Autonomous Aldosterone Production Research Articles

Evolutionary Characteristics in Primary Aldosteronism Patients.

Primary aldosteronism is predominantly caused by excessive aldosterone production from the adrenal cortex, and the aldosterone-producing structures could take many forms, like adenomas, nodules, micronodules, and so on. Most studies of primary aldosteronism were limited to the hotspot driver genes responsible for autonomous aldosterone production; however, the panoramic genetic architecture and genomic alterations of aldosterone-producing structures and their adjacent hyperplasia glands remain unknown. In this study, whole-exome sequencing and transcriptome sequencing (RNA-seq) analyses were performed using functional nodules and matched hyperplasia tissues, which were microdissected guided by aldosterone synthase immunohistochemistry. Phylogenetic trees were constructed based on the shared and unique mutations, gene mutation spectrums, and clonal characteristics. The rates of mutations represented higher means of functional nodules than hyperplasia samples, and the little mutational overlap was shown between the 2 groups on phylogenetic trees. The mutations of the aldosterone driver gene (KCNJ5 or CACNA1D) were only observed in functional nodules and indicated almost the largest values of cancer cell fraction. Moreover, the functional nodules also harbored some potential variants related to cell proliferation, which were not detected in hyperplasia tissues. Transcriptome analysis suggested that only 25.5% upregulated and 23.3% downregulated genes overlapped between functional nodules and hyperplasia tissues. This study demonstrated a genetic and transcriptome landscape of aldosterone-producing structures and adjacent hyperplasia glands in primary aldosteronism. The results indicated independent clonal origins on functional nodules and hyperplasia tissues, and little mutual evolutionary relationship was found on their phylogenetic trees.

8399 Adherens Junctions Regulate Aldosterone Production and Zona Glomerulosa Morphogenesis

Abstract Disclosure: M. Berber: None. N.A. Guagliardo: None. P.Q. Barrett: None. B. Haykir: None. V. Chortis: None. K.S. Borges: None. D.L. Carlone: None. D.T. Breault: None. Primary aldosteronism (PA) caused by the autonomous production of aldosterone by zona Glomerulosa (zG) cells of the adrenal gland is the most common cause of endocrine hypertension, which increases the risk of cardiovascular and renal disease, independent of blood pressure. Current treatments with mineralocorticoid receptor antagonists do not fully mitigate these risks. Thus, a better understanding of the mechanisms underlying PA pathogenesis is important for the development of new therapeutic strategies. zG cells are organized in multicellular rosette structures, which are the functional aldosterone-producing units. Rosettes are stabilized by intercellular adherens junctions (AJs) comprised of cadherins, beta-catenin (βCat) and alpha-catenin (αCat), which link to the actin non-muscle myosin II (NMII) network. Recent data implicate AJs and rosettes in aldosterone-producing cell clusters and an unbiased proteomic analysis of aldosterone-producing adenomas detected an upregulation of the GTPase RhoC, an important regulator of AJ stability.We hypothesized that AJs are potent modulators of aldosterone production and zG morphogenesis with important implications for PA. Using the NCI-H295R cell line with confocal microscopic and co-immunoprecipitation assays, we found that potassium (K+) and angiotensin II (AngII) stimulation markedly enhanced the formation and maintenance of AJs. In addition, treatment with inhibitors of Rho-associated kinase (ROCK) (Y-27632) and NMII (blebbistatin) largely abrogated the effects of K+ and AngII stimulation on AJs, suggesting the Rho-ROCK-NMII pathway regulates AJ stability in the zG. Moreover, disruption of AJs, using either blebbistatin or deletion of αCat using CRISPR, blunted the aldosterone response to K+ stimulation (100% and 50%, respectively). Furthermore, zG-specific deletion of αCat (αCat-LOF) in mice led to disruption of AJs, impaired rosette formation and decreased 24-hour urinary aldosterone excretion on standard (Control 5.6±1.7 ng/mg; αCat-LOF 2.9±0.7 ng/mg (aldosterone/creatinine)) or high K+ (Control: 34.7±13.7 ng/mg; αCat-LOF 19.4±9.5 ng/mg (aldosterone/creatinine)) diets. Finally, mice with zG-specific βcat-GOF demonstrated enhanced AJ stability, increased rosette size and zG hyperplasia, which was abrogated in zG-specific bigenic βCat-GOF::αCat-LOF mice, without affecting gene expression of canonical βcat targets. These data indicate that AJs modulate aldosterone production and zG morphology, revealing AJs as a novel molecular target for the treatment of high-risk patients with PA. Presentation: 6/3/2024

Adrenal Vein Sampling: Tips and Tricks.

