associate editor: rhian m. touyz emerging roles of the ... · myocardial infarction arrhytmia...

1521-0081/68/1/49–75$25.00 http://dx.doi.org/10.1124/pr.115.011106PHARMACOLOGICAL REVIEWS Pharmacol Rev 68:49–75, January 2016Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: RHIAN M. TOUYZ

Emerging Roles of the Mineralocorticoid Receptor inPathology: Toward New Paradigms in Clinical

PharmacologyF. Jaisser and N. Farman

INSERM UMR 1138 Team 1, Cordeliers Research Center, Pierre et Marie Curie University, Paris, France (F.J., N.F); and UniversityParis-Est Creteil, Creteil, France (F.J.)

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50II. Mineralocorticoid Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

A. Steroidal Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51B. Nonsteroidal Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

III. Mineralocorticoid Receptor and Cardiac Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53A. Mineralocorticoid Receptor and Extracellular Matrix Remodeling . . . . . . . . . . . . . . . . . . . . . . . . 54B. Mineralocorticoid Receptor and Electrophysiological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . 54C. Cardiac Effects of Noncardiomyocyte Mineralocorticoid Receptor . . . . . . . . . . . . . . . . . . . . . . . . . 55D. Mineralocorticoid Receptor and Cardiopathy of Metabolic Origin . . . . . . . . . . . . . . . . . . . . . . . . . 55

IV. Mineralocorticoid Receptorand Vascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56A. Role of the Endothelial Mineralocorticoid Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56B. Role of the Vascular Smooth Muscle Mineralocorticoid Receptor. . . . . . . . . . . . . . . . . . . . . . . . . . 57C. Role of the Mineralocorticoid Receptor in the Crosstalk between Endothelium

and Vascular Smooth Muscle Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58D. Mineralocorticoid Receptor Affecting Microcirculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

V. Mineralocorticoid Receptor and Metabolic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58VI. Mineralocorticoid Receptor and Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

A. Experimental Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60B. Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

VII. Mineralocorticoid Receptor and Ocular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61VIII. Mineralocorticoid Receptor and Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62IX. Paradoxical Effects of Mineralocorticoid

Receptor Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63X. Common Mechanisms Involved in Pathologic Consequences Of Mineralocorticoid

Receptor Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63A. Mineralocorticoid Receptor is a Regulator of Ion Channels in Multiple Tissues . . . . . . . . . . . 63B. Mineralocorticoids Induces Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64C. Mineralocorticoid

Receptor Activation Leads to Fibrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64D. Mineralocorticoid Receptor and Inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65E. Mineralocorticoid Receptor and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

XI. Dysregulations of Mineralocorticoid Receptor Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66A. Regulation of Mineralocorticoid Receptor Expression Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66B. Mechanisms Modulating Mineralocorticoid Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, the F-CRIN INI-CRCT: cardiovascularand Renal Clinical Trialists and the FP7-funded COST ADMIRE (BM1301) networks.

Address correspondence to: Frederic Jaisser, INSERM UMR 1138, Centre de Recherche des Cordeliers, Université Pierre et MarieCurie, 15 rue de l’Ecole de Médecine, 75006 Paris, France. E-mail: [email protected]

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XII. Conclusion and Open Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Abstract——The mineralocorticoid receptor (MR) andits ligand aldosterone are the principal modulators ofhormone-regulated renal sodium reabsorption. Inaddition to the kidney, there are several other cells andorgans expressing MR, in which its activation mediatespathologic changes, indicating potential therapeuticapplications of pharmacological MR antagonism.Steroidal MR antagonists have been used for decades tofight hypertension and more recently heart failure. Newtherapeutic indications are nowarising, andnonsteroidalMR antagonists are currently under development. Thisreview is focused on nonclassic MR targets in cardiac,vascular, renal,metabolic, ocular, andcutaneousdiseases.

The MR, associated with other risk factors, is involved inorgan fibrosis, inflammation, oxidative stress, and aging;for example, in the kidney and heart MR mediateshormonal tissue-specific ion channel regulation. Geneticand epigenetic modifications of MR expression/activitythat have been documented in hypertension may alsopresent significant risk factors in other diseases and besusceptible to MR antagonism. Excess mineralocorticoidsignaling, mediated by aldosterone or glucocorticoidsbinding, now appears deleterious in the progression ofpathologies that may lead to end-stage organ failure andcould therefore benefit from the repositioning ofpharmacological MR antagonists.

I. Introduction

The steroid aldosterone is the mainmineralocorticoidhormone; it is synthetized in the glomerular zone of theadrenal cortex in response to hyperkaliemia or sodiumdepletion as the end-point of activation of the renin-angiotensin system (Rossier et al., 2015). Local pro-duction of aldosterone may also occur in peripheraltissue (Bader, 2010; Taves et al., 2011). Aldosteronestimulates renal sodium reabsorption and potassiumexcretion, therefore playing amajor role in the control ofblood pressure and extracellular volume homeostasis.We will briefly summarize the main elements of thepathways involved in aldosterone action, because sev-eral recent reviews (Pearce et al., 2015; Penton et al.,2015; Rossier et al., 2015) have documented in detail themolecular and cellular events involved in aldosteroneeffects in the distal tubule, the connecting tubule, andthe collecting duct (referred as aldosterone-sensitivedistal nephron). Aldosterone binds to the mineralocor-ticoid receptor (MR), a ligand-dependent transcriptionfactor belonging to the nuclear receptor superfamily(Fuller and Young, 2005; Viengchareun et al., 2007;Zennaro et al., 2009; Fuller et al., 2012). The MR isexpressed in a number of tissues beside the aldosterone-sensitive distal nephron. Figure 1 illustrates MR ex-pression in a variety of human tissues.The aldosterone ligand-MR receptor complex binds to

glucocorticoid response elements within the promotorregion of aldosterone-induced (or repressed) genes tomodulate their transcription and resulting in theexpression and activation of sodium transporters or

channels. In a typical mineralocorticoid target cell, suchas the renal collecting duct principal cell, sodium ionsenter the cell through the amiloride-sensitive apicalsodium channel (ENaC, for epithelial sodium channel),and are extruded into the peritubular space by thesodium pump (Na-K-ATPase) located in the basolateralmembrane (Pearce et al., 2015; Rossier et al., 2015). Theapical Na+ channels and basolateral Na+ pumps and K+

channels are coordinately activated and upregulated bymembrane "cross-talk" in the presence of aldosterone(Harvey, 1995), leading to a sustained increase intransepithelial sodium reabsorption (Pearce et al.,2015; Penton et al., 2015; Rossier et al., 2015). TheMR binds both aldosterone and glucocorticoid hormoneswith similar high affinity (in the nanomolar range);because glucocorticoids are 100- to 1000-fold moreabundant than aldosterone in the plasma, permanentoccupancy of the MR by glucocorticoid hormones mayoccur. In the kidney, this should induce permanentmaximal sodium retention, independent of plasmaaldosterone levels. The main mechanism ensuring invivo mineralocorticoid selectivity involves coexpressionof MR and the enzyme 11b hydroxysteroid dehydroge-nase type 2 (11HSD2), that metabolizes circulatingglucocorticoid hormones (cortisol in humans, corticoste-rone in rodents) into inactive 11-dehydro-derivatives(cortisone, 11-dehydrocorticosterone) with very lowaffinity for the MR (Farman and Rafestin-Oblin, 2001;Huyet et al., 2012; Odermatt and Kratschmar, 2012).Other mechanisms are important for mineralocorticoidselectivity (i.e., the events that explain the higher

ABBREVIATIONS: AF, atrial fibrillation; AngII, angiotensin II; CIN, cyclosporine-induced nephrotoxicity; CKD, chronic kidney disease;CSCR, central serous chorioretinitis; CV, cardiovascular; DAMP, danger-associated molecular pattern molecule; EDHF, endothelium-derivedhyperpolarizing factor; ENaC, epithelial sodium channel; eNOS, endothelial NO synthase; ESRD, end-stage renal disease; ET1, endothelin 1;ET1RB, endothelin 1 receptor type B; Gal3, galectin 3; HCN, hyperpolarization-activated cyclic nucleotide; HF, heart failure; HFD, high-fatdiet; 11HSD2, 11b hydroxysteroid dehydrogenase type 2; IL, interleukin; Ito, transient outward K+ current; KO, knockout; MDR, multidrugresistance; MetS, metabolic syndrome; MI, myocardial infarction; MR, mineralocorticoid receptor; MRA, mineralocorticoid receptorantagonist; NGAL, neutrophil gelatinase-activated lipocaline; NO, nitric oxide; RGC, retinal ganglion cells; ROS, reactive oxygen species;SHR, spontaneously hypertensive Kyoto-Okamoto strain; TNF, tumor necrosis factor; VSMC, vascular smooth muscle cells.

50 Jaisser and Farman

efficacy of MR-aldosterone versus MR-glucocorticoidcomplexes) (Farman and Rafestin-Oblin, 2001; Vieng-chareun et al., 2007; Fuller et al., 2012). They includedistinct nuclear translocation kinetics and transactiva-tion activities related to the stability of the MR depend-ing on its ligand (Hellal-Levy et al., 1999).To avoid redundancy with previous reviews (Farman

and Rafestin-Oblin, 2001; Fuller and Young, 2005;Viengchareun et al., 2007; Funder, 2009; Zennaroet al., 2009; Fuller et al., 2012; Pearce et al., 2015;Penton et al., 2015; Rossier et al., 2015), detailedinformation on the mechanism of action of aldosteroneand MR are not provided here. Nongenomic mineralo-corticoid signaling has also been reviewed recently(Thomas and Harvey, 2011; Dooley et al., 2012; Meinelet al., 2014) and will not be discussed here. An in-creasing number of publications refer to the pathologicinvolvement of MR in the brain and will not beaddressed here for space limitation (Joels et al., 2008;

Geerling and Loewy, 2009; Molinari et al., 2013; deKloet, 2014; Gomez-Sanchez, 2014).

The exponential increase in the number of reports ondiseases where MR plays a role is largely based on theevidence for therapeutic efficacy of pharmacological MRantagonism (Fig. 2).Wewill focus this review on the roleof MR in pathologies other than those related to itsimpact on the aldosterone-sensitive distal nephronepithelial cells.

II. Mineralocorticoid Receptor Antagonists

MR antagonists (MRA) can be divided into steroidaland nonsteroidal compounds (Table 1).

A. Steroidal Compounds

Spironolactone was the first steroidal MR antagonistdeveloped by Searle Laboratories (Skokie, Ill) in 1959(Menard, 2004), about 30 years before the molecularcharacterization of the mineralocorticoid receptor byArriza et al. (1987). Spironolactone was approved as adiuretic and natriuretic drug for the management ofhypertension and primary aldosteronism and later on totreat heart failure (Menard, 2004). Spironolactone is apotent competitive MR antagonist but is poorly selec-tive because it also inhibits the androgen and pro-gesterone receptors, leading to side effects such asgynecomastia, impotence, and menstrual irregularities(Kolkhof and Borden, 2012). At high concentrations itmay also interfere with the glucocorticoid receptor (GR)(Kolkhof and Borden, 2012). Canrenone is an activemetabolite of spironolactone used in some countries(Armanini et al., 2014). An injectable preparation(potassium canrenoate) is more widely available. Ofnote, progesterone is a natural MRA (Huyet et al.,2012). Eplerenone (9-11a-epoxymexrenone), a secondgeneration MRA, was developed by Ciba-Geigy (Basel,Switzerland) and launched by Pfizer (New York, NY) in2002 for the treatment of hypertension and heart failure

MR antagonism for:

HEART:heart failuremyocardial infarctionarrhytmia

KIDNEY:hypertensionischemic insultglomerular injury

IMMUNE CELLS:

ADIPOSE TISSUE:obesity

BLOOD VESSELS:vasoconstrictionendothelial dysfunctionhypertensionatherosclerosisremodelling

SKIN:epidermal atrophy

RETINA:retinal edemaneoangiogenesiscentral serous chorioretinitis

Fig. 2. Beneficial effects of MR antagonism. MR activation is involved inseveral processes in different tissues. Pharmacological MR antagonism isbeneficial in various pathologic and clinical conditions.

