MR Activation Research Articles

Glycyrrhizic acid induced acquired apparent mineralocorticoid excess syndrome with a hyperadrenergic state: a case report

BackgroundSyndrome of apparent mineralocorticoid excess (AME) is characterized by excessive MR stimulation despite low levels of aldosterone. 11Beta-hydroxysteroid dehydrogenase-2 (11βDSH-2) inactivates cortisol to cortisone, preventing cortisol-induced MR activation. Genetic defects in 11βDSH-2 cause AME through accumulation of cortisol in the distal nephron, leading to MR activation induced hypertension, hypokalemia and metabolic alkalosis. Acquired AME can occur due to the ingestion of glycyrrhizic acid, found in licorice root, which inhibits 11βDSH-2 and has additional effects on cortisol homeostasis through inhibition of 11βDSH-1.Case reportWe present a case of acquired AME with a hyperadrenergic symptoms induced by ingestion of Advanced Liver Support, a nutritional supplement produced by Advanced BioNutritionals(R), in a 65-year-old Caucasian female who presented with accelerated hypertension, hypokalemia, metabolic alkalosis and adrenergic symptoms. Cessation of the licorice-containing supplement resulted in complete resolution of the patient's hypertension, symptoms and abnormal lab values. To our knowledge this is the first reported case of AME from this supplement, and the first to describe accompanying hyperadrenergic symptoms.ConclusionsGlycyrrhizic acid is increasingly being found in unregulated nutritional supplements and has the potential to induce a reversable syndrome of AME. Acquired AME should be suspected in individuals who present with hypertension along with hypokalemia, metabolic alkalosis and low plasma renin and serum aldosterone levels.

An in Vitro triple screen model for human mineralocorticoid receptor activity

The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron and other tissues. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. We developed an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussia luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45 nM and 0.30 nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6 nM and 56.0 nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20 nM (CV1-MRLuc) to ∼2000 nM, while the EC50 for aldosterone remained unchanged. The addition of 18β-glycyrrhetinic acid (18β-GA), a HSD11B2 inhibitor, restored the potency of cortisol back to ∼70 nM in CV1-hMRLuc-HSD11B2 cells. Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts.

Performance evaluation of waterborne epoxy emulsified asphalt micro-surfacing with microwave-activated waste rubber powder

As a solid renewable resource, waste rubber powder (R) applied in road engineering can help reduce the environmental pressure caused by "black pollution" and improve pavement performance. In this research, waste rubber powder was activated by microwave method, namely as MR. The content of 0%∼ 5% MR was added as a functional filler to the waterborne epoxy emulsified asphalt (WEA) micro-surfacing mixture, thereby obtaining the MRWEA micro-surfacing. Furthermore, pavement performance, durability and noise reduction property were evaluated and compared with those of WEA micro-surfacing adding non-activated R (RWEA micro-surfacing). Results show that the surface activity of MR has been enhanced effectively, and the surface hydrophobicity of MR is better than R through the contact angle test. These findings are also confirmed by Fourier transform infrared spectrometer (FTIR), scanning electron microscopy (SEM) and energy dispersive spectrometer (EDS) analysis. Then, MRWEA micro-surfacing has good workability and cohesion due to the surface activity of MR. At the same dosage MR and R, 30 min, 60 min cohesion of MRWEA micro-surfacing mixture is greater than that of RWEA micro-surfacing mixture. Furthermore, MRWEA micro-surfacing with 3% MR has better wear resistance, rutting resistance, water resistance, durability and noise reduction properties than those of RWEA micro-surfacing with 3% R.

