weeks. Importantly, the pathogenesis of cardiac fibrosis isindependent of hypertension and cardiac hypertrophy in thismodel, suggesting a direct role for MR activation in drivingthe onset and progression of cardiovascular disease.4–6

We and others have previously identified vascular inflam-mation (ie, osteopontin and plasminogen activator inhibitor

type 1 [PAI-1] expression) and an increased macrophageinfiltration in the myocardium before the onset of fibrosis,suggesting that these are key players in the initiation andprogression of MR-mediated cardiac fibrosis.7–10 Oxidativestress has also been shown to play a key role in MR-mediatedcardiac pathology.11 The NAD(P)H oxidoreductase system iswidely expressed throughout the cardiovascular system and isa major source of reactive oxygen species in the vesselwall.12,13 Expression of the NAD(P)H oxidase subunit 2(NOX2; also called gp91phox) and p22phox subunits ofNAD(P)H oxidase are increased after DOC/salt treatmentfrom 1 to 2 weeks,14 whereas a potential role specifically formacrophage-MR signaling in oxidative stress has been sug-gested by increased levels of p22phox and PAI-1 in humanmonocytes and increased macrophage NAD(P)H oxidaseactivity after aldosterone treatment in vivo.15,16 Changes inNO signaling in response to the onset of inflammation also

Received February 17, 2009; first decision March 5, 2009; revision accepted June 10, 2009.From the Prince Henry’s Institute of Medical Research (A.J.R., J.M., J.W.F., P.J.F., M.J.Y.) and Department of Nephrology, Monash Medical Centre

(G.T.), and Department of Physiology (A.J.R., M.J.Y.), Monash University, Clayton, Australia.Correspondence to Morag J. Young, Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton 3168, Australia. E-mail

[email protected]© 2009 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.131110

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contributes to the production of superoxide and vasculardysfunction in this model.11,17 It is our hypothesis that MRsignaling, specifically, in monocytes/macrophages, representsan important and novel mechanism in the pathology ofcardiovascular disease.

Macrophages contain both MRs and glucocorticoid recep-tors (GR) but not the aldosterone specificity-conferring en-zyme 11�-hydroxysteroid dehydrogenase type 2, indicatingthat MRs in macrophages will be normally occupied byglucocorticoids (cortisol in humans and corticosterone inrodents).18,19 The relative contribution of GRs and MRs andtheir respective ligands in the control of macrophage pheno-type and activation has not been determined in the context ofcardiovascular disease.

The aim of the current study is to investigate whethermineralocorticoid activation of monocytes/macrophagesplays a unique role in the cardiac pathology of the mineralo-corticoid/salt model. Conventional MR knockout (KO) miceare available but show high neonatal lethality because of theirinability to concentrate urinary sodium20; hippocampal MRselective KO mice have also been described.21 We, therefore,generated macrophage MR null (MRflox/flox/LysMCre/�) miceusing the Crelox approach and investigated cardiovascularresponse to acute (8 days) and chronic (8 weeks) administra-tion of DOC/salt.

MethodsAdditional materials and methods are provided in the online DataSupplement (please see http://hyper.ahajournals.org).

Generation of Monocyte/Macrophage MRNull MiceMice containing the MRflox allele (kindly provided by Pfizer Inc)with mice expressing Cre recombinase under the control of themyeloid lineage-specific promoter, lysozyme M (LysM), werecrossed to generate mice in which the MR was deleted in monocytes/macrophages.22 The presence of the MRflox/flox and LysM Cretransgene was determined by PCR analysis of genomic DNA from tailtips. MR deletion from the myeloid lineage was validated by Westernblot analysis of bone marrow macrophages from KO and MRflox/flox

control mice using the MR1–18 monoclonal antibody hybridomasupernatant (1:250; a gift from Prof Celso Gomez-Sanchez23).

DOC/Salt Model of Cardiac FibrosisMice �8 weeks of age (n�10 per group) were uninephrectomizedand given standard chow and 0.9% NaCl/0.4% KCl solution to drink.Mice of each genotype were randomly assigned to one of thefollowing treatments, resulting in a total of 4 groups of mice for eachtime point: (1) control treatment for 8 days, (2) continuous DOCtreatment for 8 days and control treatment for 8 weeks, and (3)continuous DOC treatment for 8 weeks.24,25 Mice receiving DOCtreatment received an SC 7-mg, 21-day release pellet that wasreplaced every 3 weeks.

Systolic Blood PressureSystolic blood pressure (SBP) was measured by tail-cuff plethys-mography (ITTC Life Science) biweekly for 3 weeks before SBPrecording.26–28

Tissue Collection and AnalysisAnimals were killed by CO2 in air at 8 days or 8 weeks with anarterial blood sample, and the heart was collected and stored foranalysis, along with plasma radioimmunoassay for aldosterone (MPBiomedical), histological analysis for collagen content (0.1% Sirius

red, Sigma-Aldrich),5,28 immunohistochemistry for CD68� mono-cytes/macrophages (1:200, Sigma-Aldrich),9,28,29 and quantitativeRT-PCR for mRNA expression, as detailed in Table S1 (available inthe online Data Supplement).7,11,28,30

StatisticsAll of the data were analyzed by 1-way ANOVA (Prism statisticalsoftware package, Graph Pad version 5.0A), and Bonferroni’scomparisons test applied to identify significant effects betweengroups. Mean differences were considered significant at P�0.05. Allof the data are reported as mean�SEM. One cohort of animals (10per group) was the subject of the current study.

