Aldosterone Response Research Articles

Salt Sensitivity of Blood Pressure and Aldosterone: Interaction Between the Lysine-specific Demethylase 1 Gene, Sex, and Age.

Salt sensitivity of blood pressure (SSBP) is associated with increased cardiovascular risk, especially in individuals of African descent, although the underlying mechanisms remain obscure. Lysine-specific demethylase 1 (LSD1) is a salt-sensitive epigenetic regulator associated with SSBP and aldosterone dysfunction. An LSD1 risk allele in humans is associated with SSBP and lower aldosterone levels in hypertensive individuals of African but not European descent. Heterozygous knockout LSD1 mice display SSBP and aldosterone dysregulation, but this effect is modified by age and biological sex. This might explain differences in cardiovascular risk with aging and biological sex in humans. This work aims to determine if LSD1 risk allele (rs587618) carriers of African descent display a sex-by-age interaction with SSBP and aldosterone regulation. We analyzed 297 individuals of African and European descent from the HyperPATH cohort. We performed multiple regression analyses for outcome variables related to SSBP and aldosterone. LSD1 risk allele carriers of African (but not European) descent had greater SSBP than nonrisk homozygotes. Female LSD1 risk allele carriers of African descent had greater SSBP, mainly relationship-driven by women with low estrogen (postmenopausal). There was a statistically significant LSD1 genotype-sex interaction in aldosterone response to angiotensin II stimulation in individuals aged 50 years or younger, with female carriers displaying decreased aldosterone responsiveness. SSBP associated with LSD1 risk allele status is driven by women with a depleted estrogen state. Mechanisms related to a resistance to develop SSBP in females are uncertain but may relate to an estrogen-modulating effect on mineralocorticoid receptor (MR) activation and/or LSD1 epigenetic regulation of the MR.

Impaired aldosterone response to ACTH without hypoaldosteronism: An unrecognized secretory pattern in search of clinical implications.

Aldosterone has been recently characterized as a 'stress hormone'. Stress per se elicits a sizable rise in aldosterone secretion, which could be replicated by the administration of a low dose (0.03-1 μg, IV) of adrenocorticotropic hormone (ACTH). Whether or not the aldosterone response to ACTH could be selectively impaired, that is, in association with intact cortisol response, is presently unknown. To determine whether or not the aldosterone response to low dose of ACTH is impaired in subjects referred to assess the hypothalamic-pituitary-adrenal axis (HPA). Retrospective analysis. Outpatient referral endocrine day care centre. One hundred and ninety-five consecutive subjects who underwent the low dose (1 μg) ACTH test, in whom decreased cortisol reserve was suspected due to former/present glucocorticoid excess, pituitary disease or/and unexplained weakness. The outcome was the detection of lack of aldosterone response, defined as a rise <111 pmol/l. In all, 46/195 subjects had subnormal aldosterone response as compared with 52/195 subjects showing diminished cortisol response. Nine subjects had combined deficient aldosterone and cortisol response. In the 37 subjects with isolated subnormal aldosterone response common associations were the use of exogenous glucocorticoids, mostly prednisone (n = 16); former Cushing disease (n = 2); nonfunctioning pituitary adenoma (n = 8); hypothyroidism (n = 11); the use of statins (n = 11), angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (n = 6), sex steroids in transgenders and orthostatic hypotension (n = 3). Twenty-seven percent (25/93) of the subjects with recent exposure to glucocorticoids had impaired aldosterone response to ACTH. Blunted aldosterone response to ACTH in the absence of hypoaldosteronism was seen in ~27% of subjects referred for HPA assessment using the low dose 1 μg ACTH test. Exposure to glucocorticoid excess was often linked to this impairment, independent of the cortisol response to ACTH.

The neuropeptide substance P regulates aldosterone secretion in human adrenals

Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes.

Abstract P237: Alterations In Renal Sodium Handling Proteins Determined By Urinary Extracellular Vesicle Analysis During Dietary Sodium Modulation And In Primary Aldosteronism

Sodium handling is altered during extremes in dietary sodium intake or in pathologic conditions such as primary aldosteronism. Urinary extracellular vesicles (uEVs) are excreted by all cell types along the nephron, are enriched for membrane proteins, and could provide insight into renal physiology in humans. We assessed uEV protein excretion of renal epithelial proteins relevant to sodium handling from 54 volunteers with pre-hypertension on high (HS, 200 mmol/d) and low (LS, 10 mmol/d) sodium diet for 5-7 days. To compare pathologic versus physiologic responses to aldosterone excess, samples were collected from 9 patients with primary aldosteronism (PA). uEVs were isolated by ultracentrifugation and trypsin digested proteins were quantified using multiple reaction monitoring mass spectrometry using isotopically labeled standards, normalized to the uEV-specific marker CD9. Wilcoxon signed rank and rank sum tests were used for within-subject and between-subject comparisons, respectively. LS diet altered uEV excretion of pendrin (4.0, expressed as fold-change vs HS), aquaporin-2 (-2.03), TRPV5 (-1.91), NCC (1.51), OSR1 (1.42; P &lt;0.01 for all), and SGLT2 (1.20, P =0.042). In primary aldosteronism, expression of ENaC-α, ENaC-β, and ENaC-γ, NCC, SGLT2, and OSR-1 were increased to a greater extent compared to during LS diet ( P &lt;0.05 for each), whereas AQP2 and TRPV5 were similar to HS diet ( Figure ). Expression of uEV proteins relevant to renal tubular sodium handling are dynamically altered by dietary sodium restriction and aldosterone excess. Further studies could identify patterns specific to pathologic versus physiologic aldosterone responses.