FRI161 An in Vitro Triple Screen Model for Human Mineralocorticoid Receptor Activity

Abstract Disclosure: H. Liu: None. S.M. Konzen: None. A. Coy: None. J. Rege: None. C.E. Gomez-Sanchez: None. W.E. Rainey: None. A.F. Turcu: None. Background: The mineralocorticoid receptor (MR, NR3C2) mediates ion and water homeostasis in epithelial cells of the distal nephron. MR is expressed in a variety of other tissues, including gastrointestinal tract, exocrine glands, central nervous system, heart, and adipose tissue. Aldosterone, the prototypical mineralocorticoid, regulates electrolyte and fluid balance. Cortisol binds to MR with equal affinity to aldosterone. In humans, cortisol concentrations far exceed those of aldosterone, but many MR-expressing tissues inactivate cortisol to cortisone via 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2). Dysregulated MR activation contributes to direct cardiovascular tissue insults. Besides aldosterone and cortisol, a variety of MR agonists and/or HSD11B2 inhibitors are putative players in the pathophysiology of low-renin hypertension (LRH), and cardiovascular and metabolic pathology. Objective: To develop an in vitro human MR (hMR) model, to facilitate screening for MR agonists, antagonists, and HSD11B2 inhibitors. Methods: The CV1 monkey kidney cells were transduced with lentivirus to stably express hMR and an MR-responsive gaussian luciferase gene. Clonal populations of MR-expressing cells (CV1-MRluc) were further transduced to express HSD11B2 (CV1-MRluc-HSD11B2). CV1-MRluc and CV1-MRluc-HSD11B2 cells were treated with aldosterone, cortisol, 11-deoxycorticosterone (DOC), 18-hydroxycorticosterone (18OHB), 18-hydroxycortisol (18OHF), 18-oxocortisol (18oxoF), progesterone, or 17-hydroxyprogesterone (17OHP). Cells were also treated with serum from patients with LRH (N=3) or primary hypertension (PH, N=3). Luminescence was measured in conditioned medium using coelenterazine. Results: In CV1-MRLuc cells, aldosterone and DOC displayed similar potency (EC50: 0.45nM and 0.30nM) and maximal response (31- and 23-fold increase from baseline) on hMR; 18oxoF and 18OHB displayed lower potency (19.6nM and 56.0nM, respectively) but similar maximal hMR activation (25- and 27-fold increase, respectively); cortisol and corticosterone exhibited higher maximal responses (73- and 52-fold, respectively); 18OHF showed no MR activation. Progesterone and 17OHP inhibited aldosterone-mediated MR activation. In the MRluc-HSD11B2 model, the EC50 of cortisol for MR activation increased from 20nM (CV1-MRLuc) to 850nM, while the EC50 for aldosterone remained unchanged. Serum from patients with both LRH and PH led to variable degrees of luminescence in the CV1-MRLuc cells, but not in the CV1-MRluc-HSD11B2 cells, suggesting a cortisol-mediated MR activation in these patients, possibly via glycyrrhetinic acid-like factors. Conclusions: Together, these two cell models will facilitate the discovery of novel MR-modulators, informing MR-mediated pathophysiology mechanisms and drug development efforts. Presentation Date: Friday, June 16, 2023

THU599 Serum And Urinary Free Cortisone As Novel Predictor Of Low-renin Arterial Hypertension

