Heart Failure Reviews, 10, 53–62, 2005

C© 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.

Effect of Aldosterone and MR Blockade on the Brainand the Kidney

Charles T. Stier, Jr., PhD1, Ricardo Rocha, MD3

and Praveen N. Chander, MD2

Departments of 1Pharmacology and 2Pathology, New YorkMedical College, Valhalla, NY 10595, USA; 3NovartisPharmaceuticals Corp., East Hanover, NJ 07936, USA

Abstract. The renin-angiotensin-aldosterone system (RAAS)

plays a central role in the development of hypertension

and the progression of end-organ damage. Although

angiotensin-I converting enzyme (ACE) inhibitors and an-

giotensin II (Ang II) subtype-1 (AT1) receptor antagonists

can initially suppress plasma aldosterone, it is now well

established that aldosterone escape may occur whereby

aldosterone levels return to, or exceed, baseline levels. The

classical effects of aldosterone relate mainly to its action

on epithelial cells to regulate water and electrolyte balance.

However, the presence of mineralocorticoid receptors (MR)

at nonepithelial sites in the brain, heart and vasculature,

is consonant with the fact that aldosterone also has direct

effects in these tissues. Substantial evidence now exists

that supports the action of aldosterone at non-epithelial

sites which in turn provokes a number of deleterious

effects on the cardiovascular system including necrosis

and fibrosis of the vasculature and the heart, vascular

stiffening and injury, reduced fibrinolysis, endothelial

dysfunction, catecholamine release and production of

cardiac arrhythmias. Several studies have now shown that

vascular and target-organ protective effects of MR antag-

onism occurs in the absence of significant blood pressure

lowering or fluid loss, which is consistent with a major role

for endogenous mineralocorticoids as direct mediators of

cardiovascular injury. Adverse cardiovascular effects may

occur in response to aldosterone alone, activation of the

RAAS or aldosterone escape during chronic ACE inhibition

or AT1 receptor antagonism. The specific blockade of

aldosterone action should prove to be of great therapeutic

value in the prevention of cerebral and renal vascular

disease and associated end-organ damage.

Introduction

The brain and the kidney are two of the major tar-get organs for aldosterone action in the body. In thebrain, a particularly high density of MR has beenidentified in the hippocampus as well as at otherneuronal sites [1]. Aldosterone actions within thecentral nervous system have been shown to resultin acute and long-term effects on vascular struc-ture and function and to produce hypertension[1]. Furthermore, intracerebroventricular admin-istration of low doses of an MR antagonist reducedsystolic blood pressure (SBP) in mineralocorticoid-

salt hypertensive rats, but its apparent absencein the systemic circulation was ineffective in pre-venting myocardial fibrosis [2]. The mechanismsfor the central effects of aldosterone are not en-tirely clear but may involve the central activa-tion of sympathetic drive to the heart and kidney[3].

The kidney displays a high density of MR alongthe tubular epithelium of the distal nephron (dis-tal convoluted tubule and collecting duct) [4]. It ishere that aldosterone governs the vectorial trans-port of water and electrolytes by acting as the prin-cipal ligand for the MR. Immunohistochemical la-beling of isolated rat preglomerular vasculature[5] and glomeruli [4] for MR has also been doc-umented. Studies by Ullian and coworkers haveshown that aldosterone can directly stimulate cit-rate synthase in isolated glomeruli from Wistarrats [6]. Aldosterone has long been known to stim-ulate citrate synthase, a Krebs cycle enzyme, inthe kidney and the heart [7] and by doing so mayenhance mitochondrial oxidative phosphorylationand oxygen free radical generation. Aldosteroneexcess can also beget hypokalemia and hypomag-nesemia, and Young and coworkers have shownthat reduced potassium ion concentration can di-rectly stimulate free radical formation in cul-tured vascular smooth muscle cells [8]. Yang andcoworkers [9] have shown that low dietary potas-sium intake with attendant hypokalemia was as-sociated with increased vascular superoxide aniongeneration. Interestingly, although Ang II has ac-tions to stimulate free radical formation, markedincreases in vascular NADPH are known to oc-cur in animals with deoxycorticosterone acetate(DOCA)-salt hypertension, a low-renin model of

Sources of support: National Institutes of Health (HL-35522to C.T. Stier).

