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Focal segmental glomerulosclerosis is a rare complication of acromegaly. A 74-year-old man was found to have acromegaly features such as enlargement of the forehead, nose, and hands. Laboratory tests showed a urine protein/creatinine ratio of 3.16 g/gCr and serum creatinine of 1.34 mg/dL. The levels of growth hormone and insulin-like growth factor I were markedly elevated, and the growth hormone level was not suppressed after 75 g oral glucose loading. Magnetic resonance imaging revealed a pituitary tumor with a diameter of 1.2 cm. Renal biopsy confirmed the diagnosis of focal segmental glomerulosclerosis. Transsphenoidal resection of the pituitary tumor led to remission of acromegaly and reduction in proteinuria highlighting the causal link between growth hormone overproduction and proteinuria. Treatment of acromegaly may be effective for acromegaly-associated focal segmental glomerulosclerosis.

The rapid progression of chronic kidney disease and higher incidence of cardiovascular complications are well known in patients with hyperaldosteronism. However, detailed renal histopathologic characteristics of this disease have remained unknown. Therefore, renal biopsy specimens of 19 cases with unilateral hyperaldosteronism were compared with 22 autopsy renal cases of estimated glomerular filtration rate-matched essential hypertension without nephropathy or endocrine disorders to explore the hyperaldosteronism-specific histopathologic renal changes in this study. Global and segmental glomerulosclerosis, interstitial fibrosis, infiltration of inflammatory cells, arteriosclerosis, hyalinization of arterioles and immunoreactivity of mineralocorticoid receptor, 11β-hydroxysteroid dehydrogenase type 1 and 2, and renin were all quantitatively evaluated. The ultrastructural analysis was added in 3 hyperaldosteronism cases. Both mineralocorticoid receptor ( P P P P P =0.08). Glomerular size was significantly larger in hyperaldosteronism ( P P =0.08), and intima-to-media ratio was significantly lower ( P =0.02) in hyperaldosteronism. Arteriolar hyalinization was significantly more pronounced ( P P

Primary aldosteronism (PA) is the most common cause of secondary hypertension, and is associated with an increased incidence of cardiovascular events. PA itself is clinically classified into the following two types: unilateral PA, mostly composed of aldosterone-producing adenoma (APA); and bilateral hyperaldosteronism, consisting of multiple aldosterone-producing micronodules (APMs) and aldosterone-producing diffuse hyperplasia. Histopathologically, those disorders above are all composed of compact and clear cells. The cellular morphology in the above-mentioned aldosterone-producing disorders has been recently reported to be closely correlated with patterns of somatic mutations of ion channels including KCNJ5, CACNA1D, ATP1A1, ATP2B3, and others. In addition, in non-pathological adrenal glands, APMs are frequently detected regardless of the status of the renin-angiotensin-aldosterone system (RAAS). Aldosterone-producing nodules have been also proposed as non-neoplastic nodules that can be identified by hematoxylin and eosin staining. These non-neoplastic CYP11B2-positive nodules could represent possible precursors of APAs possibly due to the presence of somatic mutations. On the other hand, aging itself also plays a pivotal role in the development of aldosterone-producing lesions. For instance, the number of APMs was also reported to increase with aging. Therefore, recent studies indicated the novel classification of PA into normotensive PA (RAAS-independent APM) and clinically overt PA.

Although adrenal resection is a major option to control hypercortisolemia in patients with bilateral macronodular adrenal hyperplasia, a predictive method for postoperative cortisol production has not been established. A 53-year-old man with ulcerative colitis was referred to our hospital for bilateral multiple adrenal nodules and hypertension. Physical and endocrinological examination revealed inappropriate cortisol production and suppressed secretion of adrenocorticotropic hormone with no typical signs of Cushing's syndrome. Imaging analysis revealed bilateral adrenal nodular enlargement, the nodules of which had the radiological features of adrenocortical adenomas without inter-nodular heterogeneity. In addition, computed tomography volumetry demonstrated that the left adrenal gland (70 mL) accounts for three quarters of the total adrenal volume (93 mL). The patient was diagnosed as subclinical Cushing's syndrome due to bilateral macronodular adrenal hyperplasia, and subsequently underwent a left laparoscopic adrenalectomy with the estimation of 75% decrease in the cortisol level based on the adrenal volume. The surgical treatment ultimately resulted in control of the cortisol level within the normal range, which was compatible to our preoperative prediction. However, regardless of the sufficient cortisol level, ulcerative colitis was exacerbated after the surgery, which needed a systemic therapy for remission. This case indicates successful surgical control of hypercortisolemia based on computed tomography volumetry in bilateral macronodular adrenal hyperplasia, as well as the perioperative exacerbation risk for inflammatory diseases in Cushing's syndrome. We report the potential utility of computed tomography volumetry as a quantitative method with retrospective evaluation of our historical cases.

