Mineralocorticoid Receptor Knockout Research Articles

Mineralocorticoid Receptor in Endothelial Cells Contributes to VEGF Receptor Inhibitor-Induced Vascular and Kidney Damage.

Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer patient survival by inhibiting tumor angiogenesis. However, VEGFRis induce treatment-limiting hypertension which has been associated with impaired vascular endothelial cell (EC) function and kidney damage. The mineralocorticoid receptor (MR) regulates blood pressure via effects on the vasculature and the kidney. Thus, we interrogated the role of the MR in EC dysfunction, renal impairment, and hypertension in a mouse model of VEGFRi-induced hypertension using sorafenib. EC dysfunction in mesenteric arterioles was assessed by immunoblotting for phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. Renal damage was measured by assessing glomerular endotheliosis histologically. Blood pressure (BP) was measured using implanted radiotelemetry. Six days of sorafenib treatment significantly impaired mesenteric resistance vessel EC function, induced renal damage, and increased BP. Pharmacologic MR blockade with spironolactone prevented the sorafenib-induced decline in eNOS phosphorylation and the renal glomerular endotheliosis, without affecting systolic or diastolic BP. Mice with the MR knocked out specifically in ECs (EC-MR-KO) were protected from sorafenib-induced EC dysfunction and glomerular endotheliosis, whereas smooth muscle cell-specific MR (SMC-MR) knockout mice were not. Neither EC-MR knockout nor SMC-MR knockout affected the degree to which sorafenib increased systolic or diastolic BP. These results reveal that the MR, specifically in EC but not in SMCs, is necessary for VEGFRi-induced renal and vascular injury. While ineffective at lowering SBP, these data suggest potential therapeutic benefits of MR antagonists, like spironolactone, to protect the vasculature and the kidneys from VEGFRi-induced injury.

Dendritic cell mineralocorticoid receptor controls blood pressure by regulating T helper 17 differentiation: role of the Plcβ1/4-Stat5-NF-κB pathway.

Dendritic cells (DCs) are closely related to blood pressure (BP) regulation. Mineralocorticoid receptor (MR) is an important drug target for antihypertensive treatment. However, the role of DC MR in the pathogenesis of hypertension has not been fully elucidated. This study aimed to determine the role of DC MR in BP regulation and to explore the mechanism. Renal biopsy and peripheral blood samples were collected from hypertensive patients (HTN) for immunostaining and flow cytometry. Dendritic cell MR knockout (DCMRKO) mice, DC MR overexpressing (DCMROV) mice, DCMROV/IL-17A knockout (DCMROV/IL-17AKO) mice and finerenone-treated C57BL/6 mice were infused with angiotensin II (Ang II) to establish hypertensive models. Western blotting, chromatin immunoprecipitation, co-immunoprecipitation, and in vivo DC depletion or adoptive transfer were used to delineate the functional importance of DC MR in hypertension development. Mineralocorticoid receptor antagonists (spironolactone and finerenone) suppressed DC aggregation and activation, as well as hypertension in HTN and mice. Compared with littermate control (LC) mice, dendritic cell MR knockout mice had strikingly decreased BPs and attenuated target organ damage after Ang II infusion. Flow cytometry showed that DC MR deficiency mitigated Ang II-induced DC activation and T helper 17 (Th17) cell differentiation. RNA sequencing revealed that MR-deficient DCs had elevated expression of Plcβ1 and Plcβ4, knockdown of which reversed the inhibitory effect of MR deficiency on DC activation and Th17 differentiation. Adoptive transfer of MR-deficient DCs protected Ang II-induced hypertension, whereas knockdown of Plcβ1/4 eliminated the protective effects. At the molecular level, MR negatively regulated Plcβ1/4, which recruited SHP-1 to inactivate of Stat5 activity, resulting in enhanced NF-κB activation and Th17 polarization. Furthermore, DCMROV mice manifested more elevated BPs and target organ damage than control mice after Ang II infusion, and these differences were abolished in DCMROV/IL-17AKO mice. Finally, MR antagonists decreased the aggregation of Th17 in HTN and mice. Dendritic cell MR plays important roles in the pathogenesis of hypertension by regulating Th17 through Plcβ1/4-Stat5-NF-κB signalling, and blockade of DC MR is beneficial for treating hypertension.

