Genomic and Non-Genomic Effects of Aldosterone

132 Current Signal Transduction Therapy, 2012, 7, 132-141 Genomic and Non-genomic Effects of Aldosterone

Andrea Verhovez*, Tracy A. Williams, Silvia Monticone, Valentina Crudo, Jacopo Burrello, Maddalena Galmozzi, Michele Covella, Franco Veglio and Paolo Mulatero

Department of Medicine and Experimental Oncology, Division of Internal Medicine and Hypertension, University of Torino, 10126 Torino, Italy

Abstract: The last two decades have witnessed a growing number of experimental observations regarding the mineralocorticoid physiopathology, leading to a new understanding of the molecular basis of the aldosterone-induced target organ damage. As a matter of fact, although it has long been known that the combined administration of mineralocorticoids and salt leads to extensive vascular lesions in the target organs, the recognition that aldosterone is able to induce direct toxic effects on the various cell types that make up the cardiovascular organ has built up recently. Moreover, non-genomic effects have been attributed to aldosterone, i.e. effects which do not depend on the activation of the cellular transcription machinery, whose physiopathologic relevance is still being investigated. These advances in our understanding of aldosterone physiopathology have shed light on the biological reasons which are at the base of the impressive results obtained in clinical trials of aldosterone antagonism. Keywords: Aldosterone, hyperaldosteronism, endothelium.

INTRODUCTION permeability by means of the generation of high electrochemical gradients. This action is performed by The hormone aldosterone was named after the aldehyde modulation of gene expression. group located on the carbon 18 of the steroidal skeleton which takes the place of the methyl group which Aldosterone synthase, the product of the CYP11B2 gene characterizes all the other steroidal compounds. It was and the final and rate-limiting enzyme of its synthetic isolated for the first time by Simpson and Tait in 1953 metabolic pathway, is expressed exclusively in the zona [1,2] and has since been characterized as the main glomerulosa, the outermost layer of the adrenal cortex, mineralocorticoid hormone in humans on the basis of its where it converts deoxycorticosterone to aldosterone by robust effects on the transepithelial unidirectional transport means of a three-step oxidative biochemical reaction [4,5]. of sodium [3]. Aldosterone action was originally believed to Its activity is regulated by the three main secretagogues of be limited to just few classical target organs, mainly of aldosterone, the plasma potassium concentration [6], the epithelial origin, where the hormone induced the activity of the renin-angiotensin system [7] and ACTH [8,9], biochemical modifications required to maintain volume and although many other compounds have been attributed a electrolyte homeostasis by acting through genomic minor role [10]. pathways. Nevertheless, in the last two decades a growing The genomic actions of aldosterone are mediated by its number of experimental observations has broadly expanded binding to the mineralocorticoid receptor (MR), a member of the limits of this classical model. In particular the spectrum the superfamily of ligand-regulated transcription factors. of tissues and organs involved in aldosterone action has This superfamily, featuring 49 members in humans [11] and turned out to be wider than previously expected and these including the receptors for the thyroid hormones, the retinoic targets (heart, blood vessels, brain) are now described as acid, the glucocorticoids and the sexual steroids, is made up “nonepithelial”. Moreover, aldosterone has been attributed of molecules which, in resting conditions, are localized in non-genomic effects, i.e. effects which do not depend on the the cytosol and that, following ligand binding, translocate to activation of the cellular transcription machinery. the nucleus to regulate gene expression. Further, its members CLASSICAL MOLECULAR PHYSIOLOGY OF display structural similarities, being composed of functionally ALDOSTERONE related distinct domains [12,13]: - a carboxy-terminal ligand binding domain (LBD) that Aldosterone has classically been described as a also binds heat shock proteins (HSPs) required to attain a mineralocorticoid hormone produced exclusively in the high affinity ligand-binding capacity [14], and is further adrenals and acting on a small number of epithelial target involved in receptor dimerization, nuclear targeting and organs (kidney, colon, salivary and sweat glands) exhibiting hormone-dependent transactivation; high electrical resistance and capable of regulating water - an amino-terminal domain, the most variable region within the steroid receptor superfamily, that has a ligand-

