Overproduction Of Aldosterone Research Articles

Integrative bioinformatics approach identifies novel drug targets for hyperaldosteronism, with a focus on SHMT1 as a promising therapeutic candidate

Primary aldosteronism (PA), characterized by autonomous aldosterone overproduction, is a major cause of secondary hypertension with significant cardiovascular complications. Current treatments mainly focus on symptom management rather than addressing underlying mechanisms. This study aims to discover novel therapeutic targets for PA using integrated bioinformatics and experimental validation approaches. We employed a systematic approach combining: gene identification through transcriptome-wide association studies (TWAS); causal inference using summary data-based Mendelian randomization (SMR) and two-sample Mendelian randomization (MR) analyses; additional analyses included phenome-wide association analysis, enrichment analysis, protein-protein interaction (PPI) networks, drug repurposing, molecular docking and clinical validation through aldosterone-producing adenomas (APAs) tissue. Through systematic screening and prioritization, we identified 163 PA-associated genes, of which seven emerged as potential drug targets: CEP104, HIP1, TONSL, ZNF100, SHMT1, and two long non-coding RNAs (AC006369.2 and MRPL23-AS1). SHMT1 was identified as the most promising target, showing significantly elevated expression in APAs compared to adjacent non-tumorous tissues. Drug repurposing analysis identified four potential SHMT1-targeting compounds (Mimosine, Pemetrexed, Leucovorin, and Irinotecan), supported by molecular docking studies. The integration of multiple bioinformatics methods and experimental validation successfully identified novel drug targets for hyperaldosteronism. SHMT1, in particular, represents a promising candidate for future therapeutic development. These findings provide new opportunities for developing causative treatments for PA, though further clinical validation is warranted.

131I]6ß-Iodomethyl-19-norcholesterol SPECT/CT for the Lateralization of Mineralocorticoid Overproduction in Primary Aldosteronism.

In 42 consecutive patients with confirmed primary aldosteronism, molecular imaging has been performed. After dexamethasone suppression of the non-affected adrenal tissue, 37 MBq [131I]6ß-iodomethyl-19-norcholesterol was injected i.v., and SPECT/CT images were taken 7 days later. Based on the visual evaluation of the images by two nuclear medicine specialists, patients with one-sided tracer accumulation underwent adrenalectomy. To identify a SPECT/CT parameter that best characterizes the side difference, the maximum counts and the mean counts of spherical VOIs were analyzed. Of the 42 patients, 24 had one-sided aldosterone overproduction by SPECT/CT. After surgical removal of the involved adrenal, all 24 patients with SPECT/CT-identified unilateral aldosteronism achieved biochemical cure, defined as a normalized potassium level combined with an aldosterone-to-renin ratio ≤ 30. To identify the best measurable parameter of SPECT/CT side difference, the mean counts and maximum counts of a series of spherical VOIs of different diameters were analyzed. The ratio of the mean counts of 3 cm spherical VOIs of the right and left adrenal regions (lateralization index) was the best discriminator; a ratio of ≥1.29 was characteristic of one-sided disease, without overlap between the one-sided and two-sided patient groups. [131I]6ß-iodomethyl-19-norcholesterol SPECT/CT with a count-based image interpretation and side-ratio calculation may be an equipollent non-invasive substitute for adrenal venous sampling in the lateralization of mineralocorticoid overproduction. It reliably identifies unilateral disease and facilitates patients' selection for surgical intervention. If confirmed by others, this functional imaging may replace AVS when lateralization is required for management decisions in primary aldosteronism.

Abstract 59: Beclin-1-Dependent Autophagy Prevents Hyperaldosternism-Induced Endothelial Dysfunction And Hypertension

