Increased Aldosterone Production Research Articles

Aldosterone-induced salt appetite requires HSD2 neurons.

Excessive aldosterone production increases the risk of heart disease, stroke, dementia, and death. Aldosterone increases both sodium retention and sodium consumption, and increased sodium consumption may worsen end-organ damage in patients with aldosteronism. Preventing this increase could improve outcomes, but the behavioral mechanisms of aldosterone-induced sodium appetite remain unclear. In rodents, we previously identified aldosterone-sensitive neurons, which express the mineralocorticoid receptor and its pre-receptor regulator, 11-beta-hydroxysteroid dehydrogenase 2 (HSD2). In the present study, we identified HSD2 neurons in the human brain, then used a mouse model to evaluate their role in aldosterone-induced salt intake. First, we confirmed that dietary sodium deprivation increases aldosterone production, salt intake, and HSD2 neuron activity. Next, we showed that continuous chemogenetic stimulation of HSD2 neurons causes a large and specific increase in salt intake. Finally, we use dose-response studies and genetically targeted ablation of HSD2 neurons to show that these neurons are necessary for aldosterone-induced salt intake. Identifying HSD2 neurons in the human brain and establishing their necessity for aldosterone-induced salt intake in mice improves our understanding of appetitive circuits and highlights this small cell population as a therapeutic target for moderating dietary sodium.

Baxdrostat and finerenone: new aldosterone synthase-aldosterone-mineralocorticoid receptor hormonal system inhibitors for the drug treatment of resistant arterial hypertension

Resistant arterial hypertension is characterized by failure to control target blood pressure despite long-term use of optimal or maximum tolerated doses of three different antihypertensive drugs, including diuretic. Patients with resistant hypertension are included in a group of people at high risk of cardiovascular and renal complications, including accelerated progression of chronic kidney disease with a more rapid transition to the final stage of the disease. Resistant hypertension is based on a salt-sensitive, volume-dependent form of hypertension, which usually occurs against the background of increased aldosterone production and normal or even decreased renin plasma activity. A key role in its formation is played by an increase of sodium reabsorption in the kidneys, associated with excessive activity of aldosterone-sensitive epithelial sodium channels (ENaC), which control the reabsorption of this ion in the distal segments of the nephron. Its assumed that in this pathological process, in addition to aldosterone, is also involved the small Rho GTFase Rac1 — regulatory G-protein, which can enter into a direct ligand-independent interaction with mineralcorticoid receptors, performing the function of a powerful nonsteroidal activator of the transmission of their intracellular signals. Based on controlled, randomized clinical trials, the optimal fourth drug to overcome resistance in such patients is the steroid mineralcorticoid receptor antagonist spironolactone. However, the inclusion of this drug in antihypertensive therapy not only fails to control blood pressure in a significant proportion of patients with resistant hypertension, but also significantly increases the risk of hyperkalemia, especially in people with impaired renal function. The review presents data on the pharmacodynamics and pharmacokinetics of new inhibitors of aldosterone synthase-aldosterone-mineralocorticoid receptor hormonal system baxdrostat and finerenone, as well as the results of clinical studies assessing the clinical effectiveness and safety profile of these drugs in patients with resistant hypertension.

FRI167 Implication Of Serotonin In The Physiopathology Of Aldosterone-Producing Adenoma

