Mineralocorticoid Receptor Signaling Research Articles

Mineralocorticoid receptor signaling affects therapeutic effect of enzalutamide.

Corticosteroids play important roles in prostate cancer therapeutics. However, their role when combined with enzalutamide remains obscure. Then, we aimed to elucidate the functional and clinical impact of corticosteroids on steroid receptors in androgen receptor (AR)-targeting therapy utilizing enzalutamide. The therapeutic effect was studied according to concomitant use of corticosteroids in 86 men treated with enzalutamide. The sensitivity to various agents was evaluated using cytotoxicity assays in prostate cancer cells. Gene expression levels were evaluated by quantitative real-time polymerase chain reaction in prostate cancer cells and tissues. The therapeutic effect of enzalutamide was particularly lessened with concomitant treatment with dexamethasone. Consistently, dexamethasone increased cellular resistance to enzalutamide while prednisolone and aldosterone decreased cellular resistance to enzalutamide in prostate cancer cells. Inversely, mineralocorticoid receptor (MR) knockdown augmented the activity of AR signaling and the cellular resistance to enzalutamide. MR plays a critical role in resistance to AR-targeting therapies, which may be overcome by activation of MR signaling.

Specific Activation of the Alternative Cardiac Promoter of Cacna1c by the Mineralocorticoid Receptor.

The MR (mineralocorticoid receptor) antagonists belong to the current therapeutic armamentarium for the management of cardiovascular diseases, but the mechanisms conferring their beneficial effects are poorly understood. Part of the cardiovascular effects of MR is because of the regulation of L-type Cav1.2 Ca2+ channel expression, which is generated by tissue-specific alternative promoters as a long cardiac or short vascular N-terminal transcripts. To analyze the molecular mechanisms by which aldosterone, through MR, modulates Cav1.2 expression and function in a tissue-specific manner. In primary cultures of neonatal rat ventricular myocytes, aldosterone exposure for 24 hours increased in a concentration-dependent manner long cardiac Cav1.2 N-terminal transcripts expression at both mRNA and protein levels, correlating with enhanced concentration-, time-, and MR-dependent P1-promoter activity. In silico analysis and mutagenesis identified MR interaction with both specific activating and repressing DNA-binding elements on the P1-promoter. The relevance of this regulation is confirmed both ex and in vivo in transgenic mice harboring the luciferase reporter gene under the control of the cardiac P1-promoter. Moreover, we show that this cis-regulatory mechanism is not limited to the heart. Indeed, in smooth muscle cells from different vascular beds, in which the short vascular Cav1.2 N-terminal transcripts is normally the major isoform, we found that MR signaling activates long cardiac Cav1.2 N-terminal transcripts expression through P1-promoter activation, leading to vascular contractile dysfunction. These results were further corroborated in hypertensive aldosterone/salt rodent models, showing notably a positive correlation between blood pressure and cardiac P1-promoter activity in aorta. This new vascular long cardiac Cav1.2 N-terminal transcripts molecular signature reduced sensitivity to the Ca2+ channel blocker, nifedipine, in aldosterone-treated vessels. Our results reveal that MR acts as a transcription factor to translate aldosterone signal into specific cardiac P1-promoter activation that might influence the therapeutic outcome of cardiovascular diseases.

Abstract WP418: Mineralocorticoid Receptor Signaling is Associated With Neuroinflammation and Changes in Cognitive Function in Angiotensin II-Induced Hypertension

Introduction: Mineralocorticoid receptor (MR) activation facilitates hypertension-associated cerebral artery dysfunction and reduced perfusion that may increase the risk for vascular dementia. Hypothesis: Angiotensin II (AngII)-hypertension results in neuroinflammation and oxidative stress that will impair cognition in a MR-dependent manner. Methods: 16-17 week old male C57bl/6 mice were treated with AngII (800ng/kg/min) ± the MR antagonist, eplerenone (EPL;100mg/kg/day) for 4 weeks; Sham mice served as control. Data are presented as mean ± SEM; n=4-12 per group *=different from Sham. Results: AngII reduced astrocyte density and increased microglial density; the changes in microglia were prevented by EPL. AngII reduced the mRNA expression of brain-derived neurotrophic factor (BDNF), doublecortin, glial fibrillary acidic protein (GFAP), and synaptophysin (SYP); the reductions in BDNF, GFAP and SYP mRNA expression were prevented by EPL. We assessed cognitive function using the Barnes maze; AngII treated mice spent less time in the target quadrant than the sham or AngII+EPL mice. There was a trend toward there being an increase in the latency to reach the target quadrant in the AngII mice but the changes were not significant. Changes in nest building are associated with cognitive decline, nest building was impaired in AngII mice and these changes were prevented by EPL. There was a trend for oxidized proteins to be increased in AngII mice that may be prevented by EPL but these changes were not significant. Preliminary studies suggest that AngII changes the morphology of cortical neurons suggesting that the increased blood pressure may lead to unhealthy neurons. Conclusion: AngII-hypertension results in neuroinflammation and creates an environment where markers for neuronal support and synapse formation are impaired. These changes are regulated by MR signaling and may be associated with the declined cognitive function.

