The mineralocorticoid receptor (MR) and its ligand aldosterone are the principal modulators of hormone-regulated renal sodium reabsorption. In addition to the kidney, there are several other cells and organs expressing MR, in which its activation mediates pathologic changes, indicating potential therapeutic applications of pharmacological MR antagonism. Steroidal MR antagonists have been used for decades to fight hypertension and more recently heart failure. New therapeutic indications are now arising, and nonsteroidal MR antagonists are currently under development. This review is focused on nonclassic MR targets in cardiac, vascular, renal, metabolic, ocular, and cutaneous diseases. The MR, associated with other risk factors, is involved in organ fibrosis, inflammation, oxidative stress, and aging; for example, in the kidney and heart MR mediates hormonal tissue-specific ion channel regulation. Genetic and epigenetic modifications of MR expression/activity that have been documented in hypertension may also present significant risk factors in other diseases and be susceptible to MR antagonism. Excess mineralocorticoid signaling, mediated by aldosterone or glucocorticoids binding, now appears deleterious in the progression of pathologies that may lead to end-stage organ failure and could therefore benefit from the repositioning of pharmacological MR antagonists.

Aldosterone plays a major role in the regulation of extracellular volume and blood pressure, through its renal action via the mineralocorticoid receptor (MR). The extra-renal roles of aldosterone/MR in non-epithelial cells (neurones, cardiomyocytes, vessels, adipocytes…) are now intensively investigated. Animal models (aldo/DOCA-salt) and clinical trials (RALES, EPHESUS) evidenced a major pathophysiological contribution of aldosterone/ MR in the cardiovascular system. To evaluate MR cardiac signaling, we analyzed the transcriptomic profile of 2 transgenic mouse models with conditional and inducible overexpression of the MR or of the glucocorticoid receptor (GR) in cardiomyocytes (MHCMR and MHC-GR). Using dedicated cardiochips, we identified 520 cardiac genes that are differentially expressed between control and MHC-MR mice (250 up, 270 down-regulated), and 1232 genes differentially expressed between control and MHC-GR mice (728 up, 504 down-regulated). Some of these genes were validated by real-time PCR, such as Periostin [MHC-MR vs control 10.56±2.2, p=0.02 ; MHC-GR vs control 4.36±1.72, NS], Troponin T3 [MHC-MR vs control 41.66±9.23, p=0.048 ; MHC-GR vs control 1.78±0.41, NS], Fkbp5 [MHC-MR vs control 34.51±6.16, p=0.01 ; MHC-GR vs control 3.33±0.59, p=0.006]. We have also compared the effects of aldosterone in the presence or not of different MR/GR antagonists on gene expression in a cardiomyocytes cell line (H9C2 cells) stably expressing the MR, in order to analyze hormone and antagonist specificity. Importantly, some of the MR-specific genes encode for secreted proteins that may represent novel markers of MR activation in circulating blood, including in human pathology.

BACKGROUND INFORMATION:The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells.RESULTS:In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells.CONCLUSIONS:These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.

Background—Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. Methods and Results—To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. Conclusions—Our results indicate that cardiac MR triggers cardiac arrhythmias, suggesting novel opportunities for prevention of arrhythmia-related sudden death.

The renal collecting duct plays a key role in control of ion and fluid homeostasis. Genes encoding for ion transporters, hormone receptors, or regulatory proteins specifically expressed in the collecting duct are mutated in several genetic diseases with altered blood pressure. Suitable cellular models expressing genes in a conditional way should represent attractive systems for structure-function analyses and generation of appropriate physiopathological models of related diseases. However, generation of such systems remains laborious and quite inefficient. We adapted and improved a conditional Cre-lox-inducible system in the highly differentiated aldosterone-sensitive rat cortical collecting duct (RCCD2) cell line. The inducible MerCreMer recombinase allowed tight control and high levels of transgene expression, whereas flanking a selection marker with two loxP sites strongly improved the selection procedure. We have used this system to conditionally express an enhanced green fluorescent protein-tagged human mineralocorticoid receptor. In the future, this will allow structure-function analyses as well as mineralocorticoid receptor trafficking studies in these epithelial cells, which retain the features of the native collecting duct. Improvements in the conditional Cre-lox expression system have potentially wide applications in other epithelial or nonepithelial cell lines.

Effects of aldosterone on its target cells have long been considered to be mediated exclusively through the genomic pathway; however, evidence has been provided for rapid effects of the hormone that may involve nongenomic mechanisms. Whether an interaction exists between these two signaling pathways is not yet established. In this study, the authors show that aldosterone triggers both early nongenomic and late genomic increase in sodium transport in the RCCD(2) rat cortical collecting duct cell line. In these cells, the early (up to 2.5 h) aldosterone-induced increase in short-circuit current (Isc) is not blocked by the mineralocorticoid receptor (MR) antagonist RU26752, it does not require mRNA or protein synthesis, and it involves the PKCalpha signaling pathway. In addition, this early response is reproduced by aldosterone-BSA, which acts at the cell surface and presumably does not enter the cells (aldo-BSA is unable to trigger the late response). The authors also show that MR is rapidly phosphorylated on serine and threonine residues by aldosterone or aldosterone-BSA. In contrast, the late (4 to 24 h) aldosterone-induced increase in ion transport occurs through activation of the MR and requires mRNA and protein synthesis. Interestingly, nongenomic and genomic aldosterone actions appear to be interdependent. Blocking the PKCalpha pathway results in the inhibition of the late genomic response to aldosterone, as demonstrated by the suppression of aldosterone-induced increase in MR transactivation activity, alpha1 Na(+)/K(+)/ATPase mRNA, and Isc. These data suggest cross-talk between the nongenomic and genomic responses to aldosterone in renal cells and suggest that the aldosterone-MR mediated increase in mRNA/protein synthesis and ion transport depends, at least in part, upon PKCalpha activation. E-mail: marcel.blot-chabaud@pharmacie.univ-mrs.fr

