Publications by authors named "Kazutaka Nanba"

51 Publications

Approaches to Gene Mutation Analysis Using Formalin-Fixed Paraffin-Embedded Adrenal Tumor Tissue From Patients With Primary Aldosteronism.

Front Endocrinol (Lausanne) 2021 29;12:683588. Epub 2021 Jun 29.

Department of Pathology, University of Michigan, Ann Arbor, MI, United States.

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal's capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.
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http://dx.doi.org/10.3389/fendo.2021.683588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8276099PMC
June 2021

Acute rhabdomyolysis in a young woman with moderate COVID-19.

IDCases 2021 29;25:e01212. Epub 2021 Jun 29.

Department of Infectious Diseases, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having serious medical, social, and economic impacts worldwide. COVID-19 may lead to a variety of complications, including rhabdomyolysis. Although rhabdomyolysis is a rare complication, it can lead to severe kidney damage. Recent studies suggest that rhabdomyolysis caused by SARS-CoV-2 is more common in middle-aged and older men with severe COVID-19. Herein we report a case of rhabdomyolysis in a young woman with moderate COVID-19. She had a habit of muscle training. She presented with moderate COVID-19 and acute rhabdomyolysis that required a large volume of fluid infusion in addition to dexamethasone and remdesivir. Clinicians should pay attention to the development of rhabdomyolysis in patients with COVID-19, especially those with a habit of strenuous exercise or muscle training, even if they are young and have moderate COVID-19.
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http://dx.doi.org/10.1016/j.idcr.2021.e01212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239312PMC
June 2021

Intratumoral steroid profiling of adrenal cortisol-producing adenomas by liquid chromatography- mass spectrometry.

J Steroid Biochem Mol Biol 2021 Jun 2;212:105924. Epub 2021 Jun 2.

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, 48109, USA. Electronic address:

Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.
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http://dx.doi.org/10.1016/j.jsbmb.2021.105924DOI Listing
June 2021

GENETICS IN ENDOCRINOLOGY: Impact of race and sex on genetic causes of aldosterone-producing adenomas.

Eur J Endocrinol 2021 May 21;185(1):R1-R11. Epub 2021 May 21.

Departments of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA.

Primary aldosteronism (PA) is a common cause of secondary hypertension. Recent technological advances in genetic analysis have provided a better understanding of the molecular pathogenesis of this disease. The application of next-generation sequencing has resulted in the identification of somatic mutations in aldosterone-producing adenoma (APA), a major subtype of PA. Based on the recent findings using a sequencing method that selectively targets the tumor region where aldosterone synthase (CYP11B2) is expressed, the vast majority of APAs appear to harbor a somatic mutation in one of the aldosterone-driver genes, including KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, and CLCN2. Mutations in these genes alter intracellular ion homeostasis and enhance aldosterone production. In a small subset of APAs, somatic activating mutations in the CTNNB1 gene, which encodes β-catenin, have also been detected. Accumulating evidence suggests that race and sex impact the somatic mutation spectrum of APA. Specifically, somatic mutations in the KCNJ5 gene, encoding an inwardly rectifying K+ channel, are common in APAs from Asian populations as well as women regardless of race. Associations between APA histology, genotype, and patient clinical characteristics have also been proposed, suggesting a potential need to consider race and sex for the management of PA patients. Herein, we review recent findings regarding somatic mutations in APA and discuss potential roles of race and sex on the pathophysiology of APA as well as possible clinical implications.
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http://dx.doi.org/10.1530/EJE-21-0031DOI Listing
May 2021

Primary Cultures and Cell Lines for In Vitro Modeling of the Human Adrenal Cortex.

Tohoku J Exp Med 2021 04;253(4):217-232

Department of Molecular and Integrative Physiology, University of Michigan.

The human adrenal cortex is a complex endocrine organ that produces mineralocorticoids, glucocorticoids and androgens. These steroids are produced in distinct cell types located within the glomerulosa, fasciculata and reticularis of the adrenal cortex. Abnormal adrenal steroidogenesis leads to a variety of diseases that can cause hypertension, metabolic syndrome, infertility and premature adrenarche. The adrenal cortex can also develop steroid-producing adenomas and rarely adrenocortical carcinomas. In vitro cell culture models provide important tools to study molecular and cellular mechanisms controlling both the physiologic and pathologic conditions of the adrenal cortex. In addition, the presence of multiple steroid-metabolizing enzymes within adrenal cells makes it a model for defining possible endocrine disruptors that might block these enzymes. The regulation and dysregulation of human adrenal steroid production and cell division/tumor growth can be studied using freshly isolated cells but this requires access to human adrenal glands, which are not available to most investigators. Immortalized human adrenocortical cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis and tumorigenesis. Current human adrenal cell lines include the original NCI-H295 and its substrains: H295A, H295R, HAC13, HAC15, HAC50 and H295RA as well as the recently established MUC-1, CU-ACC1 and CU-ACC2. The current review will discuss the use of primary cultures of fetal and adult adrenal cells as well as adrenocortical cell lines as in vitro models for the study of human adrenal physiology and pathophysiology.
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http://dx.doi.org/10.1620/tjem.253.217DOI Listing
April 2021

Identification of Somatic Mutations in in Aldosterone-Producing Adenomas.

J Endocr Soc 2020 Oct 1;4(10):bvaa123. Epub 2020 Oct 1.

Division of Metabolism, Endocrinology and Diabetes, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.

Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in in 2 APAs that were negative for currently known aldosterone-driver mutations. The gene encodes the voltage-gated chloride channel ClC-2. germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second mutation, which affects the same region of the gene, has not been reported previously (c.64-2_74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA.
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http://dx.doi.org/10.1210/jendso/bvaa123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528565PMC
October 2020

Prevalence of Somatic Mutations in Aldosterone-Producing Adenomas in Japanese Patients.

