Mineralocorticoid Receptor Knockout Mice Research Articles

Mineralocorticoid Receptor Antagonists Cause Natriuresis in the Absence of Aldosterone.

MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. We studied AS (aldosterone synthase)-deficient (AS-/-) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR-/-) mice. KS-MR-/- mice were used to compare MR expression and ENaC localization and cleavage with AS-/- mice. MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR-/- mice but were fully preserved along DCT2 and were partially reduced along CCD in AS-/- mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS-/- mice and were not increased along CCD after low salt. AS-/- mice exhibited transient Na+ wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS-/- mice led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR-/- mice. Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS-/- mice.

Endothelial Mineralocorticoid Receptors Constrain Arteriogenesis and Perfusion Recovery Following Chronic Arterial Ligation in a Sex‐specific Manner

Vascular occlusive diseases are major contributors to cardiovascular morbidity and mortality. Prior work has implicated mineralocorticoid receptor (MR) signaling in perfusion recovery following arterial occlusion, however, cell‐specific MR signaling in the associated angiogenic and arteriogenic responses has not been elucidated. Further, accumulating evidence has revealed sex‐specific involvement of MR signaling in a number of cardiovascular disease states. Accordingly, we examined the hypothesis that MR signaling, specifically endothelial cell (EC) MR signaling, attenuates vascular compensation and perfusion recovery following femoral artery excision and that this effect would be more pronounced in females. Unilateral femoral artery ligation and excision was performed in male and female C57BL/6J mice treated with vehicle or the MR antagonist spironolactone (Spiro; 20 mg/kg/d, sc) as well as in male and female MR Intact and EC MR knockout (KO) mice. Serial assessment of hindlimb perfusion for 28 days following surgery revealed enhanced perfusion recovery following Spiro treatment in female, but not male, mice. Interestingly, Spiro increased gastrocnemius capillary density in both the ischemic and sham limb in males and females. Following perfusion fixation, collateral remodeling was assessed by micro‐computed tomography revealing enhanced collateral expansion following Spiro treatment in female, but not male, mice. Additional mechanistic experiments similarly revealed increased perfusion recovery and enhanced collateral expansion in female, not male, EC MR KO mice independent of capillary density. At 28 days post‐surgery, distribution analysis revealed larger overall arteriolar diameters only in the collateral zone of EC MR KO females. Further analysis revealed enhanced early (7 days post‐surgery) collateral expansion in male and female EC MR KO mice. In female, but not male, EC MR KO mice this was associated with increased CD68+ macrophages and CD206+ ‘M2’‐like macrophages in the collateral zone. Together, these data demonstrate that EC MR signaling constrains early arteriogenic collateralization following arterial excision in both sexes and constrains long‐term collateralization only in females involving limitation of pro‐arteriogenic CD206+ ‘M2’‐like macrophages in the collateral zone. To our knowledge, this is the first report of a role for EC MR signaling in arteriogenesis and highlights potential use of MR blockers in the treatment of vascular occlusive events.

Eplerenone‐induced natriuresis and hyperkalemia in mice lacking aldosterone results from aldosterone‐independent mineralocorticoid receptor occupancy

