New roles of aldosterone and mineralocorticoid receptors in cardiovascular disease: translational and sex-specific effects

Abstract

Recent advances in the field of mineralocorticoid receptor (MR) and its ligand aldosterone expanded the role of this hormone and its receptor far beyond their initial function as a regulator of Na⁺ and K⁺ homeostasis in epithelial cells. The symposium “New Roles of Aldosterone and Mineralocorticoid Receptors in Cardiovascular Disease: Translational and Sex-Specific Effects” presented at the 38th World Congress of the International Union of Physiological Sciences (Rio de Janeiro, Brazil) highlighted the contribution of extrarenal MRs to cardiovascular disease. This symposium showcased how MRs expressed in endothelial, vascular smooth muscle, and immune cells plays a critical role in the development of vascular disease associated with aging, obesity, and chronic aldosterone stimulation and demonstrated that MR antagonism prevents the acute renal dysfunction and tubular injury induced by ischemia-reperfusion injury. It was also shown that the adipocyte-derived hormone leptin is a new direct regulator of aldosterone secretion and that leptin-mediated aldosterone production is a major contributor to obesity-associated hypertension in women. Sex differences in the role of aldosterone and of endothelial MR in the cardiovascular outcomes of obesity were highlighted. This review summarizes these important emerging concepts regarding the contribution of aldosterone and cell-specific MR to cardiovascular disease in male and female subjects and further supports sex-specific benefits of MR antagonist drugs to be tested in additional populations.

INTRODUCTION

The aldosterone-mineralocorticoid receptor (MR) axis has historically been described as a pathway regulating blood pressure (BP) by controlling Na⁺ retention in renal epithelial cells (14). However, discoveries from the past two decades reveal that MRs are expressed in nonepithelial cells and exerts numerous additional functions beyond electrolyte handling in the distal tubule of the kidney. MRs are expressed in adipocytes, muscle, the liver, and pancreatic β-cells and in all cells of the cardiovascular and immune systems extending the initial function of the MR to the control of the metabolic, vascular, cardiac, renal, and immune systems (10, 19, 34, 50, 73).

This symposium was organized to present the latest advances regarding the role of the MR and aldosterone in aging, metabolic, and renal disorders at the session titled “New Roles of Aldosterone and Mineralocorticoid Receptors in Cardiovascular Disease: Translational and Sex-Specific Effects” at the 38th World Congress of the International Union of Physiological Sciences at Rio de Janeiro, Brazil, in August 2017. Sponsored by the American Physiological Society, this symposium focused on the newly discovered roles of MR in endothelial, smooth muscle cells (SMCs), and immune cells in the functional and structural vascular remodeling associated with aging, cardiometabolic disease, and kidney injury. This symposium also emphasized the clinical potential of MR antagonism (MRA) for the prevention and treatment of aging and the consequences of metabolic disorders, acute kidney injury (AKI), and chronic kidney disease. The session also highlighted the new mechanisms regulating aldosterone production and demonstrated a role for MRs in the sex specificity of the mechanisms leading to endothelial dysfunction and hypertension in obesity.

The content and conclusions from this session have relevance in understanding the contribution of aldosterone and MRs to cardiovascular disease and highlight the importance of considering MR blockade as a therapeutic avenue for numerous cardiovascular diseases.

A ROLE FOR SMC-MRs IN VASCULAR AGING

Vascular Aging

Aging is a universal and powerful risk factor for cardiovascular disease. The incidence of all forms of cardiovascular disease, including myocardial infarction (MI), stroke, hypertension, heart failure, and cardiovascular death, all increase dramatically with age (32, 44). There are well-described changes in vascular structure and function with aging that contribute to cardiovascular disease (for reviews, see Refs. 98, 100, and 104). Functionally, the aging vasculature produces more reactive oxygen species (ROS) and less nitric oxide (NO), resulting in increased oxidative stress and enhanced vasoconstriction, thereby contributing to hypertension and impaired tissue perfusion. Structurally, SMCs of the aging vasculature are more migratory and proliferative with an altered extracellular matrix resulting in increased fibrosis and progressive vascular stiffening with age. This is important clinically because vascular stiffness directly correlates with the risk of MI, stroke, and cardiovascular death, independent of BP or other risk factors (102). Thus, understanding the mechanisms driving the vascular aging process has substantial clinical significance with potential to lead to novel prevention and treatment strategies for common cardiovascular disorders.