ResultsMacrophage-MR Null MiceMRflox/�/LysMCre/� and MRflox/flox (WT) mice used to breedmacrophage MR null mice showed normal fertility and littersize. Mice genotyped as MRflox/flox/LysMCre/� showed nor-mal phenotype, body, and heart weight (Figure S1A andTable S2), as well as the expected plasma aldosterone levelsat 8 days for mice drinking 1% saline (aldosterone picogramsper 100 �L: WT control, 12.4�1.6; WT DOC, 8.1�1.3; KOcontrol, 12.4�2.2; KO DOC, 6.9�0.5). Resident macrophagenumbers in the hearts of MRflox/�/LysMCre/� mice wereequivalent to those in MRflox/flox mice, indicating no obvioussystemic deletion of monocytes or macrophages (Figure 1A).Deletion of MRs from monocytes/macrophages was con-firmed by Western blot analysis of MR expression in ex-panded bone marrow macrophages (Figure S1B). An anti-body directed to the N terminus of the MR protein shows aband in WT lysates (lane 4) equivalent to the positive controlpurified human MR protein (lane 2) and whole mouse kidney(lane 3), whereas the sample from MRflox/flox/LysMCre/� miceshowed no band (lane 5).

Role of Macrophage MRs in a Model of AcuteDOC-Mediated Cardiac Pathology: 8 day Study

Macrophage Infiltration at 8 DaysTo assess the role of the macrophage MRs in acute DOC-induced macrophage recruitment, the number of infiltratingCD68-positive monocytes/macrophages was assessed by im-munohistochemistry. As shown previously in WT mice, DOCtreatment for 8 days significantly increased the number ofinfiltrating macrophages in the heart; this effect was notaltered by specific deletion of the MRs from macrophages(Figure 1A).

Cardiac Fibrosis at 8 DaysThe role of macrophage MRs in DOC-induced collagendeposition was determined by staining with Sirius red (FigureS2). As expected, DOC treatment for 8 days did not signifi-cantly alter cardiac collagen content in either genotype at 8days (Figure 1B).

Expression of Proinflammatory Genes at 8 Days(To further explore to role of macrophage MRs in macrophagerecruitment, mRNA levels of the chemoattractant monocytechemotactic protein 1 MCP-1) were assessed by quantitativeRT-PCR. DOC treatment for 8 days significantly increasedmRNA levels of MCP-1 in both genotypes (P�0.05; Figure 2A).

2 Hypertension September 2009



To investigate the role of macrophage MRs in the earlypathological events of the mineralocorticoid/salt model, ex-pression of known proinflammatory/fibrogenic genes wasdetermined (Figure S3A through S3G). For PAI-1 (Figure2B) and NOX2 (Figure S3G), gene expression DOC/salttreatment in WT, but not MR null, mice significantly in-creased mRNA levels. Values for mRNA expression ofosteopontin, transforming growth factor (TGF)-�1, eNOS,connective tissue growth factor, collagen 1, and NOX2 inuntreated MRflox/flox/LysMCre/� mice were significantly re-duced compared with values for untreated WT mice atbaseline (P�0.05; Figure S3A through S3D and S3H). Incontrast, DOC treatment for 8 days did not significantly alterthe expression of these genes over baseline for either geno-type. No significant change in mRNA levels at 8 days ofDOC/salt treatment was seen for glucose-6-phosphate dehy-drogenase, p22phox (Figure S3E and S3F), fibronectin, andprocollagen III (data not shown).

Role of Macrophage MRs in a Model of ChronicDOC-Mediated Cardiac Pathology: 8-Week Study

Macrophage InfiltrationAs expected, DOC treatment for 8 weeks significantly in-creased the number of infiltrating CD68-positive monocytes/macrophages in WT mice, and this was not altered by deletion ofMRs from macrophages (Figure 3A and Figure S2).

Cardiac FibrosisIn agreement with previous studies, 8 weeks of DOC treat-ment in WT mice significantly increased interstitial andperivascular collagen. In contrast, MR deletion from mono-cytes/macrophages protected against the DOC-induced in-crease in cardiac fibrosis (Figures 3B and S2).

Expression of Proinflammatory GenesDOC treatment for 8 weeks increased the mRNA levels ofPAI-1 (P�0.05; Figure 4A), inducible NO synthase, andconnective tissue growth factor (Figure S4A and S4B) in bothgenotypes, whereas deletion of MRs from macrophagesreduced baseline TGF-�1 mRNA levels (P�0.05; Figure 4B).Values for osteopontin for the KO DOC mice were increasedover KO control mice, whereas the equivalent values in WTmice did not reach significance (Figure S4C). In contrast,mRNA levels of collagen 1, endothelial NO synthase,glucose-6-phosphate dehydrogenase, MCP-1, NOX2 (FigureS4D through S4H), fibronectin, and procollagen III (data notshown) were not altered at 8 weeks by DOC treatment ordeletion of MRs from macrophages.

