Aldosterone-producing Adenoma Tissue Research Articles

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

Sfrp2 regulates the WNT/β-catenin pathway to slow the development of aldosterone-producing adenoma.

To explore a new drug therapy for aldosterone-producing adenoma (APA), and investigate whether Sfrp2 (secreted frizzled-related protein 2) can influence the development of adrenal APA by regulating the WNT/β-catenin pathway. Tissue samples from APA patients were collected to detect the expression of Sfrp2 and β-catenin in APA. NCI-H295R cells were cultured with WNT/β-catenin pathway inhibitors to detect cell proliferation and aldosterone secretion. Then, the expression of Sfrp2 was altered to determine the effect of Sfrp2 expression on WNT/β-catenin pathway activity and aldosterone adenocarcinoma cells. Finally, a mouse APA model was established, and the mice were intravenously injected with WNT/β-catenin pathway inhibitors or transfected with the Sfrp2 gene. The activity of the WNT/β-catenin pathway, blood pressure, aldosterone secretion, and cell growth in the mice were then observed. β-catenin was overexpressed in APA tissues, while Sfrp2 was underexpressed. Sfrp2 can negatively regulate β-catenin expression and control the activity of the WNT/β-catenin pathway. Increased Sfrp2 expression inhibited the activity of the WNT/β-catenin pathway, which suppressed aldosterone secretion and APA cell proliferation. The in vivo experiments also demonstrated that inhibition of WNT/β-catenin pathway activity in mice reduced the arterial pressure and aldosterone concentration. The increased expression of Sfrp2 can inhibit the WNT/β-catenin pathway in mice, and can also reduce arterial pressure and APA tissue growth. Sfrp2 can inhibit the WNT/β-catenin signaling pathway by suppressing the expression of β-catenin, thus controlling the concentration of aldosterone and hindering APA development. This study provides a novel therapeutic target for the treatment of APA and a new direction for future research.

ACE2 AND ANGIOTENSIN-(1-7) AND ALDOSTERONE BIOSYNTHESIS IN HUMAN ADRENOCORTICAL TISSUES

Objective: The angiotensin converting enzyme 2 (ACE2) and its product angiotensin-(1–7) (Ang-(1–7)) were suggested to play cardiovascular protective effects via angiotensin type 2 (AT2R), MasR and alamandine receptors. Whether this involves blunting of aldosterone biosynthesis in humans remains unknown. Design and method: We quantified ACE2 mRNA in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained ex vivo from primary aldosteronism (PA) patients with real-time and ddPCR. We also investigated the effects of Ang-(1–7) and diminazene aceturate (DIZE), a putative ACE2 activator, on aldosterone synthase (CYP11B2) and 11b-hydroxylase (CYP11B1) as proxy for aldosterone and cortisol secretion, respectively, in the absence and presence of the AT1R antagonist irbesartan, or the MasR antagonist A779. Results: We detected ACE2 mRNA at higher amounts in APA-adjacent tissue than in APA. Functional in vitro studies with LC-MS showed active conversion of Ang II into Ang 1–7, albeit at low level, through ACE-2-independent mechanisms. At [10–8 M] Ang-(1–7) did not affect CYP11B1 and CYP11B2 gene transcripts, while at high concentration [10–6 M] it increased both mRNAs (p < 0.001 vs vehicle) through an AT1-R-mediated mechanism as these stimulatory effects were abolished by irbesartan. To investigate if Ang-(1–7) could blunt aldosterone production when CYP11B2 is upregulated, we exposed H295 cells to [10–8 M] Ang II and found that Ang-(1–7) did not blunt Ang II-induced expression of CYP11B1 and CYP11B2 mRNA. Moreover, we discovered that [10–7 M] DIZE increased ACE2 mRNA by 1.5 fold from baseline (p = 0.01) but left CYP11B1 or CYP11B2 mRNA levels unaffected. DIZE did not blunt the AT1R mediated stimulatory effect of [10–6 M] Ang-(1–7) on CYP11B1 and CYP11B2 mRNA. Conclusions: Thus, although Ang-(1–7) can be locally generated at low levels and MasR and ACE2 mRNAs are detectable in APA and APA-adjacent tissue, neither Ang-(1–7) nor DIZE were able to blunt Ang II-mediated aldosterone synthase expression. Collectively these results suggest that the ACE2/Ang-(1–7)/MasR axis is unlikely to counteract aldosterone biosynthesis under conditions of enhanced aldosterone secretion as PA and heart failure.