Hyperaldosteronism (HA), characterized by the overproduction of aldosterone (Aldo), leads to cardiovascular damage, including endothelial dysfunction. Beclin-1 (Becn1) is a key protein regulating autophagy, a recycling process for unwanted components. Whether HA disturbs autophagic flux in endothelial cells via Becn1 is unknown. We hypothesize that Becn1-dependent autophagic flux will improve endothelial function and inhibit the hypertension (HTN) in HA. 10-to-12-week-old male C57BL/6J (WT) or Becn1 knock-in mice (Becn1 Tg ) were infused with aldosterone for 14 days (600 µg/kg/day). Vascular function was studied in mesenteric arteries (MA) and blood pressure was analyzed by radiotelemetry. HA disrupted MA autophagic flux characterized by accumulation of LC3A/B and p62 and reduction in Becn1 expression and activity followed by suppressed acetylcholine (ACh)-induced vasodilation (% relaxation) in WT [WT: 91.5±1.1 vs HA: 56.7±2.7], whereas Becn1 Tg mice were protected from HA-induced HTN [mean blood pressure (mmHg) – WT_Aldo: 149.1±1.2 vs BCN1_Aldo: 118.1±0.8] and endothelial dysfunction [HA_Becn1 Tg : 92.8±2.9]. Given the link between the immune system and cardiovascular diseases, we investigated whether HA reduces autophagic flux and BCN1 in immune cells. HA led to the accumulation of LC3A/B and p62, and decreased BCN1 activity in peritoneal cavity cells of WT mice, however bone marrow transplant from Becn1 Tg mice into WT mice did not confer protection against HA-induced changes in vascular reactivity [WT to WT_HA: 51.2±1.3 vs Becn1 Tg to WT_HA: 53.8±1.9]. As a pharmacological approach, we accelerated autophagic flux by treating mice with spermidine (3 mM for 7 days), which subsequently improved endothelial function in HA [HA: 52.7±3.9 vs HA+spermidine: 88.9±3.9]. To assess the direct impact of Aldo on disrupting autophagy flux in endothelial cells, we treated mesenteric endothelial cells (MEC) with Aldo (0.1 µM for 24 hours). This treatment resulted in the accumulation of LC3A/B and p62 proteins and a reduction in BCN1 activity, accompanied by an upregulation in the mRNA expression of pro-inflammatory cytokines. All these effects were prevented by spermidine (100 uM for 3h). These findings reveal a novel pathway in HA-associated endothelial dysfunction and HTN, where Aldo disrupts autophagic flux and inflammation by suppressing BCN1 activity, positioning BCN1 as a pharmacological target for cardiovascular outcomes in HA.

Beclin-1-Dependent Autophagy Preserves Perivascular Adipose Tissue Function and Suppresses Hypertension Development in Hyperaldosteronism

Hyperaldosteronism (HA), characterized by the independent overproduction of aldosterone (Aldo), leads to cardiovascular damage and perivascular adipose tissue (PVAT) dysfunction. Beclin-1 (Becn1) is a key protein regulating autophagy, a recycling process of unwanted components. Whether HA disturbs autophagic flux in PVAT via Becn1 is unknown. Thus, we hypothesize that Becn1-dependent autophagic flux will improve PVAT function and inhibit the genesis of hypertension (HTN) in HA. 10-12-weeks-old male C57BL/6J (WT) or Becn1 knock-in mice (Becn1Tg) mice were infused with aldosterone for 14 days (600ug/Kg/day via osmotic mini-pump). Vascular function was studied in PVAT-intact thoracic aortae and blood pressure was analyzed by radiotelemetry. Becn1Tg mice were protected from HA-induced HTN [mean blood pressure (mmHg) − before HA: 101.2±0.5 vs. after HA: 108.2±0.9]. HA disrupted PVAT autophagic flux characterized by accumulation of LC3A/B and p62 and reduction in Becn1 expression and activity. In terms of function, presence of PVAT decreased the contractile response in WT [PVAT (-): 137.2±1.9 vs. PVAT (+): 107.4±2.7, Emax (% KCl)], while HA induced loss of PVAT anti-contractile effect [PVAT (-): 159.9±2.9 vs. PVAT (+): 164.1±3.4]. Becn1Tg mice were protected from this effect. Furthermore, HA induced PVAT oxidative stress and inflammation, characterized by elevated TNF-α, IL-6, IL-1β, IL-17, and IFN-γ mRNA expression, and elevated reactive oxygen species (ROS), measured by lucigenin chemiluminescence. All these effects were prevented in Becn1Tg mice. Pharmacological induction of Becn1 activation, via TB-peptide (16mg/Kg/day for 7 days), improved PVAT function of WT mice with HA. To evaluate the molecular mechanisms, we treated brown adipocyte cells (BAC, WT1) with Aldo (100nM), which triggered a transient autophagic flux, depicted by an initial increase in LC3A/B (12 and 24 h), followed by a decrease at 48 and 96 h. Such effects were prevented by a selective mineralocorticoid receptor antagonist (Eplerenone, 1μM). Finally, Aldo induced BAC inflammation and oxidative stress, which were blunted by TB-peptide. In summary, our findings reveal that autophagy overactivation confers protection against HA-induced PVAT inflammation and dysfunction and HTN. Therefore, activation of Becn1 could be pharmacological tool to prevent HA-associated cardiovascular deleterious effects. FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA (CDA857268 and 23DIVSUP1069230), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Safety and efficacy of once-daily dexfadrostat phosphate in patients with primary aldosteronism: a randomised, parallel group, multicentre, phase 2 trial

