Search

Context The plasma aldosterone concentration (PAC), renin, and aldosterone-to-renin ratio (ARR) are used to screen for primary aldosteronism (PA). Substantial intra-individual variability of PAC and ARR using plasma renin activity in the context of usual antihypertensive therapy has been described, but there is no data on ARR variability calculated using direct renin concentration (DRC). Objective To describe the intra-individual variability of PAC, DRC, and ARR in the absence of interfering medications in patients with and without PA. Design Retrospective cohort study. Patients Hypertensive patients referred for investigation of PA, with at least 2 ARR measurements while off interfering medications. Setting Endocrine hypertension service of a tertiary center, from May 2017 to July 2021. Main outcome measures PAC, DRC, and ARR variability was calculated as coefficient of variation (CV) and percent difference (PD). Results Analysis of 223 patients (55% female, median age 52 years), including 162 with confirmed PA, demonstrated high variability with a sample CV of 22-25% in the PAC and sample CV of 41% to 42% in the DRC and ARR in both the PA and non-PA groups. The degree of variability was substantially higher than the assays’ analytical CV. Sixty-two patients (38%) with PA had at least one ARR below 70 pmol/L:mU/L (2.4 ng/dL:mU/L), a cut-off for first-line screening of PA. Conclusions Significant intra-individual variability in PAC, DRC, and hence ARR occurs in a large proportion of patients being investigated for PA. These findings support the need for at least 2 ARR before PA is excluded or further investigated.

Figure S3. Effects of XIAP inhibition alone or in combination with PPARg activation.

Table S1. Gene expression analysis by TaqMan Low-Density Array.

S1. Total number of mutated genes identified within this study that are also mutated in the studies of Caburet et al [21], Zehir et al [22], Kandoth et al [23], Lawrence et al [24] and Nik-Zainal et al [25]. Genes mutated in this current study are also presented as a percentage.

Adult granulosa cell tumor (aGCT), the most common malignant ovarian sex cord-stromal tumor, is characterized by the forkhead transcription factor FOXL2 p.C134W somatic mutation. Late recurrences are relatively common but the molecular mechanisms of relapse or aggressive behavior are not known. The mutational landscape of FOXL2 p.C134W mutation–positive tumors (n = 22) was determined using whole-exome sequencing (WES). An average of 64 coding and essential splice-site variants were identified per tumor. As the TERT promoter region is poorly covered by the WES, targeted sequencing identified the TERT -124C>T promoter mutation as the only recurrent mutation (∼40% of cases). Pathway analysis suggested an association with DNA replication/repair and the EGFR family canonical pathways. Copy number analysis confirmed that gains of chromosomes 12 and 14 occur in approximately 30% of aGCT and loss of chromosome 22 occurs in approximately 40% of cases. In summary, exome-wide analysis of the mutational landscape of aGCT revealed that, except for the TERT promoter mutation, recurrence and/or aggressive behavior is not defined by activation or loss of specific genes.Implications:This study found that although aGCTs are defined by the presence of a common FOXL2 gene mutation, recurrence and/or aggressive behavior cannot be attributed to subsequent mutation of specific gene(s) or pathways; however, there is a high frequency of the TERT -124C>T promoter mutation, which is associated with more aggressive disease.

Table S1: Somatic single nucleotide variants, indel and sequence alteration mutations identified across each sample Table S2: Mutated genes identified in samples with matched germline DNA: 1, 18 and 24 Table S3: Microarray analysis by -124C>T TERT promoter mutation status Table S4: Ingenuity Pathway Analysis on genes that differed by TERT promoter mutation status Table S5: Comparison with a series of large scale analyses looking at mutations in genes that overlap with data in this study Table S6: Mutations in genes identified by Zehir, A et al in 11 aGCT samples

Background Primary aldosteronism (PA) is the most common and potentially curable endocrine cause of secondary hypertension, and carries a worse prognosis than essential hypertension. Despite the high prevalence of hypertension in patients with chronic kidney disease (CKD), the screening rates for primary aldosteronism in CKD are unknown. Methods In this study, we retrospectively reviewed medical records of 1627 adults who presented to the nephrology clinics of 2 tertiary hospitals in Melbourne, Australia, between 2014 and 2019. In addition to assessing the pattern of screening, we also evaluated patient-specific factors associated with the decision to test for primary aldosteronism. Patients were excluded from the final analysis if they did not have CKD, had an organ transplant, had end stage renal failure, or had insufficient data or follow-up. Results Of the 600 patients included in the analysis, 234 (39%) had an indication for screening for primary aldosteronism based on recommendations made by the Endocrine Society. However, only 33 (14%) were tested. They were younger, had a higher mean systolic blood pressure, better renal function, and lower mean serum potassium than those who were indicated but not screened. Of the 33 screened patients, an elevated aldosterone-to-renin ratio was noted in 8 patients and a diagnosis of primary aldosteronism was made in 4 patients. Conclusions The screening rate for primary aldosteronism is low in a CKD population, especially in patients who are older, have a lower eGFR and normal serum potassium. The consequences of undiagnosed primary aldosteronism in this select population may be substantial due to the cardiovascular and renal sequelae associated with untreated disease. Graphical abstract

The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.

