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Home , Mineralocorticoid

International Journal of Molecular Sciences

Conference Report and Mineralocorticoid Receptors—Physiology and Pathophysiology

John W. Funder

Hudson Institute of Medical Research, Monash University, 27–31 Wright St., Clayton 3168, Australia; [email protected]

Academic Editors: Anastasia Susie Mihailidou, Jan Danser, Sadayoshi Ito, Fumitoshi Satoh and Akira Nishiyama Received: 8 March 2017; Accepted: 4 May 2017; Published: 11 May 2017

Abstract: Aldosterone is a uniquely terrestrial hormone, first appearing in lungfish, which have both gills and lungs. Mineralocorticoid receptors (MRs), on the other hand, evolved much earlier, and are found in cartilaginous and bony fish, presumptive ligand . MRs have equivalent high affinity for aldosterone, , and cortisol; in epithelia, despite much higher cortisol circulating levels, aldosterone selectively activates MRs by co-expression of the enzyme 11β-hydroxysteroid dehydrogenase, Type 11. In tissues in which the enzyme is not expressed, MRs are overwhelmingly occupied but not activated by cortisol, which normally thus acts as an MR antagonist; in tissue damage, however, cortisol mimics aldosterone and acts as an MR agonist. The risk profile for primary aldosteronism (PA) is much higher than that in age-, sex-, and pressure-matched essential hypertensives. High levels of aldosterone per se are not the problem: in chronic deficiency, as seen in the monsoon season in the highlands of New Guinea, plasma aldosterone levels are extraordinarily high, but cause neither nor cardiovascular damage. Such damage occurs when aldosterone levels are out of the normal feedback control, and are inappropriately elevated for the salt status of the individual (or experimental animal). The question thus remains of how excess salt can synergize with elevated aldosterone levels to produce deleterious cardiovascular effects. One possible mechanism is through the agency of the elusive ouabain-like factors (OLFs). Such factors are secreted from the adrenal in response to ACTH (adrenalocortical tropic hormone), to via AT2R, and—the polar opposite of aldosterone—to sodium loading. They act on blood vessels to cause vasoconstriction and thus elevate to dump excess sodium through pressure natriuresis. Their levels are chronically elevated in PA in response to the continually elevated sodium status, and they thus act to constrict coronary and systemic arteries. In the context of the elevated and total body sodium in a PA patient, this raises blood pressure and acts as the proximate cause of cardiovascular damage. If this is the case, it would appear to offer new insights into therapy for PA. One would be the use of digibindin, or its more recent successors as antagonists of OLFs acting on Na/K ATPase at the vessel wall. A second would be to routinely combine a low dose MR antagonist, an ENaC inhibitor, and sodium restriction as first-line therapy for bilateral aldosterone overproduction. Finally, for unilateral cases post-surgery, there is good reason to include low-dose MRs in therapy if required, given the ability of cortisol in damaged blood vessels to mimic aldosterone vasoconstrictor action.

Keywords: endogenous ouabain; cortisol; sodium and primary aldosteronism

1. Physiology Commonly, the physiology of aldosterone and mineralocorticoid receptors (MRs) begins (and sometimes, unfortunately ends) with the action of aldosterone via renal MRs to retain sodium (as well as ) and to excrete . This is not surprising, given that aldosterone is characterized

Int. J. Mol. Sci. 2017, 18, 1032; doi:10.3390/ijms18051032 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 1032 2 of 9 Int. J. Mol. Sci. 2017, 18, 1032 2 of 9 by the demonstration of its effects on transepithelial transport and what was initially termed by the demonstration of its effects on transepithelial electrolyte transport and what was initially “electrocortin” [1]. Similarly, the first identification of high affinity receptors for aldosterone occurred termed “electrocortin” [1]. Similarly, the first identification of high affinity receptors for aldosterone in vitro studies on rat kidney slices [2]. Again, these were initially termed “Type 1 occurred in vitro studies on rat kidney slices [2]. Again, these were initially termed “Type 1 receptors”,corticosteroid in contrast receptors”, with the in contrast -binding with the dexamethasone-binding Type 2 receptors; Type now Type2 receptors; 1 and Type now 2Type are 1 knownand Type as MRs 2 are and known as MRs and receptors glucocorticoid (GRs). Accordingly,receptors (GRs). for aAccordingly, considerably for nephrological a considerably audience,nephrological the focus audience, of the currentthe focus section of the will current be on section lesser-known will be aspects on lesser-known of aldosterone aspects and of MRaldosterone physiology. and MR physiology. In allIn all ligand– ligand–receptor systems, systems, an an obvious obvious question question is which is which came came first, first, signal signal or receptor—chickenor receptor—chicken or egg.or egg. For For aldosterone aldosterone and MRs,and theMRs, answer the answer is unequivocal: is unequivocal: the MRs precededthe MRs aldosteronepreceded aldosterone by millions by of years.millions Figure of years.1 shows Figure a 1 dendrogram shows a dendrogram of the evolution of the evolution of the tight-knitof the tight-knit MRs/GRs/progesterone MRs/GRs/progesterone receptorsreceptors (PRs)/ (PRs)/androgen receptors receptors (ARs) (ARs) subsequently subsequently from from a common a common ancestor. ancestor. The The first first of theof the four four to branchto branch off off is the is the primordial primordial MRs, MRs, as shown as shown in Figure in Figure1; the 1; inset the inset details details its presence its presence in a number in a number of species,of species, including including bony bony fish and fish cartilaginous and cartilaginous species species such assuch sharks as sharks and rays and [ 3rays]. [3].

