Mineralocorticoid Receptor Gene Research Articles

Mineralocorticoid receptor antagonism prevents coronary microvascular dysfunction in intermittent hypoxia independent of blood pressure.

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), and is associated with increased cardiovascular mortality that may not be reduced by standard therapies. Inappropriate activation of the renin-angiotensin-aldosterone system occurs in IH, and mineralocorticoid receptor (MR) blockade has been shown to improve vascular outcomes in cardiovascular disease. Thus, we hypothesized that MR inhibition prevents coronary and renal vascular dysfunction in mice exposed to chronic IH. Human and mouse coronary vascular cells and male C57BL/6J mice were exposed to IH or room air (RA) for 12 hours/day for 3 days (in vitro) and 6 weeks with or without treatments with spironolactone (SPL) or hydrochlorothiazide (HTZ). In vitro studies demonstrated that IH increased MR gene expression in human and mouse coronary artery endothelial and smooth muscle cells. Exposure to IH in mice increased blood pressure, reduced coronary flow velocity reserve (CFVR), and attenuated endothelium-dependent dilation and enhanced vasoconstrictor responsiveness in coronary, but not renal arteries. Importantly, SPL treatment prevented altered coronary vascular function independent of blood pressure as normalization of BP with HTZ did not improve CFVR or coronary vasomotor function. These data demonstrate that chronic IH, which mimics the hypoxia-reoxygenation cycles of moderate-to-severe OSA, increases coronary vascular MR expression in vitro. It also selectively promotes coronary vascular dysfunction in mice. Importantly, this dysfunction is sensitive to MR antagonism by SPL, independent of blood pressure. These findings suggest that MR blockade could serve as an adjuvant therapy to improve long-term cardiovascular outcomes in patients with OSA.

Patterns of corticosterone exposure affect the subcellular localisation of mineralocorticoid and glucocorticoid receptor complexes and gene expression

Mineralocorticoid (MR) and glucocorticoid receptors (GR) act as transcription factors and major mediators of glucocorticoid signalling, with pivotal roles in regulating the stress response and hormonal signalling, mood, cognition and memory. The MR and GR share many target genes, have a high degree of homology in their DNA binding (DBD) and ligand binding domain (LBD) but differ considerably in the N-terminal domain (NTD). Using Proximity Ligation Assay (PLA) we quantitatively assessed MR-GR complex subcellular localisation and transcriptional regulation in murine neuroblastoma (N2A) cells stimulated by constant or pulsatile corticosterone (CORT) patterns. We observe that continuous receptor activation by CORT caused localisation at the periphery of the cell nucleus. Truncation of the receptor Ligand Binding Domain (LBD) led to a stronger localisation of MR-GR complexes at the periphery of the cell nuclei. This was also observed for GR immunofluorescence (IF), while in cells expressing only MR or GR the mRNA response to pulsatile hormone treatment was substantially attenuated. However, there was no clearcut correlation between the spatial distribution of MR-GR complexes and the mRNA levels of target genes. Overall, our findings suggest that longer presence in the cell nucleus favors more peripheral nuclear localisation.

Alpha-synuclein-induced stress sensitivity renders the Parkinson’s disease brain susceptible to neurodegeneration

A link between chronic stress and Parkinson’s disease (PD) pathogenesis is emerging. Ample evidence demonstrates that the presynaptic neuronal protein alpha-synuclein (asyn) is closely tied to PD pathogenesis. However, it is not known whether stress system dysfunction is present in PD, if asyn is involved, and if, together, they contribute to neurodegeneration. To address these questions, we assess stress axis function in transgenic rats overexpressing full-length wildtype human asyn (asyn BAC rats) and perform multi-level stress and PD phenotyping following chronic corticosterone administration. Stress signaling, namely corticotropin-releasing factor, glucocorticoid and mineralocorticoid receptor gene expression, is also examined in post-mortem PD patient brains. Overexpression of human wildtype asyn leads to HPA axis dysregulation in rats, while chronic corticosterone administration significantly aggravates nigrostriatal degeneration, serine129 phosphorylated asyn (pS129) expression and neuroinflammation, leading to phenoconversion from a prodromal to an overt motor PD phenotype. Interestingly, chronic corticosterone in asyn BAC rats induces a robust, twofold increase in pS129 expression in the hypothalamus, the master regulator of the stress response, while the hippocampus, both a regulator and a target of the stress response, also demonstrates elevated pS129 asyn levels and altered markers of stress signalling. Finally, defective hippocampal stress signalling is mirrored in human PD brains and correlates with asyn expression levels. Taken together, our results link brain stress system dysregulation with asyn and provide evidence that elevated circulating glucocorticoids can contribute to asyn-induced neurodegeneration, ultimately triggering phenoconversion from prodromal to overt PD.

