Mineralocorticoid Receptor Blockade Research Articles

Eight weeks of treatment with mineralocorticoid receptor blockade does not alter vascular function in individuals with and without type 2 diabetes.

Aldosterone has been suggested to be involved in the microvascular complications observed in type 2 diabetes. We aimed to investigate the effect of mineralocorticoid receptor (MR) blockade on endothelial function in individuals with type 2 diabetes compared to healthy controls. We included 12 participants with type 2 diabetes and 14 controls. We measured leg hemodynamics at baseline and during femoral arterial infusion of acetylcholine and sodium nitroprusside before and 8 weeks into treatment with MR blockade (eplerenone). Acetylcholine infusion was repeated with concomitant n-acetylcysteine (antioxidant) infusion. No difference in leg blood flow or vascular conductance was detected before or after the treatment with MR blockade in both groups and there was no difference between groups. Infusion of n-acetylcysteine increased baseline blood flow and vascular conductance, but did not change the vascular response to acetylcholine before or after treatment with MR blockade. Skeletal muscle eNOS content was unaltered by MR blockade and no difference between groups was detected. In conclusion, we found no effect of MR blockade endothelial function in individuals with and without type 2 diabetes. As the individuals with type 2 diabetes did not have vascular dysfunction, these results might not apply to individuals with vascular dysfunction.

Subtype Identification of Surgically Curable Primary Aldosteronism During Treatment With Mineralocorticoid Receptor Blockade.

Current guidelines and consensus documents recommend withdrawal of mineralocorticoid receptor antagonists (MRAs) before primary aldosteronism (PA) subtyping by adrenal vein sampling (AVS), but this practice can cause severe hypokalemia and uncontrolled high blood pressure. Our aim was to investigate if unilateral PA can be identified by AVS during MRA treatment. We compared the rate of unilateral PA identification between patients with and without MRA treatment in large data sets of patients submitted to AVS while off renin-angiotensin system blockers and β-blockers. In sensitivity analyses, the between-group differences of lateralization index values after propensity score matching and the rate of unilateral PA identification in subgroups with undetectable (≤2 mUI/L), suppressed (<8.2 mUI/L), and unsuppressed (≥8.2 mUI/L) direct renin concentration levels were also evaluated. Plasma aldosterone concentration, direct renin concentration, and blood pressure values were similar in non-MRA-treated (n=779) and MRA-treated (n=61) patients with PA, but the latter required more antihypertensive agents (P=0.001) and showed a higher rate of adrenal nodules (82% versus 67%; P=0.022) and adrenalectomy (72% versus 54%; P=0.01). However, they exhibited no significant differences in commonly used AVS indices and the area under the receiving operating characteristic curve of lateralization index, both under unstimulated conditions and postcosyntropin. Several sensitivity analyses confirmed these results in propensity score matching adjusted models and in patients with undetectable, or suppressed or unsuppressed renin levels. At doses that controlled blood pressure and potassium levels, MRAs did not preclude the identification of unilateral PA at AVS. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01234220.

Renoprotective Effects of Mineralocorticoid Receptor Antagonists Against Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is a growing epidemic worldwide and a leading cause of end-stage kidney disease. Mineralocorticoid receptor (MR) blockade using Finerenone is a recently approved therapeutic approach to slow down the progression of DKD in patients with type 2 diabetes in addition to other therapies such as angiotensin-II converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), sodium-glucose co-transporter 2 (SGLT2) inhibitors, and glucagon-like peptide 1 (GLP-1) analogs. This review elaborates on the pathophysiologic pathways activated by aldosterone (the human mineralocorticoid) in DKD, the pharmacology of three different generations of mineralocorticoid receptor antagonists (MRAs), specifically, spironolactone, eplerenone, and finerenone, and the mechanisms by which these MRAs elicit their protective effects on the kidney under diabetic settings.