Several studies have demonstrated a bidirectional relationship between obstructive sleep apnea and primary aldosteronism (PA); however, many of these studies are limited to patients with known obstructive sleep apnea, hypertension, or PA. We evaluated the role of screening for PA in all patients referred for a diagnostic sleep study without selecting for prior diagnoses with these conditions. Plasma aldosterone and renin concentration were measured after an overnight polysomnography. Blood pressure was measured at the sleep center for all patients, while a proportion underwent 24-hour blood pressure monitoring. Of the 85 participating patients, 2 (2.4%) were identified to have likely PA based on an elevated aldosterone:renin ratio and/or clinical characteristics. Another 10 (11.8%) were identified to have possible PA based on their low or normal plasma renin concentration despite taking antihypertensive medications that are known to elevate renin. In participants with both obstructive sleep apnea and hypertension (n=40), the prevalence of likely or possible PA was 30%. However, there was no correlation between aldosterone, rennin, or aldosterone:renin ratio and the apnea-hypopnea index using multiple regression analysis adjusted for interfering medications and hypertension status. The observed high prevalence of possible PA among those with both hypertension and obstructive sleep apnea suggests that they should be routinely screened for PA.

Recent studies have suggested that the unique FOXL2C134W mutation, which is pathognomonic for adult granulosa cell tumours of the ovary, is a tumour suppressor gene. In a recent issue of The Journal of Pathology, a detailed study by Pilsworth et al. seeks to rebut the proposition that the FOXL2C134W mutation, that uniquely characterises adult granulosa cell tumours of the ovary, leads to reduced transcript levels with the implication that FOXL2 is a tumour suppressor gene. The study provides compelling evidence that both wild-type and mutant FOXL2 transcripts and protein are expressed at equivalent levels. In the context of other recent studies, one is drawn to the conclusion that FOXL2C134W is a gain of function mutation whose impact is mediated through enhanced interactions with the SMAD transcription factor complex. This article is protected by copyright. All rights reserved.

See also the editorial by Georgiades in this issue.

Background The role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective. Methods We used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments. Results Using this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARβ. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival. Conclusion Our study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.

MR activation in macrophages is critical for the development of cardiac inflammation and fibrosis. We previously showed that MR activation modifies macrophage pro-inflammatory signalling, changing the cardiac tissue response to injury via both direct gene transcription and JNK/AP-1 second messenger pathways. In contrast, MR-mediated renal electrolyte homeostasis is critically determined by DNA-binding-dependent processes. Hence, ascertaining the relative contribution of MR actions via DNA binding or alternative pathways on macrophage behaviour and cardiac inflammation may provide therapeutic opportunities which separate the cardioprotective effects of MR antagonists from their undesirable renal potassium-conserving effects. We developed new macrophage cell lines either lacking MR or harbouring a mutant MR incapable of DNA binding. Western blot analysis demonstrated that MR DNA binding is required for lipopolysaccharide (LPS), but not phorbol 12-myristate-13-acetate (PMA), induction of the MAPK/pJNK pathway in macrophages. Quantitative RTPCR for pro-inflammatory and pro-fibrotic targets revealed subsets of LPS- and PMA-induced genes that were either enhanced or repressed by the MR via actions that do not always require direct MR-DNA binding. Analysis of the MR target gene and profibrotic factor MMP12 identified promoter elements that are regulated by combined MR/MAPK/JNK signalling. Evaluation of cardiac tissue responses to an 8-day DOC/salt challenge in mice selectively lacking MR DNA-binding in macrophages demonstrated levels of inflammatory markers equivalent to WT, indicating non-DNA binding-dependent MR signalling in macrophages is sufficient for DOC/salt-induced tissue inflammation. Our data demonstrate that the MR regulates a macrophage pro-inflammatory phenotype and cardiac tissue inflammation, partially via pathways that do not require DNA binding.

BACKGROUND: Adrenal vein sampling (AVS) is crucial for accurate lateralization of aldosterone excess but it is technically challenging due to the difficulty of adrenal vein cannulation. The use of adrenocorticotropic hormone (ACTH) to improve cannulation success is controversial and can lead to discordant lateralization outcomes. OBJECTIVE: To evaluate the utility of ACTH in two centres with different levels of AVS expertise and formulate a strategy for interpreting discordant results. DESIGN: A retrospective cross-sectional analysis of AVS results and postoperative patient outcomes. SETTING: Two large tertiary hospitals with harmonized AVS protocols where adrenal venous samples are collected both before and after ACTH stimulation. MEASUREMENTS: Cannulation success (measured by selectivity index, SI), lateralization (measured by lateralization index, LI) and postoperative biochemical cure. RESULTS: Number of AVS procedures judged to have successful bilateral adrenal vein cannulation increased from 53% pre- to 73% post-ACTH. The increase in cannulation success was significantly higher in centre where AVS was performed by multiple radiologists with a lower basal success rate. In both centres, the proportion of cases deemed to display lateralization significantly decreased with the use of ACTH (70% pre- to 52% post-ACTH). Based on postoperative outcomes of patients with discordant results who underwent unilateral adrenalectomy, the combination of LI >3 pre-ACTH and LI >2 post-ACTH was predictive of a biochemical cure. CONCLUSION: Adrenocorticotropic hormone can increase the rate of cannulation success during AVS at the expense of reduced lateralization. The criteria for lateralization should be carefully determined based on local data when ACTH is used.