BackgroundMineralocorticoid receptor (MR) antagonists are recommended for patients with resistant hypertension independent of circulating aldosterone levels. Aldosterone, secreted in response to salt depletion or hyperkalemia, activates MR to increase epithelial sodium channel (ENaC) activity. While glucocorticoids can also activate MR, they are metabolized by 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), protecting MR from illicit glucocorticoid occupation. 11β‐HSD2 is expressed at increasing levels from the DCT through the collecting duct. Here, we hypothesized that glucocorticoid occupies MR to stimulate ENaC in the DCT2 and early connecting tubule.MethodsWe studied aldosterone synthase knockout (AS−/−) mice provided by Dr. JM Luther. Kidney–specific MR knockout (MR−/−) mice were used for validation of Western blot for ENaC and immunofluorescence for MR. We measured plasma and urinary electrolytes at baseline, and following dietary salt restriction with or without administration of the MR antagonist eplerenone.ResultsAS−/− mice displayed higher plasma K+ levels than AS+/+ mice but not salt wasting under baseline. Under low salt (LS) diet, AS−/− mice exhibited higher urine Na+ excretion, but preserved body weight. Cleaved αENaC and γENaC abundances were lower in AS−/− than AS+/+ mice, and were not increased in AS−/− mice after LS diet. Notably, we found nuclear localization of MR was preserved in DCT2 of AS−/− mice. Administration of the MR blocker eplerenone to AS−/− mice fed LS diet led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice.ConclusionsOur results provide evidence that MR is occupied in the absence of aldosterone, suggesting glucocorticoid‐binding to MR preserves sodium homeostasis in the DCT2 of AS−/− mice. This likely explains the milder phenotype of AS−/− mice compared with MR−/− mice.

Vascular cell-specific roles of mineralocorticoid receptors in pulmonary hypertension.

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients.

Aldosterone Regulates Pendrin and Epithelial Sodium Channel Activity through Intercalated Cell Mineralocorticoid Receptor-Dependent and -Independent Mechanisms over a Wide Range in Serum Potassium.

Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.

Two Mineralocorticoid Receptor–Mediated Mechanisms of Pendrin Activation in Distal Nephrons

Regulation of sodium chloride transport in the aldosterone-sensitive distal nephron is essential for fluid homeostasis and BP control. The chloride-bicarbonate exchanger pendrin in β-intercalated cells, along with sodium chloride cotransporter (NCC) in distal convoluted tubules, complementarily regulate sodium chloride handling, which is controlled by the renin-angiotensin-aldosterone system. Using mice with mineralocorticoid receptor deletion in intercalated cells, we examined the mechanism and roles of pendrin upregulation via mineralocorticoid receptor in two different models of renin-angiotensin-aldosterone system activation. We also used aldosterone-treated NCC knockout mice to examine the role of pendrin regulation in salt-sensitive hypertension. Deletion of mineralocorticoid receptor in intercalated cells suppressed the increase in renal pendrin expression induced by either exogenous angiotensin II infusion or endogenous angiotensin II upregulation via salt restriction. When fed a low-salt diet, intercalated cell-specific mineralocorticoid receptor knockout mice with suppression of pendrin upregulation showed BP reduction that was attenuated by compensatory activation of NCC. In contrast, upregulation of pendrin induced by aldosterone excess combined with a high-salt diet was scarcely affected by deletion of mineralocorticoid receptor in intercalated cells, but depended instead on hypokalemic alkalosis through the activated mineralocorticoid receptor-epithelial sodium channel cascade in principal cells. In aldosterone-treated NCC knockout mice showing upregulation of pendrin, potassium supplementation corrected alkalosis and inhibited the pendrin upregulation, thereby lowering BP. In conjunction with NCC, the two pathways of pendrin upregulation, induced by angiotensin II through mineralocorticoid receptor activation in intercalated cells and by alkalosis through mineralocorticoid receptor activation in principal cells, play important roles in fluid homeostasis during salt depletion and salt-sensitive hypertension mediated by aldosterone excess.

Role of Mineralocorticoid Receptor in Adipogenesis and Obesity in Male Mice.