Adrenal vein sampling (AVS) is the standard method for distinguishing unilateral from bilateral sources of autonomous aldosterone production in patients with primary aldosteronism. This procedure has been performed at limited specialized centers due to its technical complexity. With recent advances in imaging technology and knowledge of adrenal vein anatomy in parallel with the development of adjunctive techniques, AVS has become easier to perform, even at nonspecialized centers. Although rare, anatomic variants of the adrenal veins can cause sampling failure or misinterpretation of the sampling results. The inferior accessory hepatic vein and the inferior emissary vein are useful anatomic landmarks for right adrenal vein cannulation, which is the most difficult and crucial step in AVS. Meticulous assessment of adrenal vein anatomy on multidetector CT images and the use of a catheter suitable for the anatomy are crucial for adrenal vein cannulation. Adjunctive techniques such as intraprocedural cortisol assay, cone-beam CT, and coaxial guidewire-catheter techniques are useful tools to confirm right adrenal vein cannulation or to troubleshoot difficult blood sampling. Interventional radiologists should be involved in interpreting the sampling results because technical factors may affect the results. In rare instances, bilateral adrenal suppression, in which aldosterone-to-cortisol ratios of both adrenal glands are lower than that of the inferior vena cava, can be encountered. Repeat sampling may be necessary in this situation. Collaboration with endocrinology and laboratory medicine services is of great importance to optimize the quality of the samples and for smooth and successful operation. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

FRI144 Using Single Cell Technology To Predict The Cell Of Origin Of Aldosterone-producing Adrenal Adenomas

Abstract Disclosure: K. Laycock: None. C. Cabrera: None. E. Wozniak: None. C. Mein: None. E. Azizan: None. X. Wu: None. G. Argentesi: None. E. Goodchild: None. W. Drake: None. M.J. Brown: None. Background: Aldosterone-producing adrenal adenomas (APAs) are the commonest surgically curable cause of hypertension, but individual prediction of postoperative outcome is challenging. Emerging data suggest that outcomes are influenced by APA genotype; identifying the cell of origin of APAs could facilitate a greater understanding of why certain genotypes are more/less likely to be associated with residual/recurrent autonomous aldosterone production(1, 2). Aims: 1. To develop an adult adrenal single cell atlas. 2. To identify the likely cell of origin for APAs with different mutations. Methodology: Bulk RNA sequencing from 67 APAs discovered known or novel somatic genotypes in 55, and replicated or discovered distinctive transcriptomes for each genotype. Single nuclei RNA sequencing was performed on 13 adrenals adjacent to the genotyped APAs. After aggregation of all 13 datasets, cluster analysis was undertaken to identify cell types and some of the transitions between these. Similarities were sought between the genotype-enriched transcriptomes of APAs, and the transcriptomes of individual cell clusters in adjacent adrenal. Results: A single nuclei atlas of 56,442 nuclei was developed. Clusters included each of the 3 cortical zones, aldosterone-producing micronodules, capsular cells, adrenal medulla, macrophages, T cells, stromal cells, endothelial cells and neurones. Several zona glomerulosa selective transcripts in previous laser-capture studies(3, 4) clustered separately from CYP11B2-positive cells (e.g. LGR5, GABBR2) or in distinct non-steroidogenic clusters (e.g. KIAA1210, RSPO3, PTGDS).A sub-cluster of stromal cells, positive for KIAA1210, RSPO3, CDH13 and MFAP5 (each p<10-254 cf other clusters) is likely to represent the distinctive KIAA1210+ capsular/interstitial cells on adrenal immunohistochemistry (www.proteinatlas.org). Bulk RNA sequencing of a rare CLCN2-mutant APA showed 13-30-fold higher expression of these and multiple other genes localising to the single nuclei stromal-cell cluster. Conclusion: Single nuclei sequencing documents the heterogeneity of adrenal cells, and potential for multiple different cell-types to develop into APAs. Ongoing analysis is underway to replicate the CLCN2-mutant transcriptome and identify cells-of-origins for tumours with commoner mutations. 1.Wu X et al. 11C metomidate PET-CT versus AVS for diagnosing surgically curable PA: a prospective within-patient trial. Nat Med. In press 2.Zhou J et al. Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant APAs presenting in puberty, pregnancy or menopause. Nat Genet. 2021 3.Nishimoto K et al. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands. Proc Natl Acad Sci USA. 2015 4.Shaikh LH et al. LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal. J Clin Endocrinol Metab. 2015 Presentation: Friday, June 16, 2023

FRI154 Saline Suppression Testing And Acute Hypocalcemia: Impacts On Diagnostic Testing For Primary Aldosteronism