Abstract Disclosure: C. Duparc: None. M. D'agostino: None. A. Lopez: None. S. Renouf: None. E. Louiset: None. H. Lefebvre: None. In the human adrenal gland, serotonin (5-HT), released by subcapsular mast cells strongly stimulates expression of CYP11B2, encoding aldosterone-synthase and aldosterone production. The effect of 5-HT on adrenocortical cells is mediated by activation of type 4 serotonin receptor (5-HT4R) positively coupled to cAMP/protein kinase A (PKA) signaling pathway and calcium influx. Consistently, administration of cisapride, a 5-HT4R agonist, to healthy volunteers induces a significant increase in plasma aldosterone levels. In aldosterone producing adenomas (APA), the density of mast cells is enhanced in comparison with normal adrenals. Moreover, molecular studies have shown overexpression of HTR4 gene, encoding 5-HT4R, in APA tissues. Administration of cisapride to patients with APA induced more robust plasma aldosterone responses than in healthy voluntaries. These results suggest that 5-HT could play a role in physiopathology of APA. The aim of our study was to examine expression of genes and proteins involved in aldosterone production and actors of the serotonergic pathway in a series of 31 adenomas removed from patients with primary aldosteronism. We have also investigated the in vitro effect of various 5-HT4R ligands on aldosterone production by explants or cultured cells derived from adenoma tissues collected at surgery. Firstly, aldosterone synthase (AS) immunostaining allowed identification of adenoma phenotypes accordingly to the new international histopathology consensus for unilateral Primary aldosteronism. Among the 31 tissues studied, 20 were classified as classical APAs, i.e. presenting with homogenous or heterogenous AS distribution, remaining being considered as 11 non-classical APA. Expression of the gene encoding the 5-HT-synthesizing enzyme tryptophan hydroxylase was similar in APA and normal adrenals. HTR4 overexpression was more robust in classical than non-classical APAs. HTR4 mRNA levels were positively correlated to those encoding CYP11B2. In addition, 5-HT4R immunostaining was widely distributed in most APA tissues, a pattern which was not exclusively noticed in AS-positive adenomas. BIMU-8, a potent 5-HT4R agonist, strongly increased aldosterone production by perifused APA explants. This effect was blocked by administration of GR-113808, a 5-HT4R antagonist. In cultured APA cells, BIMU-8 stimulated aldosterone synthesis in a dose-dependent manner with a higher efficiency than that observed in normal adrenocortical cells. Aldosterone secretion was less strongly stimulated by prucalopride, a partial 5-HT4R agonist used in the treatment of chronic constipation. These data reveal an enhancement of the 5-HT signaling pathway in APAs in comparison with normal adrenals, suggesting that 5-HT and the 5-HT4R may participate in the pathophysiology of primary aldosteronism. Presentation: Friday, June 16, 2023

Abstract P100: Paradoxical Increase In Post-Captopril Aldosterone

Hypertension remains one of the strongest risk factors for cardiovascular disease. Primary aldosteronism, the most common form of secondary hypertension, can be confirmed using the captopril challenge test. This study aims to investigate a subset of patients who exhibit a paradoxical increase in aldosterone levels after the administration of captopril, despite the expected decrease in aldosterone production. A retrospective design was employed to evaluate patients who exhibited atypical captopril suppression test results. The cohort consisted of 120 patients who underwent the captopril suppression test as part of their diagnostic evaluation for resistant hypertension. Demographic data and clinical parameters were collected for each patient.Among the patients who tested positive on the captopril suppression test based on a post-aldosterone level criterion of >12 mg/dL, one-third exhibited an increase equal to or greater than 50% in aldosterone levels (9 out of 27).We proposed different theories to explain this phenomenon. Intraindividual variability, which has been previously reported with a laboratory coefficient of variation between 2.7% and 4.4%, was ruled out using a cut-off ≥ 50%. Another possibility is the presence of extraadrenal aldosterone production independent of the regulation provided by captopril or ATII. This could be attributed to unrecognized compensation of aldosterone production by extra-adrenal tissues in response to a decrease in adrenal aldosterone levels. Additionally, conditions such as preeclampsia, post-transplant acute rejection, autoimmune diseases, lupus, and primary hyperaldosteronism have been associated with increased aldosterone production by zona glomerulosa cells due to the presence of activating antibodies.There are certain limitations to this study. Other biochemical tests were not performed, and the sample size was relatively small, making it challenging to draw definitive conclusions. In conclusion, this study aims to contribute to our understanding of the pathogenesis of primary aldosteronism. The findings highlight the utility of the captopril suppression test as a biochemical confirmation test for the diagnosis of primary aldosteronism and resistant hypertension.

Aldosterone Biosynthesis Is Potently Stimulated by Perfluoroalkyl Acids: A Link between Common Environmental Pollutants and Arterial Hypertension.

The large environmental contamination of drinking water by perfluoroalkyl substances (PFAS) markedly increased the plasma levels of pentadecafluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in a Northern Italy population with a high prevalence of arterial hypertension and cardiovascular disease. As the link between PFAS and arterial hypertension is unknown, we investigated if they enhance the biosynthesis of the well-known pressor hormone aldosterone. We found that PFAS increased aldosterone synthase (CYP11B2) gene expression by three-fold and doubled aldosterone secretion and cell and mitochondria reactive oxygen species (ROS) production over controls (p < 0.01 for all) in human adrenocortical carcinoma cells HAC15. They also enhanced the effects of Ang II on CYP11B2 mRNA and aldosterone secretion (p < 0.01 for all). Moreover, when added 1 h before, the ROS scavenger tempol abolished the effect of PFAS on CYP11B2 gene expression. These results indicate that at concentrations mimicking those found in human plasma of exposed individuals, PFAS are potent disruptors of human adrenocortical cell function, and might act as causative factors of human arterial hypertension via increased aldosterone production.