Systolic Blood PressureThe effect of deletion of MRs from macrophages on DOC-mediated increases in SBP was assessed at 4 and 8 weeks(Figure 5). As shown previously, DOC treatment for 4 weekssignificantly increased SBP in WT mice (103�2 mm Hg forWT control versus 112�3 mm Hg for WT DOC mice;

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Figure 1. Macrophage recruitment and collagen deposition at 8 days. Treatment groups as follows: WT CON, untreated WT mice; WT DOC, WTmice treated with DOC for 8 days; KO CON, untreated macrophage-specific MR-null mice; KO DOC, macrophage-specific null mice treated withDOC for 8 days. Values are mean�SEM; n�8. A, Average number of FA/11-positive macrophages in heart tissue at 8 days. DOC treatment for 8days significantly increased the number of infiltrating FA/11-positive macrophages in WT (WT DOC) and macrophage MR-null mice (KO DOC) vsuntreated mice (*P�0.05 vs WT CON and KO CON for each). B, Cardiac collagen. DOC treatment for 8 days did not significantly alter cardiac colla-gen content in WT (WT DOC) or macrophage MR-null mice (KO DOC).

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Figure 2. mRNA levels for MCP-1 and PAI-1 at 8days, relative to 18S rRNA. Treatments are as forFigure 1. A, MCP-1, DOC treatment for 8 days sig-nificantly increased MCP-1 mRNA levels in WT (WTDOC) and macrophage MR-null mice (KO DOC) vsuntreated mice (*P�0.05 vs WT CON and KOCON); B, PAI-1; DOC treatment for 8 days signifi-cantly increased PAI-1 mRNA in WT (WT DOC) butnot in KO DOC. Data represent the average of 2separate reverse-transcription and PCR experi-ments. *P�0.05.

Rickard et al Macrophage MR Knockout and Cardiac Fibrosis 3



P�0.05; Figure 5A), whereas the specific deletion of MRsfrom macrophages attenuated this effect (103�1 mm Hg forKO DOC, not different from 100�2 mm Hg for KO controlmice; Figure 5A). DOC induced a further increase in SBP at8 weeks in WT mice (103�3 mm Hg for WT control versus121�3 mm Hg for WT DOC mice; P�0.05; Figure 5B),whereas, at 8 weeks, SBP in macrophage MR-null mice was109�3 mm Hg for KO DOC mice, not significantly differentfrom 101�2 mm Hg for KO CON (P�0.06; Figure 5B).

DiscussionThe present study shows that selective deletion of the MRsfrom the monocyte/macrophage cell lineage protects againstchronic mineralocorticoid/salt-induced cardiac fibrosis andincreased SBP after DOC administration for 8 weeks. Shorttime-course studies (8 days) investigating potential mecha-nisms underlying these responses revealed altered basalexpression of known proinflammatory/profibrotic genes re-stored to baseline at 8 weeks except for TGF-�1. Takentogether these studies demonstrate an important role formacrophage MR signaling in the maintenance of normalmacrophage phenotype and function and for the developmentof the cardiovascular sequelae of inappropriate mineralocorticoid-salt status. In support of these data, we have shown previouslythat the vascular and interstitial inflammatory and fibroticresponses to DOC/salt are MR specific in that the effects ofDOC are not altered by administration of GR antagonists.28

Cardiac collagen deposition is increased in experimentalanimals after DOC/salt treatment for periods of 6 to 8weeks.4,31,32 Tissue remodeling in this and similar models(angiotensin II/salt administration) has been clearly corre-lated with reduced cardiac function, although the present

study is focused on the mechanisms of tissue remodeling ratherthan changes in cardiac parameters. The number of macrophagesrecruited at 8 days and 8 weeks and the onset of fibrosis at 8weeks are consistent with previous rat studies of DOC/salt-induced cardiac fibrosis.4,5 We note, however, that, whereassome markers (eg, endothelial NO synthase) were regulatedin our previous rat studies by DOC-salt treatment, this wasnot always the case for the mice in the present study.Although our data are generally consistent with other reportsof DOC/salt treatment in mice, they also highlight somespecies differences.

Role of MRs in Macrophage Physiologyand PathophysiologyGiven that monocytes/macrophages express MRs but not11�-hydroxysteroid dehydrogenase type 2,18,19 these recep-tors will be predominantly occupied by endogenous glucocor-ticoids. A number of previous studies have suggested differ-ential effects of corticosteroids on macrophages and similarcells types, in particular, microglia, where very low doses ofcorticosteroids (1 nM) promote expression of inflammatoryindices, whereas higher doses (100 nM) lower expression ofthe same factors,33 reflecting MR-only occupancy at thelower concentrations and GR occupancy at higher levels. Thispattern of response contrasts with classic epithelial responsesfor MRs but is consistent with other nonepithelial, MR-expressing tissues, including cardiac myocytes and specificnuclei in the brain34–36; it is well accepted that, in nonepithe-lial tissues, glucocorticoids normally do not mimic the effectsof aldosterone mediated by the MRs but antagonize responsesto coadministered aldosterone.5 Our data for markers ofinflammation at 8 days strongly suggest that, as described for

