When first identified by Simpson and Tate in 1953, aldosterone was described as a hormone that controlled salt, volume and potassium homeostasis. In fact, several investigators working in the field at this time first gave the name ‘electrocortin’ to this compound because their research focused solely upon its role in electrolyte homeostasis. Perhaps this uni-dimensional view was a reflection of the setting in which aldosterone was first discovered. This initial research used an epithelial cell system, essentially renal tissue. Thus, this approach effectively focused research on aldosterone's role in sodium/potassium homeostasis. Over the next 40 years, investigators documented that aldosterone's primary epithelial mechanism was via a cytosolic receptor with transport to the nucleus. The molecular events induced by aldosterone leading to changes in sodium and potassium handling by the kidney were also identified. Substantial progress was made in identifying the factors that regulate aldosterone secretion and how these factors modify the biosynthetic machinery within the adrenal cortex.
In the last 10 years, it has become clear that this popular view only reflects the tip of the iceberg of aldosterone's functions and mechanisms of action. Aldosterone has more actions than controlling sodium and potassium homeostasis. The mineralocorticoid receptor has been identified in a wide variety of tissues beyond epithelial cells, including endothelial cells, vascular smooth muscle, fibroblasts, adipocytes and myocytes. The function of aldosterone in these cells is very different than the effect aldosterone has on epithelial cells. Furthermore, data support the concept that aldosterone works not only through a nuclear receptor but also through a cell-surface receptor, and intriguingly uses a common membrane mechanism – caveolae – to mediate its intracellular events. These findings have fundamentally reshaped our vision of the actions of aldosterone and its role in cardiovascular physiology and pathophysiology. Herein, we will review the role of three recently identified mediators of aldosterone's mechanism of action: lysine-specific demethylase 1 (LSD1), caveolin and striatin. Articles published in the last 18 months related to these three molecules and aldosterone and the interacting effect of salt intake will be reviewed.
LYSINE-SPECIFIC DEMETHYLASE 1, AN EPIGENETIC REGULATOR OF ALDOSTERONE AND SALT SENSITIVITY
Hypertension is a highly multigenic heritable trait (up to 40%); however, the currently identified genetic variants account for only a small fraction of the phenotypical differences and disease risk [1▪]. In the recent years, growing evidence has focused on the role of epigenetics, defined as phenotypic variations throughout life by mechanisms that alter gene expression independent of changes in DNA sequence . These epigenetic changes lead to a dynamic modulation of gene expression by several molecular processes including DNA methylation and histone modification . To date, determining the multiple environmental–genetic interactions that trigger these epigenetic changes and whether they are cause or consequence of hypertension remain to be elucidated.
A well studied epigenetic modification is DNA methylation that occurs mainly in cytosine-phosphate-guanine (CpG) dinucleotides, is maintained during cell division in mammals and generally downregulates gene transcription . Recently, Zhang et al.[4▪▪] evaluated the association of ADD1 gene promoter DNA methylation and hypertension. This gene encodes α-adducin, which regulates surface expression of multiple transporters, such as Na+/K+-ATPase, and is a well studied candidate gene for essential hypertension, despite recent meta-analyses with discrepant results [5–7]. This study showed a sexual dimorphism in DNA methylation patterns as risk factors for essential hypertension, with lower CpG1 in women and lower CpG2–5 in men. Classical risk factors for hypertension such as high-sodium diet, obesity and physical inactivity have been linked to changes in DNA methylation levels in other candidate genes, supporting a crucial interaction between environmental factors and changes in gene expression [8–10].
As hypertension is a heterogeneous disease in which several pathogenic mechanisms interplay to increase blood pressure (BP), we and others have used an intermediate phenotype approach for genetic studies . One of the best studied phenotypic traits is salt-sensitive hypertension (SSH), defined as an increase in BP in response to salt loading, with an estimated prevalence of 50–60% within hypertensive individuals . Several mechanisms have been proposed for SSH, including impaired renal sodium handling, reduced renin suppression and dysregulated rennin–angiotensin system in the vasculature, which could be mediated by epigenetic changes in response to high-salt diet [11,12].