MR Expression in Vascular SMCs Increases with Aging and Contributes to Vascular Remodeling

Data from as early as the 1970s to 1980s suggested that receptors in the arterial wall could directly respond to aldosterone and contribute to hypertension and vascular disease (61, 62). With the identification of the MR as the aldosterone-binding receptor (3), it has since been confirmed that the MR is expressed in human vascular SMCs and regulates expression of genes in SMCs that are involved in vascular fibrosis and calcification (48, 49, 78). Moreover, MR expression in SMCs has been found to increase with age in rat aortic SMCs, in the mouse aorta and mesenteric resistance vessels, and in human saphenous veins after grafting (4, 35, 63). The development of transgenic mice with MRs specifically deleted from SMCs has advanced our understanding of the specific role of SMC-MRs in vascular function and disease (for a review, see Ref. 59). With the use of these mice, SMC-MRs were found to directly contribute to vascular fibrosis in response to wire injury (86) and to vascular stiffening and integrin expression induced by hypertension (41).

Mice Lacking SMC-MRs Are Protected From Vascular Aging

The knowledge that SMC-MRs increase with age and contribute to vascular remodeling in response to injury and hypertension prompted mouse studies to explore the direct role of SMC-MRs in vascular aging. The life expectancy of laboratory mice is ~24 mo, and the aging phenotype in male mice with MRs specifically deleted from SMCs [SMC-MR knockout (KO)] was compared with MR-intact littermates as they aged to determine whether there is a role for SMC-MRs in functional and structural changes with vascular aging (35, 58, 74). In MR-intact mice, BP increased modestly with aging from 3 to 12 mo (35) along with an increase in the resistance vessel contractile response to vasoconstrictors including potassium chloride, phenylephrine, thromboxane, and angiotensin II (ANG II) (74). SMC-MR KO mice were protected from the rise in BP with aging and from the enhanced vasoconstriction to all agonists except the adrenergic agonist phenylephrine. The difference in BP in aged SMC-MR KO mice was independent of changes in renal MR function including the fractional excretion of Na⁺ under normal or low-Na⁺ conditions (74). Rather, 12-mo-old male mice lacking SMC-MRs had decreased resistance vessel myogenic tone. This was associated with decreased vascular responsiveness to activation of the L-type Ca²⁺ channel (LTCC), a channel that is critical to the mechanism of vascular tone and vasoconstriction (35, 74). ANG II-induced vascular oxidative stress and hypertension are also exacerbated with aging (66, 74), and these responses to ANG II were eliminated in aged mice lacking SMC-MRs (74). In addition to ANG II, many other factors, including elevated Na⁺, can modulate vascular oxidative stress (16, 28, 39). Overall, the new data support that in male mice, the MR, specifically in SMCs, plays a critical role in driving resistance vessel tone, oxidative stress, and vasoconstriction in the aging vasculature thereby contributing to rising BP with aging (66, 74).

SMC-MRs have also recently been found to contribute to cardiovascular structural changes with aging in mice (58). Aortic stiffness, as measured by pulse wave velocity, was found to increase with aging from 3 to 12 or 18 mo of age in male MR-intact mice. This aging-associated increase in vascular stiffness, as well as vascular fibrosis, was prevented in SMC-MR KO mice (58). There is also an aging-associated increase in cardiac stiffness. Interestingly, cardiac stiffness and coronary perivascular fibrosis were both attenuated in aging mice lacking SMC-MRs. The changes in cardiac stiffness correlated with changes in aortic stiffness, suggesting that the cardiac aging effects may in part be secondary to vascular mechanisms. As in humans, cardiovascular aging in mice was associated with a modest decline in cardiac function and exercise capacity, and this decline was attenuated in SMC-MR KO mice (58). Thus, MRs in SMCs contribute to vascular and cardiac structural changes with aging that contribute to tissue stiffness, an important determinant of adverse outcomes in aging humans.