Phospholipase D mediates very low-density lipoprotein-induced aldosterone production, in part, via lipin-1.

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low-density lipoprotein (VLDL). VLDL has been shown to induce aldosterone production in multiple adrenal zona glomerulosa models, mediated in part by phospholipase D (PLD). PLD is an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), a lipid second messenger that can also be dephosphorylated by lipin to yield diacylglycerol (DAG), yet another lipid signal. However, it is unclear which of the two lipid second messengers, PA or DAG, underlies PLD's mediation of aldosterone production. We hypothesized that the key signal produced by PLD (indirectly) is DAG such that PLD mediates VLDL-induced aldosterone production via lipin-mediated metabolism of PA to DAG. To assess the role of lipin in VLDL-induced aldosterone production, lipin-1 was overexpressed (using an adenovirus) or inhibited (using propranolol) in HAC15 cells followed by treatment with or without VLDL. Lipin-1 overexpression enhanced the VLDL-stimulated increase in CYP11B2 expression (by 75%), and lipin-1 inhibition decreased the VLDL-stimulated increase in CYP11B2 expression (by 66%). Similarly, the VLDL-stimulated increase in aldosterone production was enhanced by lipin-1 overexpression (182%) and was decreased by lipin inhibition (80%). Our results are suggestive of DAG being the key lipid signal since manipulating lipin-1 levels/activity affects VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into VLDL-stimulated steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets, such as lipin-1 and its downstream pathways, to potentially treat obesity-associated hypertension.

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.

PS-BPB05-7: UPREGULATION OF ALDOSTERONE SYNTHASE INDUCED BY LUTEINIZING HORMONE IN HUMAN ADRENOCORTICAL CELLS

Objective: Plasma aldosterone concentration (PAC) is increased in stages of elevation of luteinizing hormone (LH) during the luteal phase of the ovarian cycle, menopause, or pregnancy. PAC is elevated in patients with polycystic ovary syndrome who have elevated circulating concentration of luteinizing hormone (LH). In addition, stimulation of aldosterone production by LH has been reported. In the present study, we investigated the mechanism by which LH increases aldosterone production in human adrenocortical cells. Design and Methods: Immunohistochemical staining for CYP11B2, the aldosterone synthase, and LH receptor were performed using autopsy adrenal gland specimens. Changes in mRNA or protein expressions of CYP11B2 after treatment with LH, angiotensin (Ang) II, or LH+Ang were compared in human HAC15 adrenocortical cells. Effects of an Ang receptor blocker (ARB), adenylyl cyclase (AC) inhibitor, cyclic AMP response element binding protein (CREB) inhibitor, protein kinase A (PKA) inhibitor, or β;-catenin inhibitor on the increase in CYP11B2 mRNA expression induced by LH+Ang ll were investigated in these cells. In addition, effects of an AC agonist on CYP11B2 mRNA expression were investigated in these cells. Results: Immunostaining for LH receptor was detected in cells positive for CYP11B2 in adrenal granulosa and fasciculata region in the human autopsy specimens. CYP11B2 mRNA and protein expressions were significantly increased after treatment with LH+Ang ll than LH or Ang ll alone in HAC cells. Elevation of CYP11B2 mRNA expression induced by LH+Ang ll was completely suppressed by the ARB, AC inhibitor, or CREB inhibitor and was significantly reduced by the PKA inhibitor or β;-catenin inhibitor in these cells. CYP11B2 mRNA expression was significantly increased by the AC agonist. Conclusion: The present study showed that LH receptor is expressed in human adrenal cortical region and that LH augments CYP11B2 expression via AC-CREB pathway in the presence of Ang ll in human adrenocortical cells.

Abstract 012: L-type Calcium Channel Subunit Alpha1d (CACNA1D) Gene Polymorphisms Associate With Higher Blood Pressure And Salt Sensitivity Of Blood Pressure

Somatic activating mutations in CACNA1D can result in aldosterone producing adrenal adenomas and hypertension. Using the environmentally controlled HyperPATH cohort, we assessed whether germline mutations (variance in the CACNA1D gene) associate with blood pressure (BP), salt sensitivity of BP (SSBP) and two proximate mechanisms of SSBP--- plasma renal activity (PRA)/aldosterone (ALDO) and renal plasma flow (RPF). Participants completed a cross-over intervention of liberal and restricted Na + diets for 7 days. CACNA1D genotyping with a Multi-Ethnic Genotyping Array yielded 264 single nucleotide variants (SNVs) of which 104 met quality control. SNV rs7612148 strongly associated with systolic BP and was selected for analysis. Individuals with the SNV (n=521) were analyzed in 2 populations: 1) hypertensive, and 2) hypertensive plus normotensive. Multivariate linear regression analyses (table) identified an association with the risk allele and increased SSBP driven by a higher BP on the liberal Na + diet. Associations were also observed with reduced PRA and ALDO, and reduced RPF with increase in dietary Na + . In conclusion, we identified a novel CACNA1D SNV that associates with SSBP likely related to an impaired ability to increase RPF with increased dietary sodium intake, but not related to an increase in ALDO production.

