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

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

Association of adrenal steroids with metabolomic profiles in patients with primary and endocrine hypertension.

Introduction: Endocrine hypertension (EHT) due to pheochromocytoma/paraganglioma (PPGL), Cushing's syndrome (CS), or primary aldosteronism (PA) is linked to a variety of metabolic alterations and comorbidities. Accordingly, patients with EHT and primary hypertension (PHT) are characterized by distinct metabolic profiles. However, it remains unclear whether the metabolomic differences relate solely to the disease-defining hormonal parameters. Therefore, our objective was to study the association of disease defining hormonal excess and concomitant adrenal steroids with metabolomic alterations in patients with EHT. Methods: Retrospective European multicenter study of 263 patients (mean age 49 years, 50% females; 58 PHT, 69 PPGL, 37 CS, 99 PA) in whom targeted metabolomic and adrenal steroid profiling was available. The association of 13 adrenal steroids with differences in 79 metabolites between PPGL, CS, PA and PHT was examined after correction for age, sex, BMI, and presence of diabetes mellitus. Results: After adjustment for BMI and diabetes mellitus significant association between adrenal steroids and metabolites - 18 in PPGL, 15 in CS, and 23 in PA - were revealed. In PPGL, the majority of metabolite associations were linked to catecholamine excess, whereas in PA, only one metabolite was associated with aldosterone. In contrast, cortisone (16 metabolites), cortisol (6 metabolites), and DHEA (8 metabolites) had the highest number of associated metabolites in PA. In CS, 18-hydroxycortisol significantly influenced 5 metabolites, cortisol affected 4, and cortisone, 11-deoxycortisol, and DHEA each were linked to 3 metabolites. Discussions: Our study indicates cortisol, cortisone, and catecholamine excess are significantly associated with metabolomic variances in EHT versus PHT patients. Notably, catecholamine excess is key to PPGL's metabolomic changes, whereas in PA, other non-defining adrenal steroids mainly account for metabolomic differences. In CS, cortisol, alongside other non-defining adrenal hormones, contributes to these differences, suggesting that metabolic disorders and cardiovascular morbidity in these conditions could also be affected by various adrenal steroids.

Screening for Primary Aldosteronism by Mass Spectrometry Versus Immunoassay Measurements of Aldosterone: A Prospective Within-Patient Study.

BACKGROUND: Measurements of aldosterone by mass spectrometry are more accurate and less prone to interferences than immunoassay measurements, and may produce a more accurate aldosterone:renin ratio (ARR) when screening for primary aldosteronism (PA). METHODS: Differences in diagnostic performance of the ARR using mass spectrometry vs immunoassay measurements of aldosterone were examined in 710 patients screened for PA. PA was confirmed in 153 patients and excluded in 451 others. Disease classifications were not achieved in 106 patients. Areas under receiver-operating characteristic curves (AUROC) and other measures were used to compare diagnostic performance. RESULTS: Mass spectrometry-based measurements yielded lower plasma aldosterone concentrations than immunoassay measurements. For the ARR based on immunoassay measurements of aldosterone, AUROCs were slightly lower (P = 0.018) than those using mass spectrometry measurements (0.895 vs 0.906). The cutoff for the ARR to reach a sensitivity of 95% was 30 and 21.5 pmol/mU by respective immunoassay and mass spectrometry-based measurements, which corresponded to specificities of 57% for both. With data restricted to patients with unilateral PA, diagnostic sensitivities of 94% with specificities >81% could be achieved at cutoffs of 61 and 52 pmol/mU for respective immunoassay and mass spectrometry measurements. CONCLUSIONS: Mass spectrometry-based measurements of aldosterone for the ARR provide no clear diagnostic advantage over immunoassay-based measurements. Both approaches offer limited diagnostic accuracy for the ARR as a screening test. One solution is to employ the higher cutoffs to triage patients likely to have unilateral PA for further tests and possible adrenalectomy, while using the lower cutoffs to identify others for targeted medical therapy.German Clinical Trials Register ID: DRKS00017084.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Accurate redox state indication by in situ derivatization with N-ethylmaleimide - Profiling of transsulfuration and glutathione pathway metabolites by UPLC-MS/MS.

BACKGROUND: Reduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies. METHODS: Here we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined. RESULTS: Twelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state. CONCLUSION: The described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.

