Nicotine Reduces Reactive Oxygen Species and Enhances Cell Proliferation via the α4 Nicotinic Acetylcholine Receptor Subunit in Human Induced Pluripotent Stem Cells.

The effects of smoking on fetal development and stem cell differentiation are not fully understood. Although nicotinic acetylcholine receptors (nAChRs) are expressed in many organs of the human body, their significance in human induced pluripotent stem cells (hiPSCs) remains unclear. After expression levels of nAChR subunits in hiPSCs were determined, the effects of the nAChR agonist, nicotine, on undifferentiated hiPSCs were evaluated using a Clariom S Array. We also determined the effect of nicotine alone and with a nAChR subunit antagonist on hiPSCs. nAChR α4, α7, and ß4 subunits were strongly expressed in hiPSCs. cDNA microarray, gene ontology, and enrichment analyses showed that exposing hiPSCs to nicotine altered expression of genes associated with immune responses, neurological system, carcinogenesis, cell differentiation, and cell proliferation. Particularly affected was metallothionein, which acts to decrease reactive oxygen species (ROS). The nicotine-induced reduction of ROS in hiPSCs was canceled by an α4 subunit or nonselective nAChR antagonist. HiPSC proliferation was increased by nicotine, and this effect, too, was canceled by an α4 antagonist. In conclusion, nicotine reduces ROS and enhances cell proliferation through the α4 nAChR subunit in hiPSCs. These findings provide new insight into the significance of nAChRs on human stem cells and fertilized human ova.

Associated factors and effects of comorbid atrial fibrillation in hypertensive patients due to primary aldosteronism.

The incidence of atrial fibrillation (AF) and risk of cardiovascular events are reportedly higher in patients with primary aldosteronism (PA) than essential hypertension. However, associated factors of comorbid AF and cardiovascular events in PA patients after PA treatment remain unclear. This nationwide registration study included PA patients ≥20 years old. Incident cardiovascular events were observed with a mean follow-up of approximately 3 years. A total of 3654 patients with PA were included at the time of analysis. Prevalence of AF was 2.4%. PA patients with AF were older, more frequently male and had longer duration of hypertension than those without AF. No significant difference in basal plasma and adrenal venous aldosterone concentration, renin activity, potassium concentration, confirmatory tests of PA, laterality or surgery rate were seen between groups. Logistic regression analysis showed age, male sex, cardiothoracic ratio, past history of coronary artery disease and heart failure were independent factors associated with AF. PA patients with AF showed a higher frequency of cardiovascular events than those without AF (P < 0.001). Multivariate Cox analyses demonstrated AF in addition to older age, duration of hypertension, body mass index and chronic kidney disease as independent prognostic factors for cardiovascular events after PA treatment. Incidence of cardiovascular events were significantly lower in PA patients with AF than AF patients from the Fushimi registry during follow-up after adjusting age, sex and systolic blood pressure. Early diagnosis of PA may prevent AF and other cardiovascular events in PA patients by shortening the duration of hypertension and appropriate PA treatment.

Japan Endocrine Society clinical practice guideline for the diagnosis and management of primary aldosteronism 2021.

Primary aldosteronism (PA) is associated with higher cardiovascular morbidity and mortality rates than essential hypertension. The Japan Endocrine Society (JES) has developed an updated guideline for PA, based on the evidence, especially from Japan. We should preferentially screen hypertensive patients with a high prevalence of PA with aldosterone to renin ratio ≥200 and plasma aldosterone concentrations (PAC) ≥60 pg/mL as a cut-off of positive results. While we should confirm excess aldosterone secretion by one positive confirmatory test, we could bypass patients with typical PA findings. Since PAC became lower due to a change in assay methods from radioimmunoassay to chemiluminescent enzyme immunoassay, borderline ranges were set for screening and confirmatory tests and provisionally designated as positive. We recommend individualized medicine for those in the borderline range for the next step. We recommend evaluating cortisol co-secretion in patients with adrenal macroadenomas. Although we recommend adrenal venous sampling for lateralization before adrenalectomy, we should carefully select patients rather than all patients, and we suggest bypassing in young patients with typical PA findings. A selectivity index ≥5 and a lateralization index >4 after adrenocorticotropic hormone stimulation defines successful catheterization and unilateral subtype diagnosis. We recommend adrenalectomy for unilateral PA and mineralocorticoid receptor antagonists for bilateral PA. Systematic as well as individualized clinical practice is always warranted. This JES guideline 2021 provides updated rational evidence and recommendations for the clinical practice of PA, leading to improved quality of the clinical practice of hypertension.