SOMATIC MUTATIONS IN ADRENALS FROM PATIENTS WITH PRIMARY ALDOSTERONISM NOT CURED AFTER ADRENALECTOMY SUGGEST COMMON PATHOGENIC MECHANISMS BETWEEN UNILATERAL AND BILATERAL DISEASE

Objective: Primary aldosteronism (PA) is the most common form of secondary and curable hypertension, with a prevalence of 5% in primary care and 10% in referred patients. Different germline and somatic mutations are found in aldosterone producing adenoma (APA) and familial forms of the disease, while the causes of bilateral adrenal hyperplasia (BAH) remain largely unknown. Adrenalectomy is the recommended treatment for patients with aldosterone-producing adenoma (APA); however, 6% of patients are not cured and show persistent PA after surgery suggesting BAH. The objective of this study was to analyze clinical data of patients with APA without biochemical success after adrenalectomy and the histological and genetic characteristics of their adrenal glands. Design and method: Clinical data from 16 PA patients undergoing adrenalectomy between 2008 and 2018 without biochemical success were compared to those from 39 PA patients with hormonal cure after surgery. Histological, morphological and genetic characterization of the adrenals was carried out by CYP11B2 and CYP11B1 immunostaining and by CYP11B2 guided next generation sequencing on formalin-fixed paraffin-embedded APA tissues. Results: Patients with absent hormonal cure displayed longer duration of arterial hypertension before PA diagnosis and lower index of lateralization of aldosterone production on adrenal vein sampling. In 14 patients, APA expressing CYP11B2 were identified. No difference in histological and morphological characteristics was observed between patients with our without hormonal cure. Somatic mutations in APA driver genes were identified in 13 out of 14 CYP11B2 positive APA. Mutations in the CACNA1D gene were the most frequent genetic abnormality and associated with smaller nodules. The mutational status of APA in patients without hormonal cure was not associated with differences in CYP11B1 or CYP11B2 expression. Conclusions: In conclusion, 93% of adrenals from patients with absent hormonal cure showed CYP11B2 expressing adenomas carrying somatic mutations, confirming the diagnosis of APA. However, these patients were diagnosed later and exhibited lower lateralization index of aldosterone production, suggesting asymmetric aldosterone production in the context of BAH. Somatic mutations in adrenal glands from those patients indicate common mechanisms underlying BAH and APA.

BIOCHEMICAL, HISTOPATHOLOGICAL, AND GENETIC CHARACTERIZATION OF POSTURE RESPONSIVE AND UNRESPONSIVE ALDOSTERONE-PRODUCING ADENOMAS

Objective: Aldosterone-producing adenoma (APA) accounts for ∼30% of primary aldosteronism. Based on responses of aldosterone to upright posture, APA can be divided into posture-responsive and –unresponsive forms. Somatic gene mutations in KCNJ5, CACNA1D, ATP1A1, or ATP2B3 have been reported to exist in more than 90% of APAs. We sought to compare the biochemical, histopathological, and genetic characterization between these two types of APAs. Design and method: Posture-responsive was defined as showing a rise in plasma aldosterone concentration of at least 50% 2 - 3 h after the assumption of upright posture (10 AM) above the basal level (7 AM) following overnight recumbency. Plasma levels of aldosterone and hybrid steroids (18-oxocortisol and 18-hydroxycortisol) were measured by LC-MS/MS. Histologic analysis and immunohistochemistry for CYP11B2 (aldosterone synthase) and CYP17A1 (17aplha-hydroxylase) were performed on APA tissue sections collected from 24 posture-unresponsive and 17 posture-responsive APA patients. Sanger sequencing of KCNJ5 and gene targeted next-generation sequencing were performed to detect somatic gene mutations. Results: Compared to patients with posture-responsive APA (of which 48.7% were composed of zona glomerulosa (ZG)-like cells and 35.3% showed ZG-zona fasciculata (ZF) mixed appearance), posture-unresponsive APA patients displayed higher (P < 0.01) levels of hybrid steroids, recumbent aldosterone and cortisol, greater (P < 0.05) suppression of aldosterone secretion from the contralateral unaffected adrenal, and larger (P < 0.01) tumors mostly composed of ZF-like cells that had higher (P < 0.01) expression of CYP17A1 (but not of CYP11B2). Of 41 studied APAs, 37 (90.2%) harboured aldosterone-driving somatic mutations (KCNJ5 = 14, 34.1%; CACNA1D = 13, 31.7%; ATP1A1 = 8, 19.5%; and ATP2B3 = 2, 4.9%), including 5 that were previously unreported (3 in CACNA1D and 2 in ATP1A1). Interestingly, 64.7% (11/17) of posture-responsive APAs carried CACNA1D mutations, whereas 54.2% (13/24) of posture-unresponsive APAs harboured KCNJ5 mutations. Conclusions: The excessive production of hybrid steroids by posture-unresponsive APAs may relate to their ZF-like tumor cell composition, resulting in strong expression of CYP17A1 (in addition to somatic gene mutation-driven CYP11B2 expression), thereby allowing production of cortisol which acts as the substrate for CYP11B2-generated hybrid steroids.