*Address correspondence to this author at the Division of Internal Medicine independent transactivation function; and Hypertension, AOU San Giovanni Battista, Via Genova 3, 10126, Torino, Italy; Tel: -39-011-6336959; Fax: -39-011-6602707; - a highly conserved central DNA binding domain (DBD), E-mail: [email protected] involved in DNA binding and receptor dimerization;

- a hydrophilic hinge region between the DBD and the proteins, which are characterized by a single transmembrane LBD. domain and regulate the activity of pumps and ion transporters; it displays a 50% homology with the subunit Upon aldosterone binding, the HSPs dissociate from the + + of the Na /K -ATPase, is selectively expressed in the distal MR permitting the LBD to mediate the nuclear translocation parts of the nephron and in the epithelial cells of the distal [15,16] and the ensuing binding to specific hormone colon, and acts by increasing its affinity for the intracellular response elements in the promoter regions of target genes sodium; corticosteroid hormones, sodium restriction, low- [17]; the recruitment of the relevant transcription initiation potassium diet and metabolic acidosis have been shown to complex and coactivators or corepressors permits either to significantly up-regulate CHIF mRNA expression [32,33]. activate or repress gene transcription [18]. K-Ras is a monomeric GTPase whose activity stabilizes Since the cloning of the MR, its high sequence homology ENaC in the opening state [34]; it directly stimulates with the glucocorticoid receptor became evident [19]. This phosphoinositide 3-OH kinase (PI3K) to produce inositol homology is not limited to the DBD but also to the LBD and triphosphate which in turn acts onto the ENaC [35]. These determines a ten-fold higher affinity of cortisol for the MR data have shed light on previous observations regarding the compared to its own receptor [20]. Since cortisol circulates crucial role of PI3K for aldosterone to induce early and late at much higher concentrations than aldosterone, it would be effects on its epithelial target cells [36]. expected to preferentially occupy the MR: however, in the NON-GENOMIC EFFECTS epithelial target tissues of aldosterone and in some other tissues, aldosterone specificity is maintained by the action of Non-genomic effects are characterized by their short time the enzyme 11-hydroxysteroid dehydrogenase type 2 (11- lag of action, occurring in a few minutes or even seconds, HSD2) which converts cortisol to its inactive metabolite and their insensitivity to transcription and translation cortisone [21]. In case of genetic or pharmacological inhibitors. The first rapid non-genomic actions of aldosterone inactivation of this enzyme, the syndrome of apparent to be described date back to the sixties, when enhanced mineralocorticoid excess ensues, where the MR is constantly sodium exchange was reported in canine erythrocytes [37]. activated by circulating glucocorticoids [22]. Nevertheless, Later on, aldosterone was reported to trigger non-genomic other mechanisms that confer receptor activation specificity effects on a large number of cells types, most notably must exist in those tissues where 11-HSD2 has not been mononuclear leucocytes, endothelial cells, vascular smooth detected, such as hypothalamus and cardiomyocytes [23]. muscle cells (VSMC) and cardiomyocytes [38]. The first studies focused on the rapid modulation of the trans- The regulation of gene transcription in the classical membrane electrolyte transport consisting of a rapid and epithelial target organs occurs in a two step modality; in the transient increase in intracellular Ca2+ followed by activation early phase, which takes place in the first three hours from of the NHE [39,40]; the final effect consists in an increase in aldosterone stimulation, regulatory proteins of pre-existing cell volume that can be monitored by atomic force ion channels and transporters are modulated; in the second microscopy and intracellular alkalinization [41]. The one, new pumps, transporters and ion channels are directly intracellular metabolic pathways mediating these events rely produced with an increase in their density at the membrane on the c-Src dependent activation of EGFR and the ensuing level. These last molecules are considered the final effectors MAPK activation [42], although other authors have proposed of the mineralocorticoid action in epithelia and have been a role also for protein kinase C [43,44] and PI3K [45]. identified as the epithelial sodium channel (ENaC), acting on the