Loss of Adam10 Disrupts Ion Transport in Immortalized Kidney Collecting Duct Cells.

The kidney cortical collecting duct (CCD) comprises principal cells (PCs), intercalated cells (IC), and the recently discovered intermediate cell type. Kidney pathology in a mouse model of the syndrome of apparent aldosterone excess revealed plasticity of the CCD, with altered PC:intermediate cell:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs, but displays a range of cell types, including intermediate cells, making it ideal to study plasticity. We knocked out Adam10, a key component of the Notch pathway, in mCCDcl1 cells, using CRISPR-Cas9 technology, and isolated independent clones, which exhibited severely affected sodium transport capacity and loss of aldosterone response. Single-cell RNA sequencing revealed significantly reduced expression of major PC-specific markers, such as Scnn1g (γ-ENaC) and Hsd11b2 (11βHSD2), but no significant changes in transcription of components of the Notch pathway were observed. Immunostaining in the knockout clone confirmed the decrease in expression of γ-ENaC and importantly, showed an altered, diffuse distribution of PC and IC markers, suggesting altered trafficking in the Adam10 knockout clone as an explanation for the loss of polarization.

Somatic Mutations of GNA11 and GNAQ in CTNNB1-Mutant Aldosterone-Producing Adenomas Increases Aldosterone and Aldosterone Synthase (CYP11B2)

Abstract Most aldosterone-producing adenomas (APA) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell-clusters of normal adrenals suggests the existence of co-driver mutations which influence the development or phenotype of APAs. Gain-of-function mutations in both CTNNB1 and GNA11 were found by whole exome sequencing in 3 of 41 APAs from a UK/Irish cohort. Targeted sequencing for exon 3 mutations of CTNNB1 and p.Gln209 mutations of either GNA11 or closely homologous GNAQ confirmed these and 7 further double mutant APAs in this discovery cohort. The presence of GNA11/Q p.Gln209 mutations in CTNNB1 mutant APAs were replicated in 2 cohorts from France (n=14) and Sweden (n=3). In total, 16 (59%) of the 27 CTNNB1 mutant APAs investigated had a mutation at p.Gln209 of GNA11 (n=11) or GNAQ (n=5). Interestingly, CTNNB1-mutant APAs were more commonly present in women (23/27), and of these, those with GNA11/Q mutations were all women except for a pubertal boy. To also note, 9 of 10 of the UK/Irish double mutant APAs in the discovery cohort presented in puberty, pregnancy, or menopause. Mutation of p.Gln209, or homologous p.Gln in GNAS, GNA12-14, impair hydrogen bonds between G-protein α and β subunits. Transfection of H295R cells, an immortalised adrenocortical cell line heterozygous for the p.Ser45Pro mutation of CTNNB1 but wild-type for GNA11-14/Q/S, by each of the GNA11/Q mutations increased aldosterone secretion and CYP11B2 expression (encoding aldosterone synthase) by 1.93-6.1-fold and 8.0-9.8-fold respectively, compared to vector or wild-type -transfected cells. In ZG, GNA11/Q mediate the aldosterone response to angiotensin II, via stimulation of intracellular Ca2+ release by inositol trisphosphate. In the mutant-transfected cells, the stimulatory effect of angiotensin II 10 nM was retained. In order to determine whether the p.Gln209 mutations stimulate aldosterone production even in the absence of CTNNB1 activation, the transfections of H295R cells were repeated after either 24-h treatment with the CTNNB1 inhibitor, ICG-001, or silencing of CTNNB1 using the ONTARGETplus SMARTpool SiRNAs (Dharmacon). Both interventions reduced the aldosterone production relative to vehicle/control-treated cells; however neither ICG-001 nor silencing of CTNNB1 blunted the fold-increase in aldosterone secretion seen in mutant-transfected cells compared to wild-type. In summary, we report the discovery of gain-of-function mutations of the G-protein, GNA11, or its close homologue, GNAQ, in multiple APAs which majority presented during periods of high LH/HCG. To date, the mutation is always residue p.Gln209, and associated with a gain-of-function mutation of CTNNB1. These GNA11/Q p.Gln209 mutations increase aldosterone and CYP11B2 production both in the presence and in the absence of CTNNB1 activation.

