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.

Mineralocorticoid Receptor Antagonism Prevents Type 2 Familial Partial Lipodystrophy Brown Adipocyte Dysfunction.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in diabetic tubulopathy.

Diabetic tubulopathy (DT) is a recently recognized key pathology of diabetic kidney disease (DKD). The mitochondria-centric view of DT is emerging as a vital pathological factor in different types of metabolic diseases, such as DKD. Finerenone (FIN), a novel non-steroidal mineralocorticoid receptor antagonist, attenuates kidney inflammation and fibrosis in DKD, but the precise pathomechanisms remain unclear. The role of mineralocorticoid receptor (MR) in perturbing mitochondrial function via the PI3K/Akt/eNOS signaling pathway, including mitochondrial dynamics and mitophagy, was investigated under a diabetic state and high glucose (HG) ambiance. To elucidate how the activation of MR provokes mitochondrial dysfunction in DT, human kidney proximal tubular epithelial (HK-2) cells were exposed to HG, and then mitochondrial dynamics, mitophagy, mitochondrial ROS (mitoROS), signaling molecules PI3K, Akt, Akt phosphorylation and eNOS were probed. The above molecules or proteins were also explored in the kidneys of diabetic and FIN-treated mice. FIN treatment reduced oxidative stress, mitochondrial fragmentation, and apoptosis while restoring the mitophagy via PI3K/Akt/eNOS signaling pathway in HK-2 cells exposed to HG ambiance and tubular cells of DM mice. These findings linked MR activation to mitochondrial dysfunction via PI3K/Akt/eNOS signaling pathway in DT and highlight a pivotal but previously undiscovered role of FIN in alleviating renal tubule injury for the treatment of DKD.

Mineralocorticoid receptor antagonism in diabetes reduces albuminuria by preserving the glomerular endothelial glycocalyx.

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier. Previously, we showed that mineralocorticoid receptor (MR) activation caused GEnGlx damage and albuminuria. In this study, we investigated whether MR antagonism could limit albuminuria in diabetes and studied the site of action. Streptozotocin-induced diabetic Wistar rats developed albuminuria, increased glomerular albumin permeability (Ps'alb), and increased glomerular matrix metalloproteinase (MMP) activity with corresponding GEnGlx loss. MR antagonism prevented albuminuria progression, restored Ps'alb, preserved GEnGlx, and reduced MMP activity. Enzymatic degradation of the GEnGlx negated the benefits of MR antagonism, confirming their dependence on GEnGlx integrity. Exposing human glomerular endothelial cells (GEnC) to diabetic conditions in vitro increased MMPs and caused glycocalyx damage. Amelioration of these effects confirmed a direct effect of MR antagonism on GEnC. To confirm relevance to human disease, we used a potentially novel confocal imaging method to show loss of GEnGlx in renal biopsy specimens from patients with diabetic nephropathy (DN). In addition, patients with DN randomized to receive an MR antagonist had reduced urinary MMP2 activity and albuminuria compared with placebo and baseline levels. Taken together, our work suggests that MR antagonists reduce MMP activity and thereby preserve GEnGlx, resulting in reduced glomerular permeability and albuminuria in diabetes.

Combined antagonist treatment of glucocorticoid and mineralocorticoid receptor does not affect weight loss of fasting rainbow trout but inhibits a fasting-induced elevation of cortisol secretion.

The gastrointestinal system of fish reacts rapidly to food deprivation. The relative masses of digestive organs and activities of digestive enzymes decrease within days of fasting. This is believed to be an energy-conserving strategy as the metabolic cost of maintaining digestive capacity is high. Cortisol is known for its role in energy mobilization following stress exposure, and prolonged elevated cortisol levels have been shown to reduce growth rates in fish. Fish experiencing chronic cortisol elevations show structural changes to their digestive tissues and overall reductions in relative digestive tissue masses. In fish fasting for prolonged periods, circulating cortisol levels have been reported to be downregulated, upregulated, or unchanged compared to feeding fish. This study aimed to investigate if RU486 and spironolactone, antagonists of the glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), respectively, alone or in combination affect circulating cortisol levels during prolonged starvation. In addition, we tested the effects of blocking GR and MR, on the down-regulation of relative digestive tissue mass during starvation, and its effects on weight loss. Three treatment groups of rainbow trout were intraperitoneally implanted with either GR, MR, or GR and MR blockers. A fourth group was implanted with cortisol, while a fifth group served as a control. All treatment groups were sampled over a course of four weeks of food deprivation and compared against each other and fed control fish at day 0 of the trial. Starvation for 2 weeks and longer significantly increased circulating cortisol levels in all groups except for the group implanted with GR and MR antagonists. Loss of body mass occurred most rapidly during the first week of starvation. Spironolactone treatment resulted in significantly reduced loss of mass during the first week, however, over the following weeks, no differences in mass loss were observed in the groups implanted with blockers, while cortisol-treated fish showed the highest decrease in body mass over time. Relative digestive tissue mass decreased in all groups but apparently, the fasting-induced elevation in plasma cortisol levels did not affect the relative weight loss of digestive tissues as no differences were observed between control fish and GR + MR antagonist treated fish. Very high cortisol levels caused by cortisol treatment however caused a faster decrease in the relative mass of some digestive organs, particularly the stomach.