Adrenal Venous Sampling for Subtype Diagnosis of Primary Hyperaldosteronism.

Adrenal venous sampling (AVS) is the key procedure for lateralization of primary hyperaldosteronism (PA) before surgery. Identification of the adrenal veins using computed tomography (CT) and intraoperative cortisol assay facilitates the success of catheterization. Although administration of adrenocorticotropic hormone (ACTH) has benefits such as improving the success rate, some unilateral cases could be falsely diagnosed as bilateral. Selectivity index of 5 with ACTH stimulation to assess the selectivity of catheterization and lateralization index (LI) >4 with ACTH stimulation for unilateral diagnosis is used in many centers. Co-secretion of cortisol from the tumor potentially affects the lateralization by the LI. Patients aged <35 years with hypokalemia, marked aldosterone excess, and unilateral adrenal nodule on CT have a higher probability of unilateral disease. Patients with normokalemia, mild aldosterone excess, and no adrenal tumor on CT have a higher probability of bilateral disease. Although no methods have 100% specificity for subtype diagnosis that would allow bypassing AVS, prediction of the subtype should be considered when recommending AVS to patients. Methodological standardization and strict indication improve diagnostic quality of AVS. Development of non-invasive imaging and biochemical markers will drive a paradigm shift in the clinical practice of PA.

Should Adrenal Venous Sampling Be Performed in PA Patients Without Apparent Adrenal Tumors?

Introduction: Some aldosterone-producing micro-adenomas cannot be detected through image inspection. Therefore, adrenal venous sampling (AVS) is often performed, even in primary aldosteronism (PA) patients who have no apparent adrenal tumors (ATs) on imaging. In most of these cases, however, the PA is bilateral. Objective: To clarify the clinical need for AVS in PA patients without apparent ATs, taking into consideration the rates of adrenalectomy. Methods: This is a retrospective cross-sectional study assessing 1586 PA patients without apparent ATs in the multicenter Japan PA study (JPAS). We analyzed which parameters could be used to distinguish unilateral PA patients without apparent ATs from bilateral patients. We also analyzed the prevalences of adrenalectomy in unilateral PA patients. Results: The unilateral subtype without an apparent AT was diagnosed in 200 (12.6%) of 1586 PA patients. Being young and female with a short hypertension duration, normokalemia, low creatinine level, low plasma aldosterone concentration, and low aldosterone-to-renin ratio (ARR) was significantly more common in bilateral than unilateral PA patients. If PA patients without apparent ATs were female and normokalemic with a low ARR (<560 pg/ml per ng/ml/h), the rate of unilateral PA was only 5 (1.1%) out of 444. Moreover, 77 (38.5%) of the 200 did not receive adrenalectomy, despite being diagnosed with the unilateral subtype based on AVS. Conclusion: The low prevalence of the unilateral subtype in PA patients without apparent ATs suggests AVS is not indicated for all of these patients. AVS could be skipped in female normokalemic PA patients without apparent ATs if their ARRs are not high. However, AVS should be considered for male hypokalemic PA patients with high ARRs because the rates of the unilateral subtype are high in these patients.

Adrenal Venous Sampling-Guided Adrenalectomy Rates in Primary Aldosteronism: Results of an International Cohort (AVSTAT).