Knock‐out of Adam10 in mCCD cl1 cells gives insight into the effects of Notch pathway disruption for kidney collecting duct cell plasticity

The kidney cortical collecting duct (CCD) is responsible for the fine tuning of sodium, volume, and acid-base homeostasis, and comprises of principal and intercalated cells (PCs and ICs) as well as a newly discovered ‘intermediate’ cell type (1). Plasticity between PCs and ICs has been reported previously in mice in which genes for factors implicated in the Notch pathway, such as Adam10, have been knocked out (KO) (2). In vivo, disruption of the Notch pathway leads to a decreasing PC:IC ratio. The self-immortalized mouse CCD cell line, mCCDcl1, shows functional characteristics of PCs and a response to physiological concentrations of hormones such as aldosterone; it also has been shown to express both PC and IC markers, a phenotype transmitted through single cell cloning (3). These characteristics make mCCDcl1 cells a useful platform for the study of plasticity in the CCD. Adam10 was knocked out in mCCDcl1 cells using CRISPR-Cas9 technology and independent clones were isolated and expanded for study. Despite mCCDcl1 cells being polyploid, our strategy enabled all alleles for each gene to be successfully targeted. Clones were cultured on Transwell filters over 10 days and their electrophysiological properties measured (trans-epithelial resistance Rte, voltage Vte, calculated short-circuit current Isc). Both Adam10 KO clones showed a slightly reduced baseline Rte compared to control (0.90±0.19 kΩ.cm2 vs 1.12±0.18 kΩ.cm2) but a complete reduction in baseline Isc (0.3±0.1 μA/cm2 vs -8.1±0.4 μA/cm2 for control) as well as aldosterone response, decreased by 95% (fold change 0.19±0.03 down 3.70±0.11 for control). Single-cell RNA sequencing principal component analysis shows different clustering of KO clones compared to WT. Potentially explaining the loss of sodium transport function, a significant drop in expression for the gamma subunit of the epithelial sodium channel (γ-ENaC) (between ~40% and ~60% depending on the cluster) was detected in the KO clones, as well as a decrease in the expression of 11ßHSD2 (between ~20% and ~60%). Immunostaining in KO clones confirmed the decrease in expression of γ-ENaC and showed an altered distribution and localisation of γ-ENaC, Aqp2, and V-ATPase B1 compared to WT mCCDcl1, overall suggesting a loss of polarisation. These data suggest that genomic modification of mCCDcl1 cells provides an effective means to investigate the mechanisms through which CCD cells mediate their plasticity and can identify new pathways involved in regulating cell phenotype as well as specific effects of pathway disruption on collecting duct cells functions. (1) Park et al.; Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease; Science; 360: 758-763, 2018. (2) Guo et al.; Adam10 Mediates the Choice between Principal Cells and Intercalated Cells in the Kidney; JASN; 26: 149-159, 2015. (3) Assmus et al.; mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells; AJP-renal; 314 (5): 820-831, 2018.

Effect of Increased Potassium Intake on Adrenal Cortical and Cardiovascular Responses to Angiotensin II: A Randomized Crossover Study.

BackgroundIncreased potassium intake lowers blood pressure in patients with hypertension, but increased potassium intake also elevates plasma concentrations of the blood pressure‐raising hormone aldosterone. Besides its well‐described renal effects, aldosterone is also believed to have vascular effects, acting through mineralocorticoid receptors present in endothelial and vascular smooth muscle cells, although mineralocorticoid receptors‐independent actions are also thought to be involved.Methods and ResultsTo gain further insight into the effect of increased potassium intake and potassium‐stimulated hyperaldosteronism on the human cardiovascular system, we conducted a randomized placebo‐controlled double‐blind crossover study in 25 healthy normotensive men, where 4 weeks treatment with a potassium supplement (90 mmol/day) was compared with 4 weeks on placebo. At the end of each treatment period, we measured potassium and aldosterone in plasma and performed an angiotensin II (AngII) infusion experiment, during which we assessed the aldosterone response in plasma. Hemodynamics were also monitored during the AngII infusion using ECG, impedance cardiography, finger plethysmography (blood pressure‐monitoring), and Doppler ultrasound. The study showed that higher potassium intake increased plasma potassium (mean±SD, 4.3±0.2 versus 4.0±0.2 mmol/L; P=0.0002) and aldosterone (median [interquartile range], 440 [336–521] versus 237 [173–386] pmol/L; P<0.0001), and based on a linear mixed model for repeated measurements, increased potassium intake potentiated AngII‐stimulated aldosterone secretion (P=0.0020). In contrast, the hemodynamic responses (blood pressure, total peripheral resistance, cardiac output, and renal artery blood flow) to AngII were similar after potassium and placebo.ConclusionsIncreased potassium intake potentiates AngII‐stimulated aldosterone secretion without affecting systemic cardiovascular hemodynamics in healthy normotensive men.RegistrationEudraCT Number: 2013‐004460‐66; URL: https://www.ClinicalTrials.gov; Unique identifier: NCT02380157.