luminal side, and the Na+/K+-ATPase, acting on the Debate still persists on two main points: 1) what is the basolateral side [24]. Recently, also the thiazide-sensitive receptor that mediates these effects; 2) what are their in vivo NaCl cotransporter (NCC) [25], in the distal convoluted physiopathologic implications. On the first point, a whole tubule, and the NaH exchanger (NHE) [26], in the proximal series of evidences exists hinting to the presence of a colon, have been reported to be controlled by aldosterone. membrane receptor distinct from the classical MR; these Their increased expression in the late phase produces an evidences consist in the persistence of the non-genomic overall increase in renal and colonic sodium and water actions of aldosterone in epidermal cells of MR knock-out reabsorption and an increased potassium excretion. mice [46], in the inability of classical MR antagonists to inhibit them [47] and in the finding that aldosterone coupled Serum and glucocorticoid-regulated kinase 1 (sgk1) is the to large molecules like albumin still retains the ability to most studied early signaling molecule that gets up-regulated elicit the same rapid effects [48]. Yet, other authors reported after only 30 minutes from stimulation. It is a serine- contrasting data on the sensitivity of these actions to more threonine kinase that increases the cell surface density of flexible and water-soluble MR antagonists [49] and ENaC by the inhibitory phosphorylation of Nedd 4-2, a Grossmann et al. [50,51] have shown that, in a heterologous ubiquitin-ligase responsible for the degradation of ENaC expression system of HEK cells lacking classical MR, non- [27]. According to other authors, sgk1 increases both the + + genomic effects can only be induced by aldosterone after Na /K -ATPase expression and its activity [28,29]. transient transfection with the classical MR. Nevertheless, as sgk1 knock-out mice display a milder phenotype than adrenalectomized mice, it is assumed that As far as it regards the second point, the only data other redundant signaling factors are at stake [30]. Among suggesting that aldosterone might influence human these, glucocorticoid-induced leucine zipper protein (GILZ) physiology by means of non-genomic effects derive from stimulates ENaC activity by a similar mechanism of vascular reactivity studies showing a rapid aldosterone- regulation of ubiquitination [31]. Corticosteroid-Hormone dependent arterial vasodilation mediated by increased NO Induced Factor (CHIF) belongs to the family of FXYD synthesis in healthy subjects [52] and, on the contrary, a 134 Current Signal Transduction Therapy, 2012, Vol. 7, No. 2 Verhovez et al. direct effect of vasoconstriction on the smooth muscle pressure modifications in comparison to control littermates vascular layer [53] mediated by activation of the PLC and [60-62]. PKC pathway [54]. It derives that in a condition of endothelial dysfunction, characterized by low nitric oxide The same group subsequently described the occurrence (NO) bioavailability, aldosterone could putatively increase of identical vascular alterations in the coronary district; blood pressure values by means of a direct and acute Rocha et al. [63] reported the development of vascular modulation of vascular resistance [53]. inflammation in the coronary arterioles of aldosterone/salt- treated rats, characterized by fibrinoid necrosis of the tunica ALDOSTERONE EFFECTS ON BLOOD VESSELS media and perivascular infiltration by mononuclear leucocytes. These lesions were evident after only two weeks The position of the endothelial layer at the interface of treatment and could be attenuated by treatment with between the blood stream and the vascular wall makes it a eplerenone, a selective MR antagonist. At the same time, the crucial organ for maintaining vascular homeostasis in the face of continuous exogenous and endogenous stimuli. same authors demonstrated that aldosterone was to blame for Nowadays, it is well known that the endothelium is not just those myocardial lesions known to develop in the rat model an inert cellular monolayer, but plays a key role in vascular of angiotensin II / salt – dependent hypertension (cardiac physiopathology, finely balancing vasoconstriction and hypertrophy and vascular coronary inflammation), as both vasodilation, inhibition and stimulation of VSMC proliferation eplerenone and adrenalectomy were able to revert them [64]. and migration, adhesion of leucocytes, thrombogenesis and Also in this case, eplerenone effect was independent from fibrinolysis [55,56]. The acquired inability of the endothelium blood pressure reduction. These same coronary vascular to properly accomplish