Abstract Disclosure: A. Tapia-Castillo: None. J. Perez: None. A. Sandoval: None. F. Allende: None. S. Solari: None. C.E. Fardella: None. C. Carvajal: None. A large proportion of low-renin hypertensive (LRH) patients correspond to primary aldosteronism (PA) caused by excessive MR activation mediated by excess of aldosterone. However, some of these LRH patients have low to normal serum aldosterone levels, where is thought the MR activation is aldosterone-independent and more likely due to glucocorticoid-mediated MR activation, as occurs in the nonclassical apparent mineralocorticoid excess (NCAME), which is characterized by high cortisol/cortisone ratio and low cortisone, affecting not only blood pressure (BP) control and urinary potassium excretion but also causing cardiac and renal damage, endothelial dysfunction and subclinical inflammation. Objective: To evaluate cortisone as a predictor of LRH and its association with parameters of kidney and vascular damage. Design: A cross-sectional study was carried out in 206 adult subjects. The subjects were classified according to low-renin phenotype (plasma renin activity (PRA) <1ng/ml*h) and low serum cortisone (<p25th). We measured clinical, biochemical, renal and vascular parameters. Statistical analyses as group comparisons were performed by Mann Whitney, and discriminatory analyses by ROC curves. Results: We identified 43% of subjects with low-renin phenotype. PRA was associated with serum aldosterone (r=0.36; p<0.001) and serum cortisone (r=0.22; p=0.001). A binary logistic regression analysis shows that cortisone predicts low-renin phenotype (OR=0.4 [95% CI, 0.21 – 0.78]). The ROC curves show an AUC for serum cortisone was 0.62 (95% CI: 0.54 to 0.69; p = 0.004) and urinary cortisone 0.6 (95% CI: 0.52 to 0.67; p = 0.02) to discriminate subjects with low-renin from controls. Subjects with low serum cortisone showed a higher albuminuria, PAI-1 and lower sodium renal excretion. Urinary free cortisone was also associated to blood pressure and serum potassium (p<0.05). Conclusion: These findings highlight the concept that the low-renin phenotype is dictated not only by serum aldosterone levels but also by low cortisone, suggesting it should be considered in endocrine arterial hypertension screening, complementary in the diagnosis of primary aldosteronism since it could generate false positives in the calculation of the aldosterone to renin ratio. Moreover, our study suggests that low cortisone levels could be associated with future occurrence of vascular and kidney damage. Presentation: Thursday, June 15, 2023

C-atrial natriuretic peptide (ANP)4-23 attenuates renal fibrosis in deoxycorticosterone-acetate-salt hypertensive mice

Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. In this study, mice were randomly divided into SHAM group, DOCA-salt group and DOCA-salt + C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which were attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from cytosol to nucleus without change of MR expression. Furthermore, C-ANP4-23 rescued the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

C-ATRIAL NATRIURETIC PEPTIDE (ANP)4-23 ATTENUATES RENAL FIBROSIS IN DEOXYCORTICOSTERONE-ACETATE-SALT HYPERTENSIVE MICE

Objective: Epithelial-mesenchymal transition (EMT) plays a critical role in hypertension-induced renal fibrosis, a final pathway that leads to end-stage renal failure. C-Atrial natriuretic peptide (ANP)4-23, a specific agonist of natriuretic peptide receptor-C (NPR-C), has been reported to have protective effects against hypertension. However, the role of C-ANP4-23 in hypertension-associated renal fibrosis has not yet been elucidated. Design and method: In this study, mice were randomly divided into SHAM group, DOCA-salt group, DOCA-salt + C-ANP4-23 group and C-ANP4-23 group. Renal morphology changes, renal function and fibrosis were detected. Human proximal tubular epithelial cells (HK2) stimulated by aldosterone were used for cell function and mechanism study. Results: The DOCA-salt treated mice exhibited hypertension, kidney fibrosis and renal dysfunction, which was attenuated by C-ANP4-23. Moreover, C-ANP4-23 inhibited DOCA-salt treatment-induced renal EMT as evidenced by decrease of the mesenchymal marker alpha-smooth muscle actin (ACTA2) and vimentin and increase of epithelial cell marker E-cadherin. In HK2 cells, aldosterone induced EMT response, which was also suppressed by C-ANP4-23. The key transcription factors (twist, snail, slug and ZEB1) involved in EMT were increased in the kidney of DOCA-salt-treated mice, which were also suppressed by C-ANP4-23. Mechanistically, C-ANP4-23 inhibited the aldosterone-induced translocation of MR from the cytosol to the nucleus without change of MR expression. Furthermore, C-ANP4-23 prevented the enhanced expression of NADPH oxidase (NOX) 4 and oxidative stress after aldosterone stimulation. Aldosterone-induced Akt and Erk1/2 activation was also suppressed by C-ANP4-23. Conclusions: Our data suggest that C-ANP4-23 attenuates renal fibrosis, likely through inhibition of MR activation, enhanced oxidative stress and Akt and Erk1/2 signaling pathway.