Primary aldosteronism (PA) is caused by autonomous aldosterone overproduction and characterised by uncontrolled hypertension. There are currently no treatments that targetaldosterone synthesis. We evaluated the safety and efficacy of a novel aldosterone synthase inhibitor, dexfadrostat phosphate, in patients with PA. This multi-centre, randomised, phase 2 trial was conducted between November 2019 and May 2022 (NCT04007406; EudraCT code 2019-000919-85). Adults with PA and an office systolic blood pressure of 145-190mmHg were included. After a 2-week single-blind placebo run-in period, participants were randomised 1:1:1 to receive oral dexfadrostat phosphate 4, 8, or 12mg once daily for an 8-week double-blind treatment period, followed by a 2-week single-blind placebo withdrawal period. Randomisation was conducted centrally and stratified by centre and sex. At the beginning and end of the treatment period, 24h ambulatory systolic blood pressure (aSBP) was recorded. Blood samples were taken every 2 weeks. Primary endpoints were the change in aldosterone-to-renin ratio (ARR) and mean 24h aSBP from baseline to the end of the treatment period in the combined dose group of all participants receiving any dose of dexfadrostat phosphate. Safety endpoints were the occurrence of treatment-emergent adverse events (TEAEs) and serious adverse events over the entire study in all randomised participants who received at least one dose of dexfadrostat phosphate. In total, 35 participants received dexfadrostat phosphate and all participants completed the study. Twenty-six participants (74.3%) were male, the mean age was 51.9 years (SD 8.7), and most were White (n=32, 91.4%). The median ARR and the mean 24h aSBP significantly decreased from the beginning to the end of the treatment period in the combined dose group (ARR: 15.3 vs 0.6, least-squares mean [LSM] change in log-normal values-2.5, p<0.0001; aSBP: 142.6 vs 131.9mmHg, LSM change-10.7mmHg, p<0.0001). There were no safety concerns; all TEAEs were mild or moderate and there were no serious TEAEs. Dexfadrostat phosphate corrected the ARR and aSBP and was well tolerated in patients with PA, demonstrating the benefit of pharmacologically targeting the source of hyperaldosteronism. DAMIAN Pharma AG.

Primary Hyperaldosteronism: A Rare Potential Risk Factor for Acute Aortic Dissection, an Interesting Case Report

Primary hyperaldosteronism is overproduction of aldosterone from the adrenal cortex with wide electrolyte imbalance and cardiovascular complications like aortic dissection. While the association between primary hyperaldosteronism and aortic dissection has been a subject of interest, the exact relationship and potential causative mechanisms remain unclear. We present here a 51-year-old male with no significant past medical history who came to the emergency department with severe chest pain, sudden-onset radiating to the back, shortness of breath (SOB), and diaphoresis. A chest-to-abdominal Computed Tomography (CT) angiography showed a small tear in the inner lining of the aortic arch, just below the left subclavian artery. The descending aorta had a large mural thrombosis up to the T7 level. The pressure was controlled with intravenous drugs, and then oral antihypertensive drugs and electrolytes were corrected. The patient was recommended to go outside where cardiovascular surgery is available and is now doing well in cardiology outpatient follow-up with well-controlled blood pressure.

2023 Korean Endocrine Society Consensus Guidelines for the Diagnosis and Management of Primary Aldosteronism.

Primary aldosteronism (PA) is a common, yet underdiagnosed cause of secondary hypertension. It is characterized by an overproduction of aldosterone, leading to hypertension and/or hypokalemia. Despite affecting between 5.9% and 34% of patients with hypertension, PA is frequently missed due to a lack of clinical awareness and systematic screening, which can result in significant cardiovascular complications. To address this, medical societies have developed clinical practice guidelines to improve the management of hypertension and PA. The Korean Endocrine Society, drawing on a wealth of research, has formulated new guidelines for PA. A task force has been established to prepare PA guidelines, which encompass epidemiology, pathophysiology, clinical presentation, diagnosis, treatment, and follow-up care. The Korean clinical guidelines for PA aim to deliver an evidence-based protocol for PA diagnosis, treatment, and patient monitoring. These guidelines are anticipated to ease the burden of this potentially curable condition.