The influence of pharmacological mineralocorticoid and glucocorticoid receptor blockade on the cortisol response to psychological stress

The glucocorticoid cortisol is the end product of the hypothalamic–pituitary–adrenal (HPA) axis and crucial for the stress response in humans. Cortisol regulates numerous biological functions by binding to two different types of receptors: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Both receptors are found in the brain where they are crucially involved in various mental functions and in feedback inhibition of cortisol release. The precise role of both receptors in the human stress response is not completely understood. In this study, we examined the effects of pharmacological blockade of the MR or the GR on stress-induced cortisol release in a sample of 318 healthy young men (M = 25.42, SD = 5.01). Participants received the MR antagonist spironolactone (300 mg), the GR antagonist mifepristone (600 mg), or a placebo and were subjected 90 min later to a social-evaluative stressor (Trier Social Stress Test) or a non-stressful control condition. We found significantly higher stress-induced cortisol release in the spironolactone group, whereas participants after mifepristone administration did not differ from the control groups. These results suggest that MR blockade results in attenuated fast negative feedback processes and emphasize the important role of the MR during the early phase of the stress response.

Cortisol Sensitizes Cochlear Hair Cells to Gentamicin Ototoxicity Via Endogenous Apoptotic Pathway.

The widespread use of aminoglycosides is a prevalent cause of sensorineural hearing loss. Patients receiving aminoglycosides usually have elevated levels of circulating stress hormones due to disease or physiological stress; however, whether the stress hormone cortisol impacts aminoglycoside-mediated injury of cochlear hair cells has not been fully investigated. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with or without cortisol pretreatment were exposed to gentamicin, we investigated the effect of cortisol pretreatment on gentamicin ototoxicity by assessing cell viability. Molecular pathogenesis was explored by detecting apoptosis and oxidative stress. Meanwhile, by inhibiting glucocorticoid receptors (GR) and mineralocorticoid receptors (MR), the potential roles of receptor types in cortisol-mediated sensitization were evaluated. Cortisol concentrations below 75 μmol/l did not affect cell viability. However, pretreatment with 50 μmol/l cortisol for 24 hours sensitized hair cells to gentamicin-induced apoptosis. Further mechanistic studies revealed that cortisol significantly increased hair cell apoptosis and oxidative stress, and altered apoptosis-related protein expressions induced by gentamicin. In addition, blockade of either GR or MR attenuated cortisol-induced hair cell sensitization to gentamicin toxicity. Cortisol pretreatment increased mammalian hair cell susceptibility to gentamicin toxicity. Sensitization was related to the activation of the intrinsic apoptotic pathway and excessive generation of reactive oxygen species. Cortisol may exacerbate aminoglycoside ototoxicity.

A selective mineralocorticoid receptor blocker, esaxerenone, attenuates vascular dysfunction and inhibits atherogenesis in apolipoprotein e-deficient mice

Abstract Introduction Pharmacological blockade of mineralocorticoid receptors (MR) is a potential therapeutic approach to reduce cardiovascular complications because MR play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vascular function. We investigated the effects of a novel non-steroidal selective MR blocker, esaxerenone (ESAX), recently approved for hypertension treatment in Japan, on vascular function and the development of atherosclerosis. Methods and results ESAX (3mg/kg/day) or vehicle was orally administered to western type diet-fed apolipoprotein E-deficient mice for 20 weeks or for 8 weeks to investigate the effects on atherogenesis or vascular dysfunction. Administration of ESAX for 20 weeks suppressed the development of atherosclerotic plaques in the aortic arch compared with the vehicle group (p&amp;lt;0.001) without alteration of blood pressure. In addition, ESAX reduced the expression of intercellular cell adhesion molecule-1 (p&amp;lt;0.05), macrophage infiltration (p&amp;lt;0.01), lipid deposition (p&amp;lt;0.05) and tended to decrease the expression of vascular cell adhesion molecule-1 as determined by immunohistochemical analysis. Moreover, phosphorylation of eNOSSer1177 (p&amp;lt;0.05) and Akt (p&amp;lt;0.05) were increased in the aorta of ESAX-treated mice. Administration of ESAX for 8 weeks attenuated western type diet-induced endothelial dysfunction (p&amp;lt;0.05) accompanied with the decrease in expression of ICAM1 (p&amp;lt;0.05) and phosphorylation of JNK (p&amp;lt;0.05) in the descending aorta. In in vitro experiment, aldosterone decreased the phosphorylation of eNOSSer1177 and increased the phosphorylation of eNOSThr495 in a dose dependent manner, which were attenuated by ESAX in HUVECs. In ex vivo vascular reactivity assay, ESAX diminished the aldosterone-induced endothelial dysfunction. Conclusion ESAX ameliorated hyperlipidemia-induced atherosclerosis and vascular dysfunction in mice, suggesting that ESAX is a promising therapeutic approach for cardiovascular complications.