Figure 1. Dendrogram of evolution of mineralocorticoid receptors (MRs). Glucocorticoid receptors Figure 1. Dendrogram of evolution of mineralocorticoid receptors (MRs). Glucocorticoid receptors (GRs), progesterone receptors (PRs), and androgen receptors (ARs) from common ancestral . (GRs), progesterone receptors (PRs), and androgen receptors (ARs) from common ancestral protein. Inset: MRs across different species expanded. Redrawn from Kassahn et al., 2011 [3]. Inset: MRs across different species expanded. Redrawn from Kassahn et al., 2011 [3]. In contrast, Figure 2 shows the first creature in which aldosterone appears, the lungfish. These animals,In contrast, as their Figure name2 implies,shows thehave first both creature gills and in lungs, which marking aldosterone the transition appears, from the lungfish. an obligate Theseaquatic animals, to a as terrestrial their name milieu. implies, The have first both thing gills andthatlungs, one needs marking to theknow transition about frommineralocorticoid an obligate aquaticreceptors to a terrestrial is that they milieu. evolved The firstmillions thing of that years one be needsfore aldosterone to know about did, mineralocorticoid that their early receptorsphysiologic is thatroles they are evolvednot yet well-defined, millions of years that beforetheir probable aldosterone but not did, certain that their ligand early was physiologic cortisol, and roles that are we notavoid yet well-defined, these insights that on theirevolution probable at our but peril. not certain ligand was cortisol, and that we avoid these insights on evolution at our peril.

Int. J. Mol. Sci. 2017, 18, 1032 3 of 9 Int. J. Mol. Sci. 2017, 18, 1032 3 of 9

FigureFigure 2. 2.The The lungfish, lungfish, firstfirst creaturecreature to to make make aldosterone. aldosterone.