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

Objectives This study compared cardiac function, morphology, and tissue characteristics between two common subtypes of primary aldosteronism (PA) using a 3T MR scanner. Design A retrospective, single-center, observational study. Methods We retrospectively reviewed 143 consecutive patients with PA, who underwent both adrenal venous sampling and cardiac magnetic resonance. We acquired cine, late gadolinium enhancement, and pre- and postcontrast myocardial T1-mapping images. Results PA was diagnosed as unilateral aldosterone-producing adenoma (APA) in 70 patients and bilateral hyperaldosteronism (BHA) in 73. The APA group showed significantly higher plasma aldosterone concentration (PAC) and aldosterone to renin rate (ARR) than the BHA group. After controlling for age, sex, antihypertensive drugs, systolic and diastolic blood pressure, and disease duration, the parameters independently associated with APA were: left ventricular end-diastolic volume index (EDVI: adjusted odds ratio (aOR) = 1.06 (95% CI: 1.030–1.096), P < 0.01), end-systolic volume index (ESVI: 1.06 (1.017–1.113), P < 0.01), stroke index (SI: 1.07 (1.020–1.121), P < 0.01), cardiac index (CI: 1.001 (1.000–1.001), P < 0.01), and native T1 (1.01 (1.000–1.019), P = 0.038). Weak positive correlations were found between PAC and EDVI (R = 0.28, P < 0.01), ESVI (0.26, P < 0.01), and SI (0.18, P = 0.03); and between ARR and EDVI (0.25, P < 0.01), ESVI (0.24, P < 0.01), and native T1 (0.17, P = 0.047). Conclusions APA is associated with greater LV volumetric parameters and higher native T1 values, suggesting a higher risk of volume overload and myocardial damage.

Recent development of genetic analysis has revealed the molecular characteristics of aldosterone-producing adrenocortical lesions, including gene mutations of KCNJ5, ATP1A1, ATP2B3, CACNA1D, CACNA1H, CTNNB1, and CLCN2. In addition, CYP11B2-based immunohistochemical analysis has made it possible to classify non-neoplastic lesions of primary aldosteronism (PA) into two distinct histological subtypes based on the localization of aldosterone-producing cells: multiple adrenocortical micronodules (MN) and diffuse hyperplasia of zona glomerulosa (DH). Among MNs, CACNA1D somatic mutation was most frequently detected in 60–80% of micronodules. The number and size of those CYP11B2-positive cell clusters/micronodules could therefore contribute to the pathogenesis or development of hyperaldosteronism. In addition, this newly developed histology- and genetics-based classification of non-neoplastic PA lesions have enabled the precise identification of the responsible lesions of aldosterone excess and enormous improvement of postoperative clinical management of patients with adrenocortical disorders.

Hypothalamic obesity is a clinical syndrome characterized by severe and refractory obesity that is caused by hypothalamic function impairment. Recently, bariatric surgery has been attempted for patients with hypothalamic obesity after craniopharyngioma, but experiences have not yet been accumulated in other hypothalamic disorders. Here, we report the case of a 39-year-old male patient with panhypopituitarism who received laparoscopic sleeve gastrectomy (LSG) after intracranial germinoma treatment. The patient was diagnosed with intracranial germinoma at age 15 and achieved complete remission after radiotherapy (total 50 Gy). He was obese during diagnosis [body mass index (BMI), 29.2 kg/m2], and his obesity gradually worsened after the intracranial germinoma treatment, and LSG was considered when his BMI was 48.6 kg/m2. After 1 month of hospitalized diet-exercise program, LSG was performed. After LSG, his BMI gradually decreased and reached 38.8 kg/m2 on the day of discharge (6 weeks after the surgery). Five months after LSG, his insulin resistance improved, but insulin hypersecretion remained. Fifteen months after the surgery, his BMI was 31.2 kg/m2, with marked decrease in visceral and subcutaneous fat areas (from 393.8 cm2 and 168.2 cm2 before the surgery to 111.5 cm2 and 56.3 cm2, respectively.). To our knowledge, this is the first case of LSG for hypothalamic obesity after intracranial germinoma treatment. Although the pathophysiology of hypothalamic obesity is different from that of primary obesity, LSG could be a successful therapeutic choice for patients with hypothalamic obesity after the intracranial germinoma treatment.

Aldosterone-producing adenoma (APA) is histologically composed of clear and compact tumor cells. KCNJ5- mutated APAs were reported to be associated with higher plasma aldosterone concentration and more abundant clear tumor cells containing lipid droplets than non-KCNJ5- mutated APAs. However, the association among cholesterol uptake and/or synthesis, cellular morphology and genotypes has remained unknown. Therefore, in order to explore these differences, 52 APA cases (KCNJ5 mt: n = 33, non-KCNJ5 mt: n = 19; ATP1A1: n = 3, ATP2B3: n = 3, CACNA1D: n = 5, CTNNB1: n = 1, tumors without any mutation above: n = 7), zona glomerulosa (ZG) tissue adjacent to APA and 10 non-pathological adrenal glands (NAs) were examined for quantitative histopathological analysis of tumor morphology and immunohistochemical analysis of cholesterol receptors (SR-B1, LDL-R), cholesterol metabolic enzymes (ACAT1, ACAT2, HSL, DHCR24, StAR), and the enzymes required for steroid synthesis (CYP11A1, CYP17A, 3βHSD, CYP11B1, CYP11B2). Gas chromatography-mass spectrometry (GC-MS) analysis was further performed to profile cholesterol precursors and metabolites in 21 APA cases (KCNJ5 mt: n = 16, non-KCNJ5 mt: n = 5) and 14 adrenal cortex of adjacent adrenal tissues. Results demonstrated that both SR-B1 and DHCR24 were significantly lower in the ZG than in fasciculata or reticularis of NAs but LDL-R was not significantly different among them in immunohistochemical analysis. SR-B1 and DHCR24 were both significantly higher in APAs than in ZG tissue adjacent to APA. In GC-MS analysis, most cholesterol precursors and metabolites, except for lanosterol, and their metabolic ratios (= concentration of cholesterol/ precursor) were higher in APAs than in the adjacent adrenal cortex tissue. LDL-R, ACAT1/2, HSL, DHCR24 were all significantly lower in clear than in compact tumor cells of APA. LDL-R was significantly lower and cholesterol/lanosterol ratio was significantly higher in KCNJ5- mutated than non-KCNJ5- mutated APAs. We demonstrated SR-B1 mediated selective uptake of cholesterol ester and de novo cholesterol synthesis were both enhanced in APAs. In addition, cholesterol uptake and metabolism were different between clear and compact tumor cells. KCNJ5- mutated APAs were predominantly composed of clear tumor cells containing abundant cholesteryl ester but less activated LDL-R mediated uptake and increased de novo synthesis. Those findings above indicated their more pronounced functional deviation from the normal ZG cells in terms of their steroidogenic and intracellular cholesterol metabolism.