Abstract 18220: Does Mineralocorticoid Receptor Blockade Affect the Occurrence of Perioperative Atrial Fibrillation in Cardiac Surgery Patients? - A Systematic Review and Meta-Analysis

Background: Mineralocorticoid receptor antagonists (MRAs) have emerged as potential therapy to prevent atrial fibrillation (AF). However, inconsistent results of MRAs effect on perioperative AF have been reported in the literature in patients undergoing cardiac surgery. To our knowledge, this is the first metanalysis to assess the effect of MRAs on occurrence of perioperative AF in patients undergoing cardiac surgery. Method: We searched different electronic databases from inception to April 30th, 2023, for randomized controlled trials (RCTs) and observational studies that evaluated the use of MRAs in patients with heart failure and left ventricular ejection fraction (LVEF) 30-50% who were referred for Coronary Artery Bypass Grafting (CABG) and/or valve surgery without any AF history. Primary outcome was occurrence of perioperative AF. Three independent reviewers selected and appraised the data. A random-effects meta-analysis was performed to estimate pooled odds ratios (OR) and 95% confidence intervals (CI). Result: We included 4 studies, 1 RCT and 3 observational cohorts, with a cumulative number of 1,128 patients with mean age 65.15 years. 412(36.5%) received an MRA (spironolactone or eplerenone). The follow up duration was 5 days to 1 month. During follow-up, 118 (28.64%) patients treated with MRAs, developed AF, compared to 224 (31.28%) patients, without MRA treatment. However, metanalysis showed that there is no significant reduction in occurrence of perioperative AF in MRA treated patients (OR: 0.88 CI: 0.58-1.33 p=0.55), I2 53%; Figure] Conclusion: Experimental data have shown that MRA blockade prevents perioperative AF in animal models but our metanalysis demonstrated that MRAs were not protective for perioperative AF in patients undergoing cardiac surgery. Larger trials in humans are warranted to evaluate its efficacy, particularly in patients undergoing cardiac surgery.

Effects of Mineralocorticoid Receptor Antagonists for Chronic Kidney Disease: A Systemic Review and Meta-Analysis

Background: Mineralocorticoid receptor blockade could be a potential approach for the inhibition of chronic kidney disease (CKD) progression. The benefits and harms of different mineralocorticoid receptor antagonists (MRAs) in CKD are inconsistent. Objectives: The aim of the study was to summarize the benefits and harms of MRAs for CKD patients. Methods: We searched MEDLINE, EMBASE, and the Cochrane databases for trials assessing the effects of MRAs on non-dialysis-dependent CKD populations. Treatment and adverse effects were summarized using meta-analysis. Results: Fifty-three trials with 6 different MRAs involving 22,792 participants were included. Compared with the control group, MRAs reduced urinary albumin-to-creatinine ratio (weighted mean difference [WMD], −90.90 mg/g, 95% CI, −140.17 to −41.64 mg/g), 24-h urinary protein excretion (WMD, −0.20 g, 95% CI, −0.28 to −0.12 g), estimated glomerular filtration rate (eGFR) (WMD, −1.99 mL/min/1.73 m², 95% CI, −3.28 to −0.70 mL/min/1.73 m²), chronic renal failure events (RR, 0.86, 95% CI, 0.79–0.93), and cardiovascular events (RR, 0.84, 95% CI, 0.77–0.92). MRAs increased the incidence of hyperkalemia (RR, 2.04, 95% CI, 1.73–2.40) and hypotension (RR, 1.80, 95% CI, 1.41–2.31). MRAs reduced the incidence of peripheral edema (RR, 0.65, 95% CI, 0.56–0.75) but not the risk of acute kidney injury (RR, 0.94, 95% CI, 0.79–1.13). Nonsteroidal MRAs (RR, 0.66, 95% CI, 0.57–0.75) but not steroidal MRAs (RR, 0.20, 95% CI, 0.02–1.68) significantly reduced the risk of peripheral edema. Steroidal MRAs (RR, 5.68, 95% CI, 1.26–25.67) but not nonsteroidal MRAs (RR, 0.52, 95% CI, 0.22–1.22) increased the risk of breast disorders. Conclusions: In the CKD patients, MRAs, particularly in combination with angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, reduced albuminuria/proteinuria, eGFR, and the incidence of chronic renal failure, cardiovascular and peripheral edema events, whereas increasing the incidence of hyperkalemia and hypotension, without the augment of acute kidney injury events. Nonsteroidal MRAs were superior in the reduction of more albuminuria with fewer peripheral edema events and without the augment of breast disorder events.