The second thing one needs to know is that MRs are truly promiscuous. We all know that they Thebind secondaldosterone thing with one high needs affinity: to in know fact, they is that have MRs equivalent are truly high promiscuous.affinity for a range We of — all know that theyaldosterone, bind aldosterone cortisol, with , high affinity: deoxycor in fact,ticosterone they have (DOC), equivalent and progesterone. high affinity Given for athe range of steroids—aldosterone,emergence of aldosterone cortisol, as the corticosterone,physiologic mine deoxycorticosteroneralocorticoid, our terrestrial (DOC), MRs and might progesterone. have Givenundergone the emergence mutation of aldosteroneso that they no as longer the physiologic recognized mineralocorticoid,cortisol. This did not ourhappen terrestrial over the MRs eons, might haveand undergone we must mutation thus face sowhy that contemporary they no longer MRs recognized remain promiscuous cortisol. This and didwhat not this happen means overfor the eons,physiology and we must and thuspathophysiology. face why contemporary MRs remain promiscuous and what this means for For some years before characterization of aldosterone DOC was the ‘gold standard’ physiology and pathophysiology. mineralocorticoid; in terms of physiology, it appears to be of no real importance in this role. Under For some years before characterization of aldosterone DOC was the ‘gold standard’ normal circumstances its plasma levels are low, about the same as those of aldosterone, but the secretion mineralocorticoid;of DOC, unlike that in termsof aldosterone, of physiology, is not in response it appears to sodium to be deficiency of no real or importancevolume depletion in this [4]. role. UnderIn normaladdition, circumstances although DOC itsand plasma aldosterone levels have are simi low,lar about high affinity the same for MRs as those [5], whereas of aldosterone, aldosterone but the secretioncirculates of DOC, ~50% bound unlike to that plasma of aldosterone,protein, DOC is is 94–99% not in protein-bound: response to sodiumin vivo it deficiencyhas approximately or volume depletionone-tenth [4]. Into addition,one-fortieth although the mineralocorticoid DOC and aldosterone potency of have aldosterone similar highon a affinitydose basis for MRs[6]. [5], whereasPathophysiologically, aldosterone circulates however, ~50% it boundmay come to plasma into play protein, in the DOC ectopic is 94–99% secretion protein-bound: of ACTH fromin vivo it hasmalignancies, approximately where one-tenth the normal to one-fortieth feedback loop the between mineralocorticoid adrenal and potencyanterior pituitary of aldosterone is no longer on a dose basisoperant. [6]. Pathophysiologically, however, it may come into play in the ectopic secretion of ACTH Corticosterone is the physiologic glucocorticoid in rats and most strains of mice, where its roles from malignancies, where the normal feedback loop between adrenal and is no are (presumably) parallel those of cortisol in humans. Particular roles for circulating corticosterone longer operant. distinct from those of cortisol in humans have been suggested but are yet to be proven. Progesterone is Corticosteronealso highly protein is bound, the physiologic but in pregnancy glucocorticoid circulates at in concentrations rats and most sufficient strains to of cause mice, a natriuresis, where its roles are (presumably)elevating levels parallel of aldosterone those 3–10-fo of cortisolld in response. in humans. Much Particular lesser elevat rolesions for of circulating circulating progesterone corticosterone distinctin the from luteal those phase of cortisolof the menstrual in humans cycle have have been beensuggested shown to raise but plasma are yet aldosterone to be proven. concentrations Progesterone is also highlyin some protein women bound, to false-positive but in pregnancy aldosterone/ circulatesrenin ratios at concentrationsfor primary aldosteronism sufficient [7]. tocause a natriuresis, elevatingWhether levels of the aldosterone progesterone 3–10-fold of pregnancy in response. has a physio Muchlogic lesser role elevations as an MR ofantagonist circulating has progesteronenot been in theexplored. luteal phase It may of thebe that menstrual its MR cycleantagonist have acti beenon, shown the aldosterone to raise plasma elevation aldosterone in response, concentrations and the in someconsequent women reset to false-positive of fluid and electrolyte aldosterone/renin homeostasis, ratios is nothing for primary more than aldosteronism the biologic equivalent [7]. of collateral damage. It may also, however, speak to an as yet unrecognized physiologic role, again Whether the progesterone of pregnancy has a physiologic role as an MR antagonist has not been given the maintained high affinity of MRs for progesterone over evolution. What has been reported explored.is a single It may nucleotide be that itsmutation MR antagonist in the action, encoding the aldosterone the human elevationMR; the inmutant response, MR sees and the consequentprogesterone reset ofas fluidan MR and agonist, electrolyte resulting homeostasis, in early onset is nothing hypertension more thanvery themuch biologic exacerbated equivalent by of collateralpregnancy damage. [8]. It may also, however, speak to an as yet unrecognized physiologic role, again given the maintainedWhich highbrings affinity us to ofcortisol, MRs forcirculating progesterone at plas overma total evolution. levels ~1000-fold What has those been of reported aldosterone, is a single nucleotideand plasma mutation levels in “only” the gene ~100-fold encoding higher, the given human the MR; much the higher mutant levels MR of sees cortisol progesterone binding (~95%) as an MR agonist,to plasma resulting protein. in early The onset remarkable hypertension thing about very cortisol much exacerbatedin its relationship by pregnancy with MRs [ 8is]. that it is Whichbivalent. brings Normally, us to it cortisol,is antagonist, circulating in that it at binds plasma but totaldoes levelsnot activate ~1000-fold MRs in thosethe same of aldosterone,way as and plasma levels “only” ~100-fold higher, given the much higher levels of cortisol binding (~95%) to plasma protein. The remarkable thing about cortisol in its relationship with MRs is that it is bivalent. Normally, it is antagonist, in that it binds but does not activate