Mechanisms by Which SMC-MRs Contributes to Vascular Aging

The detailed molecular mechanism by which SMC-MRs contribute to vascular aging has begun to be elucidated (Fig. 1). Vascular RNA expression profiling revealed profound global changes in mRNA and microRNA (miR) expression in the aging vasculature that is dramatically altered in the absence of SMC-MRs (35, 58). The most downregulated miR in the aging mouse aorta was miR-155, which did not decline with aging SMC-MR KO vessels. miR-155 has been previously shown to decrease with aging in human peripheral leukocytes (79) and to correlate inversely with BP in humans (24). Mechanistic in vitro studies revealed that the MR negatively regulates transcription of the miR-155 host gene promoter, independent of aldosterone. In aging mice, rising MR mRNA in mesenteric resistance vessels correlated with a decrease in miR-155. The decline in miR-155 with age is further associated with increased vascular expression of predicted miR-155 targets, including the LTCC subunit Ca_v1.2 and the ANG II type 1 receptor (AT₁R). These gene expression changes were further associated with increased LTCC-mediated vasoconstriction and ANG II-induced oxidative stress (35). This aging mechanism is lost in SMC-MR KO mice, implicating a direct role for SMC-MRs in driving the mechanism (Fig. 1).

Fig. 1.

Mechanisms by which the smooth muscle cell mineralocorticoid receptor (SMC-MR) contributes to the adverse outcomes of vascular aging. MR expression rises with aging in vascular SMCs, resulting in suppressed transcription of microRNA (miR)-155 and enhanced expression of profibrotic genes including connective tissue growth factors (CTGF), matrix metaloprotease (MMP)-2, and bone morphogenetic proteins (BMP). Inhibition of miR-155 by MRs leads to an increase in miR-155 targets, including the angiotensin II (ANG II) type 1 receptor (AT₁R) and the L-type calcium channel (LTCC) subunit Ca_v1.2, which together promote vasoconstriction by increasing reactive oxygen species (ROS) and Ca²⁺ influx in SMCs. Together, these effects of SMC-MRs contribute to changes in vascular function and structure with aging including increased vascular; oxidative stress, vasoconstriction, fibrosis, and stiffness. By blocking these pathways, MR antagonists may improve outcomes be preventing the adverse effect of SMC-MRs on vascular aging pathology including high blood pressure, vascular stiffness, and cardiac fibrosis leading to heart attack, stroke, and heart and renal failure.

Regarding the mechanism for the structural changes with aging, lack of SMC-MRs produced a totally distinct mRNA expression signature with aging that was predicted to oppositely regulate pathways involved in cardiovascular development and function. Specifically, in the absence of SMC-MRs, there was profound suppression of a profibrotic gene expression program, including a downregulation of connective tissue growth factor (CTGF), matrix metalloproteinase (MMP)-2, and bone morphogenetic protein 4 (BMP-4). The mechanism for these global gene expression changes remains to be determined but as the MR is a transcription factor that is known to regulate CTGF (78) and other BMPs (49) in SMCs, transcriptional mechanisms are a focus of investigation.

Translational Implications and Future Directions for the Role of SMC-MRs in Vascular Aging

Alterations in Na⁺ and K⁺ metabolism related to renal mineralocorticoid effects may lead to possible confounding effects on the direct vascular actions of mineralocorticoids via the generation of ROS. Additional studies in low-Na⁺, high-K⁺ conditions will help address this concern. All of the published studies exploring the role of MRs in aging have been performed in male subjects; thus, future studies are warranted to determine whether there are sex differences in the role of MRs in vascular aging. Substantial investigation is also needed to determine whether this enhanced understanding of the role of SMC-MRs in the mechanisms driving vascular aging can be used therapeutically. Preclinical studies revealed that in aged mice, restoration of vascular miR-155 by a SMC-targeted lentivirus decreased vascular miR-155 target gene expression (Ca_v1.2 and AT₁Rs) and attenuated resistance vessel vasoconstriction (35). Treatment of 12-mo-old mice with the MR antagonist spironolactone for 4 mo decreased vascular fibrosis, stiffness, and expression of CTGF and BMP-4 (58), suggesting that MR inhibition might be used to prevent or slow vascular aging. Whether these pathways contribute to hypertension in aging humans remains to be determined, but there are some suggestive data. A single-nucleotide polymorphism in the AT₁R 3′-untranslated region that prevents miR-155 binding is associated with hypertension in humans (24). In a small study in 16 older humans, changes in serum miR-155 levels in response to MRA correlated with an improved BP response to therapy (35). In a study of 11 men (average age: 64 yr), 1 mo of eplerenone treatment also decreased serum MMP-2 with a trend toward decreased CTGF and BMP-4, suggesting an inhibitory effect on fibrosis (58). Larger studies with a longer duration of treatment that also include female subjects are needed to accurately test the potential of MR inhibition as an antiaging therapy.