J Clin Endocrinol Metab 2020 11;105(11)

Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan.

Context: Results of previous studies demonstrated clear racial differences in the prevalence of somatic mutations among patients with aldosterone-producing adenoma (APA). For instance, those in East Asian countries have a high prevalence of somatic mutations in KCNJ5, whereas somatic mutations in other aldosterone-driving genes are rare.

Objectives: To determine somatic mutation prevalence in Japanese APA patients using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided sequencing approach.

Method: Patients with a unilateral form of primary aldosteronism who underwent adrenalectomy at the Tohoku University Hospital were studied. Based on CYP11B2 immunolocalization of resected adrenals, genomic DNA was isolated from the relevant positive area of 10% formalin-fixed, paraffin-embedded tissue of the APAs. Somatic mutations in aldosterone-driving genes were studied in APAs by direct Sanger sequencing and targeted next-generation sequencing.

Results: CYP11B2 IHC-guided sequencing determined APA-related somatic mutations in 102 out of 106 APAs (96%). Somatic KCNJ5 mutation was the most frequent genetic alteration (73%) in this cohort of Japanese patients. Somatic mutations in other aldosterone-driving genes were also identified: CACNA1D (14%), ATP1A1 (5%), ATP2B3 (4%), and CACNA1H (1%), including 2 previously unreported mutations. KCNJ5 mutations were more often detected in APAs from female patients compared with those from male patients [95% (36/38) vs 60% (41/68); P < 0.0001].

Conclusion: IHC-guided sequencing defined somatic mutations in over 95% of Japanese APAs. While the dominance of KCNJ5 mutations in this particular cohort was confirmed, a significantly higher KCNJ5 prevalence was detected in female patients. This study provides a better understanding of genetic spectrum of Japanese APA patients.
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http://dx.doi.org/10.1210/clinem/dgaa595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947976PMC
November 2020

Reassessment of Urinary Aldosterone Measurement After Saline Infusion in Primary Aldosteronism.

J Endocr Soc 2020 Sep 22;4(9):bvaa100. Epub 2020 Jul 22.

Department of Endocrinology and Metabolism, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

Context: Urinary aldosterone levels (Uald) are widely measured in the oral sodium-loading test to confirm primary aldosteronism (PA), but reliable studies on their diagnostic value are limited. This may be due to the difficulty in collecting urine with reliable accuracy, keeping oral sodium intake constant between patients. Therefore, we focused on 24-hour Uald after intravenous saline infusion in a hospitalized setting, which provides a reliable sodium load in consistent amounts.

Objective: Comparing plasma aldosterone concentrations (PAC) and Uald after saline infusion in the sitting position, to evaluate the accuracy in determining PA subtypes and the correlation of both measurements.

Design And Setting: This was a retrospective cross-sectional study in a single referral center.

Patients: Of 53 patients without renal dysfunction who were diagnosed with PA and underwent adrenal venous sampling, 16 and 37 were diagnosed with unilateral and bilateral PA, respectively.

Main Outcome Measures: Uald collected for 24 hours and PAC after saline infusion.

Results: The area under the receiver operating characteristic curve for diagnosing unilateral PA was not significantly different between Uald and PAC after saline infusion (0.921 and 0.958, respectively;  = 0.370). The predicted optimal cutoff value of Uald was 16.5 μg/day (sensitivity, 87.5%; specificity, 100%), and that of PAC after saline infusion was 19.3 ng/dL (sensitivity, 87.5%; specificity, 97.3%). In studied patients with PA, Uald was positively correlated with PAC after saline infusion ( = 0.617;  < 0.001).

Conclusions: We reassessed Uald in PA patients under sufficient sodium loading and demonstrated the correlation between Uald and PAC after saline infusion.
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http://dx.doi.org/10.1210/jendso/bvaa100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417007PMC
September 2020

The Concordance Between Imaging and Adrenal Vein Sampling Varies With Aldosterone-Driver Somatic Mutation.

J Clin Endocrinol Metab 2020 10;105(10)

Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan.

Background: Correct subtyping of primary aldosteronism (PA) is critical for guiding clinical management. Adrenal imaging is less accurate than adrenal vein sampling (AVS); nonetheless, AVS is invasive, technically challenging, and scarcely available.

Objective: To identify predictors of concordance between cross-sectional imaging and lateralized AVS in patients with PA that could help circumvent AVS in a subset of patients.

Methods: We retrospectively studied all patients with PA who underwent AVS in a tertiary referral center from 2009 to 2019. AVS was performed before and after cosyntropin stimulation. Patients with lateralized AVS in at least one condition were included. Aldosterone synthase-guided next-generation sequencing was performed on available adrenal tissue. Logistic regression was implemented to identify predictors of imaging-AVS lateralization concordance.

Results: A total of 234 patients (62% men), age 20 to 79 years, 73% white, 23% black, and 2% Asian were included. AVS lateralization was found: 1) both pre- and post-cosyntropin (Uni/Uni) in 138 patients; 2) only at baseline (Uni/Bi) in 39 patients; 3) only after cosyntropin stimulation (Bi/Uni) in 29 patients. Catheterization partially failed in 28 patients. AVS-imaging agreement was higher in patients with KCNJ5 versus other aldosterone-driver somatic mutations (90.3% versus 64.6%; P < 0.001); in Asian and white versus black Americans (75%, 70%, and 36%, respectively); in younger patients; and those with left adrenal nodules and contralateral suppression. Conversely, AVS-imaging agreement was lowest in Uni/Bi patients (38% vs. 69% in Uni/Uni, and 62% in Bi/Uni; P = 0.007).

Conclusions: While AVS-imaging agreement is higher in young white and Asian patients, who have KCNJ5-mutated aldosterone producing adenomas, no predictor confers absolute imaging accuracy.
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http://dx.doi.org/10.1210/clinem/dgaa482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437239PMC
October 2020

Biochemical, Histopathological, and Genetic Characterization of Posture-Responsive and Unresponsive APAs.