Neutrophil Gelatinase-Associated Lipocalin from immune cells is mandatory for aldosterone-induced cardiac remodeling and inflammation

Immune system activation is involved in cardiovascular (CV) inflammation and fibrosis, following activation of the mineralocorticoid receptor (MR). We previously showed that Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a novel target of MR signaling in CV tissue and plays a critical role in aldosterone/MR-dependent hypertension and fibrosis. We hypothesized that the production of NGAL by immune cells may play an important part in the mediation of these deleterious mineralocorticoid-induced effects. We analyzed the effect of aldosterone on immune cell recruitment and NGAL expression in vivo. We then studied the role of NGAL produced by immune cells in aldosterone-mediated cardiac inflammation and remodeling using mice depleted for NGAL in their immune cells by bone marrow transplantation and subjected to mineralocorticoid challenge NAS (Nephrectomy, Aldosterone 200μg/kg/day, Salt 1%). NAS treatment induced the recruitment of various immune cell populations to lymph nodes (granulocytes, B lymphocytes, activated CD8+ T lymphocytes) and the induction of NGAL expression in macrophages, dendritic cells, and PBMCs. Mice depleted for NGAL in their immune cells were protected against NAS-induced cardiac remodeling and inflammation.We conclude that NGAL produced by immune cells plays a pivotal role in cardiac damage under mineralocorticoid excess. Our data further stressed a pathogenic role of NGAL in cardiac damages, besides its relevance as a biomarker of renal injury.

Endothelial Mineralocorticoid Receptor Mediates Parenchymal Arteriole and Posterior Cerebral Artery Remodeling During Angiotensin II-Induced Hypertension.

The brain is highly susceptible to injury caused by hypertension because the increased blood pressure causes artery remodeling that can limit cerebral perfusion. Mineralocorticoid receptor (MR) antagonism prevents hypertensive cerebral artery remodeling, but the vascular cell types involved have not been defined. In the periphery, the endothelial MR mediates hypertension-induced vascular injury, but cerebral and peripheral arteries are anatomically distinct; thus, these findings cannot be extrapolated to the brain. The parenchymal arterioles determine cerebrovascular resistance. Determining the effects of hypertension and MR signaling on these arterioles could lead to a better understanding of cerebral small vessel disease. We hypothesized that endothelial MR signaling mediates inward cerebral artery remodeling and reduced cerebral perfusion during angiotensin II (AngII) hypertension. The biomechanics of the parenchymal arterioles and posterior cerebral arteries were studied in male C57Bl/6 and endothelial cell-specific MR knockout mice and their appropriate controls using pressure myography. AngII increased plasma aldosterone and decreased cerebral perfusion in C57Bl/6 and MR-intact littermates. Endothelial cell MR deletion improved cerebral perfusion in AngII-treated mice. AngII hypertension resulted in inward hypotrophic remodeling; this was prevented by MR antagonism and endothelial MR deletion. Our studies suggest that endothelial cell MR mediates hypertensive remodeling in the cerebral microcirculation and large pial arteries. AngII-induced inward remodeling of cerebral arteries and arterioles was associated with a reduction in cerebral perfusion that could worsen the outcome of stroke or contribute to vascular dementia.