Address for correspondence: Charles T. Stier, Jr., PhD, Depart-ment of Pharmacology, Basic Science Building, New York Med-ical College, Valhalla, New York 10595, USA. Tel.: (914) 594-4138; Fax: (914) 594-4273; E-mail: Charles Stier@NYMC.edu

53

54 Stier et al.

hypertension [10]. In many cases, the extent towhich aldosterone mediates the known adversecardiovascular effects of Ang II remains unknownand needs to be addressed.

The role of aldosterone in promoting cardio-vascular injury is highlighted by the random-ized Aldactone evaluation study (RALES) [11] andthe Eplerenone Post-Acute Myocardial InfarctionHeart Failure Efficacy and Survival Study (EPH-ESUS) [12] trials, which showed that the addi-tion of an MR antagonist to standard therapy inheart failure patients reduces cardiac morbidityand mortality that could not be accounted for byBP reduction or fluid loss [11]. Numerous recentstudies document that aldosterone has actionsat nonepithelial sites which may be the primarytarget for production of cardiovascular end-organdamage [13]. The existence of MR has been es-tablished in vascular smooth muscle cells, cardiacmyocytes and endothelial cells and suggests thatdirect antagonism of non-classical actions of min-eralocorticoids on cardiovascular tissues may alsobe responsible for the resultant beneficial effects[14]. The present review will focus on the brainand the kidney as targets of the adverse cardio-vascular effects of aldosterone and the beneficialeffects of MR antagonism.

Aldosterone and Stroke

Hyperaldosteronism

In 1972, clinical studies by Brunner and cowork-ers identified plasma renin activity (PRA) and al-dosterone as independent risk factors for heart at-tack and stroke [15]. They found that among thepatients who developed strokes and myocardial in-farctions, all had high PRA, high aldosterone se-cretion, or both. Several other studies have alsodescribed an association between cerebrovasculardisease and primary aldosteronism (PA) [16,17].Nishimura and collaborators [16] identified car-diovascular complications in 34% of the patientswith PA secondary to aldosterone-producing ade-noma: stroke was found in 16%, and protein-uria in 24%. Unlike other types of hypertension,PA is associated with elevated aldosterone lev-els with suppression of other components of theRAAS [18]. It has generally been assumed thatthese patients do not display significant cardio-vascular complications, an assumption which isnot supported by the available data. In a classicreport, Conn analyzed 145 cases of PA secondaryto an aldosterone-producing adenoma [19]; everypatient demonstrated hypertension. More impor-tantly, cardiomegaly was detected in 41% of thepatients, retinopathy in 50%, and proteinuria in85%. A high incidence of adverse cardiovascularevents has also been identified in other studies of

patients with PA [20,21]. Thus, there is a signif-icant body of evidence that cardiovascular injuryis common in patients with chronic PA. Exogenousadministration of mineralocorticoids can producelesions of malignant nephrosclerosis and strokeand have classically been described in rats withmineralocorticoid hypertension induced by thechronic administration of DOCA and salt [22,23].Lesion development in DOCA-salt hypertensiverats is thought to be due to an intrinsic actionof mineralocorticoids since these animals exhibitmarkedly suppressed levels of PRA [18]. Clinicalstudies have revealed a correlation between highaldosterone secretion and a prominent history ofhemorrhagic stroke among patients with gluco-corticoid remediable aldosteronism, a rare formof primary aldosteronism [24]. In this condition,aldosterone is produced and secreted under thesole control of adrenocortico-tropic harmone, andalthough PRA is generally suppressed, patientsexhibit a prominent history of hemorrhagic strokeat an early age.