Treatment with siRNAs is commonly associated with GPX4 up-regulation and target knockdown-independent sensitization to ferroptosis.

Small interfering RNAs (siRNAs) are widely used in biomedical research and in clinical trials. Here, we demonstrate that siRNA treatment is commonly associated with significant sensitization to ferroptosis, independently of the target protein knockdown. Genetically targeting mitochondrial antiviral-signaling protein (MAVS) reversed the siRNA-mediated sensitizing effect, but no activation of canonical MAVS signaling, which involves phosphorylation of IkBα and interferon regulatory transcription factor 3 (IRF3), was observed. In contrast, MAVS mediated a noncanonical signal resulting in a prominent increase in mitochondrial ROS levels, and increase in the BACH1/pNRF2 transcription factor ratio and GPX4 up-regulation, which was associated with a 50% decrease in intracellular glutathione levels. We conclude that siRNAs commonly sensitize to ferroptosis and may severely compromise the conclusions drawn from silencing approaches in biomedical research. Finally, as ferroptosis contributes to a variety of pathophysiological processes, we cannot exclude side effects in human siRNA-based therapeutical concepts that should be clinically tested.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Asymmetric Adrenals: Sexual Dimorphism of Adrenal Tumors.

CONTEXT: Sexual dimorphism has direct consequences on the incidence and survival of cancer. Early and accurate diagnosis is crucial to improve prognosis. OBJECTIVE: This work aimed to characterized the influence of sex and adrenal asymmetry on the emergence of adrenal tumors. METHODS: We conducted a multicenter, observational study involving 8037 patients with adrenal tumors, including adrenocortical carcinoma (ACC), aldosterone-producing adenoma (APA), cortisol-secreting adrenocortical adenomas (CSAs), non-aldosterone-producing adrenal cortical adenoma (NAPACA), pheochromocytoma (PCC), and neuroblastoma (NB), and investigated tumor lateralization according to sex. Human adrenal tissues (n = 20) were analyzed with a multiomics approach that allows determination of gene expression, catecholamine, and steroid contents in a single sample. In addition, we performed a literature review of computed tomography and magnetic resonance imaging-based studies examining adrenal gland size. RESULTS: ACC (n = 1858); CSA (n = 68), NAPACA (n = 2174), and PCC (n = 1824) were more common in females than in males (female-to-male ratio: 1.1:1-3.8:1), whereas NBs (n = 2320) and APAs (n = 228) were less prevalent in females (0.8:1). ACC, APA, CSA, NAPACA, and NB occurred more frequently in the left than in the right adrenal (left-to-right ratio: 1.1:1-1.8:1), whereas PCC arose more often in the right than in the left adrenal (0.8:1). In both sexes, the left adrenal was larger than the right adrenal; females have smaller adrenals than males. CONCLUSION: Adrenal asymmetry in both sexes may be related to the pathogenesis of adrenal tumors and should be considered during the diagnosis of these tumors.

False-positive results for pheochromocytoma associated with norepinephrine reuptake blockade.

Measurements of plasma metanephrines and methoxytyramine provide a sensitive test for diagnosis of pheochromocytoma/paraganglioma. False-positive results remain a problem, particularly in patients taking norepinephrine reuptake-blocking drugs. Therefore, in this retrospective observational study, we measured plasma metanephrines and methoxytyramine in 61 patients taking norepinephrine reuptake blockers (tricyclic antidepressants or serotonin-norepinephrine reuptake inhibitors) and 17 others taking selective serotonin reuptake inhibitors, all without pheochromocytoma/paraganglioma. We highlight a singular case with strongly elevated plasma normetanephrine and methoxytyramine concentrations associated with norepinephrine reuptake blockade. Data were compared to results from 252 and 1804 respective patients with and without tumors. Plasma normetanephrine was 40% higher (P < 0.0001) in patients on norepinephrine reuptake blockers and methoxytyramine was 127% higher (P = 0.0062) in patients taking tricyclic antidepressants compared to patients not taking uptake blockers and without tumors. The corresponding false-positive rates rose (P < 0.0001) from 4.8% to 23.0% for normetanephrine and from 0.9% to 28.6% for methoxytyramine. Selective serotonin reuptake inhibitors did not increase plasma concentrations of metabolites. In the highlighted case, plasma normetanephrine and methoxytyramine were elevated more than six times above upper reference limits. A pheochromocytoma/paraganglioma, however, was excluded by functional imaging. All biochemical test results normalized after discontinuation of norepinephrine reuptake blockers. These findings clarify that norepinephrine reuptake blockers usually result in mild elevations of normetanephrine and methoxytyramine that, nevertheless, significantly increase the number of false-positive results. There can, however, be exceptions where increases in normetanephrine and methoxytyramine reach pathological levels. Such exceptions may reflect failure of centrally mediated sympathoinhibition that normally occurs with the norepinephrine reuptake blockade.