these functions is typical of a state of inflammatory damages and the beneficial action of high risk of damage to the vascular wall that goes under the eplerenone have been reported in uninephrectomized rats name of endothelial dysfunction. subjected to oral saline load and simultaneously treated with carbenoxolone, an 11-HSD2 inhibitor [65]. Given the low Despite it has long been known that combining the concentration of circulating mineralocorticoid hormones administration of exogenous mineralocorticoids and high salt determined by the saline load, the vascular MR would be induces the development of widespread vascular lesions in activated by glucocorticoids under circumstances of several target organs [57-59], the recognition that anomalous intracellular redox state produced by the mineraloactive compounds can induce damage of the enzymatic block. vascular wall independently from the blood pressure values is recent (Fig. 1). Since the early nineties, a group of authors At the same time, taking advantage of the improvements has produced histopathologic observations, obtained in a in the cell culture and molecular biology techniques, other model of spontaneously hypertensive rats, supporting a authors investigated the direct effects of the administration direct vasculotoxic role of the mineralocorticoid compounds of aldosterone to endothelial and smooth muscle cells. when associated to saline load; these studies highlighted ENDOTHELIUM the anatomical modifications of the renal vasculature (thrombotic microangiopathy, proliferative lesions, capillary Many aspects of the endothelial physiology are affected ischemia) and described how they could be prevented by the by aldosterone (Fig. 1). As regards the modulation of gene administration of MR antagonists in the absence of blood transcription, Sugiyama et al. reported an early increase in

BLOOD VESSELS ALDOSTERONE HEART Cell adhesion Myocyte hypertrophy NO bioavailability Myocyte apoptosis ROS production EC matrix production VSMC migration Myofibroblast proliferation VSMC proliferation KIDNEY ROS production VSMC senescence After-load VSMC calcification ROS production Altered ion fluxes Mesangial proliferation Mesangial deformability Podocyte damage

Perivascular inflammation Cardiac hypertrophy Vascular fibrosis Cardiac fibrosis Hyalinization Arrhythmias Fibrinoid necrosis Glomerular ischemia Cardiac ischemia Thickening of the tunica media Glomerular sclerosis Heart failure Endothelial dysfunction Capsular fibrosis Proteinuria Chronic renal failure

Fig. (1). Effects of aldosterone on target organs. EC: extracellular; NO: nitric oxide; ROS: reactive oxygen species; VSMC: vascular smooth muscle cells. Genomic and Non-genomic Effects of Aldosterone Current Signal Transduction Therapy, 2012, Vol. 7, No. 2 135

ACE [66] and osteopontin [67] expression in rat aortic induction of a short time-lag (14 days) artificial hyper- endothelial cells following stimulation with aldosterone, mineralocorticoid state in healthy subjects does increase the suggesting an inflammatory modulation of the cardio- endothelium-dependent vasodilation in the forearm vascular renin-angiotensin system. Further, intercellular vasculature due to a higher basal NO bioavailability [83], adhesion molecule 1 (ICAM1) has been shown to be suggesting that, in order for aldosterone to determine modestly but significantly overexpressed at 24 hours in detrimental effects on the endothelial function, concomitant aldosterone-treated human coronary endothelial cells endothelial damage deriving from pre-existing cardio- (HCAEC) with a resulting increase in leucocyte adhesion vascular risk factors should be present. As a partial [68]. Nevertheless, also shorter incubation times seem to confirmation of this theory, in a study on mice with reduced produce the same effect in human umbilical vein endothelial monocyte/macrophage function, the ability of aldosterone to cells (HUVEC) [69]. induce vascular inflammation and oxidative stress was lost, as to indicate the need for multiple contemporaneous patho- As concerns NO bioavailability, Nagata et al. proposed a genic factors to provoke the mineralocorticoid-dependent double mechanism by which aldosterone causes its reduction vascular damage [84]. in HUVEC: 1) activation of the protein phosphatase PP2A with ensuing endothelial nitric oxide synthase (eNOS) The physical equivalent of the above described molecular dephosphorylation on Ser 1177; 2) eNOS uncoupling due to and physiopathologic alterations consists in a modification the reactive oxygen species (ROS)-induced tetrahydro- of the biomechanical properties of endothelial cells measured biopterin deficiency [70]. On