MRs in the same way as aldosterone Int. J. Mol. Sci. 2017, 18, 1032 4 of 9 does; in the context, however, of tissue damage, reactive oxygen species generation and redox change cortisol becomes an MR agonist, mimicking aldosterone, as detailed below. In epithelia, and in a few non-epithelial tissues (vessel wall, nucleus tractus solitarius), Int. J. Mol. Sci. 2017, 18, 1032 4 of 9 Int. J. Mol. Sci. 2017, 18, 1032 4 of 9 MRs are protected from activation by cortisol via the twin actions of the enzyme 11βhydroxysteroid dehydrogenasealdosteronealdosterone (11 does;does;βHSD2). inin thethe In context,context, addition however,however, to converting ofof tisstissueue damage,damage, cortisol reactivereactive to receptor-inactive oxygenoxygen speciesspecies generationgeneration , andand for every moleculeredoxredox of cortisone changechange cortisolcortisol so produced, becomesbecomes anan a MRMR molecule agonist,agonist, of mimickingmimicking NAD is aldosterone,aldosterone, reduced to asas NADH. detaileddetailed Metabolismbelow.below. of cortisol debulks aldosteroneInIn epithelia,epithelia, target andand inin tissues aa fewfew non-epithelial bynon-epithelial a factor of tissuestissues ~10, still(vessel(vessel a ~10-fold wall,wall, nucleusnucleus excess tractustractus of cortisol solitarius),solitarius), over MRsMRs aldosterone, areare protected from activation by cortisol via the twin actions of the enzyme 11βhydroxysteroid such thatprotected the majority from activation of MRs areby cortisol occupied via butthe nottwin activatedactions of bythe cortisol.enzyme 11 Whatβhydroxysteroid appears to hold dehydrogenasedehydrogenase (11 (11ββHSD2).HSD2). In In addition addition to to converting converting cortisol cortisol to to receptor-inactive receptor-inactive cortisone, cortisone, for for every every cortisol-MR complexes inactive is in high levels of NADH generated, as previously reported for moleculemolecule ofof cortisonecortisone soso produced,produced, aa moleculemolecule ofof NADNAD isis reducedreduced toto NADH.NADH. MetabolismMetabolism ofof cortisolcortisol β the corepressordebulksdebulks aldosterone aldosterone c-terminal-binding target target tissues tissues by proteinby a a factor factor (CtBP)of of ~10, ~10, still still [9 ].a a ~10-fold ~10-fold When excess excess 11 HSD2of of co cortisolrtisol is over over deficient aldosterone, aldosterone, or blocked, both ofsuchsuch these thatthat ‘protective’ thethe majoritymajoritymechanisms ofof MRsMRs areare occupiedoccupied fail, and butbut cortisol notnot activatedactivated activates byby cortisol.cortisol. principal WhatWhat cellappearsappears MRs. toto holdhold In tubular intercalatedcortisol-MRcortisol-MR cells, MRscomplexescomplexes but not inactiveinactive 11β isHSD2is inin highhigh is levelslevels expressed, ofof NADHNADH and generated,generated, the MRs asas ‘protected’ previouslypreviously reportedreported by phosphorylation forfor thethe corepressor c-terminal-binding protein (CtBP) [9]. When 11βHSD2 is deficient or blocked, both of at serinecorepressor843 [10]. When, c-terminal-binding however, theyprotein are (CtBP) dephosphorylated [9]. When 11βHSD2 in responseis deficient toor angiotensin,blocked, both of they are thesethese ‘protective’‘protective’ mechanismsmechanisms failfail,, andand cortisolcortisol activatesactivates principalprincipal cellcell MRs.MRs. InIn tubulartubular intercalatedintercalated activated (experimentally) by aldosterone or cortisol—and, physiologically speaking, presumably843 by cells,cells, MRsMRs butbut notnot 1111ββHSD2HSD2 isis expressed,expressed, andand thethe MRsMRs ‘pro‘protected’tected’ byby phosphorylationphosphorylation atat serineserine843 cortisol given its orders of magnitude higher levels [11]. [10].[10]. When,When, however,however, theythey areare dephosphorylateddephosphorylated inin responseresponse toto angiotensin,angiotensin, theythey areare activatedactivated An(experimentally)(experimentally) illustration of howbyby aldosteronealdosterone cortisol mimics oror cortisol—and,cortisol—and, aldosterone physiologicallyphysiologically is the study speaking,speaking, by Mihailidou presumablypresumably et al,byby cortisolexploitingcortisol the Langendorfgivengiven ratitsits ordersorders heart ofof model magnitudemagnitude of experimental higherhigher levelslevels [11].[11]. ischemia followed by reperfusion. Cardiomyocytes express MRsAnAn but illustrationillustration not 11 β ofofHSD2, howhow cortisolcortisol so the mimicsmimics possibility aldosteronealdosterone of a isis physiologic thethe studystudy byby roleMihailidouMihailidou for aldosterone etet al,al, exploitingexploiting is remote. the Langendorf rat heart model of experimental ischemia followed by reperfusion. Cardiomyocytes Experimentally,the Langendorf as shown rat heart in Figuremodel of3, experimental aldosterone is aggravateschemia followed ischemia–reperfusion by reperfusion. Cardiomyocytes induced infarct express MRs but not 11βHSD2, so the possibility of a physiologic role for aldosterone is remote. size, as hasexpress been MRs previously but not 11 reportedβHSD2, [so12 the]. What possibility is novel, of a however,physiologic is role that for cortisol aldosterone at low is doses remote. similarly Experimentally,Experimentally, asas shownshown inin FigureFigure 3,3, aldosteronealdosterone aggravatesaggravates ischemia–reperfusionischemia–reperfusion inducedinduced infarctinfarct aggravatessize,size, tissue asas hashas damage beenbeen previouslypreviously (Figure reported4reported); that this[12].[12]. action WhatWhat isis novel,novel, via MRs however,however, and not isis thatthat GRs cortisolcortisol is attested atat lowlow to dosesdoses not merely by the dose,similarlysimilarly in that aggravatesaggravates 10 and tissuetissue 100 damagenMdamage are (Figure equipotent,(Figure 4);4); thatthatbut thisthis also actionaction by isis the viavia MRs abilityMRs andand of notnot —not GRsGRs isis attestedattested toto the GR or thenotnot PR merelymerely antagonist byby thethe RU486, dose,dose, inin to thatthat reverse 1010 andand the 100 effect100 nMnM of areare cortisol equipotent,equipotent, [13]. butbut alsoalso byby thethe abilityability ofof spironolactone—notspironolactone—not thethe GRGR oror thethe PRPR antagonistantagonist RU486,RU486, toto reversereverse thethe effecteffect ofof cortisolcortisol [13].[13].