Aldosterone and cortisol synthesis regulation by angiotensin-(1-7) and angiotensin-converting enzyme 2 in the human adrenal cortex.

The branch of the renin--angiotensin system constituting angiotensin-(1-7) [Ang-(1-7)], the Ang II type 2 receptor, the Mas receptors and the Ang-(1-7)-forming enzyme ACE-2, by counteracting the Ang II type 1 receptor (AT1R)-mediated effects, are held to be cardiovascular protective in several conditions. However, whether Ang-(1-7) and ACE-2 are detectable in human adrenocortical tissues and whether they affect aldosterone and cortisol biosynthesis was unknown. We measured angiotensin peptides with liquid chromatography tandem-mass spectrometry and ACE-2 mRNA with digital droplet (dd)PCR in human aldosterone-producing adenoma (APA) and APA-adjacent tissue obtained from patients with primary aldosteronism. We also investigated the effects of Ang-(1-7) and the ACE-2 activator diminazene aceturate (DIZE) on aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) gene expression, in the absence or presence of the AT1R antagonist irbesartan, or of the MasR antagonist A779. APA and APA-adjacent adrenocortical tissues express ACE-2 mRNA and contain detectable amounts of Ang II and Ang-(2-8), but not of Ang I, Ang-(1-5), Ang (3-8) and Ang-(1-7). Under unstimulated and Ang II- stimulated conditions Ang-(1-7) did not blunt CYP11B1 and CYP11B2 mRNA. At supraphysiological concentrations (10-4 mol/l), Ang-(1-7) stimulated both CYP11B1 and CYP11B2 mRNA via the AT1R. The ACE-2 activator DIZE increased by 1.5-fold ACE-2 mRNA but did not blunt Ang II- upregulated CYP11B1 and CYP11B2 expression. These results do not support the hypothesis that the ACE-2/Ang-(1-7)/MasR axis play a protective role by counteracting enhanced aldosterone secretion in humans.

Adrenal (Pro)renin Receptor Expression and Serum Soluble (Pro)renin Receptor Concentration in Primary Aldosteronism

The (pro)renin receptor [(P)RR] is a multifunctioning protein playing roles in various pathological conditions. A soluble form of (P)RR [s(P)RR] has been considered a biomarker for (P)RR expression in tissues. Expression of (P)RR has been described in aldosterone-producing adenoma (APA), but the roles of (P)RR have yet to be fully determined. This study investigated the significance of (P)RR and serum s(P)RR concentrations in patients with APA. We evaluated associations between (P)RR expression and expression of CYP11B2, an aldosterone synthase, and aldosterone production by the adrenal glands and assessed the relationships between serum s(P)RR concentration and background factors. (P)RR colocalized with CYP11B2 and expression levels of (P)RR were positively associated with those of CYP11B2 in APA tissues. (P)RR immunoreactivity in these tissues correlated positively with plasma aldosterone concentrations (PAC) and urinary aldosterone excretion. Also, in APA, (P)RR mRNA abundance was positively correlated with β-catenin mRNA abundance. Significant positive correlations were identified between serum s(P)RR concentration and plasma glucose, hemoglobin A1c, and serum creatinine levels, but not with PAC (in either peripheral vein or adrenal vein) or adrenal (P)RR expression level. This study showed that (P)RR expression level correlates with CYP11B2 expression in APA tissues and PAC and urinary aldosterone excretion, suggesting that (P)RR expression may contribute to aldosterone synthesis via CYP11B2 activation in APAs, although serum s(P)RR concentration failed to show any significant relationship with adrenal (P)RR expression. Adrenal (P)RR activity might offer a therapeutic target in the treatment of PA, although this issue needs to be investigated in future studies.