the contrary, aldosterone by atomic force microscopy; few minutes after adding acutely induces NO production by a non-genomic aldosterone to the culture medium, endothelial cells swell mechanism involving PI3K both in ex vivo-treated vascular and relax as a consequence of the non-genomically-regulated rings with intact endothelium [71] and in in vitro-treated entry of sodium and water through the epithelial sodium endothelial cells [72]. channels and the release of NO [85]. Subsequently, a second The increase in vascular ROS generation has been phase takes over characterized by a regression of the cell to acknowledged since the studies by Somers et al. [73] and the original volume and a parallel increase in stiffness [86]. Beswick et al. [74,75], who reported a NADPH oxidase- According to some authors, such persisting stiffness, dependent sustained production of superoxide anion in the sustained by a genomically-determined increment in the vasculature of rats made hypertensive by the association of membrane density of ENaC [87], alters the physiologic deoxycorticosterone and salt. The causal link between functions of the endothelium, and particularly NO production mineralocorticoid stimulation and ROS production was [88], with a following increase in the vascular tone. reaffirmed by Iwashima et al. [76], who described a time- VASCULAR SMOOTH MUSCLE CELLS and dose-dependent increase in superoxide anion production mediated by a Rac1-dependent stimulation of NADPH A direct effect of aldosterone on VSMC has been oxidase. Beside this pathway, in bovine aortic endothelial originally supposed on the basis of in vitro experimental data cells aldosterone reduces the expression and activity of the reporting the ability of spironolactone to inhibit their enzyme glucose 6-phosphate dehydrogenase, with a proliferation induced by angiotensin II [89] and further in consequent depletion of NADPH and reduced glutathione vivo evidences of an increased arterial stiffness and thickness that determines vascular oxidative stress, reduced NO due to accumulation of fibronectin in aldosterone/salt bioavailability and a worsened vascular reactivity [77]. uninephrectomized rats [90]. However, these detrimental actions do not seem to affect In the wake of these findings Jaffe et al. gave a the apoptotic rate in HUVEC, as demonstrated by Williams comprehensive description of the in vitro genomic effects of et al. in a paper where the anti-apoptotic effect of aldosterone on the VSMC demonstrating that they express spironolactone was attributed to the antagonism toward the both a functional MR and the enzyme 11HSD2, and, by progesterone receptor [78]. using an expression microarray with qRT-PCR confirmation, On the contrary, debate still persists on a possible role of that aldosterone up-regulates a panel of genes involved in aldosterone on the function of endothelial progenitor cells vascular fibrosis, calcification and inflammation [91]. The (EPC), a bone marrow-derived cell population involved in same group subsequently confirmed that MR activation endothelial repair and vascular homeostasis. While an early promotes the osteoblastic differentiation and mineralization report did not find any significant in vivo and in vitro effect of VSMC [92]. exerted by aldosterone on EPC senescence and Nonetheless, non-genomic signaling seems to be the bioavailability [79], recently another group reported on the predominant way of modulation of VSMC function by functional impairment of this population induced by an MR- aldosterone. Of particular interest is the ability to give rise to mediated increase in ROS production [80]. ROS generation via c-Src dependent NADPH oxidase Although a different experimental setting, the in vivo activation to produce perivascular fibrosis [93]; the activation evaluation of endothelial function in humans has produced of this non-genomic pathway seems to be particularly intense results that fit into the general model of aldosterone-induced in VSMC derived from spontaneously hypertensive rats [94]. endothelial dysfunction. Patients affected by both primary Another study suggested that the so-induced increased [81] and secondary [82] aldosteronism display a worsening oxidant stress promotes a posttranslational oxidative of endothelial function and a reduction in NO bioavailability modification of the soluble guanylyl cyclase that converts it that recover following treatment with MR antagonists. to an NO-insensitive state resulting in disruption of normal Nevertheless, more recently it has been observed that the vasodilatory signaling pathways in VSMC [95]. 136 Current Signal Transduction Therapy, 2012, Vol. 7, No. 2 Verhovez et al.