Figure 3. Aldosterone increases infarct size in Langendorf ischemia-reperfusion rat heart Figure 3.FigureAldosterone 3. Aldosterone increases increases infarct sizeinfarct in Langendorfsize in Langendorf ischemia-reperfusion ischemia-reperfusion rat heart rat preparations.heart preparations.preparations. nn valuesvalues inin parentheses;parentheses; Ald:Ald: aldosterone;aldosterone; SPIRO:SPIRO: spironolactone;spironolactone; fromfrom MihailidouMihailidou etet n values in parentheses; Ald: aldosterone; SPIRO: spironolactone; from Mihailidou et al., 2009 [13]. al.,al., 20092009 [13].[13].

FigureFigure 4.4. CortisolCortisol increasesincreases infarctinfarct sizesize inin LangendorfLangendorf ischemia-reperfusionischemia-reperfusion ratrat heartheart preparations.*preparations.* pp Figure 4.<< 0.05,0.05,Cortisol nn valuesvalues increases inin parentheses;parentheses; infarct SPIROSPIRO size spironolinspironol Langendorfactone;actone; fromfrom ischemia-reperfusion MihailidouMihailidou etet al.,al., 20092009 [13].rat[13]. heart preparations. p n * < 0.05, values in parentheses; SPIRO spironolactone; from Mihailidou et al., 2009 [13]. Int. J. Mol. Sci. 2017, 18, 1032 5 of 9