PENDRIN REDUCTION IS ASSOCIATED WITH ACUTE INTRAVENOUS SODIUM CHLORIDE LOADING IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is a common, potentially curable form of hypertension characterised by excessive, autonomous aldosterone production. In the distal renal tubule, aldosterone is involved in volume homeostasis by modulation of NaCl reabsorption via the epithelial Na+ channel (ENaC), the NaCl cotransporter (NCC) and the Cl-/HCO3- exchanger (pendrin). Na+ loading suppresses plasma aldosterone but its effects on these proteins in humans remain unclear. Design and method: Urine samples were collected before and 1 h after a 4 h 2L 0.9% saline infusion from 13 (F9/M5) subjects including 10 confirmed with PA and 3 in which PA was excluded [herein designated low renin hypertension (LRH)]. Urinary exosomes were harvested by ultracentrifugation and analysed by LC-MS/MS. Results: Plasma aldosterone decreased (P &lt; 0.05) from 623 [312, 2210] (median [range]) to 338 [197, 2330] pmol/L in confirmed PA and from 223 [180, 256] to 117 [110, 133] pmol/L in LRH. There were no changes in plasma K+ (pre 3.4 [2.6, 4.3] vs post 3.6 [2.6, 3.9] mmol/L in PA; pre 4.1 [3.6, 4.1] vs post 4.0 [3.9, 4.0] mmol/L in LRH). LC-MS/MS demonstrated decreases in abundance of pendrin (21.5%, P &lt; 0.01) and aquaporin-2 (AQP2) (42.9%, P &lt; 0.01) in urinary exosomes and a trend towards a decrease in NCC (20%, P = 0.07) in patients with PA. No changes were seen in those with LRH. PA patients had higher baseline pendrin abundance (1.2 [1.0, 2.1]) than LRH patients (0.7 [0.5, 1.0]) (P &lt; 0.05), but there was no difference in baseline AQP2. In all 13 subjects combined, baseline pendrin correlated positively with plasma aldosterone (P = 0.02, r2 = 0.32) and negatively with plasma K+ (P = 0.02, r2 = 0.35), whilst no correlations were observed between AQP2 and aldosterone or K+. Delta Pendrin (P = 0.04, r2 = 0.3) and delta AQP2 (P = 0.01, r2 = 0.58) positively correlated with delta plasma aldosterone. Conclusions: In PA, reduction in pendrin during saline infusion may be due to falling plasma aldosterone in response to NaCl loading. Although also correlating with reduction in aldosterone, a more likely explanation for the reduction in AQP2 may be a fall in plasma vasopressin induced by water loading. Different observations in LRH require validation with larger numbers.

BIOCHEMICAL, HISTOPATHOLOGICAL, AND GENETIC CHARACTERIZATION OF POSTURE RESPONSIVE AND UNRESPONSIVE ALDOSTERONE-PRODUCING ADENOMAS

Objective: Aldosterone-producing adenoma (APA) accounts for ∼30% of primary aldosteronism. Based on responses of aldosterone to upright posture, APA can be divided into posture-responsive and –unresponsive forms. Somatic gene mutations in KCNJ5, CACNA1D, ATP1A1, or ATP2B3 have been reported to exist in more than 90% of APAs. We sought to compare the biochemical, histopathological, and genetic characterization between these two types of APAs. Design and method: Posture-responsive was defined as showing a rise in plasma aldosterone concentration of at least 50% 2 - 3 h after the assumption of upright posture (10 AM) above the basal level (7 AM) following overnight recumbency. Plasma levels of aldosterone and hybrid steroids (18-oxocortisol and 18-hydroxycortisol) were measured by LC-MS/MS. Histologic analysis and immunohistochemistry for CYP11B2 (aldosterone synthase) and CYP17A1 (17aplha-hydroxylase) were performed on APA tissue sections collected from 24 posture-unresponsive and 17 posture-responsive APA patients. Sanger sequencing of KCNJ5 and gene targeted next-generation sequencing were performed to detect somatic gene mutations. Results: Compared to patients with posture-responsive APA (of which 48.7% were composed of zona glomerulosa (ZG)-like cells and 35.3% showed ZG-zona fasciculata (ZF) mixed appearance), posture-unresponsive APA patients displayed higher (P &lt; 0.01) levels of hybrid steroids, recumbent aldosterone and cortisol, greater (P &lt; 0.05) suppression of aldosterone secretion from the contralateral unaffected adrenal, and larger (P &lt; 0.01) tumors mostly composed of ZF-like cells that had higher (P &lt; 0.01) expression of CYP17A1 (but not of CYP11B2). Of 41 studied APAs, 37 (90.2%) harboured aldosterone-driving somatic mutations (KCNJ5 = 14, 34.1%; CACNA1D = 13, 31.7%; ATP1A1 = 8, 19.5%; and ATP2B3 = 2, 4.9%), including 5 that were previously unreported (3 in CACNA1D and 2 in ATP1A1). Interestingly, 64.7% (11/17) of posture-responsive APAs carried CACNA1D mutations, whereas 54.2% (13/24) of posture-unresponsive APAs harboured KCNJ5 mutations. Conclusions: The excessive production of hybrid steroids by posture-unresponsive APAs may relate to their ZF-like tumor cell composition, resulting in strong expression of CYP17A1 (in addition to somatic gene mutation-driven CYP11B2 expression), thereby allowing production of cortisol which acts as the substrate for CYP11B2-generated hybrid steroids.