Novel Evolution of Mineralocorticoid Receptor in Humans Compared to Chimpanzees, Gorillas, and Orangutans.

We identified five distinct full-length human mineralocorticoid receptor (MR) genes containing either 984 amino acids (MR-984) or 988 amino acids (MR-988), which can be distinguished by the presence or absence of Lys, Cys, Ser, and Trp (KCSW) in their DNA-binding domain (DBD) and mutations at codons 180 and 241 in their amino-terminal domain (NTD). Two human MR-KCSW genes contain either (Val-180, Val-241) or (Ile-180, Val-241) in their NTD, and three human MR-984 genes contain either (Ile-180, Ala-241), (Val-180, Val-241), or (Ile-180, Val-241). Human MR-KCSW with (Ile-180, Ala-241) has not been cloned. In contrast, chimpanzees contain four MRs: two MR-988s with KCSW in their DBD, or two MR-984s without KCSW in their DBD. Chimpanzee MRs only contain (Ile180, Val-241) in their NTD. A chimpanzee MR with either (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD has not been cloned. Gorillas and orangutans each contain one MR-988 with KCSW in the DBD and one MR-984 without KCSW, and these MRs only contain (Ile-180, Val-241) in their NTD. A gorilla MR or orangutan MR with either (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD has not been cloned. Together, these data suggest that human MRs with (Val-180, Val-241) or (Ile-180, Ala-241) in the NTD evolved after humans and chimpanzees diverged from their common ancestor. Considering the multiple functions in human development of the MR in kidney, brain, heart, skin, and lungs, as well as MR activity in interaction with the glucocorticoid receptor, we suggest that the evolution of human MRs that are absent in chimpanzees may have been important in the evolution of humans from chimpanzees. Investigation of the physiological responses to corticosteroids mediated by the MR in humans, chimpanzees, gorillas, and orangutans may provide insights into the evolution of humans and their closest relatives.

Lysine-Cysteine-Serine-Tryptophan inserted into the DNA-binding domain of human mineralocorticoid receptor increases transcriptional activation by aldosterone

Due to alternative splicing in an ancestral DNA-binding domain (DBD) of the mineralocorticoid receptor (MR), humans contain two almost identical MR transcripts with either 984 amino acids (MR-984) or 988 amino acids (MR-988), in which their DBDs differ by only four amino acids, Lys,Cys,Ser,Trp (KCSW). Human MRs also contain mutations at two sites, codons 180 and 241, in the amino terminal domain (NTD). Together, there are five distinct full-length human MR genes in GenBank. Human MR-984, which was cloned in 1987, has been extensively studied. Human MR-988, cloned in 1995, contains KCSW in its DBD. Neither this human MR-988 nor the other human MR-988 genes have been studied for their response to aldosterone and other corticosteroids. Here, we report that transcriptional activation of human MR-988 by aldosterone is increased by about 50 % compared to activation of human MR-984 in HEK293 cells transfected with the TAT3 promoter, while the half-maximal response (EC50) is similar for aldosterone activation of MR-984 and MR-988. Transcriptional activation of human MR also depends on the amino acids at codons 180 and 241. Interestingly, in HEK293 cells transfected with the MMTV promoter, transcriptional activation by aldosterone of human MR-988 is similar to activation of human MR-984, indicating that the promoter has a role in the regulation of the response of human MR-988 to aldosterone. The physiological responses to aldosterone and other corticosteroids in humans with MR genes containing KCSW and with differences at codons 180 and 241 in the NTD warrant investigation.