In situ cortisol excess was previously reported to promote cellular senescence, a cell response to stress, in cortisol-producing adenomas (CPA). The aim of this study was to explore senescence pathways in aldosterone-producing cells and related disorders, and the influence of aldosterone overproduction on in situ senescence. We analyzed 30 surgical cases of aldosterone-producing adenoma (APA), 10 idiopathic hyperaldosteronism (IHA) and 19 normal adrenals (NA). CYP11B2 and senescence markers p16 and p21 were immunolocalized in all those cases above and results were correlated with histological/endocrinological findings. In the three cohorts examined, the zona glomerulosa (ZG) was significantly more senescent than other corticosteroid-producing cells. In addition, the ZG of adjacent non-pathological adrenal glands of APA and IHA had significantly higher p16 expression than adjacent non-pathological zona fasciculata (ZF), reticularis (ZR) and ZG of NA. In addition, laboratory findings of primary aldosteronism (PA) were significantly correlated with p21 status in KCNJ5-mutated tumors. Results of our present study firstly demonstrated that non-aldosterone-producing cells in the ZG were the most senescent compared to other cortical zones and aldosterone-producing cells in PA. Therefore, aldosterone production, whether physiological or pathological, could be maintained by suppression of cell senescence in human adrenal cortex.

Objective: Laparoscopic adrenalectomy (LADX) improves hypertension in patients with primary aldosteronism (PA). However, the antihypertensive impact of LADX appears restricted in older patients with PA. In this study, we evaluated the impact of LADX in older patients focusing on the health-related quality of life (HRQoL). Methods: A total of 156 patients with PA who underwent LADX in a single institution were enrolled in this prospective cohort study. The patients were divided into 2 groups, with a boundary of 60 years. The HRQoL was evaluated using the Medical Outcomes Study’s 36-Item Short-Form Health Survey version 2 (SF-36v2) questionnaire before and after LADX. Demographics, clinical features, antihypertensive drugs before and after surgery, and perioperative evaluation were recorded. We compared all scale scores and summed scores between groups. Multivariate regression models were used to determine the associations between various covariables and the HRQoL. Results: In the older PA patients, most subscales of HRQoL at baseline were lower than the national standard values. The antihypertensive drug-free rate by LADX was only 21% in older patients, compared to 58% in younger patients. However, a significant improvement in mental HRQoL was observed after LADX (p = 0.002). The much preoperative antihypertensive drugs, lower preoperative potassium level, and smaller degree of comorbidities were predictors of improved mental HRQoL by LADX on multivariate analyses. Conclusion: The older PA patients showed lower mental HRQOL than the national standard populations. Although antihypertensive effects were limited for these patients, LADX was beneficial as PA treatment via improvement of mental HRQoL.

Voltage-gated L-type calcium channel (CaV) isoforms are well known to play pivotal tissue-specific roles not only in vasoconstriction but also in adrenocortical steroidogenesis including aldosterone biosynthesis. Alpha-1C subunit calcium channel (CC) (CaV1.2) is the specific target of anti-hypertensive CC blockers (CCBs) and its Alpha-1D subunit (CaV1.3) regulates depolarization of cell membrane in aldosterone-producing cells. Direct effects of CCBs on aldosterone biosynthesis were previously postulated but their intra-adrenal distribution and effects on steroid production in primary aldosteronism (PA) patients have remained virtually unknown. In this study, frozen tissue specimens constituting tumor, adjacent adrenal gland and peri-adrenal adipose tissues of nine aldosterone-producing adenoma (APA) cases were examined for visualization of amlodipine and aldosterone themselves using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Liquid chromatography-mass spectrometry (LC-MS) analysis was also performed to quantify amlodipine and 17 adrenal steroids in those cases above and compared the findings with immunohistochemical analysis of steroidogenic enzymes and calcium channels (CaV1.2 and CaV1.3). Effects of amlodipine on mRNA level of aldosterone biosynthetic enzymes were also explored using human adrenocortical carcinoma cell line (H295R). Amlodipine-specific peak (m/z 407.1318.1) was detected only in amlodipine treated cases. Accumulation of amlodipine was marked in adrenal cortex compared to peri-adrenal adipose tissues but not significantly different between APA tumors and adjacent adrenal glands, which was subsequently confirmed by LC-MS quantification. Intra-adrenal distribution of amlodipine was generally consistent with that of CCs. In addition, quantitative steroid profiles using LC-MS and in vitro study demonstrated the lower HSD3B activities in amlodipine treated cases. Immunoreactivity of CaV1.2 and HSD3B2 were also correlated. We report the first demonstration of specific visualization of amlodipine in human adrenal tissues by MALDI-MSI. Marked amlodipine accumulation in the adrenal glands suggested its direct effects on steroidogenesis in PA patients, possibly targeting on CaV1.2 and suppressing HSD3B activity.