Further, a large evidence in the literature underlines the modulation of the fibrogenic process by aldosterone; in a tight synergistic interaction between the non-genomic study on rats where a myocardial infarction was signaling pathways of aldosterone and angiotensin II in experimentally determined by ligation of the left coronary VSMC. These metabolic pathways include EGFR trans- artery, eplerenone administration reduced the collagen activation, c-Src and ERK phosphorylation and the deposition and the cardiac remodelling of the viable activation of redox-sensitive transcription factors such as myocardium without affecting infarct healing [105]. In the AP-1 and NF-kB [96,97], and regulate several aspects of canine microembolism model of ischemic heart failure, VSMC physiopathology affecting the overall process of eplerenone-treated animals displayed better systolic and vascular remodelling. Among these, of particular relevance diastolic function and a reduction of left ventricular are the VSMC migration through the activation of redox- remodeling compared to vehicle-treated animals [106]. In a sensitive RhoA/Rho kinase [98], the mitotic index [99] and murine model of chronic pressure overload-induced heart cell senescence [100] (Fig. 1). failure, eplerenone both attenuated the progressive left ventricular dilation and deterioration of systolic function and ALDOSTERONE EFFECTS ON THE HEART reduced the interstitial fibrosis and the oxidative response The main pathologic processes induced by the [107]. On the contrary, in a recent publication Lother et al. mineralocorticoid excess (plus saline load in the in vivo [108] reported that selective genetic MR deletion in mouse studies) in the heart are the vascular and perivascular cardiomyocytes in vivo (but not in fibroblasts) protected inflammation, fibrosis and myocardial hypertrophy; further- from left ventricular dilatation and dysfunction induced by more, electrophysiological modifications have been detected transverse aortic constriction with no influence on in the cardiomyocytes, which are potentially able to cause perivascular and interstitial fibrosis, proinflammatory gene rhythm disturbances (Fig. 1). expression, myocyte hypertrophy and apoptosis. These discrepancies could be settled in case of confirmation of Inflammation of the coronary vasculature seems to play a recent findings hinting at MR activation in macrophages as a crucial role in the aldosterone-induced physiopathologic crucial step in the process of cardiac remodelling and blood cascade in the heart; the interstitial fibrosis and cardio- pressure control itself [109,110]. myocyte damage would then follow as a mere consequence of this previous event. This theory derives from the already- Other experimental observations concerning a direct mentioned study by Rocha et al. [63], where the effect of aldosterone on both cardiac fibroblasts and administration of aldosterone+salt to uninephrectomized rats cardiomyocytes strengthened these evidences. According to led to the development of severe hypertension and inflamma- Stockland et al. [111] aldosterone produces proliferation of tory coronary lesions characterized by fibrinoid necrosis of adult rat cardiac myofibroblasts by activation of the K-Ras the tunica media and a perivascular mononuclear leukocyte and MAPK pathways. Moreover, these cells act as mediators infiltrate. Immunohistochemistry revealed an increased of the elastogenic process by a putative MR-independent expression of markers of inflammation such as cell adhesion mechanism, contributing to the progression of the fibrotic molecules (mainly VCAM-1) at the endothelial level and damage [112]. As for the cardiomyocytes, Tsybouleva et al. inducible cyclo-oxygenase, cytokines and chemokines (TGF- [113] observed that the expression of hypertrophy markers 1, osteopontin, MCP-1) at the tunica media level. These (atrial natriuretic peptide and alpha-actin) and myocyte lesions were evident after an average of only two weeks of growth are induced by incubation of rat neonatal cardio- treatment, while progression to fibrosis and myocardial myocytes with aldosterone. In a microarray study performed damage was observed after at least four weeks. Treatment on a cell line of cardiomyocytes stably expressing the MR, with eplerenone partially reversed these findings with no the authors found an early (2 hours) regulation of a panel of significant blood pressure effects. Also the myocardial genes potentially involved in the regulation of the extra- lesions that develop in the rat model of angiotensin II / salt – cellular matrix, in inflammation and in signal