FRI139 Identification Of Robust Aldosterone-Producing Adenomas-related Genes Using Adrenal Genome-wide Transcriptome Analysis

Abstract Disclosure: S. Lee: None. T. Lee: None. G. Yoo: None. J. Lim: None. Primary aldosteronism (PA), the most common cause of secondary hypertension, is caused by aldosterone overproduction in one or two adrenal glands. It carries a higher risk of cardiovascular disease and mortality compared to essential hypertension. In an effort to identify the underlying molecular mechanisms regarding aldosterone secretion and cell proliferation in aldosterone-producing adenomas (APA), genome-wide transcriptome analyses have been conducted; however, there are still some limitations that need to be addressed since a relatively small number of samples were analyzed at a single center in most studies. In this study, we performed integrative and systematic analysis using adrenal transcriptome datasets to verify candidate genes robustly associated with APA and obtain more generalized signatures involved in the pathophysiology of PA. Three mRNA datasets for adrenal tissues (GSE8514, GSE60042, and GSE64957) were obtained from the Gene Expression Omnibus Database. For the first two datasets, normal adrenal samples from patients with renal cell carcinoma and adrenal tissue samples adjacent to APA were included as controls, respectively. In GSE64957, zona glomerulosa (ZG) and zona fasciculata (ZF) samples were analyzed separately in the current study (GSE64957ZG and GSE64957ZF). Differentially expressed gene (DEG) investigation and comparative analysis were conducted using the limma method. The selection criteria for APA-related genes were as follows: those that are significantly dysregulated in more than three of the four datasets and show a consistent trend of up- or down-regulation in four datasets. 5,375 genes were present in all transcriptome datasets; for the GSE64957ZG, 57 DEGs were identified in the APA group, among which 14, 7, and 37 genes were also dysregulated in the APA group of GSE64957ZF, GSE8514, and GSE60042, respectively. All comparisons among four lists of DEGs exhibited significant enrichment results. The highest degree of overlap was shown in the DEG pair of GSE64957ZG-GSE60042. As a result, approximately 20 genes (12 upregulated and 12 downregulated DEGs), including CYP11B2, PCP4, and ATP2A3, were selected as the final APA-related genes. This study demonstrated several candidate genes that are strongly relevant to APA by examining whole genome disease-related signatures. Besides CYP11B2, it seems that alteration in PCP4 and ATP2A3 genes would be much more involved in the pathogenesis of PA, which warrants further investigation along with validation for the role of the remaining genes. Presentation: Friday, June 16, 2023

OR02-05 Differences In Genomic Characteristics Of Small Aldosterone-producing Lesions Between Normal And Pathologic Adrenals

Abstract Disclosure: J. Lim: None. S. Plaska: None. &amp;. Liu: None. J. Baker: None. A. Blinder: None. W.E. Rainey: None. A.M. Udager: None. Introduction: Primary aldosteronism (PA) – the most common form of secondary hypertension – is characterized by inappropriate adrenal aldosterone production under suppressed renin conditions. The majority of PA is caused by unilateral aldosterone-producing adenomas (APA) and bilateral hyperaldosteronism (BHA). Emerging evidence suggests that BHA is associated with the accumulation of small aldosterone-producing lesions (less than 1 cm in diameter), including aldosterone-producing micronodules (APM) and/or aldosterone-producing nodules (APN). However, previous studies have shown that APM may also be observed in normal adrenals, and thus, the impact of APM and APN on the development and progression of aldosterone overproduction is not well established. In this study, we compare the frequency and distribution of somatic aldosterone-driver mutations in small aldosterone-producing lesions from normal and pathologic adrenals. Methods: Twenty-eight normal adrenals from deceased renal donors (n=24) and patients undergoing radical nephrectomy for renal cell carcinoma (n=4) were compared with 24 pathologic adrenals from patients with PA who underwent adrenalectomy at the University of Michigan. CYP11B2 immunohistochemistry-guided capture was performed to selectively obtain APM and APN DNA from formalin-fixed paraffin embedded tissue, and mutation status for all known aldosterone-driver genes was determined by Ion Torrent-based targeted next-generation sequencing. Results: From normal adrenals, 57 APM from 12 female and 16 male subjects (median age = 49 years) were analyzed, and approximately 46% of these lesions harbored an aldosterone-driver mutation. The most commonly mutated gene in normal samples was CACNA1D (n = 23; 40.4%), but no KCNJ5 mutations were identified, and no significant sex differences were observed. From pathologic adrenals, 56 APM and 7 APN from 17 female and 7 male subjects (median age = 46.5 years) were analyzed, and the frequency of aldosterone-driver mutations (approximately 70%) was significantly higher than normal adrenals (p-value &amp;lt; 0.05). Although CACNA1D mutations were still predominant in pathologic adrenals (n = 33; 52.4%), a number of KCNJ5 mutations were also observed (n = 5; 7.9%) – particularly in APN. Finally, there was a higher rate of aldosterone-driver mutations in small aldosterone-producing lesions in pathologic adrenals from men compared to women (93.3% vs. 62.5%; p-value = 0.051). Conclusions: The frequency and distribution of aldosterone-driver mutations in small aldosterone-producing lesions (APM and APN) in pathologic adrenals are different from those in normal adrenals and may also vary by sex. Further research is needed on how these different APM patterns affect autonomous aldosterone production in patients with PA. Presentation: Thursday, June 15, 2023