MR AND ALDOSTERONE IN THE CARDIOVASCULAR CONSEQUENCES OF OBESITY: SEX DIFFERENCES

The recent epidemic of obesity, which affects more women than men worldwide (40), is one of the major risk factors for cardiovascular disease. While women are generally protected from cardiovascular disease until menopause, recent evidence suggests that obesity, particularly with diabetes, eliminates the protective effects of female sex (93, 103) and is the cause of the threefold increase in the risk for cardiovascular disease in premenopausal women over the last three decades (97) as well as of the rising number of schoolgirls diagnosed with hypertension (72, 92). These alarming data raise the urgency of understanding the mechanisms whereby obesity impairs vascular function, raises BP, and induces cardiovascular disease, particularly in female subjects.

Sex Differences in the Role of Aldosterone in Obesity and Associated Cardiovascular Disease

Several lines of evidence indicate that aldosterone, a major contributor to both cardiovascular and metabolic dysfunctions, is produced in excess in obesity (22) and correlates directly not only with visceral adipose mass and body mass index but also with BP in women only (43). In addition, blockade of aldosterone action via MRA appears more efficacious as a cardiovascular therapy regimen in women compared with men (56), which suggests an important relationship between aldosterone and cardiovascular disease in women. However, the origin of the elevated plasma aldosterone levels in obesity remains incompletely understood. Based on the observations that increases in aldosterone levels in obesity correlate directly with adipose mass and are independent of increases in renin and ANG II levels (15, 45, 57), Ehrhart-Bornstein et al. (36) investigated the contribution of the adipose tissue and identified the adipocyte as a source of aldosterone-releasing factors.

In this context, recent studies have been conducted to determine whether the adipocyte-derived hormone leptin could be the missing link between high adipose mass and elevated circulating aldosterone levels. Studies have shown that, despite obesity, mice, rats (13, 46), and humans (82, 83) deficient in leptin or harboring impaired leptin signaling do not exhibit high circulating aldosterone levels, whereas endogenous increases in leptin levels with obesity, and exogenous leptin supplementation raise adrenal aldosterone synthase [cytochrome P-450 11B2 (CYP11B2)] expression and aldosterone production (46), which suggests a central role for leptin in obesity-associated increases in aldosterone levels. Inhibition of ANG II, the primary regulator of aldosterone production, or of α- and β-adrenergic signaling in vivo does not abolish the stimulating effects of leptin on adrenal CYP11B2 expression and aldosterone production, which suggests that leptin exerts direct action on the adrenal glands. This was confirmed by demonstrating that leptin dose dependently increases CYP11B2 expression and aldosterone production via Ca²⁺-dependent mechanisms in human adrenal cortical cells in culture (Fig. 2) (46). Therefore, it is suggested that leptin, but not increases in adipose mass per se, is the source of the elevated aldosterone levels in obesity.

Fig. 2.

Mechanisms via which leptin increases cytochrome P-450 11B2 (CYP11B2) and aldosterone production in adrenal zona glomerulosa cells. Shown is an illustration of a new mechanism whereby activation of the leptin receptor in adrenal zona glomerulosa cells increases intracellular Ca²⁺ levels and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) expression, leading to an increase in aldosterone synthase expression (CYP11B2) and an increase in aldosterone production.

This newly discovered leptin-aldosterone axis emerges as a key contributor to cardiovascular disease in obese female mice predominantly. Hyperleptinemia in obesity and leptin sensitization in mice deficient in protein tyrosine phosphatase 1B deletion, a molecular “break” on leptin signaling (106), induce hypertension in male and female mice (12, 47). However, female mice only present with marked increases in adrenal aldosterone synthase (CYP11B2) expression and plasma aldosterone levels and blockade of aldosterone action with spironolactone restores BP in female but not male animals (47). Male animals, on the other hand, present with sympathoactivation with obesity and leptin sensitization (11, 47), whereas female animals do not (47). This supports the new concept that leptin induces hypertension via sex-specific mechanisms in obesity: leptin activation of the aldosterone-mineralocorticoid axis in female mice (47) and leptin-induced sympathoactivation in male mice (12) (Fig. 3).