Loss of Female Sex Hormones Does Not Further Elevate Blood Pressure in Obese Female Mice but Preserves the Contribution of Leptin to Hypertension

Obesity, which is a major risk factor for hypertension and cardiovascular disease, abolishes the cardioprotective effects of female sex hormones and predisposes women to hypertension. Although 40% of postmenopausal women are obese and 75% are hypertensive, the mechanisms whereby obesity and menopause interact to elevate blood pressure (BP) remains unknown. Formerly, our laboratory demonstrated that hypertension involves leptin‐mediated increases in aldosterone production in obese female mice of reproductive age. Herein, we hypothesized that loss of female sex hormones with ovariectomy (OVX) will further elevate BP and impairs vascular function in obese female mice. Obese agouti yellow mice (Ay) on a KK background and lean controls underwent OVX or sham surgery at 12‐week of age. At 15 weeks, mice were implanted with radiotelemeters to record BP under baseline conditions and in response to leptin receptor blockade (Allo‐Aca, 0.05mg/kg/day s.c. osmotic minipump). To ascertain the effects of OVX on autonomic control of BP, we recorded BP and heart rate (HR) responses to ganglionic blockade (hexamethonium), atropine, and propranolol. At 18 weeks, mice were euthanized and mesenteric arteries isolated to measure endothelial function via wire myography. Obesity significantly increased mean arterial pressure (MAP), systolic BP, diastolic BP, and HR but OVX did not further elevate BP. Interestingly, obesity also disrupted circadian rhythm. Unexpectedly, OVX significantly reduced MAP response to ganglionic blockade (2‐way ANOVA, P&lt;0.05) in obese mice but preserved HR responses to propranolol and atropine respectively suggesting lower neurogenic control of BP. Allo‐Aca treatment significantly decreased (2‐way ANOVA, P&lt;0.05) MAP in both sham and OVX obese mice, indicating BP elevation remains leptin‐dependent. Obesity significantly impaired relaxation to acetylcholine (2‐way ANOVA, P&lt;0.05) but OVX did not further impair relaxation. Compared to lean controls, LNAME further reduced (2‐way ANOVA, P=0.054) relaxation responses to acetylcholine in obese OVX mice suggesting greater contribution of nitric oxide synthase activity to vascular relaxation. Quantitative real‐time PCR showed a trend for decreased adrenal aldosterone synthase (CYP11B2) expression in obese OVX mice. Interestingly, menopause induced a similar decrease in CYP11B2 expression in human adrenal tissue collected from kidney donors with elevated BMIs (postmenopausal 56 yo female: BMI 27.4, vs. premenopausal 43 yo female, BMI 32). Quantification of mouse plasma aldosterone levels via LC‐MS revealed no significant difference in circulating aldosterone levels, suggesting that tissue(s) other than the adrenals could potentially produce aldosterone in obese OVX mice. Plasma analysis also revealed Allo‐Aca treatment increased ANG II, ANG I and ANG 1‐7 in obese mice. In conclusion, these data suggest that obesity‐associated hypertension remains leptin dependent in the absence of female sex hormone and that female sex steroids play limited role in the control of the development of hypertension associated with obesity.

Polyphenol-rich extract of Ocimum gratissimum leaves prevented toxic effects of cyclophosphamide on the kidney function of Wistar rats