AW-APSH-2: “MINERALOCORTICOID RECEPTOR (MR)-ASSOCIATED HYPERTENSION” IN “GREEDY ORGAN HYPOTHESIS”

Spironolactone has been used for more than 60 years. Eplerenone is MR-specific MRB and esaxerenone possesses non-steroidal structure with high affinity and specificity to MR and exhibits long half-life. We conducted a double-blind phase III study of esaxerenone compared with eplerenone in patients with essential hypertension in 2020. Esaxerenone 2.5 mg elicited comparable hypotensive effect to eplerenone 50 mg and 5 mg exhibited significantly stronger effect than 2.5 mg. We also investigated the efficacy of dosage-escalation of esaxerenone added to a RAS inhibitor in hypertensive patients with type 2 diabetes (DM) (HbA1c:6.8%) and albuminuria (212 mg/gCr). 5 mg esaxerenone induced 20/8mmHg blood pressure reduction and reduced albumin excretion significantly. Interestingly, in these studies, esaxerenone exerted similar hypotensive action in the patients with RAS inhibitors, compared to the groups without them. This observation indicates that MR can be activated being independent of renin-angiotensin-aldosterone system. In 2012, we proposed the concept of “MR-associated hypertension”, hypertension caused by over-activation of MR with wide range of plasma aldosterone level. Primary aldosteronism is the typical example of MR-associated hypertension, but even in low plasma aldosterone concentration state, such as obesity, DM, CKD, MR can be over-activated. There are several postulated mechanisms of over-activation of MR. Increased expression of MR, increased sensitivity of MR and MR protein stabilization. We are interested in the activation of MR by salt and sugar themselves. We focus on MR expressed in the intestine for sodium absorption. Administration of DOCA and high salt induced up-regulation of epithelial sodium channel, ENaC, the target molecule of MR. And intestine specific knock-out of MR mice exhibited blunted increase of blood pressure by DOCA and high salt loading. We also demonstrated that high sugar induces MR protein stabilization by PKC activation, O-GlcNac modification or EGFR-ERK activation. More recently, in relation to excessive salt and sugar intake and occurrence of non-communicable diseases (NCDs), including hypertension, we have proposed “Greedy Organ Hypothesis”. Excessive intake of salt and sugar are sensed by the kidney and the intestines, and these organs up-regulate SGLT, sodium-glucose co-transporters and come to absorb salt and sugar greedily. Excessive salt intake through SGLT2 makes the kidney greedy to induce hypertension/CKD. Excessive food intake through SGLT1 make the intestines greedy to induce obesity/DM. Furthermore, there is “crossing” relationships between “greedy organs” and NCDs. Greedy kidney and intestines over-activate MR and induce MR-associated hypertension. Therefore, MRB are effective to alleviate Greedy Organs and to suppress NCDs.