The long non-coding RNA OTX2-AS1 promotes tumor growth and predicts response to BCL-2 inhibition in medulloblastoma.

PURPOSE: Primary brain tumors are a leading cause of cancer-related death in children, and medulloblastoma is the most common malignant pediatric brain tumor. The current molecular characterization of medulloblastoma is mainly based on protein-coding genes, while little is known about the involvement of long non-coding RNAs (lncRNAs). This study aimed to elucidate the role of the lncRNA OTX2-AS1 in medulloblastoma. METHODS: Analyses of DNA copy number alterations, methylation profiles, and gene expression data were used to characterize molecular alterations of OTX2-AS1 in medulloblastoma tissue samples. In vitro analyses of medulloblastoma cell models and orthotopic in vivo experiments were carried out for functional characterization of OTX2-AS1. High-throughput drug screening was employed to identify pharmacological inhibitors, while proteomics and metabolomics analyses were performed to address potential mechanisms of drug action. RESULTS: We detected amplification and consecutive overexpression of OTX2 and OTX2-AS1 in a subset of medulloblastomas. In addition, OTX2-AS1 promoter methylation was linked to OTX2-AS1 expression. OTX2-AS1 knockout reduced medulloblastoma cell viability and cell migration in vitro and prolonged survival in the D283 orthotopic medulloblastoma mouse xenograft model. Pharmacological inhibition of BCL-2 suppressed the growth of OTX2-AS1 overexpressing medulloblastoma cells in vitro. CONCLUSIONS: Our study revealed a pro-tumorigenic role of OTX2-AS1 in medulloblastoma and identified BCL-2 inhibition as a potential therapeutic approach to target OTX2-AS1 overexpressing medulloblastoma cells.

Correction: Sigala et al. A Comprehensive Investigation of Steroidogenic Signaling in Classical and New Experimental Cell Models of Adrenocortical Carcinoma. Cells 2022, 11, 1439.

The authors made the following changes to their paper [...].

Liquid chromatography-tandem mass spectrometry based simultaneous quantification of tryptophan, serotonin and kynurenine pathway metabolites in tissues and cell culture systems.

BACKGROUND: Kynurenine and respective metabolites exhibit bioactivity as well as tryptophan, an essential amino acid, and the neurotransmitter serotonin. Dysregulations in the kynurenine pathway are involved in neurodegenerative/neuropsychiatric disorders and diabetes mellitus type 2 but also in cancer. Therefore, measurements of kynurenine-related metabolites will improve the general understanding for kynurenine pathway relevance in disease pathogenesis. METHODS: Tryptophan, serotonin, picolinic acid, quinolinic acid, 3-OH-kynurenine, kynurenine, 3-OH-anthranilic acid, kynurenic acid, anthranilic acid as well as nicotinic acid and the redox cofactor NAD+ were analyzed in heterogeneous matrices by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After validation, the described method was applied for measurements of native metabolite concentrations in murine tissues and cellular systems including pathway-shift monitoring after treatment with the tryptophan-2,3-dioxygenase-inhibitor 680C91. In addition, the method was evaluated for its ability for integration into multi-omics approaches using a single sample metabolite extraction procedure. RESULTS: A simple and sensitive UPLC-MS/MS method for simultaneous quantification of up to 10 kynurenine-related metabolites in four biological matrices was developed. Within a run time of 6.5 min, chromatographic separation of kynurenine-related metabolites, including the isomers nicotinic acid and picolinic acid, was achieved without derivatization. Validation parameters, including interday precision (<14.8%), mean accuracy (102.4% ± 12.9%) and linear detection ranges of more than three orders of magnitude, indicate method reliability. Depending the investigated sample matrix, the majority of metabolites were successfully detected and quantified in native murine and cell culture derived sample materials. Furthermore, the method allowed to monitor the impact of a tryptophan-2,3-dioxygenase-inhibitor on kynurenine pathway in a cellular system and is suitable for multi-assay analyses using aliquots from the same cell extract. CONCLUSION: The described UPLC-MS/MS method provides a simple tool for the simultaneous quantification of kynurenine pathway metabolites. Due to its suitability for many physiological matrices, the method provides wide application for disease-related experimental settings.