Fig. 3.

Potential mechanisms via which obesity leads to hypertension in male and female subjects. The illustration shows that leptin elevates blood pressure via sex-specific mechanisms in the context of obesity: elevation in sympathetic activity in male subjects and increased in aldosterone production in female subjects.

Further evidence to support the predominance of the leptin-aldosterone axis in the development of cardiovascular disease in obese female mice is presented by demonstrating that leptin deficiency in obese ob/ob female mice protects from obesity-induced endothelial dysfunction and cardiac fibrosis, while restoration of leptin levels in these mice impairs endothelial function and stimulates cardiac collagen deposition (46). Consistent with the work by the Sowers and colleagues (2, 18, 52, 53, 67) in obese diabetic mice, MR blockade with spironolactone abolished leptin-induced endothelial dysfunction and collagen deposition, providing further arguments to support the aldosterone-MR dependence of the mechanisms whereby leptin induces cardiovascular disease in the context of obesity (46).

All of these observations have been made in rodent models of obesity; therefore, additional experiments in humans would be required to confirm the predominance of the aldosterone-MR axis in the development of cardiovascular disease in obese women and determine whether leptin is also a major activator of the aldosterone-MR axis in humans.

Role of Endothelial Cell MRs

In addition to the role of MRs in SMCs in vascular remodeling and aging discussed in the first section of this review, MRs are also expressed in the vascular endothelium (23). Several mechanisms have been associated with endothelial dysfunction induced by obesity (for a review, see Ref. 37), and, recently, attention has been given to MR activation in endothelial cells (ECs) as a potential mediator. Short-term incubation of ECs with aldosterone increases endothelial NO synthase (eNOS) phosphorylation and NO release, but this effect is not observed with long-term application (71, 77). Instead, chronic aldosterone reduces eNOS-derived NO synthesis and increases ROS generation in a dose-dependent manner in ECs, which contributes to a decrease in NO bioavailability (69) and endothelial dysfunction in the presence of several cardiovascular risk factors including obesity (for a review, see Ref. 30). This is consistent with the beneficial effect of MR blockade in coronary microvascular function of individuals with type 2 diabetes (42).

One mechanism involved in the EC-MR-induced endothelial dysfunction involves oxidative stress and reduced NO synthesis and bioavailability (27). MR blockade attenuates eNOS uncoupling and decreased the expression of the NADPH oxidase subunits p22^phox and p40^phox, which renders oxidative stress and increases NO production in ECs (26, 90). In addition, aldosterone-induced epithelial Na⁺ channel expression increases EC stiffness, resulting in impaired NO release by inhibition of phosphatidylinositol 3-kinase/Akt/eNOS pathway (38, 65). Enhanced endothelial epithelial Na⁺ channel activation drives coronary endothelium remodeling and permeability in obese female mice, which was associated with cardiac diastolic dysfunction (51). These experimental data are consistent with the benefit of MR antagonist in patients with reduced ejection fraction (81).

There are sex differences in the mechanisms driving microvascular endothelial dysfunction in response to cardiometabolic risk factors (Fig. 4). This is clinically significant because dysfunction of resistance microvessels, rather than conduit vessels, predicts 5-yr cardiovascular disease risk in humans (70). Resistance vessels dilate in response to environmental changes and physiological needs to modulate blood flow to specific organs. Endothelium-dependent vasodilation is measured experimentally by quantifying the vascular relaxation response to acetylcholine. Two major components contribute to this endothelium-derived vasodilatory response of resistance vessels: eNOS-derived NO and endothelium-derived hyperpolarization (EDH). EDH is mediated by endothelial K⁺ channels, specifically IK1 and SK3 channels, which account for EDH in resistance microvessels. Microvessels from male subjects exhibit a substantial loss of NO-dependent relaxation in response to obesity, which can be compensated for initially by increased EDH (25, 31). When obesity is associated with hyperlipidemia in male subjects, the compensatory increase in EDH component, with enhanced expression of endothelial SK3 K⁺ channels, is lost resulting in resistance vessels endothelial dysfunction (31). In female subjects, obesity with or without hyperlipidemia impairs the EDH component of endothelium-dependent vasodilatation in small arteries by reducing expression of endothelial IK1 K⁺ channels.