PS-B08-4: CHRONIC INTERMITTENT HYPOXIA INDUCES RENAL FIBROSIS BY MR ACTIVATION

Purpose: Obstructive sleep apnea is mainly characterized by chronic intermittent hypoxia (CIH), which is associated with chronic kidney disease. Macrophage is known to play an important role in renal fibrosis. However, little is known of the potential role of macrophage-to-myofibroblast transition (MMT) in hypoxic conditions. The aim of this study was to understand how infiltrating macrophages may contribute to renal fibrosis in hypoxic conditions. Methods: In this study, 30 adult male sprague dawley (SD) rats were randomly divided into normoxic control(NC), chronic intermittent hypoxia(CIH), and eplerenone(EPL) groups-each containing ten rats. The CIH and EPL groups were exposed to hypoxia in a commercial hypoxic chamber for 5 weeks. Rats in the EPL group were administered EPL orally via diet for 5 weeks. All rats were then sacrificed and serum, 24-hour urine and tissue specimens were collected for renal function, histopathology, western blot, immunohistochemistry. The RAW264.7 macrophages were subjected to 24 hours of hypoxia and 12 hours of reoxygenation. Cells were collected for immunofluorescence staining. Results: In addition to blood pressure, renal aldosterone level was also increased in CIH rats. Massons trichrome and Sirius red staining showed significantly higher collagen deposition and fibrosis in CIH rats. A markedly increased co-expression of α-SMA and CD68 in vitro and in vivo after CIH exposure. MR nuclear translocation was seen in hypoxic macrophages. Western blot showed that the expression of SGK-1 and p-SGK-1 was upregulated. These effects were reversed by MR blocker (EPL). Conclusions: CIH-induced macrophage infiltration, an important source of myofibroblasts, might participate in the development of renal interstitial fibrosis. These effects were reversed by EPL. Therefore, we speculate MR activation may be related to CIH-induced renal fibrosis.

Transcriptional glucocorticoid effects in the brain: Finding the relevant target genes.

Glucocorticoids are powerful modulators of brain function. They act via mineralocorticoid and glucocorticoid receptors (MR and GR). These are best understood as transcription factors. Although many glucocorticoid effects depend on the modulation of gene transcription, it is a major challenge to link gene expression to function given the large-scale, apparently pleiotropic genomic responses. The extensive sets of MR and GR target genes are highly specific per cell type, and the brain contains many different (neuronal and non-neuronal) cell types. Next to the set "trait" of cellular context, the "state" of other active signaling pathways will affect MR and GR transcriptional activity. Here, we discuss receptor specificity and contextual factors that determine the transcriptional outcome of MR/GR signaling, experimental possibilities offered by single-cell transcriptomics approaches, and reflect on how to make sense of lists of target genes in relation to understanding the functional effects of steroid receptor activation.

Implication of MR Activity in Posttreatment Arterial Stiffness Reversal in Patients With Primary Aldosteronism.

Primary aldosteronism (PA) patients have a higher degree of arterial stiffness, which can be reversed after adrenalectomy. We aimed to compare the reversal of arterial stiffness between surgically and medically treated PA patients and to identify the predictors of effective medical treatment. We prospectively enrolled 445 PA patients and collected data on baseline clinical characteristics, biochemistry, blood pressure, and pulse wave velocity (PWV) before treatment and 12 months after treatment. In the mineralocorticoid receptor antagonist (MRA)-treated patients, the relationship between the change in PWV after 1 year (ΔPWV) and posttreatment renin activity was explored using the restricted cubic spline (RCS) method. Of the 445 enrolled PA patients, 255 received adrenalectomy (group 1) and 190 received MRAs. In the RCS model, posttreatment plasma renin activity (PRA) 1.5 ng/mL/h was the best cutoff value. Therefore, we divided the MRA-treated patients into 2 groups: those with suppressed PRA (< 1.5 ng/mL/h, group 2), and those with unsuppressed PRA (≥ 1.5 ng/mL/h, group 3). Only group 1 and group 3 patients had a statistically significant improvement in PWV after treatment (both P < .001), whereas no significant improvement was noted in group 2 after treatment (P = .151). In analysis of variance and post hoc analysis, group 2 had a significantly lower ΔPWV than group 1 (P = .007) and group 3 (P = .031). Multivariable regression analysis of the MRA-treated PA patients identified log-transformed posttreatment PRA, age, and baseline PWV as independent factors correlated with ΔPWV. The reversal of arterial stiffness was found in PA patients receiving adrenalectomy and in medically treated PA patients with unsuppressed PRA.