SAT-553 Use of Optimal Cutting Temperature Compound (OCT)-Embedded Adrenal Tumor Tissue for Intratumoral Steroid Hormone Profiling

Background: Primary aldosteronism (PA) is the most common cause of secondary hypertension, accounting for 5-8% of all hypertension. PA is most commonly attributed to an aldosterone-producing adenoma (APA) or to bilateral hyperaldosteronism (BHA). Mutations in the inward-rectifying K+ channel (mKCNJ5), which increase autonomous aldosterone production, are most frequently detected in APAs. APAs with mKCNJ5 display aberrant expression of aldosterone synthase (CYP11B2) and 17α-hydroxylase (CYP17A1), which are involved in aldosterone and cortisol synthesis, respectively. Co-expression of these enzymes results in the production of a set of “hybrid” steroids, which have been proposed as serum biomarkers. The relative production of hybrid steroids in adrenal tumors vs. adjacent normal adrenal (NA) tissue has not been investigated. Objectives: To determine the utility of OCT-embedded adrenal tumor tissue for steroid profiling. To use immunohistochemistry (IHC)-guided OCT tumor capture for intratumoral hybrid steroid profiling in mKCNJ5 APA and NA tissue. Methods: OCT-embedded adrenal tissue from 9 patients (8 women, Age 45.9 ± 3.3 years) with APAs harboring known KCNJ5 mutations were used for the study. Where available OCT-embedded normal adrenal (NA) tissue adjacent to APAs were used as controls (n=4). IHC was performed for CYP11B2 and CYP17A1 on OCT tissue allowing guided APA capture from serial sections. Steroids were extracted from APA and adjacent NA tissue, and quantified by liquid chromatography/tandem mass spectrometry. Steroids measured were normalized to the protein content of the extracted tissue. Results: Compared to NA, APA tissue demonstrated 23-, 5.6- and 6.4-fold higher levels of aldosterone, 11-deoxycorticosterone, and 18-hydroxycorticosterone, respectively (P<0.05). In addition, the “hybrid” steroid products, 18-oxocortisol and 18-hydroxycortisol, were significantly elevated in APA vs. NA (P<0.01). Conversely, the adrenal androgens dehydroepiandrosterone and 11-hydroxyandrostenedione were lower in APA as compared with NA (P<0.05). All mKCNJ5 APAs were also found to co-express CYP11B2 and CYP17A1. Conclusion: IHC-guided mKCNJ5 APA capture and steroid extraction identified a distinct intratumoral hybrid steroid signature that associated with co-expression of CYP11B2 and CYP17A1.These findings also demonstrate that OCT-embedded tissue can be used to accurately define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.

MiRNA299 involvement in CYP11B2 expression in aldosterone-producing adenoma.

The pathophysiology of aldosterone-producing adenomas (APAs) has been intensively investigated using genetic and epigenetic approaches. However, the role of miRNAs in APA is not fully understood. The present study profiled miRNAs in APAs as an exploratory approach to elucidate their pathophysiological roles in APAs. Tissues of APAs and other adrenocortical adenomas were obtained from patients who underwent adrenalectomy. Candidate miRNAs differentially detected from samples were examined by whole miRNA sequencing. The expression of candidate miRNAs in APA tissues were further validated by real-time quantitative polymerase chain reaction (qPCR). Further, differential miRNA expression between APAs with and without KCNJ5 somatic mutations was examined. Prediction of miRNA target genes was performed by bioinformatics analysis. For specific miRNAs, correlation analysis between the levels of their target genes and CYP11B2 was analyzed in APA tissues. Our study determined differential expression of six miRNAs in APA or APA with KCNJ5 mutations. We further demonstrated that miR299 levels were negatively correlated with mRNA levels of CACNB2, which encodes the beta-subunit of the L-type calcium channel. Additionally, we found significant correlations among miR299, CACNB2, and CYP11B2 levels in APA tissues. Our study suggests the possible pathophysiological involvement of specific miRNAs in calcium signaling and aldosterone hypersecretion in APAs. Further studies, including in vitro analyses, are required to clarify these findings.

The angiotensin type 2 receptor in the human adrenocortical zona glomerulosa and in aldosterone-producing adenoma: low expression and no functional role.