Context The Endocrine Society Guidelines for the diagnosis of primary aldosteronism (PA) suggest that confirmatory tests (CFT) are not required when the following criteria are met: plasma aldosterone concentration (PAC) is >20 ng/dL, plasma renin is below detection levels, and hypokalemia is present. The evidence for the applicability of the guideline criteria is limited. Objective To develop and validate optimized criteria for sparing CFT in the diagnosis of PA. Design and Setting The optimized criteria were developed in a Chinese cohort using the captopril challenge test, verified by saline infusion test (SIT) and fludrocortisone suppression test (FST), and validated in an Australian cohort. Participants Hypertensive patients who completed PA screening and CFT. Main Outcome Measure Diagnostic value of the optimized criteria. Results In the development cohort (518 PA and 266 non-PA), hypokalemia, PAC, and plasma renin concentration (PRC) were selected as diagnostic indicators by multivariate logistic analyses. The combination of PAC >20 ng/dL plus PRC Conclusions Hypertensive patients with PAC >20 ng/dL, PRC

Background A total of 44.7% adults in China have hypertension, but the prevalence of primary aldosteronism (PA) in Chinese hypertensive patients is unknown. Objectives This study prospectively investigated the prevalence, characteristics, and outcomes of PA in newly diagnosed hypertensive patients. Methods In a large community health center, consecutive hypertensive patients with an aldosterone-renin ratio >20 ng/mIU and plasma aldosterone concentration >10 ng/dl underwent captopril challenge test and/or saline infusion test for confirmation of PA. Adrenal computed tomography scan and adrenal vein sampling were used for subtyping. PA patients treated with surgery or medication were followed up for 1 year, and outcomes after treatment were evaluated. Results In total, 1,020 newly diagnosed hypertensive patients were screened over 16 months, of whom 40 were diagnosed with PA, 948 with non-PA, 32 with probable PA, resulting in a prevalence of more than 4.0%. Compared with non-PA, PA patients more frequently displayed microalbuminuria (p = 0.031), but the incidence of cardiovascular events was not different (p = 0.927). For surgically treated patients (n = 7), a complete biochemical success rate was 100% and a complete clinical success rate was 85.7%. For medically treated patients (n = 29), the proportion with optimal blood pressure control was 79%, and among them, 91% (21 of 23) only needed 1 antihypertensive drug: the mineralocorticoid receptor antagonist. Conclusions The prevalence of PA in patients with newly diagnosed hypertension in China was at least 4%. PA screening in newly diagnosed hypertensive patients leads to good clinical outcomes. (Primary Aldosteronism In Hypertensive Patients in China [PA-China]; NCT03155139)

Aims We aimed at determining the rate of drug-resistant arterial hypertension in patients with an unambiguous diagnosis of primary aldosteronism (PA). Moreover, we sought for investigating the diagnostic performance of adrenal vein sampling (AVS), and the effect of adrenalectomy on blood pressure (BP) and prior treatment resistance in PA patients subtyped by AVS in major referral centres. Methods and results The Adrenal Vein Sampling International Study-2 (AVIS-2) was a multicentre international study that recruited consecutive PA patients submitted to AVS, according to current guidelines, during 15 years. The patients were over 18 years old with arterial hypertension and had an unambiguous diagnosis of PA. The rate of resistant hypertension was assessed at baseline and after adrenalectomy using the American Heart Association (AHA) 2018 definition. Information on presence or absence of resistant hypertension was available in 89% of the 1625 enrolled PA patients. Based on the AHA 2018 criteria, resistant hypertension was found in 20% of patients, of which about two-thirds (14%) were men and one-third (6%) women (χ2 = 17.1, P Conclusions (i) Resistant hypertension is a common presentation in patients seeking surgical cure of PA; (ii) AVS is key for the optimal management of patients with PA due to resistant hypertension; and (iii) AVS-guided adrenalectomy allowed resolution of treatment-resistant hypertension.

Background While both renin‐dependent and renin‐independent aldosterone secretion contribute to aldosteronism, their relative associations with cardiovascular disease (CVD) risk has not been investigated. Methods and Results A total of 2909 participants from the FOS (Framingham Offspring Study) with baseline, serum aldosterone concentration, and plasma renin concentration who attended the sixth examination cycle and were followed up until 2014 and who were free of CVD were included. We further recruited 2612 hypertensive participants from the CONPASS (Chongqing Primary Aldosteronism Study). Captopril challenge test was performed to confirm renin‐dependent or ‐independent aldosteronism in CONPASS. Among 1433 hypertensive subjects of FOS, when compared with those with serum aldosterone concentration −1 (normal aldosterone), participants who had serum aldosterone concentration ≥10 ng dL −1 and plasma renin concentration ≤15 mIU L −1 (identified as renin‐independent aldosteronism) showed a higher risk of CVD (hazard ratio, 1.40 [95% CI, 1.08–1.82]), while those who had serum aldosterone concentration ≥10 ng dL −1 and plasma renin concentration >15 mIU L −1 (identified as renin‐dependent aldosteronism) showed an unchanged CVD risk. In CONPASS, renin‐independent aldosteronism carried a significantly higher risk of CVD than normal aldosterone (odds ratio, 2.57 [95% CI, 1.13–5.86]), while the CVD risk remained unchanged in renin‐dependent aldosteronism. Elevation of the urinary potassium‐to‐sodium excretion ratio, reflective of mineralocorticoid receptor activity, was only observed in participants with renin‐independent aldosteronism. Conclusions Among patients with hypertension, renin‐independent aldosteronism is more closely associated with CVD risk than renin‐dependent aldosteronism.