J Clin Endocrinol Metab 2020 09;105(9)

Endocrine Hypertension Research Centre, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, Australia.

Context And Objective: Posture-responsive and posture-unresponsive aldosterone-producing adenomas (APAs) account for approximately 40% and 60% of APAs, respectively. Somatic gene mutations have been recently reported to exist in approximately 90% of APAs. This study was designed to characterize the biochemical, histopathologic, and genetic properties of these 2 types of APA.

Methods: Plasma levels of aldosterone and hybrid steroids (18-oxocortisol and 18-hydroxycortisol) were measured by liquid chromatography-tandem mass spectrometry. Immunohistochemistry for CYP11B2 (aldosterone synthase) and CYP17A1 (17α-hydroxylase) and deoxyribonucleic acid sequencing (Sanger and next-generation sequencing) were performed on APA tissue collected from 23 posture-unresponsive and 17 posture-responsive APA patients.

Results: Patients with posture-unresponsive APA displayed higher (P < 0.01) levels of hybrid steroids, recumbent aldosterone and cortisol, larger (P < 0.01) zona fasciculata (ZF)-like tumors with higher (P < 0.01) expression of CYP17A1 (but not of CYP11B2) than patients with posture-responsive APA (most of which were not ZF-like). Of 40 studied APAs, 37 (92.5%) were found to harbor aldosterone-driving somatic mutations (KCNJ5 = 14 [35.0%], CACNA1D = 13 [32.5%], ATP1A1 = 8 [20.0%], and ATP2B3 = 2 [5.0%]), including 5 previously unreported mutations (3 in CACNA1D and 2 in ATP1A1). Notably, 64.7% (11/17) of posture-responsive APAs carried CACNA1D mutations, whereas 56.5% (13/23) of posture-unresponsive APAs harbored KCNJ5 mutations.

Conclusions: The elevated production of hybrid steroids by posture-unresponsive APAs may relate to their ZF-like tumor cell composition, resulting in 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.
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http://dx.doi.org/10.1210/clinem/dgaa367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426003PMC
September 2020

Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma.

Hypertension 2020 04 2;75(4):1034-1044. Epub 2020 Mar 2.

From the PARCC, INSERM, Université de Paris, France (K.D.S., S. Boulkroun, A.R., I.G.-D., L.A., F.L.F.-R., M.-C.Z.).

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated β-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in -mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098445PMC
April 2020

Somatic Mutation As a Cause of Aldosterone-Producing Adenoma.

Hypertension 2020 03 27;75(3):645-649. Epub 2020 Jan 27.

From the Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor (K.N., A.R.B., J.R., W.E.R.).

Driver somatic mutations for aldosterone excess have been found in ≈90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. In the present study, we identified a novel somatic mutation (c.T4289C, p.I1430T) in an APA without any currently known aldosterone-driver mutations using CYP11B2 immunohistochemistry-guided whole exome sequencing. The gene encodes a voltage-dependent T-type calcium channel alpha-1H subunit. Germline variants in this gene are known as a cause of familial hyperaldosteronism IV. Targeted next-generation sequencing detected identical variants in 2 additional APAs in a cohort of the University of Michigan, resulting in a prevalence of 4% (3/75) in APAs. We tested the functional effect of the variant on adrenal cell aldosterone production and mRNA expression using the human adrenocortical HAC15 cell line with a doxycycline-inducible mutation. Doxycycline treatment increased mRNA levels as well as aldosterone production, supporting a pathological role of the p.I1430T mutation on the development of primary aldosteronism. In conclusion, somatic mutation is a genetic cause of APAs. Although the prevalence of this mutation is low, this study will provide better understanding of molecular mechanism of inappropriate aldosterone production in APAs.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.119.14349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059016PMC
March 2020

Seated saline infusion test in predicting subtype diagnosis of primary aldosteronism.

Clin Endocrinol (Oxf) 2019 12 6;91(6):737-742. Epub 2019 Nov 6.

Department of Endocrinology and Metabolism, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

Context: Although saline infusion test is widely used as a confirmatory test for primary aldosteronism (PA), it is reportedly less sensitive in patients in whom aldosterone is responsive to the upright position by performing it in recumbent position. Based on a single-centre experience, seated saline infusion test (SSIT) has been reported to be highly sensitive and superior to recumbent testing in identifying both unilateral and bilateral forms of PA. However, due to limited participants number, the utility of SSIT needs to be validated in other series.

Objective: This study aimed to evaluate the accuracy of SSIT in determining the PA subtypes compared with adrenocorticotropic hormone stimulation test under dexamethasone suppression (Dex-AT).

Patients And Setting: Sixty-four patients with PA who underwent both SSIT and Dex-AT were included. Subtype diagnosis of PA was determined by adrenal venous sampling (AVS) (16 unilateral and 48 bilateral forms).

Main Outcome Measure: Plasma aldosterone concentrations (PACs) were measured after SSIT and Dex-AT.

Results: The area under the receiver operating characteristic (ROC) curve for diagnosing unilateral PA was greater in SSIT than that in Dex-AT (0.907 vs. 0.755; P = .023). ROC curve analysis predicted optimal cut-off PACs of 13.1 ng/dL (sensitivity, 93.8%; specificity, 79.2%) for SSIT and 34.2 ng/dL (sensitivity, 75.0%; specificity, 68.8%) for Dex-AT.

Conclusions: Seated saline infusion test has superior accuracy in subtype diagnosis of PA compared with Dex-AT. SSIT can be a sensitive test for determining patients who require AVS prior to surgery.
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http://dx.doi.org/10.1111/cen.14111DOI Listing
December 2019

Three Discrete Patterns of Primary Aldosteronism Lateralization in Response to Cosyntropin During Adrenal Vein Sampling.

J Clin Endocrinol Metab 2019 12;104(12):5867-5876

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan.