The angiotensin II (Ang II) type 2 receptor (AT2R) and the angiotensin-(1-7) (Ang-(1-7)) receptor (MasR) play a cardiovascular protective role by counter-regulating Ang II type 1 receptor (AT1R)-mediated effects, but whether this involves blunting of adrenocortical hormone secretion is unknown. We investigated the presence of AT1R, AT2R, and MasR in aldosterone-producing adenoma (APA), a condition featuring hyperaldosteronism, and in APA-adjacent tissue. The effect of Compound 21 (C21), an AT2R agonist, on CYP11B1 (cortisol synthase) and CYP11B2 (aldosterone synthase) gene expression in NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, was also assessed using the AT1R antagonist irbesartan to ascertain the specificity of C21 effect. We found that the AT1R, AT2R, and MasR were expressed in APA and APA-adjacent tissue, albeit heterogeneously. The gene expression of AT1R and AT2R was lower, and that of the MasR higher in APAs than in APA-adjacent tissue. In steroid-producing NCI-H295R and HAC15 cell lines, and in APA and APA-adjacent tissue, C21 was ineffective at nanomolar concentrations, but increased CYP11B1 and CYP11B2 gene expression at micromolar concentrations through AT1R, as this effect was blunted by irbesartan. The scant expression of the AT2R, along with the lack of any effect of C21 at low concentrations on CYP11B2, do not support the contention that the protective arm of renin-angiotensin system (RAS) blunts aldosterone synthase in the normal adrenal cortex and primary aldosteronism.

KCNJ5 mutation as a predictor for resolution of hypertension after surgical treatment of aldosterone-producing adenoma

To investigate the effect of KCNJ5 mutations on the cure of hypertension in patients with aldosterone-producing adenoma (APA) after unilateral adrenalectomy. Our study included 142 patients with APA, who were detected with an endocrinological abnormality and diagnosed with hypertension, as confirmed by pathological analysis. We sequenced KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 from APA tissue samples, and performed a retrospective analysis to determine correlations between wild-type or mutated KCNJ5 and patient clinical characteristics. Somatic KCNJ5 mutations were identified in 106 of 142 patients with APA, 136 of whom had resolution of hyporeninemic-hyperaldosteronemia 1 year after surgery. Of the 136 patients, 81 patients had resolution of hypertension ('Cured group' vs. 'Improved group'). We found increased prevalence of KCNJ5 mutations in the Cured group compared to the Improved group (85.2% vs. 60.0%, respectively; P = 0.002), which was associated with younger age, shorter duration of hypertension, fewer antihypertensive medications, lower BMI, higher aldosterone level, higher estimated glomerular filtration rate, and milder vascular complications. In both groups we found that harbouring a KCNJ5 mutation, taking fewer antihypertensive medications, and the duration of hypertension were independently associated with resolution of hypertension by unilateral adrenalectomy. In patients with KCNJ5-mutated APA, left ventricular hypertrophy was significantly decreased by surgical treatment in patients from either Cured or Improved groups, although those patients with wild-type KCNJ5 showed no change. Testing for KCNJ5 mutations in young patients with APA may provide a prognostic indication for resolution of hypertension and severity of vascular complications.

Macrolides Blunt Aldosterone Biosynthesis: A Proof-of-Concept Study in KCNJ5 Mutated Adenoma Cells Ex Vivo.

Aldosterone-producing adenoma (APA), a major subtype of primary hyperaldosteronism, the main curable cause of human endocrine hypertension, involves somatic mutations in the potassium channel Kir3.4 (KCNJ5) in 30% to 70% of cases, typically the more florid phenotypes. Because KCNJ5 mutated channels were reported to be specifically sensitive to inhibition by macrolide antibiotics, which concentration dependently blunts aldosterone production in HAC15 transfected with the G151R and L168R mutated channel, we herein tested the effect of clarithromycin on aldosterone synthesis and secretion in a pure population of aldosterone-secreting cells obtained by immunoseparation (CD56+ cells) from APA tissues with/without the 2 most common KCNJ5 mutations. From a large cohort of patients with an unambiguous APA diagnosis, we recruited those who were wild type (n=3) or had G151R (n=2) and L168R (n=2) mutations. We found that clarithromycin concentration dependently lowered CYP11B2 gene expression (by 60%) and aldosterone secretion (by 70%; P<0.001 for both) in CD56+ cells isolated ex vivo from KCNJ5 mutated APAs, although it was ineffective in CD56+ cells from wild-type APAs. By proving the principle that the oversecretion of aldosterone can be specifically blunted in APA cells ex vivo with G151R and L168R mutations, these results provide compelling evidence of the possibility of specifically correcting aldosterone excess in patients with APA carrying the 2 most common KCNJ5 somatic mutations.