The adrenal cortex plays pivotal roles in the maintenance of blood volume, responsiveness to stress and the development of gender characteristics. Gender differences of human adrenal cortex have been recently reported and attracted increasing interests. Gender differences occur from the developing stage of the adrenal, in which female subjects had more activated stem cells with higher renewal capacity resulting in gender-associated divergent structures and functions of cortical zonations of human adrenal. Female subjects generally have the lower blood pressure with the lower renin levels and ACE activities than male subjects. In addition, HPA axis was more activated in female than male, which could possibly contribute to gender differences in coping with various stressful events in our life. Of particular interest, estrogens were reported to suppress RAAS but activate HPA axis, whereas androgens had opposite effects. In addition, adrenocortical disorders in general occur more frequently in female with more pronounced adrenocortical hormonal abnormalities possibly due to their more activated WNT and PRK signaling pathways with more abundant activated adrenocortical stem cells present in female adrenal glands. Therefore, it has become pivotal to clarify the gender influence on both clinical and biological features of adrenocortical disorders. We herein reviewed recent advances in these fields.

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 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.

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

Background Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents anddetecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results even if measured by inaccurateradioimmunoassay, or we must accept high-cost LC/MS, and development of a more rapid and accurate substitute has been long hoped. We havedeveloped a novel, fully-automated, high-quantitative noncompetitive chemiluminescence immunoassay (NC-CLEIA) for detecting aldosterone inserum and plasma, and its performance is evaluated as compared to LC/MS measurement. Methods Recently a unique anti-metatype antibody,which recognizes the immunocomplex of aldosterone and its monoclonal antibody, was established. Using this antibody for sensing permittedthe construction of non-competitive assay for the detection of aldosterone. The reaction protocol of novel aldosterone assay is the following. Inthe 1st reaction, aldosterone in patient’s sample is captured on anti-body coated magnetic particles. Alkaline phosphatase-conjugated antimetatypeantibody is added and incubated as 2nd reaction following a wash. Then substrate solution is added after washing immunocomplex.The resulting reaction signals are proportional to the amount of aldosterone in the sample allowing quantitative determination of in serum orplasma sample. The overall reaction is completed within 30 min. Results Limit of blank (LoB), limit of detection (LoD) and limit of quantitation(LoQ) of our NC-CLEIA aldosterone assay were 0.09 ng/dL, 0.21 ng/dL and 0.57 ng/dL, respectively. NC-CLEIA aldosterone measurements werelinearly well correlated with LC/MS aldosterone measurements (N = 130, y = 1.027x - 0.23 ng/dL, Spearman’s ρ = 0.996, P< 0.0001). Bland-Altmanplot analysis between NC-CLEIA and LC-MS/MS of aldosterone revealed a bias of 0.40 ng/dL with the limits of agreement of -4.60 and 5.41 ng/dLwith 95% confidence interval. Conclusion Our novel NC-CLEIA aldosterone assay was well-correlated and had only a very low bias with LC-MS/MSmethod and also was able to accurately quantify low level samples even in essential hypertension patients. This aldosterone assay can be a most equivalent to LC-MS/MS measurement with a low cost of 12 $ and a short measuring time of 30 minutes.

Introduction Aging is associated with the pathogenesis of many endocrine disorders such as cardiovascular diseases and diabetes. Cell senescence has been reported as one of their mechanisms. In addition, stress responsiveness has been reported to be associated with cell senescence. In addition, some genetic abnormalities such as mitochondrial DNA (mtDNA) damages or telomere shortening, have been detected in some endocrine disorders. Cortisol is a well-known stress-induced hormone and closely associated with aging. We previously reported that cortisol-producing adenoma (CPA) more abundantly expressed cell senescent markers such as p16 and p21 than other hormone-producing adrenocortical adenomas. However, the detailed pathophysiology of cell senescence and its association with histological features in CPAs have remained virtually unknown. Therefore, we analyzed cell senescent markers (telomere length, mtDNA copy number, mtDNA deletion and p16 and p21 immunoreactivity) and analyzed their correlation with clinicopathological factors in CPA patients. Methods & Materials Forty CPA cases was immunohistochemically evaluated. Twenty CPA, ten adjacent ZF and six non-functional adenoma (NFA) were examined for mtDNA abnormalities. mtDNA deletion was evaluated by nested-PCR and mtDNA copy number and telomere length were measured using real-time PCR. Results p21 immunoreactivity was significantly higher in CPA than that of adjacent ZF (P=0.0001) and significantly inversely correlated with tumor size (P=0.0004). Telomere length was much longer in CPA than that in adjacent ZF(P=0.0038), and NFA (P=0.0018). mtDNA copy number of NFA was significantly higher than that of CPA and adjacent ZF (P=0.0038). mtDNA copy number of compact cells was significantly higher than that of clear cells (P=0.0432). mtDNA copy number of compact cells was positively correlated with urinary free cortisol (UFC) (P=0.0428) and plasma cortisol (F) (P=0.0609). mtDNA copy number of clear cells were inversely correlated with F (0.0497). 4977 bp mtDNA deletion was more frequently detected in CPA (54%) and in adjacent ZF (50%) than in NFA (17%). Discussion Results of our present study did reveal that CPA harbored more senescent phenotype as demonstrated by abundant p16 and p21, marked telomere shortening, frequent mtDNA 4977bp deletion and relatively low mtDNA copy number, possibly caused by long-term exposure of excessive cortisol in situ compared to NFA. In addition, clear tumor cells could represent more senescent histological phenotype because of their lower mtDNA copy numbers. This is the first study to demonstrate that compact tumor cells were biologically more active than clear tumor cells and could reflect clinical cortisol biosynthesis, resulting in marked functional intratumoral heterogeneity in CPAs.