Context: Cosyntropin [ACTH (1-24)] stimulation during adrenal vein (AV) sampling (AVS) enhances the confidence in the success of AV cannulation and circumvents intraprocedure hormonal fluctuations. Cosyntropin's effect on primary aldosteronism (PA) lateralization, however, is controversial.

Objectives: To define the major patterns of time-dependent lateralization, and their determinants, after cosyntropin stimulation during AVS.

Methods: We retrospectively studied patients with PA who underwent AVS before, 10, and 20 minutes after cosyntropin stimulation between 2009 and 2018. Unilateral (U) or bilateral (B) PA was determined on the basis of a lateralization index (LI) value ≥4 or <4, respectively. Available adrenal tissue underwent aldosterone synthase-guided next-generation sequencing.

Results: PA lateralization was concordant between basal and cosyntropin-stimulated AVS in 169 of 222 patients (76%; U/U, n = 110; B/B, n = 59) and discordant in 53 patients (24%; U/B, n = 32; B/U, n = 21). Peripheral and dominant AV aldosterone concentrations and LI were highest in U/U patients and progressively lower across intermediate and B/B groups. LI response to cosyntropin increased in 27% of patients, decreased in 33%, and remained stable in 40%. Baseline aldosterone concentrations predicted the LI pattern across time (P < 0.001). Mutation status was defined in 61 patients. Most patients with KCNJ5 mutations had descending LI, whereas those with ATP1A1 and ATP2B3 mutations had ascending LI after cosyntropin stimulation.

Conclusion: Patients with severe PA lateralized robustly regardless of cosyntropin use. Cosyntropin stimulation reveals intermediate PA subtypes; its impact on LI varies with baseline aldosterone concentrations and aldosterone-driver mutations.
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http://dx.doi.org/10.1210/jc.2019-01182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800532PMC
December 2019

Genetics of aldosterone-producing adenomas with pathogenic KCNJ5 variants.

Endocr Relat Cancer 2019 04 1;26(4):463-470. Epub 2019 Feb 1.

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan, USA.

Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APA). Although the mechanisms leading to an increased aldosterone production in APA cells has been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on eleven APA, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variant, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.
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http://dx.doi.org/10.1530/ERC-18-0364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869655PMC
April 2019

Genetic Characteristics of Aldosterone-Producing Adenomas in Blacks.

Hypertension 2019 04;73(4):885-892

From the Department of Molecular and Integrative Physiology (K.N., W.E.R.), University of Michigan, Ann Arbor.

Somatic mutations have been identified in aldosterone-producing adenomas (APAs) in genes that include KCNJ5, ATP1A1, ATP2B3, and CACNA1D. Based on independent studies, there appears to be racial differences in the prevalence of somatic KCNJ5 mutations, particularly between East Asians and Europeans. Despite the high cardiovascular disease mortality of blacks, there have been no studies focusing on somatic mutations in APAs in this population. In the present study, we investigated genetic characteristics of APAs in blacks using a CYP11B2 (aldosterone synthase) immunohistochemistry-guided next-generation sequencing approach. The adrenal glands with adrenocortical adenomas from 79 black patients with primary aldosteronism were studied. Seventy-three tumors from 69 adrenal glands were confirmed to be APAs by CYP11B2 immunohistochemistry. Sixty-five of 73 APAs (89%) had somatic mutations in aldosterone-driver genes. Somatic CACNA1D mutations were the most prevalent genetic alteration (42%), followed by KCNJ5 (34%), ATP1A1 (8%), and ATP2B3 mutations (4%). CACNA1D mutations were more often observed in APAs from males than those from females (55% versus 29%, P=0.033), whereas KCNJ5 mutations were more prevalent in APAs from females compared with those from males (57% versus 13%, P<0.001). No somatic mutations in aldosterone-driver genes were identified in tumors without CYP11B2 expression. In conclusion, 89% of APAs in blacks harbor aldosterone-driving mutations, and unlike Europeans and East Asians, the most frequently mutated aldosterone-driver gene was CACNA1D. Determination of racial differences in the prevalence of aldosterone-driver gene mutations may facilitate the development of personalized medicines for patients with primary aldosteronism.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.12070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416065PMC
April 2019

Targeted Molecular Characterization of Aldosterone-Producing Adenomas in White Americans.

J Clin Endocrinol Metab 2018 10;103(10):3869-3876

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan.

Context: Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown.

Objective: To investigate the prevalence and spectrum of somatic mutations in APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)‒guided next-generation sequencing (NGS) approach.

Methods: Formalin-fixed paraffin-embedded adrenal tissue from white American patients with primary aldosteronism who underwent adrenalectomy at the University of Michigan was used. Genomic DNA was isolated from 75 APAs (identified by CYP11B2 IHC). NGS was performed to identify somatic mutations by sequencing the entire coding region of a panel of genes mutated in APAs.

Results: Somatic mutations were identified in 66 of 75 APAs (88%). Of the APAs with somatic mutations, six were smaller than coexisting CYP11B2-negative adrenocortical adenomas. The most frequently mutated gene was KCNJ5 (43%), followed by CACNA1D (21%), ATP1A1 (17%), ATP2B3 (4%), and CTNNB1 (3%). In addition to identification of previously reported mutations, we identified five previously unreported mutations (two in KCNJ5, one in ATP1A1, one in ATP2B3, and one in CACNA1D genes). KCNJ5 mutations were more frequent in women (70% vs 24% in men).

Conclusion: Comprehensive NGS of CYP11B2-expressing adrenal tumors identified somatic mutations in aldosterone-driving genes in 88% of APAs, a higher rate than in previous studies using conventional approaches.
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http://dx.doi.org/10.1210/jc.2018-01004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179168PMC
October 2018

Development and validation of subtype prediction scores for the workup of primary aldosteronism.