Fig. 1. MR expression in human tissues. MR immunodetection wasperformed with the 6G1 antibody from C. Gomez-Sanchez. (A and B)Kidney removed for tumor: cortex (A) and outer medulla (B); nuclear MRsignal is in distal tubule and collecting duct cells (star), in podocytes[arrow in (A)] and glomerular parietal epithelial cells [arrowhead in (A)].(B) Vascular MR is located in VSMC (arrow) and endothelial cells(arrowhead) of an artery (art) and vein surrounding the tumor. MR is alsofound in the nuclei of atrial cardiomyocytes (C), in subcutaneousadipocytes (D), in epidermis (epi) and hair follicle (hf) from skin (E and F),as well as in sweat glands (sg), and venous endothelium (F); also note MRexpression in capillaries surrounding the hair follicles (F). bar : 25 micrometer

The Mineralocorticoid Receptor in Pathology 51

(Menard, 2004; Kolkhof and Borden, 2012). ThisMRA ismuch more selective for MR than spironolactone, butless potent (40� less), requiring higher dosage toachieve similar MR antagonism. The different pharma-cokinetic and pharmacodynamic properties of spirono-lactone and eplerenone, however, result in a differencein efficacy in the treatment of hypertension: 100 mgeplerenone is almost 50 to 75% as potent as 100 mgspironolactone in patients with essential hypertension(Weinberger et al., 2002; Parthasarathy et al., 2011).Spironolactone is rapidly metabolized into severalactive metabolites with a half-life of about 15 hours,whereas eplerenone has no active metabolite and has ashorter half-life of less than 4 hours. The half-life of thesteroidal MRA has not been explored in disease subjects(Kolkhof and Borden, 2012), except for eplerenone inchronic kidney disease (Schwenk et al., 2015). This isimportant to consider when an MRA is selected forclinical trials. The use of MRAs that do not need to bemetabolized in active moieties for full efficacy, such aseplerenone, may be preferred when effects are expectedrelatively rapidly after administration. Short half-life isimportant to consider for safety concerns to yield a rapiddecrease of activemoieties when halting therapy in caseof toxic side effects. Racial difference in response to MRblockade may exist: African Americans with heartfailure are less responsive to the renal effect of spiro-nolactone and develop less hyperkaliemia (Cavallariet al., 2004); this is not the case with eplerenone (Flacket al., 2003). It has been reported that spironolactone (atmicromolar concentrations) inhibits aldosterone bio-synthesis (Netchitailo et al., 1985) and blocks enzymesinvolved in steroidogenesis (Penhoat et al., 1988; Yeet al., 2009). In contrast, eplerenone (1–30 mM) did notimpair basal and angiotensin II-induced cortisol andaldosterone production in human adrenocortical H295Rcells (Ye et al., 2009). Thus spironolactone and epler-enone have differential effects on steroidogenesis. Re-cently, spironolactone was reported to be effective inantitumor therapy, an effect independent of the classicmineralocorticoid pathway, requiring retinoid X recep-tor coactivation (Leung et al., 2013). This may highlightnovel aldosterone and/or MR-independent effects ofspironolactone.The “hormonal related" side effects of spironolactone

are due to its inhibitory actions on other steroid receptorssuch as the androgen and progesterone receptors.Hyperkaliemia is a potential life-threatening sideeffect of MRAs, related to their efficacy to decreaseurinary potassium excretion. Indeed these drugs are

potassium-sparing diuretics, and their “propensity” tolead to hyperkaliemia has been observed in mostclinical studies, especially when renal function is im-paired. It should be emphasized that clinically relevanthyperkaliemia (requiring discontinuation of MRA oradministration of potassium chelator) is rare. Closefollow up of kaliemia is required, especially at the onsetof MRA administration, as well as regular monitoring ofrenal function. Some of the beneficial effects of MRAmay be related to a decreased occurrence of potassiumdepletion and hypokaliemia often observed in patientswith heart failure and treated with diuretics. Neverthe-less, the risk of hyperkaliemia clearly accounts for theunderuse of this efficient therapeutic class (Albert et al.,2009; Maron and Leopold, 2010; Jaisser et al., 2011;Rossignol et al., 2012). Despite this risk, even inpatients with impaired renal function, the benefit/riskbalance is in favor of MRA administration (Rossignolet al., 2014).

Excessive/inappropriate MR activation in pathologyis often inferred from the benefits provided by MRAadministration. Is it conceivable that spironolactonecould have other targets? Some studies suggest compe-tition between spironolactone (or other MRAs) anddigitalin analogs through the Na-K-ATPase. Indeedthere is a strong structural similarity between thesedrugs. Hans Selye et al. reported in 1969 that spirono-lactone "protects the rat against the production ofmyocardial necroses and other manifestations of digi-toxin poisoning"; this pioneering work proposed spiro-nolactone as an antidote to digitonin cardiac toxicity(Selye et al., 1969). Canrenone interacts with the cellmembrane of cultured macrophages and vascularsmooth muscle, and it partially reversed the distur-bance of cation handling induced by high concentrationsof ouabain, a response observed as early as 30 minutesafter addition of drugs (Hannaert et al., 1986). A laterstudy (Finotti and Palatini, 1981) concluded that can-renone is a partial agonist of brain Na-K-ATPase at theouabain binding site. The question of direct interactionof canrenone and Na-K-ATPase has been explored (Taland Karlish, 1988): although canrenone could displaceouabain binding to partially purified sodium pumps, itwas concluded that canrenone does not react within theouabain binding site itself but rather interacts alloste-rically with this site. Because spironolactone acts onNa-K-ATPase within minutes, one can wonder whetherinteraction with the sodium pump could explain, atleast in part, the rapid effects of spironolactone (usuallyreferred as nongenomic mineralocorticoid effects). More

TABLE 1Characteristics of the mineralocorticoid receptor antagonists

MRA Generation Structure Example/Class Selectivity Potency Half-Life Tissue Distribution Tissue Specificity

I steroidal Spironolactone low high long 6 � higher in kidney noII steroidal Eplerenone high low Short (4 hours) 3 � higher in kidney noIII nonsteroidal Finerenone high high ? equal CV . kidney ?


recently, it has been reported that spironolactone canantagonize ouabain or analogs such as marinobufage-nin; these observations suggest that some of the effectsof spironolactone (reduction of experimental cardiacfibrosis) could rely on antagonism of marinobufageninthrough the Na-K-ATPase (Balzan et al., 2003; Tianet al., 2009).

B. Nonsteroidal Compounds

In the last decade, the broader therapeutic indica-tions of MRA in heart failure and possibly in kidneydiseases, as well as the underuse of classic steroidalMRAs due to the risk of hyperkaliemia has stimulatedthe search for novel MR antagonists with higherselectivity, higher potency, and, if possible, a reducedrisk of hyperkaliemia. Several nonsteroidal compoundsacting as MRAs were identified. Dihydropyridines(L-type calcium channel antagonists) act as MRAs invitro and in vivo (Kolkhof and Borden, 2012). Successfuloptimization of antagonists devoid of L-type calciumchannel antagonist properties led to several new com-pounds and derivatives. For example, the Bayer BR-4628 compound was identified as a potent and selectiveMRA. Its mode of action differs from classic steroidalMRA, acting as a bulky antagonist, leading to theprotrusion of MR helix 12 [which plays a major role inMR activation upon ligand binding (Hellal-Levy et al.,2000)]. This causes the formation of an unstable re-ceptor complex unable to recruit coregulators and israpidly degraded. Optimization of BR-4628 led to BAY94-8862 (Finerenone); this third generation MRA wasused in phase 2a clinical trials (Pitt et al., 2013) inpatients with heart failure and mild renal dysfunction.Finerenone was shown to be safe and led to lower rate ofhyperkaliemia than spironolactone, for a similar effecton the N terminal-proBNP surrogate marker (ARTS)(Pitt et al., 2013). Two clinical trials have been launchedrecently in heart failure (ARTS-HF) (Pitt et al., 2015)and in diabetic nephropathy (ARTS-DN) (Ruilope et al.,2014; Bakris et al., 2015) to further test safety andefficacy in phase 2b trials. The relative benefit ofFinerenone over the classic steroidal antagonist spiro-nolactone, regarding increased plasma potassium lev-els, may rely on a differential tissue distribution of thetwo compounds; while spironolactone (and eplerenone)accumulated three- to sixfold more in the kidney than inthe heart, the distribution of finerenone appears equiv-alent in rat heart and kidney (Kolkhof et al., 2014).Therefore, low doses may allow sufficient MR antago-nism outside of the kidney with less renal MR blockadeand reduced K+ sparing effect. Other nonsteroidalMRAs such as PF-03882845 (Pfizer) and SM-368229(Dainippon Sumitomo Pharma, Osaka, Japan), forexample, have been generated and proven to be efficientin preclinical models and are currently going throughclinical trials (Collin et al., 2014).

III. Mineralocorticoid Receptor andCardiac Diseases

The combined inhibition of receptors of vasoactivehormones (angiotensin II, aldosterone, catecholamines)that are chronically activated in heart failure (HF) arenow considered as main therapeutic tools. Severalclinical studies have demonstrated that antagonism ofMR is highly beneficial in mild to severe HF, suggestingthat an excessive activation of the MR occurs over thecourse of the disease and plays an important role in thepathophysiology of HF. The mechanisms that explainthe efficacy of MRAs in HF are likely multiple. Phar-macological blockade of MR limits the transition toheart failure in models of systolic left ventriculardysfunction (Kuster et al., 2005) and myocardial in-farction (Wang et al., 2004a; Galuppo and Bauersachs,2012) aswell as inmodels of diastolic dysfunction in rats(Ohtani et al., 2007) and mice (Di Zhang et al., 2008).Clinical trials showed unambiguously that this benefittranslates in patients with HF; the RALES, EPHESUS,and EMPHASIS trials demonstrated clear benefit ofMR antagonists in HF, reducing morbidity and mortal-ity, paving the way for broader clinical use of MRAs incardiovascular (CV) diseases (Pitt et al., 1999, 2003;Zannad et al., 2011). The benefit of MRAs now extend toearly phases of myocardial infarction (MI) (REMINDERstudy) (Montalescot et al., 2014) and possibly toHFwithpreserved ejection fraction (post hoc study of the TOP-CAT trial) (Pfeffer et al., 2015).

In these trials, the beneficial effects of MR blockadeare often observed in the absence of elevated plasmaaldosterone levels, raising the question of the nature ofthe ligand that activates the MR excessively or in-appropriately. It is generally considered that cortisol isthe main MR ligand in the heart, because of the lowlevel of the MR protecting enzyme 11 HSD2 (Funder,2009). However, this view is challenged by reportsshowing that serum aldosterone and cortisol are in-dependent predictors of increased mortality risk inheart failure (Guder et al., 2007, 2015). To assess thein vivo differential effects of aldosterone and glucocor-ticoids in heart, we compared the molecular cardiacsignature of mouse heart after infusion of mice withaldosterone or with corticosterone inmoderate amounts(fivefold increase in plasma aldosterone and twofoldincrease in corticosterone). We identified several genesthat are specifically modified by aldosterone and un-affected by glucocorticoid treatment. For example, theconnective tissue growth factor, involved in fibrosis, wasupregulated specifically by aldosterone and not byglucocorticoid (Messaoudi et al., 2013; Gravez et al.,2015). Of note, aldosterone and not corticosterone,enhanced cell cycle-related gene networks and endothe-lial cell proliferation in myocardial capillaries, reveal-ing a potential benefit for aldosterone stimulated-MRpathway to fight capillary rarefaction.


A. Mineralocorticoid Receptor and ExtracellularMatrix Remodeling

Since the pioneeringwork ofWeber and collaborators,it is now accepted that chronic MR activation isassociated with fibrosis, extracellular matrix remodel-ing and cell growth and survival (Brilla and Weber,1992; Brilla et al., 1993; Robert et al., 1994; Dooley et al.,2011). The onset of fibrosis may involve a number of celltypes including fibroblasts but also other cells prone tosecrete profibrotic factors [cardiomyocytes, vascularsmooth muscle cells (VSMC), and inflammatory cellsincluding macrophages and dendritic cells] that second-arily stimulate extracellular matrix production.Chronic coadministration of aldosterone and NaCl

in uninephrectomized rats (aldosterone-salt ordeoxycorticosterone-salt (DOCA -salt) models) stimu-lates perivascular and interstitial cardiac fibrosis in leftand right ventricles. This occurs independently of anincrease in blood pressure, because MR blockade atsubhypotensive doses could prevent fibrosis (Brillaet al., 1993; Robert et al., 1994). The development ofcardiac fibrosis seems to start around vessels (associ-ated with coronary and myocardial inflammation) andextends later to the interstitium (Robert et al., 1994). Inmyocardial infarction, reparative scar and interstitialfibrosis are improved by MR antagonism (Galuppo andBauersachs, 2012). Deletion of cardiomyocyte MR ame-liorates adverse remodeling after MI (Galuppo andBauersachs, 2012). Clinical trials indicate that thebenefit of MRA in the EPHESUS trial is associatedwith reduced plasma procollagen type I amino-terminalpro-peptide, a surrogate biomarker of fibrosis (Iraqiet al., 2009). One-month treatment with Spironolactonealso reduced procollagen type I carboxyl-terminal pro-peptide in patients after stroke (Wong et al., 2013).The mechanisms underlying the beneficial effects of

MRA in cardiac diseases are complex and diverse.Aldosterone alone (ex vivo) or in association with salt(in vivo) stimulates the expression of proinflammatorymolecules that may contribute to the pathogenesis ofcardiac remodeling. For example, aldosterone increasesendothelin 1 (ET1), transforming growth factor b, and(plasminogen activator inhibitor) PAI through MR-dependent mechanisms as well as collagen andmetallo-proteases (Marney and Brown, 2007). Oxidative stressis also induced, mainly through activation of theNADPH oxidases (Johar et al., 2006). Concomitantadministration of NaCl is a prerequisite to inducecardiac fibrosis: aldosterone administration alone (orchronic increased aldosteronemia in models of hyper-aldosteronism) is not sufficient to induce cardiac fibro-sis in rats or mice (Brilla, 2000; Wang et al., 2004b).Cardiomyocyte-specific MR or aldosterone synthaseoverexpression did not induce fibrosis in the mouse(Garnier et al., 2004; Ouvrard-Pascaud et al., 2005),suggesting that one or more cofactors (salt, oxidative

stress,…) are required to induce the profibrotic effects ofaldosterone.