Aldosterone-producing adenoma (APA) is well known to harbor marked intratumoral heterogeneity in terms of morphology and CYP11B2 (aldosterone synthase) localization. In histology, APA is generally characterized by two distinct cell subtypes, namely “clear cells” and “compact cells”. Clear tumor cells harbor abundant lipid droplets in their cytoplasms and compact tumor cells generally featuring small round shape have abundant intracytoplasmic organelles including mitochondria. Relatively close correlation between these histological characteristics (morphology and CYP11B2 immunohistochemistry) and genotypes of aldosterone-driver gene somatic mutation has been reported. Among them, KCNJ5-mutated APAs have been reported to harbor clear cell predominant features, while APAs with other rare somatic mutations including ATP1A1, ATP2B3 and CACNA1D harbor heterogenous or relatively compact cell predominant morphometry. However, these previous evaluation were based on eyeball analysis with relatively low reproducibility. Therefore, we developed the more quantitative methods using digital image software in order to analyze the widespread area, which can reflect intratumoral heterogeneity, with high reproducibility to analyze the further detailed correlation between histopathological characteristics and genotype in APA. We explored the utility of immunohistochemistry including CYP11B2 and KCNJ5. We further attempted to propose histopathological scoring system to predict the presence of KCNJ5 somatic mutation in APAs. Results of our present study revealed that KCNJ5 was predominantly immunolocalized in zona glomerulosa among adrenal cortex (vs. ZF, P=0.0002, vs. ZR, P=0.0002), furthermore, predominantly in APCCs than in non-APCCs (P=0.0019). Among the tumors, KCNJ5 immunoreactivity was significantly higher in KCNJ5-wild type APAs than in mutated ones (P=0.0037). KCNJ5-mutated APAs had significantly lower nuclear / cytoplasm ratio and abundant clear cell components than those with wild type, harboring large tumor size. In conclusion, we firstly proposed a novel histopathological predicting scoring system for the presence of KCNJ5 somatic mutation, including the following histopathological findings; N/C ratio, clear cell (%), tumor size, CYP11B2 immunoreactivity and KCNJ5 immunoreactivity. It is true that no single histological factors above could precisely predict the presence of KCNJ5 somatic mutation but this newly developed combined histopathological predicting scoring system could provide relatively high accuracy to predict KCNJ5 somatic mutation in APAs (AUC=96%, sensitivity:100%, specificity:90%, 4 points or more). However, further prospective validation by large number of cases is required for clarification.

Because of its rarity, our understanding of steroidogenesis in estrogen-producing adrenocortical adenoma, including the response to adrenocorticotropic hormone (ACTH) stimulation, remains limited. A 65-year-old man was referred to us because of primary aldosteronism and a right adrenal tumor. Endocrinological evaluations revealed secondary hypogonadism due to hyperestrogenemia. Adrenal venous sampling (AVS) and subsequent liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated bilateral hyperaldosteronism and a right estrogen-producing adrenocortical tumor. He subsequently underwent right unilateral adrenalectomy, which resulted in clinical remission of hypogonadism. Subsequent histopathological analysis identified a right estrogen-producing adrenocortical adenoma and multiple, concomitant adrenocortical micronodules. Sequential evaluation of steroid profiles using LC-MS/MS revealed unique hormone production, including adrenal androgens, and less responsiveness to ACTH in the right estrogen-producing adrenocortical adenoma as compared to the nonneoplastic adrenal cortex. This case study revealed unique profiles of steroid production in estrogen-producing adrenocortical adenoma associated with concomitant primary aldosteronism. Sequential steroid profiling analysis using LC-MS/MS in combination with AVS can contribute to the diagnosis of various adrenal disorders.

In chromaffin cells, tyrosine hydroxylase (TH), aromatic l-amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are mainly involved in catecholamine synthesis. In this study, we evaluated the association between the status of catecholamine-synthesizing enzymes and histopathological features of pheochromocytoma and extraadrenal paraganglioma with special emphasis upon their postoperative clinical behavior. Immunohistochemical evaluation of TH, DBH, AADC, PNMT, Ki 67, and S-100 was performed in 29 pheochromocytoma and 10 extraadrenal paraganglioma and one lymph node harboring metastatic pheochromocytoma. Among these cases, metastasis was subsequently developed in three cases. Urinary normetanephrine (U-NM) levels were significantly higher in clinical metastatic cases than non-metastatic ones. Ki 67 labeling index was significantly higher in both clinical metastatic cases and the Adrenal Gland Scaled Score (PASS) score of ≧ 4 cases than PASS

Objectives To compare image quality and radiation dose of right adrenal vein (RAV) imaging computed tomography (CT) among conventional, low kV, and low kV with reduced contrast medium protocols. Methods One-hundred-and-twenty patients undergoing adrenal CT were randomly assigned to one of three protocols: contrast dose of 600 mgI/kg at 120-kV tube voltage setting (600-120 group), 600 mgI/kg at 80 kV (600-80 group), and 360 mgI/kg at 80 kV (360-80 group). Iterative reconstruction was used for 80-kV groups. Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the RAV and size-specific dose estimates (SSDE) were measured. Three radiologists evaluated 4-point visualisation scores of RAV by consensus reading. Results The RAV detectability was 95%, 97.2%, and 97.3% for 600-120, 600-80, and 360-80 groups, respectively (p = 1.000). Visualisation scores were not significantly different among the groups (p = 0.152). There were no significant differences in CNR or SNR between the 600-120 and 360-80 groups. SSDE of the 360-80 group was significantly lower than that of the 600-120 group (5.86 mGy ± 1.44 vs. 7.27 mGy ± 1.81, p Conclusions 80-kV scans with 360 mgI/kg contrast media showed comparable detectability of RAV to conventional scans, while reducing 19% of SSDE.