MR Antagonism in Stroke-Prone

Spontaneously Hypertensive Rats

Spontaneously hypertensive rats of the stroke-prone substrain (SHRSP) develop severe hyper-tension, cerebrovascular lesions and malignantnephrosclerosis. Placing SHRSP on a 1% NaCldrinking solution in combination with a Stroke-Prone Rodent Diet of reduced potassium and pro-tein content can markedly accelerate the onset ofmicrovascular damage in these animals [25]. Pre-vious studies from our laboratory have shown thatchronic treatment of SHRSP with either an ACEinhibitor [25] or an AT1 receptor antagonist [26],can provide marked cerebrovascular protective ef-fects. Because BP was not markedly reduced bythese treatments, we postulated that the role ofAng II in these animals was not related to a directvascular action but might be secondary to the pow-erful action of Ang II to release aldosterone fromthe adrenal gland. In a separate experimental se-ries, SHRSP were fed Stroke-Prone Rodent Dietand 1% NaCl drinking solution at 8.8 weeks of ageand were chronically treated with either spirono-lactone or vehicle [27]. Five out of the 6 SHRSPfrom the vehicle-treated group displayed signs ofstroke beginning at 13.4 weeks of age and diedbefore 16 weeks of age. In contrast, none of thesix spironolactone-treated SHRSP showed strokesigns and all survived until 19 weeks of age, atwhich point the study was ended. Microscopic ex-amination revealed cerebrovascular lesions in thebrains of all vehicle-treated SHRSP, commensu-rate with the demonstration of stroke signs inthese animals. Brains from spironolactone-treatedanimals showed occasional lesions of rarefaction

Aldosterone in Stroke and Renal Disease 55

and edema that were not as marked as in the con-trols. As observed in our previous study, animalsin both groups became severely hypertensive andthere was no significant difference in SBP betweenthe groups over the course of the study.

The EPHESUS trial showed that eplerenonecould increase survival among patients who re-cently had a myocardial infarction [12]. We alsoexamined the selective aldosterone antagonist,eplerenone, which lacks the anti-androgenic andprogestational effects of spironolactone and foundthat it exerted a marked cerebrovascular protec-tive action in saline-drinking SHRSP [28]. In thesestudies, SHRSP were placed on a 1% NaCl/Stroke-Prone Rodent Diet at 9 weeks of age. At thesame time, treatment with either eplerenone(100 mg/kg/day po; n = 8) or vehicle (0.5% methyl-cellulose; n = 9) was started. SHRSP were main-tained on these treatments until 19 weeks of age,at which time the animals that survived were sac-rificed. Tail-cuff SBP measured before death wassimilarly elevated in the 2 groups (Fig. 1(a)). Start-ing at 13 weeks of age, vehicle-treated SHRSP be-gan to show neurological signs of stroke and sub-sequently died (Fig. 1(b)). All of the vehicle-treatedSHRSP were dead by 18 weeks of age. In contrast,none of the eplerenone-treated littermates exhib-ited signs of stroke and all survived through 19weeks of age, with the exception of 1 animal, whichdeveloped stroke signs and died at 18 weeks ofage. Figures 2(a) and (b) illustrate the represen-tative cerebral histological changes in untreatedand eplerenone-treated saline-drinking SHRSP,respectively. Histopathologic scoring of the brainsrevealed severe cerebral vascular and parenchy-mal lesions characteristic of ischemic and hemor-rhagic stroke in untreated SHRSP whereas cere-bral injury in animals receiving eplerenone waslargely prevented (Fig. 1(c)). Studies by McLeodet al., have demonstrated that chronic infusionof aldosterone can reverse the ability of capto-pril to prevent mortality and cerebrovascular in-jury in salt-loaded SHRSP [29]. Together, thesestudies provide strong evidence to support arole for endogenous mineralocorticoids in the de-velopment of cerebral lesions in saline-drinkingSHRSP.

Studies have also been conducted to examinethe effect of MR antagonism on experimental is-chemic infarct size in SHRSP [30]. Spironolactonewas administered at 3.3 mg/day over a period ofsix weeks and had no effect on body weight orSBP over the treatment period. However, spirono-lactone reduced the size of cerebral infarcts af-ter middle cerebral artery occlusion in placebo-treated vs. spironolactone-treated SHRSP (52 ±4 vs. 22 ± 7% of the hemisphere infarcted, respec-tively, P < 0.05). In addition, spironolactone re-duced epidermal growth factor receptor, but not

Fig. 1. Cerebral injury and survival with eplerenone. (a)Tail-cuff systolic blood pressure in saline-drinking,stroke-prone spontaneously hypertensive rats (SHRSP)measured before death. (b) Survival curves for SHRSP treatedwith vehicle (unfilled circles, n = 8) or eplerenone (filled circles,n = 7; 100 mg/kg/day, P < 0.001). The arrow indicates whentreatment began. (c) Histopathological scores for cerebralinjury in SHRSP receiving either vehicle or eplerenonetreatment (P < 0.001). (From Rocha and Stier, 2001) [28].

epidermal growth factor, mRNA expression. Thesefindings further support a role for aldosterone inprovoking cerebrovascular damage in SHRSP andidentify the potential involvement of epidermalgrowth factor receptor activation.