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.

Mineralocorticoid receptor knockout in Schwann cells alters myelin sheath thickness.

Myelination allows fast and synchronized nerve influxes and is provided by Schwann cells (SCs) in the peripheral nervous system. Glucocorticoid hormones are major regulators of stress, metabolism and immunity affecting all tissues. They act by binding to two receptors, the low-affinity glucocorticoid receptor (GR) and the high-affinity mineralocorticoid receptor (MR). Little is known about the effect of glucocorticoid hormones on the PNS, and this study focuses on deciphering the role of MR in peripheral myelination. In this work, the presence of a functional MR in SCs is demonstrated and the expression of MR protein in mouse sciatic nerve SC is evidenced. Besides, knockout of MR in SC (SCMRKO using Cre-lox system with DesertHedgeHog (Dhh) Cre promoter) was undertaken in mice. SCMRKO was not associated with alterations of performance in motor behavioral tests on 2- to 6-month-old male mice compared to their controls. No obvious modifications of myelin gene expression or MR signaling gene expression were observed in the SCMRKO sciatic nerves. Nevertheless, Gr transcript and GR protein amounts were significantly increased in SCMRKO nerves compared to controls, suggesting a possible compensatory effect. Besides, an increase in myelin sheath thickness was noted for axons with perimeters larger than 15 µm in SCMRKO illustrated by a significant 4.5% reduction in g-ratio (axon perimeter/myelin sheath perimeter). Thus, we defined MR as a new player in peripheral system myelination and in SC homeostasis.

T-Cell Mineralocorticoid Receptor Deficiency Attenuates Pathologic Ventricular Remodelling After Myocardial Infarction.

Mineralocorticoid receptor (MR) antagonists have been widely used to treat heart failure (HF). Studies have shown that MR in T cells plays important roles in hypertension and myocardial hypertrophy. However, the function of T-cell MR in myocardial infarction (MI) has not been elucidated. In this study, we used T-cell MR knockout (TMRKO) mouse to investigate the effects of T-cell MR deficiency on MI and to explore the underlying mechanisms. Echocardiography and tissue staining were used to assess cardiac function, fibrosis, and myocardial apoptosis after MI. Flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect immune cell filtration and inflammation. T-cell MR deficiency significantly improved cardiac function, promoted myocardial repair, and inhibited myocardial apoptosis, fibrosis, and inflammation after MI. Luminex assays revealed that TMRKO mice had significantly lower levels of interferon-gamma (IFN-γ) and interleukin-6 (IL-6) in serum and infarcted myocardium than littermate control mice. In cultured splenic T cells, MR deficiency suppressed IL-6 expression, whereas MR overexpression enhanced IL-6 expression. Chromatin immunoprecipitation (ChIP) assay demonstrated that MR bound to the MR response element on the promoter of IL-6 gene. Finally, T-cell MR deficiency significantly suppressed accumulation of macrophages in infarcted myocardium and differentiation of proinflammatory macrophages, thereby alleviating the consequences of MI. T-cell MR deficiency improved pathologic ventricular remodelling after MI, likely through inhibition of accumulation and differentiation of proinflammatory macrophages. At the molecular level, MR may work through IFN-γ and IL-6 in T cells to exert functions in MI.

The mineralocorticoid receptor modulates timing and location of genomic binding by glucocorticoid receptor in response to synthetic glucocorticoids in keratinocytes.

Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.

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.