The Mineralocorticoid Receptor Plays a Crucial Role in Macrophage Development and Function.

Stress and the attendant rise in glucocorticoids (GCs) results in a potent suppression of the immune system. To date, the anti-inflammatory role of GCs, via activation of the glucocorticoid receptor, has been well-characterized. However, cortisol, the primary GC in both fish and humans, also signals through the high-affinity mineralocorticoid receptor (MR), of which the immunomodulatory role is poorly understood. Here, we tested the hypothesis that MR is a key modulator of leukocyte function during inflammation. Using transgenic MR knockout zebrafish with fluorescently labelled leukocytes, we show that a loss of MR results in a global reduction in macrophage number during key development stages. This reduction was associated with impaired macrophage proliferation and responsivity to developmental distribution signals, as well as increased susceptibility to cell death. Using a tail fin amputation in zebrafish larvae as a model for localized inflammation, we further showed that MR knockout larvae display a reduced ability to produce more macrophages under periods of inflammation (emergency myelopoiesis). Finally, we treated wild-type larvae with an MR antagonist (eplerenone) during definitive hematopoiesis, when the macrophages had differentiated normally throughout the larvae. This pharmacological blockade of MR reduced the migration of macrophages toward a wound, which was associated with reduced macrophage Ccr2 signalling. Eplerenone treatment also abolished the cortisol-induced inhibition of macrophage migration, suggesting a role for MR in cortisol-mediated anti-inflammatory action. Taken together, our work reveals that MR is a key modulator of the innate immune response to inflammation under both basal and stressed conditions.

Aldosterone Antagonism Is More Effective at Reducing Blood Pressure and Excessive Renal ENaC Activity in AngII-Infused Female Rats Than in Males.

AngII (angiotensin II)-dependent hypertension causes comparable elevations of blood pressure (BP), aldosterone levels, and renal ENaC (epithelial Na+ channel) activity in male and female rodents. Mineralocorticoid receptor (MR) antagonism has a limited antihypertensive effect associated with insufficient suppression of renal ENaC in male rodents with AngII-hypertension. While MR blockade effectively reduces BP in female mice with salt-sensitive and leptin-induced hypertension, MR antagonism has not been studied in female rodents with AngII-hypertension. We hypothesize that overstimulation of renal MR signaling drives redundant ENaC-mediated Na+ reabsorption and BP increase in female rats with AngII-hypertension. We employ a combination of physiological, pharmacological, biochemical, and biophysical approaches to compare the effect of MR inhibitors on BP and ENaC activity in AngII-infused male and female Sprague Dawley rats. MR blockade markedly attenuates AngII-hypertension in female rats but has only a marginal effect in males. Spironolactone increases urinary sodium excretion and urinary sodium-to-potassium ratio in AngII-infused female, but not male, rats. The expression of renal MR and HSD11β2 (11β-hydroxysteroid dehydrogenase type 2) that determines the availability of MR to aldosterone is significantly higher in AngII-infused female rats than in males. ENaC activity is ≈2× lower in spironolactone-treated AngII-infused female rats than in males. Reduced ENaC activity in AngII-infused female rats on spironolactone correlates with increased interaction with ubiquitin ligase Nedd4-2, targeting ENaC for degradation. MR-ENaC axis is the primary determinant of excessive renal sodium reabsorption and an attractive antihypertensive target in female rats with AngII-hypertension, but not in males.