Abstract Disclosure: W.W. Parksook: None. J.M. Brown: Consulting Fee; Self; Bayer, Inc. J. Milks: None. A. Moore: None. Y. Niebuhr: None. B. Honzel: None. A. Vaidya: Consulting Fee; Self; HRA Pharmaceuticals, Mineralys. Background: Calcium ion signaling is a critical factor required for the physiologic production of aldosterone. Emerging evidence has revealed that the majority of cases of primary aldosteronism involve pathogenic somatic mutations that ultimately increase calcium signaling and autonomous aldosterone production. Therefore, testing for primary aldosteronism that modulates calcium homeostasis may influence the diagnostic interpretation of the results. Method: Overweight to obese participants with metabolic risk factors and pre-hypertension or stage 1 hypertension (n=56) underwent a standard seated saline suppression test (SSST). Measurement of blood pressure, plasma aldosterone, serum calcium, as well as other factors, was performed at times 0, 120, and 240 minutes during SSST. Linear regression models were used to analyze the association between changes in calcium and each parameter. Results: Following 2 liters of saline infusion during SSST, all 56 participants (100%) had a significant decrease in serum calcium levels from baseline, with 26 (47%) developing new-onset frank hypocalcemia (calcium <8.6 mg/dL). The mean baseline serum calcium was 9.2 ± 0.3 mg/dL, which declined to 8.9 ± 0.4 mg/dL at 120 mins (P<0.001), and to 8.6 ± 0.4 mg/dl (P<0.001) at 240 mins when the SSST concluded. Consistent with the known vasopressor effect of calcium, greater drops in serum calcium levels were associated with lower blood pressure at 240 minutes (β;= 13.5, P<0.01 for DBP; β;= 10.7, P= 0.06 for SBP). Among those with post-SSST aldosterone levels <10 ng/dl (a categorical threshold used to exclude primary aldosteronism), greater reductions in serum calcium levels were associated with larger drops in aldosterone levels (β;= 9.9, P<0.05). Conclusion: The standard SSST induced a substantial decline in serum calcium levels in 100% of participants, with nearly half of all participants developing frank hypocalcemia at the end of saline infusion. Since calcium is known to play a critical role in physiologic and pathophysiologic production of aldosterone, these findings raise the question whether hypocalcemia induced by the acute loading of 2 liters of saline may confound the interpretation of aldosterone changes. Further research should focus on better understanding the diagnostic implications of these results and potential limitations of the SSST to exclude primary aldosteronism. 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. &. 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 < 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

Primary aldosteronism: molecular medicine meets public health.

Primary aldosteronism is the most common single cause of hypertension and is potentially curable when only one adrenal gland is the culprit. The importance of primary aldosteronism to public health derives from its high prevalence but huge under-diagnosis (estimated to be <1% of all affected individuals), despite the consequences of poor blood pressure control by conventional therapy and enhanced cardiovascular risk. This stateofaffairs is attributable to the fact that the tools used for diagnosis or treatment are still those that originated in the 1970-1990s. Conversely, molecular discoveries have transformed our understanding of adrenal physiology and pathology. Many molecules and processes associated with constant adrenocortical renewal and interzonal metamorphosis also feature in aldosterone-producing adenomas and aldosterone-producing micronodules. The adrenal gland has one of the most significant rates of non-silent somatic mutations, with frequent selection of those driving autonomous aldosterone production, and distinct clinical presentations and outcomes for most genotypes. The disappearance of aldosterone synthesis and cells from most of the adult human zona glomerulosa is the likely driver of the mutational success that causes aldosterone-producing adenomas, but insights into the pathways that lead to constitutive aldosterone production and cell survival may open up opportunities for novel therapies.

CXCR4-DIRECTED [68GA]GA-PENTIXAFOR PET/CT AS A NOVEL DIAGNOSTIC MODALITY IN PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is caused by autonomous production of aldosterone by either a unilateral aldosterone-producing adenoma (APA) or by bilateral adrenal hyperplasia (BAH). Distinction (‘subtyping’) is crucial, PA is cured by adrenalectomy in unilateral PA and is treated by medication in bilateral PA. Subtyping is currently done by adrenal vein sampling (AVS). We propose a new diagnostic imaging modality employing the PET-tracer [68Ga] Ga-PentixaFor. This tracer reportedly has high uptake in APAs, whereas uptake is low in BAH or normal adrenals. The goal of this study is to determine concordance of [68Ga] Ga-PentixaFor PET/CT and AVS. Secondary outcomes are optimal timepoint for scanning and quantitative criteria for lateralization at the optimal timepoint. Design and method: This study is step 1 of a two-step randomized controlled diagnostic trial. We included patients with PA from Radboudumc Nijmegen and UMC Utrecht, confirmed by an intravenous salt-loading test. Patients underwent AVS and [68Ga]Ga-PentixaFor PET/CT. A dynamic scan was performed to determine optimal timepoint for scanning in the first 6 patients. The main outcome was the concordance of AVS and [68Ga]Ga-PentixaFor PET/CT. The SUVmax was used to determine lateralization in [68Ga]Ga-PentixaFor PET/CT. Results: Nineteen patients underwent adrenal vein sampling and a [68Ga]Ga-PentixaFor PET/CT. The optimal timepoint of scanning was 1h post injection. AVS showed lateralization in 13/19 patients. Using a cut-off of SUV¬max-ratio of 1.4, concordance was 68%. (figure 1) Using a Bayesian prediction model, the predicted concordance is: 67% (CI80% = 53 - 79). Figure 1: Scatter dot plot of the SUVmax Ratio in patients who had lateralization or no lateralization on AVS with a cut-off value of 1.4. Conclusions: [68Ga]Ga-PentixaFor PET/CT is a new imaging modality with high concordance with AVS. It is safe, readily available and as an out-patient procedure has the potential to improve subtyping in PA. With these preliminary results we have sufficient evidence of concordance to proceed to step 2 of the two-step trial: A pragmatic randomized controlled diagnostic trial, in which patients will be randomized in either [68Ga]Ga-PentixaFor PET/CT or AVS.