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B Net collagen levels at 8 weeksFigure 3. Macrophage recruitment and colla-gen deposition at 8 weeks. Treatments are asfor Figure 1. Values are mean�SEM; n�8. A,Average number of FA/11-positive macrophagesin heart tissue at 8 weeks. DOC treatment for 8weeks increased the number of infiltratingFA/11-positive macrophages in WT (WT DOC)and macrophage MR-null mice (KO DOC) vsuntreated mice. B, Cardiac collagen. DOC treat-ment for 8 weeks significantly increased cardiaccollagen content in WT mice (WT DOC) but notmacrophage MR-null mice (KO DOC). *P�0.05vs WT CON and KO CON.

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Figure 4. Expression of genes associated withinflammation, oxidative stress, and tissue remodel-ing, relative to 18S rRNA, at 8 weeks. Treatmentsare as for Figure 1. A, PAI-1. B, TGF-�1. DOC treat-ment for 8 weeks increased PAI-1 mRNA in WT (WTDOC) and macrophage MR-null mice (KO DOC) vsuntreated controls. Deletion of the MR from macro-phages reduced baseline mRNA levels of TGF-�1 at8 weeks (WT CON vs KO CON). *P�0.05 vs WTCON and KO CON.




an increasing number of tissues, MRs may not only play animportant role in regulating basal functions previously as-cribed solely to the GR, but that the role of MRs ininflammation is in direct contrast to that of GR.

Macrophages and FibrosisThe recruitment of macrophages is clearly important for theonset of the cardiac remodeling processes and the develop-ment of fibrosis, as indicated by studies in osteopontin nullmice and MCP-1 null mice. In these animals, a markedlyreduced monocyte/macrophage infiltration in cardiac andrenal disease models is accompanied by a substantial reduc-tion in the fibrosis37,38; in contrast, our data show no tissueremodeling with normal macrophage recruitment. The pro-tection from tissue fibrosis observed in the MRflox/flox/Ly-sMCre/� mice is evidence for a direct role for MR activationin macrophage function.

Our data show that MR signaling in macrophages isrequired to elicit a full fibrotic response in the heart. Macro-phages play a key role in initiating fibroblast differentiationinto myofibroblasts, important collagen-producing cells, viasecretion of profibrotic stimuli, including angiotensin II,TGF-�, and cytokines.39–41 Examination of a panel of mark-ers for inflammation and tissue remodeling showed signifi-cant induction of MCP-1, macrophage number, NOX2, andPAI-1 at 8 days of DOC treatment in the WT mice; increasedNOX2 and PAI-1 expression were blocked by loss of MRsignaling in macrophages, consistent with a role for inflam-mation and activated macrophages in the early stages of thepathology. PAI-1 levels were, however, increased at 8 weeksin the MRflox/flox/LysMCre/� mice, the reason for whichremains to be explored. Moreover, a key finding at 8 days wasthat, for most markers, the baseline expression of mRNA wasconsistently significantly lower in MRflox/flox/LysMCre/�

mice. The combination of lower levels of marker expressionmay represent a loss of the classic activation phenotype (M1)in both the infiltrating and resident macrophages.41,42

Whether macrophages in these mice are able to fully differ-entiate into one or both of the classic polarized phenotypesremains to be determined.

A second key finding in the current study is that there is amismatch between the normal recruitment of macrophagesand loss of the fibrotic response. These data indicate that MRsignaling in other cell types (ie, endothelial cells, vascularsmooth muscle cells, and cardiac myocytes) remains intact,

enabling normal recruitment of macrophages to the myocar-dium; in contrast, the ability of macrophages to mount anormal inflammatory response to DOC/salt-mediated tissueinjury is lost. This observation is important in that it indicatesthat the initial tissue response to DOC/salt, including macro-phage recruitment, is mediated by a cell type(s) other than themacrophage. The cardiovascular injury response and macro-phage recruitment have been well described by our laboratoryand others and appear to involve, at least in part, 11�-hydroxysteroid dehydrogenase type 2–protected MRs, asfound in vascular smooth muscle cells and endothelial cells.Analysis of remodeling and inflammatory responses afterselective deletion of the MRs from the other cell types in theheart will be informative in this regard.

Macrophages and SBP: Regulation byInflammatory CellsThe unexpected finding that loss of MR signaling in macro-phages blocked the DOC-induced rise in SBP suggests a rolefor inflammatory cells in blood pressure regulation. In thecurrent study, the magnitude of response to DOC/salt admin-istration at 8 weeks is less than that demonstrated previouslyfor rat studies (�180 to 200 mm Hg at 8 weeks). Althoughthis may reflect the fact that C57Bl6 mice are less susceptibleto hypertension than some other strains, eg, 129/sv mice,43,44

the data are consistent with previous mouse studies usingDOC pellets over 6 to 8 weeks.24,25 Vascular inflammation,however, does not necessarily result in elevated blood pres-sure; eg, overexpression of endothelin 1 in endothelial cells ischaracterized by increased vascular inflammation but not byincreased SBP.45