Mineralocorticoid receptor blockade protects the kidneys but does not affect inverted blood pressure rhythm in hypertensive transgenic (mRen-2)27 rats

Aldosterone regulates blood pressure (BP) through water and sodium balance. In our study, we studied if continuous treatment with a mineralocorticoid receptor antagonist, spironolactone (30 mg/kg/day) for 20 days can: 1) attenuate hypertension development and restore inverted 24-h BP rhythm in hypertensive transgenic (mRen-2)27 rats (TGR) measured by telemetry; 2) improve function of the kidneys and heart; 3) be protective against high salt load (1% in water) by mitigating oxidative injury and improving kidney function. Spironolactone decreased albuminuria and 8-isoprostane in normal and salt load conditions in BP-independent effects. Salt load increased BP, impaired autonomic balance, suppressed plasma aldosterone level and increased natriuresis, albuminuria and oxidative injury in TGR. Spironolactone did not restore the inverted 24-h rhythm of BP in TGR, therefore, mineralocorticoids are not crucial in regulation of BP daily profile. Spironolactone improved kidney function, decreased oxidative stress and was protective against high salt load in the BP-independent manner.

Recent progress in the diagnosis and treatment of primary aldosteronism.

Primary aldosteronism (PA) is caused by excessive secretion of aldosterone from the adrenal glands, with subsequent changes in the renin-angiotensin system. In Japan, chemiluminescent enzyme immunoassay is currently performed for aldosterone assay rather than the earlier method of radioimmunoassay. This change in aldosterone measurement methods has resulted in faster and more accurate measurement of blood aldosterone levels. Since 2019, esaxerenone, a mineralocorticoid receptor antagonist (MRA) with a non-steroidal skeleton, has been available in Japan for the treatment of hypertension. Esaxerenone has been reported to have various effects, such as strong antihypertensive and anti-albuminuric/proteinuric effects. Treatment of PA with MRAs has been reported to improve the patient's quality of life and to suppress the onset of cardiovascular events independent of their effects on blood pressure. Measuring renin levels is recommended for monitoring the extent of mineralocorticoid receptor blockade during MRA treatment. Patients receiving MRAs are prone to developing hyperkalemia, and combining MRAs with sodium/glucose cotransporter 2 inhibitors is expected to prevent severe hyperkalemia and provide additional cardiorenal protection. Mineralocorticoid receptor-associated hypertension is a broad concept of hypertension that includes not only PA, but also hypertension caused by borderline aldosteronism, obesity, diabetes, and sleep apnea syndrome. New findings on primary aldosteronism, which is part of MR-associated hypertension. Aldosterone measurements have been changed to the CLEIA method. Treatment of primary aldosteronism with MRAs has a variety of positive effects. CT-guided radiofrequency ablation and transarterial embolization are alternatives to surgery for aldosterone-producing adenomas. BP blood pressure, CLEIA chemiluminescent enzyme immunoassay, CT computed tomography, K serum potassium, MR mineralocorticoid receptor, MRA mineralocorticoid receptor antagonist, QOL quality of life, SGLT2i sodium/glucose cotransporter 2 inhibitor.

Therapeutic perspective: evolving evidence of nonsteroidal mineralocorticoid receptor antagonists in diabetic kidney disease.

Approximately 30%-40% of patients with type 1 or type 2 diabetes (T1D/T2D) develop diabetic kidney disease (DKD), which can lead to end-stage kidney disease (ESKD). Angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, and sodium-glucose cotransporter 2 (SGLT2) inhibitors have been investigated as treatments for DKD. However, these drugs do not prevent overactivation of the mineralocorticoid receptor (MR). Studies have shown a correlation between MR hyperactivation, renal injury, and DKD. Finerenone, a novel and selective nonsteroidal mineralocorticoid receptor antagonist (NS-MRA), was approved for the treatment of patients with DKD, and is associated with lower rates of hyperkalemia. Other NS-MRAs (such as KBP-5074, BR-4628, esaxerenone, and apararenone) may also be effective drugs for the treatment of DKD. This review summarizes the effects of pharmacological MR blockade on diabetes and diabetes-associated CKD, with a particular focus on the therapeutic mechanisms of NS-MRAs in preclinical studies and ongoing clinical studies. Further investigation of combined treatment with renoprotective drugs and NS-MRAs to improve the treatment of DKD is needed.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obesity