J Hypertens 2018 11;36(11):2269-2276

Department of Endocrinology and Metabolism, National Hospital Organization Kyoto Medical Center, Kyoto.

Objectives: A subtype prediction score for primary aldosteronism has not yet been developed and validated using a large dataset. This study aimed to develop and validate a new subtype prediction score and to compare it with existing scores using a large multicenter database.

Methods: In total, 1936 patients with primary aldosteronism were randomly assigned to the development and validation datasets, constituting 1290 and 646 patients, respectively. Three prediction scores were generated with or without confirmatory tests, using logistic regression analysis. In the validation dataset, new and existing prediction scores were compared using receiver operating characteristic curve, net reclassification improvement, and integrated discrimination improvement analyses.

Results: The new prediction score is simply calculated using serum potassium levels [>3.9 mmol/l (four points); 3.5-3.9 mmol/l (three points)], the absence of adrenal nodules during computed tomography (three points), a baseline plasma aldosterone concentration of <210.0 pg/ml (two points), a baseline aldosterone/renin ratio of less than 620 (two points), and female sex (one point). Using the validation dataset, we found that a new subtype prediction score of at least 8 had a positive predictive value of 93.5% for bilateral hyperaldosteronism. The new prediction score for bilateral hyperaldosteronism was better than the existing prediction scores in the receiver operating characteristic curve and net reclassification improvement analyses.

Conclusion: The new prediction score has clear advantages over the existing prediction scores in terms of diagnostic accuracy, feasibility, and the potential for generalization in a large population. These data will help healthcare professionals to better select patients who require adrenal venous sampling.
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http://dx.doi.org/10.1097/HJH.0000000000001855DOI Listing
November 2018

High-Resolution Tissue Mass Spectrometry Imaging Reveals a Refined Functional Anatomy of the Human Adult Adrenal Gland.

Endocrinology 2018 03;159(3):1511-1524

Department of Internal Medicine, Division of Endocrinology and Diabetology, University Hospital Würzburg, University of Würzburg, Würzburg, Germany.

In the adrenal gland, neuroendocrine cells that synthesize catecholamines and epithelial cells that produce steroid hormones are united beneath a common organ capsule to function as a single stress-responsive organ. The functional anatomy of the steroid hormone-producing adrenal cortex and the catecholamine-producing medulla is ill defined at the level of small molecules. Here, we report a comprehensive high-resolution mass spectrometry imaging (MSI) map of the normal human adrenal gland. A large variety of biomolecules was accessible by matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI, including nucleoside phosphates indicative of oxidative phosphorylation, sterol and steroid metabolites, intermediates of glycolysis and the tricarboxylic acid cycle, lipids, and fatty acids. Statistical clustering analyses yielded a molecularly defined adrenal anatomy of 10 distinct molecular zones including a highly structured corticomedullary interface. By incorporating pathway information, activities of carbohydrate, amino acid, and lipid metabolism as well as endocrine bioactivity were revealed to be highly spatially organized, which could be visualized as different molecularly defined zones. Together, these findings provide a molecular definition of human adult adrenal gland structure beyond classical histological anatomy.
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http://dx.doi.org/10.1210/en.2018-00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839739PMC
March 2018

Aging and Adrenal Aldosterone Production.

Hypertension 2018 02 11;71(2):218-223. Epub 2017 Dec 11.

From the Department of Molecular and Integrative Physiology (K.N., W.E.R.), and Department of Internal Medicine (W.E.R.), University of Michigan, Ann Arbor; and Center for Adrenal Disorders, Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, MA (A.V.).

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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.10391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839673PMC
February 2018

Subtype prediction of primary aldosteronism by combining aldosterone concentrations in the left adrenal vein and inferior vena cava: a multicenter collaborative study on adrenal venous sampling.

J Hum Hypertens 2017 12 24;32(1):12-19. Epub 2017 Nov 24.

Department of Endocrinology, Metabolism, and Hypertension, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

Subtype diagnosis of primary aldosteronism (PA) by adrenal vein sampling (AVS) is recommended as a mandatory step for indicating adrenal surgery. It is a technically demanding procedure, especially in the right adrenal vein. The aim of the study was to predict the subtype diagnosis in the absence of values from the right AVS. From the databases of nine centers (WAVES-J), 308 patients with PA who underwent successful AVS were studied. Based on the ipsilateral ratio (IR) (aldosterone/cortisol ratio of the left adrenal vein [A/C] / aldosterone/cortisol ratio of the inferior vena cava [A/C]), the patients were divided into two groups: the patients with IR ≥ 1.0 (n = 262) and those with IR < 1.0 (n = 46). In patients with IR > 1.0, the A/C was significantly higher in patients with the left unilateral subtype than in patients with the bilateral subtype. Receiver operating characteristic (ROC) curve analysis revealed that an A/C cutoff >68 showed 70.8% sensitivity and 93.5% specificity for the left unilateral subtype. On the other hand, in patients with IR < 1.0, the A/C was significantly lower in patients with the right unilateral subtype. ROC analysis revealed that an A/C cutoff <9 showed 86.7% sensitivity and 75.0% specificity for the right unilateral subtype. Hence, the combination of the IR and A/C ratio in the left adrenal vein is useful for predicting the subtype. The present results provide important information for patients with PA in whom AVS was unsuccessful in the right adrenal vein.
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http://dx.doi.org/10.1038/s41371-017-0015-0DOI Listing
December 2017

Development of monoclonal antibodies against the human 3β-hydroxysteroid dehydrogenase/isomerase isozymes.

Steroids 2017 11 31;127:56-61. Epub 2017 Aug 31.

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA.