Although the MR-mediated inflammatory response ap-pears to be involved in the genesis of the hypertension seen inthis model, numerous studies have shown that cardiac fibrosisin the DOC/salt model is not secondary to hypertension.5,46 Inaldosterone/salt-treated rats, increased blood pressure wasblocked by intracerebroventricular infusion of the MR antag-onist RU28318, despite volume expansion consequent to theepithelial actions of administered aldosterone.46 Subse-quently, in DOC/salt-treated rats infused with RU28318intracerebroventricular, the cardiac fibrosis response to sys-temic DOC/salt was equivalent to that in intracerebroventric-ular vehicle–infused rats, despite no elevation of bloodpressure.5 Moreover, it is also well accepted that equivalenttissue remodeling and inflammatory responses occur not only

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Figure 5. SBP determined by tail-cuff pleth-ysmography at 4 and 8 weeks. Treatmentsare as for Figure 1. DOC treatment for 4and 8 weeks significantly increased SBP inWT mice (WT DOC) vs untreated (WT CON;no significant differences were seen for KOmice given DOC/salt at either time point vsKO CON and WT CON. *P�0.05 vs WTCON, KO CON, and KO DOC.




in the left ventricle but also in the right ventricle, which is notsubject to changes in systemic blood pressure,4,5,7,11 and thatsubpressor doses of MR antagonists provide marked cardio-vascular protection.1,2,47 Thus, the fact that the macro-phage-MR null mice were protected from elevated SBPsuggests that the hypertension typically seen in the DOC/saltmodel may have a macrophage component in its etiology.

Recently, a critical role for T cells has been demonstratedin the angiotensin II/salt and DOCA/salt models.48 Replace-ment of T- and B-cell populations in RAG1�/� (T- and B-cellnull) mice, which are resistant to angiotensin II/salt andDOCA/salt-induced hypertension, identified T cells as thecritical cell type for restoration of the hypertensive pheno-type. It is also clear from these studies that low-gradeinflammatory changes in the adventitia and perivascularspaces of resistance vessels can regulate blood pressure. Giventhat subpopulations of T cells play an important role in theactivation of macrophages, it remains to be determined in thepresent study whether the MR null macrophages remain respon-sive to T cells.

PerspectivesMice in which MRs have been selectively deleted frommacrophages show baseline differences in gene expressioncompared with WT mice, evidence for a putative role ofglucocorticoid-bound MRs in maintaining normal macro-phage function. These mice also show cardiovascular protec-tion from the administration of DOC/salt, consistent with adistinct role for these receptors in the resultant phenotype inWT mice. Specifically, our data show that mice with selectivedeletion of macrophage MRs do not, as anticipated, respondto DOC/salt by increasing cardiac inflammation and fibrosis.Interestingly, the effect of macrophage-MR deletion extendedto the SBP response, which, in contrast to WT mice, was notdifferent from control mice, suggesting that MR signaling inmacrophages may contribute to blood pressure responses.Although elevated blood pressure in response to DOC/salttreatment is commonly held to reflect volume expansion,central and vascular mechanisms have also been shown toplay key roles. Thus, the present studies suggest critical rolesfor macrophage MRs under basal conditions, in terms ofmacrophage function, and in determining the inflammatoryand fibrotic responses to DOC/salt. They also suggest ahitherto-unexpected role for macrophage MRs in the hyper-tensive effects of DOC/salt by mechanisms that await furtherexploration.

AcknowledgmentsWe are grateful to Pfizer Inc for the generous gift of MRflox/flox miceand to Prof Celso Gomez-Sanchez (University of Mississippi) for thekind gift of the MR 1-18 antibody. We thank Dr David Nikolic-Paterson (Monash University) for his constructive input into thepreparation of this article.

Sources of FundingThis work is supported by grant 388914 from the National Healthand Medical Research Council of Australia. A.J.R. is supported by aMonash Graduate Scholarship.

DisclosuresJ.W.F. has received consulting fees from Merck, Pfizer, Sankyo,Lilly, Schering-Plough, Bayer, and CBio. P.J.F. has received con-sulting fees from Merck and lecture fees from Bayer, Pfizer, andNovartis. M.J.Y., P.J.F., and J.W.F. have been the recipients of aprevious research grant from Pfizer Inc and M.J.Y. and J.W.F. fromMerck. The present study does not relate to these activities.

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1

Supplementary Information Title: Deletion of mineralocorticoid receptors from macrophages protects against DOC/salt-induced cardiac fibrosis and hypertension. Amanda J. Rickard1,3, James Morgan1, Greg Tesch2, John W. Funder1, Peter J. Fuller1 and Morag J. Young1,3,4. 1. Prince Henry's Institute of Medical Research 2. Department of Nephrology, Monash Medical Centre, Monash University 3. Department of Physiology, Monash University 4. Address for correspondence: Dr Morag Young Prince Henry's Institute of Medical Research. P.O. Box 5152, Clayton 3168, Australia. Ph +61 3 9594 4286 Fax +61 3 9594 6125 [email protected]