An Abbreviated History of Aldosterone Metabolism, Current and Future Challenges.

The initial isolation of adrenal steroids from large quantities of animal adrenals resulted in an amorphous fraction resistant to crystallization and identification and had potent effects on electrolyte transport. Aldosterone was eventually isolated and identified in the fraction and was soon shown to cause hypertension when in excess. The autonomous and excessive production of aldosterone, primary aldosteronism, is the most common cause of secondary hypertension. Aldosterone is metabolized in the liver and kidney, and its metabolites are conjugated with glucuronic acid for excretion. The most common liver metabolite is 3α,5β-tetrahydroaldosterone-3-glucuronide, while that of the kidney is aldosterone-18-oxo-glucuronide. In terms of their value, especially the aldosterone-18-oxo-glucuronide, is commonly used for the diagnosis of primary aldosteronism because they provide an integrated value of the total daily production of aldosterone. Conversion of aldosterone to 18-oxo-glucuronide is impeded by drugs, like some common non-steroidal anti-inflammatory drugs that compete for UDP-glucuronosyltransferase-2B7, the most important glucuronosyltransferase for aldosterone metabolism. Tetrahydroaldosterone is the most abundant metabolite and the most reliable for the diagnosis of primary aldosteronism, but it is not commonly measured.

Autophagy induction protects the perivascular adipose tissue during hyperaldosteronism-induced hypertension

Hyperaldosteronism (HA), characterized by an autonomous production of aldosterone, promotes increased blood pressure and cardiovascular damage. The perivascular adipose tissue (PVAT) participates of the control of vascular function and is inflamed in HA. Autophagy is a recycling process of unnecessary or dysfunctional components and it is important for inflammatory tolerance, but whether the autophagic flux is dysregulated in the PVAT during HA, which would contribute to the inflammation and dysfunction of this tissue, is still to be elucidated. Herein, we hypothesize that treatment with autophagy inducer will rescue the autophagic unbalance, subsequently decreasing inflammation, and improving PVAT function during HA. 10-12-weeks-old male C57BL/6J 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 the tail-cuff. Brown adipocyte cells (WT1) were used to evaluate the molecular mechanisms by which HA induces PVAT dysfunction. HA promoted an increase in systolic blood pressure [mmHg – before HA: 123.8±0.9 vs. after HA: 149.5±1.1]. Furthermore, PVAT decreased the contractile response [Emax (% KCl)] in control arteries [Control PVAT (-): 137.2±1.9 vs. Control PVAT (+): 107.4±2.7], whereas HA promoted loss of PVAT anti-contractile effect [HA PVAT (-): 159.9±2.9 vs. HA PVAT (+): 164.1±3.4]. We found that PVAT from HA mice presents an unbalance in LC3II/LC3I ratio, an important autophagy marker, inflammation, characterized by elevated TNF-α, IL-6, IL-1β, IL-17, and IFN-γ gene expression, and elevated reactive oxygen species (ROS). To induce autophagy, we treated mice with TB-peptide (16mg/Kg/day via ip for 7 days). TB-peptide partially improved the PVAT function of mice with HA [HA PVAT (+): 122.5±4.1]. On mechanistic levels, aldosterone (100nM, 12-96 h) induced an oscillatory autophagy flux in WT1 cells, characterized by an initial increase in LC3II/LC3I ratio at 12 and 24 h, followed by a decrease at 48 and 96 h. These effects were not observed in the presence of the mineralocorticoid receptor antagonist (Eplerenone, 1μM). Aldosterone also led to the expression of pro-inflammatory genes (TNF-α, IL-6, IL-1β, IL-17, and IFN-γ) and elevated ROS, such effects were prevented by inducing autophagy with TB-peptide. In summary, these findings reveal a new mechanism in vascular injury associated with HA, aldosterone impairs autophagic flux in PVAT, inducing inflammation, oxidative stress, and dysfunction. Our study places PVAT and autophagic process as attractive targets to reduce cardiovascular risk in HA. FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), 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 2023 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.

Phosphoproteomics Reveals the Wolframin-Calcium Axis as an Important Pathogenic Signaling Node in Primary Aldosteronism.