Cortisol Co-Secretion and Clinical Usefulness of ACTH Stimulation Test in Primary Aldosteronism: A Systematic Review and Biases in Epidemiological Studies.

The hypothalamus-pituitary-adrenal (HPA) axis plays an important role in primary aldosteronism. Aldosterone biosynthesis is regulated not only by angiotensin II in the renin-angiotensin-aldosterone system, but also by adrenocorticotropic hormone (ACTH), one of the key components of the HPA axis. Although previous studies have reported cortisol cosecretion in primary aldosteronism, particularly aldosterone-producing adenoma (APA), the clinical relevance of such aldosterone and cortisol cosecretion from APA and hypertension or other metabolic disorders has not been fully established. Several somatic mutations including KCNJ5 and CACNA1D are known to induce autonomous production of aldosterone in APA, and the aldosterone responsiveness to ACTH may vary according to each mutation. The ACTH stimulation test has been reported to be a useful tool to distinguish the subtypes of primary aldosteronism (e.g., unilateral vs bilateral) in some studies, but it has not been commonly applied in clinical practice due to limited evidence. Given the recent advancement of imaging, omics research, and computational approach, it is important to summarize the most updated evidence to disentangle the potential impact of cortisol excess in primary aldosteronism and whether the ACTH stimulation test needs to be considered during the diagnostic process of primary aldosteronism. In this article, we conducted a systematic review of epidemiological studies about (i) cortisol cosecretion in primary aldosteronism and (ii) the ACTH stimulation test for the diagnosis of primary aldosteronism (including subtype diagnosis). Then, we discussed potential biases (e.g., confounding bias, overadjustment, information bias, selection bias, and sampling bias) in the previous studies and introduced some advanced epidemiological/statistical methods to minimize these limitations. A better understanding of biases and epidemiological perspective on this topic would allow us to produce further robust evidence and balanced discussion about the causal mechanisms involving the HPA axis and clinical usefulness of the ACTH stimulation test among patients with primary aldosteronism.

Aldosterone is Aberrantly Regulated by Various Stimuli in a High Proportion of Patients with Primary Aldosteronism

In primary aldosteronism (PA), aldosterone secretion is relatively independent of the renin-angiotensin system, but can be regulated by several other stimuli. To evaluate aldosterone response to several stimuli in a series of patients with PA secondary either to bilateral adrenal hyperplasia (BAH) or unilateral aldosterone-producing adenoma (APA). Prospective cohort study conducted in a university teaching hospital research center. Forty-three patients with confirmed PA and subtyped by adrenal vein sampling (n = 39) were studied, including 11 with BAH, 28 with APA, and 4 with undefined etiology. We also studied 4 other patients with aldosterone and cortisol cosecretion. We systematically explored aberrant regulation of aldosterone using an in vivo protocol that included the following stimulation tests performed over 3 days under dexamethasone suppression: upright posture, mixed meal, adrenocorticotropin (ACTH) 1-24, gonadotropin-releasing hormone (GnRH), vasopressin, and serotonin R4 agonist. Positive response was defined as >50% renin or ACTH-independent increase in plasma aldosterone/cortisol concentration following the various stimulation tests. Renin-independent aldosterone secretion increased in response to several aberrant stimuli (upright posture, GnRH) in up to 83% of patients with APA or BAH in whom ACTH 1-24 and HT4R agonists also produced aldosterone oversecretion in all patients. The mean significant aberrant responses per patient was similar in BAH (4.6) and in APA (4.0). Aldosterone secretion in PA is relatively autonomous from the renin-angiotensin system, but is highly regulated by several other stimuli, which contributes to the large variability of aldosterone levels in PA patients.