The interaction between MR and angiotensin II(AngII) receptor AT1R signaling cascades plays a keyrole in aldosterone-induced fibrosis. Spironolactoneprevents AngII-induced cardiac and vascular remodel-ing (Virdis et al., 2002; Johar et al., 2006); AngII canactivate the MR pathway indirectly via the EGFRpathway (Rautureau et al., 2011); expression of AT1Ris obligatory for aldosterone activation of VSMC(Lemarie et al., 2009). These observations support thebenefit of adjunct therapies employing MR and AT1Rantagonists in cardiovascular pathologies.

Novel profibrotic targets modulated by MR wererecently identified, including enzymes such as myocar-dial lysyl oxidase involved in the maturation of procol-lagen (Lopez et al., 2009) or profibrotic molecules ascardiotrophin 1 CT1 (Lopez-Andres et al., 2008, 2011),galectin 3 (Gal3) (Calvier et al., 2013), or lipocalin 2, alsoreferred as neutrophil gelatinase-activated lipocaline(NGAL) (Newfell et al., 2011; Latouche et al., 2012;Gilet et al., 2015). Indeed, gene inactivation of CT1(Lopez-Andres et al., 2011), Gal3 (Calvier et al., 2013;2015), and lipocalin 2 (Tarjus et al., 2015c) blunted thecardiac remodeling and inflammation induced by min-eralocorticoids in experimental mouse models. Theseobservations highlight the underlying essential roleof NGAL in mineralocorticoid-mediated extracellu-lar matrix remodeling (Leopold, 2015). Pharmacologi-cal antagonism of Gal3 using modified citrus pectinalso blunted mineralocorticoid-induced cardiovascularremodeling in rats (Calvier et al., 2015), indicating thatprofibrotic targets downstream to MR activation mayprovide alternative therapeutical targets when MRAcannot be used, for example in patients at high risk fordeveloping hyperkaliemia.

B. Mineralocorticoid Receptor andElectrophysiological Disorders

Recently, the deleterious role of aldosterone (and thebeneficial effect of MR antagonists) was highlighted inthe occurrence of atrial and ventricular arrhythmias.

Spironolactone prevents atrial remodeling (fibrosisand dilation) as well as apoptosis in an experimentalmodel of atrial fibrillation (AF) and prevents the in-creased inducibility and duration of tachypacing-induced AF (Zhao et al., 2010a) as well as atrialremodeling and AF inducibility in a rat hypertensivemodel (Kimura et al., 2011). Fibrotic remodeling ob-served in a genetic mouse model of AF (constitutivelyactive Rac1 under the control of the a myosin heavychain) is prevented byMR antagonism (spironolactone ornonsteroidal BR-4628) (Lavall et al., 2014). An increasedrate of AF was reported in patients with hyperaldoster-onism (Milliez et al., 2005). Aldosteronemia is increasedin patients with chronic AF and decreases rapidly afterelectrical cardioconversion (Goette et al., 2001). Atrial


MR expression was increased in patients with AF (Tsaiet al., 2010), and MRA was associated with reduced AF-related hospitalization (Williams et al., 2011). Eplere-none improved maintenance of sinus rhythm in patientswith long standing AF (Ito et al., 2013), and post hocanalysis of the EMPHASIS-HF trial indicated thateplerenone reduced the incidence of new onset of AF(Swedberg et al., 2012).MR activation can also lead to ventricular arrhyth-

mias. Ex vivo, aldosterone increases T-type calciumchannel expression and beating frequency in neonatalrat ventricular cardiomyocytes (Lalevee et al., 2005;Rossier et al., 2008). In vivo, aldosterone infusion oroverexpression ofMR in cardiomyocytes induces cardiacion channel remodeling: the transient outward K+

current (Ito) is decreased, whereas the L type calciumcurrent activity is increased; the activity of the ryano-dine receptor is also impaired (Gomez et al., 2009;Ouvrard-Pascaud et al., 2005). This has importantconsequences in the control of calcium homeostasis,modulation of calcium transients, sarcoplasmic reticu-lum diastolic leaks, and initiating cardiac rhythmdisorders. Indeed, conditional overexpression of theMR in cardiomyocytes is associated with ventricularextrasystoles and increased sensitivity to triggeringventricular arrhythmias (Ouvrard-Pascaud et al.,2005). In cardiomyopathic hamsters, eplerenone re-duces cardiac remodeling and decreases the rate ofspontaneous ventricular tachycardia. The spatial dis-persion of QT interval is restored, whereas the STsegment depression and action potential propagationand conduction velocity are considerably improved(De Mello, 2006). Similarly, spironolactone treatmentprevents gap junction remodeling and restores thedecreased transverse conduction velocity in a thoracicaortic constriction model (Qu et al., 2009). Likewise,eplerenone reduces fibrosis-related arrhythmias inaged mice (Stein et al., 2010). Both spironolactone andeplerenone reduce the prolongation of QT intervals anddiminishes the occurrence of ventricular prematurebeats or nonsustained ventricular tachycardia inaldosterone-infused rats (Dartsch et al., 2013). Spiro-nolactone has similar effects on inducible ventriculartachyarrhythmia in rats with aldosterone-salt chal-lenge (Deshmukh et al., 2011).The beneficial effects of MRA on electrical remodeling

may underlie the impressive beneficial effect of MRantagonism on lowering the rate of sudden death in theRALES and EPHESUS trials (about 50% of the totalbenefit of MRA in these trials) (Pitt et al., 1999, 2003).Indeed, ameta-analysis of seven clinical trials including8,635 patients showed that MRA reduced the risk ofsudden death by 21% in patients with HF and lessenedthe risk of ventricular tachycardia by 72% (Wei et al.,2010). A recent dedicated clinical trial showed thatadministration of MRA at the early stage of MI (canre-none bolus on admission for primary percutaneous

coronary intervention followed by spironolactone 25 mg/day) was associated with decreased life-threateningarrhythmia and cardiac arrest (Beygui et al., 2013).

C. Cardiac Effects of NoncardiomyocyteMineralocorticoid Receptor

The MR is expressed in cardiomyocytes and also inother cardiac cell types: coronary endothelial andsmooth muscle cells (VSMC) and inflammatory cellsas macrophages, which participate in the remodelingprocess of cardiac pathologies (Nguyen Dinh Cat andJaisser, 2012). The benefits of MRA in cardiac diseasesmay thus also be accounted for by MR blockade in thesecellular targets.

The specific knockout of MR inmacrophages preventsthe development of cardiac interstitial fibrosis in theDOCA-salt model, although the cardiac recruitment ofthese cells was not prevented (Rickard et al., 2009).Myeloid MR can induce proinflammatory M1 macro-phage polarization, and deletion of macrophage MRprevents interstitial fibrosis upon AngII infusion andchronic nitric oxide (NO) inhibition (Usher et al., 2010).These observations indicate that MR signaling inmacrophages is required to elicit a full fibrotic responsein the heart.

The importance of the coronary circulation in cardiacpathophysiology is well known, although its specificcontribution to the harmful effects of mineralocorti-coids has been scantily addressed. In a model ofcardiomyocyte-specific overexpression of the aldoste-rone synthase, aldosterone concentration in cardiactissue ismoderately increased and there is no alterationof cardiac structure and function (Ambroisine et al.,2007). In contrast, coronary arteries develop a majorNO-independent vascular dysfunction due to an al-tered expression and activity of the potassium channelBKCa expressed in VSMC (Ambroisine et al., 2007).Cardiomyocyte-specific overexpression of the MR isassociated with local oxidative stress leading to coro-nary endothelial dysfunction (Favre et al., 2007). MR-dependent and aldosterone-induced coronary vesselalterations could increase the sensitivity of the heartto ischemia and predispose cardiac tissue to heartdysfunction. Unpublished data from our group showedthat deletion of MR in VSMC is crucial in MI-inducedheart dysfunction: deletion of the VSMC MR drasticallyimproved coronary function after MI, as well as cardiacperfusionmeasured by magnetic resonance imaging, andMI-induced cardiac remodeling.MR antagonismwith thenonsteroidal MRA finerenone had a similar effect, im-proving coronary reserve and coronary function in MI.

D. Mineralocorticoid Receptor and Cardiopathy ofMetabolic Origin

The beneficial effects of MR blockade could extend tocardiopathies related to metabolic diseases. In anexperimental model of type 2 diabetes (db/db mice),


eplerenone normalized the cardiac expression of severaladipokines (Guo et al., 2008). Eplerenone or spirono-lactone attenuated cardiac steatosis, apoptosis, anddiastolic dysfunction in Zucker Diabetic Fatty rats(Ramirez et al., 2013; Bender et al., 2015a). Conversely,increased cardiac production of aldosterone is beneficialand protects against reduced capillarization in type Iand type II diabetes (Messaoudi et al., 2009; Fazal et al.,2014). This response appears to be mediated by anincrease in Akt phosphorylation (Fazal et al., 2014).Eplerenone improves coronary circulation (Joffe et al.,2007) and spironolactone likewise improves coronaryreserve in type 2 diabetic patients (Garg et al., 2015).Adipocytes are found in close proximity to blood

vessels, where adipocyte-derived cytokines (adipokines)can directly influence vascular function (Nguyen DinhCat and Jaisser, 2012). Bender et al. (2015b) recentlyuncovered a specific role for MR as a mediator ofcoronary (not peripheral) vascular dysfunction in pa-tients with obesity and diabetes. It is evident from theseobservations that the role of MR in epicardial orperivascular fat on coronary function deserves furtheranalysis.

IV. Mineralocorticoid Receptor andVascular Diseases

The pharmacological blockade of MR using spirono-lactone or eplerenone provides benefits for vasculartone and remodeling of the vascular wall (Virdis et al.,2002). Several studies in preclinical models have shownbeneficial effects of MRA on endothelial dysfunctioninduced by diabetes (Schafer et al., 2010, 2013; Adelet al., 2014), by a high-fat diet (Schafer et al., 2013), ormyocardial infarction (Sartorio et al., 2007). Eplerenoneprevented the potentiation of agonist-induced vasocon-striction induced by aldosterone (Michea et al., 2005). Inrats treated with aldosterone (Lacolley et al., 2002),eplerenone prevented blood pressure increase and re-duced pulse pressure and elastic modulus of largevessels, two markers of vascular stiffness (Mitchell,2014). In human hypertensive patients, MRA improvedflow-mediated dilation, a mechanism contributing tovascular tone in hypertension (Fujimura et al., 2012). Inpatients with end-stage renal disease (ESRD), MRAalso had favorable effects on intima-media remodeling(Vukusich et al., 2010). Of note, two studies reported anondiuretic effect on blood pressure in patients withESRD treated with spironolactone or eplerenone (Grosset al., 2005; Shavit et al., 2012).More recently, the benefit of MRA has been reported

in experimental models of pulmonary arterial hyper-tension, improving pulmonary vascular remodeling,right ventricular systolic pressure (Preston et al., 2013),and pulmonary artery systolic pressure (Maron et al.,2012). Importantly, this may translate to clinics, becausein patients with pulmonary arterial hypertension,

spironolactone as an adjunct to classic therapeutics hasbeneficial effects with improved exercise tolerance anddecreased BNP plasmatic concentration (a marker ofcardiac dysfunction) (Maron et al., 2013).

Although MR-mediated effects on arteries have beenlargely documented, only in abnormal situations suchas venous arterialization do veins express functionalMR and HSD2. MR expression may potentiate AngIIactivity in grafted veins (Bafford et al., 2011), whereasremodeling of venous wall is reduced by MRA (Ehsanet al., 2013). Veins exhibit a higher sensitivity tovasoconstrictor agents compared with arteries, whichdid not differ in their response between normal miceand DOCA-salt hypertension model (Perez-Riveraet al., 2004). Venous smooth muscle tone is increasedin DOCA-salt hypertensive rats (Fink et al., 2000).Whether changes in venous contractility are secondaryto the rise in blood pressure or depend directly on MRfunction is currently unknown.