Aldosterone-producing adenoma (APA) is a benign adrenal tumor with autonomous aldosterone production which causes hypertension and excess cardiovascular risk. Protein phosphorylation regulates aldosterone secretion from adrenal cortical cells, but how signaling networks are remodeled in APA remains unknown. We performed an integrated proteomic and phosphoproteomic profiling of 15 APA and 10 matched nonfunctioning adrenocortical tumors (NFAT) based on the 4-dimensional label-free technique. We further validated our main findings in enlarged APA samples, mice, and adrenocortical cell line. The proteomic and phosphoproteomic profiling of APA and NFAT quantified 5989 proteins and 9011 phosphopeptides. We highlighted differentially expressed and phosphorylated proteins which modulated aldosterone synthesis and secretion from APA. As intracellular calcium is the central signal for aldosterone synthesis, our integrated calcium signaling network implicated wolframin in the control of calcium influx and CYP11B2 (aldosterone synthase) activation in APA (ratio of wolframin expression in APA to NFAT: 6.411, P<0.001). Among 97 APA cases for validation, a higher expression level of wolframin was associated with a higher plasma aldosterone concentration postcaptopril challenge test and a higher systolic blood pressure. In vitro, the secretion of aldosterone was enhanced by wolframin overexpression, while aldosterone secretion in response to potassium or angiotensin II was inhibited by the knockdown of wolframin. Further in vivo and in vitro data demonstrated the wolframin-calcium axis as an important regulator of CYP11B2 expression and aldosterone production. Wolframin is a regulatory protein in aldosterone hypersecretion. Remodeled calcium transportation and mitochondrial function are involved in wolframin-related aldosterone secretion.

Ketosis, Salt, and Water: Novel Mechanistic Insights into Diet and Mineralocorticoid Metabolism.

The association between dietary macronutrient composition and cardiovascular health has been studied extensively for decades (1, 2). High dietary sodium intake has traditionally been linked with cardiovascular disease and mortality, although recent data suggest that this association may be negligible where mean intake is less than 5 g/day. The renin-angiotensin-aldosterone system (RAAS) is a key regulator of circulating blood volume, as well as sodium and potassium homeostasis. Renin production from the renal juxtaglomerular apparatus is stimulated by reduced circulating blood volume, thereby stimulating aldosterone secretion from zona glomerulosa cells of the adrenal cortex. Primary aldosteronism, due to a unilateral aldosterone-producing adenoma (Conn's syndrome) or bilateral hyperplasia of the glomerulosa, is an important cause of secondary hypertension and has independent associations with cardiovascular disease (3). In the absence of autonomous aldosterone production, antagonism of the mineralocorticoid receptor is a key therapeutic approach in hypertension and heart disease by lowering blood pressure and improving cardiac remodeling.

Primary Aldosteronism and COVID-19-related Management, Disease Severity, and Outcomes: A Retrospective Cohort Study.

The SARS-CoV-2 virus is dependent on components of the renin-angiotensin-aldosterone system for infectivity. Primary aldosteronism (PA) is a form of secondary hypertension mediated by autonomous aldosterone production. The intersection of COVID-19 and PA, both which may involve components of the renin-angiotensin-aldosterone system, remains unknown. We assessed PA as a risk factor for COVID-19 infection and compared management, severity of disease, and outcomes during COVID-19 with a matched population of patients with essential hypertension (EH) by conducting a retrospective observational cohort study. Of the patients with PA, 81 had a negative PCR test for COVID-19, whereas 43 had a documented positive PCR test for COVID-19. Those patients with PA who tested positive for COVID-19 tended to be female (P = .08) and the majority of those with COVID-19 infection identified as non-White race (P = .02) and Hispanic ethnicity (P = .02). In a subanalysis, 24-hour urine aldosterone on initial PA diagnosis tended to be higher those in the PA group who developed COVID-19 compared with those in the PA group who did not develop COVID-19 [median (interquartile range): 36.5 (16.9, 54.3) vs 22.0 (15.8, 26.8) mcg, P = .049] and was an independent predictor of COVID-19 infection controlling for sex, race, and ethnicity. Angiotensin-converting enzyme inhibitor, angiotensin II receptor blocker, and mineralocorticoid receptor antagonist use did not differ between those patients with PA who did and did not have COVID-19 infection. Comparing those patients with PA and matched patients with EH (n = 286) who were COVID-19 PCR positive, there was a significantly higher incidence of cardiovascular complications (12 vs 2%, P = .004) in the PA vs EH group. These data begin to inform us as to whether PA should be a newly identified subpopulation at risk for COVID-19-related cardiovascular disease sequelae.

Adrenal Anion Channels: New Roles in Zona Glomerulosa Physiology and in the Pathophysiology of Primary Aldosteronism.

The mineralocorticoid aldosterone is produced in the zona glomerulosa of the adrenal cortex. Its synthesis is regulated by the serum concentrations of the peptide hormone angiotensin II and potassium. The primary role of aldosterone is to control blood volume and electrolytes. The autonomous production of aldosterone (primary aldosteronism, PA) is considered the most frequent cause of secondary hypertension. Aldosterone-producing adenomas and (micro-)nodules are frequent causes of PA and often carry somatic mutations in ion channels and transporters. Rare familial forms of PA are due to germline mutations. Both somatic and germline mutations in the chloride channel gene CLCN2, encoding ClC-2, have been identified in PA. Clinical findings and results from cell cultureand animal models have advanced our knowledge about the role of anions in PA. The zona glomerulosa of the adrenal gland has now been firmlyestablished as a tissue in which anions play a significant role for signaling. In this overview, we aim to summarize the current knowledge and highlight novel concepts as well as open questions.