The enzyme 11-beta hydroxysteroid dehydrogenase type 2 plays a major role in blood pressure regulation. It metabolizes glucocorticoid hormones into derivatives with low affinity for the mineralocorticoid receptor, preventing its permanent occupancy by circulating cortisol, which is 100- to 1000-fold more abundant than aldosterone in the plasma. Inactivating mutations of the enzyme result in severe hypertension, as seen in children with apparent mineralocorticoid excess syndrome. In patients with essential hypertension, however, attempts to evidence enzyme deficiency have been inconclusive. In this pilot study, its catalytic activity was measured directly in aldosterone-sensitive sweat gland ducts collected from skin biopsy samples of 10 male normotensive subjects and 10 subjects with essential hypertension (more than 140 to 90 mm Hg) with no sign of hypermineralocorticism. Isolated ducts were assayed for nicotinamide-dinucleotide-dependent dehydrogenase activity (transformation of tritiated corticosterone into tritiated-11 dehydrocorticosterone, as measured by high-pressure liquid chromatography). Hypertensive patients exhibited significantly lower 11-beta hydroxysteroid dehydrogenase type 2 activity (9.7+/-4.7 femtomoles per 3 mm length of duct and per 10 minutes incubation, median+/-SD) than did normotensive subjects (15.9+/-2.6). Such defect was undetectable using the classical urinary corticosteroid metabolism indexes, probably because of compensatory mechanisms. Relations between these findings and blood pressure levels should benefit from direct enzyme measurements in the vasculature. In conclusion, this cross-sectional study points to partial 11-beta hydroxysteroid dehydrogenase type 2 deficiency as a novel feature of essential hypertension, which should stimulate search for new signaling pathways and therapeutical targets.

It has been recently proposed that 11,8-hydroxysteroid dehydrogenase (1 1,-OHSD) is responsible for aldosterone tissue specificity. A 1 1#-OHSD deficiency has been invoked as a cause of the syndrome of apparent mineralocorticoid excess, and I If-OHSD inhibition by liquorice has been invoked to explain the hypertension induced by this drug. Since the renal tubule is composed of aldosterone-sensitive and insensitive segments, we determined the distribution of 11f-OHSD along the rabbit tubule. Pools of tubular segments isolated by microdissection were incubated for 2 h at 370C in the presence of I3Hlcorticosterone (3H-B, 8.10-9 M). Afterwards, the amounts of 3H-B and of the metabolite 11-dehydrocorticosterone (3H-A) were determined using HPLC analysis. In the proximal tubule, in either its convoluted or straight portion, and in the medullary thick ascending limb, the amount of 3H-A was 19.6±3.8% (n = 12), 17.9±3.4 (a = 8), and 15.0±2.2 (n = 4), respectively, of the sum of 3H-A + 3H-B. In the cortical ascending limb and the collecting tubule in its cortical and medullary parts, it was 74.7±6.8% (n = 4), 74.1±4.9 (n = 9) and 64.6±14.1 (n = 3), respectively. In both proximal and cortical collecting tubule, addition of carbenoxolone 8.10-4 M, an in-hibitor of 11fl-OHSD, almost completely inhibited the conversion of 3H-B to 3H-A. Thus, 11#-OHSD activity was high in the aldosterone-sensitive segments, and low in the aldoste-rone-insensitive segments. These results strongly favor the hypothesis that 1 1-OHSD is a key enzyme in mineralocorticoid tissue specificity along the rabbit nephron. They reinforce the notion that a defect in 11j-OHSD plays a major role in the syndrome of apparent mineralocorticoid excess and liquorice-induced hypertension. (J. Clin. Invest. 1990. 86:832-837.)

Cardiac failure is a common feature in the evolution of cardiac disease. Among the determinants of cardiac failure, the renin-angiotensin-aldosterone system has a central role, and antagonism of the mineralocorticoid receptor (MR) has been proposed as a therapeutic strategy. In this study, we questioned the role of the MR, not of aldosterone, on heart function, using an inducible and cardiac-specific transgenic mouse model. We have generated a conditional knock-down model by expressing solely in the heart an antisense mRNA directed against the murine MR, a transcription factor with unknown targets in cardiomyocytes. Within 2-3 mo, mice developed severe heart failure and cardiac fibrosis in the absence of hypertension or chronic hyperaldosteronism. Moreover, cardiac failure and fibrosis were fully reversible when MR antisense mRNA expression was subsequently suppressed.