A. Role of the Endothelial Mineralocorticoid Receptor

MR expression was demonstrated in the endothelialcells of rabbit aorta in the early 1990s (Lombes et al.,1992) and then later in bovine aortic endothelial cells(Leopold et al., 2007) and in endothelial cells fromhuman coronary arteries and aorta (Caprio et al.,2008). Endothelial MR expression is increased in themicrovasculature of spontaneously hypertensive Kyoto-Okamoto strain (SHR) rats (DeLano and Schmid-Schonbein, 2004). A mouse model with conditional andinducible endothelium-specific MR overexpressionhighlighted the involvement of the endothelial MR inblood pressure regulation (Nguyen Dinh Cat et al.,2010). Basal blood pressure was increased, as well asthe blood pressure response to the acute infusion of thevasoconstrictors AngII and endothelin 1 (ET1), accom-panied by an increased contractile response of mesen-teric arteries to AngII and ET1 (Nguyen Dinh Cat et al.,2010). However, endothelial MR overexpression had noeffect on endothelium-dependent or independent re-laxation (Nguyen Dinh Cat et al., 2010). Endothelium-specific deletion of MR indicated that endothelial MR isnot involved in deoxycorticosterone-salt (DOCA-salt)-mediated increase in systolic blood pressure (Rickardet al., 2014). Endothelial MR deletion could bluntobesity-induced endothelial dysfunction (Schafer et al.,2013); however, blood pressure was not assessed in thisstudy. Both studies reported beneficial effects of endo-thelial MR inactivation on endothelial dysfunction(estimated as an altered dilatory response to acetylcho-line) induced by high-fat diet or DOCA-salt challenge(Schafer et al., 2013; Rickard et al., 2014). MR deletionin endothelium has no consequences for systolic bloodpressure under basal conditions, in contrast to thehypertensive responses observed with endothelial MRoverexpression. This observation was confirmed re-cently in another model targeting MR in endothelial


cells (Mueller et al., 2015). Interestingly, endothelialMR deletion prevented DOCA-salt-induced endothelialdysfunction in aorta but not in mesenteric arteries,suggesting a distinct role in different vascular beds(Rickard et al., 2014). Indeed Mueller et al. (2015)recently reported that endothelial-MR participates inregulation of vasomotor function in a vascular bed-specific manner. Inducing angiotensin II-dependenthypertension caused an impaired endothelial-dependent relaxation that was prevented in endothelialMR-deficient mice in mesenteric vessels but not incoronary vessels. This vascular bed-specific contributionof endothelial MR is also suggested by the MR-inducedvasoconstrictor phenotype reported in large arteries,contrasting with its vasodilatory effect on the choroidcapillary network of the posterior eye (see below).Aldosterone via MR activation modulates several

endothelial functions and pathways, such as enhancedexocytosis of Weibel-Palade bodies containing proin-flammatory cytokines (Jeong et al., 2009), cell adhesionof inflammatory cells (Caprio et al., 2008), and theproduction of reactive oxygen species (ROS). MR acti-vation increases the activity of NADPH oxidase andreduces the degradation of ROS through reduced ex-pression and activity of the antioxidant enzyme glucose-6-phosphate dehydrogenase (Leopold et al., 2007). Thisalters nitric oxide (NO) bioavailability, an importantsecond messenger in endothelial cells. MR antagonismprevents the effects on oxidative stress and theirconsequences on vascular reactivity (Leopold et al.,2007). In pulmonary artery endothelial cells, the highproduction of hydrogen peroxide induced by aldosteroneinduces a sulfenic posttranslational modification of theendothelin receptor type B, leading to reduced NObioavailability in the pulmonary vasculature (Maronet al., 2012). MR-dependent ROS production also im-paired the differentiation and migration of bonemarrow-derived endothelial progenitor cells, whichare crucial for endothelial repair and vascular homeo-stasis (Thum et al., 2007).MR-modulated pathways in the endothelium may

impact blood pressure or vascular properties in patholo-gies such as atherosclerosis or injury-induced remodeling(McGraw et al., 2013). Another recent issue is the in-volvement ofMR signaling in vascular thrombosis. Inmicewith arterial injury or in normotensive rats, aldosteronewas reported to favor thrombosis, whereas MR over-expression in endothelium impaired thrombus formation(Lagrange et al., 2014). Endothelial MR activation mayhave a beneficial antithrombotic action in healthy vessels,whereas deleterious prothrombotic effects may occur onlywhen the endothelium is injured (Lagrange et al., 2014).

B. Role of the Vascular Smooth MuscleMineralocorticoid Receptor

MR is expressed in the vascular smooth muscle cells(VSMC) that play a crucial role in the regulation of

vascular tone (Jaffe and Mendelsohn, 2005; NguyenDinh Cat et al., 2010). VSMC MR expression is in-creased in aging rats (Krug et al., 2010). VSMC MR isinvolved in blood pressure regulation, as shown in twodifferent models of VSMC-specific MR gene inactivation(McCurley et al., 2012; Galmiche et al., 2014). Thegenetic inactivation of MR in 4-month-old adult miceprevented the increase in blood pressure occurring withaging in 9-month-old mice (McCurley et al., 2012). In aconstitutive model of VSMC-specific MR inactivation,basal blood pressure was reduced in 4-month-old MRknockout (KO) mice (Galmiche et al., 2014). Interest-ingly, VSMC MR inactivation prevented the in vivorise in blood pressure induced by AngII infusion butnot by aldosterone-salt treatment (McCurley et al.,2012; Galmiche et al., 2014). VSMC MR gene inacti-vation blunted the contractile response to pressureand agonists (phenylephrine, AngII) in aged mice only(McCurley et al., 2012). This difference in the effect ofVSMCMR deletion on blood pressure in mouse modelswith either constitutive or inducible MR deletionindicates that long-lasting MR inactivation is requiredto affect blood pressure control and decrease basalblood pressure: in both models, MR inactivationoccurring during 4–5 months is required to result inlower basal blood pressure. Whether this reflectsadaptation/compensatory mechanisms rather than aprimary role of VSMMR in the control of blood pressureremains to be evaluated. In vivo studies showed thatVSMC MR plays a role in the remodeling of theextracellular matrix of the vascular wall as demon-strated using VSMC MR KO mice with aldosterone-salt challenge (Galmiche et al., 2014). As detailedbelow, activation of MR impacts on VSMC through ionchannel regulation (McCurley et al., 2012; Tarjus et al.,2015a). MR activation in VSMC induces the expressionof profibrotic markers (Jaffe and Mendelsohn, 2005;Kiyosue et al., 2011; Zhu et al., 2012; Calvier et al.,2013).

In pulmonary arteries, MR activation induced theproliferation of smooth muscle cells, an effect preventedby spironolactone (Preston et al., 2013). These effectscould likely contribute to the development of pulmonaryarterial hypertension and to the benefit of MRA inpatients.

VSMCMRparticipates in vascular calcification (Jaffeet al., 2007) by regulating the expression of the phos-phate transporter Pit1, which has an osteogenic func-tion in the smooth muscle (Voelkl et al., 2013). In vivoadministration of spironolactone blunts the vascularcalcification observed in the Klotho deficient mousemodel (Voelkl et al., 2013) and in uremic rats (Tatsu-moto et al., 2015).

In conclusion, MR-mediated effects in VSMC maycontribute to a variety of disease processes includingvascular remodeling and stiffness, hypertension, andlong-term consequences of aging.


C. Role of the Mineralocorticoid Receptor in theCrosstalk between Endothelium and Vascular SmoothMuscle Cells

The crosstalk between endothelium and vascularsmooth muscle is crucial for vascular function. IndeedVSMC responses are controlled by endothelial cells viacomplex intercellular signaling processes. Endothelial-dependent vascular relaxation is mediated by the re-lease of nitric oxide (NO) or vasoactive prostanoids.Another pathway is associated with the hyperpolariza-tion of both the endothelial cells and VSMCmediated byendothelium-derived hyperpolarizing factor(s) (EDHF)(Edwards et al., 2010). EDHF-mediated responses in-volve epoxyeicosatrienoic acids, potassium ions andchannels, reactive oxygen species (ROS), and myoendo-thelial junctions (Edwards et al., 2010) Moreover,endothelial-derived contractile factors are also impor-tant (Virdis et al., 2010).MR activation clearly affects endothelium-VSMC

crosstalk. Endothelial MR activation decreases NOsynthesis by reducing the activity of endothelial NOsynthase (eNOS) either directly or via eNOS uncoupling(Bauersachs and Fraccarollo, 2006). It also increaseslocal ROS production and therefore decreases NO bio-availability. Aldosterone/MR also affects EDHF: endo-thelial MR increases calcium-activated potassiumchannel expression and activity (Zhao et al., 2012),resulting in membrane hyperpolarization leadingto vasorelaxation. Aldosterone/MR also increasedendothelial-derived contractile factors via increasedsynthesis of endothelin (Park and Schiffrin, 2001;Nguyen Dinh Cat et al., 2010). A novel mechanism ofaction of aldosterone/MR/MRA on the endothelin path-way has been recently highlighted: oxidative stressinduced by mineralocorticoid activation resulted in aninactivating posttranslational modification of the endo-thelin 1 receptor type B (ET1RB) related to a cysteinsulfenication (Maron et al., 2012; Barrera-Chimal et al.,2015b) MRAs prevented such inactivating modificationof the ET1RB, allowing vasodilation to counterbalancethe vasoconstriction induced by endothelin 1-mediatedendothelin 1 receptor type A stimulation, leading tobetter local hemodynamics. These beneficial effects ofMRA have been reported in pulmonary hypertension(Maron et al., 2012) as well as in renal ischemiareperfusion injury (Barrera-Chimal et al., 2015b).

D. Mineralocorticoid Receptor AffectingMicrocirculation

Although most studies demonstrated MR-mediatedeffects on large vessels, for technical reasons (size ofvessels), there is limited information on the microcircu-lation. However the microcirculation plays a major rolein tissue pathology (Granger et al., 2010). There is alongitudinal gradient of expression of MR, with higherexpression in large arteries (Lombes et al., 1992), but

MR is also expressed in arterioles and capillaries(DeLano and Schmid-Schonbein, 2004; Zhao et al.,2012), although to a lower extent, and perhaps essen-tially only in pathologic situations. Moreover, MRexpression may depend on the precise location of thevessels within an organ (for example, in the renalglomerulus afferent versus efferent arteries or peritub-ular capillaries).

V. Mineralocorticoid Receptor andMetabolic Diseases

Experimental and clinical studies have highlightedaldosterone as a potential risk factor for diabetes andmetabolic syndrome (MetS), through mechanisms atleast partially independent of hypertension (Whaley-Connell et al., 2010). A new role for aldosterone/MR ac-tivation in adipose tissue has been highlighted (Zennaroet al., 2009; Marzolla et al., 2014; Gomez-Sanchez,2015 ). MR is involved in the plasticity of whiteadipocyte and MR antagonism promotes “browning” ofthe white adipose tissue (i.e., increased presence ofbrown adipocytes within the white adipose tissue)through direct control of autophagy promoting in-creased metabolic activity of adipose depots (Armaniet al., 2014). Ex vivo experiments indicate that activa-tion of MR by aldosterone (Caprio et al., 2007) orglucocorticoids (Hirata et al., 2012) influence adipocytedifferentiation and the secretion of adipokines asadiponectin and leptin as well as proinflammatorymarkers. Experimental studies in rodent models [db/db, ob/ob, or high-fat diet (HFD)-induced obese mice]indicate a specific role of aldosterone and/or MR acti-vation. Indeed, MRA improves glucose tolerance anddecreases insulin resistance, plasma levels of triglycer-ides, and proinflammatory cytokines (Guo et al., 2008;Hirata et al., 2009; Wada et al., 2010). In db/db mice,Guo et al. (2008) reported that pharmacological treat-ment with the selective MR antagonist eplerenone for16 weeks reversed obesity-related changes in adiposetissue gene expression (as the increased expression ofPAI-1, leptin, and proinflammatory cytokines tumornecrosis factor (TNF)-a and MCP-1 with a concomitantreduction of PPARc and adiponectin observed in db/dbmice. Short-term eplerenone administration (3 weeks)in db/db and ob/ob mice also showed improved insulinsensitivity and MR antagonism restored the dysre-gulation of adipose gene expression in both models(Hirata et al., 2009). Eplerenone improved endothelialdysfunction induced by HFD (Schafer et al., 2013) andin streptozotocin-induced diabetic rats (Schafer et al.,2010) Spironolactone prevented HFD-induced arterialstiffening (DeMarco et al., 2015). Pharmacologicalapproaches however do not discriminate between directconsequences of MR blockade in the adipose tissue andthose related to global MR antagonism in other organsaffected in MetS such as the heart, the vasculature, the


pancreas, the liver, or muscle, where MR is expressedand/or involved in insulin secretion/sensitivity and/ororgan damage. The specific role of adipocyte MR wasrecently addressed using a novel transgenic mousemodel, allowing inducible expression of MR in adipo-cytes only. Increased MR expression mimicking theincreased expression observed in adipose tissues ofexperimental models of obesity (db/db, ob/ob, HFD)and human obese patients was associated with in-creased body weight, insulin resistance, and featuresof MetS (Urbanet et al., 2015). A novel MR target wasidentified: MR activation modulated adipocyte expres-sion of the prostaglandin D2 synthase that was anabsolute requirement for the ex vivo adipogenic effectsof aldosterone (Urbanet et al., 2015). Interestingly,adipose tissue expression of MR correlated with pros-taglandin D2 synthase expression in human fat depots(Urbanet et al., 2015).Aldosterone-inducedMR activation is associated with

impaired insulin sensitivity in adipocytes, skeletalmuscle, and the vasculature (Garg and Adler, 2012;Bender et al., 2013). Plasma aldosterone is correlatedwith body mass index and insulin resistance and Connand Fajans (1956) already reported in the 1950s thatpatients with primary aldosteronism had increasedrisk of diabetes. A high prevalence (10–50%) of glu-cose intolerance and/or diabetes has been reported inprimary aldosteronism, and these metabolic distur-bances could be corrected by surgical removal of thealdosterone-producing adenoma (Fallo et al., 2012). Theunderlying mechanisms remain partially known andmay converge on the insulin receptor/insulin growthfactor receptor/insulin receptor substrate/AKT path-ways (Garg andAdler, 2012; Bender et al., 2013; Luther,2014). Locally synthesized aldosterone may also in-tervene; aldosterone production by adipocytes has beendemonstrated (Briones et al., 2012) and may affectvascular function and insulin response by a paracrinemechanism. Cultured human adipocytes can secretefactors that stimulate adrenal production of aldosterone(Ehrhart-Bornstein et al., 2003).Aldosterone may also affect insulin secretion (Luther,