PS-BPC02-2: CLINICAL IMPACTS OF ENDOTHELIUM-DEPENDENT FLOW-MEDIATED VASODILATION ASSESSMENT ON PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is defined as autonomous aldosterone production from the adrenal glands and represents the most common and treatable cause of hypertension. Also, it is well known that the incidence of cardiovascular morbidity is higher among patients with PA than among those with essential hypertension (EH). Histologically, PA is divided into two major subtypes, aldosterone-producing adenoma (APA) and bilateral idiopathic hyperplasia (IHA). However, the nature of vascular function in PA patients remains to be determined. The aim of this study was to determine the vascular function and investigate the implications of vascular function assessments in the patients with PA. Design and Method: All patients were diagnosed with PA according to the guidelines of the Japan Endocrine Society. Flow-mediated dilation (FMD), as an index of endothelial function, and cardio-ankle vascular index (CAVI), as an index of arterial stiffness, were retrospectively compared between 42 patients with APA, 37 patients with IHA, and 42 patients with EH. These values were also compared with background factors, KCNJ5 mutation and clinical outcome in terms of blood pressure reduction after adrenalectomy in the APA group. Results: No differences were found among the three groups (APA, IHA and EH) with regard to age, sex, duration of hypertension, body mass index, blood pressure, renal function and hemoglobin A1c. FMD was significantly lower in the APA group (4.8 ± 2.1%) and IHA group (4.1 ± 1.9%) than in the EH group (5.7 ± 2.1%). CAVI did not differ significantly among APA, IHA and EH groups (7.9 ± 1.3, 7.8 ± 1.0, 7.8 ± 1.1, respectively). Although no significant correlations were seen between FMD and background factors (i.e. age, body mass index, renal function, glucose and lipid metabolic parameters, and hormonal data) in the IHA group, FMD correlated negatively with BMI and plasma aldosterone concentration in the APA group (rs = -0.313, rs = -0.342, respectively). No significant difference in FMD (4.8 ± 1.4%, 4.5 ± 2.1%, respectively) or CAVI (8.1 ± 1.8, 8.0 ± 1.1, respectively) was seen between the KCNJ5-mutant group and the wild-type group. A cut-off FMD of 4.3% was selected for complete clinical success after adrenalectomy, offering 78% sensitivity and 80% specificity. Conclusions: Patients with PA displayed impaired endothelial function. Complete clinical success after adrenalectomy was associated with preserved endothelial function. This study provides a better understanding of FMD assessment in patients with PA.

PS-C37-5: NEW THERAPEUTIC PERSPECTIVES FOR BLOOD PRESSURE CONTROL: DEXFADROSTAT PHOSPHATE, A NOVEL ALDOSTERONE SYNTHASE INHIBITOR, IN PATIENTS WITH PRIMARY ALDOSTERONISM

Objective: Aldosterone, the primary mineralocorticoid produced by the adrenal gland, regulates fluid homeostasis by stimulating sodium reabsorption in exchange for potassium in the distal tubules and collecting duct of the kidney. Aldosterone synthase (CYP11B2) is the rate-limiting enzyme for aldosterone biosynthesis. Patients with primary aldosteronism (PA) are characterized by an uncontrolled, excess production of aldosterone and are at higher risk of stroke, atrial fibrillation, and heart failure compared to patients with essential hypertension. Furthermore, PA increases the risk of death even after initiation of medical treatment to control blood pressure. Therefore, PA is the compelling indication to demonstrate the value for an aldosterone synthase inhibitor (ASI) as targeted treatment. Previous attempts to eliminate the excessive spontaneous aldosterone synthesis defining PA have been unsuccessful, particularly due to the unintended inhibition of other steroid hormones. Dexfadrostat phosphate is a novel ASI that acts directly on CYP11B2, targeting the root cause of primary aldosteronism. Design and method: Dexfadrostat phosphate was first investigated in patients with PA diagnosed within 1 year of study entry according to medical guidelines including a suppression test to demonstrate autonomous aldosterone production. Eligibility was verified and approved by a Central Review panel. Following a 2-week placebo-controlled run-in period, enrolled patients were randomized to one of three doses of dexfadrostat taken orally, once daily. After 8 weeks of treatment, patients were switched to placebo and followed for an additional 2 weeks. Blood samples were taken at 2-week intervals and evaluated in a central laboratory for steroid and peptide hormone as well as electrolyte levels. Blood pressure was measured in the office during each clinic visit. Ambulatory systolic blood pressure (aSBP) was monitored over 24-hours at baseline and after 8 weeks of dexfadrostat treatment. Results: All endpoints of the study were met with high significance. Dexfadrostat phosphate treatment was safe and well tolerated. We present the effects on the aldosterone-to-renin ratio (ARR) and subsequent blood pressure reduction, the correction of hypokalemia, and the persistence and reversal of therapeutic effects upon drug withdrawal. Conclusions: The phase 2 study in patients evaluated the ability of dexfadrostat phosphate to deliver both biochemical (ARR, potassium) and subsequent clinical (aSBP) correction of the consequences of uncontrolled aldosterone production. By demonstrating its clinical utility in a rarely diagnosed indication of extreme aldosterone dysregulation, the potential of dexfadrostat phosphate may well expand to address essential hypertension, chronic kidney disease, hypokalemia, volume expansion and organ remodeling.