Fig. 4.

Sex differences in mechanisms of resistance vessel endothelial dysfunction in obesity. Changes in endothelial function and the effect of endothelial mineralocorticoid receptor (EC-MR) deletion in small mesenteric arteries of high fat diet-induced male and female obese mice are shown. Female mice develop endothelial dysfunction associated with reduced expression of endothelial K_Ca3.1 and intermediate-conductance Ca²⁺-activated K⁺ (IK1) channels and impaired endothelium-dependent hyperpolarization (EDH). In male mice, high fat diet-induced obesity leads to a reduction in nitric oxide (NO) levels, which is compensated for by increased expression of endothelial K_Ca2.3 and small-conductance Ca²⁺-activated K⁺ (SK3) channels and EDH. EC-MR deletion restores endothelial function only in female mice by enhancing NO release.

Use of mice with MRs specifically deleted from ECs (EC-MR KO) revealed the endothelial MR as a key mechanism driving sex differences in the vasodilatory endothelial dysfunction of resistance arteries (31, 52), further supporting a role for EC-MRs in obesity-induced endothelial dysfunction in female subjects. MR deletion in ECs protects female subjects from endothelial dysfunction induced by obesity and dyslipidemia by increasing NO availability, but this mechanism was not observed in male subjects (Fig. 4). We cannot exclude that an interplay between EC-MRs and caveolin-1 prevents an increase in NO in male subjects, as in the absence of caveolin-1 MR activation upregulates eNOS expression and activity (84, 85). However, in obese male subjects, rather than endothelial MR signaling, sympathetic activation appears to be the driver of cardiovascular damage as endothelial dysfunction is attenuated by the treatment with β-blockers (29, 95). These results support the concept that different molecular mechanisms drive endothelial dysfunction in male and female subjects and suggest that sex-specific therapies are likely to be needed to prevent the adverse cardiovascular consequences of obesity.

ROLE OF IMMUNE CELLS IN ALDOSTERONE-INDUCED VASCULAR DAMAGE

Aldosterone has proinflammatory effects in various cell types, including vascular cells and cells of the innate and adaptive immune systems: it increases the DNA-binding activity of transcription factors such as NF-κB and activator protein (AP)-1, increases the expression of adhesion molecules such as ICAM-1 and VCAM-1, and increases the expression of other inflammatory markers (cyclooxygenase-2, macrophage chemoattractant protein-1, osteopontin, TNF-α, IL-1β, and IL-6) (94). Aldosterone (as well as the aldosterone precursor DOCA) also induces accumulation of macrophages and T cells in the kidneys (96), heart (88), and vasculature. These proinflammatory effects of aldosterone usually rely on MR activation and contribute to end-organ damage in many pathological conditions. In addition, many experimental and clinical studies have demonstrated that MRA produces beneficial outcome in patients with cardiovascular and metabolic diseases, mainly due to prevention of inflammatory responses.

Accordingly, cells of the immune system express MRs, and MR activation modulates immune cell functions. For example, MR expression has been reported in primitive blood- and bone marrow-derived progenitor cells (CD34⁺ hematopoietic stem cells), monocytes/macrophages, neutrophils, dendritic cells, and peripheral T and B lymphocytes from humans and experimental animals. In many of these immune cells, aldosterone-induced MR activation induces cytokine secretion and activates phagocytic/humoral responses. Furthermore, deletion of MRs in macrophages protects from cardiac fibrosis and increased BP induced by DOCA-salt treatment (87), and inhibition of T lymphocytes polarization by MRA reduces mineralocorticoid-induced end-organ damage (1). For more details, please refer to the following excellent and recent review articles in Refs. 6, 21, 76, 91, and 101.

To better understand the mechanisms involved in the proinflammatory phenotype of aldosterone or how the innate immune system contributes to aldosterone-mediated vascular injury, our group investigated whether aldosterone activates the NOD-like receptor (NLR) pyrin-domain-containing protein 3 (NLRP3) inflammasome (20). NLRP3 is a member of the NLR family. It regulates the assembly of a multimolecular complex (the inflammasome) that activates inflammatory caspases and generate proinflammatory cytokines such as IL-1β and IL-18. Cells of the innate immunity express pattern recognition receptors (PRRs), which include the large families of Toll-like receptors, C-type lectin receptors, RIG-I-like receptors, and NLRs. PRRs are activated by pathogen-associated molecular patterns, which are molecules expressed by microbial pathogens and by damage-associated molecular patterns, i.e., cell components that are released during cell damage or death. Activation of PRRs triggers signaling pathways that control the expression and release of cytokines, cell adhesion molecules, and migration. Activation of the NLRP3 occurs in response to a variety of signals that are indicative of damage, including bacterial DNA, ATP, glucose, uric acid and cholesterol crystals, and mitochondria-derived ROS.