Cell-type specific effects of mineralocorticoid receptor gene expression suggest intercellular communication regulating fibrosis in skeletal muscle disease.

Introduction: Duchenne muscular dystrophy (DMD) is a fatal striated muscle degenerative disease. DMD is caused by loss of dystrophin protein, which results in sarcolemmal instability and cycles of myofiber degeneration and regeneration. Pathology is exacerbated by overactivation of infiltrating immune cells and fibroblasts, which leads to chronic inflammation and fibrosis. Mineralocorticoid receptors (MR), a type of nuclear steroid hormone receptors, are potential therapeutic targets for DMD. MR antagonists show clinical efficacy on DMD cardiomyopathy and preclinical efficacy on skeletal muscle in DMD models. Methods: We have previously generated myofiber and myeloid MR knockout mouse models to dissect cell-specific functions of MR within dystrophic muscles. Here, we compared skeletal muscle gene expression from both knockouts to further define cell-type specific signaling downstream from MR. Results: Myeloid MR knockout increased proinflammatory and profibrotic signaling, including numerous myofibroblast signature genes. Tenascin C was the most highly upregulated fibrotic gene in myeloid MR-knockout skeletal muscle and is a component of fibrosis in dystrophic skeletal muscle. Surprisingly, lysyl oxidase (Lox), canonically a collagen crosslinker, was increased in both MR knockouts, but did not localize to fibrotic regions of skeletal muscle. Lox localized within myofibers, including only a region of quadriceps muscles. Lysyl oxidase like 1 (Loxl1), another Lox family member, was increased only in myeloid MR knockout muscle and localized specifically to fibrotic regions. Discussion: This study suggests that MR signaling in the dystrophic muscle microenvironment involves communication between contributing cell types and modulates inflammatory and fibrotic pathways in muscle disease.

First Evidence of Mineralocorticoid Receptor Gene and Protein Expression in Rat and Human Thyroid Tissues and Cell Cultures.

Aldosterone (Aldo) exerts its action through binding with the mineralocorticoid receptor (MR). Clinically, a link between primary aldosteronism (PA) and thyroid diseases has been hypothesised. However, the presence and activity of MR on the thyroid have not yet been demonstrated. We investigated the gene/protein expression and activation of MR in primary thyroid cell cultures (normal rat thyroid [FRTL-5] and human papillary thyroid cancer [PTC] cell lines, BCPAP and K1) through qRT-PCR analysis, immunofluorescence, and confocal microscopy. We also studied the effects of Aldo on thyroid-specific and inflammation genes in vitro. Paired human normal and neoplastic thyroid tissues were also studied. We demonstrated both gene and protein expression and activation of MR in normal rat thyroid and human PTC lines. Incubation with Aldo induced an acute increase in IL-6 expression in both the FRTL-5 and BCPAP lines, which was antagonised by spironolactone, and an acute and late upregulation of thyroid-specific genes in FRTL-5. MR was also expressed at both gene and protein levels in normal human thyroid tissues and in PTC, with a progressive decline during neoplastic tumourigenesis, particularly in more aggressive histotypes. We present the first evidence of MR gene and protein expression in both normal and pathological thyroid cells and tissues. We have shown that MR is present and functionally activated in thyroid tissue. Binding of Aldo to MR induces the expression of inflammatory and thyroid-specific genes, and the thyroid may thus be considered a novel mineralocorticoid target tissue.

Mild weight loss and activation of RAAS in gene-edited mice with a E958A knock-in mutation in the AF2-LBD domain of the mineralocorticoid receptor gene

Mild weight loss and activation of RAAS in gene-edited mice with a E958A knock-in mutation in the AF2-LBD domain of the mineralocorticoid receptor gene