2

Detailed methods Experiments were performed according to the NHMRC of 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. Generation of monocyte/macrophage MR null mice Deletion of the MR in monocytes/macrophages was achieved by breeding mice harboring the MRflox allele (kindly provided by Pfizer Inc.) with mice expressing Cre recombinase under the control of the myeloid lineage-specific promoter, lysozyme M (LysM). The LysM gene is expressed in all cells of the myeloid lineage including monocytes, macrophages, neutrophils, some dendritic cells and T cells. All mice used for breeding tissue-selective knockout of the MR were on the C57bl/6J background. The presence of the MRflox/flox and Lysozyme M Cre transgene was determined by PCR analysis of genomic DNA from tail tips. Primers are listed in Supplementary table S1. Validation of MR deletion from the myeloid lineage Western blot detection of macrophage MR was performed on cultured bone marrow cells extracted from the femur and tibia of freshly killed wild type and MRflox/flox/LysMCre/- mice. Cells were cultured in 6 well plates with DMEM containing 20% L-cell conditioned media, 10% fetal calf serum, 10ng/mL rm-MCSF and antibiotics (penicillin/streptomycin). Purified bone marrow macrophages (>97% F4/80+) were obtained after 6 days in culture and 2 adherence steps (day 0 and day 3). On day 6, one well/mouse was lysed directly in 200uL of lysis buffer (50mM Tris-HCL pH 8.0, 10nM β-mercaptethanol, 100mM KCl, 1% NP-40, 1:200 protease inhibitor, 50mM NaF and 50mM β-glycerophosphate). After 10 min on ice, the sample was centrifuged, the lysate mixed with an equal volume of 2xSDS reducing sample buffer and heated for 5 min at 99C. For the western blot, lysate samples from macrophages (30uL) and MR-transfected SF-9 cells (5uL) were run on a 4-20% gradient SDS-PAGE gel at 30mA for 2 hr. Gels were then electroblotted onto nitrocellulose at 0.6A for 6 hr in tris-glycine transfer buffer with 10% methanol and 0.02% SDS. Blots were rinsed in tris-saline buffer (TSB) and incubated for 2 hr in blocking solution (1:1 Lycor block/TSB) and incubated overnight at 4C with MR1-18 mAb hybridoma supernatant (1:250; a gift from Professor Celso Gomez-Sanchez) in blocking solution with 0.05% Tween-20. After washing 5 times with TBS/0.1% Tween-20, blots were then incubated in darkness for 1 hr with donkey anti-mouse IR-800 (1:5000) in blocking solution with 0.05% Tween-20, washed a further 5 times with TBS/0.1% Tween-20 and then stored in TBS at 4C until scanning. Blots were visualized on the LI-COR IR-scanner (LI-COR, NA) and viewed with Odyssey software (LI-COR, NA). Western blot analysis was performed twice on independent samples. DOC/salt model of cardiac fibrosis Mice approximately 8 weeks of age (n=10/group) were anesthetized with Ilium Xylazil (8mg/kg; Troy Laboratories, NSW, Australia) plus Ketamine (60mg/kg; Pfizer Pty. Ltd., N.S.W. Australia) for uninephrectomy via dorsal incision. All mice were subsequently maintained on standard chow with 0.9% sodium chloride (NaCl) plus 0.4% potassium chloride (KCl) solution to drink. Mice of each genotype were randomly assigned to one the following treatments resulting in a total of 8 groups of mice: control treatment 8 days, deoxycorticosterone (DOC) treatment 8 days and control treatment 8 weeks and DOC treatment for 8 weeks. One cohort of a total of 80 animals was the subject of the current studies. Mice receiving DOC treatment (Sigma-Aldrich, St Louis, MO. USA) were given a subcutaneous 7 mg 21 day release pellet made in house, which was replaced every three weeks throughout the eight week study1, 2.



3

Systolic blood pressure Systolic blood pressure (SBP) was measured by tail-cuff plethysmography (ITTC Life Science, Woodland Hills, CA, USA), by an experienced operator using a procedure adapted from the ITTC Life Science manual. Mice were trained biweekly for 3 weeks prior to SBP recording. On the day of measurement, they were acclimated to the preheated chamber (29°C) for 15min; pressure was then read over 3 consecutive manual inflation-deflation cycles. If the pressure readings differed by more than 5 mmHg they were discarded, the mice allowed to rest and the procedure repeated until 3 consistent readings were obtained. Tissue collection Animals were killed by CO2 in air at 8 days or 8 weeks with an arterial blood sample and the heart