Many of the patients with primary aldosteronism (PA) are denied curative adrenalectomy because of limited availability or failure of adrenal vein sampling. It has been suggested that adrenal vein sampling can be omitted in young patients with a unilateral adrenal nodule, who show a florid biochemical PA phenotype. As this suggestion was based on a very low quality of evidence, we tested the applicability and accuracy of imaging, performed by computed tomography and/or magnetic resonance, for identification of unilateral PA, as determined by biochemical and/or clinical cure after unilateral adrenalectomy. Among 1625 patients with PA submitted to adrenal vein sampling in a multicenter multiethnic international study, 473 were ≤45 years of age; 231 of them had exhaustive imaging and follow-up data. Fifty-three percentage had a unilateral adrenal nodule, 43% had no nodules, and 4% bilateral nodules. Fifty-six percentage (n=131) received adrenalectomy and 128 were unambiguously diagnosed as unilateral PA. A unilateral adrenal nodule on imaging and hypokalemia were the strongest predictors of unilateral PA at regression analysis. Accordingly, imaging allowed correct identification of the responsible adrenal in 95% of the adrenalectomized patients with a unilateral nodule. The rate raised to 100% in the patients with hypokalemia, who comprised 29% of the total, but fell to 88% in those without hypokalemia. Therefore, a unilateral nodule and hypokalemia could be used to identify unilateral PA in patients ≤45 years of age if adrenal vein sampling is not easily available. However, adrenal vein sampling remains indispensable in 71% of the young patients, who showed no nodules/bilateral nodules at imaging and/or no hypokalemia. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01234220.

ObjectivesPrimary aldosteronism (PA), the most common endocrine cause of hypertension, is associated with a higher risk of cardiovascular disease (CVD) than blood pressure (BP)-matched essential hypertension (EH). We aimed to compare the calculated risks of CVD in patients who had hypertension with PA or EH using CVD risk calculators, hypothesising that they will fail to recognise the increased CVD risk in PA.DesignCross-sectional analysis.SettingAn endocrine hypertension service in Victoria, Australia.ParticipantsPatients who had hypertension without CVD referred for the investigation of hypertension.Outcome measuresCalculated 5-year or 10-year CVD risk as predicted by the National Vascular Disease Prevention Alliance (NVDPA) algorithm, Framingham Risk Score, Pooled Cohort Equations and QRISK3.ResultsThose with PA (n=128) and EH (n=133), did not differ significantly in their calculated CVD risks with the NVDPA algorithm (moderate-to-high 5-year risk 36/100 vs 45/99, p=0.17); the Framingham Risk Score (median 10-year risk 7.72% (4.43%–12.95%) vs 6.84% (3.85%–10.50%), p=0.14); the Pooled Cohort Equations (median 10-year risk 9.45% (4.36%–15.37%) vs 7.90% (2.09%–14.73%), p=0.07); and QRISK3 (median 10-year risk 11.31% (7.22%–20.29%) vs 12.47% (5.10%–19.93%), p=0.51). Similarities persisted on regression analyses accounting for systolic BP.ConclusionsCVD risk algorithms do not reflect the increased risk of CVD in patients with PA, and likely underestimate the true risk of CVD among those with PA. Screening for PA, in addition to using the CVD risk algorithm in patients who had hypertension, may facilitate the targeted treatment of PA and minimisation of cardiovascular risk in affected individuals.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15540. Nuclear hormone receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Steroid hormone receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [74, 215, 3]) are nuclear hormone receptors of the NR3 class, with endogenous agonists that may be divided into 3-hydroxysteroids (estrone and 17β-estradiol) and 3-ketosteroids (dihydrotestosterone [DHT], aldosterone, cortisol, corticosterone, progesterone and testosterone). For rodent GR and MR, the physiological ligand is corticosterone rather than cortisol.

Ovarian sex cord-stromal tumors (SCST) are uncommon tumors accounting for approximately 8% of all ovarian malignancies. By far, the most common are granulosa cell tumors (GCT) which represent approximately 90% of SCST. SCST are also found in the hereditary syndromes: Peutz-Jeghers syndrome, Ollier disease and Maffucci syndrome, and DICER1 syndrome. Key genomic and genetic events contributing to their pathogenesis have been the focus of recent studies. Most of the genomic studies have been limited to GCT which have identified a number of recurring chromosomal abnormalities (monosomy and trisomy), although their contribution to pathogenesis remains unclear. Recurrent DICER1 mutations are reported in non-hereditary cases of Sertoli cell and Sertoli–Leydig cell tumors (SLCT), while recurrent somatic mutations in both the juvenile (jGCT) and adult forms of GCT (aGCT) have also been reported. Approximately 30% of jGCT contain a somatic mutation in the gsp oncogene, while a further 60% have activating mutations or duplications in the AKT gene. For aGCT, a well characterized mutation in the FOXL2 transcription factor (FOXL2 C134W) is found in the majority of tumors (primary and recurrent), arguably defining the disease. A further mutation in the human telomerase promoter appears to be an important driver for recurrent disease in aGCT. However, despite several studies involving next generation sequencing, the molecular events that determine the stage, behavior and prognosis of aGCT still remain to be determined. Further, there is a need for these studies to be expanded to other SCST in order to identify potential targets for personalized medicine.

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.