Mineralocorticoid Receptor Signaling in the Inflammatory Skeletal Muscle Microenvironments of Muscular Dystrophy and Acute Injury

Duchenne muscular dystrophy (DMD) is a striated muscle degenerative disease due to loss of functional dystrophin protein. Loss of dystrophin results in susceptibility of muscle membranes to damage, leading to muscle degeneration and continuous inflammation and fibrosis that further exacerbate pathology. Long-term glucocorticoid receptor (GR) agonist treatment, the current standard-of-care for DMD, modestly improves prognosis but has serious side effects. The mineralocorticoid receptor (MR), a ligand-activated transcription factor present in many cell types, has been implicated as a therapeutic target for DMD. MR antagonists (MRAs) have fewer side effects than GR agonists and are used clinically for heart failure. MRA efficacy has recently been demonstrated for DMD cardiomyopathy and in preclinical studies, MRAs also alleviate dystrophic skeletal muscle pathology. MRAs lead to improvements in muscle force and membrane stability and reductions in degeneration, inflammation, and fibrosis in dystrophic muscles. Myofiber-specific MR knockout leads to most of these improvements, supporting an MR-dependent mechanism of action, but MRAs additionally stabilize myofiber membranes in an MR-independent manner. Immune cell MR signaling in dystrophic and acutely injured normal muscle contributes to wound healing, and myeloid-specific MR knockout is detrimental. More research is needed to fully elucidate MR signaling in striated muscle microenvironments. Direct comparisons of genomic and non-genomic effects of glucocorticoids and MRAs on skeletal muscles and heart will contribute to optimal temporal use of these drugs, since they compete for binding conserved receptors. Despite the advent of genetic medicines, therapies targeting inflammation and fibrosis will be necessary to achieve optimal patient outcomes.

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.

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.

Myeloid mineralocorticoid receptors contribute to skeletal muscle repair in muscular dystrophy and acute muscle injury.

Suppressing mineralocorticoid receptor (MR) activity with MR antagonists is therapeutic for chronic skeletal muscle pathology in Duchenne muscular dystrophy (DMD) mouse models. Although mechanisms underlying clinical MR antagonist efficacy for DMD cardiomyopathy and other cardiac diseases are defined, mechanisms in skeletal muscles are not fully elucidated. Myofiber MR knockout improves skeletal muscle force and a subset of dystrophic pathology. However, MR signaling in myeloid cells is known to be a major contributor to cardiac efficacy. To define contributions of myeloid MR in skeletal muscle function and disease, we performed parallel assessments of muscle pathology, cytokine levels, and myeloid cell populations resulting from myeloid MR genetic knockout in muscular dystrophy and acute muscle injury. Myeloid MR knockout led to lower levels of C-C motif chemokine receptor 2 (CCR2)-expressing macrophages, resulting in sustained myofiber damage after acute injury of normal muscle. In acute injury, myeloid MR knockout also led to increased local muscle levels of the enzyme that produces the endogenous MR agonist aldosterone, further supporting important contributions of MR signaling in normal muscle repair. In muscular dystrophy, myeloid MR knockout altered cytokine levels differentially between quadriceps and diaphragm muscles, which contain different myeloid populations. Myeloid MR knockout led to higher levels of fibrosis in dystrophic diaphragm. These results support important contributions of myeloid MR signaling to skeletal muscle repair in acute and chronic injuries and highlight the useful information gained from cell-specific genetic knockouts to delineate mechanisms of pharmacological efficacy.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

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.

Coordinated Action of Corticotropin-Releasing Hormone and Cortisol Shapes the Acute Stress-Induced Behavioural Response in Zebrafish