collected and stored for analysis. The heart was immediately sectioned in to 2 parts. The upper half of the heart was immersion-fixed in 4% paraformaldehyde for histology, and the apex snap frozen in liquid nitrogen for RNA extraction and quantitative PCR. Plasma radioimmunoassay for aldosterone Serum was isolated from arterial blood and aldosterone levels were determined by radioimmunoassay (MP Biomedicals, NSW Australia). The sensitivity of the assay was 9.1 pg/mL and the intra-assay variability 5.9%. Histological analysis The extent of fibrosis was determined by dewaxing and staining 5m heart sections with 0.1% Sirius red (Sigma-Aldrich, St Louis, MO) in saturated picric acid (BDH AnalaR, UK) and quantifying the collagen content with the Analytical Imaging Station (AIS) software package (Version 4.0 Beta 1.5, Imaging Research Inc. Canada), as previously described 3. Immunohistochemistry The number of infiltrating macrophages was determined by immunohistochemistry using the primary antibody FA/11, a monoclonal antibody against mouse CD68+ monocytes/macrophages (1:200 dilution in 1%TBS) (Serotec, UK), on 5m heart sections as previously described 3. Infiltrating CD68-positive macrophages were quantified by an optical dissector method 4, which provides a value for the average number of macrophages per frame (826890μm²) rather than per section; more than 80 CD68-positive macrophages were counted for each mouse to allow accurate statistical comparisons between groups. One section per heart was analyzed where the investigator was blinded to the treatment and genotype of each sample. Reverse Transcription-PCR Total mouse heart RNA was prepared with Ultraspec (Fisher Scientific, Pittsburgh, PA). First strand

cDNA synthesis from 500ng total RNA was performed following DNAase treatment with avian myeloblastosis virus (AMV) reverse transcriptase (Roche, Indianapolis, IN) and priming with random hexamers. PCR reactions were carried out with the primer sets listed in Supplementary Table S1. Expression levels were normalized to those of the 18S ribosomal subunit (V01270). To validate the

real-time PCR protocol, standard curves were generated for each gene by 1:10 serial dilutions of previously prepared standards. Standards were diluted from 10 to 0.1 pg/µl for 18S and from 500 to

0.5 fg/µl for other transcripts. Real-time PCR amplification was performed on the LightCycler (Roche, Indianapolis, IN) using SYBR Green reaction mix (Roche, Indianapolis, IN). Samples of cDNA for 18S rRNA analysis were diluted 1:20 in water immediately before use and all remaining samples analyzed undiluted. Relative amounts of mRNA were calculated by normalizing values to 18S rRNA values. Data presented are the average of 2 separate RT and PCR experiments.



4

Statistics All data were analyzed by one-way analysis of variance (Prism statistical software package, Graph Pad version 5.0A, USA), and Bonferroni’s comparisons test applied to identify significant effects between groups; mean differences were considered significant at p0.05. All data are reported as means ±SEM. One cohort of animals (10 per group) was the subject of the current study.



5

Supplementary Table S1. Primers used for genotyping and analysis of gene expression. Gene name Accession number Forward Primer Reverse Primer

MR genotyping NM_001083906 5’-ttctttccccagctccacctttacga-3’ 5’-agcaagagacaactgcagcgtttta-3’ 5’-atgtggaatgtgtgcgaggccagag-3’

LysM Cre genotyping

NM_017372 (Lys M mRNA)

5’-cccagaaatgccagattacg-3’ 5’-cttgggctgccagaatttctc-3’ 5’-ttacagtcggccaggctgac-3’

MCP-1 NM_011333 5’-agcaccagccaactctcact-3’ 5’-tcattgggatcatcttgctg-3’

COL1 NM_007742 5’-cctcagggtattgctggacaac-3’ 5’-ttgatccagaaggaccttgtttg-3’

CTGF NM_010217 5’-tgacccctgcgacccaca-3’ 5’-tacaccgacccaccgaagacacag-3’

PAI-1 NM_008871 5’-agtctttccgaccaagagca-3’ 5’-gccgaaccacaaagagaaag-3’

TGFβ1 NM_011577 5’-tgcgcttgcagagattaaaa-3’ 5’-ctgccgtacaactccagtga-3’

eNOS NM_008713 5’-caggacaacctcatccctgt-3’ 5’-ctggccttctgctcattttc-3’

Nox2 NM_007807 5’-actccttgggtcagcactgg-3’ 5’-gttcctgtccagttgtcttcg-3’

G6PD Z11911 5’-actcacattccacccaggag-3’ 5’-gggcagtgactcaccgttat-3’

iNOS NM_010927 5’-caccttggagttcacccagt-3’ 5’-accactcgtacttgggatgc-3’

COL III NM_009930 5’-gtccacgaggtgacaaaggt-3’ 5’-gatgcccacttgttccatct-3’

Fibronectin NM_010233 5’-aatggaaaaggggaatggac-3’ 5’-ctcggttgtccttcttgctc-3’

p22phox EU791539 5’-aaagaggaaaaaggggtcca-3’ 5’-taggctcaatgggagtccac-3’



6

Supplementary Results Table S2. Index of cardiac and renal hypertrophy at 8 days and 8 weeks (mg/g; means±SEM).

Tissue WT Control WT DOC MRKO Control MRKO DOC Heart weight/body weight 8 days

5.9±0.1 6.0±0.1 5.8±0.1 6.2±0.2

Kidney weight/body weight 8 days

9.4±0.1 10.0±0.3 9.7±0.2 10.2±0.3

Heart weight/body weight 8 weeks

6.0±0.4 6.5±0.6 5.8±0.3 7.0±0.5

Kidney weight/body weight 8 weeks

8.8±0.5 9.2±0.7 10.0±0.4 10.1±0.9

1. Artunc F, Amann K, Nasir O, Friedrich B, Sandulache D, Jahovic N, Risler T, Vallon V,

Wulff P, Kuhl D, Lang F. al. Blunted DOCA/high salt induced albuminuria and renal tubulointerstitial damage in gene-targeted mice lacking SGK1. J Mol Med. 2006;84:737-746.