Salivary aldosterone and cortisone respond differently to high- and low-psychologically stressful soccer competitions

ABSTRACT Aldosterone and cortisone are released in response to physical and psychological stress. However, aldosterone and cortisone responses in children engaged in physical competition have not been described. We examined salivary aldosterone and salivary cortisone responses among Hong Kongese boys, aged 8-11 years, during (1) a soccer match against unknown competitors (N = 84, high psychological stress condition) and (2) an intrasquad soccer scrimmage against teammates (N = 81, low psychological stress condition). Aldosterone levels increased during the soccer match and intrasquad soccer scrimmage conditions, consistent with the view that aldosterone responds to physical stress. During the soccer match, winning competitors experienced larger increases in aldosterone compared to losing competitors, indicating that the degree of aldosterone increase was attenuated by match outcome. Cortisone increased during the soccer match and decreased during the intrasquad soccer scrimmage. Competitors on teams that resulted in a tie had larger cortisone increases compared to winners or losers. These findings highlight that the degree of cortisone change is related to boy's cognitive appraisal of the competitor type (i.e., teammates vs. unknown competitors) and the competitive nature of the game (e.g., tie). These results shed new light on adrenal hormone mediators of stress and competition during middle childhood.

Juvenile Children’s Salivary Aldosterone and Cortisone Decrease during Informal Math and Table-Tennis Competitions

Among adults, aldosterone and cortisone increases are reported in response to physically taxing forms of competition, enabling individuals to rapidly adapt to variable sociocompetitive contexts. Yet, aldosterone and cortisone responses in juvenile children engaged in less strenuous forms of competition have not been investigated. Here, we sought to measure aldosterone and cortisone responses in children who participated in math and table-tennis competitions. We hypothesized that the responses would significantly vary with respect to the type of competition. Pre- and post-match salivary aldosterone and cortisone were measured in Hong Kongese children, aged 8–11 years, during (1) a mixed-sex, team-based, math competition (N = 45) and (2) a dyadic, table-tennis competition against peers (N = 22). In the math competition, aldosterone and cortisone levels decreased in boys and girls, while members on losing teams had greater match decreases in cortisone levels compared to individuals on winning teams. In the table-tennis competition, time of day led to significant diurnal differences in competitors’ pre-match aldosterone and cortisone concentrations. As a result, each sample was analyzed independently according to match time (8:30 AM and 11:00 AM). Aldosterone levels decreased among the competitors who participated in the 11:00 AM table-tennis matches. Cortisone levels decreased for the majority of competitors, but only significantly decreased in the 8:30 AM sample. These findings highlight that juvenile competitors’ hypothalamic-pituitary-adrenal (HPA) axis and renin-angiotensin-aldosterone system (RAAS) are sensitive to less physically strenuous forms of competition. Further, the differences in the competitive environment likely stimulate the direction of aldosterone (RAAS) and cortisone (HPA) reactive change.

Influence of an Isotonic Sports Drink during Exercise and Recovery on Subsequent Endurance Capacity and Aldosterone Response in the Heat in Well-Trained Endurance Athletes

Influence of an Isotonic Sports Drink during Exercise and Recovery on Subsequent Endurance Capacity and Aldosterone Response in the Heat in Well-Trained Endurance Athletes

The neuropeptide substance P regulates aldosterone secretion in human adrenals

Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes.

SUN-LB91 The Arg16/Gln27 Polymorphism of the Beta2-Adrenergic Receptor Impacts Blood Pressure Levels in a Transgenic Mouse Model via Sex-Specific Mechanisms

Background: The β2-adrenergic receptor (β2-AR) has been implicated in blood pressure (BP) homeostasis via its effects on the sympathetic nervous system, cardiac function, peripheral vascular resistance, and renin release. We also have documented that a β2-AR diplotype (Arg16/Gln27), carried by 15% of hypertensives, have salt sensitive hypertension (SSBP) and increased aldosterone (ALDO). Gap in knowledge: it has been reported that hypertensive men and women respond differently to beta blockers. However, information regarding sex differences in carriers of the β2-AR risk diplotype is limited. Hypothesis: In mice, the phenotype in female carriers of the mutant β2-AR risk diplotype will differ from male carriers. Methods: The CRISPR/Cas9 approach was used to generate transgenic mice carrying 0, 1 or 2 Arg16/Gln27 variant alleles (i.e. wild-type (WT), heterozygous (Het) or mutant (Mut)). The experimental design included twelve weeks old mice divided into 6 genotype/gender groups (male and female; WT, Het and Mut) maintained on low (0.03% Na+, LS) and high sodium diets (1.6% Na+, HS) for a week each. Results: 1) Both male and female mice displayed significantly increased BP on LS and HS with increasing number of mutated alleles.2) Only Mut females displayed SSBP (P<0.05).3) As compared to WT, urine and plasma ALDO levels were lower in male (but not female) carriers of the mutated allele (P<0.05).4) As anticipated, urine K+ excretion was significantly lower in the Mut male (but not female) mice (P<0.05).5) Doppler ultrasound measurements of renovascular function shows that the resistive index was significantly lower in Mut vs WT males (P<0.05), consistent with an appropriate increase in renal blood flow (RBF) in the face of an elevated BP. However, in the Mut females the RBF was inappropriately decreased compared to the WT (P<0.05).6) β2-AR expression was significantly lower in female vs. male WT mice; however, this difference was lost in the carriers of the mutated allele, as B2-AR expression levels were significantly higher in female carriers of the mutated allele, as compared to WT (P<0.05). Interestingly, the B2-AR genotype had no effect on the receptor expression levels in males. Conclusion: Both male and female Mut animals have increased BP compared to WT mice, but the mechanisms underlying their increased BP differ by sex. Female Mut mice have SSBP, inappropriately non-suppressed ALDO and decreased RBF. Male Mut mice do not have SSBP and have appropriate ALDO and RBF response to the salt load.