The human 3β-hydroxysteroid dehydrogenase/isomerase (HSD3B) enzymes catalyze the conversion of 3β-hydroxy Δ5-6 steroids into 3-keto Δ4-5 steroids, which is required for the synthesis of the mature steroid hormones secreted by the adrenal and gonads. The human has 2 isozymes, the HSD3B1 that is traditionally located in placenta and extra-adrenal tissues and the HSD3B2 that is expressed in the adrenal and gonads. Mice with both cryptochrome 1 and 2 genes deletion were recently found to have salt-sensitive hypertension and hyperaldosteronism. These deletions were also associated with overexpression of the Hsd3b6 enzyme, the homolog of the human HSD3B1, in the zona glomerulosa which was believed to explain the hyperaldosteronism. A report using antibodies against human HSD3B1 suggested that it was expressed in the zona glomerulosa of normal human adrenals and in patients with idiopathic hyperaldosteronism and the HSD3B2 expressed in both the zona fasciculata and glomerulosa. We have developed specific monoclonal antibodies against the human HSD3B1 and HSD3B2 isozymes and found that the main enzyme expressed in the zona glomerulosa was the HSD3B2. Faint staining of the adrenal was also obtained using the anti-HSD3B1antibody only at high concentrations of antibody. This study fails to confirm that HSD3B1 expression in the human zona glomerulosa and double immunofluorescence clearly shows that the HSD3B2 is expressed in the zona glomerulosa and fasciculata and in the zona glomerulosa HSD3B2 is co-expressed with aldosterone synthase (CYP11B2).
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http://dx.doi.org/10.1016/j.steroids.2017.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628156PMC
November 2017

Discordance between imaging and immunohistochemistry in unilateral primary aldosteronism.

Clin Endocrinol (Oxf) 2017 Dec 4;87(6):665-672. Epub 2017 Sep 4.

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, MI, USA.

Objective: Correct subtyping of primary aldosteronism (PA) is essential for good surgical outcomes. Adrenal vein sampling (AVS) and/or computed tomography (CT) are used for PA subclassification. Clinical and/or biochemical improvement after surgery, however, is not always achieved in patients with presumed unilateral PA. We aimed to identify the pitfalls in PA subclassification leading to surgical treatment failures.

Patients And Design: We retrospectively studied 208 patients who underwent adrenal vein sampling (AVS) for PA subclassification in a tertiary referral centre, between January 2009 and August 2016. Simultaneous bilateral AVS was performed before and after cosyntropin administration. We implemented immunohistochemistry for aldosterone synthase (CYP11B2) and 17α-hydroxylase/17,20 lyase (CYP17A1) in adrenal glands resected from patients without improvement of PA after surgical treatment and from those with limitations in AVS interpretation.

Results: Of 55 patients who underwent adrenalectomy, three (5.5%) had no improvement of PA. All three patients underwent partial adrenalectomy to remove a CT-detected nodule present on the same side with AVS lateralization. Immunohistochemistry revealed a CYP11B2-negative nodule in both cases available. All patients who underwent total adrenalectomy based on AVS lateralization benefitted from surgery, including three patients with unilateral unsuccessful AVS and aldosterone suppression in the catheterized side vs inferior vena cava.

Conclusions: Radiographically identified adrenal nodules are not always a source of PA, even when ipsilateral with AVS lateralization. These data caution against reliance on imaging findings, either alone or in conjunction with AVS, to guide surgery for PA.
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http://dx.doi.org/10.1111/cen.13442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698145PMC
December 2017

Age-Related Autonomous Aldosteronism.

Circulation 2017 Jul 31;136(4):347-355. Epub 2017 May 31.

From Departments of Molecular and Integrative Physiology & Internal Medicine, University of Michigan, Ann Arbor (K.N., I.Z., W.E.R.); Center for Adrenal Disorders, Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, MA (A.V., G.H.W.); and Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor (T.E., W.E.R.).

Background: Both aging and inappropriate secretion of aldosterone increase the risk for developing cardiovascular disease; however, the influence of aging on aldosterone secretion and physiology is not well understood.

Methods: The relationship between age and adrenal aldosterone synthase (CYP11B2) expression was evaluated in 127 normal adrenal glands from deceased kidney donors (age, 9 months to 68 years). Following immunohistochemistry, CYP11B2-expressing area and areas of abnormal foci of CYP11B2-expressing cells, called aldosterone-producing cell clusters, were analyzed. In a separate ancillary clinical study of 677 participants without primary aldosteronism, who were studied on both high and restricted sodium diets (age, 18-71 years), we used multivariable linear regression to assess the independent associations between age and renin-angiotensin-aldosterone system physiology.

Results: In adrenal tissue, the total CYP11B2-expressing area was negatively correlated with age (=-0.431, <0.0001), whereas the total aldosterone-producing cell cluster area was positively correlated with age (=0.390, <0.0001). The integrated ratio of aldosterone-producing cell cluster to CYP11B2-expressing area was most strongly and positively correlated with age (=0.587, <0.0001). When participants in the clinical study were maintained on a high sodium balance, renin activity progressively declined with older age, whereas serum and urinary aldosterone did not significantly decline. Correspondingly, the aldosterone-to-renin ratio was positively and independently associated with older age (adjusted β=+5.54 ng/dL per ng/mL per hour per 10 years, <0.001). In contrast, when participants were assessed under sodium-restricted conditions, physiological stimulation of aldosterone was blunted with older age (β=-4.6 ng/dL per 10 years, <0.0001).

Conclusions: Aging is associated with a pattern of decreased normal zona glomerulosa CYP11B2 expression and increased aldosterone-producing cell cluster expression. This histopathologic finding parallels an age-related autonomous aldosteronism and abnormal aldosterone physiology that provides 1 potential explanation for age-related cardiovascular risk.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.028201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568806PMC
July 2017

Double adrenocortical adenomas harboring independent KCNJ5 and PRKACA somatic mutations.

Eur J Endocrinol 2016 Aug 10;175(2):K1-6. Epub 2016 May 10.