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Emerging evidence suggests development of heart failure with preserved ejection fraction in females with CMD and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the hypothesis that smooth muscle cell (SMC)-specific MR deletion prevents obesity-associated coronary and cardiac diastolic dysfunction in females. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Initial studies revealed that global MR blockade with spironolactone prevented impaired coronary vasodilation and diastolic dysfunction in obese females. Importantly, specific deletion of SMC-MR similarly prevented obesity-associated coronary and cardiac dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in multiple non-myocyte populations (B/T cells, macrophages, and endothelium), independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling broadly impacting the cardiac cellulome and underlying obesity-associated coronary and cardiac dysfunction thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity. Supported by NIH R01 HL136386 (Bender), NIH R01 HL119290 (Jaffe), the University of Missouri Research Core Facilities Grant Program (Bender), NHMRC Ideas Grant GNT1188503 (Pinto), VA Merit I01 BX004220 and RCS IK2 6004016 (Chandrasekar). The work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mineralocorticoid Receptor Antagonists for Preventing Chronic Kidney Disease Progression: Current Evidence and Future Challenges.

Regulation and action of the mineralocorticoid receptor (MR) have been the focus of intensive research over the past 80 years. Genetic and physiological/biochemical analysis revealed how MR and the steroid hormone aldosterone integrate the responses of distinct tubular cells in the face of environmental perturbations and how their dysregulation compromises fluid homeostasis. In addition to these roles, the accumulation of data also provided unequivocal evidence that MR is involved in the pathophysiology of kidney diseases. Experimental studies delineated the diverse pathological consequences of MR overactivity and uncovered the multiple mechanisms that result in enhanced MR signaling. In parallel, clinical studies consistently demonstrated that MR blockade reduces albuminuria in patients with chronic kidney disease. Moreover, recent large-scale clinical studies using finerenone have provided evidence that the non-steroidal MR antagonist can retard the kidney disease progression in diabetic patients. In this article, we review experimental data demonstrating the critical importance of MR in mediating renal injury as well as clinical studies providing evidence on the renoprotective effects of MR blockade. We also discuss areas of future investigation, which include the benefit of non-steroidal MR antagonists in non-diabetic kidney disease patients, the identification of surrogate markers for MR signaling in the kidney, and the search for key downstream mediators whereby MR blockade confers renoprotection. Insights into these questions would help maximize the benefit of MR blockade in subjects with kidney diseases.

Neutral Effect of Skeletal Muscle Mineralocorticoid Receptor on Glucose Metabolism in Mice.

The mineralocorticoid receptor (MR) is able to regulate the transcription of a number of genes in the myotube, although its roles in skeletal muscle (SM) metabolism still await demonstration. SM represents a major site for glucose uptake, and its metabolic derangements play a pivotal role in the development of insulin resistance (IR). The aim of this study was to investigate the contribution of SM MR in mediating derangements of glucose metabolism in a mouse model of diet-induced obesity. We observed that mice fed a high-fat diet (HFD mice) showed impaired glucose tolerance compared to mice fed a normal diet (ND mice). Mice fed a 60% HFD treated with the MR antagonist Spironolactone (HFD + Spiro) for 12 weeks revealed an improvement in glucose tolerance, as measured with an intraperitoneal glucose tolerance test, compared with HFD mice. To investigate if blockade of SM MR could contribute to the favorable metabolic effects observed with pharmacological MR antagonism, we analyzed MR expression in the gastrocnemius, showing that SM MR protein abundance is downregulated by HFD compared to ND mice and that pharmacological treatment with Spiro was able to partially revert this effect in HFD + Spiro mice. Differently from what we have observed in adipose tissue, where HDF increased adipocyte MR expression, SM MR protein was down-regulated in our experimental model, suggesting a completely different role of SM MR in the regulation of glucose metabolism. To confirm this hypothesis, we investigated the effects of MR blockade on insulin signaling in a cellular model of IRin C2C12 myocytes, which were treated with or without Spiro. We confirmed MR protein downregulation in insulin-resistant myotubes. We also analyzed Akt phosphorylation upon insulin stimulation, and we did not observe any difference between palmitate- and palmitate + Spiro-treated cells. These results were confirmed by in vitro glucose uptake analysis. Taken together, our data indicate that reduced activity of SM MR does not improve insulin signaling in mouse skeletal myocytes and does not contribute to the favorable metabolic effects on glucose tolerance and IR induced by systemic pharmacological MR blockade.