Finally,Int. J. Mol. Sci. one 2017 of, 18 the, 1032 enigmas of the RALES study [14] showing the remarkable effects5 of 9 of spironolactone added to standard of care on mortality and morbidity in congestive heart failure Finally, one of the enigmas of the RALES study [14] showing the remarkable effects of was the very modest average dose of spironolactone (26 mg/day) used. The effects were dramatic spironolactone added to standard of care on mortality and morbidity in congestive heart failure was (a 30% reduction in mortality, leading to the trial being stopped halfway through), and 35% the very modest average dose of spironolactone (26 mg/day) used. The effects were dramatic (a 30% fewerreduction hospitalizations. in mortality, Plasma leading aldosterone to the trial concentrations being stopped were halfway in the through), low–normal and range,35% fewer and the unprotectedhospitalizations. MRs were Plasma activated aldosterone by normal concentrations levels ofwere cortisol: in the howlow–normal is it, then, range, that and low-dose the spironolactoneunprotected proved MRs were so efficacious? activated by normal levels of cortisol: how is it, then, that low-dose Aspironolactone clue to theanswer proved so may efficacious? lie in Figure 5, where the effects of spironolactone absent any other in infarctA clue to size the and answer area may at risk lie arein Figure shown 5, in wher thee Langendorf the effects of model spironolactone [10]. Whether absent the any Langendorf other preparationsteroid wasin infarct from size intact and rats area maintained at risk are on shown tap water, in the or Langendorf from rats adrenalectomizedmodel [10]. Whether one the week previouslyLangendorf and maintained preparation on was 0.9% from NaCl intact to rats drink, maintained spironolactone on tap water, reduced or from both rats infarct adrenalectomized area (Figure 5A) one week previously and maintained on 0.9% NaCl to drink, spironolactone reduced both infarct and apoptotic index (Figure5B). The rats adrenalectomized (to exclude any residual corticosteroid) area (Figure 5A) and apoptotic index (Figure 5B). The rats adrenalectomized (to exclude any residual are understandably more fragile than intact rats, but still show significantly lower levels with corticosteroid) are understandably more fragile than intact rats, but still show significantly lower spironolactone:levels with spironolactone: in both cases, in the both effect cases, is the due effe toct spironolactone is due to spironolactone acting as acting an inverse as an inverse agonist to reduceagonist cell death to reduce at the cell infarct death at margin. the infarct What margin. this meansWhat this is thatmeans spironolactone is that spironolactone does not does act not merely act as a “blocker”,merely as a there “blocker”, is no corticosteroidas there is no corticosteroid to block, but to acts block, putatively but acts asputatively an intracellular as an intracellular antagonist of MR activationantagonistbeyond of MR activation the receptor. beyond the receptor.

Figure 5. Spironolactone lowers infarct size (A) and apoptotic index (B) in Langendorf ischemia- Figure 5. A B reperfusionSpironolactone rat heart preparations, lowers infarct in both size adrenal ( ) and intact apoptotic rats (Columns index 3 & ( 4)) inand Langendorf adrenalectomized ischemia- reperfusionrats (Columns rat heart 5 & preparations, 6) * significantlydifferent in both adrenal from intact IR baseline rats (Columns ischemia-reperfusion, 3 & 4) and adrenalectomized† significantly rats (Columnsdifferent from 5 & adrx 6) * alone; significantlydifferent from Mihailidou et from al., 2009 IR baseline[13]. ischemia-reperfusion, † significantly different from adrx alone; from Mihailidou et al., 2009 [13]. 2. Pathophysiology 2. PathophysiologyOver a decade ago, two studies reported patients with primary aldosteronism as having a higher Overcardiovascular a decade risk ago, profile two studies [15] or reportedwith evidence patients of cardiovascular with primary damage aldosteronism [16] than as age-, having sex- aand higher blood pressure-matched essential hypertensives. The first was from Parisian inpatients with long- cardiovascular risk profile [15] or with evidence of cardiovascular damage [16] than age-, sex- and blood standing blood pressure elevation; those with primary aldosteronism had a 4.2 times higher risk of pressure-matchedstroke, 6.5 times essential the risk hypertensives. of non-fatal myocardial The first wasinfarct, from and Parisian 12.1 times inpatients the prevalence with long-standing of atrial bloodfibrillation pressure [15]. elevation; those with primary aldosteronism had a 4.2 times higher risk of stroke, 6.5 times theThe risk second of non-fatal study, myocardialpublished at infarct, the same and 12.1 time, times addresses the prevalence the effects of atrial of fibrillationvery early [15]. Thehyperaldosteronism second study, [16]. published Michael Stowasser at the and same his colleagues time, addresses compared eight the young, effects normotensive of very early hyperaldosteronismpatients with proven [16]. Michaelfamilial Stowasserhyperaldosteroni and hissm, colleagues Type 1, case-matched compared eight with young,24 normotensive normotensive patientscontrols. with provenThose with familial primary , aldosteronism had Typea thicker 1, case-matched left ventricular with wall 24and normotensive reduced diastolic controls. Thosefunction with primary compared aldosteronism with the controls, had evidence a thicker that left inappropriate ventricular wallaldosterone and reduced levels are diastolic deleterious, function even in the absence of elevated blood pressure. compared with the controls, evidence that inappropriate aldosterone levels are deleterious, even in the absence of elevated blood pressure. Int. J. Mol. Sci. 2017, 18, 1032 6 of 9