Abstract P275: Peripheral 18-oxocortisol Concentration Reflects Intra-tumoral Activities of Both CYP11B1 and CYP11B2 in Aldosterone-producing Adenoma

Background: The 18-oxocortisol (18oxoF) is an expected biomarker for detection of patients with aldosterone-producing adenoma (APA) among patients with primary aldosteronism. However, the mechanism of 18oxoF synthesis in APA and differences of the production amount among APAs remain unclear. Objective: To examine the associations between peripheral 18oxoF concentration (p18oxoF) and clinical or pathological parameters in APA cases. Method: We included 48 APA cases who were diagnosed in our center. The p18oxoF and tissue steroid levels were measured by highly sensitive LC-MS/MS measurement. We performed immunohistochemical (IHC) staining of 11β-hydroxylase (CYP11B1), aldosterone synthase (CYP11B2) and 17α-hydroxylase (c17) for all the cases. The IHC images were analyzed by HALO digital image software. Moreover, we performed immunofluorescence staining to evaluate the number of CYP11B1/B2 double positive cells (B1/B2 cells). Result: The characteristics of 48 cases were as follows; Age 51.7 ±11.8 years; Male 54.2% (26/48); Plasma aldosterone (A) 52.2 ±45.8 ng/dl; Plasma renin activity 0.35 ±0.43 ng/ml/h; Serum cortisol (F) 10.0 ±3.5 μg/dl; Cross-sectional area (CSA) of APA 63.9 ±52.7 mm 2 . The p18oxoF showed a lognormal distribution from 0.19 to 183.13 ng/dl (Median; 11.92 ng/dl). The p18oxoF, a derivative of F, was positively correlated with plasma A (r=0.728, P&lt;0.0001) but not serum F (P=N.S.). However, there were significant correlations between p18oxoF and intra-tumoral A or F levels in 15 frozen APA tissues which were available (A: r=0.750, P&lt;0.01, F: r=0.575, P&lt;0.05). On the other hand, p18oxoF was positively correlated with CSA (r=0.734, P&lt;0.0001). Divided them into low and high groups based on p18oxoF, high group had larger CSA (36.1 ±37.9 vs 91.8 ±51.2 mm 2 , P&lt;0.0001) and immunoreactive positive areas of CYP11B1 (7.9 ±12.5 vs 17.9 ±18.4 mm 2 , P=0.003), CYP11B2 (9.0 ±8.8 vs 20.7 ±9.9 mm 2 , P&lt;0.0001) and c17 (6.1 ±10.1 vs 12.6 ±7.6 mm 2 , P=0.0004) than low group. High group also had more B1/B2 cells than low group (106.5 ±93.1 vs 49.3 ±59.3 /mm 2 , P=0.009). Conclusion: These results suggest 18oxoF synthesis could be influenced by the size of APA, which reflects intra-tumoral activities of not only CYP11B2 but also CYP11B1, a rate-limiting enzyme of F.

Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation

BackgroundThe adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. MethodsWe evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. ResultsWe revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. ConclusionThe present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs.

Clinical and Steroidogenic Characteristics of Aldosterone-Producing Adenomas With ATPase or CACNA1D Gene Mutations.

This comparative study clarified the clinical characteristics and in vitro steroidogenic activities of aldosterone-producing adenomas (APAs) harboring ATPase or CACNA1D gene mutations. Genetic testing was performed on 159 unilateral APAs. Somatic ATPase and CACNA1D gene mutations were analyzed in 42 APA tissues without KCNJ5 gene mutations. ATP1A1, ATP2B3, and CACNA1D mutations were detected in one, four, and four patients, respectively. Compared with patients without KCNJ5, ATPase, or CACNA1D mutations (wild type), ATPase mutations tended to have more severe hyperaldosteronism and smaller tumors; those with CACNA1D mutations had clinical characteristics and tumor sizes similar to those with wild-type genes. APAs with ATPase mutations were composed mainly of compact eosinophilic tumor cells, whereas CACNA1D mutations resulted in predominantly clear tumor cells. Aldosterone production in APA cells with ATP2B3 mutations were more responsive to dibutyryl cAMP, whereas those with CACNA1D mutations were more responsive to adrenocorticotropic hormone than the wild-type cells. APAs with ATPase mutations demonstrated a potentially severe primary aldosteronism phenotype, whereas those with CACNA1D mutations displayed characteristics similar to wild-type APAs. The status of stimulated aldosterone production was also different according to the cell types, suggesting that the regulatory effects of adrenocorticotropic hormone on aldosterone synthesis could possibly vary according to the intracellular signaling involved in hormone production.