Abstract 120: Endothelial Specific Sodium Channel Activation in Endothelium Dysfunction and Vascular Stiffness in Obese Female Mice

Excessive activation of endothelial cell (EC) mineralocorticoid receptor (ECMR) signaling induces EC epithelial sodium channel (EnNaC) activity to promote cardiovascular stiffness. Our previous study has demonstrated that activated ECMR signaling prompts expression and translocation of EnNaC to the EC surface inducing fibrosis, inflammation, and macrophage infiltration in the vasculature of female mice fed a western diet (WD). As ECMR KO also prevented these abnormalities, we posit that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC-specific EnNaC activation would mediate endothelium dysfunction, vascular stiffness, and impair flow-mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO and wild-type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC, significantly inhibited inward Na+ currents and EnNaC activity in the cultured endothelial cells. Amiloride treatment significantly attenuated WD-induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. In addition, amiloride improved flow mediated dilation in mesenteric arteries and endothelium-dependent relaxation in response to acetylcholine (10 -9 -10 -4 mol/L). Furthermore, amiloride prevented WD-induced increases in coronary endothelium permeability that were associated with decreased expression of claudin-5 and occluding. This also resulted in reduction of total macrophage recruitment (CD11b) and M1 polarization (CD11c). Importantly, genetic knock-out EnNaC KO also prevented aldosterone-induced endothelium stiffening and impairment of endothelium-dependent relaxation. These data indicate that EC specific EnNaC activation decreases bioavailable NO, increases vascular endothelium dysfunction, and prompts vascular stiffening in obese female mice.

Abstract P277: Mineralocorticoid Receptor Signaling Regulates TRPV4-mediated Vasodilation of Parenchymal Arterioles

Hypertension impairs endothelium-dependent dilation of the parenchymal arterioles (PA) that regulate white matter perfusion. In the periphery, mineralocorticoid receptor (MR) activation facilitates hypertension-associated artery dysfunction; the role of MR signaling in PA dysfunction has not been defined. We hypothesized that Angiotensin II (AngII)-hypertension impairs TRPV4-mediated dilation in an MR-dependent manner. 16 week old male C57bl/6 mice were treated with AngII (800ng/kg/min) ± the MR antagonist, eplerenone (EPL; 100mg/kg/day) for 4 weeks; Sham mice served as control. PAs were isolated and studied by pressure myography. Data are presented as mean ± SEM; n=4-6 per group. AngII increased systolic blood pressure, an effect not blunted by EPL (Sham:147 ± 3; AngII: 177* ± 3; AngII+EPL: 182* ± 6mmHg; *=p<0.05 vs. Sham). However, EPL prevented the increased myogenic tone (31±1 vs 48*±4 vs 35±3 % tone; *=p<0.05 vs. Sham) and the impaired carbachol (CbCh)-induced dilation (51±3 vs 24*±2 vs 49±3 %dilation; Sham vs. AngII vs. AngII+EPL; *=p<0.05 vs. Sham) caused by AngII infusion. CbCh-induced dilation was unaltered by L-NAME (10 -5 M) and indomethacin (10 -4 M) in all groups (Sham: 51±3 vs. 53±2; AngII:24±2 vs. 28±2; AngII+EPL 49±3 vs. 47±3% dilation; CbCh vs. CbCh+L-NAME+Indomethacin) implicating endothelium-derived hyperpolarization (EDH). In contrast, the TRPV4 antagonist, GSK2193874 (GSK2, 10 -7 M) blunted the CbCh-induced dilation in all groups (Sham: 51±3 vs 13*±8; AngII: 24±2 vs 7*±2; AngII+EPL: 49±3 vs 16*±4 % dilation; CbCh vs CbCh+GSK2 ;*=p<0.05). In the AngII and AngII+EPL mice, TRPV4 inhibition reduced myogenic tone (AngII: 16±3; AngII+EPL: 17±6 %tone loss; p<0.05), an effect that was not observed in the Sham mice (2±3 %tone loss). AngII infusion decreased cerebral perfusion; EPL prevented this decrease (Sham: 938±32; AngII:596*±53; AngII+EPL:793±36 perfusion units; *=p<0.05 vs. Sham). MR activation regulates TRPV4 signaling in PAs during AngII-infusion, independent of hypertension. Impaired TRPV4 function plays a critical role in AngII-hypertension-associated changes in PA reactivity and may increase the risk of small vessel disease.

Deoxycorticosterone/Salt-Mediated Cardiac Inflammation and Fibrosis Are Dependent on Functional CLOCK Signaling in Male Mice.