Increased visceral adiposity and hyperglycemia, 2 characteristics of metabolic syndrome, are also present in conditions of excess glucocorticoids (GCs). GCs are hormones thought to act primarily via the glucocorticoid receptor (GR). GCs are commonly prescribed for inflammatory disorders, yet their use is limited due to many adverse metabolic side effects. In addition to GR, GCs also bind the mineralocorticoid receptor (MR), but there are many conflicting studies about the exact role of MR in metabolic disease. Using MR knockout mice (MRKO), we find that both white and brown adipose depots form normally when compared with wild-type mice at P5. We created mice with adipocyte-specific deletion of MR (FMRKO) to better understand the role of MR in metabolic dysfunction. Treatment of mice with excess GCs for 4 weeks, via corticosterone in drinking water, induced increased fat mass and glucose intolerance to similar levels in FMRKO and floxed control mice. Separately, when fed a high-fat diet for 16 weeks, FMRKO mice had reduced body weight, fat mass, and hepatic steatosis, relative to floxed control mice. Decreased adiposity likely resulted from increased energy expenditure since food intake was not different. RNA sequencing analysis revealed decreased enrichment of genes associated with adipogenesis in inguinal white adipose of FMRKO mice. Differentiation of mouse embryonic fibroblasts (MEFs) showed modestly impaired adipogenesis in MRKO MEFs compared with wild type, but this was rescued upon the addition of peroxisome proliferator-activated receptor gamma (PPARγ) agonist or PPARγ overexpression. Collectively, these studies provide further evidence supporting the potential value of MR as a therapeutic target for conditions associated with metabolic syndrome.

Mineralocorticoid receptor negatively regulates angiogenesis through repression of STAT3 activity in endothelial cells.

The mineralocorticoid receptor (MR) plays important roles in cardiovascular pathogenesis. The function of MR in angiogenesis is still controversial. This study aimed to explore the role of endothelial MR in angiogenesis and to delineate the underlying mechanism. Endothelial-hematopoietic MR knockout (EMRKO) mice were generated and subjected to hindlimb ischemia and injection of melanoma cells. Laser Doppler measurements showed that EMRKO mice had improved blood flow recovery and increased vessel density in ischemic limbs. In addition, EMRKO accelerated growth and increased the vessel density of tumors. Matrigel implantation, aortic ring assays, and tube formation assays demonstrated that MRKO endothelial cells (ECs) manifested increased angiogenic potential. MRKO ECs also displayed increased migration ability and proliferation. MRKO and MR knockdown both upregulated gene expression, protein level, and phosphorylation of signal transducer and activator of transcription 3 (STAT3). Stattic, a selective STAT3 inhibitor, attenuated the effects of MRKO on tube formation, migration, and proliferation of ECs. At the molecular level, MR interacted with CCAAT enhancer-binding protein beta (C/EBPβ) to suppress the transcription of STAT3. Furthermore, interactions between MR and STAT3 blocked the phosphorylation of STAT3. Finally, stattic abolished the pro-angiogenic phenotype of EMRKO mice. Taken together, endothelial MR is a negative regulator of angiogenesis, likely in a ligand-independent manner. Mechanistically, MR downregulates STAT3 that mediates the impacts of MR deficiency on the angiogenic activity of ECs and angiogenesis. Targeting endothelial MR may be a potential pro-angiogenic strategy for ischemic diseases. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The effects of different rearing conditions on sexual maturation and maternal care in heterozygous mineralocorticoid receptor knockout mice

Sexual and social development is affected by a complex interplay between genetic makeup and the early-life rearing environment. While many rodent studies focused primarily on the detrimental effects of early-life stress, human literature suggests that genetic susceptibility may not be restricted to negative environments; it may also enhance the beneficial effects of positive rearing conditions. To examine this interaction in a controlled setting, heterozygous mineralocorticoid receptor knockout (MR+/−) mice and control litter mates were exposed to a limited nesting/bedding (LN, impoverished), standard nesting (SN, control) or communal nesting (CN, enriched) paradigm from postnatal day 2–9 (P2-P9). Offspring was monitored for puberty onset between P24-P36 and, in females, maternal care-giving (i.e. as F1) during adulthood, after which basal corticosterone was measured. Different home-cage environments resulted in profound differences in received maternal care and offspring body weight. In male offspring, LN resulted in delayed puberty onset that was mediated by body weight and unpredictability of maternal care received during early development. In female offspring, rearing condition did not significantly alter sexual maturation and had little effect on their own maternal care-giving behavior. Genotype did affect maternal care: female MR+/− offspring exhibited a less active nursing style and upregulated fragmentation during adulthood, irrespective of early life conditions. Basal corticosterone levels were highest in MR+/− mice with a background of LN. Overall, we found a gene-by-environment interaction with respect to basal corticosterone levels, but not for sexual maturation or maternal behavior.