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy.

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Mineralocorticoid Receptor Antagonists Cause Natriuresis in the Absence of Aldosterone.

BACKGROUND: 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 11ßHSD2 (11ß-hydroxysteroid dehydrogenase type 2). 11ßHSD2 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. METHODS: 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. RESULTS: MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11ßHSD2 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. CONCLUSIONS: 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.

Identification of Mineralocorticoid Receptor Modulators with Low Impact on Electrolyte Homeostasis but Maintained Organ Protection.

The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.

Pharmacokinetics, Metabolism, and Excretion of [14C]Esaxerenone, a Novel Mineralocorticoid Receptor Blocker in Humans.

Esaxerenone (CS-3150) is a novel, nonsteroidal, selective mineralocorticoid receptor blocker. The absorption, metabolism, distribution, and excretion of esaxerenone were assessed in in vitro studies and in a clinical study, where [14C]esaxerenone (150 µCi/20 mg) was administered orally to six healthy male subjects. The plasma concentrations of esaxerenone and its metabolites (M4, M11, and M1) were measured using liquid chromatography-tandem mass spectrometry. The recovery of radioactivity was 92.5%, with 38.5% and 54.0% excreted in the urine and feces, respectively. The half-life of radioactivity in blood and plasma was approximately 30 hours, similar to that of the unchanged form in plasma. The blood-to-plasma ratio was 0.628, demonstrating low partitioning to blood components. In plasma, esaxerenone was the most abundant moiety (40.8%), followed by O-glucuronide (21.4%; M4), acyl-glucuronide of amide-bond hydrolysate (8.0%; M11), and the deshydroxyethyl form (1.7%; M1). In vitro studies showed that esaxerenone was a substrate of CYP3A and multiple UDP-glucuronosyltransferase isoforms. Oxidation contributed approximately 30% to its clearance, as indicated by the excretion ratio of oxidized metabolites into urine and feces. Caco-2 studies showed that esaxerenone was a substrate of P-glycoprotein and breast cancer resistance protein; however, the excretion ratios of the unchanged form in the feces and urine were 18.7% and 1.6%, respectively, indicating that these transporters were not important for the absorption and elimination of esaxerenone. Low urinary excretion of esaxerenone suggested that the plasma exposure of esaxerenone was not affected by renal dysfunction. Multiple elimination pathways including oxidation, glucuronidation, and hydrolysis, and the low contribution of transporters, indicated limited drug-drug interaction potential.

Biotransformation of Finerenone, a Novel Nonsteroidal Mineralocorticoid Receptor Antagonist, in Dogs, Rats, and Humans, In Vivo and In Vitro.

Mass balance and biotransformation of finerenone, a nonsteroidal mineralocorticoid receptor antagonist, were investigated in four healthy male volunteers following a single oral administration of 10 mg (78 µCi) of [14C]finerenone and compared with data from studies in dogs and rats. The total recovery of the administered radioactivity was 101% in humans, 94.7% in dogs, and 95.2% in rats. In humans, radioactivity was mainly excreted renally (80%); in rats, it was primarily the biliary/fecal route (76%); and in dogs, excretion was more balanced. Finerenone was extensively metabolized in all species by oxidative biotransformation, with minor amounts of unchanged drug in excreta (humans: 1%; dogs, rats: <9%). In vitro studies suggested cytochrome P450 3A4 was the predominant enzyme involved in finerenone metabolism in humans. Primary metabolic transformation involved aromatization of the dihydronaphthyridine moiety of metabolite M1 as a major clearance pathway with a second oxidative pathway leading to M4. These were both prone to further oxidative biotransformation reactions. Naphthyridine metabolites (M1-M3) were the dominant metabolites identified in human plasma, with no on-target pharmacological activity. In dog plasma, finerenone and metabolite M2 constituted the major components; finerenone accounted almost exclusively for drug-related material in rat plasma. For metabolites M1-M3, axial chirality was observed, represented by two atropisomers (e.g., M1a and M1b). Analysis of plasma and excreta showed one atropisomer (a-series, >79%) of each metabolite predominated in all three species. In summary, the present study demonstrates that finerenone is cleared by oxidative biotransformation, mainly via naphthyridine derivatives.

Patiromer: A Review in Hyperkalaemia.