MON-246 A Diagnostic Dilemma of Primary Versus Secondary Thyroid and Adrenal Hormone Insufficiencies: A Unique Case of Widely Metastatic Renal Cell Carcinoma After Brain Radiation

Background Identifying the etiology of hormonal insufficiency is important for correct diagnosis and for appropriate hormone supplementation. Usually multiple hormone deficiencies are driven by pituitary pathology, but here we present a case that poses a unique diagnostic and therapeutic dilemma. Clinical Case A 55 y/o lady with HTN and recently diagnosed metastatic Renal Cell Carcinoma (RCC) presented with weakness, dizziness, altered mental status, polydipsia and polyuria. Her metastases included the bilateral adrenal glands, thyroid, brain, and she had a sellar lesion displacing the pituitary as well as infundibular thickening. She had been treated with dexamethasone due to vasogenic edema, whole brain radiation, and was going to start immunotherapy (Ipilumimab&Nivolumimab). On exam she had relative hypotension and tachycardia (BP 115/58, HR 108). She was diagnosed with non-PTH mediated hypercalcemia (Ca 13.9/ 15.3 corrected for albumin 2.3, PTH 6.5, PTH-RP 69, Vitamin D-25 19.4). Her calcium normalized after fluid resuscitation and bisphosphonate treatment, but weakness and hypotension persisted. We tested thyroid and adrenal function given the location of her lesions, recent whole brain radiation, and recent steroid use. Her TSH was 0.017 (0.270 - 4.200 µIU/mL), fT4 0.84 (0.80 - 1.50 ng/dL), T3 0.59 (72.0 - 153.0 ng/dL), ACTH was 1.7 (7.2-63.3 pg/ml), cortisol 1.3 (6.0 - 18.4 ug/dL), aldosterone 3 (< or = 28 ng/dL), renin 7.87 (0.25 - 5.82 ng/mL/h), renin/aldosterone 0.4 (0.9 - 28.9). At 30 minutes after cosyntropin administration cortisol was 7.7 ug/dL, aldosterone 26 ng/dL, and at 60 minutes cortisol was 10.3 ug/dL. Hydrocortisone was initiated while tapering off dexamethasone, and levothyroxine was offered but declined. She opted to postpone further workup. Conclusion This patient presents a dilemma in identifying the primary causes of hormonal abnormalities given potential pituitary and primary thyroid and adrenal disease. The thyroid abnormalities could represent sick euthyroid physiology with lower T4 to T3 conversion or the effects of steroids which can also suppress TSH vs. hypothalamic or pituitary disease. Primary hypothyroidism from gland destruction was unlikely. The low ACTH and cortisol levels were expected since she was on dexamethasone, but low aldosterone with raised renin activity was concerning for primary adrenal insufficiency. However, after cosyntropin the suboptimal cortisol yet preserved aldosterone response supported secondary or tertiary adrenal insufficiency, and only cortisol was supplemented. Understanding her endocrine disease had implications for longer term management given that replacement may only need to be temporary and adrenal recovery might be possible. Further, the use of a 30 minute aldosterone level can be helpful in cases where multiple factors and discrepant laboratory findings may exist.

SAT-224 Recurrent Co-Driver Mutation in CTNNB1-Mutant Aldosterone-producing Adenomas (APA), Causing Reversible Hypertension in Puberty, Pregnancy or Menopause