The keyword here is inappropriate. In the highlands of New Guinea, yams are the staple food, and in the monsoon season, the rains leach the minerals from the soil. As a result, the average daily intake of sodium is very low, and excretion commonly 2–3 meq/day. These subjects have low normal blood pressure, no cardiovascular damage, and extraordinarily high plasma aldosterone concentrations. What this means is that physiologically circulating aldosterone levels can be very high, in homeostatic mode responding to sodium deficiency, without the deleterious effects seen in primary aldosterone patients with much lower levels of plasma aldosterone. Here, the keyword again is appropriate. When very high aldosterone levels are appropriate for the degree of sodium deficiency, they are homeostatic—“a good thing”. When aldosterone levels are out of the normal feedback control loop, and inappropriate for sodium status, cardiovascular damage ensues—“a bad thing”. Currently, our clinical focus is on aldosterone as the culprit, with posited direct effects on the heart and on blood vessels, producing early and late indices of cardiovascular damage detailed above [12,13]. What has been neglected is the obvious crucial role of salt—and the fact that, without salt, even sky-high aldosterone levels are benign. One possible mechanism to explain this apparent dilemma is the agency of the elusive endogenous ouabain (EO), or ouabain-like compounds (e.g., marinobufagenin), hereafter referred to as EO. Secretion of these compounds is from the at very low levels, similar to those of aldosterone. ACTH increases the secretion of EO, and ACTH-driven experimental hypertension is reversed by the administration of digibindin [17], as its name implies an EO blocker. Aldosterone is similarly elevated by ACTH, to which it is particularly sensitive in ~25% of hypertensives, elevating aldosterone at doses too low to increase cortisol [18]. The two diverge to a degree in terms of their responsiveness to angiotensin. Angiotensin II stimulates secretion of both, but crucially through different receptors—AT1R for aldosterone, AT2R for EO [19], an important difference given the order of magnitude of lower affinity of the AT2R. The final divergence is total, and crucial to the presumed physiologic rule of EO, and its possible role in primary aldosteronism, EO secretion is increased by sodium loading, whereas that of aldosterone is decreased, a polar opposite effect [20]. + + Like exogenous ouabain, EO inhibits the α2 subunit of the Na /K pump. In kidney tubules, this reduces sodium reabsorption; in vascular smooth muscle cells, it induces Na+ and Ca2+ accumulation, vasoconstriction and systemic hypertension [21]. In terms of physiology then, its role would appear to be to increase natriuresis, pressure-induced via vasoconstriction, directly at the level of the renal tubule, in a homeostatic response to sodium loading. Pathophysiologically, however, when aldosterone is out of its normal homeostatic feedback control—which is the case in primary aldosteronism—the inappropriate sodium retention indicates the continued elevation of EO in an attempt at natriuresis. In addition to seeking direct effects of aldosterone on blood vessels—for which there is clear evidence [22,23]—and on the cardiomyocyte—for which (given unprotected MRs) there is not—we thus need to factor in the lessons from New Guinea. When aldosterone levels are elevated in sodium deficiency, they serve to constrict blood vessels to offset, at least in part, the concomitant volume contraction: homeostasis again [24]. Under such circumstances, the secretion of EO is attenuated, and aldosterone does no cardiovascular damage. If this is the case, in part or in full, there are a number of things that flow in terms of the ultimate treatment of PA. Young female patients in whom an aldosterone-producing adenoma (APA) is detected by prompt screening and lateralization have the best chance of completing clinical remission post-operatively. In males and with age, the percentage of APA patients cured by unilateral adrenalectomy falls, so that in a yet unpublished study, the average cure rate was 37% across 12 expert centers using the same criteria for complete clinical remission. Across a range of studies, a biochemical cure—i.e., plasma [K+] and plasma aldosterone concentration (PAC) returning to normal levels—is, commonly almost 100%. In some patients, clinical remission is partial—blood pressure is lower but is still elevated and necessitates . Int. J. Mol. Sci. 2017, 18, 1032 7 of 9