Patiromer (Veltassa®) for oral suspension is a non-absorbed, sodium-free potassium binding polymer that exchanges calcium for potassium in the gastrointestinal (GI) tract, thereby increasing faecal potassium excretion and reducing serum potassium levels. Patiromer was approved in the USA in 2015 and is now approved in several other countries, including those of the EU, for the treatment of hyperkalaemia in adults. In clinical trials, patiromer reduced serum potassium levels and the risk of recurrent hyperkalaemia in patients with chronic kidney disease (CKD) and/or diabetic nephropathy with or without heart failure (HF), allowing the majority of patients to continue receiving renin-angiotensin-aldosterone system (RAAS) inhibitors (drugs that inhibit the renal excretion of potassium) for up to 52 weeks. Patiromer also maintained normokalaemia in patients with HF and a propensity for hyperkalaemia, enabling concomitant administration and up-titration of spironolactone. Patiromer was generally well tolerated, with a low risk of hypokalaemia. GI disorders and hypomagnesaemia were the most common adverse events; these were generally of mild or moderate severity. Therefore, oral patiromer is a valuable treatment option for the long-term management of hyperkalaemia.

Smooth Muscle Cell-Mineralocorticoid Receptor as a Mediator of Cardiovascular Stiffness With Aging.

Stiffening of the vasculature with aging is a strong predictor of adverse cardiovascular events, independent of all other risk factors including blood pressure, yet no therapies target this process. MRs (mineralocorticoid receptors) in smooth muscle cells (SMCs) have been implicated in the regulation of vascular fibrosis but have not been explored in vascular aging. Comparing SMC-MR-deleted male mice to MR-intact littermates at 3, 12, and 18 months of age, we demonstrated that aging-associated vascular stiffening and fibrosis are mitigated by MR deletion in SMCs. Progression of cardiac stiffness and fibrosis and the decline in exercise capacity with aging were also mitigated by MR deletion in SMC. Vascular gene expression profiling analysis revealed that MR deletion in SMC is associated with recruitment of a distinct antifibrotic vascular gene expression program with aging. Moreover, long-term pharmacological inhibition of MR in aged mice prevented the progression of vascular fibrosis and stiffness and induced a similar antifibrotic vascular gene program. Finally, in a small trial in elderly male humans, short-term MR antagonism produced an antifibrotic signature of circulating biomarkers similar to that observed in the vasculature of SMC-MR-deleted mice. These findings suggest that SMC-MR contributes to vascular stiffening with aging and is a potential therapeutic target to prevent the progression of aging-associated vascular fibrosis and stiffness.

Differential effects of imipramine and CORT118335 (Glucocorticoid receptor modulator/mineralocorticoid receptor antagonist) on brain-endocrine stress responses and depression-like behavior in female rats.

Depression is commonly associated with hypothalamic-pituitary adrenal (HPA) axis dysfunction that primarily manifests as aberrant glucocorticoid secretion. Glucocorticoids act on Type I mineralocorticoid (MR) and Type II glucocorticoid receptors (GR) to modulate mood and endocrine responses. Successful antidepressant treatment normalizes HPA axis function, in part due to modulatory effects on MR and GR in cortico-limbic structures. Although women are twice as likely to suffer from depression, little is known about how antidepressants modulate brain, endocrine, and behavioral stress responses in females. Here, we assessed the impact of CORT118335 (GR modulator/MR antagonist) and imipramine (tricyclic antidepressant) on neuroendocrine and behavioral responses to restraint or forced swim stress (FST) in female rats (n=10-12/group). Increased immobility CORT118335 in the FST is purported to reflect passive coping or depression-like behavior. CORT118335 dampened adrenocorticotropic hormone (ACTH) and corticosterone responses to the FST, but did not affect immobility. Imipramine suppressed ACTH, but had minimal effects on corticosterone responses to FST. Despite these marginal effects, imipramine decreased immobility, suggesting antidepressant efficacy. In an effort to link brain-endocrine responses with behavior, c-Fos was assessed in HPA axis and mood modulatory regions in response to the FST. CORT118335 upregulated c-Fos expression in the paraventricular nucleus of the hypothalamus. Imipramine decreased c-Fos in the basolateral amygdala and hippocampus (CA1 and CA3), but increased c-Fos in the central amygdala. These data suggest the antidepressant-like (e.g., active coping) properties of imipramine may be due to widespread effects on cortico-limbic circuits that regulate emotional and cognitive processes.

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.

Signal Enhancement in the HPLC-ESI-MS/MS analysis of spironolactone and its metabolites using HFIP and NH4F as eluent additives.