Impact of mineralocorticoid receptor gene NRC2 on the prediction of arrhythmia after acute myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Patients with acute myocardial infarction (AMI) suffer from distress that causes a rise in cortisol (stress hormone) which acts through glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Both receptors are present in cardiomyocyte nuclei (1-4). The animal model study in 2017 showed that activated MR increases cardiomyocyte oxidative stress leading to adverse electrophysiological remodelling that causes arrhythmias. 1.4% of tachyarrhythmias which is the main cause of cardiac death occur within the first month in AMI patients. MR are encoded by the human gene NR3C2 (nuclear receptor subfamily 3 group C member (5-6). Thus, we hypothesized that NR3C2 rs2070950, rs4635799 and rs5522 gene polymorphisms might have an extensive impact on rhythm disorders that occur in a cohort of AMI patients. Aim The aim is to evaluate the impact of the NR3C2 rs2070950, rs4635799 and rs5522 genes polymorphism in patients that developed rhythm and conduction disorders after suffering AMI. Methods The study included 301 patients treated at our hospital after suffering from AMI; all study subjects underwent primary percutaneous coronary intervention (PCI) and guideline-directed medical treatment (1). Patients' blood samples that were collected upon arrival had cortisol and troponin I levels checked and SNPs of the NR3C2 gene (rs2070950, rs4635799 and rs5522) assessed. To avoid inaccuracies all patients with a previous history of coronary syndrome or PCI were excluded. Rhythm conduction disorders and NR3C2 gene polymorphism were analyzed using the exact Fisher’s test. All the statistical analyses were performed with SPSS 27.0 software. The value of p < 0.05 was considered statistically significant. Results Patients with ventricular and atrial events in the early phase of AMI had higher levels of serum cortisol than the patients that did not have rhythm disorders (p=0.001) (Table 1). Rhythm disorders such as ventricular tachycardia (VT), ventricular flutter (VF) and atrial flutter or atrial fibrillation (AF/AFL) that occurred during hospitalization for AMI were notably associated with rs5522 gene polymorphism. During a hospital stay for AMI higher rs5522 TT genotype frequency was noticed in patients with AF/AFL than in patients without. C allele recurrence was 19.6%. Moreover, the protective effect was detected for AF/AFL there is also a protective effect in VA. (Table 2). Despite this, the gene polymorphism and high-grade atrioventricular block (HAVB) were unrelated. Genes rs2070950 and rs4635799 were not found significant to rhythm and conduction disorders. Conclusions Patients with the rs5522 TT allele are more prone to experience a higher frequency of arrhythmias. However, if at least one rs5522 C is found it associates with a smaller risk of arrhythmias post-AMI, meaning that this gene possibly limits damaging effects on cardiomyocytes that are caused by cortisol.

Is chronic stress a causal mechanism for small mammal population cycles? Reconciling the evidence.

Chronic stress has long been hypothesized to play a role in driving population cycles. Christian (1950) hypothesized that high population density results in chronic stress and mass "die-offs" in small mammal populations. Updated variations of this hypothesis propose that chronic stress at high population density may reduce fitness, reproduction, or program aspects of phenotype, driving population declines. We tested the effect of density on the stress axis in meadow voles (Microtus pennsylvanicus) by manipulating population density in field enclosures over three years. Using fecal corticosterone metabolites as a non-invasive measure of glucocorticoid (GC) concentrations, we found that density alone was not associated with GC differences. However, we found that the seasonal relationship of GC levels differed by density treatment, with high-density populations having elevated GC levels early in the breeding season and decreasing towards late summer. We additionally tested hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression in juvenile voles born at different densities, with the hypothesis that high density may reduce receptor expression, altering negative feedback of the stress axis. We found that females had marginally higher glucocorticoid receptor expression at high density, no effect in males, and no detectable effect of density on mineralocorticoid receptor expression in either sex. Hence, we found no evidence that high density directly impairs negative feedback in the hippocampus, butrather female offspring may be better equipped for negative feedback. We compare our findings with prior studies to attempt to disentangle the complicated relationship between density, seasonality, sex, reproduction and the stress axis.

The mineralocorticoid receptor gene (NR3C2) is linked to and associated with polycystic ovarian syndrome in Italian families.