BackgroundCyclophosphamide (CP) is one of the potent and low cost chemotherapy used in clinical setting against a variety of tumors. However, its association with nephrotoxicity limits its therapeutic use. Ocimum gratissimum leaf is a medicinal plant with numerous pharmacological and therapeutic efficacies, such as antioxidant, anti-inflammation, and anti-apoptotic properties.MethodsThe present study was designed to evaluate the protective effect of Ocimum gratissimum (OG) against CP-induced kidney dysfunction in rats. Rats were pre-treated with 400 mg/kg b.w. of leave extract of Ocimum gratissimum (Ocimum G.) for 4 days and then 50 mg/kg b.w. of CP was co-administered from day 5 to day 7 along with Ocimum G. Markers of renal function and oxidative stress, food and water intake, electrolytes, aldosterone, leukocytes infiltration, inflammation and histopathological alteration were evaluated.ResultsObvious renal inflammation and kidney injuries were observed in CP treated groups. However, administration of leave extract of Ocimum G. prevented oxidative stress, kidney injuries, attenuated inflammation, increased aldosterone production and reduced sodium ion and water loss in rats. The plasma creatinine, urea and urine albumin concentration were normalized after the administration of Ocimum G. extract in rats treated with CP. Ocimum G. also decreased the plasma concentrations of Interleukin-(IL)-6, C-reactive protein and activity of myeloperoxidase and malondialdehyde in CP treated rats.ConclusionOcimum G. prevented kidney injury and enhanced renal function via inhibiting inflammation and oxidant-induced CP toxicity. The efficacy of Ocimum G. is related to the presence of various phytochemicals in the plant.

Diseases caused by mutations in the Na+/K+ pump α1 gene ATP1A1.

Human cell survival requires function of the Na+/K+ pump; the heteromeric protein that hydrolyzes ATP to extrude Na+ and import K+ across the plasmalemma, thereby building and maintaining these ions' electrochemical gradients. Numerous dominant diseases caused by mutations in genes encoding for Na+/K+ pump catalytic (α) subunit isoforms highlight the importance of this protein. Here, we review literature describing disorders caused by missense mutations in ATP1A1, the gene encoding the ubiquitously expressed α1 isoform of the Na+/K+ pump. These various maladies include primary aldosteronism with secondary hypertension, an endocrine syndrome, Charcot-Marie-Tooth disease, a peripheral neuropathy, complex spastic paraplegia, another neuromuscular disorder, as well as hypomagnesemia accompanied by seizures and cognitive delay, a condition affecting the renal and central nervous systems. This article focuses on observed commonalities among these mutations' functional effects, as well as on the special characteristics that enable each particular mutation to exclusively affect a certain system, without affecting others. In this respect, it is clear how somatic mutations localized to adrenal adenomas increase aldosterone production without compromising other systems. However, it remains largely unknown how and why some but not all de novo germline or familial mutations (where the mutant must be expressed in numerous tissues) produce a specific disease and not the other diseases. We propose hypotheses to explain this observation and the approaches that we think will drive future research on these debilitating disorders to develop novel patient-specific treatments by combining the use of heterologous protein-expression systems, patient-derived pluripotent cells, and gene-edited cell and mouse models.

Single-Center Prospective Cohort Study on the Histopathology, Genotype, and Postsurgical Outcomes of Patients With Primary Aldosteronism.

[Figure: see text].

Aldosterone Insufficiency Contributes to Calcineurin Inhibitor‐induced Hyperkalemia

The mineralocorticoid aldosterone promotes renal K+ excretion and Na+ reabsorption; thereby it is critical for the regulation of ion homeostasis and blood pressure. Interestingly, immunosuppression therapy with protein phosphatase 3 (calcineurin) inhibitors often results in rather low plasma aldosterone levels despite a concomitant hyperkalemia and hyperreninemia. Calcineurin (Cn) is a calcium and calmodulin-dependent protein phosphatase expressed in the adrenal cortex. We tested the hypothesis that Cn participates in the signal transduction pathway mediating the K+-dependent stimulation of aldosterone production. To address this question, we used the adrenocortical cell model NCI-H295R, mouse and human ex vivo adrenal preparations and a ZG-specific and inducible Cn-beta subunit 1 (CnB1) knockout mouse model (ZG-CnB1-KO). Inhibition of Cn with tacrolimus abolished the K+-stimulated expression of CYP11B2 in NCI-H295R cell line as well as in mouse and human adrenal pieces, ex vivo. Moreover, high K+ diet-dependent increase in aldosterone production was blunted in wild type mice treated with tacrolimus. Furthermore, male ZG-CnB1-KO mice fed a high K+ diet exhibited a decreased aldosterone excretion compared to control animals while ZG-CnB1-KO females became hyperkalemic. Using a phosphoproteomics analysis, we identified the nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a downstream factor regulated by Cn. The genetic deletion of NFATc4 in NCI-H295R cells reduced the basal expression of CYP11B2 and blunted the K+-stimulated expression of this gene. Conversely, the expression of a constitutively active form of NFATc4 in NCI-H295R cells drastically increased the expression of CYP11B2 which remained unaltered upon treatment with K+ or tacrolimus. Altogether, our data indicate that the calcineurin-NFATc4 pathway is activated by K+ to promote adrenal aldosterone production. Moreover, our data indicate that insufficient aldosterone production contributes to Cn inhibitor-induced hyperkalemia.