As described with DNA, histones can undergo epigenetic modifications such as methylation or acetylation, changing the chromatin structure that packs the DNA, thereby playing an important role in gene regulation. By contrast to DNA methylation, the relaxation of condensed histones is a key step for activating gene expression . Pojoga et al.  and Williams et al. [15▪▪] recently described that LSD1 is involved in the pathogenesis of SSH in both animals and humans. LSD1 is a flavin-dependent amine oxidase that can act either as a transcription corepressor, if demethylation occurs at the lysine 4 of histone H3 (H3K4) site, or as a transcription coactivator if demethylation is switched towards the H3K9 site . In a translational approach, Williams et al. [15▪▪] showed that LSD1 heterozygote knockout mice (LSD1+/−) and African-American individuals with polymorphic variants of LSD1 had a similar phenotype that included SSH and decreased aldosterone levels on a high-salt diet. These results support a role of LSD1 as an epigenetic mediator of sodium's effects on BP and renal sodium excretion, although the underlying molecular pathway warrants further investigation (Fig. 1). In addition, the study showed that LSD1 protein levels in kidney and heart tissue were modified by salt intake in wild-type mice, providing new evidence that dietary salt can also modify the expression of a gene known to be an epigenetic regulator per se[15▪▪]. Further, Krug et al.[17▪] showed that LSD1 modulates the interaction of ageing and salt intake on BP. The study included data on LSD1+/− mice that showed a more pronounced age-associated increase in SBP when compared with wild-type after a 1-week high-salt diet. In addition, normotensive white individuals with LSD1 polymorphism rs7548692 showed a significant age–genotype interaction. The observed BP response to dietary salt intake strongly suggested that LSD1 polymorphism not only increased the prevalence of SSH but also modified the ageing effect on salt sensitivity [17▪]. As age and dietary salt intake are well described predictors of hypertension, this study supports the role of LSD1 as an epigenetic regulator that could explain, at least in part, how environmental stressors and physiological processes interact to modify gene expression and the resultant phenotype. Also, this study explored a potential mechanism in the interaction between salt intake and LSD1, showing that LSD1+/- mice had increased total plasma volume in association with increased BP, suggesting that LSD1 is involved in the regulation of whole body sodium homeostasis. Consistent with these results, Mu et al. added new epigenetic information in SSH by reporting that WNK lysine-deficient protein kinase 4 (WNK4) transcription could be downregulated by histone modification. The proposed mechanism is that sodium intake stimulates β2-adrenergic receptor resulting in cyclic AMP inhibition and histone acetylation. Thus, this study suggests that salt loading could also trigger another epigenetic modification resulting in decreased renal WNK4 expression and subsequent sodium retention and hypertension .
CAVEOLIN AS A MODULATOR OF ALDOSTERONE ACTIONS
Caveolae, small invaginations in the cellular membrane, are not only a structural platform for receptors, channels and enzymes but also act as functional modulators of a wide selection of cellular pathways. In cardiovascular and renal tissues, caveolae are particularly abundant and mediate a number of critical signal transduction mechanisms. The main component of plasma membrane caveolae, caveolin 1 (cav-1), has recently drawn growing attention owing to its effects on cellular physiology, from receptor and ion channel activation to signal transduction and intracellular trafficking.