Introduction: The stress response mediated by the hypothalamus-pituitary-adrenal (HPA) axis activation is highly conserved in vertebrates. Hyperactivity is one such established acute stress response, and corticotropin-releasing hormone (CRH), the primary step in HPA activation, signalling has been implicated in this stressor-mediated behaviour. However, whether CRH mediates the acute behavioural effects either alone or in conjunction with glucocorticoids (GCs) are far from clear. We hypothesized that the CRH receptor 1 (CRHR1)-mediated rise in GCs post-stress is necessary for the initiation and maintenance of the acute stress-related behaviour. Methods: We first generated zebrafish (Danio rerio) with a mutation in the CRHR1 gene (CRHR1-KO) to assess the function of CRH. The behavioural readout utilized for this study was the locomotor activity of larval zebrafish in response to an acute light exposure, a protocol that freezes the larvae in response to the light stimulus. To test whether cortisol signalling is involved in the stress-mediated hyperactivity, we treated wildtype fish with metyrapone (MET), an inhibitor of 11β-hydroxylase, to suppress cortisol production. The temporal role for cortisol signalling in the stress-related hyperactivity was tested using the glucocorticoid receptor knockout (GRKO) and mineralocorticoid receptor knockout (MRKO) zebrafish mutants. Results: CRHR1-KO larvae did not increase cortisol, the principal GC in teleosts, post-stress, confirming a functional knockout. An acute stress resulted in the hyperactivity of the larvae in light at 15, 60, and 240 min post-stress, and this was absent in CRHR1-KO larvae. Addition of MET effectively blocked the attendant rise in cortisol post-stress; however, the stress-mediated hyperactivity was inhibited only at 60 and 240 min but not at 15 min post-stress. Addition of human CRH peptide caused hyperactivity at 15 min, and this response was also abolished in the CRHR1-KO mutants. The stress-induced hyperactivity was absent in the MRKO fish, while GRKO mutants showed transient effects. Conclusions: The results suggest that the stress-induced hyperactivity is induced by the CRH/CRHR1 system, while the temporal activation of cortisol production and the associated GR/MR signalling is essential for prolonging the stressor-induced hyperactivity. This study underscores the importance of systems-level analysis to assess stress responsivity.

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.

Postnatal triglyceride accumulation is regulated by mineralocorticoid receptor activation under basal and stress conditions.

Glucocorticoids (GCs) either enhance or reduce obesity in mammals, but limited information exists on the role of corticosteroid receptors in mediating the effect of GCs on lipid metabolism during postnatal development. Mineralocorticoid receptor (MR) activation leads to triglyceride (TG) accumulation post-feeding, whereas glucocorticoid receptor (GR) activation reduces TG levels. The TG profile was inversely related to the lipoprotein lipase (lpl) transcript abundance, and this gene was downregulated by MR activation. Cortisol plays an important role in adipogenesis during postnatal development in zebrafish, and this includes gene/pathway-specific signalling by GR, MR and GR/MR interactions. Ubiquitous MR and GR knockout in zebrafish provides an excellent model to study the mode of action of GCs in regulating lipid metabolism. Glucocorticoids (GCs) act through two receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), which differ in both their affinity to bind GCs and their function. As MR has 10-fold higher affinity for GCs compared to GR, it has been postulated that MR activation occurs at basal levels, while stress levels of these steroid hormones activate GR signalling. There is a growing body of evidence that both these receptors are involved in GC-mediated lipid metabolism. However, the role of GCs in lipogenesis and lipolysis is controversial, as these steroids appear to both enhance and reduce obesity. As lipid synthesis is a critical part of early development, we hypothesized that both MR and GR contribute to lipid regulation by GCs during postnatal growth. Using MR and GR knockout zebrafish, we demonstrate that MR activation, but not GR activation, is involved in triglyceride (TG) accumulation during the larval development post feeding. Lack of MRs did not affect the gene expression of fatty acid synthase (fas), or acyl-CoA:diacylglycerol acyltransferase 2 (dgat2), but increased lipoprotein lipase (lpl) transcript abundance. Activation of GR with exogenous cortisol decreased TG levels and increased lpl mRNA levels, but these responses require the presence of MR. Larval transcriptome revealed that MR was the primary regulator of genes involved in lipid synthesis, while GR activation favoured lipid catabolism. Our results underscore a key role for MR activation in mediating postnatal lipid accumulation, as well as cooperatively regulating GR-mediated lipolysis during postnatal stress.

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.