2014). MR is expressed in rodent pancreatic islets andaldosterone impairs glucose-stimulated insulin secretionin vivo in mice and in murine islets (Luther et al., 2011).The potential role of inflammatory cells (essentially

macrophages) in the adipose tissue and the implicationof MR activation in macrophages has been raisedrecently (Marzolla et al., 2014). Indeed MR activation,affecting macrophages polarization toward a proinflam-matory phenotype, may participate in the developmentof dysfunctional adipose tissue upon corticosteroidstimulation. Conversely, MR blockade may affect adi-pose metabolic function by reducing adipose tissueinflammation, now recognized as an important factorin metabolic diseases contributing to insulin resistanceand dysfunctional adipocytes (Toubal et al., 2013).

Despite strong evidence for a deleterious role of MRactivation and a beneficial role of MR antagonism inmetabolic diseases in preclinical models, results fromhuman studies remain equivoqual and the benefit ofpharmacological MR antagonism to improve metabolicdiseases has not been established. For example, al-though spironolactone improves coronary microvascu-lar function in type 2 diabetic patients without clinicalischemic disease, MRA has no effect on lipids, bodymass index, or fasting glucose (Garg et al., 2015). This isconsistent with a pilot study from the same group usingeplerenone (Joffe et al., 2007). However, in patientswith moderate chronic kidney disease and increasedHOmeostatic Model Assessement (HOMA) index, thereis a significant reduction of HOMA index and fastinginsulin after 6 months spironolactone treatment(Hosoya et al., 2015).

VI. Mineralocorticoid Receptor andRenal Diseases

In 1964, Conn et al. (1964) described the first 145cases of proven primary hyperaldosteronism associatedwith hypertension and where proteinuria was presentin 85% of the patients. The proteinuria was consideredas a consequence of hypertension until the 1990s, whenexperiments on the remnant kidney model in ratsrevealed that mineralocorticoid hormones can induceproteinuria in the absence of hypertension. In the salt-sensitive hypertensive Dahl rat, renal failure, protein-uria, and histologic renal lesions could be fullyprevented by low-dose eplerenone, whereas blood pres-sure remained very high (230 mmHg). The benefit ofMRA in renal damage occurred independently of bloodpressuremodulation (Kobayashi et al., 2005). The groupof T. Fujita highlighted recently a novel pathwayinvolved in renal MR signaling whereby the smallGTPase Rac1 potentiates the activity of MR, contribut-ing to ligand-independent MR activation in preclinicalmodels of kidney injury (Nagase and Fujita, 2013).Therefore, although aldosterone exerts primarily phys-iologic homeostatic responses, allowing maintenanceof extracellular volume in response to acute volume lossor salt depletion, a sustained MR activation, in somepathologic situations, can lead to pressure-independentrenal damage.

In addition to functional MR in the epithelial cells ofthe distal nephron, MR is also expressed in vascularendothelial cells and, to a lesser extent, in vascularsmooth muscle cells of interlobar renal arteries in themouse (Nguyen Dinh Cat et al., 2010). Under physio-logic conditions, MR expression is not detectable in theglomerulus (Farman et al., 1982a,b, 1991) but MRexpression has been demonstrated ex vivo in culturedpodocytes, mesangial cells, and renal fibroblasts(Nishiyama et al., 2005; Shibata et al., 2008). In vivo, itis possible that MR, in nonclassic target tissues such as


podocytes or mesangial cells, reaches a significant levelof expression only during pathologic situations as intype I diabetes observed in the rat (Lee et al., 2009) andin spontaneous hypertensive rats with metabolic syn-drome (Nagase et al., 2006). Increased MR expressionalso occurs in patients with chronic renal disease ofvarious origins (Quinkler et al., 2005), but the affectedcell type is unknown.

A. Experimental Studies

Experimental evidence points to a benefit of MRantagonism in models of chronic kidney diseases (neph-ron reduction, diabetic nephropathy, glomerulopathies)and has been reviewed (Bertocchio et al., 2011; Ritz andTomaschitz, 2014). Potential novel therapeutic indica-tions of MRA have emerged from situations of renalischemia (Juncos and Juncos, 2015). MRAs have provento be highly efficient in preventing ischemic reperfusioninjury; MRAs administered before or just after ischemicinjury fully prevented acute renal injury in rat (Mejia-Vilet et al., 2007; Sanchez-Pozos et al., 2012; Barrera-Chimal et al., 2013). This has been confirmed recently inthe mouse in which the nonsteroidal antagonist BR-4628 was also shown to be efficient (Barrera-Chimalet al., 2015b). Of major interest, short-term MRAtreatment, flanking the ischemic period, also had long-term beneficial effects after the therapeutic interven-tion, because it prevented the delayed occurrence ofchronic renal failure and interstitial fibrosis (Barrera-Chimal et al., 2013, 2015a). The role of renal inflam-mation and activation of MR in macrophages has beenunderlined in an elegant study using mouse modelswith genetic MR inactivation in the myeloid lineage.Glomerulopathy induced by antiglomerular basementmembrane antibody is blunted inmicewithMRdeletionin the myeloid lineage (Huang et al., 2014), underlyingthe role of macrophage MR activation in the inflamma-tory process associated with renal injury. Whether thisnotion could apply to renal injury of other originsremains to be tested. The role of vascular MR in therenal vascular bed has been highlighted recently byobservations that MRA can limit cyclosporine-inducednephrotoxicity (CIN) (Bobadilla and Gamba, 2007). Thenephrotoxicity of cyclosporine involves vasoconstrictionand altered renal hemodynamics (Amador, 2015). CINwas prevented in mice with genetic VSMC MR in-activation but not when endothelial MR was deleted(Amador, 2015). The hemodynamic alterations relatedto MR activation may have important impact in renaltransplantation outcomes and MRA may prevent orslow down the progression of CIN. Indeed, spironolac-tone improves transplant vasculopathy in rats withrenal transplant (Waanders et al., 2009). MRA maytherefore limit delayed graft dysfunction by moderatingthe ischemia-reperfusion injury occurring during organpreservation/transplantation.

B. Clinical Studies

An increase in plasma aldosterone has been reportedto be a risk factor for kidney injury in clinical studies;chronic kidney disease (CKD) can be considered as astate of relative hyperaldosteronism (Schwenk et al.,2015), and possible benefit of MRA has been explored.In 2001, a brief description of the use of MRA in eightproteinuric patients reported 54% decrease in protein-uria after 4 weeks administration of spironolactone(Chrysostomou and Becker, 2001). Since then, severalstudies have questioned the role of aldosterone and MRin proteinuria and in the progression of CKD, asreviewed recently (Bertocchio et al., 2011; Ritz andTomaschitz, 2014; Schwenk et al., 2015). A meta-analysis showed that MRA, in addition to angiotensi-nogen converting enzyme inhibitors and angiotensinreceptor blockers (or both), reduced proteinuria(Bolignano et al., 2014); however, their consequenceson progression toward end-stage renal disease (ESRD)or major cardiovascular events need to be furtherinvestigated. A small decrease in estimated glomerularfiltration rate (eGFR) was frequently reported, espe-cially in diabetic patients, probably reflecting reversalof hyperfiltration (Bolignano et al., 2014). The decreasein eGFR has also been reported in large CV trials(RALES, EPHESUS, or EMPHASIS-HF) but did notlead to harmful alteration of kidney function, even inpatients with pre-existing altered renal function. Im-portantly, the long-term benefit of MRAs in thesepatients occurred despite eGFR reduction (Vardenyet al., 2012; Rossignol et al., 2014). Several clinicaltrials are now ongoing or were recently completed indiabetic nephropathy: EVALUATE (Ando et al., 2014)and ARTS-DN trials (Bakris et al., 2015). The variableeffects of MRA on renal function in diabetic patients toslow down CKD progression were recently reviewed(Mavrakanas et al., 2014).

Another target of MRA in CKD are the CV eventsassociated with CKD progression or ESRD. Patientswith CKD present an increased risk of CV events, LVhypertrophy, increased vascular stiffness, stroke, andsudden death. Of note, the 4D study indicated thataldosterone levels were powerful predictors of suddendeath in hemodialyzed patients with type 2 diabetes(Drechsler et al., 2013). Because MRA treatment hasnow been clearly established as beneficial in CVdiseases, the added value of MRAs on CV events inCKD and ESRD patients has been proposed. A positiveimpact of MRA on CV events has been reported inhemodialyzed patients (Matsumoto et al., 2014), but thenumber of patients with CV events in the placebo groupwas low; therefore a larger clinical trial is needed (Pittand Rossignol, 2014). Such a clinical trial (Alchemist,NCT 01848639) is ongoing and will assess CV eventsandmorbidity-mortality in hemodialized patients treat-ed or not with spironolactone.


Only scarce data are available in transplanted pa-tients. Addition of spironolactone to the blockade of therenin-angiotensin system with angiotensinogen con-verting enzyme inhibitors and angiotensin receptorblockers provided added value, decreasing severe pro-teinuria in 11 renal transplant patients, without signif-icant effect on renal function (Gonzalez Monte et al.,2010). Of interest, spironolactone given 1 day before and3 days after transplantation can reduce oxidative stressin renal transplant patients from living donors withoutaffecting renal function (Ojeda-Cervantes et al., 2013).When using MRA in patients prone to hyperkaliemia

(CKD, ESRD, or renal graft), a major issue is the safetyconcern regarding the increase in plasma potassiumconcentration. Hyperkaliemia is often observed inpatients treated with MRAs, reflecting efficacy ofMRA in reducing urinary K+ secretion. Life-threatening hyperkaliemia have been reported, partic-ularly when using high dosage of MRAs (Schwenk et al.,2015). Therefore close monitoring of kaliemia in patientswith impaired renal function is required to avoid clini-cally meaningful hyperkaliemia above 5.5 mmol/l. Thebenefit of MRAs may nevertheless be greater than therisk associated with increased plasma potassium levels.

VII. Mineralocorticoid Receptor andOcular Diseases

Recent reports have demonstrated that the retina is atarget tissue for mineralocorticoids, with specific involve-ment in eye pathology (Wilkinson-Berka et al., 2012;Zhao et al., 2012). Whether MR antagonists may exertbeneficial effects in retinal diseases is a novel concept inophthalmology. In clinical ophthalmology, high doses ofglucocorticoids (injected into the vitreous cavity of pa-tients with macular edema) are currently used for anti-inflammatory and antiedematous effects on the retina;this treatment is accompanied by frequent and some-times severe side effects such as intraocular hypertension(glaucoma), cataract, or toxicity. In addition to GR-mediated events, excess glucocorticoids may activatethe ocular MR, leading to pathology, and inappropriateMR signaling may be deleterious for the retina.Retinal Muller glial cells are essential for retinal water

andK+ homeostasis. These cells express theMR, togetherwith the GR and the MR-protector enzyme 11 HSD2(Zhao et al., 2010b, 2011). In retinal Muller glial cells, theMR is functional, as 24-hour aldosterone treatmentpromotes upregulation of ion and water channels (potas-sium channel Kir 4.1, epithelial sodium channel ENaC,and water channel AQP4); aldosterone also increasesretinal thickness, reminiscent of a proedematous effect(Zhao et al., 2010b). The retinal pigmented epithelium(RPE) controls the movements of fluid from the innerretina toward the choroid vessels and blood stream, andretinal detachment may occur in pathology between theapical side and the neighboring photoreceptors. We

showed that retinal pigmented epithelium cells expressboth MR and GR (Zhao et al., 2010b), but the role of MR/GR pathways in this epithelium is unknown.