Owing to its role in water and sodium homeostasis, aldosterone is a prime candidate to explain phenotypic abnormalities associated with SSH. Apart from the classical, genomic effects of aldosterone via transcriptional effects of the mineralocorticoid receptor in the nucleus, new evidence suggests that aldosterone can have rapid, nongenomic effects in cardiovascular and renal tissues. Such effects have been characterized to occur via mineralocorticoid receptor activation at the plasma membrane, or via interaction with other receptor pathways typically initiated in caveolae, including the angiotensin II type 1 receptor (AT1R), the epidermal growth factor receptor (EGFR) or the novel G protein coupled receptor GPR30 . Interestingly, Ricchiuti et al.  and Gildea et al.  have shown that during sodium loading, cav-1 and mineralocorticoid receptor levels are increased in both polar and nonpolar aldosterone target cells (such as cardiac and renal tissues), whereas mineralocorticoid receptor expression is decreased in cardiac and vascular tissues from cav-1 knockout mice , consistent with impaired mineralocorticoid receptor signalling in the absence of cav-1. In addition, Pojoga et al.  have shown that the mineralocorticoid receptor (like other steroid receptors) colocalizes and coimmunoprecipitates with cav-1 in these tissues and that mineralocorticoid receptor–caveolin complexes are more abundant during sodium loading . These data are consistent with an interplay between cav-1 and mineralocorticoid receptor in modulating mechanisms of salt sensitivity in aldosterone target tissues (Fig. 1). Indeed, Pojoga et al. [22,24] demonstrated that cav-1 deficient mice display profound vascular dysfunction that can be ameliorated by dietary sodium restriction or mineralocorticoid receptor blockade.
Importantly, other pathways modulated by cav-1 have also been implicated in mechanisms of aldosterone signalling and/or salt sensitivity, such as enzymes involved in oxidative stress responses, G-protein coupled receptors, as well as ion channels and transporters. Thus, it was recently shown that mineralocorticoid receptor blockade rescues the cardiovascular phenotype in Dahl salt-sensitive rats via improvements in oxidative stress mediated by the endothelial nitric oxide synthase (eNOS) and NADPH oxidase . These enzymes are not only known regulators of the redox potential inside cells (via their interaction with cav-1) but also key players in the cause of cardiovascular  and renal  diseases. Furthermore, recent evidence suggests that dietary sodium activates the small GTPase Rac1 in Dahl rat kidney tissues, leading to aldosterone-independent mineralocorticoid receptor activation, oxidative stress and target organ damage [28,29]. Importantly, cav-1 has been shown to be a key player in polyubiquitylation events that control Rac1 intracellular levels and activity .
Salt sensitivity is also often associated with abnormal ion transport in cardiovascular and renal tissues. There is now abundant evidence that cav-1 targets to caveolae and modulates aldosterone-sensitive molecules such as the epithelial sodium channel (ENaC) , the Na+/K+-ATPase [21,32,33], the Na+/H+ exchanger NHE-1 , the Na+/K+/2Cl− cotransporter (NKCC2) [35▪] or the large conductance Ca2+ and voltage-activated K+ channels (BKCa) . Initially demonstrated in kidney cells, these effects of cav-1 on aldosterone/mineralocorticoid receptor signalling have been recently extended to nonrenal tissues. Although many of these influences are exerted via changes in cav-1 mediated endocytosis, it has become clear that cav-1 can also facilitate direct signalling at the membrane. Interestingly, a recent study  suggests that the alpha-subunit of the Na+/K+-ATPase acts as a cell surface receptor for the nongenomic action of steroids, in a cav-1 dependent manner. Although the study was focused on the effects of progesterone in cultured oocytes, it can be speculated that similar interactions hold true for aldosterone in cardiovascular and renal tissues in vivo. Furthermore, cav-1 plays a critical role in maintaining appropriate urinary sodium excretion and BP homeostasis under conditions of sodium loading . Taken together, these data suggest that caveolae and cav-1 act as modulators of salt sensitivity via an interplay with mineralocorticoid receptor activation in aldosterone target tissues.
STRIATIN AND THE MINERALOCORTICOID RECEPTOR
Striatin, a scaffolding protein that is abundant in neurons, interacts with mediators of vesicular trafficking and cav-1 and is localized to caveolae. There is growing evidence that striatin facilitates the formation and cross-talk of a membrane signalling complex. Striatin contains a caveolin-binding motif, a coiled-coil structure, a Ca2+-calmodulin-binding site and a large WD-repeat domain . This WD-repeat domain interacts with GPCR-Gαi protein and protein phosphatase 2A, allowing for rapid activation of several transduction molecules including eNOS and mitogen-activated protein kinase.