An ovarian phenotype of alpha 7 nicotinic receptor knockout mice.

In brief: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Abstract: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.

Succinate mediates inflammation-induced adrenocortical dysfunction.

The hypothalamus-pituitary-adrenal (HPA) axis is activated in response to inflammation leading to increased production of anti-inflammatory glucocorticoids by the adrenal cortex, thereby representing an endogenous feedback loop. However, severe inflammation reduces the responsiveness of the adrenal gland to adrenocorticotropic hormone (ACTH), although the underlying mechanisms are poorly understood. Here, we show by transcriptomic, proteomic, and metabolomic analyses that LPS-induced systemic inflammation triggers profound metabolic changes in steroidogenic adrenocortical cells, including downregulation of the TCA cycle and oxidative phosphorylation, in mice. Inflammation disrupts the TCA cycle at the level of succinate dehydrogenase (SDH), leading to succinate accumulation and disturbed steroidogenesis. Mechanistically, IL-1ß reduces SDHB expression through upregulation of DNA methyltransferase 1 (DNMT1) and methylation of the SDHB promoter. Consequently, increased succinate levels impair oxidative phosphorylation and ATP synthesis and enhance ROS production, leading to reduced steroidogenesis. Together, we demonstrate that the IL-1ß-DNMT1-SDHB-succinate axis disrupts steroidogenesis. Our findings not only provide a mechanistic explanation for adrenal dysfunction in severe inflammation, but also offer a potential target for therapeutic intervention.

Fatty acid desaturase 2 determines the lipidomic landscape and steroidogenic function of the adrenal gland.

Corticosteroids regulate vital processes, including stress responses, systemic metabolism, and blood pressure. Here, we show that corticosteroid synthesis is related to the polyunsaturated fatty acid (PUFA) content of mitochondrial phospholipids in adrenocortical cells. Inhibition of the rate-limiting enzyme of PUFA synthesis, fatty acid desaturase 2 (FADS2), leads to perturbations in the mitochondrial lipidome and diminishes steroidogenesis. Consistently, the adrenocortical mitochondria of Fads2-/- mice fed a diet with low PUFA concentration are structurally impaired and corticoid levels are decreased. On the contrary, FADS2 expression is elevated in the adrenal cortex of obese mice, and plasma corticosterone is increased, which can be counteracted by dietary supplementation with the FADS2 inhibitor SC-26192 or icosapent ethyl, an eicosapentaenoic acid ethyl ester. In humans, FADS2 expression is elevated in aldosterone-producing adenomas compared to non-active adenomas or nontumorous adrenocortical tissue and correlates with expression of steroidogenic genes. Our data demonstrate that FADS2-mediated PUFA synthesis determines adrenocortical steroidogenesis in health and disease.

Crosstalk between androgen receptor and WNT/ß-catenin signaling causes sex-specific adrenocortical hyperplasia in mice.

Female bias is highly prevalent in conditions such as adrenal cortex hyperplasia and neoplasia, but the reasons behind this phenomenon are poorly understood. In this study, we show that overexpression of the secreted WNT agonist R-spondin 1 (RSPO1) leads to ectopic activation of WNT/ß-catenin signaling and causes sex-specific adrenocortical hyperplasia in mice. Although female adrenals show ectopic proliferation, male adrenals display excessive immune system activation and cortical thinning. Using a combination of genetic manipulations and hormonal treatment, we show that gonadal androgens suppress ectopic proliferation in the adrenal cortex and determine the selective regulation of the WNT-related genes Axin2 and Wnt4. Notably, genetic removal of androgen receptor (AR) from adrenocortical cells restores the mitogenic effect of WNT/ß-catenin signaling. This is the first demonstration that AR activity in the adrenal cortex determines susceptibility to canonical WNT signaling-induced hyperplasia.