Polycystic ovarian syndrome (PCOS) is a complex heterogeneous disorder characterized by hyperandrogenism, irregular menses, and subfertility and often accompanied by other related comorbid disorders such as insulin resistance, obesity, and type 2 diabetes. Several genetic risk factors predispose to PCOS, but most are still unknown. Up to 30% of women with PCOS may have hyperaldosteronism. Blood pressure and the ratio of blood levels of aldosterone to renin are higher in women with PCOS compared to healthy controls, even if still in the normal range; and the aldosterone antagonist spironolactone has been used as therapy for PCOS, mainly due to its antiandrogenic activity. Thus, we aimed to investigate the potential pathogenetic role of the mineralocorticoid receptor gene (NR3C2) as the encoded NR3C2 product binds aldosterone and plays a role in folliculogenesis, fat metabolism, and insulin resistance. Within 212 Italian families with T2D and phenotyped for PCOS, we analyzed 91 single nucleotide polymorphisms in the NR3C2 gene. We tested the NR3C2 variants for linkage and linkage disequilibrium to the PCOS phenotype by using parametric analysis. We found 18 novel risk variants significantly linked to and/or associated with the risk of PCOS. We are the first to report NR3C2 as a risk gene in PCOS. However, our findings need to be replicated in other ethnic groups in order to reach more solid conclusions.

Novel linkage and association of the mineralocorticoid receptor gene (NR3C2) with familial type 2 diabetes and depression and their comorbidity

IntroductionThe mineralocorticoid receptor gene (NR3C2) appears to modulate stress and cognitive performance in patients with major depressive disorder (MDD). In addition, abnormalities in NR3C2 are associated in rodents with type 2 diabetes (T2D) and in humans with features of metabolic syndrome. Of note, NR3C2 antagonists are approved treatments in heart failure and chronic kidney disease with T2D. The NR3C2 gene is therefore a candidate gene for studying T2D-MDD comorbidity. To our knowledge, no study has so far reported risk variants in the NR3C2 gene with either MDD and/or T2D. Materials and methodsIn 212 multigenerational Italian families with enriched family history of T2D and with MDD, we analyzed 86 single nucleotide polymorphisms (SNPs) within the NR3C2 gene for parametric linkage to and/or linkage disequilibrium (LD) with T2D and MDD. ResultsWe identified a total of 7 independent SNPs significantly linked to/in LD with MDD only, 20 SNPs significantly linked to/in LD with T2D only, and 9 SNP significantly linked to/in LD with both T2D and MDD. The SNPs were statistically significant across different models. Two sets of LD blocks were MDD-specific, and one set was T2D-specific. In silico analysis of the risk variants predicted 3 variants with potential functional effects. ConclusionsThis is the first study to report NR3C2 as a novel risk gene in T2D and MDD comorbidity. However, our results need to be replicated in other ethnicities.

Impact of Mineralocorticoid Receptor Gene NR3C2 on the Prediction of Functional Classification of Left Ventricular Remodeling and Arrhythmia after Acute Myocardial Infarction.

Background: The NR3C2 gene encodes the mineralocorticoid receptor, which is present on cardiomyocytes. Prior studies reported an association between the presence of NR3C2 single-nucleotide polymorphisms (SNPs) and an increased cortisol production during a stress response such as acute myocardial infarction (AMI), which may lead to adverse cardiac remodeling. Objective: To study the impact of the NR3C2 rs2070950, rs4635799 and rs5522 gene polymorphisms on left ventricular (LV) remodeling, rhythm and conduction disorders in AMI patients. Methods: A cohort of 301 AMI patients who underwent revascularization was included. SNPs of the NR3C2 gene (rs2070950, rs4635799 and rs5522) were evaluated. A total of 127 AMI patients underwent transthoracic echocardiography follow-up after 72 h and 6 months. Results: The rs2070950 GG genotype and rs4635799 TT genotype were most common in patients who had LV end-diastolic volume increase < 20% and the same or increased LV ejection fraction, indicating a possible protective effect of these SNPs. The rs5522 TT genotype was associated with a higher frequency of arrhythmias, while the presence of at least one rs5522 C allele was associated with a lower risk of arrhythmias. Conclusion: SNPs of the NR3C2 gene appear to correlate with better ventricular remodeling and a reduced rate of arrhythmias post-AMI, possibly by limiting the deleterious effects of cortisol on cardiomyocytes.

Mechanism by Which Inflammation and Oxidative Stress Induce Mineralocorticoid Receptor Gene Expression in Aging Vascular Smooth Muscle Cells.