Relationship to Sodium Transport

Antagonism

ACE inhibitors [25], AT1 receptor antagonists[26], and MR antagonists (spironolactone and

56 Stier et al.

Fig. 2. Cerebral histopathology with or without eplerenone.(a) Photomicrograph of cerebral cortex of a stroke-pronespontaneously hypertensive rat (SHRSP) receiving vehicletreatment, showing fibrinoid necrotic lesions in cerebralarteries (arrows) and liquefaction necrosis in the parenchyma.(b) Cerebral cortex of a SHRSP receiving eplerenone treatmentand showing no damage. Scale bars = 80 µm. (From Rochaand Stier, 2001) [28].

eplerenone) [27,28], have been shown to markedlyreduce proteinuria and vascular injury in saline-drinking, SHRSP. The latter agents work by com-petitively inhibiting the binding of aldosterone toits intracellular receptor and hence reduce thesubsequent translocation of the hormone recep-tor complex into the nucleus. One of the majorevents stimulated by aldosterone is an increasedgenomic expression of epithelial sodium channels(ENaC). Aldosterone has also been reported to in-duce the formation of proteins that stabilize andenhance the activity of sodium channels. Sincethe action of anti-aldosterone agents may also re-late to interfering with these events, we exam-ined whether amiloride, a potent and directly act-ing ENaC inhibitor, would mimic the anti stroke

activity of MR antagonism in SHRSP [31]. Wefound that chronic treatment with amiloride at1 mg/kg/day markedly increased the survival ofSHRSP compared with untreated saline-drinkingSHRSP littermate controls. Control SHRSP dis-played neurological signs of stroke prior to deathwith 71% and 100% mortality by 13 and 16.4weeks of age, respectively. In contrast, six of theeight amiloride-treated SHRSP showed no signsof stroke and survived through 20 weeks of age.Amiloride increased longevity of saline-drinkingSHRSP by more than 6 weeks (P < 0.001). SBPin the control and amiloride-treated SHRSPgroups progressively increased with no signifi-cant difference between them (228 ± 6 mmHg vs.217 ± 4 mmHg in control vs. amiloride) at 11.7weeks of age. Amiloride-treated SHRSP remainedseverely hypertensive thereafter with SBP rang-ing from 240 to 260 mmHg. These data sug-gest that the cerebrovascular protective effectsof MR antagonists are, at least in part, me-diated by amiloride-sensitive sodium transportinhibition.

Aldosterone and Renal Disease

Aldosterone Excess

Evidence to support the renal vasculotoxic effectof high circulating mineralocorticoid levels comesfrom clinical and experimental studies. In patientstudies, of moment is the high incidence of protein-uria that is associated with hyperaldosteronismas a consequence of an aldosterone producing ade-noma [19]. Also, Grady and colleagues publisheda report of 32 renal biopsies obtained from pa-tients with aldosterone-producing adenomas andshowed that 56% had mild to severe arteriolarsclerosis, and 46% presented with interstitial fi-brosis and tubular atrophy [21]. In animal studies,renal lesions of malignant nephrosclerosis havebeen produced following unilateral nephrectomyand prolonged treatment with DOCA and 1% NaCldrinking water [22]. In these rats, Gavras andcolleagues reported dramatic and significant re-ductions in PRA, thus documenting a dissoci-ation between elevated circulating renin levelsand malignant nephrosclerosis [18]. It was sug-gested that vascular damage was a consequenceof excess amounts of salt and that reduced renalmass and DOCA greatly increase the susceptibil-ity to salt. In keeping with these observations,pharmacological studies indicate that Ang II doesnot contribute to the development of renal injuryin mineralocorticoid-salt hypertension, as chronictreatment with enalapril or losartan did not re-duce the occurrence of pathologic changes underthese conditions [32].