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

TSPAN12 (Tetraspanin 12) Is a Novel Negative Regulator of Aldosterone Production in Adrenal Physiology and Aldosterone-Producing Adenomas.

BACKGROUND: Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism, a condition of low-renin hypertension, in which aldosterone overproduction is usually driven by a somatic activating mutation in an ion pump or channel. TSPAN12 is differentially expressed in different subgroups of APAs suggesting a role in APA pathophysiology. Our objective was to determine the function of TSPAN12 (tetraspanin 12) in adrenal physiology and pathophysiology. METHODS: APA specimens, pig adrenals under dietary sodium modulation, and a human adrenocortical cell line HAC15 were used for functional characterization of TSPAN12 in vivo and in vitro. RESULTS: Gene ontology analysis of 21 APA transcriptomes dichotomized according to high versus low TSPAN12 transcript levels highlighted a function for TSPAN12 related to the renin-angiotensin system. TSPAN12 expression levels in a cohort of 30 APAs were inversely correlated with baseline plasma aldosterone concentrations (R=-0.47; P=0.009). In a pig model of renin-angiotensin system activation by dietary salt restriction, TSPAN12 mRNA levels and TSPAN12 immunostaining were markedly increased in the zona glomerulosa layer of the adrenal cortex. In vitro stimulation of human adrenocortical human adrenocortical cells with 10 nM angiotensin II for 6 hours caused a 1.6-fold±0.13 increase in TSPAN12 expression, which was ablated by 10 µM nifedipine (P=0.0097) or 30 µM W-7 (P=0.0022). Gene silencing of TSPAN12 in human adrenocortical cells demonstrated its inverse effect on aldosterone secretion under basal and angiotensin II stimulated conditions. CONCLUSIONS: Our findings show that TSPAN12 is a negative regulator of aldosterone production and could contribute to aldosterone overproduction in primary aldosteronism.

Long-Term Excessive Dietary Phosphate Intake Increases Arterial Blood Pressure, Activates the Renin-Angiotensin-Aldosterone System, and Stimulates Sympathetic Tone in Mice.

Increased dietary phosphate intake has been associated with severity of coronary artery disease, increased carotid intima-media thickness, left ventricular hypertrophy (LVH), and increased cardiovascular mortality and morbidity in individuals with normal renal function as well as in patients suffering from chronic kidney disease. However, the underlying mechanisms are still unclear. To further elucidate the cardiovascular sequelae of long-term elevated phosphate intake, we maintained male C57BL/6 mice on a calcium, phosphate, and lactose-enriched diet (CPD, 2% Ca, 1.25% P, 20% lactose) after weaning them for 14 months and compared them with age-matched male mice fed a normal mouse diet (ND, 1.0% Ca, 0.7% P). Notably, the CPD has a balanced calcium/phosphate ratio, allowing the effects of elevated dietary phosphate intake largely independent of changes in parathyroid hormone (PTH) to be investigated. In agreement with the rationale of this experiment, mice maintained on CPD for 14 months were characterized by unchanged serum PTH but showed elevated concentrations of circulating intact fibroblast growth factor-23 (FGF23) compared with mice on ND. Cardiovascular phenotyping did not provide evidence for LVH, as evidenced by unchanged LV chamber size, normal cardiomyocyte area, lack of fibrosis, and unchanged molecular markers of hypertrophy (Bnp) between the two groups. However, intra-arterial catheterization revealed increases in systolic pressure, mean arterial pressure, and pulse pressure in mice fed the CPD. Interestingly, chronically elevated dietary phosphate intake stimulated the renin-angiotensin-aldosterone system (RAAS) as evidenced by increased urinary aldosterone in animals fed the CPD, relative to the ND controls. Furthermore, the catecholamines epinephrine, norepinephrine, and dopamine as well as the catecholamine metabolites metanephrine. normetanephrine and methoxytyramine as measured by mass spectrometry were elevated in the urine of mice on CPD, relative to mice on the ND. These changes were partially reversed by switching 14-month-old mice on CPD back to ND for 2 weeks. In conclusion, our data suggest that excess dietary phosphate induces a rise in blood pressure independent of secondary hyperparathyroidism, and that this effect may be mediated through activation of the RAAS and stimulation of the sympathetic tone.