Vascular MR (mineralocorticoid receptor) expression increases with age driving aging-associated vascular stiffness and hypertension. MR has two isoforms (1α and 1β) with distinct 5'-untranslated and promoter sequences (P1 and P2), but the gene regulatory mechanisms remain unknown. We investigated mechanisms driving MR gene transcriptional regulation in aging human smooth muscle cells (SMC). MR was quantified in aortic tissue and primary human aortic SMC (HASMC) comparing adult and aged donors and adult HASMC treated with H2O2, to induce aging. Predicted transcription factor (TF) binding sites in the MR gene were validated using chromatin immunoprecipitations and reporter assays. The impact of TF inhibitors on MR isoforms and fibrosis target gene expression was examined. Expression of both MR mRNA isoforms increased with donor age or H2O2 treatment in HASMCs. HIF1α (hypoxia-inducible factor) and the inflammatory TF NFκB (nuclear factor kappa B) both increased with age in HASMCs and are predicted to bind MR promoters. H2O2 induced HIF1α and NFκB expression and DNA binding of HIF1α to the MR P1 promoter and of NFκB to both MR promoters in HASMCs. HIF1α inhibition decreased MR-1α isoform expression while NFκB inhibition decreased both MR isoforms. HIF1α, NFκB, and MR inhibition decreased the expression of a SMC-MR target gene implicated in vascular fibrosis. In human aortic tissues, expression of HIF1α and NFκB each positively correlated with donor age and MR expression (P<0.0001). These data implicate the inflammatory TF, NFκB, and oxidative stress-induced TF, HIF1α, in regulating SMC MR transcription in aging HASMCs, which drives aging-related vascular stiffness and cardiovascular disease.

PMON316 Novel Mutation Causing Pseudohypoaldosteronism Type 1 and Transient Hypercalcemia: A Patient Report

Abstract Background Autosomal dominant pseudohypoaldosteronism Type 1 (PHA-1) is a salt-wasting syndrome due to mutation in the renal mineralocorticoid receptor. Here, we report an infant with a novel mutation in NR3C2 causing PHA and associated with transient hypercalcemia. Clinical Case An ex-28 weeks (birth weight 1.35 kg) male infant admitted to the NICU had increased urine output of 6 mL/kg/hr on days of life (DOL) 9-10. Initial laboratory tests showed hyponatremia (121-127 mEq/L), hyperkalemia (5.4-8.4 mEq/L), hypochloremia (89-94 mEq/L), and hypercalcemia (11.2-11.6 mg/dL). Weight had decreased 4% from birth. On exam he was normotensive with no midline defect or hyperpigmentation; he had palpable testes bilaterally and 2 cm penile length. Sodium chloride (NaCl) supplementation 2.5 mEq/day was started on DOL 10. State newborn screen 17-OHP levels were normal. His family history was unremarkable. To further investigate the hyponatremia and hyperkalemia, serum ACTH, cortisol, aldosterone, and renin were obtained. ACTH and cortisol were 33 pg/mL and 11.4 mcg/dL, respectively. While awaiting the results of the aldosterone and renin assays, fludrocortisone was started with minimal improvement in serum sodium and hypercalcemia worsening to 12.0 mg/dL. Laboratory tests obtained to investigate the hypercalcemia included phosphorus 5.5 mg/dL, hypercalciuria (urine calcium to creatinine ratio 0.4), 25-OH vitamin D 21.0 ng/mL (normal 30-100 ng/mL), and PTH 29 pg/mL (normal 10-65 pg/mL). PTH was inappropriately normal, concerning for primary hyperparathyroidism. While awaiting the aldosterone level, it was therefore recommended to decrease fludrocortisone dose. Plasma renin activity resulted at 319 ng/mL/hr (normal 2-37 ng/mL/hr) and aldosterone at 612 ng/dL (normal 5-90 ng/dL), suggesting a diagnosis of PHA. Fludrocortisone was discontinued and NaCl supplementation was increased. A heterozygous pathogenic variant, c.2457C&amp;gt;A (p.Tyr819Ter), was identified in the mineralocorticoid receptor gene, NR3C2, confirming the diagnosis of autosomal dominant PHA-1. This variant has not been previously reported but meets the American College of Medical Genetics and Genomics and the Association for Molecular Pathology's criteria for pathogenicity. At his outpatient follow up at 4 months, sodium was 134 mEq/L and calcium was 10.5 mg/dL. He was continued on 1.5 grams NaCl supplementation. Conclusion We report a patient with a novel mutation in the NR3C2 gene resulting in autosomal dominant PHA-1 and associated with transient hypercalcemia. Further exacerbation of hypercalcemia was seen with addition of fludrocortisone. We suspect the etiology of the transient hypercalcemia to be secondary to excess mineralocorticoids (exogenous or endogenous) that drives PTH to increase serum calcium levels, as previously described by Vaidya et al. (1) Reference (1) Vaidya A, Brown JM, Williams JS. The renin-angiotensin-aldosterone system and calcium-regulatory hormones. J Hum Hypertens. 2015 Sep;29(9): 515-21. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