Introduction: Primary aldosteronism (PA) is the most common cause of secondary hypertension. More than 70% of APAs have been reported to have KCNJ5 somatic mutation in Asian countiries. Patients with KCNJ5 mutated APAs generally harbor high plasma aldosterone concentration (PAC), and are mainly composed of clear tumor cells containing abundant lipid droplets. However, an association among intracellular cholesterol metabolism, morphological features and genotypes in tumor cells has remained virtually unknown.[Materials and methods]Three cholesterol receptors (SR-B1, LDL-R, VLDL-R) were immunolocakized in 25 APA cases (KCNJ5 mt: n= 15, WT: n=10) who underwent adrenalectomy at Tohoku University Hospital. Image analysis software (HALO, India Labs) was used to quantitatively analyze the intracellular localization and immunoreactivity and expored their correlation with genotype and clinical factors.[Results]LDL-R immunoreactivity was significantly lower in KCNJ5 mt group than WT group (P = 0.0369). In KCNJ5 mt group, a significant correlation was detected between LDL-R immunoreactivity and CYP11B2 (Aldosterone synthase), (P = 0.0271, ρ = 0.5684) but not in WT group. In addition, LDL-R immunoreactivity was significantly inversely correlated with tumor size (P = 0.0142, ρ = -0.6176) and PAC (P =

Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assays has been long expected. We have developed chemiluminescent enzyme immunoassays enabling the simultaneous measurement of both aldosterone and renin concentrations in 10 minutes by a fully automated assay using antibody-immobilized magnetic particles with quick aggregation and dispersion. We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism from 97 patients with essential hypertension. Results of this novel assay significantly correlated with the results of radioimmunoassay (aldosterone, active renin concentration, and renin activity) and liquid chromatography–tandem mass spectrometry (aldosterone). The analytic sensitivity of this particularly novel active renin assay was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ratio of aldosterone-to-renin concentrations of 6.0 (ng/dL per pg/mL) provided 92.0% sensitivity and 76.3% specificity as a cutoff for differentiating primary aldosteronism from essential hypertension. This novel measurement is expected to be a clinically reliable alternative for conventional radioimmunoassay and to provide better throughput and cost effectiveness in diagnosis of hyperaldosteronism from larger numbers of hypertensive patients in clinical settings.

[Introduction] Primary hyperparathyroidism (PHPT) occasionally presents a hyperparathyroid crisis which provides a pivotal cue for its diagnosis. However, the perioperative risk for exacerbation of hypercalcemia has been unknown in PHPT. We herein report 2 cases of PHPT which presented a hyperparathyroid crisis during the perioperative period for other complications. [Case presentaion] Case 1; A 81-year-old man was referred to our center due to urinary stone and hypercalcemia. Baseline laboratory data were as follows; corrected serum Ca 11.2 mg/dl; serum P 2.0 mg/dl; whole PTH 89.6 pg/ml, eGFR 49 ml/min/1.73m2. CT revealed a neck tumor of 10 mm with 99mTc-MIBI uptake, which was considered as a cause of PHPT. Simultaneously, CT also pointed out thoracic aortic aneurysm of 70 mm. Therefore, he underwent endovascular stent graft therapy prior to tumorectomy of PHPT. The procedure of stent graft was successfully performed but also caused subarachnoid hemorrhage and acute kidney injury (AKI). Corrected Ca level was subsequently elevated to 12.4 mg/dl, implying an exacerbation of PHPT. He received 60 mg denosumab administration in order to control Ca level complicated with AKI, resulted in improvement of hypercalcemia. However, PTH level was not significantly changed (57.4 pg/ml) during the period, indicated that lower urinary excretion of calcium due to AKI was the main cause of Ca level elevation. Case 2; A 57-year-old woman was admitted to our center due to incidental hypercalcemia. Baseline laboratory data were as follows; corrected serum Ca 12.5 mg/dl; serum P 2.1 mg/dl; whole PTH 140 pg/ml, eGFR 74 ml/min/1.73m2. CT revealed a neck tumor of 17 mm with 99mTc-MIBI uptake, considered as a cause of PHPT. Unexpectedly, MR imaging revealed meningioma of 48 mm on the Turkish saddle. She underwent embolization and removal of meningioma prior to tumorectomy of PHPT. After surgery, she had a persistent disturbance of consciousness because of hypernatremia (over 160 mM) and hypercalcemia (17.2 mg/dl) complicated with AKI. As a hyperparathyroid crisis, we administered 60 mg denosumab and continued fluid replacement therapy, leading to gradual improvement of electrolyte imbalances. PTH level was significantly elevated to 443 pg/ml compatible with a hyperparathyroid crisis. Her serum Ca level was kept lower than 12 mg/dl by denosumab for more than 3 months. [Conclusion] Our cases suggested that serum Ca level in PHPT could be influenced by perioperative conditions, although the trigger for exacerbation of PHPT remains unclear. Management of PHPT should be considered before surgery for other complications. Denosumab is useful for management of PHPT regardless of renal function.