CONTEXT: Adrenal venous sampling (AVS) is the current criterion standard lateralization technique in primary aldosteronism (PA). Japanese registry data found that 30% of patients with unilateral PA did not undergo adrenalectomy, but the reasons for this and whether the same pattern is seen internationally are unknown. OBJECTIVE: To assess the rate of AVS-guided adrenalectomy across an international cohort and identify factors that resulted in adrenalectomy not being performed in otherwise eligible patients. DESIGN, SETTING, AND PARTICIPANTS: Retrospective, multinational, multicenter questionnaire-based survey of management of PA patients from 16 centers between 2006 and 2018. MAIN OUTCOME MEASURES: Rates of AVS implementation, AVS success rate, diagnosis of unilateral PA, adrenalectomy rate, and reasons why adrenalectomy was not undertaken in patients with unilateral PA. RESULTS: Rates of AVS implementation, successful AVS, and unilateral disease were 66.3%, 89.3% and 36.9% respectively in 4818 patients with PA. Unilateral PA and adrenalectomy rate in unilateral PA were lower in Japanese than in European centers (24.0% vs 47.6% and 78.2% vs 91.4% respectively). The clinical reasoning for not performing adrenalectomy in unilateral PA were more likely to be physician-derived in Japan and patient-derived in Europe. Physician-derived factors included non-AVS factors, such as good blood pressure control, normokalemia, and the absence of adrenal lesions on imaging, which were present before AVS. CONCLUSION: Considering the various unfavorable aspects of AVS, stricter implementation and consideration of surgical candidacy prior to AVS will increase its diagnostic efficiency and utility.

Nadir Aldosterone Levels After Confirmatory Tests Are Correlated With Left Ventricular Hypertrophy in Primary Aldosteronism.

Left ventricular hypertrophy (LVH) is often seen in patients with primary aldosteronism (PA), and the prevalence of LVH is reportedly higher among patients with PA than patients with essential hypertension. However, the correlation between aldosterone levels and LVH is undefined, and how aldosterone affects LVH in patients with PA remains unclear. We, therefore, retrospectively assessed a large PA database established by the multicenter JPAS (Japan Primary Aldosteronism Study) to reveal the factors associated with LVH in patients with PA without suspected autonomous cortisol secretion. In the 1186 patients with PA studied, the basal plasma aldosterone concentration, plasma renin activity, and the aldosterone-to-renin ratio did not significantly correlate with left ventricular LV mass index (LVMI) in single or multiple regression analyses. However, the plasma aldosterone concentration after the captopril challenge test or saline-infusion test, which are associated with autonomous aldosterone secretion, correlated significantly with LVMI, even after adjusting for patients' backgrounds, including age and blood pressure. In addition, hypokalemia and the unilateral subtype also correlated with LVMI. Longitudinal subanalysis of medically or surgically treated patients with PA showed significant reductions in LVMI in both the surgery (63.0±18.1 to 55.3±19.5 g/m2.7, P<0.001) and drug treatment (56.8±14.1 to 52.1±13.5 g/m2.7, P<0.001) groups. Our results suggest the autonomous aldosterone secretion level, not the basal aldosterone level itself, is relevant to LVH in patients with PA. In addition, the elevated LVMI seen in patients with PA is at least partially reversible with surgical or medical treatment.

Latent Autonomous Cortisol Secretion From Apparently Nonfunctioning Adrenal Tumor in Nonlateralized Hyperaldosteronism.

CONTEXT: Adrenal tumors (ATs), even those diagnosed as nonfunctioning, may cause metabolic disorders. Some primary aldosteronism (PA) patients with ATs are diagnosed with bilateral PA based on adrenal venous sampling (AVS), and their ATs are apparently nonfunctioning. OBJECTIVE: To clarify the influence of apparently nonfunctioning ATs, we compared hormone levels and clinical complications between bilateral PA cases with and without ATs. DESIGN, SETTING, AND PARTICIPANTS: After retrospectively assessing 2814 patients with PA in the multicenter Japan PA study, bilateral PA cases on AVS were divided into cases with and without ATs by computed tomography findings. Importantly, patients with cortisol levels >1.8 µg/dL after the 1-mg dexamethasone suppression test (DST) were excluded. Clinical characteristics and biochemical data were compared between them. The correlation between AT size and hormone levels was also analyzed. MAIN OUTCOME MEASURES: Analyzed were 196 bilateral PA patients with ATs and 331 those without ATs. Although basal cortisol and aldosterone levels were similar between them, cortisol levels after the 1-mg DST and the prevalences of diabetes mellitus and proteinuria were significantly higher and ACTH levels and plasma renin activity were significantly lower in cases with ATs than in those without. After adjusting for patients' backgrounds, cortisol levels after the 1-mg DST and plasma renin activity remained significantly different between them. Moreover, cortisol levels after the 1-mg DST and ACTH levels correlated with AT size. CONCLUSIONS: Apparently nonfunctioning ATs in bilateral PA cases may cause latent autonomous cortisol secretion, inducing diabetes and proteinuria.