Adult granulosa cell tumor (aGCT), the most common malignant ovarian sex cord-stromal tumor, is characterized by the forkhead transcription factor FOXL2 p.C134W somatic mutation. Late recurrences are relatively common but the molecular mechanisms of relapse or aggressive behavior are not known. The mutational landscape of FOXL2 p.C134W mutation–positive tumors (n = 22) was determined using whole-exome sequencing (WES). An average of 64 coding and essential splice-site variants were identified per tumor. As the TERT promoter region is poorly covered by the WES, targeted sequencing identified the TERT -124C>T promoter mutation as the only recurrent mutation (∼40% of cases). Pathway analysis suggested an association with DNA replication/repair and the EGFR family canonical pathways. Copy number analysis confirmed that gains of chromosomes 12 and 14 occur in approximately 30% of aGCT and loss of chromosome 22 occurs in approximately 40% of cases. In summary, exome-wide analysis of the mutational landscape of aGCT revealed that, except for the TERT promoter mutation, recurrence and/or aggressive behavior is not defined by activation or loss of specific genes. Implications: This study found that although aGCTs are defined by the presence of a common FOXL2 gene mutation, recurrence and/or aggressive behavior cannot be attributed to subsequent mutation of specific gene(s) or pathways; however, there is a high frequency of the TERT -124C>T promoter mutation, which is associated with more aggressive disease.

Context Adrenal gland imaging is recommended by the current guidelines for the workup of primary aldosteronism (PA). However, its diagnostic performance has not been established in large, multiethnic cohorts of patients who undergo adrenal vein sampling (AVS) and adrenalectomy. Objective This work aims to assess the diagnostic accuracy of cross-sectional adrenal imaging. Methods This international multicenter study took place in tertiary referral centers. A total of 1625 PA patients seeking surgical cure were enrolled in an international study involving 19 centers in North America, Europe, Asia, and Australia. Of these, 1311 (81%) had imaging data available and 369 (23%), who received a final diagnosis of surgically cured unilateral PA, were examined. Patients underwent AVS and imaging by computed tomography and/or magnetic resonance imaging. The accuracy of detection of unilateral PA at imaging was estimated by the area under the receiver operator characteristics curve using cure (biochemical and/or full clinical success) as the reference at follow-up after unilateral adrenalectomy. Results In the cohort of 1311 patients with imaging data available, 34% and 7% of cases showed no detectable or bilateral nodules, respectively. Imaging did not detect the culprit adrenal in 28% of the surgically cured unilateral PA patients. Moreover, the clinical outcome did not differ significantly between the imaging-positive and imaging-negative patients. Conclusion Cross-sectional imaging did not identify a lateralized cause of disease in around 40% of PA patients and failed to identify the culprit adrenal in more than one-fourth of patients with unilateral PA.

Excess aldosterone promotes pathological remodeling of the heart and imbalance in cardiac ion homeostasis of sodium, potassium and calcium. Novel treatment with proanthocyanidins in aldosterone-treated rats has resulted in downregulation of cardiac SGK1, the main genomic aldosterone-induced intracellular mediator of ion handling. It therefore follows that proanthocyanidins could be modulating cardiac ion homeostasis in aldosterone-treated rats. Male Wistar rats received aldosterone (1 mg kg−1 day−1) +1% NaCl for three weeks. Half of the animals in each group were simultaneously treated with the proanthocyanidins-rich extract (80% w/w) (PRO80, 5 mg kg−1 day−1). PRO80 prevented cardiac hypertrophy and decreased calcium content. Expression of ion channels (ROMK, NHE1, NKA and NCX1) and calcium transient mediators (CAV1.2, pCaMKII and oxCaMKII) were reduced by PRO80 treatment in aldosterone-treated rats. To conclude, our data indicate that PRO80 may offer an alternative treatment to conventional MR-blockade in the prevention of aldosterone-induced cardiac pathology.

Background: Hypertension tracks throughout childhood into adulthood. Aldosterone excess, or primary aldosteronism, has been reported as the most common secondary cause of hypertension in adults. Elevated aldosterone in the setting of low renin predicts incident hypertension in normotensive adults. However, the relationship between aldosterone and blood pressure in adolescents and young adults is unclear. Objectives: To evaluate the relationship between aldosterone, renin and the aldosterone:renin ratio (ARR) and blood pressure (BP) at age 17y as well as BP at age 27y in a community-based population. Methods: This is a prospective birth cohort study. Young adult offspring (Gen2) of women enrolled during pregnancy into the Raine Study, with 1239 at age 17y and 1006 at age 27y, were evaluated. Females taking hormonal contraception and participants without BP data were excluded from the current analysis. A generalised linear model was used to examine the relationship between BP and aldosterone, renin and ARR over time. The median aldosterone, renin and ARR was compared between sexes using quantile regression. Results: At 17y, females had similar aldosterone (349 vs 346 pmol/L, p=0.833) but significantly lower renin (20.6 vs 25.7 mU/L, p