transduction dependent hypertension (cardiac hypertrophy and vascular by physiological concentrations of aldosterone [114]. Some coronary inflammation) are to blame on aldosterone, as both of these transcripts (PAI1, Adamts1) were confirmed to be eplerenone (with no blood pressure effect) and under regulation by aldosterone also in the hearts of double- adrenalectomy are able to revert them [64]. Further, the transgenic mice with conditional, cardiomyocyte-specific advantage deriving from the use of mineralocorticoid overexpression of the MR [115], although this experimental antagonists is not limited to the prevention of the above- model does not permit to ascertain the endogenous ligand of mentioned myocardial lesions, but also to the regression of the MR and no major morphological alterations could be the fibrosis and the vascular alterations which are already observed in the hearts of these mice. Further, transgenic present, notwithstanding the continuous exposure to the mice with cardiomyocyte-specific overexpression of 11- fibrotic stimulus [101]. Consequently to these findings, it HSD2 develop fibrosis, hypertrophy and, at last, heart failure was initially thought that the development of myocardial [116]; under normal conditions cardiomyocytes do not interstitial fibrosis would represent the reparative evolution express this enzyme, so that cardiac MR are constitutively of these perivascular inflammatory lesions [102], even more occupied and tonically inhibited by glucocorticoids; so in the light of the fact that perivascular and interstitial therefore, the pathological effects observed in this paper are areas were the most affected by the fibrotic process [103] in accordance with a direct myotoxic effect of aldosterone. and that fibroblasts were often found to co-localize with Beyond the discrepancies in the scientific literature, the inflammatory cells [104]. thread connecting all the publications on this topic remains Nevertheless, a whole series of evidences in other the concept that the cardiotoxic action of aldosterone, experimental models points to a more general detrimental whether direct or indirect, MR-dependent or independent, Genomic and Non-genomic Effects of Aldosterone Current Signal Transduction Therapy, 2012, Vol. 7, No. 2 137 associated with an inappropriate saline status or not, cannot The relationship between mineralocorticoids and be addressed exclusively to the increased blood pressure. nephropathy was subsequently investigated by using animal This takes us back to one of the first works on the cardiac models; Greene et al. [124] utilized the so-called “remnant- effects of aldosterone where Brilla et al. [103] showed that kidney model”, consisting in subjecting normotensive rats to aldosterone/salt-treated rats displayed fibrosis not only in the subtotal nephrectomy, which leads to chronic renal failure, pressure-overloaded left ventricle, but also in the right one. arterial hypertension and glomerular sclerosis. In this study, the exogenous administration of aldosterone reversed the As far as it concerns the electrophysiological ability of the combined treatment with enalapril and losartan modifications induced in the cardiomyocytes, these depend to attenuate the appearance of the renal lesions. Although the on the effects of aldosterone on the membrane ion balances. The importance of this subject is underlined by the results of final development of glomerular sclerosis was not prevented, clinical trials demonstrating that adding low doses of MR chronic administration of high doses of spironolactone antagonists to patients affected by advanced congestive heart significantly delayed the appearance of proteinuria. failure (NYHA III and IV) [117] or acute myocardial The model of the spontaneously hypertensive rat-stroke infarction complicated by left ventricular dysfunction [118] prone (SHR-SP) was then used; these animals develop significantly reduces the sudden cardiac death rate. malignant hypertension 7 to 10 weeks after starting a diet It was long thought that hypokalemia was the main rich in salt and poor in potassium. This is coupled to determinant of the cardiac rhythm disturbances induced by paradoxical hyperactivation of the renin-angiotensin- aldosterone. Actually, aldosterone directly alters the ion aldosterone system, the early development of progressive fluxes at the cardiomyocyte plasmalemmal level by acting on nephroangiosclerosis with worsening proteinuria, and transport proteins. In contrast to what happens in the kidneys ischemic stroke that leads these animals to death by week 13 and the colon, in the cardiomyocytes