In bone marrow-derived macrophages from wild-type mice, aldosterone increases mitochondrial ROS and caspase-1 activation. Caspase-1 activation is blunted in aldosterone-stimulated bone marrow-derived macrophages from NLRP3^−/− mice. Aldosterone infusion in wild-type mice activates the NLRP3 inflammasome (it increases NLRP3 expression, caspase-1 activity, and mature IL-1β) in cells from the peritoneal cavity (peritoneal macrophages). Aldosterone also increases plasma IL-1β and induces vascular dysfunction (abnormal vascular reactivity, remodeling, and increased expressed of adhesion molecules). NLRP3 deletion almost completely prevented all effects of aldosterone: changes in vascular reactivity, increased expression of cell adhesion molecules, vascular remodeling (increased cross-sectional area and increased wall-to-lumen ratio), and increased systolic BP (20).

Of clinical importance, patients with aldosterone-producing adenomas and resistant arterial hypertension exhibit high levels of IL-1β and other cytokines (IL-6 and TNF-α), and MR blockade with spironolactone or eplerenone, as well as adrenalectomy, decreases cytokine levels to levels seen in healthy control subjects (64). Our study showed that leukocytes from patients with hyperaldosteronism exhibit NLRP3 inflammasome activation and increased serum IL-1β levels compared with healthy human volunteers (20). Additional data from our laboratory show that aldosterone-induced NLRP3 activation relies on MR activation (N. S. Ferreira, T. Buder-Nascimento, C. A. Pereira, C. Z. Zanotto, D. S. Prado, J. F. Silva, D. M. Rassi, M. C. Foss-Freitas, J. C. F. Alves-Filho, D. C. Sartori, and R. C. Tostes, unpublished observations). Aldosterone seems to be capable of activating both the priming process (it increases the expression of NLRP3 and pro-IL-1β and activates NF-κB) and the assembly of the inflammasome (mainly via generation of mitochondria-derived ROS) (20). However, further studies are needed to clarify how aldosterone/MR activation triggers the activation of the immune system.

Aldosterone-induced NLRP3 activation also contributes to an inflammatory phenotype and end-organ damage in other pathological conditions including kidney injury leading to chronic kidney disease progression/nephrotic syndrome (5, 33, 55) and obesity-associated adipose tissue and liver dysfunction (99), reinforcing that the NLRP3 inflammasome plays a key role on aldosterone/MR-induced inflammation and target-organ abnormalities in cardiovascular, renal, and metabolic diseases.

MRA: A PROMISING THERAPEUTIC APPROACH TO TREAT AKI AND AKI-MEDIATED CHRONIC KIDNEY DISEASE

AKI is a frequent complication in hospitalized patients with higher incidence rates in patients in intensive care units. It is associated with unfavorable outcomes such as increased short- and long-term mortality rates, longer hospital stay, cardiovascular complications and chronic kidney disease development (17).

During the past decade, evidence indicating that MRA may be a useful strategy to protect against AKI has been accumulating (54). The prophylactic administration of spironolactone prevented the acute renal dysfunction and tubular injury induced by ischemia-reperfusion (I/R) (75). The beneficial effects of spironolactone were associated with the preservation of a normal renal blood flow and reduction in oxidative stress. Importantly spironolactone is also able to efficiently treat kidney I/R injury (IRI) when administered up to 3 h after reperfusion (89). This protective effect was also observed with nonsteroidal MRAs (7, 9), which have been suggested to have a better therapeutic index for hyperkalemia in patients with renal dysfunction (60). The mechanisms underlying the deleterious effects of MR activation during IRI highlight the critical role of MR-mediated oxidative stress; MR-mediated oxidative stress has been shown to lead to a specific imbalance of vascular endothelin signaling through an oxidative stress-dependent posttranslational modification of the vasodilatory endothelin B receptor, leading to functional inactivation of the endothelin B receptor and sustained decrease of the renal blood flow (9, 68). MRAs can prevent oxidative stress and its deleterious consequences (7, 68). The genetic deletion of the MR in SMCs was associated with weaker Rac1 signaling and oxidative stress production (7), and the role of Rac1 was confirmed by the in vivo deletion of Rac-1 in SMCs, which also protected against AKI (7). Decreased oxidative stress production in mice with genetic deletion of the MR in SMCs was associated with reduced posttranslational modification of the endothelin B receptor leading to increased NO production and improved renal perfusion (7). Importantly, the data obtained in both mice and rats can be translated to the Large White pig: soludactone, a soluble MRA, prevented acute IRI after bilateral IRI. This was associated to decreased urinary excretion of an oxidative stress marker (7).