Primary aldosteronism (PA) is a major cause of secondary hypertension, accounting for 5 to 10% of all of the patients with hypertension. PA is also well known to harbor relatively higher incidence of cardiovascular complications than essential hypertension (EH) despite similar blood pressure levels. Among these complications, renal injuries are considered clinically important because up to 15% of APA (Aldosterone-producing adenoma) patients had already been chronic kidney disease at the time of its initial diagnosis (Iwakura et al., JCEM, 2014). In addition, PA patients are relatively younger than EH, which makes early clinical intervention pivotal. Many pathways of aldosterone-induced renal injuries have been proposed but the actual mechanism of aldosterone related injuries in human kidney has remained virtually unknown. Therefore, in this study, we performed the detailed histopathological analysis for kidneys and adrenal glands of the same PA patients and compared with those of EH. We analyzed 7 APA cases undergoing renal biopsy or nephrectomy with adrenalectomy. As a control group, 40 age-matched EH cases (31 autopsy, 9 biopsy) were examined. Digital image software (HALO, Indica Labs) was employed to perform quantitative histological analysis. There were no significant differences of eGFR between EH and PA before adrenalectomy (p=0.8111), but eGFR become lower in post-operative PA than in EH (p=0.0511). The prevalence of global glomerular sclerosis (GGS) and focal glomerular sclerosis (FGS) was significantly higher in PA than in EH (p=0.0031, 0.0035). Mesenchymal fibrosis was also more marked in PA than in EH (p=0.0091). Among the increased GGS groups (over 10%), the ratio of GGS/mesenchymal fibrosis tended to be lower in PA than in EH(p=0.0653). CYP11B2 positive area in adrenal cortex was more extended in autopsy EH than in adjacent cortex of PA (p=0.0209), and a significant correlation was also detected between CYP11B2 and renin immunoreactivity in autopsy EH adrenals (p=0.0067). In PA, CYP11B2 immunoreactivity in adenomas tended to be correlated with glomerular swelling (p=0.0539), probably reflecting the status of glomerular hyperfiltration. 11βHSD2 immunoreactivity of cortical tubules was significantly higher in PA than in EH (p=0.0001) and significantly correlated with the degree of renal arteriole hyalinization in PA (p

Recent advances of molecular analysis and the development of specific monoclonal antibody against aldosterone synthase (CYP11B2) have indeed established the histological subtypes based on the localization of aldosterone overexcess and revealed the association between genotype and clinicopathological phenotype. In particular, several kinds of aldosterone-driver gene somatic mutation have been reported, including KCNJ5, ATP1A1, ATP2B3, CACNA1D and CTNNB1. Among them, KCNJ5 somatic mutation was most frequently detected in aldosterone-producing adenoma although the prevalence of PA genotypes was markedly different among ethnic groups. The precise prevalence of genotype and histological subtypes in Japanese PA patients in large cohort has virtually remained unknown. Therefore, in this study, consecutive 125 unilateral PA cases entirely composed of Japanese patients who underwent surgery from 2012 to 2017 were histologically and genetically evaluated in order to clarify the precise prevalence of genotypes and histological subtypes among Japanese PA patients. 125 cases were precisely classified as follows based on detailed histopathological examination; single APA (84.0%), multiple (more than two) APAs (1.6%), APA + NFA (non-functional adenoma) (5.6%), MN (multiple adrenocortical micronodules) (5.6%), MN+NFA (2.4%) and DH (diffuse hyperplasia of the zona glomerulosa) (0.8%). Adrenal glands harboring single APA were further examined for genotyping. Among them, 67% harbored KCNJ5 mutated (G151R: 39.6%, L168R: 26.4%, T158A: 1.1%) and 33% harbored wild type. 5.4% of single APA clinically combined with Subclinical Cushing's syndrome, all harbored KCNJ5 mutated APAs. The results of our large-scaled analysis did firstly reveal the precise prevalence of histological subtypes and genotypes among Japanese PA patients who underwent surgery. Approximately 10% of unilateral resected PA cases histologically harbored non-neoplastic subtypes such as MN and DH. In addition to those non-neoplatic subtypes, 15% of unilateral resected PA cases other than single APA definitely required CYP11B2 immunohistochemistry in order to precisely detect the responsible lesions for aldosterone overexcess, distinguishing from the non-functional adrenocortical lesions or/and detecting more than two responsible lesions.

[Introduction] Control of uric acid level (UA) plays an important role in the protection of organs in hypertensive (HTN) patients. Newly usable xanthine oxidase inhibitors, febuxostat (FBX) and topiroxostat (TPX) are expected to lead to more reduction of organ damages. However, the effects of them in a clinical situation remain unclear. We hypothesized these drugs have different effects based on the difference in actionmechanism. [Objectives] To reveal the effects of FBX and TPX on renal functions and compare them between the drugs. [Methods] We retrospectively collected HTN patients with hyperuricemia (HU) who newly received a prescription of FBX or TPX and continued 24 weeks. Those who had other uric acid-lowering drugs, diabetes mellitus or severe proteinuria were excluded. 252 Participants were divided into FBX (128 patients) and TPX (124 patients) groups matched for age and sex. The changes of uric acid (UA), blood pressure, eGFR (defined by Japanese Society of Nephrology) and urinary albumin-to-creatinine ratio (UACR) were evaluated between the baseline and the 24-week-treatment by FBX and TPX. [Results] The almost baseline characteristics were not significantly different between both groups except the usage of renin-angiotensin inhibitors. Administration of FBX or TPX lowered UA significantly (p