of hyperaldosteronemic to 18 [125]. Chronic treatment with ACE inhibitors [126], rabbits a reduced affinity of the Na/K-ATPase pump for angiotensin II receptor antagonists [127] and spironolactone sodium was reported with ensuing intracellular hyper- [128] causes a dramatic reduction in both the vascular renal natremia and hypercalcemia [119]. Also potassium fluxes damage and proteinuria with no significant blood pressure during cardiomyocyte repolarization are altered with a lowering or modifications in ion and water balances. consequent lengthening of the QT interval on the electro- Administering aldosterone to captopril-treated SHR-SP cardiogram [120]. This effect is reversed by spironolactone restores the renal lesions without blood pressure modifications and has to be most probably attributed to the block of the [61]. Chandler PN, et al. [129] summed up these observations rapid component of the delayed rectifier K+ current carried and separated the pathogenetic role of angiotensin II and by human ether-a-go-go–related gene (HERG) channels. aldosterone in a study on saline-loaded SHR-SP where they According to other authors, aldosterone acts by inducing the demonstrated that bilateral adrenalectomy prevents the expression of those voltage-gated sodium channels which are development of proteinuria and thrombotic microangiopathy responsible of the so-called “funny current”; following notwithstanding the persistence of high blood pressure; the electrophysiological analysis of cultured neonatal rat chronic administration of angiotensin II only in the operated ventricular myocytes treated with aldosterone showed an rats does not restore the organ damage notwithstanding the accelerated rate of spontaneous beating due to increased high blood pressure values, while aldosterone does. excitability [121]. In another animal model, aldosterone is directly infused As a partial in vivo confirmation of these data, Ouvrad- in the subcutaneous tissue of uninephrectomized and salt- Pascaud et al. [122] demonstrated that, in a transgenic mouse loaded Sprague-Dawley rats. These animals display severe model with conditional cardiac-specific overexpression arterial hypertension and renal lesions, comparable to those of the human MR, embryonic and early post-natal lethality previously described, which are markedly reduced by the was superior to the controls in the absence of structural oral administration of eplerenone [130]. Vascular inflamma- cardiac alterations; such lethality was prevented by the tion is sustained by the activation of mediators like administration of spironolactone or propanolol to the osteopontin, MCP-1, interleukin-6 and 1, whose expression pregnant mother and had to be charged on a pro- is reduced by MR antagonism. arrhythmogenic propensity presenting as prolonged Other groups focused on the direct effects of mineralo- ventricular repolarization and severe ventricular arrhythmias. active compounds on various renal cell types. Nishiyama et ALDOSTERONE EFFECTS ON THE KIDNEY al. [131] reported that aldosterone directly induces rat mesangial cells proliferation and deformability through MR The effects determined on the kidney by high activation in vitro, possibly contributing to the pathogenesis concentrations of mineralocorticoid hormones have been of glomerular mesangial injury. According to the same known since the study by Selye et al. [123], who in 1943 authors, aldosterone acts in mesangial cells by increasing published his observations regarding the malignant ROS generation through MR-mediated membranous trans- nephrosclerosis induced in rats by the administration of location of specific components of NADPH oxidase [132]. DOCA and salt. This treatment induces thrombotic microangiopathy of both the medium-sized arteries and the Other authors investigated the capability of eplerenone to arterioles, associated with thickening of the tunica media, mitigate the damage to podocytes (and the ensuing fibrinoid necrosis and inflammatory vascular modifications worsening of proteinuria) observed in salt sensitive Dahl rats with marked leukocyte infiltration; the glomerular tufts subjected to massive salt loading [133]. Also in this case, the display an ischemic retraction with marked hyalinization and mineralocorticoid-induced podocyte damage is mediated by capsular fibrosis that result in proteinuria (Fig. 1). the activation of ROS-generating molecules [134]. 138 Current Signal Transduction Therapy, 2012, Vol. 7, No. 2 Verhovez et al.

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Received: June 29, 2011 Revised: August 10, 2011 Accepted: September 14, 2011