Episodes of AKI lead to increased risk of chronic kidney disease progression and renal failure (105). MRA administration during the acute phase of IRI also prevents the decline of long-term renal function and tubulo-interstitial fibrosis in rats (8, 68) and mice 7a. Steroidal and nonsteroidal MRAs are equally efficient in preventing the progression of chronic kidney disease (8, 68). The underlying mechanism of the benefit of MRAs relies on the prevention of low-grade inflammation and polarization of macrophages toward an M2 repair phenotype (7a). Whether prevention of the progression of chronic kidney disease after IRI using MRAs also on living-donor renal transplantation (80) demonstrated a beneficial effect of spironolactone on renal oxidative stress when administered to the recipients 1 day before and 3 days after transplantation. However, this was not associated with improved short-term renal function since renal function was already good in the placebo group, as expected for living-donor transplantation. A multicenter clinical trial is ongoing in France to study the impact of short-term administration of eplerenone (25 mg twice a day, just before and 4 days after the transplant) on 3-mo renal graft function in patients receiving a graft from an expanded-criteria donor, which is more susceptible to ischemia insult [Eplerenone in Patients Undergoing Renal Transplant (EPURE): ClinicalTrials.gov NCT02490904, supported by the French Programme Hospitalier de Recherche Clinique].

In conclusion, MRA is a promising therapeutic approach to treat AKI and AKI-mediated CKD. The benefit relies on decreased oxidative stress and improved renal perfusion associated with reduced low-grade inflammation and increased macrophage-mediated repair, leading to reduced progression to chronic kidney disease.

CONCLUSIONS

In summary, the symposium presentations reflected growing insights into the new roles for extrarenal MR signaling in aging and cardiovascular diseases. The focus was on the sex differences in MR activation in SMCs and ECs. There are two main conclusions from the presentations. First, MR activation in SMCs plays a critical role driving vascular aging phenotype and AKI in male subjects. SMC-MR deletion attenuates aging-induced aortic stiffness, increased resistance vessel tone and vasoconstriction, and hypertension. Moreover, mice lacking SMC-MRs are protected against AKI associated with weaker Rac1 signaling and oxidative stress, improving NO production and renal perfusion. Second, a possible role of SMC-MR in female subjects still need to be addressed: endothelial MR signaling is a key mechanism for sex differences in obesity-induced endothelial dysfunction, mediating endothelial stiffness and impairing endothelium-dependent relaxation of conduit and resistance arteries of obese female but not male subjects. Leptin is a major activator of the aldosterone-MR axis, contributing to the cardiovascular damage in female obesity. MR antagonist drugs are efficient in preventing cardiovascular injury associated with EC- and SMC-MR signaling and may be beneficial in additional populations that remain to be tested in clinical trials.

GRANTS

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo 2017/10771-5 and 2014/26192-6 (to A. P. Davel), National Heart, Lung, and Blood Institute (NHLBI) Grant 1R0-1HL-130301-01 and American Heart Association (AHA) Grant 16IRG27770047 (to E. J. Belin de Chantemèle), and NHLBI Grants R01-HL-095590 and R01-HL-119290 and AHA Grant EIA-18290005 (to I. Z. Jaffe).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. conceived and designed research; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. performed experiments; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. analyzed data; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. interpreted results of experiments; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. prepared figures; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. drafted manuscript; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. edited and revised manuscript; A.P.D., I.Z.J., R.C.T., F.J., and E.J.B.d.C. approved final version of manuscript.