Primary aldosteronism (PA) is the main cause of secondary hypertension, accounting for approximately 5–10% of all hypertension. Amlodipine, a third-generation calcium channel blocker, is one of the most frequently administered pharmaceuticals medications of hypertension, binds specifically to Cav1.2, a calcium channel primarily localized in the cardiovascular system, and exerts antihypertensive effects through inhibiting calcium influx into the vascular smooth muscle cells. In addition, calcium influx also plays important roles in aldosterone production and amlodipine was also reported to influence in vitro functions of Cav1.3, a calcium channel involved in aldosterone secretion. Ca channel blockers were also reported to reduce plasma aldosterone concentration by some clinical studies although with mild degrees. However, in vivo effects of amlodipine to aldosterone secretion has remained virtually unknown. A novel technique “Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI)” has been recently developed, which did make it possible to visualize non-labeled small molecules on tissue sections. Therefore, in this study, we firstly applied MALDI-MSI to visualize amlodipine on human adrenal glands including aldosterone producing adenoma (APA). We performed selective imaging of amlodipine using MALDI-MSI on the resected adrenal tissues from APA patients. Frozen sections containing whole representative tumor area were coated with a matrix called CHCA (α-Cyano-4-hydroxycinnamic acid) by deposition as a pretreatment. We subsequently analyzed and detected a precursor ion with MS at m/z 407.1 and then an amlodipine-specific ion with MS/MS at m/z 318.1. We also examined the concordance of amlodipine distribution obtained by this method with immunohistochemistry. Human resected adrenal tissues obtained from the patients APAs treated with and without amlodipine before adrenalectomy were examined. Periadrenal adipose tissues were also analyzed as a control tissue of non-aldosterone-producing tissues. Amlodipine was specifically detected and visualized only in the administered cases. Amlodipine was more abundantly detected in adrenal tissues than periadrenal adipose tissues. On the other hand, significant different was not detected between tumors and adjacent adrenal glands by semi-quantification using MALDI-MSI. In this study, we firstly visualized amlodipine directly in human tissue sections using MALDI-MSI. Increased accumulation of amlodipine in APAs treated with amlodipine did indicate direct effects of amlodipine on aldosterone production but further investigations are required for clarification between neoplastic and non-neoplastic aldosterone producing cells.

Adrenal Cushing syndrome (CS) is caused by the overproduction of cortisol in adrenocortical tumors including adrenal cortisol-producing adenoma (CPA). In CS, steroidogenic enzymes such as 17α-hydroxylase/17, 20-lase (CYP17A1), 3β-hydroxysteroid dehydrogenase (HSD3B), and 11β-hydroxylase (CYP11B1) are abundantly expressed in tumor cells. In addition, several transcriptional factors have been reported to play pivotal roles in the regulation of these enzymes in CPA, but their correlations with those enzymes above have still remained largely unknown. Therefore, in this study, we examined the status of steroidogenic enzymes and their transcriptional factors in 78 and 15 CPA cases by using immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR), respectively. Immunoreactivity of HSD3B2, CYP11B1, CYP17A1, steroidogenic factor-1 (SF1[NR5A1]), GATA6, and nerve growth factor induced-B (NGFIB[NR4A1]) was detected in tumor cells. Results of qPCR analysis revealed that expression of HSD3B2 mRNA was significantly higher than that of HSD3B1, and CYP11B1 mRNA was significantly higher than CYP11B2. In addition, the expression of CYP11B1 mRNA was positively correlated with those of NR5A1, GATA6, and NR4A1. These results all indicated that HSD3B2 but not HSD3B1 was mainly involved in cortisol overproduction in CPA. In addition, NR5A1, GATA6, and NR4A1 were all considered to play important roles in cortisol overproduction through regulating CYP11B1 gene transcription.

The renal resistive index (RI) calculated by Doppler ultrasonography has been reported to be correlated with renal structural changes and outcomes in patients with essential hypertension or renal disease. However, little is known about this index in primary aldosteronism. In this prospective study, we examined the utility of this index to predict blood pressure (BP) outcome after adrenalectomy in patients with primary aldosteronism. We studied 94 patients with histopathologically proven aldosteronoma who underwent surgery. Parameters on renal function, including renal flow indices, were examined and followed up for 12 months postoperatively. The renal RI of the main, hilum, and interlobar arteries was significantly higher in patients with aldosteronoma compared with 100 control patients. BP, estimated glomerular filtration rate, and urinary albumin excretion significantly decreased after adrenalectomy. The resistive indices of all compartment arteries were significantly reduced 1 month after adrenalectomy and remained stable for 12 months. Patients whose interlobar RI was in the highest tertile at baseline had higher systolic BP after adrenalectomy than those whose RI was in the lowest tertile. Logistic regression analysis demonstrated that the RI of the interlobar and hilum arteries could be an independent predictive marker for intractable hypertension (systolic BP ≥140 mm Hg, increased BP, taking ≥3 antihypertensive agents, or increased number of agents) even after adrenalectomy. Therefore, in patients with aldosteronoma, the renal RI indicates partially reversible renal hemodynamics and renal structural damages that would influence postoperative BP outcome.