Aldosterone in Stroke and Renal Disease 57

Renal Protective Effects of Mineralocorticoid

Receptor Antagonism

Stroke-Prone Spontaneously HypertensiveRats. Saline-drinking SHRSP develop renal le-sions of malignant nephrosclerosis that are vir-tually indistinguishable from those observed inDOCA-salt hypertensive rats. Unlike the situationin DOCA-salt hypertension, ACE inhibition [25] orAT1 receptor blockade [26] prevented the develop-ment of kidney damage in saline-drinking SHRSP.To evaluate the possibility that aldosterone me-diates the renal injury in these animals, wechronically treated male saline-drinking SHRSPvia subcutaneously implanted time-release pel-lets containing either spironolactone, at a doseof 13–17 mg/kg/day, or placebo [27]. SHRSP fromboth groups showed a progressive increase in SBPwith age. By the end of the study, animals in bothgroups were severely hypertensive with, no signifi-cant differences in SBP. Urinary protein excretionincreased markedly in SHRSP receiving placeboand averaged 150 ± 6 mg/day at 10.4 weeks of age.In contrast, urinary protein excretion remainedat low levels and averaged 22 ± 5 mg/day at10.4 weeks of age in the spironolactone-treatedgroup (P < 0.0001). Body weight increased pro-gressively in both groups through 9.5 weeks of age,when the weight of placebo-treated animals be-gan to decline as these animals started to showsigns of stroke and became debilitated. Figures3(A) and (B) illustrate the representative renalhistologic changes in placebo- and spironolactone-treated littermates, respectively. Histologic anal-ysis of the kidneys from placebo-treated SHRSPshowed prominent glomerular and vascular le-sions of thrombotic microangiopathy, character-istic of malignant nephrosclerosis. In contrast,spironolactone-treated SHRSP exhibited markedreductions in both renal vascular and glomeru-lar lesions. The values for both parameters werehighest for the placebo-treated SHRSP and low-est for the spironolactone-treated SHRSP, with nooverlap between the groups. Treatment of saline-drinking SHRSP with spironolactone was not as-sociated with a diuretic or natriuretic response. Insimilar studies conducted in SHRSP chronicallyinstrumented with radiotelemetry BP probes andmaintained on a 1% NaCl drinking solution andstandard rodent chow (Purina), treatment withspironolactone substantially reduced proteinuriaand renal damage score. Under these conditions,the development of hypertension was delayed, butnot prevented, and there was no effect on urinarysodium or potassium excretion [33].

Radiation-Induced Renal Injury and MouseModels. A renal protective effect of spironolac-tone against the development of nephrosclerosis

Fig. 3. Photomicrographs of representative hematoxylin andeosin-stained mid-coronal kidney sections from two 10.4week-old stroke-prone spontaneously hypertensive ratsmaintained on Stroke-Prone Rodent Diet and 1% NaClstarting at 7.5 weeks of age (original magnification, × 130).(A) Renal cortex from an animal into which a placebo pelletwas implanted at 7.5 weeks of age. The photomicrographillustrates the presence of lesions of thromboticmicroangiopathy affecting several glomeruli and bloodvessels. A relatively normal glomerulus is seen towards theright lower quadrant. Ischemic retraction and obliteration ofcapillary tufts is seen in 1 glomerulus (small arrow).Segmental capillary tuft necrosis, widespread thrombosis, andcellular swelling, are evident in two additional swollenglomeruli (large arrows). Microvascular lesions consist ofmarked concentric medial hypertrophy (small arrowhead),and circumferential, transmural fibrinoid necrosis asobserved in two small arteries (large arrowheads), oneshowing fragmented and extravasated erythrocytes. (B) Renalcortex from an animal into which a time-release pelletcontaining 200 mg of spironolactone was implantedsubcutaneously at 7.5 weeks of age. The photomicrographillustrates the absence of significant glomerular or vascularpathology as seen above in the age-matched, placebo-treatedlittermate control. (From Rocha et al., 1998) [27].

has also been observed in rats following bilateralirradiation of the kidneys [34]. Radiation causeda marked increase in proteinuria and glomeru-lar sclerosis vs. nonradiated controls and this was