Expression and functional role of the prorenin receptor in the human adrenocortical zona glomerulosa and in primary aldosteronism.

Prorenin can be detected in plasma of hypertensive patients. If detected in patients with primary aldosteronism could implicate prorenin in the development of primary aldosteronism. To address this issue, we measured the plasma prorenin levels in primary aldosteronism patients, the expression of the prorenin receptor (PRR) in the normal human adrenocortical zona glomerulosa and aldosterone-producing adenoma (APA), and we investigated the functional effects of PRR activation in human adrenocortical cells. Plasma renin activity, aldosterone, and active and total trypsin-activated renin were measured in primary aldosteronism patients, essential hypertensive patients, and healthy individuals, and then prorenin levels were calculated. Localization and functional role of PRR were investigated in human and rat tissues, and aldosterone-producing cells. Primary aldosteronism patients had detectable plasma levels of prorenin. Using digital-droplet real-time PCR, we found a high PRR-to-porphobilinogen deaminase ratio in both the normal adrenal cortex and APAs. Marked expression of the PRR gene and protein was also found in HAC15 cells. Immunoblotting, confocal, and immunogold electron microscopy demonstrated PRR at the cell membrane and intracellularly. Renin and prorenin significantly triggered both CYP11B2 expression (aldosterone synthase) and ERK1/2 phosphorylation, but only CYP11B2 transcription was prevented by aliskiren. The presence of detectable plasma prorenin in primary aldosteronism patients, and the high expression of PRR in the normal human adrenal cortex, APA tissue, CD56+ aldosterone-producing cells, along with activation of CYP11B2 synthesis and ERK1/2 phosphorylation, suggest that the circulating and locally produced prorenin may contribute to the development or maintenance of human primary aldosteronism.

Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas.

Adrenal mast cells can stimulate aldosterone secretion through the local release of serotonin (5-HT) and activation of the 5-HT4 receptor (5-HT4). In aldosterone-producing adenomas (APAs), 5-HT4 receptor is overexpressed and the administration of 5-HT4 receptor agonists to patients with APA increases plasma aldosterone levels. These data and the well-documented role of mast cells in tumorigenesis suggest that mast cells may be involved in the pathophysiology of APA. The study aimed at investigating the occurrence of mast cells in a series of APA tissues and to examine the influence of mast cells on aldosterone secretion. The occurrence of mast cells in APAs was investigated by immunohistochemistry. Mast cell densities were compared with clinical data. The influence of mast cells on aldosterone production was studied by using cultures of human mast cell and adrenocortical cell lines. In APA tissues, the density of mast cells was found to be increased in comparison with normal adrenals. Mast cells were primarily observed in adrenal cortex adjacent to adenomas or in the adenomas themselves, distinguishing two groups of APAs. A subset of adenomas was found to contain a high density of intratumoral mast cells, which was correlated with aldosterone synthase expression and in vivo aldosterone secretory parameters. Administration of conditioned medium from cultures of human mast cell lines to human adrenocortical cells induced a significant increase in aldosterone synthase (CYP11B2) mRNA expression and aldosterone production. APA tissues commonly contain numerous mast cells that may influence aldosterone secretion through the local release of regulatory factors.

Prevalence and Characterization of Somatic Mutations in Chinese Aldosterone-Producing Adenoma Patients

Recently somatic mutations of KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been identified in patients with aldosterone-producing adenoma (APA). The present study sequenced the DNA in the tissues and blood samples from Chinese patients with APA for KCNJ5, ATP1A1, ATP2B3, and CACNA1D gene mutations.Among the 114 patients, 86 (75.4%) were identified with KCNJ5 somatic mutations, including 3 previously reported (G151R, L168R, T158A) and 2 other unreported mutations. One patient presented with both a point mutation (E147) and an insertion mutation, whereas another had a 36-base duplication, G153_G164dup. No mutation of ATP1A1 and ATP2B3 in the known hotspots was identified and only 1 male patient was detected with a novel CACNA1D mutation, V748I. Unlike other studies, male and female patients had similar KCNJ5 mutation rates (76.9% vs 74.2%). Mutation carriers were younger and had lower preoperative potassium level, whereas male (but not female) mutation carriers had higher preoperative plasma aldosterone concentration and preoperative blood pressures. Mutation carriers also had higher LV mass index (LVMI) than nonmutation carriers. After surgery, LVMI improved significantly in the KCNJ5 mutation group but not in the nonmutation group. The mRNA expression of KCNJ5, CYP11B2, and ATP2B3 was higher in the KCNJ5-mutated APA tissues. Functional characterization of the 2 novel KCNJ5 mutations showed that they were associated with decreased proliferation, membrane depolarization, elevated secretion of aldosterone, and increased expression of CYP11B1 and CYP11B2.In conclusion, Chinese APA patients appear to have a high frequency of somatic KCNJ5 mutation. Mutation prevalence rates are similar among men and women and 2 novel mutations are identified. KCNJ5-mutated patients benefit more from surgical resection of APA than nonmutated patients.