Few reports have addressed the functional role ofMR of retinal vessels in pathology. Activation (eithersystemic or intraocular) of the renin-angiotensin-aldosterone system is a key feature of diabetes, andblockers of the renin-angiotensin-aldosterone systemappear to be effective in reducing the risk of ophthal-mological complications of diabetes in some patients(Fletcher et al., 2010; Wilkinson-Berka et al., 2012). MRantagonism attenuates pathologic angiogenesis of ratretinal vessels in neonatal oxygen-induced retinopathy,amodel of retinopathy of prematurity (Wilkinson-Berkaet al., 2009), indicating a direct pathologic role ofaldosterone (Wilkinson-Berka et al., 2009). It shouldbe noted that, in the absence of induced pathology,aldosterone per se did not promote retinal angiogenesis.Low-salt diet appeared protective for the retina inischemic retinopathy, despite elevated plasma aldoste-rone (Deliyanti et al., 2014).

Choroid vessels form a rich vascular network plexuslying behind the retina and choroid blood flow is one ofthe highest of the body (together with the renal bloodflow, and 10-fold higher than in the brain) (Nickla andWallman, 2010). The choroidal endothelium expressesthe MR and this tissue is mineralocorticoid sensitive(Zhao et al., 2012; Bousquet et al., 2013). We haveshown that a single injection of aldosterone into the ratvitreous cavity leads to choroidal vasodilation (Zhaoet al., 2012). A similar phenotype was observed in micewith overexpression of the MR in endothelial cells (ourunpublished results). In choroid vessels, MR activationenhanced the expression of the endothelial calcium-activated KCa2.3 channel and intravitreous injection ofthe KCa2.3 channel inhibitor apamine prevented thealdosterone-induced vasodilation. In addition to retinaldetachment, dilation of choroid vessels is a feature ofcentral serous chorioretinitis (CSCR), an eye diseaseaffecting stressed young men, and triggered by oraggravated by glucocorticoids. CSCR can becomechronic and possibly lead to blindness, with no currentgold standard treatment. We showed in pilot clinicalstudies that short-term (1–3 months) treatment of thepatients with MR antagonist leads to spectacularresolution of the disease and recovery of vision (Zhaoet al., 2012; Bousquet et al., 2013). These pilot studieswere confirmed in a randomized clinical trial usingspironolactone (Bousquet et al., 2015). CSCR disease isthe first indication of an effective MRA therapy inophthalmology (Daruich et al., 2015).

The retinal ganglion cells (RGC) are neurons that relayvisual information to the central nervous system. TheMRis expressed by retinal ganglion cells (Wilkinson-Berkaet al., 2009; Zhao et al., 2010b) but its function remainslargely unknown. Interestingly it has been reported thatintravitreal injection of aldosterone reduces the number


of RGC (Liu et al., 2012). Chronic aldosterone infusionleads to RCG cell death and degeneration of the opticnerve (without affecting intraocular pressure), raising thequestion of a role for MR in normotensive glaucoma(Nitta et al., 2013). A subset of retinal ganglion cells (themelanopsin light-sensitive RGC) participates in thesynchronization of circadian rhythms in response to light.Whether nonvisual functions such as endogenous clockand chronobiology (Lahouaoui et al., 2014) may beinfluenced by MR activation is currently unknown.

VIII. Mineralocorticoid Receptor andSkin Diseases

Evidence for MR expression in the human epidermisand hair follicle (as well as in sebaceous and sweatglands) is available but with limited knowledge of itscutaneous role (Kenouch et al., 1994; Zennaro et al.,1997; Farman et al., 2010). Glucocorticoids are efficientdrugs to treat chronic inflammatory or autoimmuneskin diseases. The skin may be damaged by glucocorti-coid excess (Schoepe et al., 2006), as observed withlocally applied dermocorticoids or per os administra-tion, endogenous secretion (Cushing disease), or en-hanced local biosynthesis (Slominski et al., 2013).Glucocorticoid excess can lead to a thin and fragile skinand delayedwound healing, which is of great concern forpatients (Schoepe et al., 2006). Such side-effects havesimilarities with skin defects occurring during aging(Tiganescu et al., 2013). Glucocorticoids may bind to thecutaneous MR, in addition to the GR, and contribute toglucocorticoid-induced side effects in the skin (SainteMarie et al., 2007; Farman et al., 2010; Perez, 2011).Aldosterone and MR antagonists modulate elastin

and collagen content of human skin (Mitts et al., 2010).Restoration of dermal homeostasis by MR antagonismmay rely on antifibrotic and anti-inflammatory actions,leading to improved collagen structural organizationand remodeling and cellularity. In a mouse model ofaging skin, associating ultraviolet irradiation and met-abolic syndrome, MR blockade could improve someaging-like features (Nagase et al., 2013). Genetic in-activation of the glucocorticoid-activating enzyme11HSD1 (or its local blockade) leads to reduced en-dogenous glucocorticoid production, thus limiting thepossibility ofMR occupancy. In this situation, the aging-dependent dermal atrophy and delayed wound healingin mice were reversed (Tiganescu et al., 2013).The epidermis is a mineralocorticoid-sensitive epithe-

lium. MR-mediated sodium absorption through the epi-dermis is a key feature of amphibian skin to adapt fromaqueous to terrestrial environment. Pioneer studiesdemonstrated aldosterone-stimulated sodium absorptionacross the skin of Rana temporaria and Bufo marinus(Crabbe andDeweer, 1964). This functionwas lost duringevolution of higher vertebrates. Thus one can wonderwhether the mammalian epidermal MR may exert

another function in normal states or in pathology. Ofnote, the activity of theMR-protecting enzyme 11HSD2 isminimal in human epidermis (Kenouch et al., 1994),permitting MR occupancy by glucocorticoids. Excessglucocorticoid activation of the MR is most likely to occurin the face of local application of dermocorticoids.Whether and how this mechanism could contribute tosome of the deleterious side effects of dermocorticoids,such as skin atrophy and delayed wound healing, is animportant therapeutical issue, because it could be limitedby local coadministration of MR antagonists.

Unexpected roles of MR in keratinocytes have beenhighlighted by a conditional mouse model expressingthe human MR under the control of a keratinocyte-specific promotor (K5-MR mice) (Sainte Marie et al.,2007). Gestational expression of the MR led to pre-mature skin barrier establishment and epidermal atro-phy reminiscent of the glucocorticoid-induced atrophy(Sainte Marie et al., 2007). The pathways regulated byepidermal MR are currently unknown. Of note, thethree subunits of the epithelial sodium channel ENaCare overexpressed in the skin of K5-MR mice at birth(Maubec et al., 2015). Enhanced ENaC expression inK5-MR pups may depend on glucocorticoid-activatedMR, because of the perinatal rise in plasma glucocorti-coid concentration. In addition, blockade of MR duringgestation (canrenoate given to the mother) preventedthe neonatal epidermal atrophy and ENaC overex-pression (Sainte Marie et al., 2007; Maubec et al.,2015), pointing at mineralocorticoid signaling as anovel epidermal modulator. The hypothesis thatinappropriate/excessive activation of the cutaneousMR by glucocorticoids leading to atrophy may beextended to human skin. The potent glucocorticoidclobetasol induces epidermal atrophy in cultured hu-man skin explants and in subjects with local applicationof the glucocorticoid as a gel. Importantly, the coadmin-istration of MRA could limit the glucocorticoid-inducedatrophy (Maubec et al., 2015). Although the recoverywas partial, the improvement may be important forpatients, because there is no alternative treatmentother than glucocorticoid withdrawal. Therefore, thecombination of MR antagonists with local corticoidtreatment in humans presents a novel promisingtherapeutical approach to limit epidermal atrophy(Maubec et al., 2015). The mechanisms at the origin ofthis beneficial effect include correction of impairedkeratinocyte proliferation and are far from being eluci-dated. The blockade of the activity of ENaC alsoimproved epidermal atrophy and fully restored kerati-nocyte proliferation rate, suggesting that ENaC activa-tion may participate in the glucocorticoid-stimulatedepidermal MR signaling cascade (Maubec et al., 2015).

Postnatal induction of MR in K5-MR mice wasassociated with progressive loss of fur (alopecia) anddysmorphy of hair follicles (Sainte Marie et al., 2007).Elevated plasma aldosterone levels were noted in


subjects with androgenetic alopecia (Arias-Santiagoet al., 2009), suggesting that excessive MR signal-ing may alter hair growth. On the other hand, someclinical reports mentioned that spironolactone can beused to treat hirsutism or, conversely, female patternhair loss. Future studies should help to elucidate theseeffects.

IX. Paradoxical Effects of MineralocorticoidReceptor Activation

In contrast with the above-mentioned deleteriouseffects of MR activation, some reports indicate bene-ficial effects of aldosterone in specific pathologicconditions.The increased cardiac production of aldosterone in

the mouse heart is accompanied by beneficial effects onperipheral capillarization in type I and type II diabetes(Messaoudi et al., 2009; Fazal et al., 2014). Aldosteronealso improved neovascularization in a model of limbischemia in mice (Michel et al., 2004). The expressionof the MR in neutrophils may transduce an anti-inflammatory response to aldosterone mediated byinhibition of nuclear factor kB, leading to reducedneutrophil adhesion to endothelium (Bergmann et al.,2010). In a rat model of endotoxin-induced uveitis,intraocular injection of aldosterone reduced the clinicalintensity of the early phase of uveitis and restored thedownregulated MR brought on by inflammation in theiris and ciliary body (Bousquet et al., 2012). Activationof MR appears to have anticoagulant properties in micewith intact endothelium (Lagrange et al., 2014). Thus,although MR activation is generally considered asdetrimental, it is important to realize that this pathwaymay be beneficial in some specific pathologic situations.

X. Common Mechanisms Involved in PathologicConsequences Of Mineralocorticoid

Receptor Activation

A. Mineralocorticoid Receptor is a Regulator of IonChannels in Multiple Tissues

Aldosterone and MR modulate various ion channels(Table 2) (Pearce et al., 2015; Penton et al., 2015). Theprototype of aldosterone action in the kidney collectingduct principal cell is upregulation of the epithelialsodium channel ENaC, the rate-limiting step of Na+

reabsorption. Aldosterone and MR also control potas-sium secretion through the regulation of apical K+

channel ROMK and fluid absorption through aquaporin2 water channels. Disturbance of these signaling path-ways is responsible for altered Na+ and K+ homeostasiaand hypertension. These regulatory pathways will notbe reviewed here (Frindt et al., 2008; Soundararajanet al., 2010). The notion that aldosterone regulates ionchannels now extends far beyond classic MR epithelialcells targets (kidney, distal colon, sweat and salivarygland ducts).

Evidence has been provided that MR signaling leadsto changes in ion channel expression or activity invascular endothelium and smooth muscle (DuPontet al., 2014). Ion channels may be direct MR targets inthe vascular endothelium. The Ca2+-activated K+ chan-nel (KCa2.3) is upregulated in human umbilical veinendothelial cells by aldosterone. In the retinal choroidvascular bed, the activity of the KCa2.3 K+ channel isrequisite for the vasodilatory effect of aldosterone (Zhaoet al., 2012). The epithelial sodium channel (ENaC) isalso modulated by aldosterone via MR in the endothe-lium (Kusche-Vihrog et al., 2008; Warnock et al., 2014).Because endothelial ENaC contributes to the stiffening

TABLE 2Impact of aldosterone/MR on ion channel expression/activity

Ion Channel Tissue Reference

Na channelENaC Kidney Pearce et al., 2015

Distal colon Escoubet et al., 1997Endothelium (HUVEC/EaHy) Kusche-Vihrog et al., 2008Epidermis Maubec et al., 2015

K channelROMK Kidney Penton et al., 2015Kir 4.1 Eye (retina) Zhao et al., 2010bKCa 2.3 Endothelium (choroid, HUVEC) Zhao et al., 2012; Jaisser, unpublished dataKCa 2.2 Endothelium (choroid) Farman, unpublished dataTransient

outward (Ito)Ventricular cardiomyocyte Benitah and Vassort, 1999; Benitah

et al., 2001; Ouvrard-Pascaud et al., 2005BKCa Coronary artery (vascular smooth

muscle cell)Ambroisine et al., 2007

Ca channelCav 1.2 Ventricular cardiomyocyte Benitah and Vassort, 1999;