58 Stier et al.

significantly decreased by either spironolactone orAT1 receptor inhibition (P < 0.001 vs. placebofor either drug). Levels of plasminogen activa-tor inhibitor-1 (PAI-1) were increased eight foldby radiation but this effect was significantly re-duced by chronic treatment with either spirono-lactone or an AT1 receptor antagonist. Althoughexposure to radiation moderately increased BP(152 ± 11 mmHg), this response was unaffectedby spironolactone (149 ± 6 mmHg, P = NS)and was fully reversed by AT1 receptor block-ade (102 ± 6 mmHg, P < 0.001). In studies per-formed in PAI-1 knockout mice, the renal vas-cular damage produced by aldosterone infusionwas undiminished, but rather tended to increase[35], whereas the more chronic changes associ-ated with aldosterone infusion were markedly di-minished [34]. These results support a role foraldosterone or a related factor with mineralo-corticoid activity, in the development of renalvascular injury and implicate the participationof PAI-1 in mediating some of the late adverseconsequences of aldosterone on the kidney. In-terestingly, aldosterone infusion for three daysinto otherwise normal rats increased the uri-nary excretion of transforming growth factor-β1by two fold without changes in SBP or evidenceof kidney damage [36]. The effect was blockedby spironolactone but not by amiloride, whichsuggests that it is dependent on the MR butindependent of sodium transport. Elevated lev-els of transforming growth factor-β1 derived fromglomerular or extraglomerular sources are capa-ble of increasing albumin permeability in iso-lated glomeruli via superoxide and hydroxyl rad-icals and may lead to proteinuria in vivo [37].In studies conducted in vivo, we have identi-fied reactive oxygen species as playing a role inthe production of aldosterone-induced renal injuryin saline-drinking SHRSP, as free radical scav-engers were able to markedly diminish protein-uria and renal pathologic effects in these animals[38]. These findings suggest that increased vascu-lar (endothelial) permeability as a consequence ofaldosterone-induced increases in reactive oxygenspecies may be the initial event leading throm-botic microangiopathy and the progression of re-nal vascular damage and its complications. In-creased oxidative stress induced by aldosterone inthe kidney may also be a triggering mechanism bywhich MR activation induces renal inflammatorychanges in saline-drinking, uni-nephrectomizedrats infused with aldosterone [39]. In this study,aldosterone-induced renal inflammation and dam-age was associated with significant activation ofthe cytokines, osteopontin, IL-1β, IL-6 and MCP-1 and these changes were markedly attenuated byeplerenone.

Aldosterone and the Renal Protective Effect of

ACE Inhibition

Animal Studies. Although the beneficial effectsof ACE inhibitors have typically been attributedto reductions in the vascular actions of Ang II,administration of these agents can also result ininhibition of aldosterone release. If the beneficialrenal vascular effect provided by ACE inhibitionis a consequence of interference with endogenousaldosterone formation, chronic exogenous infusionof aldosterone should restore lesion development.To evaluate this possibility in saline-drinkingSHRSP, we performed experiments in animalsthat were treated for two weeks according to thefollowing five regimens: an infusion of the vehicle(0.5% ethanol) used to dissolve aldosterone withno captopril (n = 8); captopril alone (50 mg/kg/day,orally n = 10); combined dosing with captopriland infusion of aldosterone at 20 (n = 6); or 40µg/kg/day, sc (n = 7); or aldosterone infusion alone(40 µg/kg/day, sc) with no captopril (n = 7) [40].Captopril was added to the 1% sodium chloridedrinking solution to provide a daily dose of 50mg/kg because the authors previously found thatthis dose would prevent stroke and renal injurythrough 26 weeks of age [25]. All groups of SHRSPdemonstrated a progressive increase in SBP andwere severely hypertensive after two weeks. Atthat time, SBP of SHRSP infused with aldos-terone alone (40 µg/kg/day) was approximately 20mmHg higher than in those given captopril aloneor captopril plus aldosterone at 20 µg/kg/day. Nosignificant differences in SBP were found amongthe other groups. At the end of the two-week treat-ment period, aldosterone levels were markedlyreduced and the development of proteinuria wasprevented in captopril compared with vehicle-control SHRSP. Histopathologic analysis ofkidneys from SHRSP treated with captopril aloneshowed neither glomerular nor renal vascular le-sions. When aldosterone was infused at either 20or 40 µg/kg/day, plasma aldosterone levels werefully restored and the antiproteinuric effect ofcaptopril was reversed. Histopathologic analysisof the kidneys revealed prominent glomerular andvascular lesions of thrombotic microangiopathycharacteristic of malignant nephrosclerosis in cap-topril plus low- or high-dose aldosterone-infusedSHRSP that were comparable to those in vehicleor aldosterone-infused SHRSP. Despite concur-rent ACE inhibitor therapy, captopril-treatedSHRSP infused with aldosterone at 40 µg/kg/day,developed similar levels of proteinuria and renalinjury as SHRSP infused with aldosterone aloneat 40 µg/kg/day. Thus, the protective effects ofcaptopril were absent when plasma aldosteronelevels were not subject to modulation. The results