Abstract P304: Coronary Flow Reserve In Primary Aldosteronism

Introduction: Primary aldosteronism (PA) is characterized by excess cardiovascular risk, underpinned by evidence of microvascular dysfunction. Whether this extends to the coronary vasculature and, as such, regresses after appropriate treatment remains to be determined. Aim: To investigate the presence of coronary microvascular dysfunction and its reversibility in PA patients. Methods: Fifteen PA patients with evidence of lateralized aldosterone overproduction at adrenal vein sampling underwent Doppler echocardiography for the assessment of Coronary flow reserve (CFR: diastolic peak flow on the anterior descending coronary artery during pharmacological [dipyridamole, n=14] or physical stress [n=1] -induced hyperaemia/basal flow) and the ratio between diastolic peak flow and left ventricle mass (FMR), at the time of diagnosis (after withdrawal of RAAS-interfering drugs and normalization of kalemia with K supplementation) and 4-6 months after specific treatment. In case CFR was ≤ 2.5 or the stress protocol suggested potential ischemia, regardless of CFR, a coronary CT angiography (coroCT) was performed to exclude underlying macrovascular coronary artery disease. Results: Out of 15 PA patients (5F; 52±10 yo; BP:149/91±17/11 mmHg; sK + =3.5±0.6 at diagnosis and 3.8±0.3 mmol/L on KCl supplements; DRC=3.0 [2.0-5.5] mIU/L; PAC=747 [417-1430] pmol/L), 14 were cured by unilateral adrenalectomy (127/81±13/11 mmHg; sK + =4.3±0.4 mmol/L; DRC=11.8 [6.7-19.2] mIU/L; PAC=200 [136-244] pmol/L, p&lt;0.05 for all); one patient experienced incomplete biochemical cure and was managed with long-term mineralocorticoid receptor antagonism. At the time of diagnosis, n=7 (46%), n=3 (20%) and n=1 (7%) patients showed CFR ≤ 3.0, 2.5 and 2.0, respectively; none had evidence of macrovascular disease at coroCT. In parallel to a reduction in left ventricular mass (trend; p=0.084), both CFR and FRM significantly increased after treatment (CFR=3.1 [2.6-3.3] vs. 3.4 [3.1-4.6], p=0.013; FMR=0.71±0.33 vs. 0.91±0.32 cm x g/sec x m 2 , p=0.038). Conclusions: Treatment of PA improves coronary microvascular function, as assessed by established prognostic indices like CFR and FMR.

Integration of clinical parameters and CT-based radiomics improves machine learning assisted subtyping of primary hyperaldosteronism.

The aim of this study was to investigate an integrated diagnostics approach for prediction of the source of aldosterone overproduction in primary hyperaldosteronism (PA). 269 patients from the prospective German Conn Registry with PA were included in this study. After segmentation of adrenal glands in native CT images, radiomic features were calculated. The study population consisted of a training (n = 215) and a validation (n = 54) cohort. The k = 25 best radiomic features, selected using maximum-relevance minimum-redundancy (MRMR) feature selection, were used to train a baseline random forest model to predict the result of AVS from imaging alone. In a second step, clinical parameters were integrated. Model performance was assessed via area under the receiver operating characteristic curve (ROC AUC). Permutation feature importance was used to assess the predictive value of selected features. Radiomics features alone allowed only for moderate discrimination of the location of aldosterone overproduction with a ROC AUC of 0.57 for unilateral left (UL), 0.61 for unilateral right (UR), and 0.50 for bilateral (BI) aldosterone overproduction (total 0.56, 95% CI: 0.45-0.65). Integration of clinical parameters into the model substantially improved ROC AUC values (0.61 UL, 0.68 UR, and 0.73 for BI, total 0.67, 95% CI: 0.57-0.77). According to permutation feature importance, lowest potassium value at baseline and saline infusion test (SIT) were the two most important features. Integration of clinical parameters into a radiomics machine learning model improves prediction of the source of aldosterone overproduction and subtyping in patients with PA.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

Cardiovascular and metabolic characters of KCNJ5 somatic mutations in primary aldosteronism.