OR26-2 mTOR Interacts with the Mineralocorticoid Receptor to Regulate its Activation by Ligand

Abstract Background Phosphorylation of the mineralocorticoid receptor (MR) at serine 843 inhibits its activity. mTOR phosphorylation of ULK1 prevents this MR phosphorylation. Purpose To study the relationship between mTOR and MR and its role in the modulation of aldosterone signaling. Methods Mouse cortical collecting duct M1 cells stably expressing the rat wild type MR or mutated MR (mu/S843A) and a Gaussia luciferase gene reporter under a hormone response promoter tyrosine aminotransferase (TAT) were incubated with mTOR activator and inhibitors in the presence and absence of aldosterone or corticosterone. Co-immunoprecipitation was performed to study protein interactions with the MR, and a phospho-serine antibody was used to demonstrate MR and ULK1 phosphorylation. Effects of mTOR knockdown in M1 cells by lentiviral transduction with CRISPR/gRNA for Raptor and Rictor were also studied. Results mTOR and the adaptor proteins of mTOR complex 1 and 2, Raptor and Rictor, respectively, co-precipitated with the MR of M1-rMR cells. Expression levels of these proteins were not changed by aldosterone. mTOR inhibitors significantly reduced ligand-induced MR reporter gene transactivation, however the mTOR activator MHY1485 had no effect. CRISPR/gRNA gene knockdown of Raptor or Rictor also inhibited MR activation stimulated by ligands, supporting a role of mTOR in maintaining the active MR. The mTOR inhibitor AZD8055 attenuated ligand-mediated MR gene transcription assessed by the reporter gene product and measurement of products of the endogenous MR target genes ACSBG1, LCN2 and Psca. AZD8055 also decreased phospho-ULK1 levels in a dose-dependent manner. We thus assumed that mTOR decreases ULK1 activity and prevents ULK1 phosphorylation of the MR at Serine (S843). Indeed, phosphorylation of the MR was increased in M1-rMR cells treated with AZD8055. However, AZD8055 still inhibited ligand-induced MR activity in cells carrying the mutated MR in which the Serine at 843 was replaced with an Alanine. These results suggest that mTOR has an additional role in MR activity unrelated to the specific phosphorylation at S843 by ULK1. This merits further investigation. Presentation: Monday, June 13, 2022 11:15 a.m. - 11:30 a.m.

Syndromes of Pseudo-Hyperaldosteronism.

Syndromes of Pseudo-Hyperaldosteronism.

Mineralocorticoid receptor antagonist treatment of established pulmonary arterial hypertension improves interventricular dependence in the SU5416-hypoxia rat model.