Aldosterone in Stroke and Renal Disease 59

of this study indicate that the vascular protectiveeffects of captopril are associated with diminishedaldosterone levels and that aldosterone infusioncan completely reverse the renal protective actionof this ACE inhibitor in saline-drinking SHRSP.Consonant with this observation is the findingthat aldosterone can restore the development ofcerebral vascular injury in salt-loaded SHRSPchronically treated with captopril [29], vide supra.Together, these observations are consistent with acentral role for aldosterone in the evolution of ma-lignant nephrosclerosis and stroke in SHRSP andimplicate activation of the RAAS as an initiatingevent.

Aldosterone has also been reported to play arole in the development of renal injury in the rem-nant kidney model of chronic renal failure [41]. Inthat study, the ability of combined treatment withenalapril and losartan to attenuate proteinuria,hypertension, and glomerular sclerosis was com-pletely reversed by aldosterone infusion. In a sep-arate experimental series of rats, treatment withspironolactone did not reduce glomerular sclerosisbut did transiently reduce proteinuria, lowered ar-terial BP, and lessened cardiac hypertrophy. Theability of aldosterone infusion to reproduce the re-nal damage in this experimental model, despiteblockade of both the formation and the action ofAng II provided some of the initial evidence fora role of this steroid in provoking chronic renaldisease.

Patient Studies. The inability of ACE inhibi-tion to provide sustained suppression of plasmaaldosterone has been termed aldosterone escape.The randomized evaluation of strategies for leftventricular dysfunction (RESOLVD) trial showedthat 8 mg of candesartan and 20 mg of enalaprilproduced greater suppression of plasma aldos-terone levels than enalapril alone at 17 weeks oftreatment, but at 43 weeks the ability of combinedtreatment to suppress aldosterone levels was lost.Similar aldosterone escape has been seen in al-most every clinical study of an ACE inhibitor or anAT1 receptor antagonist. These observations, cou-pled with the inability of ACE inhibition to pre-vent the adverse cardiovascular effects of aldos-terone as described in the animal studies above,underscore the need for additional pharmacolog-ical intervention directed at aldosterone antago-nism to provide optimal cardiovascular protection.Among patients with stage 1 and stage 2 hyper-tension, eplerenone produced significantly greaterlowering of the urinary albumin:creatinine ratioat six months than enalapril (−61.5% vs. –25.7%,respectively, P < 0.01), but was as effective asenalapril in reducing both SBP and diastolic BP[42]. Among patients with renal disease whose

urinary protein excretion was poorly controlleddespite ACE inhibitor therapy for more than 12months, the addition of spironolactone produceda 54% further reduction in proteinuria [43]. Thelow dose of spironolactone (25 mg/day) adminis-tered to these patients was also associated with afurther lowering of mean arterial BP, ∼10 mmHg,which may have contributed to the antiproteinuricresponse. Interestingly, a meta-analysis of the ef-fect of blood-pressure-lowering agents on protein-uria (41 studies, 1124 patients) revealed that de-spite equivalent BP lowering (∼12%), there was a39.9% reduction in proteinuria if ACE inhibitorswere given and a 17% reduction in proteinuria ifagents other than ACE inhibitors were given [44].Numerous randomized clinical trials in patientswith renal disease (with or without diabetes) indi-cate renoprotective effects of RAAS inhibition in-dependent of an effect on systemic BP [45]. Theseobservations are consistent with the notion thatMR antagonists may have renal vascular protec-tive effects beyond what is observed with loweringof BP.

Alterations in Aldosterone Responsiveness

Studies have also been conducted in Wistar-Furthrats, an inbred strain resistant to actions ofmineralocorticoids, to evaluate a role for aldos-terone in renal injury following 5/6 nephrectomy[46]. Renal damage and albuminuria in responseto 5/6 nephrectomy were greatly decreased inWistar-Furth rats as compared with Wistar rats.Glomerular damage consisting of mesangial pro-liferation, mesangial lysis, and segmental necro-sis was observed in 42% of glomeruli from Wis-tar rats but in 0% of glomeruli from Wistar-Furthrats with reduced renal mass. Although mean ar-terial BP was 20 mmHg lower in Wistar-Furth ratsthan Wistar rats with 5/6 nephrectomy, albumin-uria remained more than 10-fold higher in Wistarrats with 5/6 nephrectomy that had BP controlledto an equal level by a hydralazine-reserpine-hydrochlorothiazide treatment regimen. Isolatedglomeruli from Wistar-Furth rats, unlike Wistarrats, showed no increase in citrate synthase inresponse to direct application of aldosterone [6].These findings further support the hypothesis thataldosterone itself has a role in mediating progres-sive renal disease through a mechanism that doesnot involve alterations in BP.