The mineralocorticoid receptor (MR) has an established role in blood pressure control and cardiovascular homeostasis via many actions in the heart and kidney. We recently identified a role for the MR in controlling the circadian clock in cardiac cells and demonstrated that time-of-day impacts MR activation in the heart. While time dependent behaviours such as upright posture and fluid intake control aldosterone release via the renin-angiotensin-aldosterone system (RAAS), we hypothesise that the circadian clock controls aldosterone signalling by modifying MR transcriptional outcomes. Two established MR target genes and core circadian clock genes are period 1 (Per1) and period 2 (Per2). We have previously shown that a bolus dose of aldosterone (i.p.) induced cardiac expression of Per1 and Per2 in wildtype mice treated at 8AM (start of rest period) but not when administered at 8PM (start of active period). Whether MR activation in the kidney is similarly dependent on time of day and aligns with MR actions in the heart remains to be assessed. We also sought to determine if the MR directly regulates the molecular clock in a ligand-dependent manner. In contrast to the heart, renal Per1 and Per2 expression was not upregulated at four hours following aldosterone administration at either 8AM or 8PM. Interestingly, aldosterone administered at 8AM, but not at 8PM, significantly down-regulated expression of the circadian clock gene, nuclear receptor reverse strand c-ERBA (ReverbA); ReverbA was not regulated in the heart following aldosterone treatment. Investigation of renal-MR target genes involved in sodium ion transfer, showed a down-regulation of epithelial sodium channel 1 alpha (ENaC-α), by aldosterone administered only at 8AM. Conversely, aldosterone administered at 8AM induced gene expression of FKBP Prolyl Isomerase 5 (Fkbp5), an immunophilin that is important in nuclear trafficking of the MR. These data provide new insights for tissue sensitivity to MR activation in the heart and the kidney, which may play differing roles in contributing to regulating circadian patterns of several cardiovascular and renal parameters. Moreover, our data suggest that in addition to the accepted entrainment role of the glucocorticoid receptor (GR), the MR may also play a role in the entrainment of the circadian clock in peripheral tissues. Ongoing mechanistic studies will determine whether the MR can directly control the periodicity of the renal cellular clock, as it does in the heart. Key outcomes will be assessed in population databases to determine the clinical relevance of these findings. We propose that understanding of time-of-day dependent vulnerability to MR signalling in the heart versus the kidney may offer the rationale for the development of novel temporal or tissue specific-MR modulators in the management of cardiovascular disease.

Background Hypertensive patients with primary aldosteronism (PA) have a higher risk of cardiovascular complications than those with blood pressure-matched essential hypertension. The excess cardiovascular consequences of PA can be attributed to the pro-inflammatory effect of excessive aldosterone and mineralocorticoid receptor activation in a range of peripheral tissues and cell types. The neutrophil-to-lymphocyte ratio (NLR) is a widely available markers of inflammation which has been shown to predict cardiovascular outcome in the general population. This study aims to evaluate the use of NLR as a potential biomarker of PA and PA severity. Methods Patients with PA (n=355) were identified from two large PA databases in Australia and China, while controls (n=222) were patients with hypertension who were referred for assessment but did not meet the diagnostic criteria for PA. The NLR was retrospectively collected from routine full blood examination, prior to commencement of targeted treatment for PA. Results The NLR did not differ between PA patients and hypertensive controls (median 2.3 and 2.4, p=0.563). However, amongst patients with PA, the NLR was positively correlated with baseline and post-saline aldosterone levels (r=0.22 and p Conclusion Whilst the NLR did not distinguish PA from controls, it was a marker of PA severity, being associated with aldosterone concentration as well as the presence of CKD. A prospective study is needed to further clarify the role of NLR in predicting end-organ damage associated with PA.

Context Adrenal venous sampling (AVS) is the key test for subtyping primary aldosteronism (PA), but its interpretation varies widely across referral centers and this can adversely affect the management of PA patients. Objectives To investigate in a real-life study the rate of bilateral success and identification of unilateral aldosteronism and their impact on blood pressure outcomes in PA subtyped by AVS. Design and settings In a retrospective analysis of the largest international registry of individual AVS data (AVIS-2 study), we investigated how different cut-off values of the selectivity index (SI) and lateralization index (LI) affected rate of bilateral success, identification of unilateral aldosteronism, and blood pressure outcomes. Results AVIS-2 recruited 1625 individual AVS studies performed between 2000 and 2015 in 19 tertiary referral centers. Under unstimulated conditions, the rate of biochemically confirmed bilateral AVS success progressively decreased with increasing SI cut-offs; furthermore, with currently used LI cut-offs, the rate of identified unilateral PA leading to adrenalectomy was as low as Conclusion Commonly used SI and LI cut-offs are associated with disappointingly low rates of biochemically defined AVS success and identified unilateral PA. Evidence-based protocols entailing less restrictive interpretative cut-offs might optimize the clinical use of this costly and invasive test. (J Clin Endocrinol Metab XX: 0-0, 2020)