Activation of the mineralocorticoid receptor (MR) promotes inflammation, fibrosis, and hypertension. Clinical and experimental studies show that MR antagonists have significant therapeutic benefit for all-cause heart failure; however, blockade of renal MRs limits their widespread use. Identification of key downstream signaling mechanisms for the MR in the cardiovascular system may enable development of targeted MR antagonists with selectivity for pathological MR signaling and lower impact on physiological renal electrolyte handling. One candidate pathway is the circadian clock, the dysregulation of which is associated with cardiovascular diseases. We have previously shown that the circadian gene Per2 is dysregulated in hearts with selective deletion of cardiomyocyte MR. We therefore investigated MR-mediated cardiac inflammation and fibrosis in mice that lack normal regulation and oscillation of the circadian clock in peripheral tissues, that is, CLOCKΔ19 mutant mice. The characteristic cardiac inflammatory/fibrotic response to a deoxycorticosterone (DOC)/salt for 8 weeks was significantly blunted in CLOCKΔ19 mice when compared with wild-type mice, despite a modest increase at "baseline" for fibrosis and macrophage number in CLOCKΔ19 mice. In contrast, cardiac hypertrophy in response to DOC/salt was significantly greater in CLOCKΔ19 vs wild-type mice. Markers for renal inflammation and fibrosis were similarly attenuated in the CLOCKΔ19 mice given DOC/salt. Moreover, increased CLOCK expression in H9c2 cardiac cells enhanced MR-mediated transactivation of Per1, suggesting cooperative signaling between these transcription factors. This study demonstrates that the full development of MR-mediated cardiac inflammation and fibrosis is dependent on intact signaling by the circadian protein CLOCK.

Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae

Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3days post-fertilization were exposed to waterborne cortisol for 24h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11β-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae.

HuR-Dependent Editing of a New Mineralocorticoid Receptor Splice Variant Reveals an Osmoregulatory Loop for Sodium Homeostasis

Aldosterone and the Mineralocorticoid Receptor (MR) control hydroelectrolytic homeostasis and alterations of mineralocorticoid signaling pathway are involved in the pathogenesis of numerous human diseases, justifying the need to decipher molecular events controlling MR expression level. Here, we show in renal cells that the RNA-Binding Protein, Human antigen R (HuR), plays a central role in the editing of MR transcript as revealed by a RNA interference strategy. We identify a novel Δ6 MR splice variant, which lacks the entire exon 6, following a HuR-dependent exon skipping event. Using isoform-specific TaqMan probes, we show that Δ6 MR variant is expressed in all MR-expressing tissues and cells and demonstrate that extracelullar tonicity regulates its renal expression. More importantly, this splice variant exerts dominant-negative effects on transcriptional activity of the full-length MR protein. Collectively, our data highlight a crucial role of HuR as a master posttranscriptional regulator of MR expression in response to osmotic stress. We demonstrate that hypotonicity, not only enhances MR mRNA stability, but also decreases expression of the Δ6 MR variant, thus potentiating renal MR signaling. These findings provide compelling evidence for an autoregulatory feedback loop for the control of sodium homeostasis through posttranscriptional events, likely relevant in renal pathophysiological situations.

The role of mineralocorticoid receptor signaling in the cross-talk between adipose tissue and the vascular wall.

Vascular dysfunction and impaired endothelial mediated relaxation are powerful underlying abnormalities in the pathogenesis of hypertension, coronary heart disease, and stroke. Obesity, type 2 diabetes mellitus, and other metabolic abnormalities are associated with activation of mineralocorticoid receptor (MRs) in the vasculature and adipose tissue. While MR signaling is involved in the normal physiological differentiation and maturation of adipocyte, enhanced activation of MRs also contributes to increase oxidative stress, release of pro-inflammatory adipokines, and dysregulation of adipocyte autophagy. This, in turn, increases the maladaptive expansion of subcutaneous, visceral and perivascular adipose tissue, resulting in systemic and cardiovascular (CV) insulin resistance and increased CV stiffness and impaired vascular and cardiac relaxation. This review summarizes the normal role of MR activation in adipose tissues and explores the mechanisms by which excessive MR activation mediates adipose tissue inflammation and vascular dysfunction. Potential preventative and therapeutic strategies directed in the prevention of MR activation and CV disease are also discussed.

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Coregulators as mediators of mineralocorticoid receptor signalling diversity.