Primary aldosteronism (PA) is the leading cause of curable endocrine hypertension, which is associated with a higher risk of cardiovascular and metabolic insults compared to essential hypertension. Aldosterone-producing adenoma (APA) is a major cause of PA, which can be treated with adrenalectomy. Somatic mutations are the main pathogenesis of aldosterone overproduction in APA, of which KCNJ5 somatic mutations are most common, especially in Asian countries. This article aimed to review the literature on the impacts of KCNJ5 somatic mutations on systemic organ damage. PubMed literature research using keywords combination, including "aldosterone-producing adenoma," "somatic mutations," "KCNJ5," "organ damage," "cardiovascular," "diastolic function," "metabolic syndrome," "autonomous cortisol secretion," etc. APA patients with KCNJ5 somatic mutations are generally younger, female, have higher aldosterone levels, lower potassium levels, larger tumor size, and higher hypertension cure rate after adrenalectomy. This review focuses on the cardiovascular and metabolic aspects of KCNJ5 somatic mutations in APA patients, including left ventricular remodeling and diastolic function, abdominal aortic thickness and calcification, arterial stiffness, metabolic syndrome, abdominal adipose tissue, and correlation with autonomous cortisol secretion. Furthermore, we discuss modalities to differentiate the types of mutations before surgery. KCNJ5 somatic mutations in patients with APA had higher left ventricular mass (LVM), more impaired diastolic function, thicker aortic wall, lower incidence of metabolic syndrome, and possibly a lower incidence of concurrent autonomous cortisol secretion, but better improvement in LVM, diastolic function, arterial stiffness, and aortic wall thickness after adrenalectomy compared to patients without KCNJ5 mutations.

PS-BPR01-1: SUCCESSFUL TREATMENT WITH ESAXERENONE IN A PREGNANT WOMAN WITH PRIMARY ALDOSTERONISM AND SUPERIMPOSED PREECLAMPSIA

Background: During pregnancy, there is no established treatment for idiopathic hyperaldosteronism (IHA), the most common form of primary aldosteronism (PA) due to bilateral adrenal hyperplasia. Here, we report the case of a pregnant patient with IHA who was successfully treated with esaxerenone, a non-steroidal mineralocorticoid receptor (MR) antagonist. Case: A 39-year-old woman was referred to our outpatient clinic for hypertension with high plasma aldosterone concentration (PAC). Her PAC was 204 pg/mL (radioimmunoassay) and plasma renin activity was 1.2 ng/mL/h at the time of referral. She was diagnosed with PA since the saline-loading test for PA was positive. She had no hypokalemia and computed tomography scans showed no adrenal tumors. Adrenal vein sampling indicated overproduction of aldosterone in both adrenal glands leading to the diagnosis of IHA. She was commenced on nifedipine controlled release (CR) 20 mg daily because she desired to be pregnant. After one year, she became pregnant. Her blood pressure was well controlled until 34 weeks of gestation when her home blood pressure became elevated up to 140/90 mmHg. Although the dose of nifedipine CR was increased to 80 mg daily, her blood pressure increased to 151/97 mmHg and urinary test showed proteinuria of 2 + in 35 weeks of gestation. She was diagnosed with superimposed preeclampsia (SPE) and additionally treated with esaxerenone. Her blood pressure decreased to 120–140/98–100 mmHg and the proteinuria improved to +/-. A successful cesarean section at 37 weeks resulted in the delivery of a healthy baby boy. Esaxerenone was discontinued 2 weeks after delivery, because she was eager to breast-feed the baby and no safety data of esaxerenone in breast-feeding woman was available. Her blood pressure has been well controlled by nifedipine CR 80 mg daily even after 100 days of postpartum. Conclusions: This is the first case of a pregnant woman who was successfully treated with esaxerenone despite being an elderly pregnant woman who had been diagnosed with IHA and developed SPE. Further studies are needed to investigate the efficacy and safety of non-steroidal selective MR antagonist in similar pregnant patients with IHA, to establish better treatment strategy for these patients.

PS-R07-8: A CASE OF PRIMARY ALDOSTERONISM CAUSED BY BILATERAL ALDOSTERONE-PRODUCING MICROTUMORS WITH A UNILATERAL ADRENAL MASS