CAV1.3, HSD3B2 and CYP11B2 immunoexpressions in aldosterone-producing adenoma are not correlated with post-adrenalectomy outcome in primary aldosteronism

Somatic mutations of KCNJ5, ATP1A1, CACNA1D, ATP2B3, have been shown to be involved in the pathophysiology of aldosterone-producing adenoma (APA). This study aimed to investigate immunolocalization of CaV1.3 coded by CACNA1D, as well as steroidogenic enzymes HSD3B2 and CYP11B2 (rate-limiting enzyme for aldosterone production) in paraffin-embedded APA tissues and to relate these findings to mutation status, histopathology and outcome following adrenalectomy. APA tissues were divided into subgroups based on their different somatic mutations. Size and cell composition of tumors, immunolocalization and semi-quantitative expression of CYP11B2, HSD3B2 and CaV1.3 were analyzed. Scoring staining intensity was determined along with clinical findings. Our results showed that a majority of APA consist of both zona fasciculata-like and zona glomerulosa (ZG)-like cells. CACNA1D-mutated APA were significantly smaller than KCNJ5-mutated (P < 0.05). APA with a somatic mutation presented CYP11B2 positive clusters or scattered cells with variable degree of staining while non-mutated APA were weakly stained for CYP11B2 or presented only clusters in adjacent ZG. H-Score for CYP11B2 even when adjusted for tumor size, was significantly different between non-mutated and KCNJ5-mutated APA (P < 0.05). Immunoreactivity of CaV1.3 and HSD3B2 was present in all APA tissues analyzed. Potassium levels of all patients were normalized after adrenalectomy. Better clinical outcome only correlated with aldosterone renin ratio at diagnosis and shorter duration of presurgical hypertension. Our findings suggest that KCNJ5 and not ATP1A1, CACNA1D or ATP2B3 mutated APA present a significantly different CYP11B2 immunoreactivity, compared with non-mutated APA. However immunoreactivities of CYP11B2, CaV1.3 or HSD3B2 are not correlated with outcome.

Steroidogenic Activity of Aldosterone-Producing Adenoma with and without KCNJ5 Gene Mutations, Comparing with that in Each Adherent Normal Tissue

Introduction: The steroidogenic activity in a KCNJ5-gene mutated aldosterone-producing adenoma (APA) was reported to be much stronger than that in an APA of the wild type, while it is still controversial. Objectives: We examined whether the normal adrenal tissue attached to APA also possesses KCNJ5 gene mutations, and also investigated steroidogenic activity of mutated and non-mutated APA tissue cells with that of each normal tissue cell attached to APA in order to clarify the nature of steroidogenic activity. Methods: We selected 12 patients with and without KCNJ5 gene mutation-harboring APA in whom normal adrenal tissue adhered to the APA. We analyzed the presence of KCNJ5 gene mutations by using those tissues. Results: APA tissues of 6 patients possessed KCNJ5 gene mutations, whereas those mutations were not detected in any of the adjacent normal tissue samples as well as another 6 cases of APA. In two APA tissues with or without KCNJ5 gene mutation, we also compared the steroidogenic activity of isolated cells from each APA tissue with that from the normal adherent tissues. The cortisol production of the mutated APA cells was markedly decreased compared with that of the normal cells, suggesting that the aldosterone production pathway becomes dominant and the cortisol production pathway is downregulated in that APA tissue. Furthermore, the ratio of aldosterone to cortisol in the normal adherent tissue was about 1/4th of that of the wild-type APA tissue, while it was about 1/10th in the mutated APA. Conclusion: KCNJ5 gene mutations in the normal tissue adhering to KCNJ5 gene mutation-harboring APA was not observed, suggesting that somatic mutations of the KCNJ5 gene only occur in APA tissue. It is also suggested that KCNJ5 gene mutations dominantly induce the aldosterone production pathway as well as concomitant decline in cortisol production, resulting in highly autonomous aldosterone production.