Background: Three patients with a syndrome of LH/HCG-activated primary aldosteronism in pregnancy or menopause carrying somatic CTNNB1 mutations were reported four years ago (Teo et al. NEJM 2015). This proved but half the story. Diagnosis of an APA in a 12-year old boy with severe hypertension revealed an apparently essential co-driver mutation. Methods: WES of tumour and blood was performed in the pubertal boy. Candidate genes were Sanger sequenced in other APAs from GB/Ireland, and France with known or suspected CTNNB1 mutations. LHCGR, GNRHR and CYP11B2 expression were measured in all available patients’ APAs and the adjacent adrenal gland (AAG) by RT-PCR. RNA and gDNA from the zona glomerulosa (ZG) of the proband’s AAG were collected by laser capture microdissection for Sanger sequencing of GNA11 and CTNNB1. Function of mutant genes was assessed by measurement of aldosterone production and LHCGR expression by immunofluorescence (IFC) in NCI-H295R adrenocortical cells and primary human APA cells. Results: The proband’s APA contained a p.(S45F) somatic mutation in CTNNB1, and a p.(Q209P) somatic mutation of the GTPase-activating residue (Q209) in GNA11. Mutations of Q209, to P or H, were also found in six other GB/Irish patients with previously identified mutations of CTNNB1 (S33C, G34R, T41A, S45F, or S45P). All seven patients remain normotensive 2-12 years post-adrenalectomy, including some with long-standing pre-operative hypertension. Four of the 13 French patients with CTNNB1 exon 3 mutant APAs have somatic mutation of Q209 of either GNA11 (n=3) or GNAQ. In comparison with their own AAG, the GB/Irish double mutant APAs showed an increase in expression of LHCGR, CYP11B2 and GNRHR by 32-166, 158-18980, and 1174-6642 fold, respectively. All four French double-mutants had >10 fold higher LHCGR than APAs with single mutations of CTNNB1 or other genes. Hyperplasia of ZG was observed in the ZG of the boy’s AAG but no APCC was detected. Homozygous or heterozygous Q209P mutation of GNA11 was detected in multiple ZG samples in RNA and/or gDNA but WT in CTNNB1 exon 3. H295R cells (CTNNB1 S45P) were GNA11 WT. Overexpression of GNA11 Q209 mutation increased aldosterone secretion to 465% of GNA11 WT overexpressing cells (n=6, P<0.001) and CYP11B2 expression was also increased several-fold. Smaller increases were seen in primary human adrenal cells after double-transfection by GNA11 and CTNNB1 mutants (n=3, P<0.001). This also caused membrane expression of LHCGR, visualised by IFC. Conclusions: APAs with double mutation of GNA11/GNAQ Q209 and CTNNB1 have a distinct phenotype, in which hypertension is triggered by high LH or HCG, and cured in all cases by adrenalectomy. GNA11/Q mediates the aldosterone response to ANGII, and the Q209 codon is analogous to the Q227 of GNAS, mutated in McCune Albright. Mosaicism for GNA11 may cause ZG hyperplasia.

MON-209 Identification of a New Heterozygous Germline ARMC5 Deletion in a Familial Case of Primary Bilateral Macronodular Adrenal Hyperplasia Co-Secreting Cortisol and Aldosterone

Context. Approximately 50% of familial cases of primary bilateral macronodular adrenal hyperplasia (PBMAH) are caused by mutations in the ARMC5 gene. Case report. We report the case of a 37 year-old patient of Haitian origin, who presented with resistant hypertension. His workup showed high aldosterone (410 pmol/L) with suppressed renin levels (0.2 ng/mL/h) with an aldosterone to renin ratio (ARR) of 2050. Patient also had suppressed ACTH levels (<0.5 pmol/L (N: 2-12)) and high UFC (1103 nmol/d (N: <330)). He had an aberrant cortisol and aldosterone response to catecholamines and vasopressin (V1R). An abdominal CT scan showed bilateral enlargement of adrenal glands and a 3.3cm dominant nodule on the right gland. Moreover, a 2.8cm mass on the pancreatic tail was present. Patient underwent left laparoscopic adrenalectomy and distal pancreatectomy; the pathology confirmed PBMAH and a pancreatic neuroendocrine tumor (NET). Following surgery, ARR and UFC remained high and patient was treated successfully with B-blockers and MR antagonists. A head MRI showed no sign of intracranial meningiomas. Genetic analyses. Following genetic counselling, MEN1 gene analysis was performed using sequencing/MLPA techniques but did not reveal a mutation. Initial genetic testing included ARMC5 gene analysis using direct Sanger sequencing which was negative. However, using Next-Generation Sequencing (NGS) and MLPA analysis, a heterozygous germline ARMC5 deletion of exons 5-8 was identified. The deletion is predicted to prematurely truncate the protein product and cause loss of function. The ARMC5 deletion segregated with the disease in his 24 yo son who had bilateral adrenal adenomas that appeared to be non-functional. The patient’s father was also known for having bilateral adrenal masses and hypertension.To our knowledge we report the second case of ARMC5 deletion in familial PBMAH. Suzuki et al. reported two patients, a mother and her son, carrying ARMC5 deletion of exons1-5 and interestingly they were also affected by PBMAH co-secreting cortisol and aldosterone (1). As in this case report, the ARMC5 deletion was missed using Sanger sequencing initially. Conclusion. These cases demonstrate that large deletions may be missed by Sanger sequencing and that the real prevalence of ARMC5 mutations may have been underestimated. The link between deletion of ARMC5 and correlation with PBMAH co-secreting aldosterone and cortisol remains to be determined but may be a step forward for genotype-phenotype correlation.1.Suzuki S, et al. Endocrine practice: official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists. 2015;21 (10):1152-60.