Prevention of Obesity‐Associated Coronary and Cardiac Diastolic Dysfunction by Deletion of Smooth Muscle Cell Mineralocorticoid Receptor in Females

Coronary microvascular dysfunction is independently predictive of cardiac diastolic dysfunction and both conditions are common in obesity and associated metabolic syndrome. Recent work by us and others indicates that mineralocorticoid receptor (MR) antagonism treats obesity‐associated coronary and cardiac diastolic dysfunction. Vascular cell‐specific MR involvement in these obesity‐related defects remains unclear. Thus, we evaluated the hypothesis that deletion of smooth muscle cell (SMC) MR would prevent obesity‐associated coronary and cardiac diastolic dysfunction in females. Female wild‐type (WT) and SMC MR knockout (KO) mice were fed a standard chow or western diet (WD) to induce obesity for 16 weeks. WD feeding induced obesity, hyperinsulinemia, hypercholesterolemia, and visceral adipose inflammation in both WT and SMC MR KO mice. Assessment of coronary vascular function via wire myography revealed WD‐induced impairment of endothelium‐dependent vasodilation and enhanced vasoconstriction to the thromboxane A2 analog U46619 in WT but not SMC MR KO mice. Vasodilation to the nitric‐oxide donor sodium nitroprusside was unchanged by WD feeding. Global quantitative proteomics of aortas from each group revealed a number of differentially expressed proteins following WD feeding including upregulation of the oxidative stress‐related small GTPase Rac‐1 in WT but not SMC MR KO aortas. Accordingly, pretreatment of coronary arteries with the superoxide dismutase mimetic Tempol restored endothelium‐dependent vasodilation in WD‐fed WT mice with no effect in SMC MR KO vessels. Furthermore, WD feeding induced cardiac diastolic dysfunction assessed by echocardiography (i.e., increased E/E′, reduced E′/A′, increased diastolic stiffness). Coincident with prevention of WD‐induced coronary dysfunction, SMC MR deletion prevented WD‐induced cardiac diastolic dysfunction. Importantly, this cardioprotective effect of SMC MR deletion occurred independent of changes in left ventricular hypertrophy, blood pressure, aortic stiffening, and proteinuria. Taken together, our data demonstrate a central role of SMC MR signaling underlying WD‐induced coronary and cardiac diastolic dysfunction in females. Importantly, this study adds to an emerging body of evidence suggesting a vascular origin of cardiac dysfunction in obesity.Support or Funding InformationSupported by NIH (HL136386 to SBB, HL119290 to IZJ) and the Department of Veterans Affairs BLR&D (CDA‐2 IK2 BX002030). BC is supported by Department of Veterans Affairs (IK6 BX004016 and I01 BX002255).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanistic Insights in NeuroD Potentiation of Mineralocorticoid Receptor Signaling.

Mineralocorticoid receptor (MR)-mediated signaling in the brain has been suggested as a protective factor in the development of psychopathology, in particular mood disorders. We recently identified genomic loci at which either MR or the closely related glucocorticoid receptor (GR) binds selectively, and found members of the NeuroD transcription factor family to be specifically associated with MR-bound DNA in the rat hippocampus. We show here using forebrain-specific MR knockout mice that GR binding to MR/GR joint target loci is not affected in any major way in the absence of MR. Neurod2 binding was also independent of MR binding. Moreover, functional comparison with MyoD family members indicates that it is the chromatin remodeling aspect of NeuroD, rather than its direct stimulation of transcription, that is responsible for potentiation of MR-mediated transcription. These findings suggest that NeuroD acts in a permissive way to enhance MR-mediated transcription, and they argue against competition for DNA binding as a mechanism of MR- over GR-specific binding.