Introduction: Primary aldosteronism (PA) is defined as a disease in which overproduction of aldosterone causes various complications such as hypertension, cardiovascular disorder and kidney disease. We herein report a case of PA caused by bilateral aldosterone-producing microtumors with a unilateral adrenal mass. Case presentation: A 54-year-old man was referred to our hospital with a clinical suspicion of PA. He had a history of hypertension and chronic kidney disease for 10 years and had experienced paresthesia that was caused by putaminal hemorrhage in the past. In the initial examination, he exhibited hypertension (150/90 mmHg), high aldosterone (pg/mL)-to-renin (ng/mL/h) ratio (1,744), and overt hypokalemia (K, 2.9 mEq/L). The serum creatinine level was 1.89 mg/dL. The captopril challenge test showed no changes in his blood pressure or plasma aldosterone concentration. These results led to a clinical diagnosis of PA. Computed tomography (CT) revealed the presence of left adrenal mass, so that we suspected that the mass should be responsible for overproduction of aldosterone. We performed adrenal venous sampling. Unexpectedly, the adrenal venous sampling highly suggested the presence of aldosterone-producing microtumors in bilateral adrenal glands (lateralized ratio, 1.3; left contralateral ratio, 0.2). After evaluation, we decided that there was no indication for the removal of left adrenal mass. Then, a low dose of a mineralocorticoid receptor (MR) antagonist was started. Although the dose of an MR antagonist could not be increased sufficiently due to poor renal function, he exhibited an improved control of blood pressure and was followed up thereafter. Discussion and Conclusion: Although PA is one of the most common causes of secondary hypertension, making an accurate diagnosis and appropriate treatment is sometimes problematic due to the difficulty in assessing the site of aldosterone overproduction. The Japanese Endocrine Society clinical guideline states that PA with unilateral adrenal tumor in young patients may not require adrenal venous sampling. From the current case, however, we should always consider the possibility of bilateral microtumors or hyperplasia even in the presence of a unilateral adrenal mass.

PS-B02-14: DISCOVERY OF A NEW DRUG FOR PRIMARY ALDOSTERONISM TARGETING THE CYP11B2 AND KCNJ5 MUTANTS

Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β;-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examing the detailed mechanism of action of the compounds. In addition, we would like to further experiment the effects of the compounds in vivo using hypertensive model mice, Task1 /Task3 double KO mice.

S-13-5: UPDATES ON FAMILIAL HYPERALDSOTERONISM

The first reported (Sutherland et al. 1966) form of familial primary aldosteronism (PA) involved a father and son and was glucocorticoid-remediable (familial hyperaldosteronism type I, FH-I). In 1992, Lifton and colleagues elucidated its causation, a hybrid gene mutation composed of CYP11B1 (encoding 11beta-hydroxylase) regulatory sequences and CYP11B2 (aldosterone synthase) coding sequences causing ACTH-regulated aldosterone overproduction and hypertension, both readily controlled by low-dose glucocorticoid administration. Expression of aldosterone synthase activity within zona fasciculata (ZF) explained overproduction of hybrid steroids, 18-hydroxy- and 18-oxocortisol. Since 2011, PA-causing germline mutations in another four genes (three associated with FH) have been reported. While three encode cation channels (KCNJ5, CACNA1H and CACNA1D), one encodes an anion channel (CLCN2). FH-III is caused by inheritance of gain-of-function mutations in KCNJ5, encoding an inwardly rectifying potassium channel (GIRK-4). Depending on the exact mutation involved, phenotype varies from very severe (profound PA, hybrid steroid elevation and ZF hyperplasia and requiring bilateral adrenalectomy to control hypertension) to mild (absence of above with PA controllable with spironolactone). Most pedigrees demonstrate early-onset hypertension. Mutations reduce selectivity of GIRK-4 to potassium, permitting entry of sodium into the adrenal cortical cell, which results in cell depolarization and calcium influx, leading to increased expression of CYP11B2 and aldosterone synthesis. A gain-of-function mutation in CACNA1H, encoding a T-type voltage-gated calcium channel, was found in five of 40 subjects with early-onset PA and three relatives with early-onset hypertension (FH-IV). Adrenals appeared normal on imaging. The mutation shifts activation of zona glomerulosa (ZG) cells to a more hyperpolarized potential and slows inactivation, putatively resulting in increased calcium influx and aldosterone production. Germline mutations in CACNA1D, encoding an L-type voltage-gated calcium channel highly expressed in ZG, were identified in two of 100 individuals with early-onset PA. Both occurred de novo, and were associated with seizures and a variety of neurological abnormalities (PASNA). Both mutations cause channel activation at less depolarized membrane potentials, and one nearly abolished channel inactivation. In a large Australian family with FH-II, a mutation was found in CLCN2 encoding a chloride channel, ClC-2. CLCN2 mutations were found in another seven of 80 individuals with early-onset PA. A concurrent report found a CLCN2 mutation in one of 12 subjects with early-onset PA. PA due to CLCN2 mutations responds well to medical treatment. In the adrenal, ClC-2 localizes predominantly to ZG and mutations predispose cell depolarization, leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production.

TSPAN12 (Tetraspanin 12) Is a Novel Negative Regulator of Aldosterone Production in Adrenal Physiology and Aldosterone-Producing Adenomas.