The cloning of the mineralocorticoid receptor (MR) 30 years ago was the start of a new era of research into the regulatory processes of MR signalling at target genes in the distal nephron, and subsequently in many other tissues. Nuclear receptor (NR) signalling is modified by interactions with coregulatory proteins that serve to enhance or inhibit the gene transcriptional responses. Over 400 coregulatory proteins have been described for the NR super family, many with functional roles in signalling, cellular function, physiology and pathophysiology. Relatively few coregulators have however been described for the MR although recent studies have demonstrated both ligand and/or tissue selectivity for MR-coregulator interactions. A full understanding of the cell, ligand and promoter-specific requirements for MR-coregulator signalling is an essential first step towards the design of small molecular inhibitors of these protein-protein interactions. Tissue-selective steroidal or non-steroidal modulators of the MR are also a desired therapeutic goal. Selectivity, as for other steroid hormone receptors, will probably depend on differential expression and recruitment of coregulatory proteins.

Mineralocorticoid Receptor Signaling as a Therapeutic Target for Renal and Cardiac Fibrosis.

Activation of the mineralocorticoid receptor (MR) plays important roles in both physiological and pathological events. Blockade of MR signaling with MR antagonists (MRAs) has been used clinically to treat kidney and cardiac disease associated with hypertension and other chronic diseases, resulting in suppression of fibrosis in these organs. However, the current use of steroidal MRAs has been limited by off target effects on other hormone receptors or adverse effects on kidney tubular function. In this review, we summarize recent insights into the profibrotic roles of MR signaling in kidney and cardiovascular disease. We review experimental in vitro data identifying the pathological mechanisms associated with MR signaling in cell types found in the kidney (mesangial cells, podocytes, tubular cells, macrophages, interstitial fibroblasts) and heart (cardiomyocytes, endothelial cells, vascular smooth muscle cells, macrophages). In addition, we demonstrate the in vivo importance of MR signaling in specific kidney and cardiac cell types by reporting the outcomes of cell type selective MR gene deletion in animal models of kidney and cardiac disease and comparing these findings to those obtained with MRAs treatment. This review also includes a discussion of the potential benefits of novel non-steroidal MRAs for targeting kidney and cardiac fibrosis compared to existing steroidal MRAs, as well as the possibility of novel combination therapies and cell selective delivery of MRAs.

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor null mice: informing cell-type-specific roles.

The mineralocorticoid receptor (MR) mediates the actions of two important adrenal corticosteroid hormones, aldosterone and cortisol. The cell signalling roles of the MR in vivo have expanded enormously since the cloning of human MR gene 30 years ago and the first MR gene knockout in mice nearly 20 years ago. Complete ablation of the MR revealed important roles postnatally for regulation of kidney epithelial functions, with MR-null mice dying 1-2 weeks postnatally from renal salt wasting and hyperkalaemia, with elevated plasma renin and aldosterone. Generation of tissue-selective MR-deficient mice using Cre recombinase-LoxP gene targeting has made it possible to analyse mice lacking MR only in specific cell types. Targeting renal-specific MR has differentiated roles in specific compartments of the kidney. Ablating MR in neurons of the forebrain reinforced important roles of the MR in response to stress, behaviour and anxiety, but suggested a minimal role in maintaining basal HPA axis tone. Deletion of the MR in macrophages and other cell types of the cardiovascular system clearly defined important roles for the regulation of cardiovascular physiology and pathophysiology. Knockdown of MR mRNA in vivo using antisense/siRNA approaches, and similarly MR overexpression, has provided useful rodent models to study physiological roles of MR signalling in vivo More recently, targeted mutation of specific domains of the MR such as the DBD has defined genomic vs non-genomic roles in vivo New tissue-selective MR-null models are required to define roles of MR signalling in other regions of the brain, the eye, gastrointestinal tract, lung, skin, breast and gonadal organs.

Interaction of the Mineralocorticoid Receptor With RACK1 and Its Role in Aldosterone Signaling.