Regulation of Atrial Fibrosis by the Bone.

MR (mineralocorticoid receptor) antagonists have been demonstrated to provide beneficial effects on preventing atrial fibrosis. However, the underlying cellular and molecular mechanisms remain unclear. We aim to determine the role of osteoblast MR in atrial fibrosis and to explore the underlying mechanism. Using osteoblast MR knockout mouse in combination with mutant TGF (transforming growth factor)-β1 transgenic mouse, we demonstrated that MR deficiency in osteoblasts significantly attenuated atrial fibrosis. Mechanistically, MR directly regulated expression of OCN (osteocalcin) in osteoblasts. Both carboxylated and undercarboxylated OCNs (ucOC) were less secreted in osteoblast MR knockout mice. Mutant TGF-β1 transgenic mice supplemented with recombinant ucOC showed aggravated atrial fibrosis. In cultured atrial fibroblasts, ucOC treatment promoted proliferation and migration of atrial fibroblasts, whereas cotreatment with an antagonist for a GPRC6A (G-protein-coupled receptor, family C, group 6, member A) abolished these effects. Western blotting analysis revealed upregulation of PKA (protein kinase A) and CREB (cAMP-response element-binding protein) phosphorylation after ucOC treatment. Inhibition of PKA with its antagonist reduced ucOC-induced proliferation and migration of atrial fibroblasts. Finally, the impact of osteoblast MR deficiency on atrial fibrosis was abolished by ucOC administration in mutant TGF-β1 transgenic mice. Taken together, MR deficiency in osteoblasts attenuated atrial fibrosis by downregulation of OCN to promote proliferation and migration of atrial fibroblasts.

Leptin‐Induced Endothelial Dysfunction is Mediated by Endothelial Mineralocorticoid Receptor Epithelial Sodium Channel Activation in Female Mice

Rising rates of hypertension are significantly associated with rising rates of obesity. While the mechanisms of obesity‐associated hypertension have been heavily investigated, these studies have overwhelmingly favored men and male rodents as research subjects. Therefore, the sex‐specific mechanisms of obesity‐associated hypertension require investigation. Our lab has previously shown that the adipocyte‐derived hormone leptin, whose levels are drastically increased in obesity, is required for hypertension and endothelial dysfunction in obese female mouse models, the development of which is dependent on leptin‐induced aldosterone secretion and subsequent mineralocorticoid receptor (MR) activation. The epithelial sodium channel alpha (ENaC), whose transcription is directly upregulated by MR activation, has also been implicated in endothelial dysfunction in female mice. We therefore sought to test the hypothesis that endothelial MR activation mediates leptin‐induced endothelial dysfunction via increased activation of ENaC. To test that endothelial MR activation, specifically, is required for leptin‐induced endothelial dysfunction we infused female endothelial cell‐specific MR knockout mice (ECMR KO) with leptin (0.9 mg/kg/day) via subcutaneous osmotic minipump. Wild‐type mice (WT) developed endothelial dysfunction, as assessed by vascular (aortic rings) relaxation to acetylcholine (10−9 M–10−5 M, nonlinear fit curve), in response to leptin infusion (P<0.05, two‐way ANOVA, n=4–6). However, ECMR KO female mice infused with leptin were protected from endothelial dysfunction (n=6–7), indicating that endothelial MR activation mediates leptin‐induced endothelial dysfunction in female mice. In contrast, no effect of leptin nor endothelial MR deletion was observed on endothelial function in male WT and ECMR KO mice (n=2–5). To assess if MR‐mediated ENaC activation plays a role in leptin‐induced endothelial dysfunction, leptin‐infused female mice were treated with amiloride (ENaC antagonist, 100 ug/day in drinking water) which ablated endothelial dysfunction in female leptin‐infused mice (P<0.05, two‐way ANOVA, n=4), indicating that leptin‐induced endothelial dysfunction requires ENaC activation. In addition, we sought to assess the potential for leptin to upregulate endothelial cell ENaC expression in female mice. Isolated endothelial cells, which were obtained from the aorta via CD31‐specific magnetic bead sorting, were extracted from female Agouti hyperleptinemic obese mice and RT‐PCR analysis for mRNA expression of ENaC demonstrated elevated ENaC expression in hyperleptinemic mice (10.8±0.5‐fold change from lean control female mice, n=2–3). These elevations in endothelial ENaC mRNA expression are associated with elevated plasma aldosterone in hyperleptinemic female mice, indicating that leptin‐induced aldosterone induces endothelial dysfunction via increased MR activation, which ultimately leads to elevated ENaC expression and activation. Therefore, MR‐stimulated ENaC upregulation may play a crucial role in the development of leptin‐mediated cardiovascular diseases in obese females.Support or Funding InformationThis work was supported by the following: NIH 1R01HL130301‐01, AHA 16IRG27770047, NIH 5F32HL136191‐0This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mineralocorticoid receptor blockade improves glucocorticoid-induced skin atrophy but partially ameliorates anti-inflammatory actions in an irritative model in human skin explants.