N-glycosylation of mannose receptor (CD206) regulates glycan binding by C-type lectin domains

The macrophage mannose receptor (MR, CD206) is a transmembrane endocytic lectin receptor, expressed in selected immune and endothelial cells, and is involved in immunity and maintaining homeostasis. Eight of the ten extracellular domains of the MR are C-type lectin domains (CTLDs) which mediate the binding of mannose, fucose, and GlcNAc in a calcium-dependent manner. Previous studies indicated that self-glycosylation of MR regulates its glycan binding. To further explore this structure-function relationship, we studied herein a recombinant version of mouse MR CTLD4-7 fused to human Fc-portion of IgG (MR-Fc). The construct was expressed in different glycosylation-mutant cell lines to study the influence of differential glycosylation on receptor glycan-binding properties. We conducted site-specific N- and O-glycosylation analysis and glycosylation site characterization using mass spectrometry by which several novel O-glycosylation sites were identified in mouse MR and confirmed in human full-length MR. This information guided experiments evaluating the receptor functionality by glycan microarray analysis in combination with glycan-modifying enzymes. Treatment of active MR-Fc with combinations of exoglycosidases, including neuraminidase and galactosidases, resulted in the loss of trans-binding (binding of MR CTLDs to non-MR glycans), due to unmasking of terminal, nonreducing GlcNAc in N-glycans of the MR CTLDs. Regalactosylation of N-glycans rescues mannose binding by MR-Fc. Our results indicate that glycans within the MR CTLDs act as a regulatory switch by masking and unmasking self-ligands, including terminal, nonreducing GlcNAc in N-glycans, which could control MR activity in a tissue- and cell-specific manner or which potentially affect bacterial pathogenesis in an immunomodulatory fashion.

Cortisol Receptor Sensitivity as a Risk Factor for Depression

In 2020, the World Health Organization reported over 264 million people across the world were suffering from depression. Studies have demonstrated that one source of depression is a hormonal imbalance involved in the stress response. Cortisol is a stress hormone regulated by the Hypothalamic‐Anterior‐Pituitary (HPA) Axis. Its effects on the stress response and other metabolic activities in the body are exerted through the glucocorticoid and mineralocorticoid receptors (GR and MR respectively).Our research has examined mutations known as single‐nucleotide‐polymorphisms (SNPs) relating to cortisol‐receptor sensitivity and the behavior of cortisol in the body to investigate the link between cortisol activity and depression. The relationship between SNPs relating to receptor sensitivity and depression in patients will be established by analysis of SNPs in coordination with data from psychological inventory assessments collected for each patient. Patient samples and clinical information was collected from adult patients diagnosed with depression by collaborating physicians. Quantitative polymerase chain reaction (qPCR) was used for genotyping. Analysis of the data yielded no significant correlation between SNPs relating to GR or MR and depression as scored by the Beck Depression Inventory (BDI). However one SNP in GR (rs33389) and one SNP on the 11β‐hydroxysteroid dehydrogenase type 1 gene (rs12086634) correlated with scores on the Mindful Attention Awareness Scale and State Trait Anxiety scale, respectively.Current studies focus on SNPs involved in the regulation of GR and MR activity that are not directly found in those receptors such as the FKBP5 GR co‐regulator protein. Investigating other non‐GR and MR SNPs could provide alternate explanations for cortisol sensitivity beyond the receptors themselves. In analyzing genetic differences relating to cortisol sensitivity in patients clinically diagnosed with depression, we aim to determine if an imbalance in an individual’s response to cortisol is linked to depression. If such a relationship can be established, this can provide physicians with a screening tool to better inform them of a patient’s risk factors for depression.