Very Low‐Density Lipoprotein Signals through Scavenger Receptor Class B Type I And Sphingosine‐1‐Phosphate Receptor 1 to Induce Aldosterone Production, in part, via Lipin‐1 in Human Adrenocortical Cells

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of both very low-density lipoprotein (VLDL) and sphingosine-1-phosphate (S1P). S1P is transported in blood bound to lipoproteins such as VLDL, as well as low-density lipoprotein and high-density lipoprotein (HDL); the VLDL particle contains the highest levels of S1P. S1P in HDL has been shown to promote interactions between scavenger receptor class B type I (SR-BI) and S1P receptor 1 (S1PR1), suggesting the possibility that VLDL, which can also act through SR-BI, may stimulate S1PR1. VLDL has been shown to induce aldosterone production in multiple zona glomerulosa models, mediated in part by phospholipase D (PLD). PLD is an enzyme that hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA), a lipid second messenger that can also be dephosphorylated by lipin-1 to yield diacylglycerol (DAG), yet another lipid second messenger. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD's mediation of aldosterone production. We hypothesized that upon binding to SR-BI, VLDL signals through S1PR1 to induce aldosterone production, in part, via lipin-1 mediated metabolism of PA to DAG. To determine whether VLDL functions through SR-BI and S1PR1, human adrenocortical (HAC15) cells were treated with VLDL and/or an S1PR1 antagonist (Ex26) for 24 h. The expression of steroidogenic genes and aldosterone production were monitored by qRT-PCR and radioimmunoassay, respectively. Ex26 inhibited VLDL-induced increases in CYP11B2 (22-fold) and StAR (1.5-fold) expression by 43% and 10%, respectively. In addition, the VLDL-induced 5-fold increase in aldosterone levels was significantly inhibited by Ex26 (36%). Our results suggest that VLDL signals by binding to SR-BI and activating S1PR1, since the S1PR1 antagonist reduced VLDL-induced aldosterone production. To assess the role of lipin-1 in VLDL-induced aldosterone production, lipin-1 was overexpressed (using an adenovirus) or inhibited (using propranolol) in HAC15 cells followed by treatment with or without VLDL for 24 h. While lipin-1 overexpression enhanced the VLDL-stimulated 55-fold increase in CYP11B2 expression by 75%, lipin-1 inhibition decreased the VLDL-stimulated 21-fold increase in CYP11B2 expression by 66%. Similarly, the VLDL-stimulated increase in aldosterone production was enhanced by lipin-1 overexpression (182%) and was decreased by lipin-1 inhibition (80%). Our results are suggestive of DAG being the key lipid signal since manipulating lipin-1 levels/activity affects VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into VLDL-stimulated steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets, such as S1PR1, lipin-1, or their downstream pathways, to potentially treat obesity-associated hypertension.

In the Refractory Hypertension “Labyrinth”. Focus on Primary Hyperaldosteronism

Primary hyperaldosteronism is an existence of a functional autonomous source with increased aldosterone production (full or partial) in relation to the renin-angiotensin system. Increased production of aldosterone by the adrenal cortex is the most common form of a secondary hypertension despite the low attention of internists to the problem. The success of a treatment and a prognosis of these patients depend on correct choice of screening (aldosterone/renin ratio) and clarifying diagnostic methods. There are clear algorithms for conducting these tests in accordance with Russian and International recommendations in the respective groups of patients. The purpose of this case report is to demonstrate the long way to diagnosis of primary hyperaldosteronism in a young patient with refractory hypertension, right adrenal adenoma, and clinical (convulsions, weakness) and laboratory signs of hypokalemia. It should not only have made the diagnosis easy, but it could have also absolutely justified the surgical tactics. Unfortunately, the final verification of the disease by carrying out a saline test was accomplished 13 years after the debut of hypertension and 10 years after the primary visualization of the adrenal adenoma.

Abstract P250: Sphingosine-1-phosphate Receptor Agonist And Very Low-density Lipoprotein Regulate Aldosterone Production Via Lipin-1 In Human Adrenocortical Cells

Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of both sphingosine-1-phosphate (S1P) and very low-density lipoprotein (VLDL). S1P has been reported to be a novel stimulator of aldosterone secretion and phospholipase D (PLD) activity. VLDL has also been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via PLD. PLD is an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA) which can then be converted to diacylglycerol (DAG) by lipin-1. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s mediation of aldosterone production. We hypothesized that the S1P1 receptor (S1PR1) agonist, SEW2871, (and VLDL) induces steroidogenesis and therefore aldosterone production via lipin-1-mediated metabolism of PA to DAG, with our hypothesis focusing on DAG as the key lipid signal produced by PLD (indirectly). In HAC15 cells, lipin-1 was overexpressed using an adenovirus or inhibited using propranolol followed by treatment with or without SEW2871 (or VLDL) for 24 h. Steroidogenic gene expression and aldosterone levels were monitored by qRT-PCR and radioimmunoassay, respectively. We demonstrated that lipin-1 overexpression (OE) enhanced the SEW2871-stimulated 109-fold increase in CYP11B2 expression by 26% while lipin-1 inhibition decreased the SEW2871-stimulated 56-fold increase in CYP11B2 expression by 74%. While lipin-1 OE had no further effect, propranolol reduced SEW2871-stimulated increases in NR4A1 (2-fold) and NR4A2 (9-fold) mRNA levels by 22% and 52% respectively. The SEW2871-stimulated increase in aldosterone production was inhibited by propranolol (53%), although it was not enhanced by lipin-1 OE. Similar results were obtained with VLDL. Our results are, therefore, suggestive of DAG being the key lipid signal since regulating lipin-1 affects S1PR1 agonist- and VLDL-stimulated steroidogenic gene expression and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin-1, or its downstream pathways, to potentially treat obesity-associated hypertension.

SUN-220 High Salt Intake May Paradoxically Drive Autonomous Aldosterone Production

Many modifiable factors contribute to the high prevalence rates of hypertension, among them is the consumption of too much salt (sodium). Another curable cause of hypertension is the excess of the hormone aldosterone. Aldosterone is normally produced by the zona glomerulosa (ZG) of the adrenal glands in response to a lack of salt and conversely suppressed by salt excess. We hypothesize that [i] suppression of aldosterone production induces apoptosis of ZG cells, as occurs following genetic deletion1; [ii] this sets up a maladaptive response to chronic salt overload by conferring a survival advantage to cells in which mutations drive autonomous aldosterone production. To address [i], we measured apoptosis of cells in which aldosterone synthesis was inhibited; to address [ii] we undertook a cross-sectional clinical study of aldosterone and sodium excretion, hypothesising that aldosterone excretion will be highest in the outside quartiles of sodium excretion. Aldosterone was inhibited by modification, in human adrenocortical H295R cells, of either CADM1 expression (mutated in aldosterone-producing adenomas2) or intracellular calcium concentration3. Apoptosis was measured by flow cytometric analysis of annexin V conjugates. 24-hour urinary aldosterone excretion (24h-Ualdo) was correlated with 24-hour urinary sodium (24h-Usodium) in 24h-urine samples collected for a Malaysian population-based salt intake study (MyCoSS). The prevalence of autonomous aldosterone production was estimated from the proportion of subjects with serum measurement whose “SUSSPUP” ratio (= serum sodium to urinary sodium)/(serum potassium2 to urinary potassium) was >5.34. Modification of CADM1 in human adrenocortical H295R cells decreased aldosterone production by half compared to vector control, and this was associated with a 3 to 5-fold increase of apoptotic cells (p<0.05; n>3). Pilot investigation of a Cav1.3 inhibitor decreased aldosterone production by 70%, and increased apoptosis by 7-fold (p<0.10; n=2). In 767 subjects, 24h-urine samples from the high urinary sodium quartile (>150mmol/d) had higher urinary aldosterone (4+0.18 ug/d) than other quartiles (p=0.00001). Overall, the estimated prevalence of autonomous aldossterone production using SUSSPUP ratio was 4.5% (8 of 179 subjects). In 63 subjects with 24h-Usodium>200 mmol/day, autonomous aldosterone secretion (conventionally >10µg/d) was found in 9.5%. Our results support the hypothesis that initial suppression of aldosterone production by salt excess may create a selective advantage for cells which autonomously produce aldosterone, and hence an inappropriate long-term increase in aldosterone production. 1Lee et al., Endocrinology. 2005;146:2650-6. 2Wu et al., 21st European Congress of Endocrinology. Vol. 63. BioScientifica, 2019. 3Xie et al., Sci Rep. 2016;6:24697. 4Willenberg et al., Eur J Clin Invest. 2009;39:43-50.