Treatment options for radioiodine-refractory epithelial thyroid cancer (ETC) are limited to tyrosine kinase inhibitors (TKIs), which are associated with significant adverse effects and are not curative. The inhibitors of apoptosis (IAP) family have oncogenic properties and overexpression of X-linked IAP (XIAP) in papillary thyroid carcinoma (PTC) is associated with a poor prognosis. Our group has previously reported a synergistic interaction between SMAC mimetics (SM) (IAP antagonists with XIAP and cIAP1 specificity), and PPARgamma agonists in granulosa cell tumors (1). Such an approach has not been explored in ETC. We hypothesize that SM may be efficacious alone or in combination with a secondary agent to inhibit proliferation, induce cell death and/or promote differentiation to resensitize cells to radioiodine. Four ETC-derived cell lines (K1, Nthy-ori 3-1, TPC-1 and SW-1736) were examined for cIAP1, cIAP2 and XIAP expression by RT-PCR. The K-1 and TPC-1 cell lines (PTC origin, BRAFV600E and PI3KCA mutation positive) were chosen to investigate the effects of an SM in combination with either a PPARgamma agonist (rosiglitazone) or a broad-spectrum TKI (sorafenib). Cell proliferation was examined using xCELLigence Real-Time Cell Analysis. Viability was assessed using total cell counts at 24 and 48 hours. In the four cell lines, we found abundant cIAP1 and XIAP expression and low cIAP2 expression. When an SM was used in combination with either rosiglitazone or sorafenib, we observed significant impairment of cell proliferation and viability with a clear morphological response. These effects appear SM-dependent, as SM treatment alone also showed significant effects on proliferation. However, these effects were enhanced when combined with sorafenib, which was ineffective alone. Markers of differentiation are currently being examined. Our findings suggest a novel role for SM in treating or redifferentiating radioiodine-refractory ETC. Clinically, this may involve lower doses of individual agents than when used alone, thereby reducing adverse effects. References1. Leung, D.T.H., et al., Combined PPARgamma Activation and XIAP Inhibition as a Potential Therapeutic Strategy for Ovarian Granulosa Cell Tumors. Mol Cancer Ther, 2019. 18(2): p. 364-375.

Background: Primary aldosteronism (PA) accounts for 3.2-12.7% of hypertension in primary care but is often diagnosed late, if at all. A delayed or missed diagnosis leads to poor blood pressure control and greater cardiovascular risk that could be averted with targeted treatment. An Endocrine Hypertension Service (EHS), encompassing an education program, streamlined diagnostic tests and dedicated PA clinic, was developed to address this issue. Aims: To analyse the impact of Victoria’s first dedicated EHS on the pattern of PA diagnoses. Methods: Socio-demographic and clinical data from all patients who attended the EHS since July 2016 (N=267) was collected prospectively. Patients were divided into Year 1 (Y1), Year 2 (Y2), and Year 3 (Y3), based on their first visit. Results: The proportion of referrals from primary care increased (20% in Y1 to 52% in Y3) with more referrals being made for treatment-naive hypertension (3% in Y1 to 19% in Y3). Patients with a hypertension diagnosis of 5 years or less at the time of referral to EHS increased from 34% in Y1 to 45% in Y3 whilst the percentage of patients with a hypertension diagnosis of more than 10 years decreased from 50% in Y1 to 35% in Y3. Consistent with an earlier presentation, the proportion of patients with end-organ damage at the time of referral decreased from 44% in Y1 to 29% in Y3. Almost a third of the PA patients had unilateral disease; all of those who underwent adrenalectomy had biochemical cure. Patients with bilateral PA were treated with spironolactone. Their systolic/diastolic blood pressure decreased by 15/12 mmHg in Y1, 17/13 mmHg in Y2 and 23/11 mmHg in Y3; while the mean number of antihypertensive medications decreased from 2.9 to 1.8 in Y1, 2.7 to 2.0 in Y2 and 2.2 to 1.6 in Y3 Conclusion: The EHS has facilitated an increase in referrals for PA screening from primary care, resulting in the earlier diagnosis of PA, when less complications are present, and optimised patient outcomes. A broader uptake of such a clinical service, integrated with education outreach, will bridge the gap between the reported high prevalence of PA and the actual low diagnostic rates.

Background: Primary aldosteronism is the most common specifically treatable and potentially curable cause of hypertension. Objective: The aim of this study was to analyse the referral pattern and disease characteristics of patients with hypertension and suspected primary aldosteronism. Methods: We prospectively collected clinical data from patients who attended the Endocrine Hypertension Service (EHS) at Monash Health from May 2016 to May 2017. Results: Of 87 patients, 69.2% had indications for primary aldosteronism screening in the primary care setting, although only 3.7% were actually screened. At the time of referral to the EHS, 61% had hypertension for over 10 years and 42% of the patients with primary aldosteronism had associated end-organ damage. Targeted treatment of primary aldosteronism led to biochemical and clinical improvement in all patients. Discussion: The diagnosis of primary aldosteronism is currently delayed and associated with significant end-organ damage. Increased awareness of primary aldosteronism and its screening indications in primary and tertiary care is needed for earlier diagnosis and improved outcomes.