Spontaneous Remission of Primary Aldosteronism with Mineralocorticoid Receptor Antagonist Therapy: A Review.

In this review, we describe previous basic and clinical studies on autonomous aldosterone production. Over the past decades, mineralocorticoid receptor antagonists (MRAs) have been found to concentration-dependently inhibit steroidogenesis in different degrees. However, many studies have proven the suppressive effects of MRAs on the activities of hormone synthase. The probable factors of cytochrome P-450 reduction, both in microsomes and mitochondria, have also been considered: (1) one of the spironolactone metabolite forms had destructive function, except canrenone, (2) 7α-thio-spironolactone was an obligatory intermediate in the spironolactone-induced CYP450 decrease, and (3) the contributing steroids should have 7α-methylthio or 7α-methylsulfone groups. In previous clinical research, spironolactone-body-containing cells showed a type II pattern of enzyme activity (i.e., enhanced 3β-hydroxysteroid dehydrogenase, glucose-6-phosphate, and NADP-isocitrate dehydrogenase activities and weaken succinate dehydrogenase activity), and the subcapsular micronodules composed of spironolactone-body-containing cells also exhibited a type II pattern and excess aldosterone secretion, indicating that the subcapsular micronodules might be the root of aldosterone-producing adenoma. Moreover, combined with the potential impeditive function to aldosterone secretion, a few cases of spontaneous remission of primary aldosteronism, with normal ranges of blood pressure, plasma potassium, plasma renin activity, and aldosterone renin ratio, have been reported after long-term treatment with MRAs.

LBMON299 Aldosterone Production Is Regulated By Gap Junctions In Human Adrenal Cells

Abstract Aldosterone plays a critical role in blood pressure regulation via the renin-angiotensin-aldosterone system. Autonomous production of aldosterone, primary aldosteronism (PA), is the commonest curable cause of hypertension worldwide. Two cell types in the adrenal cortex produce aldosterone: zona glomerulosa (ZG) cells and aldosterone producing nodules (APN), and in cells of aldosterone producing adenomas (APA). Adrenal cortex cells undergo phenotypic metamorphosis as they migrate from the subcapsular region, centripetally, towards the corticomedullary junction, changing from aldosterone- to cortisol- producing cells as they go. Gap junctions (GJs) are specialised channels of cytoplasmic communication between cells, permitting the regulated transfer of substrates. Loss-of-function mutations in the CADM1 gene, which affects GJ protein CX43, have been found in APAs, implicating CX43 in aldosterone regulation. This study aims to demonstrate the dysregulation of aldosterone production by CX43 inhibition in human adrenal cell line, H295R, and elucidate GJ distribution in human adrenal cortex. Methodology H295R cells were treated with 1×10-8M angiotensin II and connexin mimetic GJ inhibitor, GAP27, at 0µM, 25µM, 83µM and 250µM. Immunofluorescence (IF) was used to identify the abundance of CX43 in different cell types of the adrenal cortex, performed on ex-vivo para-APA adrenal tissue, using antibodies against CX43 (GJA1), colocalised with markers of aldosterone-producing cells (aldosterone synthase, CYP11B2), cells of the zona glomerulosa (ZG) (VSNL1, and DAB2) and zona fasciculata (ZF) (CYP7A1). Results Inhibition of CX43 with Gap27 results in a dose-dependent increase in CYP11B2 mRNA expression and aldosterone production. At the highest concentration of 250µM GAP27, CYP11B2 mRNA expression increased by 133-fold (p=&amp;lt;0. 00001) and aldosterone production by 29-fold (p=0. 0006). IF studies demonstrate a crescendo gradient of CX43 in a centripetal direction from cells of APN to ZF. The abundance was lowest in aldosterone-producing APN cells, moderate in non-aldosterone-producing ZG cells and highest in cortisol-producing ZF cells. GJ distribution was pan-membranous in ZF, but, where present, mostly cytoplasmic in APN. Evidence of recent active communication, in the form of annular gap junctions (AJG), was present in all three cell types. Conclusion The CX43 inhibition-induced upregulation of CYP11B2 expression and aldosterone synthesis indicates that it has an important regulatory role in physiological aldosterone production via CYP11B2 expression. The distribution of CX43 in adrenal tissue suggests its transport substrate not only regulates aldosterone synthesis, but also in adrenal cortex cell phenotypic metamorphosis/migration. Future work will investigate the substrate of CX43 in adrenal cells and its possible effect on adrenal cell phenotype. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