Mineralocorticoid receptors contribute to ethanol-induced vascular hypercontractility through reactive oxygen species generation and up-regulation of cyclooxygenase 2

The effects on blood pressure produced byethanol consumption include both vasoconstriction and activation of the renin-angiotensin-aldosterone system (RAAS), although the detailed relationship between these processes is yet to be accomplished. Here, we sought to investigate the contribution of mineralocorticoid receptors (MR) to ethanol-induced hypertension and vascular hypercontractility. We analyzed blood pressure and vascular function of male Wistar Hannover rats treated with ethanol for five weeks. The contribution of the MR pathway to the cardiovascular effects of ethanol was evaluated with potassium canrenoate, a MR antagonist (MRA). Blockade of MR prevented ethanol-induced hypertension and hypercontractility of endothelium-intact and -denuded aortic rings. Ethanol up-regulated cyclooxygenase (COX)2 and augmented vascular levels of both reactive oxygen species (ROS) and thromboxane (TX)B2, a stable metabolite of TXA2. These responses were abrogated by MR blockade. Hyperreactivity to phenylephrine induced by ethanol consumption was reversed by tiron [a scavenger of superoxide (O2∙–)], SC236 (a selective COX2 inhibitor) or SQ29548 (an antagonist of TP receptors). Treatment with the antioxidant apocynin prevented the vascular hypercontractility, as well as the increases in COX2 expression and TXA2 production induced by ethanol consumption. Our study has identified novel mechanisms through which ethanol consumption promotes its deleterious effects in the cardiovascular system. We provided evidence for a role of MR in the vascular hypercontractility and hypertension associated with ethanol consumption. The MR pathway triggers vascular hypercontractility through ROS generation, up-regulation of COX2 and overproduction of TXA2, which will ultimately induce vascular contraction.

A Selective Mineralocorticoid Receptor Blocker, Esaxerenone, Attenuates Vascular Dysfunction in Diabetic C57BL/6 Mice.

Pharmacological blockade of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications since MRs play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vessel function. We investigated the effect of a novel nonsteroidal selective MR blocker, esaxerenone, on diabetes-induced vascular dysfunction. Diabetes was induced by a single dose of streptozotocin in 8-week-old male C57BL/6 mice. Esaxerenone (3 mg/kg/day) or a vehicle was administered by gavage to diabetic mice for 3 weeks. Metabolic parameters, plasma aldosterone levels, and parameters related to renal function were measured. Endothelium-dependent or -independent vascular responses of the aortic segments were analyzed with acetylcholine or sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study. Induction of diabetes elevated plasma aldosterone level (P＜0.05) and impaired endothelium-dependent vascular relaxation (P＜0.05). The administration of esaxerenone ameliorated the endothelial dysfunction (P＜0.01) without the alteration of metabolic parameters, blood pressure, and renal function. Esaxerenone improved the eNOSSer1177 phosphorylation in the aorta obtained from diabetic mice (P＜0.05) compared with that in the vehicle-treated group. Furthermore, a major MR agonist, aldosterone, decreased eNOSSer1177 phosphorylation and increased eNOSThr495 phosphorylation in HUVECs, which recovered with esaxerenone. Esaxerenone ameliorated the endothelium-dependent vascular relaxation caused by aldosterone in the aortic segments obtained from C57BL/6 mice (P＜0.001). Esaxerenone attenuates the development of diabetes-induced endothelial dysfunction in mice. These results suggest that esaxerenone has potential vascular protective effects in individuals with diabetes.