The association between type 1 diabetes mellitus (T1DM) and bone health has garnered interest over the years. Fracture risk is known to be increased in individuals with T1DM, although bone health assessment is not often performed in the clinical setting. We describe the case of a 21-year-old male with longstanding T1DM with multilevel vertebral fractures on imaging, after presenting with acute back pain without apparent trauma. Dual-energy X-ray absorptiometry (DXA) revealed significantly reduced bone mineral density at the lumbar spine and femoral neck. Extensive investigations for other secondary or genetic causes of osteoporosis were unremarkable, apart from moderate vitamin D deficiency. High-resolution peripheral quantitative computed tomography and bone biospy revealed significant alterations of trabecular bone microarchitecture. It later transpired that the patient had sustained vertebral fractures secondary to unrecognised nocturnal hypoglycaemic seizures. Intravenous zoledronic acid was administered for secondary fracture prevention. Despite anti-resorptive therapy, the patient sustained a new vertebral fracture after experiencing another hypoglycaemic seizure in his sleep. Bone health in T1DM is complex and not well understood. There are significant challenges in the assessment and management of osteoporosis in T1DM, particularly in young adults, where fracture prediction tools have not been validated. Clinicians should be aware of hypoglycaemia as a significant risk factor for fracture in patients with T1DM. Learning points: Type 1 diabetes mellitus (T1DM) is a secondary cause of osteoporosis, characterised by reduced bone mass and disturbed bone microarchitecture. Hypoglycaemic seizures generate sufficient compression forces along the thoracic column and can cause fractures in individuals with compromised bone quality. Unrecognised hypoglycaemic seizures should be considered in patients with T1DM presenting with fractures without a history of trauma. Patients with T1DM have increased fracture risk and risk factors should be addressed. Evaluation of bone microarchitecture may provide further insights into mechanisms of fracture in T1DM. Further research is needed to guide the optimal screening and management of bone health in patients with T1DM. Background Type 1 diabetes mellitus (T1DM) is an immune-mediated condition culminating in the destruction of pancreatic beta cells, which are necessary for insulin production. T1DM is commonly diagnosed in childhood and young adults, thus reflecting the burden of this chronic disease in a young population, who are ultimately at risk of the long-term complications of diabetes. In recent years, there has been growing interest and awareness of the pathophysiology and mechanisms behind diabetic bone disease. Although T1DM is an established risk factor for osteoporosis and fracture, bone health in patients with T1DM is not routinely assessed. This may be in part due to the lack of guidelines for fracture risk assessment and management of such patients, particularly in children and young adults. We present the challenges in diagnosis and management of recurrent fractures secondary to hypoglycaemic seizures in a patient with T1DM.

Asymptomatic urolithiasis is common and of mixed composition in patients with β-thalassaemia major. Twenty-seven subjects were imaged using dual-energy computer tomography to determine the presence and composition of urolithiasis. The prevalence of urolithiasis was 59% and affected patients generally had multiple stones, often with more than one component: struvite (33%), calcium oxalate (31%) and cystine (22%). Hypercalciuria was present in 78% of subjects and calcium-containing urolithiasis was associated with reduced femoral neck Z scores.

The cloning of the mineralocorticoid receptor (MR) 30 years ago was the start of a new era of research into the regulatory processes of MR signalling at target genes in the distal nephron, and subsequently in many other tissues. Nuclear receptor (NR) signalling is modified by interactions with coregulatory proteins that serve to enhance or inhibit the gene transcriptional responses. Over 400 coregulatory proteins have been described for the NR super family, many with functional roles in signalling, cellular function, physiology and pathophysiology. Relatively few coregulators have however been described for the MR although recent studies have demonstrated both ligand and/or tissue selectivity for MR-coregulator interactions. A full understanding of the cell, ligand and promoter-specific requirements for MR-coregulator signalling is an essential first step towards the design of small molecular inhibitors of these protein-protein interactions. Tissue-selective steroidal or non-steroidal modulators of the MR are also a desired therapeutic goal. Selectivity, as for other steroid hormone receptors, will probably depend on differential expression and recruitment of coregulatory proteins.

Background & Aims: Options for the prevention of short-bowel syndromeâassociated liver disease (SBS-ALDs) are limited and often ineffective. The farnesoid X receptor (FXR) is a newly emerging pharmaceutical target and FXR agonists have been shown to ameliorate cholestasis and metabolic disorders. The aim of this study was to assess the efficacy of obeticholic acid (OCA) treatment in preventing SBS-ALDs. Methods: Piglets underwent 75% small-bowel resection (SBS) or sham surgery (sham) and were assigned to either a daily dose of OCA (2.4 mg/kg/day) or were untreated. Clinical measures included weight gain and stool studies. Histologic features were assessed. Ultraperformance liquid chromatography tandem mass spectrometry was used to determine bile acid composition in end point bile and portal serum samples. Gene expression of key FXR targets was assessed in intestinal and hepatic tissues via quantitative polymerase chain reaction. Results: OCA-treated SBS piglets showed decreased stool fat and altered liver histology when compared with nontreated SBS piglets. OCA prevented SBS-associated taurine depletion, however, further analysis of bile and portal serum samples indicated that OCA did not prevent SBS-associated alterations in bile acid composition. The expression of FXR target genes involved in bile acid transport and synthesis increased within the liver of SBS piglets after OCA administration whereas, paradoxically, intestinal expression of FXR target genes were decreased by OCA administration. Conclusions: Administration of OCA in SBS reduced fat malabsorption and altered bile acid composition, but did not prevent the development of SBS-ALDs. We postulate that extensive small resection impacts the ability of the remnant intestine to respond to FXR activation. Keywords: Short-Bowel Syndrome, Liver Disease, Intestinal FailureâAssociated Liver Disease, Obeticholic Acid, Bile Acids, Farnesoid X Receptor

The physiological mechanisms that confer different outcomes in morbidity and mortality of the fetus exposed to stressful environments may be driven by significant differences in the expression and function of the placental glucocorticoid receptor (GR). The recent discovery that the placenta contains at least 8 different isoforms of the GR raises questions about the regulation and physiological relevance of the many GR variants expressed in the placenta. The current data also highlights that individual differences in glucocorticoid sensitivity, variations in the effect of different complications of pregnancy on birth outcomes and sex differences in the response to stress, may all be dependent on a specific GR isoform expression profile. This review will investigate the current state of knowledge of GR isoforms in the placenta and discuss the potential role of these multiple isoforms in regulating glucocorticoid sensitivity.