Striatin has been shown to be a key intermediary of the effects of oestrogen receptor-α (ERα) activation and critical for the rapid/nongenomic effects of oestrogen on Akt and eNOS activation in endothelial cells . In fact, Lu et al.  have shown that blockade of the ERα–striatin complex has no effect on ERα-mediated gene transcription in human endothelial cells. Striatin's N-terminal segment interacts with the DNA-binding domain of ERα in endothelial cells that organizes ERα–eNOS membrane signalling leading to rapid nongenomic activation of eNOS. Of interest, overexpression of striatin leads to increased ERα in membrane fractions containing EGFR. More recent information from this group shows the in-vivo relevance of the ERα–striatin complex [39▪▪]. In-vivo blockade of the ER–striatin complex was performed using a novel mouse model that was created to produce a blocking peptide against the complex. The authors demonstrate loss of oestrogen-mediated protection against vascular injury following carotid artery wire injury in these mice.
The association between ERα and striatin raises the possibility that striatin may be a mediator of the rapid/nongenomic effects of other steroids. To this end, we recently reported that activation of mineralocorticoid receptor leads to increases of striatin levels in the vasculature and that the mineralocorticoid receptor is likewise complexed with cav-1 and striatin in vascular tissue (Fig. 1) [40▪]. We determined the in-vivo effects of salt restriction on striatin levels in vascular tissue that were associated with increased striatin levels in heart, aorta and kidneys. Our results also show that striatin is present and coprecipitates with the mineralocorticoid receptor in mouse aortas, heart and endothelial cells. Of importance, we have reported that aldosterone in vitro can stimulate increases of striatin mRNA and protein levels in both human and mouse endothelial cells, events that are not observed when cells are incubated with oestrogen. The in-vivo relevance of these studies was confirmed using two mouse models of mineralocorticoid receptor activation. We studied two previously described mouse models of increased aldosterone levels: intraperitoneal aldosterone administration and a model of chronic aldosterone mediated cardiovascular damage following treatment with N(G)-nitro-L-arginine methyl ester along with angiotensin II. In both models, we observed increased abundance of striatin protein in heart tissue. These results provide evidence further supporting the contention that striatin is an important component of mineralocorticoid receptor activation in the cardiovascular system.
Other than the work of the Karas group and Romero's group, there remains little information on the physiological role of striatin in the cardiovascular system. Transcriptional profiling of heart tissue has revealed the presence of striatin . Meurs et al.  localized striatin to the intercalated discs within cardiac myocytes from boxer dogs. They also identified a deletion in the 3’ untranslated region of the STRN gene that was associated with arrhythmogenic right ventricular cardiomyopathy in these dogs, which was associated with reduced striatin expression and protein levels. However, much more remains to be done to thoroughly comprehend the role of striatin in the cardiovascular system. Taken together, the available evidence would suggest that striatin mediates a novel level of interaction between signalling molecules, steroids and the cardiovascular system that may be important to understand steroid function in the vasculature.
The recent literature summarized above underlines the emergent roles of LSD1, cav-1 and striatin in modulating aldosterone's pathways resulting in salt sensitivity of BP. Clearly, further studies are needed to elucidate the relevance of these molecules as markers of aldosterone/mineralocorticoid receptor activation in humans. Furthermore, these biomarkers may identify new molecular pathways involved in hypertension, thus improving our current strategies for treatment and prevention.
This work was supported in part by grants from the National Institutes of Health: HL104032, HL096518, RM-07-2002, T32 HL007609, HL086907, HL094452, HL085224; The Chilean National Science and Technology Research Fund 1130427 and The William F. Milton Fund Faculty Award, Harvard Medical School.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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