Treatment with mineralocorticoid receptor (MR) antagonists beginning at the outset of disease, or early thereafter, prevents pulmonary vascular remodeling in preclinical models of pulmonary arterial hypertension (PAH). However, the efficacy of MR blockade in established disease, a more clinically relevant condition, remains unknown. Therefore, we investigated the effectiveness of two MR antagonists, eplerenone (EPL) and spironolactone (SPL), after the development of severe right ventricular (RV) dysfunction in the rat SU5416-hypoxia (SuHx) PAH model. Cardiac magnetic resonance imaging (MRI) in SuHx rats at the end of week 5, before study treatment, confirmed features of established disease including reduced RV ejection fraction and RV hypertrophy, pronounced septal flattening with impaired left ventricular filling and reduced cardiac index. Five weeks of treatment with either EPL or SPL improved left ventricular filling and prevented the further decline in cardiac index compared with placebo. Interventricular septal displacement was reduced by EPL whereas SPL effects were similar, but not significant. Although MR antagonists did not significantly reduce pulmonary artery pressure or vessel remodeling in SuHx rats with established disease, animals with higher drug levels had lower pulmonary pressures. Consistent with effects on cardiac function, EPL treatment tended to suppress MR and proinflammatory gene induction in the RV. In conclusion, MR antagonist treatment led to modest, but consistent beneficial effects on interventricular dependence after the onset of significant RV dysfunction in the SuHx PAH model. These results suggest that measures of RV structure and/or function may be useful endpoints in clinical trials of MR antagonists in patients with PAH.

METABOLIC EFFECT OF A MINERALOCORTICOID RECEPTOR ANTAGONIST IN A NEW MODEL OF DIABETIC NEPHROPATHY

Objective: Diabetic nephropathy (DN) is the most prevalent form of chronic kidney disease (CKD) and is associated with cardiovascular diseases. Several studies reported beneficial effects of mineralocorticoid receptor (MR) antagonists in DN, indicating the importance of aldosterone/MR axis in this pathology We investigated the metabolic alterations associated to a new model of DN and explored the effect of the MR antagonist canrenoate and conditional MR knock-down Design and method: One month after CKD induction by 5/6 nephrectomy, mice were split in 4 groups: SHAM, CKD, CKD with 60% fat diet (CKD-HFD) and CKD-HFD treated with 30 mg/kg/day canrenoate (CKD-HFD-CAN). Another series was split in 2 groups CKD-HFD Ctrl and CKD-HFD-AdipoMRKO (in which the MR gene is deleted in adipocytes only). Glycated hemoglobin (HbA1c) was measured and glucose tolerance test (ipGTT) and insulin tolerance test (ipITT) were performed between 5 and 7 weeks after the starting of the diet/treatment. The whole body fat content was determined by MRI (Brucker). The comparisons were performed by one-way ANOVA and two-way ANOVA for the insulin and glucose tolerance tests. Results were considered significant with p &lt; 0.05. Results: The body weight, the fat content, the epididymal visceral adipose tissue (EVAT) size and HbAc1 were significantly higher in the CKD-HFD group vs SHAM or CKD, an effect prevented by the treatment with canrenoate. The glucose tolerance and the response to insulin were impaired in CKD-HFD vs SHAM or CKD, an effect ameliorated by canrenoate (Table). The impact on body weight, fat content and EVAT size was similar in CKD-HFD-adipoMRKO mice when compared to CKD-HFD Ctrl mice, however the improvement of the glucose tolerance was milder (Table). Conclusions: MR antagonism improves metabolic alterations associated to DN, partially due to its effect on adipose tissue, which could be translated in a better prognosis of the disease

Diversification of mineralocorticoid receptor genes in a subterranean rodent, the naked mole-rat.

Naked mole-rats (Heterocephalus glaber) inhabit subterranean burrows in savannas and are, thus, unable to access free water. To identify their mechanism of osmoregulation in xeric environments, we molecularly cloned and analyzed the nuclear receptor subfamily 3 group C member 2 (NR3C2) gene encoding the mineralocorticoid receptor (MR), required for hormone-dependent regulation of genes contributing to body fluid homeostasis. Most vertebrates harbor a single MR homolog. In contrast, we discovered that MR is duplicated in naked mole-rats. The amino acid sequence of naked mole-rat MR1 is 90% identical to its mouse ortholog, and MR1 is abundantly expressed in the kidney and the nervous system. MR2 encodes a truncated protein lacking DNA- and ligand-binding domains of MR1 and is expressed in diverse tissues. Although MR2 did not directly transactivate gene expression, it increased corticosteroid-dependent transcriptional activity of MR1. Our results suggest that MR2 might function as a novel regulator of MR1 activity to fine-tune MR signaling in naked mole-rats.