HIF1α and NFκB Regulate Mineralocorticoid Receptor Gene Expression in Aging Human Vascular Smooth Muscle Cells

BackgroundMineralocorticoid receptor (MR) activation drives aging‐associated cardiovascular diseases including hypertension, vascular stiffness and heart failure. Vascular MR expression increases with age and drives the aging phenotype but the mechanism of MR gene regulation is unknown. The MR has two mRNA isoforms (1α and 1β) that differ in only the 5’‐untranslated region (exon 1α or 1β) and in the promoter sequence (P1 or P2, respectively). Thus, we investigated the transcriptional mechanisms regulating MR gene expression in aging human smooth muscle cells (SMC).MethodsMR mRNA and protein expression were quantified in primary human aortic SMC (HASMC) and human aortic tissue comparing adult (average age 40s) and aged (average age 70s) male and female donors and comparing adult HASMC or Pac1 (rat SMC line) treated with H2O2 to induce aging. MR promoter activity was tested by luciferase reporter assays. In silico analysis of the MR promoters identified putative transcription factor (TF) binding sites and inhibitor studies and chromatin immunoprecipitations (ChIP) were performed to validate the predicted regulatory mechanisms.ResultsIncreased SMC donor age or H2O2 treatment were associated with significantly increased expression of both of the MR mRNA isoforms, MR protein level, and activity of both MR promoters in HASMCs. The hypoxia‐inducible TF, HIF1α, and the inflammatory TF, NFκB, were predicted in silico to bind to the MR promoters and found to increase significantly with age in HASMCs. HIF1α was induced by H2O2 in HASMCs and this was associated with significant enrichment of HIF1α binding specifically at a site in the 1a exon, near the MR P1 promoter. HIF1α inhibition attenuated expression specifically of the MR‐1α mRNA isoform resulting in decreased MR protein in HASMC. NFκB also increased with age in HASMCs and contributes to MR expression. In response to H2O2,NFκB binding is enriched at to two different binding sites, one each in the P1 and P2 promoters and NFκB inhibition decreased expression of both MR mRNA isoforms (1α and 1β) and MR protein in SMC. Finally, HIF1α and NFκB expression each positively correlated with donor age and with MR expression (p&lt;0.0001) in human aortic tissue from males and females.ConclusionThe MR gene is regulated by the inflammatory transcription factor NFkB and by the hypoxia/oxidative stress‐induced TF HIF1α, to increase MR expression in SMC of the aging human vasculature. This molecular insight may explain enhanced MR activity in cardiovascular aging and could potentially contribute to other cardiovascular conditions driven by oxidative stress and inflammation.

Endothelial sodium channel activation mediates DOCA-salt-induced endothelial cell and arterial stiffening

IntroductionHigh salt intake and aldosterone are both associated with vascular stiffening in humans. However, our preliminary work showed that high dietary salt alone did not increase endothelial cell (EC) or vascular stiffness or endothelial sodium channel (EnNaC) activation in mice, presumably because aldosterone production was significantly suppressed as a result of the high salt diet. We thus hypothesized that high salt consumption along with an exogenous mineralocorticoid would substantially increase EC and vascular stiffness via activation of the EnNaC. Methods and resultsMice were implanted with slow-release DOCA pellets and given salt in their drinking water for 21 days. Mice with either specific deletion of the alpha subunit of EnNaC or treated with a pharmacological inhibitor of mTOR, a downstream signaling molecule involved in mineralocorticoid receptor activation of EnNaC, were studied. DOCA-salt treated control mice had increased blood pressure, EC Na+ transport activity, EC and arterial stiffness, which were attenuated in both the αEnNaC−/− and mTOR inhibitor treated groups. Further, depletion of αEnNaC prevented DOCA-salt-induced impairment in EC-dependent vascular relaxation. ConclusionWhile high salt consumption alone does not cause EC or vascular stiffening, the combination of EC MR activation and high salt causes activation of EnNaC which increases EC and arterial stiffness and impairs vascular relaxation. Underlying mechanisms appear to include mTOR signaling.

Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9–80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

Aldosterone and dexamethasone activate African lungfish mineralocorticoid receptor: Increased activation after removal of the amino-terminal domain

Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50 s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50 s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR.