Effects of Mineralocorticoid Receptor Blockade and Statins on Kidney Injury Marker 1 (KIM-1) in Female Rats Receiving L-NAME and Angiotensin II.

Kidney injury molecule-1 (KIM-1) is a biomarker of renal injury and a predictor of cardiovascular disease. Aldosterone, via activation of the mineralocorticoid receptor, is linked to cardiac and renal injury. However, the impact of mineralocorticoid receptor activation and blockade on KIM-1 is uncertain. We investigated whether renal KIM-1 is increased in a cardiorenal injury model induced by L-NAME/ANG II, and whether mineralocorticoid receptor blockade prevents the increase in KIM-1. Since statin use is associated with lower aldosterone, we also investigated whether administering eiSther a lipophilic statin (simvastatin) or a hydrophilic statin (pravastatin) prevents the increase in renal KIM-1. Female Wistar rats (8-10 week old), consuming a high salt diet (1.6% Na+), were randomized to the following conditions for 14 days: control; L-NAME (0.2 mg/mL in drinking water)/ANG II (225 ug/kg/day on days 12-14); L-NAME/ANG II + eplerenone (100 mg/kg/day p.o.); L-NAME/ANG II + pravastatin (20 mg/kg/day p.o.); L-NAME/ANG II + simvastatin (20 mg/kg/day p.o.). Groups treated with L-NAME/ANG II had significantly higher blood pressure, plasma and urine aldosterone, cardiac injury/stroke composite score, and renal KIM-1 than the control group. Both eplerenone and simvastatin reduced 24-h urinary KIM-1 (p = 0.0046, p = 0.031, respectively) and renal KIM-1 immunostaining (p = 0.004, p = 0.037, respectively). Eplerenone also reduced renal KIM-1 mRNA expression (p = 0.012) and cardiac injury/stroke composite score (p = 0.04). Pravastatin did not affect these damage markers. The 24-h urinary KIM-1, renal KIM-1 immunostaining, and renal KIM-1 mRNA expression correlated with cardiac injury/stroke composite score (p < 0.0001, Spearman ranked correlation = 0.69, 0.66, 0.59, respectively). In conclusion, L-NAME/ANG II increases renal KIM-1 and both eplerenone and simvastatin blunt this increase in renal KIM-1.

Multi-omic analysis of the cardiac cellulome defines a vascular contribution to cardiac diastolic dysfunction in obese female mice

Coronary microvascular dysfunction (CMD) is associated with cardiac dysfunction and predictive of cardiac mortality in obesity, especially in females. Clinical data further support that CMD associates with development of heart failure with preserved ejection fraction and that mineralocorticoid receptor (MR) antagonism may be more efficacious in obese female, versus male, HFpEF patients. Accordingly, we examined the impact of smooth muscle cell (SMC)-specific MR deletion on obesity-associated coronary and cardiac diastolic dysfunction in female mice. Obesity was induced in female mice via western diet (WD) feeding alongside littermates fed standard diet. Global MR blockade with spironolactone prevented coronary and cardiac dysfunction in obese females and specific deletion of SMC-MR was sufficient to prevent obesity-associated coronary and cardiac diastolic dysfunction. Cardiac gene expression profiling suggested reduced cardiac inflammation in WD-fed mice with SMC-MR deletion independent of blood pressure, aortic stiffening, and cardiac hypertrophy. Further mechanistic studies utilizing single-cell RNA sequencing of non-cardiomyocyte cell populations revealed novel impacts of SMC-MR deletion on the cardiac cellulome in obese mice. Specifically, WD feeding induced inflammatory gene signatures in non-myocyte populations including B/T cells, macrophages, and endothelium as well as increased coronary VCAM-1 protein expression, independent of cardiac fibrosis, that was prevented by SMC-MR deletion. Further, SMC-MR deletion induced a basal reduction in cardiac mast cells and prevented WD-induced cardiac pro-inflammatory chemokine expression and leukocyte recruitment. These data reveal a central role for SMC-MR signaling in obesity-associated coronary and cardiac dysfunction, thus supporting the emerging paradigm of a vascular origin of cardiac dysfunction in obesity.