OR20-3 Characterization of the Pathophysiologic Spectrum of Subclinical Primary Aldosteronism in Normotension

Abstract Context Accumulating evidence suggests that primary aldosteronism can manifest across a broad phenotypic spectrum and that its origins can at times be detected among normotensive individuals. Herein, we conducted deep-phenotyping studies to interrogate the pathophysiologic continuum of the primary aldosteronism phenotype among normotensive participants. Methods Normotensive participants with obesity, metabolic syndrome, and/or high-normal untreated blood pressure underwent dietary sodium loading and restriction protocols (to maximally suppress and stimulate angiotensin II-mediated aldosterone production), dexamethasone suppression and cosyntropin stimulation protocols (to maximally suppress and stimulate ACTH-mediated aldosterone production), and 24h ambulatory blood pressure monitoring. Measurement of aldosterone and 18-hybrid steroids via LC-MS/MS was conducted to quantify the degree of dysregulated CYP11B2 expression. Urinary extracellular vesicles (uEV) were isolated to measure renal tubular protein content associated with mineralocorticoid receptor activation. In order to characterize parameters across the severity spectrum of non-suppressible and renin-independent aldosterone production, participants with suppressed renin activity were categorized by tertile of 24h urinary aldosterone production following oral sodium suppression testing. Repeated measures linear mixed models were used to analyze trends for each parameter across tertiles of autonomous aldosterone production; for ease of presentation, the mean [SD] of the first vs. third tertile are presented for selected parameters. Results Following oral sodium loading, greater non-suppressible renin-independent aldosterone production was associated with greater kaliuresis, higher daytime ambulatory systolic blood pressure within the normal range, and a progressive impairment in the ability to suppress production of serum aldosterone (P-trend=0.009), 18-hydroxycorticosterone (9.3 [5.0] vs. 19.6 [6.5] ng/dL, P-trend=0.004), 18-hydroxycortisol (45.0 [25.4] vs. 70.9 [29.9] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.03] vs. 3.4 [7.2], P-trend=0.01). Similarly, following dexamethasone suppression testing, there was a progressive inability to suppress 18-hydroxycorticosterone (8.3 [5.0] vs. 15.4 [8.2] ng/dL, P-trend=0.004), 18-hydroxycortisol (6.6 [3.8] vs. 10.2 [3.6] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.01] vs. 1.4 [0.6] ng/dL, P-trend=0.01) in parallel with greater renin-independent aldosterone production. In contrast, following stimulation with cosyntropin, greater non-suppressible and renin-independent aldosterone production was associated with progressively enhanced production of 18-hydroxycorticosterone (77.6 [25.4] vs. 121.1 [31.1] ng/dL, P-trend=0.004), 18-hydroxycortisol (68.5 [28.4] vs. 86.2 [26.3] ng/dL, P-trend=0.03), and 18-oxocortisol (3.2 [2.8] vs. 7.3 [3.5] ng/dL, P-trend=0.01). Excretion of uEV's of pendrin (P=0.002) and aquaporin 2 (P=0.002) all increased with greater non-suppressible aldosterone production. Conclusions Dynamic phenotyping studies in normotensive individuals demonstrate a continuum of non-suppressible and renin-independent aldosterone production that parallels higher blood pressure, greater kaliuresis, increased 18-hybrid steroid production indicative of dysregulated CYP11B2 expression, and increased uEV excretion indicative of renal mineralocorticoid receptor activation. This pathophysiologic continuum of normotensive primary aldosteronism provides phenotypic support for histopathology studies showing ectopic CYP11B2 expression within adrenal glands of normotensive humans, and for epidemiologic studies demonstrating renin-independent aldosterone production in normotensive populations increases risk for developing hypertension. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Update on the Genetics of Primary Aldosteronism and Aldosterone-Producing Adenomas.

Primary aldosteronism (PA) is the leading cause of secondary hypertension, accounting for over 10% of patients with high blood pressure. It is characterized by autonomous production of aldosterone from the adrenal glands leading to low-renin levels. The two most common forms arise from bilateral adrenocortical hyperplasia (BAH) and aldosterone-producing adenoma (APA). We discuss recent discoveries in the genetics of PA. Most APAs harbor variants in the KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1 genes. With the exception of β-catenin (CTNNB1), all other causative genes encode ion channels; pathogenic variants found in PA lead to altered ion transportation, cell membrane depolarization, and consequently aldosterone overproduction. Some of these genes are found mutated in the germline state (CYP11B2, CLCN2, KCNJ5, CACNA1H, and CACNA1D), leading then to familial hyperaldosteronism, and often BAH rather than single APAs. Several genetic defects in the germline or somatic state have been identified in PA. Understanding how these molecular abnormalities lead to excess aldosterone contributes significantly to the elucidation of the pathophysiology of low-renin hypertension. It may also lead to new and more effective therapies for this disease acting at the molecular level.