We recently demonstrated that blockade of the mineralocorticoid receptor (MR) effectively ameliorated GC-induced skin atrophy in healthy human skin explants and epidermal MR knockout mice. However, whether MR blockade improves the therapeutic index of glucocorticoids (GCs) in skin pathology was not investigated. We assessed the effects of GCs, MR antagonists (MRA) or both, in SDS-treated human skin explants. All treatments restored SDS-augmented epidermal thickness but only GC plus MRA restored the expression of COL1A1. However, MRA alone or in combination with GCs may exert a dual role in regulating inflammatory cytokines. Thus, although combined treatment may be beneficial to improve irritative skin, extensive in vivo testing is required to establish whether the anti-inflammatory effects of GCs are maintained in the presence of MRA.

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.

Mineralocorticoid Receptor Deficiency in T Cells Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Dysfunction Through Modulating T-Cell Activation.

Although antagonists of mineralocorticoid receptor (MR) have been widely used to treat heart failure, the underlying mechanisms are incompletely understood. Recent reports show that T cells play important roles in pathologic cardiac hypertrophy and heart failure. However, it is unclear whether and how MR functions in T cells under these pathologic conditions. We found that MR antagonist suppressed abdominal aortic constriction-induced cardiac hypertrophy and decreased the accumulation and activation of CD4+ and CD8+ T cells in mouse heart. T-cell MR knockout mice manifested suppressed cardiac hypertrophy, fibrosis, and dysfunction compared with littermate control mice after abdominal aortic constriction. T-cell MR knockout mice had less cardiac inflammatory response, which was illustrated by decreased accumulation of myeloid cells and reduced expression of inflammatory cytokines. Less amounts and activation of T cells were observed in the heart of T-cell MR knockout mice after abdominal aortic constriction. In vitro studies showed that both MR antagonism and deficiency repressed activation of T cells, whereas MR overexpression elevated activation of T cells. These results demonstrated that MR blockade in T cells protected against abdominal aortic constriction-induced cardiac hypertrophy and dysfunction. Mechanistically, MR directly regulated T-cell activation and modulated cardiac inflammation. Targeting MR in T cells specifically may be a feasible strategy for more effective treatment of pathologic cardiac hypertrophy and heart failure.