This paper describes an LC-MS/MS method to determine the concentration of spironolactone and its metabolites 7-alpha-methylthiospironolactone and canrenone in blood plasma samples. The resulting assay is simple (using protein precipitation for sample preparation) and sensitive (the lower limit of quantification is close to 0.5 ng/ml) while requiring only 50 µl of plasma, making it especially suitable for analyzing samples obtained from pediatric and neonatal patients where sample sizes are limited. The sensitivity is achieved by using ammonium fluoride as an eluent additive, which in our case amplifies the signal from our analytes in the plasma solution on average about 70 times. The method is fully validated according to the European Medicines Agency's guideline and used for the measurement of pediatric patients' samples in clinical trials for evaluating oral spironolactone's and its metabolites' pharmacokinetics in children up to 2 years of age.

Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11ß-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11ß-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11ß,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11ß-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed.

Third-generation Mineralocorticoid Receptor Antagonists: Why Do We Need a Fourth?

The first mineralocorticoid receptor (MR) antagonist, spironolactone, was developed almost 60 years ago to treat primary aldosteronism and pathological edema. Its use waned in part because of its lack of selectivity. Subsequently, knowledge of the scope of MR function was expanded along with clinical evidence of the therapeutic importance of MR antagonists to prevent the ravages of inappropriate MR activation. Forty-two years elapsed between the first and MR-selective second generation of MR antagonists. Fifteen years later, despite serious shortcomings of the existing antagonists, a third-generation antagonist has yet to be marketed. Progress has been slowed by the lack of appreciation of the large variety of cell types that express the MR and its diverse cell-type-specific actions, and also its unique complex interaction actions at the molecular level. New MR antagonists should preferentially target the inflammatory and fibrotic effects of MR and perhaps its excitatory effects on sympathetic nervous system, but not the renal tubular epithelium or neurons of the cortex and hippocampus. This review briefly describes efforts to develop a third-generation MR antagonist and why fourth generation antagonists and selective agonists based on structural determinants of tissue and ligand-specific MR activation should be contemplated.

A Mixed Glucocorticoid/Mineralocorticoid Selective Modulator With Dominant Antagonism in the Male Rat Brain.

Adrenal glucocorticoid hormones are potent modulators of brain function in the context of acute and chronic stress. Both mineralocorticoid (MRs) and glucocorticoid receptors (GRs) can mediate these effects. We studied the brain effects of a novel ligand, C118335, with high affinity for GRs and modest affinity for MRs. In vitro profiling of receptor-coregulator interactions suggested that the compound is a "selective modulator" type compound for GRs that can have both agonistic and antagonistic effects. Its molecular profile for MRs was highly similar to those of the full antagonists spironolactone and eplerenone. C118335 showed predominantly antagonistic effects on hippocampal mRNA regulation of known glucocorticoid target genes. Likewise, systemic administration of C118335 blocked the GR-mediated posttraining corticosterone-induced enhancement of memory consolidation in an inhibitory avoidance task. Posttraining administration of C118335, however, gave a strong and dose-dependent impairment of memory consolidation that, surprisingly, reflected involvement of MRs and not GRs. Finally, C118335 treatment acutely suppressed the hypothalamus-pituitary-adrenal axis as measured by plasma corticosterone levels. Mixed GR/MR ligands, such as C118335, can be used to unravel the mechanisms of glucocorticoid signaling. The compound is also a prototype of mixed GR/MR ligands that might alleviate the harmful effects of chronic overexposure to endogenous glucocorticoids.

Adrenal gland inclusions in patients treated with aldosterone antagonists (Spironolactone/Eplerenone): incidence, morphology, and ultrastructural findings.

BACKGROUND: Spironolactone is often used to treat hypertension caused by hyperaldosteronism, and as a result, can form concentrically laminated electron dense spironolactone body inclusions within the adrenal gland. Spironolactone bodies have not been investigated in a contemporary cohort or in patients treated with the more recently approved aldosterone antagonist, eplerenone. METHODS: Spironolactone bodies were retrospectively investigated in patients treated for hyperaldosteronism (n=15) from 2012-2013 that underwent a subsequent adrenalectomy. RESULTS: Inclusions were identified in 33% of patients treated with aldosterone antagonists, far less than previously reported. Remarkably, 50% of patients treated with spironolactone had inclusions while no patients using eplerenone alone had inclusions. Two patients treated with spironolactone had bodies present longer than the duration described in prior studies. Inclusions unexpectedly persisted in 1 patient despite increased duration of discontinued pharmacological treatment. A spectrum of histologic and ultrastructural findings were encountered within an adrenal cortical adenoma from a patient treated with both spironolactone and eplerenone. Ultrastructural examination revealed laminated electron dense bodies with the appearance of classic spironolactone inclusions as well as electron dense bodies without laminations and laminated bodies without electron dense cores. CONCLUSIONS: Our incidence rate of spironolactone bodies was much lower than previously reported, with no inclusions seen in patients treated solely with the newer aldosterone antagonist, eplerenone. Pathologists should be aware of these infrequently encountered inclusions, particularly as the clinical history of hyperaldosteronism and pharmacologic treatment may not be provided. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4597918761268031.