Glomerulosa Cells Research Articles

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

7290 Hippo Signaling Regulates Zona Glomerulosa Cell Fate In The Adrenal Cortex Of Mice

Abstract Disclosure: N. Abou Nader: None. N. Jakuc: None. M. Meinsohn: None. L. Charrier: None. M. Paquet: None. G. St-Jean: None. J. Brind’Amour: None. D. Boerboom: None. J. Mao: None. D. Pepin: None. G. Zamberlam: None. D.T. Breault: None. A. Boyer: None. The Hippo signaling is a major signaling pathway that regulates processes such as cell proliferation and differentiation during both embryogenesis and postnatal homeostatic maintenance in several tissues. Our recent findings, using the Nr5a1-cre model, have highlighted the essential role of Lats1 and Lats2 (large tumor suppressor homolog kinases 1/2), the main kinases of the Hippo signaling cascade, in maintaining proper differentiation of mouse developing adrenocortical cells by suppressing their differentiation into myofibroblast-like cells. However, since the abnormal transdifferentiation of Lats1/2-negative adrenocortical cells occurs before the establishment of zonation when using the Nr5a1-cre model, the role of Hippo signaling in zona glomerulosa (zG) cells could not be assessed with this model. To overcome this limitation, we inactivated Lats1/2 in aldosterone-producing zG cells using the zG cell-specific AS-cre strain (Lats1flox/flox; Lats2flox/flox; AScre/+). We report here that specific loss of Lats1/2 in steroidogenic zG cells results in the accumulation of an important extracellular matrix (ECM) in the zG an outer zona fasciculata (zF) by 4 weeks of age; a phenotype that does not seem to progress with aging. In addition, tracing experiments demonstrate that the inactivation of Lats1/2 leads to the ectopic transdifferentiation of the zG cells into the ECM-producing cells rather than into zF cells. We then performed RNAseq experiments on whole adrenals which further suggested that the ECM producing cells derived from zG cells share characteristics with chondroblast/osteoblast mesenchymal progenitors among other cell lineages and, that the ectopic transdifferentiation of zG cells triggers a significant inflammation/immune response in the adrenal cortex. Our study also demonstrates that the increased activity of the Hippo pathway effectors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) is responsible for the ectopic transdifferentiation of the zG cells as their concomitant inactivation with Lats1/2 (Yapflox/flox; Tazflox/flox; Lats1flox/flox; Lats2flox/flox; AScre/+) rescues the observed phenotype. Finally, we also demonstrate that the expression of a constitutively active form of YAP (YAP5SA) in zG cells (Rosa26YAP5SA/+; AScre/+) does not alter their identity as severely as the loss of Lats1/2 but leads to adrenal hyperplasia. Together, these novel findings underscore the crucial role of Hippo signaling in maintaining the identity of zG cells in the adrenal cortex. Presentation: 6/2/2024

6636 Primary Hyperaldosteronism Leading to Aldosterone Deficiency

Abstract Disclosure: N.G. Haverstick: None. J.R. Anthony: None. Primary Hyperaldosteronism is a disease process that is often missed, and under checked due to poor screening tools. The disease presents with a common problem, uncontrolled hypertension. It also presents with hypokalemia, fatigue and headaches. It can easily be masked with blood pressure medications, CKD, and pain medications. After treatment there are some rare reports of patients developing hypoaldosteronism. Some risk factors are age >50, duration of hypertension >10 years, pre-existing renal impairment and adrenal adenoma size >2 cm. The patient will present with the opposite: hyperkalemia and hypotension. This is due to the atrophy of the previously healthy gland. Usually this resolves within a year. However in our patient she has continued to require long standing mineralocorticoid replacement therapy. This case highlights the importance of early recognition of hyperaldosteronism. A 63 year-old-female with a medical history of hypertension and diabetes mellitus is being followed for 30+ years of uncontrolled hypertension. Patient has been on multiple agents and blood pressure remained elevated after trying triamterene/hctz, metoprolol, irbesartan, amlodipine. After changing her PCP, there was further investigation into the cause. Leading differentials were Primary Hyperaldosteronism vs Renal Artery Stenosis vs Primary Hypertension. She also had multiple labs with her Potassium below 3 mEq/l. Additional labs were obtained and her Aldosterone level 209 ng/dl and Renin <0.167 ng/mL/hr. The patient was referred to Endocrinology who performed a fludrocortisone suppression test, which did not suppress the levels. A CT scan of the abdomen was performed that showed a 1.3 cm right adrenal mass typical with adrenal adenoma. The patient had an adrenalectomy surgery in February 2022. The patient recovered well, however now developed hypotension off medication, and had consistent hyperkalemia with K > 5.5 mEq/l. The patient had now developed aldosterone deficiency. This was due to the long standing hyperaldosteronism that had caused her left adrenal gland to atrophy after 30 years. The patient was started on Fludrocortisone 0.1 mg daily and a low potassium diet. She has remained off any blood pressure medications since her right adrenalectomy. She has continued to require the mineralocorticoid replacement to date. The diagnostic work-up for primary aldosteronism involves screening, confirmatory testing, and localization studies to distinguish unilateral pathology which may be amenable to adrenalectomy from bilateral adrenal hyperplasia. Through literature review, one theory is that chronic suppression of renin may cause atrophy of the zona glomerulosa cells and hypoaldosteronism. Throughout the case report we emphasize on the importance of early diagnostic screening of secondary hypertension as well as the complications from adrenalectomy. Presentation: 6/2/2024

Comparative Anatomical Studies of Adrenal Glands in Large White Yorkshire Pigs Reared at Low and High Altitudes in Kerala

The adrenal gland plays a crucial role in the body's homeostasis and during the stress response. However, less attention was given to the structural variation of the adrenal glands in pigs reared at different altitudes. Hence, the present study was conducted to correlate structural variation in the adrenal glands in 24 ten-month-old Large White Yorkshire pigs of either sex reared at low-altitude and high-altitude regions of Kerala, India. Microscopically, the adrenal gland presented the three typical concentric cortical zones: zona glomerulosa, zona fasciculata, zona reticularis, and a central medulla. The arrangement pattern of cells of the zona glomerulosa resembled the arcs; zona fasciculata, similar to cords separated by intervening trabeculae; and zone reticularis, like irregularly anastomosing cords. Two types of cells were present in the medulla: large, granular, intensely-stained cells towards the periphery and small polyhedral cells towards the centre of the medulla. Chromaffin was identified in the medulla, while undifferentiated cells were identified within the capsule, subcapsular zone, the transitional zone between zona glomerulosa and zona fasciculata, and even medulla. Zona fasciculata formed the largest zone of the adrenal cortex, and its thickness was significantly higher in the low-altitude male and female groups than in the corresponding high-altitude groups, which was correlated to serum cortisol levels. The comparative micrometrical data revealed a larger zona fasciculata in the low-altitude male and female groups than in the corresponding high-altitude groups, indicating variation between the groups.

Evaluating the role of aldosterone synthesis on adrenal cell fate.

Hypertension affects one-third of the adult population worldwide, with primary aldosteronism (PA) accounting for at least 5-10% of these cases. The aldosterone synthase enzyme (CYP11B2) plays a pivotal role in PA manifestation, as increased expression of CYP11B2 leads to excess aldosterone synthesis. Physiological expression of CYP11B2 in humans is normally limited to cells of the adrenal zona glomerulosa under tight homeostatic regulation. In PA, however, there are CYP11B2-positive lesions in the adrenal cortex that autonomously secrete aldosterone, highlighting the dysregulation of adrenal cortex zonation and function as a key aspect of PA pathogenesis. Thus, this review aims to summarize the development of the adrenal glands, the key regulators of adrenal cortex homeostasis, and the dysregulation of this homeostasis. It also discusses the development of CYP11B2 inhibitors for therapeutic use in patients with hypertension, as well as the current knowledge of the effects of CYP11B2 inhibition on adrenal cortex homeostasis and cell fate. Understanding the control of adrenal cell fate may offer valuable insights into both the pathogenesis of PA and the development of alternative treatment approaches for PA.

Extracellular Matrix Protein Signatures of the Outer and Inner Zones of the Rat Adrenal Cortex.

This study analyzes the extracellular matrix (ECM) signatures of the outer (OF = capsule + subcapsular + zona glomerulosa cells) and inner fractions (IF = zona fasciculata cells) of the rat adrenal cortex, which comprise two distinct microenvironment niches. Proteomic profiles of decellularized OF and IF samples, male and female rats, identified 252 proteins, with 32 classified as ECM-component and ECM-related. Among these, 25 proteins were differentially regulated: 17 more abundant in OF, including Col1a1, Col1a2, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, Lama5, Lamb2, Lamc1, Eln, Emilin, Fbln5, Fbn1, Fbn2, Nid1, and Ltbp4, and eight more abundant in IF, including Col4a1, Col4a2, Lama2, Lama4, Lamb1, Fn1, Hspg2, and Ecm1. Eln, Tnc, and Nid2 were abundant in the female OF, while Lama2, Lama5, Lamb2, and Lamc1 were more abundant in the male IF. The complex protein signature of the OF suggests areas of tissue stress, stiffness, and regulatory proteins for growth factor signaling. The higher concentrations of Col4a1 and Col4a2 and their role in steroidogenesis should be further investigated in IF. These findings could significantly enhance our understanding of adrenal cortex functionality and its implications for human health and disease. Key findings were validated, and data are available in ProteomeXchange (PXD046828).

Zona Glomerulosa-Derived Klotho Modulates Aldosterone Synthase Expression in Young Female Mice.

Klotho plays a critical role in the regulation of ion and fluid homeostasis. A previous study reported that haplo-insufficiency of Klotho in mice results in increased aldosterone synthase (CYP11B2) expression, elevated plasma aldosterone, and high blood pressure. This phenotype was presumed to be the result of diminished Klotho expression in zona glomerulosa (zG) cells of the adrenal cortex; however, systemic effects on adrenal aldosterone production could not be ruled out. To examine whether Klotho expressed in the zG is indeed a critical regulator of aldosterone synthesis, we generated a tamoxifen-inducible, zG-specific mouse model of Klotho deficiency by crossing Klotho-flox mice with Cyp11b2-CreERT mice (zG-Kl-KO). Tamoxifen-treated Cyp11b2-CreERT animals (zG-Cre) served as controls. Rosa26-mTmG reporter mice were used for Cre-dependent lineage-marking. Two weeks after tamoxifen induction, the specificity of the zG-Cre line was verified using immunofluorescence analysis to show that GFP expression was restricted to the zG. RNA in situ hybridization revealed a 65% downregulation of Klotho messenger RNA expression in the zG of zG-Kl-KO female mice at age 12 weeks compared to control mice. Despite this significant decrease, zG-Kl-KO mice exhibited no difference in plasma aldosterone levels. However, adrenal CYP11B2 expression and the CYP11B2 promotor regulatory transcription factors, NGFIB and Nurr1, were enhanced. Together with in vitro experiments, these results suggest that zG-derived Klotho modulates Cyp11b2 but does not evoke a systemic phenotype in young adult mice on a normal diet. Further studies are required to investigate the role of adrenal Klotho on aldosterone synthesis in aged animals.

T- and L-Type Calcium Channels Maintain Calcium Oscillations in the Murine Zona Glomerulosa.

The zona glomerulosa of the adrenal gland is responsible for the synthesis and release of the mineralocorticoid aldosterone. This steroid hormone regulates salt reabsorption in the kidney and blood pressure. The most important stimuli of aldosterone synthesis are the serum concentrations of angiotensin II and potassium. In response to these stimuli, voltage and intracellular calcium levels in the zona glomerulosa oscillate, providing the signal for aldosterone synthesis. It was proposed that the voltage-gated T-type calcium channel CaV3.2 is necessary for the generation of these oscillations. However, Cacna1h knock-out mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. We used a combination of calcium imaging, patch clamp, and RNA sequencing to investigate calcium influx pathways in the murine zona glomerulosa. Cacna1h-/- glomerulosa cells still showed calcium oscillations with similar concentrations as wild-type mice. No calcium channels or transporters were upregulated to compensate for the loss of CaV3.2. The calcium oscillations observed were instead dependent on L-type voltage-gated calcium channels. Furthermore, we found that L-type channels can also partially compensate for an acute inhibition of CaV3.2 in wild-type mice. Only inhibition of both T- and L-type calcium channels abolished the increase of intracellular calcium caused by angiotensin II in wild-type. Our study demonstrates that T-type calcium channels are not strictly required to maintain glomerulosa calcium oscillations and aldosterone production. Pharmacological inhibition of T-type channels alone will likely not significantly impact aldosterone production in the long term.

Angiotensin II-dependent aldosterone production in the adrenal cortex.

The adrenal cortex is responsible for production of adrenal steroid hormones and is anatomically divided into three distinct zones: zona glomerulosa secreting mineralocorticoids (mainly aldosterone), zona fasciculata secreting glucocorticoids (cortisol), and zona reticularis producing androgens. Importantly, due to their high lipophilicity, no adrenal steroid hormone (including aldosterone) is stored in vesicles but rather gets synthesized and secreted instantly upon cell stimulation with specific stimuli. Aldosterone is the most potent mineralocorticoid hormone produced from the adrenal cortex in response to either angiotensin II (AngII) or elevated K+ levels in the blood (hyperkalemia). AngII, being a peptide, cannot cross cell membranes and thus, uses two distinct G protein-coupled receptor (GPCR) types, AngII type 1 receptor (AT1R) and AT2R to exert its effects inside cells. In zona glomerulosa cells, AT1R activation by AngII results in aldosterone synthesis and secretion via two main pathways: (a) Gq/11 proteins that activate phospholipase C ultimately raising intracellular free calcium concentration; and (b) βarrestin1 and -2 (also known as Arrestin-2 and -3, respectively) that elicit sustained extracellular signal-regulated kinase (ERK) activation. Both pathways induce upregulation and acute activation of StAR (steroidogenic acute regulatory) protein, the enzyme that catalyzes the rate-limiting step in aldosterone biosynthesis. This chapter describes these two salient pathways underlying AT1R-induced aldosterone production in zona glomerulosa cells. We also highlight some pharmacologically important notions pertaining to the efficacy of the currently available AT1R antagonists, also known as angiotensin receptor blockers (ARBs) or sartans at suppressing both pathways, i.e., their inverse agonism efficacy at G proteins and βarrestins.

OR08-02 In Situ Reconstruction Of Distinct Normal And Pathological Cell Populations Within The Human Adrenal Gland Using Spatial Transcriptomics

Abstract Disclosure: F. Rui: None. K. Walters: None. K. Koc: None. A. Baldwin: Stock Owner; Self; Tempus Labs. K. Kiseljak-Vassiliades: Advisory Board Member; Self; HRA Pharma. L.M. Fishbein: Consulting Fee; Self; Lantheus/Progenics Azedra. N. Mukherjee: None. The human adrenal gland is a vital endocrine organ controlling a multitude of functions through the secretion of cortical steroid and medullary catecholamine hormones. Several outstanding questions remain about the adrenal spatial organization and related differential developmental signaling pathways which have functional consequences within and between the cortex and medulla. The current model of the functional zonation of the cortex posits a centripetal differentiation process in which the cortical cells arise in the capsule and migrate inwards changing their gene expression patterns as they progress to the three different functional layers of the cortex. The evidence for this model is based on mouse lineage tracing experiments, and it is unclear to what extent it applies to human adrenal glands. To address this, we performed spatially resolved transcriptomics on four adult adrenal sections from two different human specimens to identify cell populations and their spatial relationship in this highly organized and functionally diverse organ. We identified 14 distinct cell populations; 79% of these spots represented the cortex or medulla and exhibited the concentric expression pattern reflecting the known spatial organization of the human adrenal gland. Trajectory inference analysis of the cortical cells showed continuous expression transitions from cells of the capsule to zona glomerulosa to zona fasciculata to zona reticularis. These data demonstrate for the first time that the centripetal differentiation model for functional zonation in mice is consistent for adult human adrenal tissue. We also validated the spatial expression pattern of a subset of WNT-related genes specifically expressed with distinct patterns in the capsule and zona glomerulosa cells. These genes represent novel regulatory factors that may be crucial in the differentiation of adult human adrenal cortex. Finally, we observed two CYP11B2 positive aldosterone producing cell clusters (APCC) in the adrenal tissue section from a 72-year-old woman. APCCs are thought to play a role in the pathological progression of primary aldosteronism; however, it is unclear if there are unifying expression signatures other than CYP11B2 protein expression. Our analysis found both similarities and differences to previously published data sets of APCC signatures. It is unclear to what extent these expression differences are technical (i.e. different platforms) or due to biological heterogeneity (between individuals or APCCs). The subgroup of similar gene expression in APCCs across all studies thus far may represent a core APCC signature which should be validated on a larger cohort. Altogether, our study uses spatial transcriptomics of the normal human adrenal and the results have important implications for the differentiation and functional zonation,as well as, for the early dysregulation leading to primary aldosteronism. Presentation: Friday, June 16, 2023

FRI141 Dietary Salt Regulates mTORC1 Signaling In Humans And Mice With Hypertension

Abstract Disclosure: H. Aljaibeji: None. D.L. Brooks: None. A.E. Garza: None. P. Gerges: None. M. Heydarpour: None. P. Luminita: None. J.S. Williams: None. G. Williams: None. J.R. Romero: None. Raptor and the mammalian target of rapamycin (mTOR) form a complex (mTORC1) that has been proposed to play a role in aldosterone (ALDO) production. Pharmacological inhibition of mTORC1 reduces blood pressure (BP) and ALDO levels. However, the effects of environmental factors such as dietary salt on mTORC1 in humans are unclear. We studied the impact of one-week liberal Na+ (LIB, 200 mmol/day) and restricted (RES, 10 mmol/day) Na+ diets on Raptor and mTOR expression in human peripheral blood mononuclear cells (PBMC) from hypertensive subjects. Raptor and mTOR expression were higher on PBMCs from subjects on a LIB than on RES Na+ diet (n=13; P=0.036 and P=0.0008, respectively). We also studied Caucasian subjects (n=792) from the Hypertensive Pathotype (HyperPATH) cohort using the Multi-Ethnic Genotyping Array on the Illumina platform. Candidate gene association analyses showed that the single nucleotide polymorphic (SNP) Raptor gene variant, rs99001846, was associated with BP and ALDO levels. Multivariate linear regression analyses performed on hypertensive and normotensive participants showed that in hypertensives, rs99001846 risk allele carriers (AA/GA), as compared to non-risk carriers (GG), were associated with increased systolic BP levels on bothLIB and RES Na+ diets (P=0.02, P=0.01 respectively). In addition, risk allele carriers, as compared to non-risk carriers, showed increased ALDO and reduced Na+ excretion on a LIB Na+ diet (P=0.009, P=0.025, respectively). We also studied a mouse model of low nitric oxide and high AngII-mediated hypertension and observed increased cardiac Raptor and mTOR expression (n=4, P=0.0008 and P=0.03 respectively). We then performed in silico bioinformatic analyses of rat zona glomerulosa cells following 2 hr treatment with 100 nM angiotensin II (AngII) using the Gene Expression Omnibus (GEO). AngII treatment led to the activation of mTOR signaling pathways as shown by increased expression of insulin receptor substrate type 2 (P=0.00001) and Low-density lipoprotein-related receptor 5 (P=0.03). Also, AngII reduced the expression of Eukaryotic Translation Initiation Factor 4E Binding Protein 1 by 43% (P= 0.001) and increased expression of CAP-Gly Domain Containing Linker Protein 1 by 70% (P= 0.00003) - important signaling molecules that are downstream of mTOR activation. Our results suggest that in hypertension, Na+ intake and Raptor gene variants modify mTORC1. Thus, mTOR/Raptor expression are a potential biomarker of ALDO-mediated cardiovascular injury. 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.

Regulation of the renin-angiotensin-aldosterone system by cyclic nucleotides and phosphodiesterases.

The renin-angiotensin-aldosterone system (RAAS) is one of the key players in the regulation of blood volume and blood pressure. Dysfunction of this system is connected with cardiovascular and renal diseases. Regulation of RAAS is under the control of multiple intracellular mechanisms. Cyclic nucleotides and phosphodiesterases are the major regulators of this system since they control expression and activity of renin and aldosterone. In this review, we summarize known mechanisms by which cyclic nucleotides and phosphodiesterases regulate renin gene expression, secretion of renin granules from juxtaglomerular cells and aldosterone production from zona glomerulosa cells of adrenal gland. We also discuss several open questions which deserve future attention.

Englerin A Inhibits T-Type Voltage-Gated Calcium Channels at Low Micromolar Concentrations.

Englerin A (EA) is a potent agonist of tetrameric transient receptor potential canonical (TRPC) ion channels containing TRPC4 and TRPC5 subunits. TRPC proteins form cation channels that are activated by plasma membrane receptors. They convert extracellular signals such as angiotensin II into cellular responses, whereupon Na+ and Ca2+ influx and depolarization of the plasma membrane occur. Via depolarization, voltage-gated Ca2+ (CaV) channels can be activated, further increasing Ca2+ influx. We investigated the extent to which EA also affects the functions of CaV channels using the high-voltage-activated L-type Ca2+ channel CaV1.2 and the low-voltage-activated T-type Ca2+ channels CaV3.1, CaV3.2, and CaV3.3. After expression of cDNAs in human embryonic kidney (HEK293) cells, EA inhibited currents through all T-type channels at half-maximal inhibitory concentrations (IC50) of 7.5 to 10.3 μM. In zona glomerulosa cells of the adrenal gland, angiotensin II-induced elevation of cytoplasmic Ca2+ concentration leads to aldosterone release. We identified transcripts of low- and high-voltage-activated CaV channels and of TRPC1 and TRPC5 in the human adrenocortical (HAC15) zona glomerulosa cell line. Although no EA-induced TRPC activity was measurable, Ca2+ channel blockers distinguished T- and L-type Ca2+ currents. EA blocked 60% of the CaV current in HAC15 cells and T- and L-type channels analyzed at -30 mV and 10 mV were inhibited with IC50 values of 2.3 and 2.6 μM, respectively. Although the T-type blocker Z944 reduced basal and angiotensin II-induced 24-hour aldosterone release, EA was not effective. In summary, we show here that EA blocks CaV1.2 and T-type CaV channels at low-micromolar concentrations. SIGNIFICANCE STATEMENT: In this study we showed that englerin A (EA), a potent agonist of tetrameric transient receptor potential canonical (TRPC)4- or TRPC5-containing channels and currently under investigation to treat certain types of cancer, also inhibits the L-type voltage-gated Ca2+ (CaV) channel CaV1.2 and the T-type CaV channels CaV3.1, CaV3.2, and CaV3.3 channels at low micromolar concentrations.

New insights into signal transduction pathways in adrenal steroidogenesis: role of mitochondrial fusion, lipid mediators, and MAPK phosphatases.

Hormone-receptor signal transduction has been extensively studied in adrenal gland. Zona glomerulosa and fasciculata cells are responsible for glucocorticoid and mineralocorticoid synthesis by adrenocorticotropin (ACTH) and angiotensin II (Ang II) stimulation, respectively. Since the rate-limiting step in steroidogenesis occurs in the mitochondria, these organelles are key players in the process. The maintenance of functional mitochondria depends on mitochondrial dynamics, which involves at least two opposite events, i.e., mitochondrial fusion and fission. This review presents state-of-the-art data on the role of mitochondrial fusion proteins, such as mitofusin 2 (Mfn2) and optic atrophy 1 (OPA1), in Ang II-stimulated steroidogenesis in adrenocortical cells. Both proteins are upregulated by Ang II, and Mfn2 is strictly necessary for adrenal steroid synthesis. The signaling cascades of steroidogenic hormones involve an increase in several lipidic metabolites such as arachidonic acid (AA). In turn, AA metabolization renders several eicosanoids released to the extracellular medium able to bind membrane receptors. This report discusses OXER1, an oxoeicosanoid receptor which has recently arisen as a novel participant in adrenocortical hormone-stimulated steroidogenesis through its activation by AA-derived 5-oxo-ETE. This work also intends to broaden knowledge of phospho/dephosphorylation relevance in adrenocortical cells, particularly MAP kinase phosphatases (MKPs) role in steroidogenesis. At least three MKPs participate in steroid production and processes such as the cellular cycle, either directly or by means of MAP kinase regulation. To sum up, this review discusses the emerging role of mitochondrial fusion proteins, OXER1 and MKPs in the regulation of steroid synthesis in adrenal cortex cells.

Exploring heterogeneity of tumor immune cells and adrenal cells in aldosterone-producing adenomas using single-cell RNA-seq and investigating differences by sex

The mechanism behind the higher incidence of aldosterone-producing adenoma (APA) in women compared to men is not yet understood. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to investigate the immune cell infiltration and adrenal cell characteristics in APA. Our findings revealed a high presence of immune cells in the tumor microenvironment, with macrophages and T lymphocytes being the most prevalent. Comparison of infiltrating cells between males and females showed that female CD8+T cells had stronger cytotoxic and inflammation-related functions, while female myeloid cells had more enrichment in inflammatory pathways. Additionally, we found that female adrenal cells had greater upregulation of immune-related and antigen presentation pathways. Furthermore, our analysis revealed that zona glomerulosa (ZG) cells had a higher capability for aldosterone synthesis. These results provide a deeper understanding of the APA microenvironment in patients of different sexes and offer new insights into the onset of APA.

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.

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.

Lysine specific demethylase 1 deficiency modifies aldosterone synthesis in a sex-specific manner.

Biologic sex influences the development of cardiovascular disease and modifies aldosterone (ALDO) and blood pressure (BP) phenotypes: females secrete more ALDO, and their adrenal glomerulosa cell is more sensitive to stimulation. Lysine-specific demethylase 1 (LSD1) variants in Africans and LSD1 deficiency in mice associate with BP and/or ALDO phenotypes. This study, in 18- and 40-week-old wild type (WT) and LSD1+/- mice, was designed to determine whether: 1) sex modifies ALDO biosynthetic enzymes; 2) LSD1 deficiency disrupts the effect of sex on these enzymes; 3) within each genotype, there is a positive relationship between ALDO biosynthesis (proximate phenotype), plasma ALDO (intermediate phenotype) and BP levels (distant phenotype); and 4) sex and LSD1 genotype interact on these phenotypes. In WT mice, female sex increases the expression of early enzymes in ALDO biosynthesis, but not ALDO levels or SBP. However, enzyme expressions are shifted downward in LSD1+/- females vs. males, so that early enzyme levels are similar but the late enzymes are substantially lower. In both age groups, LSD1 deficiency modifies the adrenal enzyme expressions, circulating ALDO levels and SBP in a sex specific manner. Finally, significant sex/LSD1 genotype interactions modulate the three phenotypes in mice. In conclusion, biologic sex in mice interacts with LSD1 deficiency to modify several phenotypes: 1) proximal (ALDO biosynthetic enzymes); 2) intermediate (circulating ALDO); and 3) distant (SBP). These results provide entrée to better understand the roles of biologic sex and LSD1 in: 1) hypertension heterogeneity and 2) in providing more personalized treatment.

S-13-2: PATHOLOGY UPDATE OF CUSHING'S SYNDROME AND PRIMARY ALDOSTERONISM ACCORDING TO WHO 2022

New pathology classification of endocrine and neuroendocrine tumours is currently being updated as one of WHO classification of tumours version 5. Not only pathologists but also clinicians involved in the clinical management of those patients should be familiar with novel classification including their new concepts. Therefore in this presentation, we will introduce newly updated concepts of adrenocortical diseases associated with Cushing's syndrome and primary aldosteronism. In Cushing's syndrome, primary bilateral nodular adrenal cortical disease has turned out to harbor multiple independent clonal proliferations and the term hyperplasia should not be used in these adrenocortical disorders. Hyperplasia should be only reserved for those associated with ACTH overproduction from pituitary or others. Various somatic and/or genomic mutations of the genes involved in intracellular signaling pathways have been reported in almost all bilateral micronodular adrenal cortical disease and the great majority of bilateral macronodular adrenal cortical disease associated with Cushing's syndrome. For instance, bilateral micronodular adrenocortical disease harbors pathogenic germline mutations resulting in the activation of the protein kinase A or PKA pathway including PRKAR1A, PRKAR1A, PRKACA, PDE11A, PDE8B and 2p16 CNC2 locus alterations. On the other hand, bilateral macronodular adrenal cortical disease has been linked to several germline alterations including ARMC5, MEN1, FH, APC and others.Clinical significance of those genetic abnormalities above including potential therapeutic manipulation is being studied at this juncture. In primary aldosteronism, the introduction of CYP11B2 has enormously contributed to the establishment of new concept of adrenocortical pathology. Primary aldosteronism can be generally classified into two categories. The one in which aldosterone production diffusely occurs in the hyperplastic zona glomerulosa and the other harboring localized aldosterone production. The former is generally bilateral and the latter unilateral or bilateral. The latter category also includes both nodule and adenoma, which could be differentiated according to the status of intra-lesional distribution of CYP11B2. CYP11B2 positive localized proliferation of the zona glomerulosa cells is frequently detected in non hypertensive subject and should be termed aldosterone producing nodules (APN) rather than aldosterone producing cell clusters (APCC). The potential cascade from these aldosterone producing nodules in normal subject to those associated with clinically overt primary aldosteronism is being investigated including the possible concept of pre- or subclinical primary aldosteronism.