Mineralocorticoid Receptor Deficiency in Macrophages Inhibits Atherosclerosis by Affecting Foam Cell Formation and Efferocytosis

Mineralocorticoid receptor (MR) has been considered as a potential target for treating atherosclerosis. However, the cellular and molecular mechanisms are not completely understood. We aim to explore the functions and mechanisms of macrophage MR in atherosclerosis. Atherosclerosis-susceptible LDLRKO chimeric mice with bone marrow cells from floxed control mice or from myeloid MR knock-out (MRKO) mice were generated and fed with high cholesterol diet. Oil red O staining showed that MRKO decreased atherosclerotic lesion area in LDLRKO mice. In another mouse model of atherosclerosis, MRKO/APOEKO mice and floxed control/APOEKO mice were generated and treated with angiotensin II. Similarly, MRKO inhibited the atherosclerotic lesion area in APOEKO mice. Histological analysis showed that MRKO increased collagen coverage and decreased necrosis and macrophage accumulation in the lesions. In vitro results demonstrated that MRKO suppressed macrophage foam cell formation and up-regulated the expression of genes involved in cholesterol efflux. Furthermore, MRKO decreased accumulation of apoptotic cells and increased effective efferocytosis in atherosclerotic lesions. In vitro study further revealed that MRKO increased the phagocytic index of macrophages without affecting their apoptosis. In conclusion, MRKO reduces high cholesterol- or angiotensin II-induced atherosclerosis and favorably changes plaque composition, likely improving plaque stability. Mechanistically, MR deficiency suppresses macrophage foam cell formation and up-regulates expression of genes related to cholesterol efflux, as well as increases effective efferocytosis and phagocytic capacity of macrophages.

Mineralocorticoid Receptor Deficiency in Macrophages Inhibits Neointimal Hyperplasia and Suppresses Macrophage Inflammation Through SGK1-AP1/NF-κB Pathways.

Restenosis after percutaneous coronary intervention remains to be a serious medical problem. Although mineralocorticoid receptor (MR) has been implicated as a potential target for treating restenosis, the cellular and molecular mechanisms are largely unknown. This study aims to explore the functions of macrophage MR in neointimal hyperplasia and to delineate the molecular mechanisms. Myeloid MR knockout (MMRKO) mice and controls were subjected to femoral artery injury. MMRKO reduced intima area and intima/media ratio, Ki67- and BrdU-positive vascular smooth muscle cells, expression of proinflammatory molecules, and macrophage accumulation in injured arteries. MMRKO macrophages migrated less in culture. MMRKO decreased Ki67- and BrdU-positive macrophages in injured arteries. MMRKO macrophages were less Ki67-positive in culture. Conditioned media from MMRKO macrophages induced less migration, Ki67 positivity, and proinflammatory gene expression of vascular smooth muscle cells. After lipopolysaccharide treatment, MMRKO macrophages had decreased p-cFos and p-cJun compared with control macrophages, suggesting suppressed activation of activator protein-1 (AP1). Nuclear factor-κB (NF-κB) pathway was also inhibited by MMRKO, manifested by decreased p-IκB kinase-β and p-IκBα, increased IκBα expression, decreased nuclear translocation of p65 and p50, as welll as decreased phosphorylation and expression of p65. Finally, overexpression of serum-and-glucocorticoid-inducible-kinase-1 (SGK1) attenuated the effects of MR deficiency in macrophages. Selective deletion of MR in myeloid cells limits macrophage accumulation and vascular inflammation and, therefore, inhibits neointimal hyperplasia and vascular remodeling. Mechanistically, MR deficiency suppresses migration and proliferation of macrophages and leads to less vascular smooth muscle cell activation. At the molecular level, MR deficiency suppresses macrophage inflammatory response via SGK1-AP1/NF-κB pathways.

Adult nephron-specific MR-deficient mice develop a severe renal PHA-1 phenotype.

Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR(Pax8/LC1)) using a doxycycline-inducible system. Under standard diet, MR(Pax8/LC1) mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.

A novel actor in skin biology: the mineralocorticoid receptor.

A novel actor in skin biology: the mineralocorticoid receptor.