Division of Metabolism, Endocrinology, and Diabetes

Objective: Co-secretion of cortisol and aldosterone can be observed in adrenal adenomas. The aim of this study was to investigate the molecular characteristics of a co-existing aldosterone- and a cortisol-producing adenoma (CPA) in the same patient.

Design And Methods: Two different adenomas within the same adrenal gland from a 49-year-old female patient with primary aldosteronism (PA) and Cushing's syndrome (CS) were studied. Multiple formalin-fixed paraffin-embedded tumor blocks were used for the analysis. Immunohistochemistry (IHC) was performed using a specific antibody against aldosterone synthase (CYP11B2). DNA and RNA were isolated separately from CYP11B2-positive and -negative tumor regions based on CYP11B2 IHC results.

Results: CYP11B2 IHC clearly demonstrated that three pieces from one adenoma were positive for CYP11B2 and the remaining three from the other adenoma were negative for CYP11B2. In quantitative real-time RT-PCR, CYP11B2 mRNA was upregulated in CYP11B2-positive tumor specimens (219-fold vs CYP11B2-negative tumor specimens). Targeted next-generation sequencing (NGS) detected novel KCNJ5 gene mutations (p.T148I/T149S, present in the same reads) and a PRKACA gene hotspot mutation (p.L206R) in the CYP11B2-positive and -negative tumors, respectively. Sanger sequencing of DNA from each tumor specimen (CYP11B2-positive tumor, n=3; CYP11B2-negative tumor, n=3) showed concordant results with targeted NGS.

Conclusion: Our findings illustrate the co-existence of two different adrenocortical adenomas causing the concurrent diagnosis of PA and CS in the same patient. Molecular analysis was able to demonstrate that the two diseases resulted from independent somatic mutations seen in double adrenocortical adenomas.
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http://dx.doi.org/10.1530/EJE-16-0262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030510PMC
August 2016

Adrenal Venous Sampling in Patients With Positive Screening but Negative Confirmatory Testing for Primary Aldosteronism.

Hypertension 2016 May 14;67(5):1014-9. Epub 2016 Mar 14.

From the Department of Endocrinology, Metabolism, and Hypertension, Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto, Japan (H.U., M.N., K.N., M.T.); Department of Diabetes and Endocrinology, Sapporo City General Hospital, Sapporo, Japan (N.W.); Department of Diabetes and Endocrinology, Saiseikai Yokohama City Toubu Hospital, Yokohama City, Japan (T.I.); Department of Cardiology, Akashi Medical Center, Akashi, Japan (K.K.); Department of Cardiology, Sanda City Hospital, Sanda, Japan (Y.M.); Department of Cardiology, Hiroshima General Hospital of West Japan Railway Company, Hiroshima, Japan (Y.F.); Department of Cardiology, Saiseikai Tondabayashi Hospital, Osaka, Japan (T.K.); Department of Internal Medicine, Matsuyama Red Cross Hospital, Matsuyama, Japan (T.F.); Department of Metabolism and Endocrinology, Clinical Research Institute, National Hospital Organization Kyushu Medical Center, Fukuoka, Japan (R.S., A.O.); and Department of Public Health, Kitasato University School of Medicine, Kanagawa, Japan (T.S.).

Adrenal venous sampling is considered to be the most reliable diagnostic procedure to lateralize aldosterone excess in primary aldosteronism (PA). However, normative criteria have not been established partially because of a lack of data in non-PA hypertensive patients. The aim of the study was to investigate aldosterone concentration and its gradient in the adrenal vein of non-PA hypertensive patients. We retrospectively studied the results of cosyntropin-stimulated adrenal venous sampling in 40 hypertensive patients who showed positive screening testing but negative results in 2 confirmatory tests/captopril challenge test and saline infusion test. Plasma aldosterone concentration, aldosterone/cortisol ratio, its higher/lower ratio (lateralization index) in the adrenal vein with cosyntropin stimulation were measured. Median plasma aldosterone concentration in the adrenal vein was 25 819 pg/mL (range, 5154-69 920) in the higher side and 12 953 (range, 1866-36 190) pg/mL in the lower side (P<0.001). There was a significant gradient in aldosterone/cortisol ratio between the higher and the lower sides (27.2 [5.4-66.0] versus 17.3 [4.0-59.0] pg/mL per μg/dL;P<0.001) with lateralization index ranging from 1.01 to 3.87. The aldosterone lateralization gradient was between 1 to 2 in 32 patients and 2 to 4 in 8 patients. None of the patients showed lateralization index ≥4. The present study demonstrated that plasma aldosterone concentration in the adrenal veins showed significant variation and lateralization gradient even in non-PA hypertensive patients. Adrenal venous sampling aldosterone lateralization gradients between 2 and 4 should be interpreted with caution in patients with PA because these gradients can be found even in patients with negative confirmatory testing for PA.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.06607DOI Listing
May 2016

Molecular Heterogeneity in Aldosterone-Producing Adenomas.

J Clin Endocrinol Metab 2016 Mar 14;101(3):999-1007. Epub 2016 Jan 14.

Departments of Molecular and Integrative Physiology and Internal Medicine (K.N., A.X.C., G.D.H., W.E.R.), Pathology (K.O., M.V., T.J.G., S.A.T.), and Urology (S.A.T.), Comprehensive Cancer Center (T.J.G., T.E., S.A.T.), Division of Metabolism, Endocrinology, and Diabetes (T.J.G., T.E., G.D.H.), Endocrine Oncology Program (T.E., G.D.H.), Center for Organogenesis, and Michigan Center for Translational Pathology (S.A.T.), University of Michigan, Ann Arbor, Michigan 48109.

Context: The use of next-generation sequencing has resulted in the identification of recurrent somatic mutations underlying primary aldosteronism (PA). However, significant gaps remain in our understanding of the relationship between tumor aldosterone synthase (CYP11B2) expression and somatic mutation status.

Objective: The objective of the study was to investigate tumor CYP11B2 expression and somatic aldosterone-driver gene mutation heterogeneity.