Benitah et al., 2001; Ouvrard-Pascaudet al., 2005; Perrier et al., 2005

Vascular smooth muscle McCurley et al., 2012Hyperpolarization-activated current

HCN1 Embryonic stem cell-derivedcardiomyocytes

Le Menuet et al., 2010

HCN 2, HCN4 Cardiomyocyte Muto et al., 2007


of endothelial cell membranes (Jeggle et al., 2013) andto the activity of eNOS (Perez et al., 2009), aldosterone/MR modulation of endothelial ENaC expression/activity is an important issue (Warnock et al., 2014).The ENaC channel is also expressed in the VSMC andcontributes to myogenic tone (Drummond, 2012).Whether ENaC is regulated by MR in the VSMC (inaddition to the endothelium) is unknown. The role ofVSMC-MR in the contractile response could depend onthe expression of L-type calcium (Ca2+) channel(Cav1.2) (McCurley et al., 2012). Indeed, the Cav1.2agonist BayK8644 (1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid,methyl ester) had lower vasoconstrictive effects inmesenteric arteries from aged VSMC-MR KO mice(McCurley et al., 2012). The impaired Ca2+ signalingcan affect the contractile machinery, as demonstratedby reduced phosphorylation of myosin phosphatase-targeting subunit 1, myosin light chain kinase, andmyosin light chain 2 (Tarjus et al., 2015b).Mineralocorticoid stress modifies cardiac ion chan-

nels (Laszlo et al., 2011; Gomez et al., 2013), providing acellular substratum for arrhythmia. Aldosterone in-creases L-type Ca2+ current and decreases transientoutward K+ current (Ito) in cultured ventricular myo-cytes (Benitah and Vassort, 1999; Benitah et al., 2001)and the hyperpolarization-activated cyclic nucleotide-gated (HCN2 and 4) channels (Muto et al., 2007). Incoronary artery, aldosterone regulates the calcium-activated potassium (BKCa) channel (Ambroisineet al., 2007). MR (not GR) enhances the cardiac calciumcurrent (Rougier et al., 2008). Cardiac MR overexpres-sion has a major impact on Ca2+ currents together withdecreased K+ transient outward current (Ito) (Ouvrard-Pascaud et al., 2005). Enhanced MR expression incardiomyocytes promotes cardiac ryanodine receptoropening (Gomez et al., 2009). There is a strong correla-tion between aldosterone levels and L-type Ca2+ cur-rents in ventricular myocytes freshly isolated frommouse heart overexpressing the MR and submitted toaldosterone infusion (Perrier et al., 2005).The epidermis expresses ENaC (Roudier-Pujol et al.,

1996; Brouard et al., 1999; Mauro et al., 2002) andappears as a novel target of the MR in the skin (Farmanet al., 2010;Maubec et al., 2015). TheMR-ENaC cascadeparticipates to the regulation of keratinocyte homeo-stasia through control of keratinocyte proliferation(Maubec et al., 2015).Ion channels (ENaC, Kir4.1) are also aldosterone/MR

targets in the neuroretina (as mentioned above), thusplaying a role in the hydration of the retina (Zhao et al.,2010b) and vasodilation of the choroid capillariesdepends on the activity of KCa2.3 channel (Zhao et al.,2012).Altogether, from these data (and evidence that MR

activates neuronal channels in the brain) we would liketo suggest that ion channel remodeling is a general

feature of mineralocorticoid signaling, however, thenature of affected ion channels depends on the cellcontext and pathology.

B. Mineralocorticoids Induces Oxidative Stress

Oxidative stress appears as a central mechanism inthe effects of aldosterone and MR activation. This hasbeen reviewed recently in excellent publications(Queisser and Schupp, 2012; Even et al., 2014). Asmentioned above, mineralocorticoid-induced oxidativestress may lead to reversible and irreversible post-translational modifications (carbonylation, sulfenica-tion, …) of important downstream pathways, asexemplified for the inactivating sulfenication of theET1RB occurring in renal ischemia-reperfusion injuryfor example (Barrera-Chimal et al., 2015b). Aldosteronestimulates oxidative stress in classic and nonclassictarget cells, such as collecting duct cells, cardiomyo-cytes, endothelial and vascular smooth muscle cells,adipocytes, and macrophages. Aldosterone/MR upregu-lates the expression of NADPH oxidase subunits, suchas Nox 2, Nox4 (Bayorh et al., 2011; Brown, 2013),p47phox, p67phox (Queisser and Schupp, 2012), andrac1 (Iwashima et al., 2008), thereby affecting Noxactivity. Cellular accumulation of Zn2+ and Ca2+ ionsin the heart after aldosterone-salt challenge contributesto cardiac oxidative stress and mitochondrial dysfunc-tion, an effect prevented by spironolactone (Kamalovet al., 2009). The consequences of oxidative stress aremultiple and are involved in some of the pathologiceffects of MR activity. Oxidative stress increases DNAdamage (Schupp et al., 2010) and protein carbonylation(Di Zhang et al., 2008). Uncoupling of the NO synthasedecreases the availability of NO for vasorelaxation, aswell as increased production of hydrogen peroxide, andactivation of the nuclear factor kB pathway leading toinflammation and fibrosis (Mayyas et al., 2013; Karbachet al., 2014)

C. Mineralocorticoid Receptor Activation Leadsto Fibrosis

A growing body of evidence indicates that MR playsan important role in CV and renal diseases by pro-moting fibrosis (Thomas et al., 2010; Young and Rickard,2012). In randomized clinical trials, the beneficialeffects of MRA in heart failure were associated with areduction of fibrosis surrogate markers (Iraqi et al.,2009). MRA also induced arterial destiffening (Lacolleyet al., 2009). Several profibrotic factors (NGAL, CT1,Gal3, osteopontin) are direct MR targets in the CVsystem. Inappropriate MR activation has been shownto promote CV and renal tubulointerstitial fibrosis(Bauersachs et al., 2015). Moreover, fibrosis is increas-ingly appreciated as a major player in adipose tissuedysfunction (Divoux andClement, 2011). Recent reportsindicate that spironolactone limits peritoneal fibrosis inrats, opening up the possibility to improve the efficiency


of peritoneal dialysis (Vazquez-Rangel et al., 2014;Yelken et al., 2014; Zhang et al., 2014). In addition,spironolactone may be beneficial for the cardiovascularfunction of these patients (Ito et al., 2014). MR activa-tion may be involved in liver fibrosis, as inferred fromthe beneficial effect of MRA in mice with nonalcoholicsteatohepatitis (Pizarro et al., 2015). Spironolactonewas also shown to limit skin (dermal) fibrosis (Mittset al., 2010). In the lung, hypoxia-induced local secretionof aldosterone by pulmonary artery endothelial cellsleads to pulmonary vascular fibrosis (Maron et al.,2014). Thus fibrotic actions of MR appear as a generalfeature that can be prevented by MRA.

D. Mineralocorticoid Receptor and Inflammation

Low-grade inflammation is a hallmark of cardiovas-cular, renal, and metabolic diseases. In pathology,inflammatory biomarkers (C-reactive protein, cyto-kines, and their receptors) are associated with poorclinical outcomes and prognosis (Schiffrin, 2013). Pa-tients with hypertension and CV diseases present witha chronic vascular inflammatory state that may dependon adaptive immune response mechanisms (Schiffrin,2013). Pharmacological MR blockade correlates withthe prevention/improvement of the chronic inflamma-tory state associated with CV dysfunction (Herradaet al., 2010, 2011). Chronic MR activation may interferewith inflammatory mechanisms. Macrophages, den-dritic cells, and T lymphocytes were recently identifiedas MR target cells (Bene et al., 2014). MR activationleads to MR-dependent potentiation of interleukin (IL)-6 and tumor necrosis factor-a (TNF-a) expression andnuclear factor kB activation in both immune and non-immune cells (Bene et al., 2014).Vascular inflammation and the infiltration of the

arterial wall and perivascular space by immune cells isa relatively early event after MR activation (Kasal andSchiffrin, 2012). Monocyte/macrophage deficiency inosteopetrotic mice results in the absence ofaldosterone-induced oxidative stress and endothelialdysfunction (De Ciuceis et al., 2005). T regulatory cellsprevent aldosterone-induced vascular injury in mice(Kasal et al., 2012). Macrophages participate to thehypertensive and CV remodeling effects of aldosterone-salt challenge, as shown using macrophage-specific MRKOmice (Rickard et al., 2009).MacrophageMRdeletionalso protects against cardiac fibrosis induced by L-nitro-arginine methyl ester-Ang II pharmacological treat-ment (Usher et al., 2010) or thoracic aortic constriction(Li et al., 2014). In adipose tissue, aldosterone regulatessecretion of proinflammatory adipokines and MR acti-vation stimulates production of TNFa, MCP1, PAI-1,IL-6, an effect prevented by pharmacological MR an-tagonism (Guo et al., 2008). MR activation also pro-motes a switch in macrophage polarization toward aproinflammatory phenotype (Marzolla et al., 2014).Aldosterone activation of dendritic cells increases IL-6

and transforming growth factor b expression aswell as dendritic cell-mediated Th17 polarization ofT lymphocytes (Herrada et al., 2010, 2011). Aldosteronepromotes autoimmune renal damage by enhancingTh17-mediated immunity (Herrada et al., 2010) thatwas blocked by MR antagonism (Amador et al., 2014).

These data suggest that aldosterone/MR modulatesinnate and adaptive immunity, which may have acritical role in initiating/maintaining vascular remodel-ing as well as hypertension and organ damage inresponse to exogenous aldosterone and/or MR activa-tion. Recent studies focused on a potential role ofDAMPs (danger-associated molecular pattern mole-cules), which can initiate and perpetuate immuneresponses in the noninfectious inflammatory response(Anders and Schaefer, 2014). In particular, factors suchas collagen peptides/tenascine-C/fibronectin, heatshock proteins, IL-1a, osteopontin, and uric acid havebeen identified as DAMPs related to cardiovascularmorbidity (Frantz et al., 2014). Several of these fac-tors (tenascine-C, osteopontin, galectin3, collagen/fibronectin peptides) have been clearly identified asMR targets, although the link between MR activationand DAMPs awaits clarification.

In conclusion, although aldosterone has proinflam-matory properties, there is a large body of evidenceshowing that MRA can prevent/limit the inflammationthat precedes the development of fibrosis. Because MRis expressed in innate and adaptive immune systemsand can modulate immune functions, it suggests thatthe beneficial effects of MRA in cardiovascular, renal,and metabolic diseases could in part rely on their anti-inflammatory properties.

E. Mineralocorticoid Receptor and Aging

Glucocorticoid-mediated MR activation occurring inaging persons should be considered in light of the risksfor development of heart failure and aging-associatedpathologies (Pitt, 2012). Although plasma aldosteroneconcentration declines with age, cortisol concentration,and the cortisol/cortisone ratio (an indicator of reduced11HSD2 activity) increases (Henschkowski et al., 2008;Pitt, 2012). A cross sectional study of normotensivesubjects indicated that 11HSD2 activity declines afterthe 4th decade of life, favoring cortisol activation of MRand onset of hypertension (Campino et al., 2013).Considering the bulk of aging-associated pathologies,particularly heart failure, it is worth considering MRantagonism in elderly patients.

MR activation is associated with cellular aging in thekidney, with the induction of senescence-associated bgalactosidase, of the cyclin-dependent kinase inhibitor(p21) and decreased expression of SIRT1 (Fan et al.,2011). In VSMC, aldosterone stimulates senescence andp21, p53, P16, p27, and Ki-ras2 expression (Min et al.,2007). Of note, the crosstalk between AngII and aldo-sterone plays a central role in this process (Min et al.,


2007). Aldosterone induces oxidative stress as well asDNA strand breaks and chromosomal damage that maypromote senescence (Schupp et al., 2010). Interestingly,this is independent of blood pressure as subhypotensivedoses of spironolactone could prevent renal DNA dam-age in vivo in rats challenged with aldosterone and saltwithout uninephrectomy (Queisser and Schupp, 2012).MR expression is also elevated in the aorta of old rats

(34 months), and increased MR signaling may promoteand amplify age-associated inflammation (Krug et al.,2010). VSMC MR is important for the control bloodpressure and vasoreactivity in aged mice (McCurleyet al., 2012). The role of MR in aging probably extends toother tissues besides the vasculature. In the skin, aging-like changes have been linked to excessiveMR signaling(Nagase et al., 2013).