Adrenalectomy

The adrenal gland is the major source of circu-lating aldosterone in the body. If the beneficialcardiovascular effect of MR blockade is a conse-quence of the prevention of the actions of aldos-terone, bilateral adrenalectomy should mimic thiseffect. Experimental nephropathy in rats with 5/6

60 Stier et al.

nephrectomy can be ameliorated by adrenalec-tomy and this renal protection was unaffectedby glucocorticoid replacement with corticosterone[47]. Aldosterone replacement was not performedin this study. However, in rats with 5/6 nephrec-tomy, infusion of aldosterone fully reversed theprotective effect of pharmacological blockade of al-dosterone formation by treatment with an ACEinhibitor and an AT1 receptor antagonist [41].

In saline-drinking SHRSP, we used bilateraladrenalectomy to abolish circulating aldosteronelevels and provided glucocorticoid replacementwith dexamethasone followed by subcutaneous in-fusions of vehicle, Ang II or aldosterone for 2 weeks[48]. Adrenalectomy prevented proteinuria, ab-rogated thrombotic microangiopathy and slightlylowered blood pressure but did not change bodyweight, plasma creatinine, sodium, and potas-sium and daily urinary sodium and potassiumexcretion. Infusion of aldosterone, but not AngII, completely reversed the renal protective ef-fect of adrenalectomy in saline-drinking SHRSPwhereas both agents restored SBP to levels ob-served in sham-operated control animals. Sincethe adrenal glands produce a variety of substancesincluding peptides and catecholamines, it is ofmoment that replacement of aldosterone alonewas able to recreate lesion development in theseanimals. These findings confirm and extend ourprevious observations that have identified aldos-terone as the key factor in the production of ma-lignant nephrosclerosis in saline-drinking SHRSPand identify the adrenal gland as the major sourceof this vasculotoxic steroid.

A role for aldosterone in the development of vas-cular injury in the heart and kidneys of saline-drinking Wistar rats induced by combined nitro-L-arginine methyl ester (L-NAME)/Ang II treatmenthas been established [49]. The vascular injury ob-served included lesions of segmental or circum-ferential fibrinoid necrosis of the media, panarte-rial inflammation and small foci of ischemic andnecrotic damage in the myocardium or glomeru-lar and vascular injury in the kidney. This treat-ment caused severe myocardial necrosis in bothventricles and fibrinoid necrosis of small cardiacand renal arteries and arterioles. Adrenalectomyand eplerenone reduced this damage. Aldosteronereplacement in adrenalectomized animals againinduced cardiovascular damage. Aldosterone an-tagonism with eplerenone has also been reportedto ameliorate proteinuria and nephrosclerosis inthe L-NAME SHR model of renal injury [50]. To-gether, the findings from studies that have em-ployed adrenalectomy as a means for eliminatingthe action of aldosterone, support those using MRantagonists and further identify aldosterone asa critical mediator of microangiopathic injury inboth the heart and the kidney.

Summary

Substantial evidence now exists to support theaction of aldosterone at non-epithelial sites toprovoke cerebrovascular, renal and cardiac injury.Aldosterone has acute and long-term effects onvascular structure and function and affects thecentral nervous system to produce hypertension.Aldosterone can promote end-organ damage in thebrain and the kidneys in addition to the heart. Al-dosterone escape during chronic ACE inhibitionand/or AT1 receptor antagonism may necessitateadditional MR antagonism in order to obtain op-timal end-organ protection. The specific blockadeof aldosterone action is an important target in theclinical management of patients with heart, kid-ney and cerebrovascular dysfunction.

Acknowledgments

Some of the experimental work discussed here was supportedin part by the National Heart Lung and Blood Institute GrantHL-35522 (to C.T. Stier).

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