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a condition of low-renin hypertension, in which aldosterone overproduction is usually driven by a somatic activating mutation in an ion pump or channel. TSPAN12 is differentially expressed in different subgroups of APAs suggesting a role in APA pathophysiology. Our objective was to determine the function of TSPAN12 (tetraspanin 12) in adrenal physiology and pathophysiology. APA specimens, pig adrenals under dietary sodium modulation, and a human adrenocortical cell line HAC15 were used for functional characterization of TSPAN12 in vivo and in vitro. Gene ontology analysis of 21 APA transcriptomes dichotomized according to high versus low TSPAN12 transcript levels highlighted a function for TSPAN12 related to the renin-angiotensin system. TSPAN12 expression levels in a cohort of 30 APAs were inversely correlated with baseline plasma aldosterone concentrations (R=-0.47; P=0.009). In a pig model of renin-angiotensin system activation by dietary salt restriction, TSPAN12 mRNA levels and TSPAN12 immunostaining were markedly increased in the zona glomerulosa layer of the adrenal cortex. In vitro stimulation of human adrenocortical human adrenocortical cells with 10 nM angiotensin II for 6 hours caused a 1.6-fold±0.13 increase in TSPAN12 expression, which was ablated by 10 μM nifedipine (P=0.0097) or 30 μM W-7 (P=0.0022). Gene silencing of TSPAN12 in human adrenocortical cells demonstrated its inverse effect on aldosterone secretion under basal and angiotensin II stimulated conditions. Our findings show that TSPAN12 is a negative regulator of aldosterone production and could contribute to aldosterone overproduction in primary aldosteronism.

LBODP025 A Novel Drug Discovery For Primary Aldosteronism Targeting Aldosterone Synthase (cyp11b2) And Kcnj5 Mutant

Abstract Primary aldosteronism (PA) is a common cause of secondary hypertension. PA induces damage to cardiovascular and renal blood vessels. Therefore, PA is regarded as a problem because it increases the risk of stroke, cardiomyopathy, kidney disease, etc. Aldosterone-producing adenomas (APA) are one of the major causes of PA, and gene mutations are often identified in tumor cells. KCNJ5 mutation is well known as a gene mutation that causes the loss of ion selectivity in APA. KCNJ5 is an inwardly rectifying potassium channel. KCNJ5 induces sodium ion influx due to mutations in APA. The sodium ion influx induces depolarization, and which overexpresses CYP11B2. CYP11B2 is the rate-determining enzyme for aldosterone synthesis, overexpression of CYP11B2 induces aldosterone overproduction. Examples of therapeutic drugs for PA and APA include calcium channel blockers, aldosterone antagonist and β-blockers. However, therapeutic drugs for PA and APA targeting cell membrane depolarization have not been developed yet. We aim to find compounds that suppress CYP11B2 expression by membrane depolarization to develop novel therapeutic drugs for PA and APA. By developing a therapeutic drug that targets depolarization, we hope to establish an effective therapeutic method for APA caused by gene mutations such as KCNJ5 mutation. We performed high-throughput screening (HTS) using Tohoku University Compound Library (6080 compounds). First, we generated H295R-CYP11B2 N-Luc cells in which the luciferase gene is inserted downstream of the CYP11B2 locus. The cells H295R-CYP11B2 N-Luc were stimulated with KCl to cause depolarization, after which candidate compounds were added and HTS was performed by Luc assay. As a result, 101 compounds were selected. Furthermore, 25 of 101 compounds were selected by examining the cytotoxicity and dose response. Also, 11 of the 25 compounds suppressed CYP11B2 mRNA expression in KCl stimulated H295R cells. Secondary, we generated KCNJ5 mutant cells that expressed the KCNJ5 mutant (L168R) in a Doxycycline-inducible manner. 3 of 11 compounds that suppressed the expression of CYP11B2 mRNA were selected by examination using KCNJ5 mutant cells. After that, 2 of 3 compounds decreased the aldosterone production. These compounds have a different structure from existing therapeutic drugs such as calcium channel blockers. Since these can be novel therapeutic drugs for PA and APA caused by depolarization such as KCNJ5, we are examining the detailed mechanism of action of the compounds. Presentation: No date and time listed

Primary aldosteronism: Pathophysiological mechanisms of cell death and proliferation.

Primary aldosteronism is the most common surgically curable form of hypertension. The sporadic forms of the disorder are usually caused by aldosterone overproduction from a unilateral adrenocortical aldosterone-producing adenoma or from bilateral adrenocortical hyperplasia. The main knowledge-advances in disease pathophysiology focus on pathogenic germline and somatic variants that drive the excess aldosterone production. Less clear are the molecular and cellular mechanisms that lead to an increased mass of the adrenal cortex. However, the combined application of transcriptomics, metabolomics, and epigenetics has achieved substantial insight into these processes and uncovered the evolving complexity of disrupted cell growth mechanisms in primary aldosteronism. In this review, we summarize and discuss recent progress in our understanding of mechanisms of cell death, and proliferation in the pathophysiology of primary aldosteronism.