In some patients, remission is missing, with no appreciable fall in blood pressure or in the defined daily dose of . In the former, which is the larger group, persistent hypertension is attributed to pre-existing vascular damage, eminently feasible given the common long period between the onset of hypertension and surgical intervention. In the group with no clinical remission, age clearly plays a role; in subjects with imperfect biochemical remission, the possibility of bilateral nodules unsuspected on imaging cannot be ruled out. The other group, which is much larger than that of patients with APA, is that of patients with bilateral adrenal hyperplasia (BAH). In these patients, primary aldosteronism is commonly less florid, so that, in centers using very straitened cutoffs, the ratio of APA-to-BAH is close to 1; in centers using more relaxed cut-offs, the ratio of verified primary aldosteronism is 1:2 for APA/BAH. Currently, there is mounting evidence that inappropriate aldosterone secretion—also known as primary aldosteronism—may have an overall prevalence of up to 50% in hypertensives [18,25], and of the order of 15–20% in normotensives [26,27]. The current treatment for BAH is relatively low-dose mineralocorticoid receptor antagonists (MRAs)—12.5–50 mg/day for spironolactone and 50–100 mg/day for , with additional agents if and as required to bring blood pressure into the normal range. Neither of the current MRAs is optimal: in men, spironolactone has side effects (gynecomastia and erectile dysfunction), mirroring its AR antagonist activity; in women, effects via activating PRs in the menstrual cycle, and mastodynia. Eplerenone is expensive, and in some jurisdictions reimbursable only for heart failure. Although the percentage of disabling side effects of low-dose spironolactone is not high, it is not popular with patients, and adherence is often a problem. Over the next five years, it is to be hoped that therapy for incomplete remission following APA/BAH is refined in three ways, with the possible emergence of a fourth. First is to recognize that salt is the driver, and that patients in both APA post-surgery and BAH limit their salt intake. The second is the development of potent, selective, non-steroidal MRAs, widely available and recompensable, for patients with BAH. The third is the recognition that, despite their normal aldosterone levels, APA patients with incomplete or missing clinical remission still have cortisol activating their vascular smooth muscle MR—and an MRA should be the first-line hypertensive in such patients, with conventional agents to follow if necessary. The reason that this is not currently the case is ignorance, plus a misnomer. The misnomer is the persisting “aldosterone receptor”: if you have fixed the aldosterone, why pay attention to the receptor? The ignorance is of the evolution, physiology, and pathophysiology of the MRs, even among those who are otherwise experts in managing primary aldosteronism. It is increasingly recognized that MRAs should be used in resistant hypertension and low-renin hypertension (and that in these categories 15–70% of patients may in fact have primary aldosteronism); there are compelling data for eplerenone as a potent hypotensive in essential hypertensives with normal plasma renin, aldosterone, and potassium [28]. In studies on DOCA/salt rats, blood pressure and indices of cardiovascular damage progressively increased [29,30]. Those given DOCA for the first four weeks, but in which salt intake continued until the eighth week, showed blood pressure and fibrosis levels equivalent to those of DOCA/salt rats killed at four weeks. Similarly fed rats (4 weeks DOCA, 8 weeks salt) given eplerenone from weeks 5–8 returned to a normal blood pressure with a resolution of cardiovascular damage. Unless a patient is cured by surgery—and this is likely to be a very small percentage, as inappropriate aldosterone secretion is increasingly recognized as primary aldosteronism—all other patients need to receive low-dose MRAs as a first-line antihypertensive. Now, MRA therapy is spironolactone, eplerenone if affordable, and where available. They also need to halve (probably) their salt intake at the same time. When Generation 3 MRAs (which are non-steroidal, cheap to produce, modestly priced, as potent as spironolactone, and as selective as eplerenone) emerge, they can jettison their existing MRAs. Importantly, Generation 4 MRAs (all the above plus tubule-sparing), the current focus of much of the development, are specifically contra-indicated for this growing group of subjects. Int. J. Mol. Sci. 2017, 18, 1032 8 of 9

The fourth and as yet final possible therapeutic option is that of EO blockers/antagonists. Over 20 years ago, it was reported that “aldosterone antagonists” (sic) inhibited ouabain-like factors, based on in vivo observations: it is probable that this was a secondary effect, mediated via natriuresis. A decade ago, Rostafuroxin (PST 2238) was shown to be a safe and very effective antagonist both of EO- and α-adducin induced hypertension [31]. In more recent animal studies [32], Rostafuroxin has been shown to be renoprotective, blunting EO-induced changes in renal ischemia-induced injury. Whether or not EO antagonists might have a place in the management of PA is yet to be explored.

Conflicts of Interest: The author declares no conflict of interest.

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