The mineralocorticoid receptor (MR) is a member of the steroid-thyroid hormone receptor superfamily of ligand-dependent transcription factors with diverse functions including the biological actions of aldosterone. Identification of the various transcriptional coregulators of MR is essential for understanding the complexity of MR signaling pathways under physiological and pathological conditions. We used a yeast two-hybrid system to find proteins that interact with a full-length MR and found, among other proteins, that MR interacted specifically with receptor for activated C kinase 1 (RACK1), a scaffolding protein. Overexpression of RACK1 using a tetracycline-inducible lentivirus in mouse cortical collecting duct M1 cells stably expressing the rat MR and a Gaussia luciferase gene reporter under a hormone-response element promoter resulted in enhanced agonist-dependent MR transactivation. Knockdown of RACK1 protein expression by short hairpin RNAs led to a significant reduction in MR activation of the reporter gene and the endogenous genes Ctla2α and Psca. We also demonstrated that RACK1 regulation of MR action is mediated through phosphorylation by the PKC-β signaling pathway. MR and RACK1 were coimmunoprecipitated using an MR antibody in male Sprague-Dawley brain tissue and M1-rMR cells, and colocalization in M1-rMR cells and male rat brains was confirmed by immunofluorescence and immunohistochemistry. The scaffolding protein RACK1 is associated with MR under basal and agonist-stimulated conditions and facilitates agonist-stimulated MR actions through PKC-β. These findings indicate that RACK1 is a newly described coactivator of MR.

Smooth Muscle Cell Mineralocorticoid Receptors Are Necessary for Cardiovascular Fibrosis with Aging

Vascular aging involves fibrosis leading to stiffening of the vascular wall, an independent predictor of adverse cardiovascular events. Mineralocorticoid receptors (MR) are expressed in vascular smooth muscle cells (SMC) and MR signaling has been implicated in the regulation of cardiovascular fibrosis but has not been explored in cardiovascular aging. We previously demonstrated that aged mice lacking MR in SMCs (SMC‐MR‐KO) have lower blood pressure and reduced responsiveness to angiotensin II. The aim of this study was to investigate the role of SMC‐MR in cardiovascular structural and functional changes with aging and to explore mechanisms by which SMC‐MR contributes to vascular aging by modulating global vascular gene expression in mice. Vascular and cardiac fibrosis, aortic pulse wave velocity (PWV), cardiac echocardiography, intra‐cardiac pressure‐volume loops, and endurance exercise capacity were assessed in 3, 12 and 18 month‐old male MR‐intact and SMC‐MR‐KO mice. Vascular fibrosis in aortas, carotid arteries, and renal arterioles and aortic stiffness increased with aging in MR‐intact mice. Importantly, these aging‐induced increases were attenuated in SMC‐MR‐KO mice. Cardiac hypertrophy, wall thickness, fibrosis and stiffness increased and cardiac function and endurance exercise capacity decreased with aging in MR‐intact mice. Aging‐induced changes in cardiac hypertrophy, wall thickness and function were unaffected by the absence of SMC‐MR, however, SMC‐MR‐KO mice were protected from the aging‐induced increases in cardiac fibrosis and stiffness and the decrease in endurance exercise capacity. There was a significant correlation between aortic PWV and end‐systolic pressure volume relationship, suggesting that the changes in cardiac systolic stiffness are likely secondary to changes in aortic stiffness with aging. Renal fibrosis increased with aging but this increase was independent of the presence of SMC‐MR. To explore the mechanism by which SMC‐MR contributes to vascular fibrosis and stiffness with aging, we characterized global gene expression profiles in aortas from 3 and 12 month‐old MR‐intact and SMC‐MR‐KO mice. 552 and 477 genes were differentially expressed with aging in aortas from MR‐intact and SMC‐MR‐KO, respectively. Only 79 genes were differentially expressed with aging in both MR‐intact and SMC‐MO‐KO mice. Pathway analysis revealed that changes in cardiovascular development pathways with aging are predicted to act in opposing directions in SMC‐MR‐KO mice compared to MR intact mice. We also identified a network of fibrosis‐related genes that are down‐regulated with aging in SMC‐MR‐KO mice, providing a possible mechanism for the reduced vascular fibrosis with aging in SMC‐MR‐KO mice. Our findings provide evidence that SMC‐MR plays a role in aging‐induced increase in vascular fibrosis that contributes to cardiovascular tissue stiffening and dysfunction with aging. Further studies are underway to treat aged mice with MR antagonist to attempt to reverse the aging phenotype.Support or Funding InformationNIH/NHLBI R01 HL119290 and AHA EIA18290005

Endothelial specific epithelial sodium channel activation prompts vascular stiffness in female mice