XI. Dysregulations of MineralocorticoidReceptor Activity

A. Regulation of Mineralocorticoid ReceptorExpression Levels

It has been recognized only recently that the level ofMR expression can be enhanced in pathology (Table 3).Assessment of MR is most frequently performed bydetection of its mRNA (because of the convenience ofquantitative-polymerase chain reaction assay), some-times at the protein level, and scarcely by measure-ments of MR binding capacity with radioactive ligands.In vivo enhanced MR expression has been reported in

several experimental models, explaining the efficacy ofMRA:

‐ in the heart of rodents with myocardial infarction(Milik et al., 2007; Takeda et al., 2007), withdiastolic heart failure (Ohtani et al., 2007), orwith hypertension (Silvestre et al., 2000; Konishiet al., 2003);

‐ in vessels of hypertensive animals (SHR) (DeLanoand Schmid-Schonbein, 2004) or vascular cells ina model of normal aging (30-month-old Fisher344cross-bred Brown Norway rats) (Krug et al.,2010);

‐ in hypoxic pulmonary artery vascular endothelialcells (Maron et al., 2014);

‐ in the kidney of Brown Norway rats (Cavallariet al., 2008) and in the renal collecting duct ofspontaneously hypertensive rats (Farman andBonvalet, 1985);

‐ in adipose tissue from diabetic animal models(obese db/db and ob/ob mice, HFD) (Guo et al.,2008; Hirata et al., 2009, 2012); in kidney fromdb/db mice and streptozotocin-treated rats (Guoet al., 2006);

‐ in skin of mice with ultraviolet irradiation andmetabolic syndrome (Nagase et al., 2013).

AlthoughMR expression in human tissues is difficult toassess because of limited availability of premortemorgans, some studies reported enhanced MR levels incardiac tissue from patients with heart failure (Yoshida

TABLE 3Tissue upregulation of MR expression

Species Organ Pathology Increase in MR Reference

SD rat heart infarct MR mRNA LV, RV proteinonly in LV

Milik et al., 2007

SD rat heart infarct MR and HSD2 mRNA Takeda et al., 2007SS-Dahl rat heart Hypertension diastolic heart failure MR protein Ohtani et al., 2007Wistar rat heart DOCA salt hypertension MR mRNA, protein, in LV

(no change in RV or K)Silvestre et al., 2000

Wistar rat heart Ang II infusion 7 days MR mRNA, protein, in LV(no change in RV or K)

Silvestre et al., 2000

SHR rat heart hypertension MR mRNA, protein, in LV(no change in RV or K)

Silvestre et al., 2000

SHR rat heart malignant stroke-prone rats MR and HSD2 mRNA Konishi et al., 2003human heart terminal heart failure

hypertrophic cardiomyopathyMR, not HSD2 mRNA Chai et al., 2010MR and HSD2 mRNA

human heart congestive heart failure MR immunohistochemistry Yoshida et al., 2005human cardiac atrial cells atrial fibrillation MR and HSD2 mRNA, protein De-An et al., 2010human cardiac atrial cells atrial fibrillation MR mRNA (not HSD2) Tsai et al., 2010SHR rat Endothelium

arterioles, veinshypertension MR immunohistochemistry DeLano and Schmid-Schonbein,

2004BN rats aorta aging MR mRNA, protein Krug et al., 2010BN rats kidney renal artery clip hypertension MR mRNA Cavallari et al., 2008SHR rat renal collecting duct hypertension MR binding Farman and Bonvalet, 1985human kidney cancer MR mRNA Yakirevich et al., 2008human kidney renal failure Quinkler et al., 2005db and ob mice white adipose tissue insulin resistance, obesity MR mRNA Guo et al., 2008; Hirata et al.,

2009, 2012; Urbanet et al., 2015human white adipose tissue obesity MR mRNA Urbanet et al., 2015db mice and

STZ ratskidney diabetes type I and II MR mRNA, protein Guo et al., 2006

mouse skin metabolic syndrome MR mRNA Nagase et al., 2006

LV, left ventricle ; RV: right ventricle ; K: kidney; SD, Sprague Dawley; SS-Dahl, salt-sensitive Dahl; SHR, spontaneously hypertensive Kyoto-Okamoto strain; BN, Brown-Norway; STZ, streptozotocine-induced diabetes.


et al., 2005; Chai et al., 2010) or atrial fibrillation(De-An et al., 2010; Tsai et al., 2010), in the kidney ofpatients with renal chromophobe renal cell carcinomaand oncocytoma (Yakirevich et al., 2008), and in renalbiopsies of patients with heavy proteinuria (Quinkleret al., 2005).

B. Mechanisms ModulatingMineralocorticoid Receptor

The MR may be subjected to genetic and epigeneticchanges as well as posttranscriptional/posttranslationalalterations, underlying the importance of elucidatingthe regulation of the receptor itself. The mechanismsunderlying alterations of cell/tissue-specific MRexpression or activity include genetic polymorphisms,epigenetic changes, mRNA stabilization/destabilization,and posttranslational modifications of the MR.Human mutations of the MR affecting its function

have been reported. These include loss of functionmutations (Zennaro et al., 2012) leading to neonatalsalt-wasting syndrome, featured by renal type I pseu-dohypoaldosteronism, and a gain of function mutationleading to severe hypertension (Geller et al., 2000).Mutations affecting downstream pathways also affectrenal sodium handling and blood pressure levels (Liftonet al., 2001; Shibata et al., 2013). In addition, variousgenetic determinants can contribute to variations inplasma aldosterone (Zennaro et al., 2013). Becauseexcellent reviews have detailed these features, theywill not be addressed here.Several functional MR polymorphisms (i.e., affecting

its activity or expression levels) have been identified, asreviewed by Dalila et al. (2015). The MRI180V (rs5522)polymorphism results in an amino acid change in the Nterminal domain of the MR, which is present in 7–13%of the population and affects MR activity and ligand-mediated MR activation in a reporter cell system(DeRijk et al., 2006). The MR-2GC (rs2070951) poly-morphism is located inside the Kozak translationregulatory sequence and affects MR translation effi-ciency and MR expression level (van Leeuwen et al.,2011). It is present in 50% of the population. MR genehaplotypes constituted of MR-2GC and I180V polymor-phisms strongly modulate cortisol-induced MR tran-scription and protein expression (van Leeuwen et al.,2011). Some studies analyzed the association be-tween the presence of MR polymorphisms and dis-eases: MR polymorphisms are correlated to obesityand low-density lipoprotein-cholesterol (I180V) orblood pressure (I180V and MR-2GC) (Fernandes-Rosa et al., 2010). The MRI180V polymorphism wasshown to affect blood pressure response to enalapriltreatment and may serve as a useful pharmacogenomicmarker of antihypertensive response to enalapril inessential hypertension patients (Luo et al., 2014). TheMR-2GC polymorphism was associated with higherhyperkaliemic response to spironolactone in heart failure

patients (Cavallari et al., 2010). The rs3857080 poly-morphism, localized in intron 3, was associated withurinary electrolyte excretion (Dalila et al., 2015). Thispolymorphism was previously associated with nocturnalsystolic blood pressure (Tobin et al., 2008).

Epigenetic events may modulate nuclear receptors,leading to deregulation of their activity. In uteroexposure to di-(2-ethylhexyl) phtalate leads to loss ofmethylation of MR gene promotor and reduced MRmRNA expression in testes (Martinez-Arguelles et al.,2009). Histone deacetylase 3 and 4 complexes have beenreported to regulate MR transcriptional activity incultured cells (Lee et al., 2015). Epigenetic mechanismmay regulate MR expression via expression of micro-RNAs (miRNAs/miRs) and short regulatory RNAs. Insilico prediction identified the MR as a target of miR-135a and miR-124 (Sober et al., 2010; Mannironi et al.,2013). These miRNAs were shown to decrease MRtransactivation in a reporter assay (Sober et al., 2010;Mannironi et al., 2013). Upregulation of miRNA 124was also associated with a decrease inMR expression inpodocytes upon mechanical stretch (Li et al., 2013).Interestingly a polymorphism (rs5534) located in the39 untranslated region of the MR was recently identifiedand predicted to modify hsa-miR-383 binding andpossibly involved in the decrease of MR expressionmediated by this miRNA. This polymorphism wasassociated with an increased risk of myocardial in-farction (Nossent et al., 2011). Whether and how suchphenomena could participate in pathologic MR down-regulation remain to be explored.

Posttranscriptional events modulating MR activityhave been reviewed (Faresse, 2014). These includephosphorylation, ubiquitylation, sumoylation, and oxi-dation. Posttranscriptional modifications affecting thestability of the mRNAs encoding the MR have beenreported in the kidney. Hypertonicity induces themRNA-destabilizing protein Tis11b, which interactswith the adenylate/uridylate-rich elements present inthe 39 untranslated region of the MRmRNA, increasingMR mRNA turnover and accelerating mRNA destabili-zation, therefore reducingMR expression (Viengchareunet al., 2014). Ubiquitylation of the MR has been demon-strated to be modulated by phosphorylation, leading toenhanced MR degradation and therefore affecting MRsignaling (Faresse et al., 2010, 2012). Whether thisphenomenon occurs in vivo remains to be demonstrated.A spontaneous mutation of a potential phosphorylationsite of the MR in Brown Norway rats (Y73C) has beenidentified as a gain of function mutation modulatingtransactivation activity in the presence of aldosterone(and progesterone) (Marissal-Arvy et al., 2004). Phos-phorylation of a critical residue in the ligand bindingdomain preventing ligand-mediated MR activation hasbeen identified in the renal intercalated cell but itsconsequences on MR stability have not been reported(Shibata et al., 2013).


XII. Conclusion and Open Questions

The notion of excessive MR activity leading to organpathology is expanding rapidly, raising the possibility ofseveral unexpected novel indications of MR antago-nism. However, several aspects of MR action in diseaseremain to be elucidated.It is important to recall that the transactivation

activity of the MR is highly dependent on the natureof the bound ligand (Hellal-Levy et al., 1999; Fulleret al., 2012). Each ligand (agonist or antagonist) inducesa unique conformational change that drives interac-tions with receptor coregulators and tissue-specifictranscriptional factors. Thus the ligand-receptor com-plexes may have distinct (sometimes opposite) tissue-specific target genes and therefore distinct downstreameffects depending on the steroid accommodated in theligand binding pocket of the MR.In pathologic situations, it is often considered that

glucocorticoids most likely occupy the MR rather thanaldosterone. In addition, the amount of ligand activat-ing the MR in target cells may be different from theirplasma concentrations. Indeed aldosterone or glucocor-ticoids may be secreted locally, and local productionmay be altered (reduced or increased) in pathologicsituations (Taves et al., 2011).Glucocorticoids may be inactivated within a tissue in

the presence of 11HSD2; conversely, the enzyme11HSD1 (reductase) allows regeneration of active glu-cocorticoids from dehydrogenated inactive forms. Thecellular cortisol can originate from circulating cortisone,thus providing excessive MR activation. Regulation of11HSD2-11HSD1 expression and activity, which couldcontribute significantly to altered MR signaling, are farfrom being completely elucidated, particularly in thecontext of diseases (Odermatt and Kratschmar, 2012;Chapman et al., 2013) .Another issue possibly modifying local MR ligands or

antagonists, in a specific cell context, is related to effluxpumps that limit cellular accumulation and thereforebiologic activity. Although corticosteroids exhibitlipophilic structures favoring their diffusion throughthe cell membrane, their entry or efflux into and out ofcells is influenced by ATP-binding cassette transporters(ABC transporters) including P-gP/multidrug resis-tance (MDR), or MDR-associated proteins. Severalexcellent reviews detail the characteristics of the su-perfamily of efflux pumps (Deeley et al., 2006; Klaassenand Aleksunes, 2010). There is a wide variation intissue distribution, species differences, and sex speci-ficity of efflux pump expression (Cui et al., 2009), butthere is limited literature on the functional interactionsof P-gP/MDR and corticosteroid hormones. Interest inthe role of efflux pumps to regulate corticosteroidhormone functions has been renewed by the evidencethat these pumps are important to restrain access ofcorticosteroid hormones to the brain (Geerling and

Loewy, 2009). P-gP can transport aldosterone, cortisol,dexamethasone, and corticosterone (Ueda et al., 1992).Spironolactone could interfere with efflux pumps, thusmodifying accumulation/elimination of corticosteroidsin cells. Whether and how efflux pumps modify cortico-steroids or MRA concentrations in specific tissues inpathology are open questions. The involvement of ABCpumps to regulate blood pressure and salt sensitivitywasrecently addressed with specific focus on biologic inter-actions between P-gP and drugs influencing the renin-angiotensin-aldosterone system (Bochud et al., 2011).The search for genetic variants of P-gP/MDR genes inaldosterone-related diseases, other than hypertension,may be a worthwhile avenue of investigation.

In conclusion, we reported some aspects of inappro-priate MR activation in cardiovascular, renal, meta-bolic, ocular, and skin diseases that illustrate situationswhere MR blockade is now recognized to bring clearclinical benefit or should do so in the near future. Thislist is not complete (for instance the impact of MRA inthe central nervous system and brain diseases has notbeen addressed) and we apologize for the publicationsthat have not been cited because of space limitations.Based upon the recent and impressive accumulation ofknowledge, including novelMR target diseases, a newerafor pharmacological MR antagonism is open in medicine.

Acknowledgments

This review is dedicated to our mentor and friend Jean PierreBonvalet, who sadly recently died. We are indebted to Brian Harveyfor extensive editing of the manuscript. We wish to thank JulieFaugeroux for expert immunohistochemistry experiments and CelsoGomez-Sanchez for gift of MR antibodies.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Jaisser,Farman.

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