OR24-03 GRK2 Mediates Beta-Adrenergic Receptor Crosstalk to Enhanced Adrenocortical AngII-Dependent Aldosterone Production

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to hyperkalemia or angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone synthesis and secretion via both G proteins and the GPCR adapter proteins βarrestins. AZG cells express all three subtypes of β-adrenergic receptor (AR) and respond to catecholamines by producing aldosterone. Being GPCRs, both activated βARs and AT1Rs are phosphorylated by GPCR-kinases (GRKs), followed by βarrestin binding to initiate G protein-independent signaling. Herein, we investigated whether the major adrenal GRKs, GRK2 and GRK5, are involved in catecholaminergic regulation of AngII-dependent aldosterone production. We used the human AZG cell line H295R, in which we measured aldosterone secretion via ELISA and synthesis via real-time PCR for steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA levels. Isoproterenol (Iso, a βAR full agonist) treatment significantly augmented AngII-dependent aldosterone synthesis (2.2+0.8-fold CYP11B2 & 1.6+0.5-fold StAR mRNA inductions over AngII alone; p<0.05, n=4), as well as secretion (2.3+0.8-fold of vehicle with Iso; 3.2+1.1-fold of vehicle with AngII; 7.4+1.1-fold of vehicle with Iso+AngII, p<0.05 vs. either agent alone; n=5) in H295R cells. Importantly, GRK2, but not the other major GRK isoform expressed in human adrenals GRK5, was indispensable for the catecholamine-mediated enhancement of aldosterone production in response to AngII in H295R cells. Specifically, GRK2 inhibition with the small molecule Cmpd101 abolished Iso effects on AngII-induced aldosterone synthesis and secretion (Iso+AngII-induced aldosterone secretion: 8.1+2.3-fold of vehicle without Cmpd101; 2.8+0.8-fold of vehicle with Cmpd101; p<0.05, n=5). In contrast, GRK5 knockout via CRISPR/Cas9 did not affect the synergism between isoproterenol and AngII in stimulating aldosterone production. Mechanistically, βAR-activated GRK2, but not GRK5, phosphorylated and activated the Ca2+-activated chloride channel anoctamine-1 (ANO1), also known as transmembrane member (TMEM)16A, ultimately increasing aldosterone production in H295R cells (Iso+10–6 M [Ca2+]-induced ANO1 activity of Cmpd101-pretreated cells: 55+15 % of non-Cmpd101-pretreated cells; p<0.05, n=5). AngII alone failed to stimulate GRK2 in H295R cells. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk at the level of ANO1 activation, which results in enhanced aldosterone production in H295R cells. This finding suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems in the adrenal cortex and that GRK2 inhibition might be therapeutically advantageous for aldosterone suppression.

Very Low‐Density Lipoprotein and a Sphingosine‐1‐Phosphate Receptor Agonist Regulate Aldosterone Production via Lipin‐1 in Human Adrenocortical Cells

Aldosterone plays an important role in blood pressure homeostasis, and hyperaldosteronism can result in hypertension. Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low‐density lipoprotein (VLDL). VLDL has been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA), a lipid second messenger. In addition, sphingosine‐1‐phosphate (S1P), a bioactive sphingolipid also elevated in obesity, has been reported to be a novel stimulator of aldosterone secretion and PLD activity. The mechanisms underlying the action of these non‐conventional secretagogues of aldosterone remain to be elucidated. Angiotensin II, a classical aldosterone secretagogue, not only activates PLD but also elevates the expression of lipin‐1, an enzyme that converts PA to another lipid second messenger, diacylglycerol (DAG), in human adrenocortical carcinoma (HAC15) cells. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s role in aldosterone production. Herein, we used HAC15 cells to determine whether VLDL and an S1P1 receptor (S1PR1) agonist (SEW2871) stimulate aldosterone production by increasing steroidogenic gene expression via lipin‐1‐mediated metabolism of PA to DAG. We overexpressed lipin‐1 using an adenovirus or inhibited it using propranolol followed by treatment with or without VLDL or SEW2871 for 24 h. The expression of steroidogenic genes and aldosterone secretion were monitored using qRT‐PCR and radioimmunoassay. We demonstrated that lipin‐1 overexpression enhanced the VLDL‐stimulated 55‐fold increase in CYP11B2 expression by 75% while lipin‐1 inhibition decreased the VLDL‐stimulated 21‐fold increase in CYP11B2 expression by 66%. A parallel trend was observed with aldosterone secretion levels: VLDL‐stimulated increase in aldosterone production was enhanced by lipin‐1 overexpression (182%) and was decreased by propranolol (80%). Similar results were obtained with SEW2871. Our results are, therefore, suggestive of DAG being the key lipid signal since lipin‐1 regulates VLDL‐ and S1PR1 agonist‐stimulated expression of steroidogenic genes and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin‐1, or its downstream pathways, to potentially treat obesity‐associated hypertension.Support or Funding InformationThis work was supported in part by the Augusta University Adrenal Center. WBB was also supported by VA Merit Award #BX001357.