Methods: Fifty-one adrenals from 51 PA patients were studied. Immunohistochemistry for CYP11B2 was performed. Aldosterone-producing adenomas with intratumor CYP11B2 heterogeneity were analyzed for mutation status using targeted next-generation sequencing. DNA was isolated from CYP11B2-positive, CYP11B2-negative, and adjacent normal areas from formalin-fixed, paraffin-embedded sections.

Results: Of 51 adrenals, seven (14 %) showed distinct heterogeneity in CYP11B2 by immunohistochemistry, including six adenomas with intratumor heterogeneity and one multinodular hyperplastic adrenal with both CYP11B2-positive and -negative nodules. Of the six adrenocortical adenomas with CYP11B2 heterogeneity, three had aldosterone-regulating mutations (CACNA1D p.F747C, KCNJ5 p.L168R, ATP1A1 p.L104R) only in CYP11B2-positive regions, and one had two different mutations localized to two histologically distinct CYP11B2-positive regions (ATP2B3 p.L424_V425del, KCNJ5 p.G151R). Lastly, one adrenal with multiple CYP11B2-expressing nodules showed different mutations in each (CACNA1D p.F747V and ATP1A1 p.L104R), and no mutations were identified in CYP11B2-negative nodule or adjacent normal adrenal.

Conclusions: Adrenal tumors in patients with PA can demonstrate clear heterogeneity in CYP11B2 expression and somatic mutations in driver genes for aldosterone production. These findings suggest that aldosterone-producing adenoma tumorigenesis can occur within preexisting nodules through the acquisition of somatic mutations that drive aldosterone production.
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http://dx.doi.org/10.1210/jc.2015-3239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803171PMC
March 2016

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness.

J Mol Endocrinol 2016 Feb 17;56(2):69-76. Epub 2015 Nov 17.

Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA Departments of Molecular & Integrative Physiology and Internal MedicineUniversity of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USADivision of MetabolismEndocrinology, and Diabetes, University of Michigan, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, USA

The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic-pituitary-adrenal axis.
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http://dx.doi.org/10.1530/JME-15-0230DOI Listing
February 2016

Potassium channels related to primary aldosteronism: Expression similarities and differences between human and rat adrenals.

Mol Cell Endocrinol 2015 Dec 12;417:141-8. Epub 2015 Sep 12.

Department of Molecular and Integrative Physiology, University of Michigan, 1150 W. Medical Center Dr, Ann Arbor, MI 48109, USA; Department of Internal Medicine, University of Michigan, 1150 W. Medical Center Dr, Ann Arbor, MI 48109, USA. Electronic address:

Three potassium channels have been associated with primary aldosteronism (PA) in rodents and humans: KCNK3 (TASK-1), KCNK9 (TASK-3), and KCNJ5 (Kir3.4). Mice with deficiency in Kcnk3 and Kcnk9 have elevated aldosterone production and blood pressure. In humans, adrenal tumors with somatic mutations in KCNJ5 cause PA. However, there are very few reports on the expression patterns of these genes in humans versus rodents. Herein, we compared human and rat mRNA expression (by quantitative real-time polymerase chain reaction (qPCR) and protein levels (by immunohistochemistry) across three tissues (adrenal, brain, heart) and two laser-captured adrenal zones (zona glomerulosa, zona fasciculata). Our findings show that expression patterns of KCNK3, KCNK9, and KCNJ5 are inconsistent between rats and humans across both tissues and adrenal zones. Thus, species variation in the expression of PA-related potassium channels indicates an evolutionary divergence in their role in regulating adrenal aldosterone production.
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http://dx.doi.org/10.1016/j.mce.2015.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646165PMC
December 2015

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

Proc Natl Acad Sci U S A 2015 Aug 3;112(33):E4591-9. Epub 2015 Aug 3.

Departments of Molecular and Integrative Physiology and Internal Medicine, University of Michigan, Ann Arbor, MI 48109;

Primary aldosteronism (PA) represents the most common cause of secondary hypertension, but little is known regarding its adrenal cellular origins. Recently, aldosterone-producing cell clusters (APCCs) with high expression of aldosterone synthase (CYP11B2) were found in both normal and PA adrenal tissue. PA-causing aldosterone-producing adenomas (APAs) harbor mutations in genes encoding ion channels/pumps that alter intracellular calcium homeostasis and cause renin-independent aldosterone production through increased CYP11B2 expression. Herein, we hypothesized that APCCs have APA-related aldosterone-stimulating somatic gene mutations. APCCs were studied in 42 normal adrenals from kidney donors. To clarify APCC molecular characteristics, we used microarrays to compare the APCC transcriptome with conventional adrenocortical zones [zona glomerulosa (ZG), zona fasciculata, and zona reticularis]. The APCC transcriptome was most similar to ZG but with an enhanced capacity to produce aldosterone. To determine if APCCs harbored APA-related mutations, we performed targeted next generation sequencing of DNA from 23 APCCs and adjacent normal adrenal tissue isolated from both formalin-fixed, paraffin-embedded, and frozen tissues. Known aldosterone driver mutations were identified in 8 of 23 (35%) APCCs, including mutations in calcium channel, voltage-dependent, L-type, α1D-subunit (CACNA1D; 6 of 23 APCCs) and ATPase, Na(+)/(K+) transporting, α1-polypeptide (ATP1A1; 2 of 23 APCCs), which were not observed in the adjacent normal adrenal tissue. Overall, we show three major findings: (i) APCCs are common in normal adrenals, (ii) APCCs harbor somatic mutations known to cause excess aldosterone production, and (iii) the mutation spectrum of aldosterone-driving mutations is different in APCCs from that seen in APA. These results provide molecular support for APCC as a precursor of PA.
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http://dx.doi.org/10.1073/pnas.1505529112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547250PMC
August 2015
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