Abstract 09: Mtor Inhibition Decreases Ligand-induced Mineralocorticoid Receptor Activation

Introduction: ULK1 phosphorylates the MR at S843, decreasing its ligand binding and transcriptional activity. Angiotensin II-induced mTOR phosphorylation of ULK1 inactivates ULK1, preventing its phosphorylation of MR. Aim: Further elucidate the role of mTOR in the regulation of MR transcriptional activity. Methods: M1 mouse cortical collecting duct cells stably transduced with the rat MR cDNA and a MMTV- Gaussia luciferase reporter gene, were incubated with an mTOR activator and several inhibitors, +/- aldosterone or corticosterone. Similar studies were done after lentiviral transduction of CRISPR/gRNA for raptor and rictor genes or mutated MR (mu/S843A) cDNA. Results: mTOR inhibition significantly decreased ligand activation of the MR reporter gene, while the mTOR activator MHY1485 had no effect suggesting that mTOR is tonically active. MR activation induced by aldosterone and corticosterone was also decreased by CRISPR/gRNA gene knockdown of raptor and rictor, the adaptors of mTOR complex 1 and 2, respectively, supporting a role for mTOR. The mTOR inhibitor AZD8055 (AZD) reduced phospho-ULK1 and attenuated ligand-mediated MR transactivation in a dose-dependent manner. The ULK1 inhibitor MRT68921 increased MR transactivation. We speculated that mTOR decreased ULK1 activity by phosphorylating it, thereby preventing ULK1 phosphorylation of the MR at Serine (S843). However, when M1 cells were transduced with an MR cDNA in which S843 was replaced with Alanine that cannot be phosphorylated, ligand-induced activation of the mu/S843A MR was still decreased by AZD, but unchanged by MRT68921. This suggests that mTOR has an additional effect on MR activity unrelated to ULK1 activity. AZD also decreased P70S6K and AKT phosphorylation in these cells. Conclusions: mTOR phosphorylation of ULK1 prevents its phosphorylation of the MR and reduction of MR transcriptional activity. mTOR inhibitors and deletion of raptor and rictor decreased MR transcriptional activity. mTOR has additional positive effects on MR activity possibly related to its phosphorylation of AKT and P70S6K. Inhibition of mTOR action may be a useful target for mitigating excessive MR activation.

Long Noncoding RNA Tug1 Promotes Angiotensin II-Induced Renal Fibrosis by Binding to Mineralocorticoid Receptor and Negatively Regulating MicroR-29b-3p.

[Figure: see text].

A novel mineralocorticoid receptor antagonist, 7,3',4'-trihydroxyisoflavone improves skin barrier function impaired by endogenous or exogenous glucocorticoids

Excess glucocorticoids (GCs) with either endogenous or exogenous origins deteriorate skin barrier function. GCs bind to mineralocorticoid and GC receptors (MRs and GRs) in normal human epidermal keratinocytes (NHEKs). Inappropriate MR activation by GCs mediates various GC-induced cutaneous adverse events. We examined whether MR antagonists can ameliorate GC-mediated skin barrier dysfunction in NHEKs, reconstructed human epidermis (RHE), and subjects under psychological stress (PS). In a preliminary clinical investigation, topical MR antagonists improved skin barrier function in topical GC-treated subjects. In NHEKs, cortisol induced nuclear translocation of GR and MR, and GR and MR antagonists inhibited cortisol-induced reductions of keratinocyte differentiation. We identified 7,3’,4’-trihydroxyisoflavone (7,3’,4’-THIF) as a novel compound that inhibits MR transcriptional activity by screening 30 cosmetic compounds. 7,3’,4’-THIF ameliorated the cortisol effect which decreases keratinocyte differentiation in NHEKs and RHE. In a clinical study on PS subjects, 7,3',4'-THIF (0.1%)-containing cream improved skin barrier function, including skin surface pH, barrier recovery rate, and stratum corneum lipids. In conclusion, skin barrier dysfunction owing to excess GC is mediated by MR and GR; thus, it could be prevented by treatment with MR antagonists. Therefore, topical MR antagonists are a promising therapeutic option for skin barrier dysfunction after topical GC treatment or PS.