2. Hartner A, Cordasic N, Klanke B, Veelken R, Hilgers KF. Strain differences in the development of hypertension and glomerular lesions induced by deoxycorticosterone acetate salt in mice. Nephrol Dial Transplant. 2003;18:1999-2004.

3. Young M, Funder JW. Eplerenone, but not steroid withdrawal, reverses cardiac fibrosis in deoxycorticosterone/salt-treated rats. Endocrinology. 2004;145:3153-3157.

4. Wreford NG. Theory and practice of stereological techniques applied to the estimation of cell number and nuclear volume in the testis. Microsc Res Tech. 1995;32:423-436.



Supplementary Figure S1

1 2 3 4 5

a. Representative genotyping gel

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b. MR expression in macrophages

150kD

100kD1 2 3 4 5

Supplementary Figure 1. a. Genotyping approach: detecting MR floxed alleles. Lanes 1-3 represent DNA heterozygous for the insertion of the Lox p sites and lanes 4-5 are homozygous for Lox psites and lanes 4 5 are homozygous for Lox p insertion; b. verification of deletion of the MR from bone marrow derived macrophages: western blot for the MR using an antibody raised to the MR N terminus. A band was detected in the wild type sample of the correct size for MR (Lane 4), but not in the Cre recombinase expressing mice (Lane 5). Purified human MR from an over expression systemPurified human MR from an over expression system was used as positive control (Lane 2). Lane 1 molecular weight marker; Lane 2 MR-transfected cells; Lane 3, wild type kidney; Lane 4, wild-type bone marrow macrophages; Lane 5 MR-null bone marrow macrophages.



WT CON WT DOC KO CON KO DOC

red

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Supplementary Figure S2. Photomicrographs showing examples of Picrosirius red staining, FA11 and CD11b macrophage immunostaining. Treatment groups as follows: WT CON untreated wild type mice; WT DOC wild type mice treated with

CD

11b

follows: WT CON, untreated wild-type mice; WT DOC, wild-type mice treated with deoxycorticosterone for 8 days; KO CON, untreated macrophage specific MR-null mice; KO DOC macrophage specific null mice treated with deoxycorticosterone for 8 days. Scale bar — represents 200nM. Arrows indicate macrophages in lower panels.



1.5

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Supplementary Figure S3. Expression of genes associated with macrophage recruitment, inflammation, oxidative stress and tissue remodeling at 8 days.

ona. Osteopontin

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Supplementary Figure S3. mRNA levels relative to 18S rRNA for a. pp y gOsteopontin, b. connective tissue growth factor (CTGF), c. Procollagen I (Col I), d. Endothelial nitric oxide synthase (eNOS), e. Glucose-6-phosphate dehydrogenase (G6PD), f. p22phox, g. NADPH containing oxidase 2 (NOX2) and h. transforming growth factor-1 (TGF-1) . Treatment groups as follows: WT CON, untreated wild-type mice; WT DOC, wild-type mice treated with deoxycorticosterone for 8 days; KO CON, untreated macrophage specific MR-null mice; KO DOC macrophage specific null mice treated with deoxycorticosterone for 8 days. Values are mean ± SEM; n=8. DOC treatment for 8 days significantly increased NOX2 mRNA levels in WT (WT DOC) but not macrophage MR null miceincreased NOX2 mRNA levels in WT (WT DOC) but not macrophage MR-null mice (KO DOC) compared with untreated mice (* p<0.05 vs. WT CON and KO CON).Specific deletion of MR from macrophages significantly reduced baseline mRNA levels of: osteopontin, CTGF, Col I, eNOS and TGF-1, * p<0.05 vs. WT CON . Data represent the average of 2 separate RT and PCR experiments.



* *

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Supplementary Figure s4. Expression of genes associated with macrophage recruitment, inflammation, oxidative stress and tissue remodeling at 8 weeks

a. iNOS

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• Supplementary Figure S4. Expression of genes associated with inflammation, pp y g p g ,oxidative stress and tissue remodeling, relative to 18S rRNA, at 8 weeks. Treatments are as for Figure 1.a. inducible nitric oxide (iNOS), b. connective tissue growth fa)ctor (CTGF), c. osteopontin, d. endothelial nitric oxide synthase (eNOS), e. Glucose-6-phosphate dehydrogenase (G6PD), f. procollagen I (COL1), g. macrophage chemoattractant protein I (MCP-1), h. NADPH oxidase 2 (NOX2). DOC treatment for 8 weeks increased CTGF mRNA in WT (WT DOC) and macrophage MR-null mice (KO DOC) compared with untreated controls. Values for osteopontin for the KO DOC mice were significantly increased over KO CON whereas the equivalent changes in the WT mice did not reachover KO CON whereas the equivalent changes in the WT mice did not reach significance. iNOS was significantly increased in WT DOC mice vs WT CON whereas for KO DOC vs KO CON values did not reach significance. * p<0.05

•



Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

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