Our research suggests that enhanced endothelium mineralocorticoid receptor (ECMR) signaling induces endothelium epithelial sodium channel (EnNaC) activation to promote vascular stiffness. This occurs, in part, by reduction of endothelial nitric oxide (NO) synthase (eNOS) activity and bioavailable NO. This study demonstrated that consumption of a Western Diet (WD) containing excess fat and refined sugar for 16 weeks increases aldosterone level and MR activation resulting in impaired NO mediated vascular relaxation and aortic stiffness in females. These abnormalities were prevented by low doses of the MR antagonist, spironolactone. As ECMR KO also prevented these abnormalities we posited that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC‐specific EnNaC activation would mediate vascular stiffness and impair flow‐mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO (EnNaC−/−) and wild‐type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC for 16 weeks, could significantly attenuate WD‐induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. Meanwhile, amiloride further improved flow mediated dilation in mesenteric arteries and endothelium‐dependent relaxation with acetylcholine (10−9–10−4 mol/L) stimulation in wire myograph. Importantly, EnNaC KO mice also prevent aldosterone‐induced endothelium stiffness and impairment of endothelium‐dependent relaxation. These data suggest that the sequence of activated ECMR leading to cell specific EnNaC activation and signaling decreases bioavailable NO. This cascade plays a key role in the development of vascular stiffness and dysfunction.Support or Funding InformationNIH (R01 HL73101‐01A and R01 HL107910‐01) and the Veterans Affairs Merit System (0018) to Dr. Sowers

Alterations in the Balance of Cardiomyocyte Glucocorticoid and Mineralocorticoid Receptor Signaling Lead to Heart Failure

Stress is associated with adverse cardiac outcomes; however, the mechanisms underlying these effects are poorly understood. Glucocorticoids are stress hormones that regulate cell and tissue homeostasis through two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Cardiomyocytes express both these receptors but little is known concerning their coordinated actions in heart physiology and pathology. Here, we show that patients with failing hearts have a disparity in the relative levels of GR and MR compared to non‐failing donor hearts. To examine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte‐specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioMRKO mice exhibited normal heart function whereas the cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction. Surprisingly, the cardioGRMRdKO mice were protected from cardiac disease, despite having many of the same pathological gene changes that were present in the GR deficient hearts. Microarray analysis revealed a set of cardioprotective gene changes that occurred specifically in the cardioGRMRdKO hearts. Re‐installation of MR into the cardioGRMRdKO hearts by adeno‐associated virus gene delivery reversed these cardioprotective gene changes and resulted in cardiac dysfunction. These findings reveal critical gene‐regulatory roles for both GR and MR in the heart and suggest that an imbalance in these two signaling pathways leads to heart disease. Therapies designed to modulate cardiomyocyte glucocorticoid signaling to favor more GR and less MR activity may provide new approaches for treating the failing heart.Support or Funding InformationNIH

Albuminuria is associated with an increased prostasin in urine while aldosterone has no direct effect on urine and kidney tissue abundance of prostasin.

The proteinase prostasin is a candidate mediator for aldosterone-driven proteolytic activation of the epithelial sodium channel (ENaC). It was hypothesized that the aldosterone-mineralocorticoid receptor (MR) pathway stimulates prostasin abundance in kidney and urine. Prostasin was measured in plasma and urine from type 2 diabetic patients with resistant hypertension (n=112) randomized to spironolactone/placebo in a clinical trial. Prostasin protein level was assessed by immunoblotting in (1) human and rat urines with/without nephrotic syndrome, (2) human nephrectomy tissue, (3) urine and kidney from aldosterone synthase-deficient (AS-/-) mice and ANGII- and aldosterone-infused mice, and in (4) kidney from adrenalectomized rats. Serum aldosterone concentration related to prostasin concentration in urine but not in plasma. Plasma prostasin concentration increased significantly after spironolactone compared to control. Urinary prostasin and albumin related directly and were reduced by spironolactone. In patients with nephrotic syndrome, urinary prostasin protein was elevated compared to controls. In rat nephrosis, proteinuria coincided with increased urinary prostasin, unchanged kidney tissue prostasin, and decreased plasma prostasin while plasma aldosterone was suppressed. Prostasin protein abundance in human nephrectomy tissue was similar across gender and ANGII inhibition regimens. Prostasin urine abundance was not different in AS-/- and aldosterone-infused mice. Prostasin kidney level was not different from control in adrenalectomized rats and AS-/- mice. We found no evidence for a direct relationship between mineralocorticoid receptor signaling and kidney and urine prostasin abundance. The reduction of urinary prostasin in spironolactone-treated patients is most likely the result of an improved glomerular filtration barrier function and generally reduced proteinuria.

Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1.

AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1-GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI.