Cardiovascular Outcomes of KCNJ5 Mutated Aldosterone-Producing Adenoma: A Systematic Review.

BACKGROUND: While clinical features of KCNJ5-mutated aldosterone-producing adenoma (APA) have been reported, evidence of its clinical outcomes is lacking. We aimed to synthesize available literature about the associations between KCNJ5 mutation with cardiovascular and metabolic outcomes among patients with APA. METHODS: In this systematic review of observational studies, MEDLINE and Embase were searched through August 2022. Two independent authors screened the search results and extracted data from eligible observational studies investigating cardiovascular or metabolic outcomes between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs. Risk of Bias In Non-randomized Studies of Interventions was used to assess the quality of the included studies. RESULTS: A total of 573 titles/abstracts were screened and after the expert opinion of the literature, full text was read in 20 titles/abstracts, of which 12 studies were included. Across 3 studies comparing the baseline or change in the cardiac function between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs, all studies reported the association between impaired cardiac functions and KCNJ5 mutation status. Among 6 studies evaluating the cure of hypertension after surgery, all studies showed that KCNJ5 mutation was significantly associated with the cure of hypertension. In quality assessment, 7 studies were at serious risk of bias, while the remaining studies were at moderate risk of bias. CONCLUSIONS: This systematic review provided evidence of the significant association between KCNJ5 mutation and unfavorable cardiovascular outcomes in patients with primary aldosteronism. Further research is needed to improve the quality of evidence on this topic and elucidate the underlying mechanisms of the potential burden of KCNJ5 mutation.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1hM1560V/+ ).

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1hM1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h-/- ). Adrenal morphology of both Cacna1hM1560V/+ and Cacna1h-/- mice was normal. Cacna1hM1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1hM1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h-/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1hM1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Molecular evolution of the switch for progesterone and spironolactone from mineralocorticoid receptor agonist to antagonist.

The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target.

Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Pathology of Aldosterone Biosynthesis and its Action.

Aldosterone plays pivotal roles in renin-angiotensin-aldosterone system in order to maintain the equilibrium of liquid volume and electrolyte metabolism. Aldosterone action is mediated by both mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). Its excessive actions directly induced tissue injuries in its target organs such as myocardial and vascular fibrosis in addition to chronic kidney diseases. Excessive aldosterone actions were also reported to be involved in unbalanced electrolyte metabolism in inflammatory bowel disease and development of pulmonary diseases. Hyperaldosteronism is tentatively classified into primary and secondary types. Primary aldosteronism is more frequent and has been well known to result in secondary hypertension with subsequent cardiovascular damages. Primary aldosteronism is also further classified into distinctive subtypes and among those, aldosterone-producing adenoma is the most frequent one accounting for the great majority of unilateral primary aldosteronism cases. In bilateral hyperaldosteronism, aldosterone-producing diffuse hyperplasia and aldosterone-producing micronodules or nodules are the major subtypes. All these aldosterone-producing lesions were reported to harbor somatic mutations including KCNJ5, CACNA1D, ATP1A1 and ATP2B3, which were all related to excessive aldosterone production. Among those mutations above, somatic mutation of KCNJ5 is the most frequent in aldosterone-producing adenoma and mostly composed of clear cells harboring abundant aldosterone synthase expression. In contrast, CACNA1D-mutated aldosterone-producing micronodules or aldosterone-producing nodules were frequently detected not only in primary aldosteronism patients but also in the zona glomerulosa of normal adrenal glands, which could eventually lead to an autonomous aldosterone production resulting in normotensive or overt primary aldosteronism, but their details have remained unknown.

Impaired Morphogenesis and Function of Rat Adrenal Zona Glomerulosa by Developmental Low-Dose Exposure to DDT Is Associated with Altered Oct4 Expression.

Dichlorodiphenyltrichloroethane (DDT) is a persistent organic pollutant and one of the most widespread endocrine disrupting chemicals. The impact of low-dose exposure to DDT on the morphogenesis of the adrenal gland is still poorly understood. The development and function of zona glomerulosa in rats has been found to be associated with changes in the expression of the transcription factor Oct4 (Octamer 4), which is the most important player in cell pluripotency. The aim of the study was to investigate the morphogenesis and function of rat adrenal zona glomerulosa in rats exposed to low doses of DDT during prenatal and postnatal development and to determine the possible role of Oct4 in DDT-mediated structural and functional changes. The DDT-exposed rats demonstrated slower development and lower functional activity of the zona glomerulosa during the pubertal period associated with higher expression of Oct4. Further, accelerated growth and restoration of hormone production was associated with, firstly, a decrease in Oct4 expressing cells and secondly, the loss of the inverse relationship between basal aldosterone levels and the number of Oct4 expressing cells. Thus, the transcriptional factor Oct4 exhibited an altered pattern of expression in the DDT-exposed rats during postnatal development. The results of the study uncover a novel putative mechanism by which low doses of DDT disrupt the development of adrenal zona glomerulosa.

Human cytochrome P450 11B2 produces aldosterone by a processive mechanism due to the lactol form of the intermediate 18-hydroxycorticosterone.

Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme (i.e. processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.

Adrenal cortex hypoxia modulates aldosterone production in heart failure.

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated ß3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced ß3-adrenergic receptor expression. Hypoxia-mediated ß3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Aldosterone Synthase in Peripheral Sensory Neurons Contributes to Mechanical Hypersensitivity during Local Inflammation in Rats.

BACKGROUND: Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. METHODS: In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. RESULTS: In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). CONCLUSIONS: Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.

The Effect of Extracellular Calcium Metabolism on Aldosterone Biosynthesis in Physiological and Pathological Status.

Primary aldosteronism (PA) was reported to frequently harbor not only cardiovascular diseases but also some metabolic disorders including secondary calcium metabolic diseases. Recently, the potential association between aldosterone producing cells and systemic calcium metabolism has been proposed. For instance, PA is frequently associated with hypercalciuria or hypocalcemia, which subsequently stimulates parathyroid hormone (PTH) secretion. This altered calcium metabolism in PA patients could frequently result in secondary osteoporosis and fracture in some patients. On the other hand, extracellular calcium itself directly acts on adrenal cortex and has been also proposed as an independent regulator of aldosterone biosynthesis in human adrenals. However, it is also true that both PTH and vitamin D pathways stimulate endocrine functions of adrenal cortical adenomas to co-secret both aldosterone and cortisol. Therefore, it has become pivotal to explore the potential crosstalk between aldosterone and systemic calcium metabolism. We herein reviewed recent advances in these fields.

Immunohistochemistry for aldosterone synthase CYP11B2 and matrix-assisted laser desorption ionization imaging mass spectrometry for in-situ aldosterone detection.

PURPOSE OF REVIEW: Immunohistochemistry for aldosterone synthase (CYP11B2) has markedly provided a comprehensive picture of the adrenocortical diseases, particularly primary aldosteronism. The findings from CYP11B2-immunohistochemistry are consistent with the clinical courses of most patients with primary aldosteronism. We herein review the updated pathophysiology and usefulness of the method for understanding individual patients with different subtypes of primary aldosteronism. RECENT FINDINGS: After our discovery of aldosterone-producing cell clusters (APCCs) using the immunohistochemistry for CYP11B2, we found possible APCC-to-APA transitional lesions (pAATLs) in a few cases that had been hitherto classified as unilateral hyperplasia or multiple nodules. On the basis of morphological and functional features of pAATLs as well as distributions of somatic mutations within the lesions, we have made a hypothesis that APCC grows to APA via pAATL for one of developmental courses of APA. Recently, we successfully performed in-situ detection of aldosterone on adrenal tissue sections using a state-of-the-art technique, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-imaging). This method revealed in-situ synthesis of aldosterone in APCCs and APAs in addition to several other steroids. SUMMARY: CYP11B2 immunohistochemistry revealed the pathophysiology of aldosterone production in the past decade, especially formation of APCC in normal adrenals and pAATL that is a possible lesion developing from APCC to APA. The term 'idiopathic hyperaldosteronism' may soon become obsolete.

Steroid Profiling and Immunohistochemistry for Subtyping and Outcome Prediction in Primary Aldosteronism-a Review.

PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.

Steroidogenic abnormalities in translocator protein knockout mice and significance in the aging male.

The translocator protein (TSPO) has been proposed to act as a key component in a complex important for mitochondrial cholesterol importation, which is the rate-limiting step in steroid hormone synthesis. However, TSPO function in steroidogenesis has recently been challenged by the development of TSPO knockout (TSPO-KO) mice, as they exhibit normal baseline gonadal testosterone and adrenal corticosteroid production. Here, we demonstrate that despite normal androgen levels in young male TSPO-KO mice, TSPO deficiency alters steroidogenic flux and results in reduced total steroidogenic output. Specific reductions in the levels of progesterone and corticosterone as well as age-dependent androgen deficiency were observed in both young and aged male TSPO-KO mice. Collectively, these findings indicate that while TSPO is not critical for achieving baseline testicular and adrenal steroidogenesis, either indirect effects of TSPO on steroidogenic processes, or compensatory mechanisms and functional redundancy, lead to subtle steroidogenic abnormalities which become exacerbated with aging.

Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands.

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.

Endogenous Purification of NR4A2 (Nurr1) Identified Poly(ADP-Ribose) Polymerase 1 as a Prime Coregulator in Human Adrenocortical H295R Cells.

Aldosterone is synthesized in zona glomerulosa of adrenal cortex in response to angiotensin II. This stimulation transcriptionally induces expression of a series of steroidogenic genes such as HSD3B and CYP11B2 via NR4A (nuclear receptor subfamily 4 group A) nuclear receptors and ATF (activating transcription factor) family transcription factors. Nurr1 belongs to the NR4A family and is regarded as an orphan nuclear receptor. The physiological significance of Nurr1 in aldosterone production in adrenal cortex has been well studied. However, coregulators supporting the Nurr1 function still remain elusive. In this study, we performed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins), a recently developed endogenous coregulator purification method, in human adrenocortical H295R cells and identified PARP1 as one of the top Nurr1-interacting proteins. Nurr1-PARP1 interaction was verified by co-immunoprecipitation. In addition, both siRNA knockdown of PARP1 and treatment of AG14361, a specific PARP1 inhibitor suppressed the angiotensin II-mediated target gene induction in H295R cells. Furthermore, PARP1 inhibitor also suppressed the aldosterone secretion in response to the angiotensin II. Together, these results suggest PARP1 is a prime coregulator for Nurr1.

Role of Cryptochrome-1 and Cryptochrome-2 in Aldosterone-Producing Adenomas and Adrenocortical Cells.

Mice lacking the core-clock components, cryptochrome-1 (CRY1) and cryptochrome-2 (CRY2) display a phenotype of hyperaldosteronism, due to the upregulation of type VI 3ß-hydroxyl-steroid dehydrogenase (Hsd3b6), the murine counterpart to the human type I 3ß-hydroxyl-steroid dehydrogenase (HSD3B1) gene. In the present study, we evaluated the role of CRY1 and CRY2 genes, and their potential interplay with HSD3B isoforms in adrenal pathophysiology in man. Forty-six sporadic aldosterone-producing adenomas (APAs) and 20 paired adrenal samples were included, with the human adrenocortical cells HAC15 used as the in vitro model. In our cohort of sporadic APAs, CRY1 expression was 1.7-fold [0.75⁻2.26] higher (p = 0.016), while CRY2 showed a 20% lower expression [0.80, 0.52⁻1.08] (p = 0.04) in APAs when compared with the corresponding adjacent adrenal cortex. Type II 3ß-hydroxyl-steroid dehydrogenase (HSD3B2) was 317-fold [200⁻573] more expressed than HSD3B1, and is the main HSD3B isoform in APAs. Both dehydrogenases were more expressed in APAs when compared with the adjacent cortex (5.7-fold and 3.5-fold, respectively, p < 0.001 and p = 0.001) and HSD3B1 was significantly more expressed in APAs composed mainly of zona glomerulosa-like cells. Treatment with angiotensin II (AngII) resulted in a significant upregulation of CRY1 (1.7 ± 0.25-fold, p < 0.001) at 6 h, and downregulation of CRY2 at 12 h (0.6 ± 0.1-fold, p < 0.001), through activation of the AngII type 1 receptor. Independent silencing of CRY1 and CRY2 genes in HAC15 cells resulted in a mild upregulation of HSD3B2 without affecting HSD3B1 expression. In conclusion, our results support the hypothesis that CRY1 and CRY2, being AngII-regulated genes, and showing a differential expression in APAs when compared with the adjacent adrenal cortex, might be involved in adrenal cell function, and in the regulation of aldosterone production.

[Efficacy of Screening and Confirmatory Tests of Primary Aldosteronism in Diagnosing Aldosterone Producing Adenoma].

OBJECTIVE: To assess the efficacy of screening and confirmatory tests of primary aldosteronism (PA) in diagnosing aldosterone producing adenoma (APA). METHODS: Clinical data of 167 hypertensive patients were retrospectively reviewed,including 93 patients with APA and 74 patients with essential hypertension (EH). The area under curves (AUC) of receiver operating characteristic (ROC) curves were compared among the five indicators: supine plasma aldosterone concentration (PAC),absolute PAC values and PAC drop rates post saline infusion test (SIT) and captopril challenge test (CCT). RESULTS: APA patients had higher supine PAC,higher percentage of third degree hypertension,and lower serum potassium level than EH patients (P<0.05). Compared with EH patients,APA patients had lower PAC change rates,post posture change and SIT (P<0.05),but similar post CCT (P>0.05). The AUC of supine PAC reached 0.975. Higher AUC was found in absolute PAC values post SIT compared with PAC droop rates (0.984 vs. 0.680,P<0.001). Similar results were also found with CCT (0.949 vs. 0.538,P<0.001). A cut-off of supine aldosterone renin ratio (ARR) >30 and supine PAC>17.8 ng/dL had 96.8% sensitivity and 90.5% specificity. A cut-off of 14.59 ng/dL PAC post SIT had 90.2% sensitivity and 97.3% specificity. A cut-off of 19.11 ng/dL PAC post CCT had 88.8% sensitivity and 95.9% specificity. CONCLUSION: Screening tests using supine ARR>30 plus supine PAC>17.83 ng/dL are preferred with high sensitivity and specificity. The absolute values of PAC post SIT and CCT are recommended for confirming APA.

A ubiquitin-proteasome inhibitor bortezomib suppresses the expression of CYP11B2, a key enzyme of aldosterone synthesis.

CYP11B2 is a key enzyme involved in the synthesis of the mineralocorticoid aldosterone. CYP11B2 expression in the adrenal glands is controlled by the renin-angiotensin system (RAS), and plays an important role in the maintenance of electrolyte metabolism in higher organisms. Abnormal overexpression of CYP11B2 results in the disruption of mineral balance and can lead to hypertension. Though the molecular mechanism of the regulation of CYP11B2 expression has remained elusive, we hypothesize that compounds that prevent CYP11B2 expression could represent a novel class of antihypertensive drugs. In this study, we established a high-throughput screening system to identify such compounds, and subsequently carried out chemical screening. We found that the ubiquitin-proteasome inhibitor bortezomib could suppress CYP11B2 expression and secretion of aldosterone induced by angiotensin II (Ang II) in adrenocortical H295R cells. Moreover, bortezomib down-regulated the Cyp11b2 mRNA expression facilitated in the adrenal gland of Tsukuba hypertensive mice, resulting in subsequent lowering of their blood pressures. Furthermore, we observed the characteristic alteration of H3K27ac in the adrenal CYP11B2 gene promoter induced by Ang II stimulation, which was found to be disrupted by bortezomib. Taken together, these results suggest the possibility of developing novel antihypertensive drugs that prevent CYP11B2 expression.