Obesity as a Key Factor Underlying Idiopathic Hyperaldosteronism.

Context: Recently, the relationship between primary aldosteronism (PA) and various metabolic disorders, including obesity, diabetes mellitus, and dyslipidemia, has been discussed. However, in PA, aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA) have different etiologies. Objective: Our objectives were to clarify differences in obesity and metabolic disorders between APA and IHA and to gain insight in the pathogenesis of IHA. Design, Setting, and Participants: This is a retrospective cross-sectional study. We assessed the PA database established by the multicenter Japan Primary Aldosteronism Study. For comparative analysis, data were also collected from 274 patients with essential hypertension (EHT). Main Outcome Measures: We compared prevalences of obesity and metabolic disorders between patients with APA and patients with IHA. Comparisons with sex-, age-, and blood pressure-matched patients with EHT were also performed. Correlations between metabolic parameters and plasma aldosterone concentrations (PACs) in each subtype were analyzed. Results: Analysis of 516 patients with APA and 1015 patients with IHA revealed PACs were significantly higher in patients with APA than patients with IHA. By contrast, after we adjusted for clinical backgrounds, the prevalence of obesity was significantly higher in patients with IHA than in patients with APA or EHT. Although the prevalences of diabetes mellitus and dyslipidemia did not significantly differ between patients with IHA and patients with APA, triglyceride and HbA1c were significantly higher in patients with IHA than in patients with APA. There was no significant correlation between metabolic parameters and PACs in either subtype. Conclusions: Patients with IHA tend to be obese despite lower PACs than in patients with APA. The present results suggest that obesity-related factors contribute to the pathogenesis of IHA.

Evaluation of quantitative parameters for distinguishing pheochromocytoma from other adrenal tumors.

Adrenal tumors are increasingly found incidentally during imaging examinations. It is important to distinguish pheochromocytomas from other adrenal tumors because of the risk of hypertensive crisis. Although catecholamines and their metabolites are generally used to diagnose pheochromocytoma, false-positive test results are common. An effective screening method to distinguish pheochromocytoma from adrenal incidentalomas is needed. We analyzed 297 consecutive patients with adrenal incidentalomas. Our findings included 162 non-functioning tumors, 47 aldosterone-producing adenomas, 26 metastases, 22 cases of subclinical Cushing's syndrome, 21 pheochromocytomas, 12 cases of Cushing's syndrome, and 7 adrenocortical cancers. We checked quantitative parameters such as age, blood, and urine catecholamines and their metabolites, neuron-specific enolase, size and computed tomography (CT) attenuation values. Among catecholamine-related parameters, the sum of urine metanephrine and normetanephrine (urineMNM) levels produced the highest area under the receiver operating characteristic curve regarding discrimination of pheochromocytoma from other lesions. Size and CT attenuation values also differed significantly. However, size was correlated with catecholamine levels. CT attenuation was not correlated with other factors. The optimal thresholds were 19 Hounsfield units (HU) for CT attenuation (sensitivity, 100%; specificity, 60%) and 0.43 mg/24 h for urineMNM (sensitivity, 89%; specificity, 96%). No pheochromocytomas were evident when CT attenuation values were under 19 HU. Even in adrenal tumors with CT attenuation values ≥ 19 HU, when urineMNM was < 0.43 mg/24 h, the frequency of pheochromocytoma was only 4.3%, when urineMNM was ≥ 0.43 mg/24 h, the frequency of pheochromocytoma was 93% and when urineMNM was > 0.77 mg/24 h the frequency of pheochromocytoma was 100%. CT attenuation value and urineMNM represented the most useful combination for diagnosis of pheochromocytoma.

Prevalence of Cardiovascular Disease and Its Risk Factors in Primary Aldosteronism: A Multicenter Study in Japan.

There have been several clinical studies examining the factors associated with cardiovascular disease (CVD) in patients with primary aldosteronism (PA); however, their results have left it unclear whether CVD is affected by the plasma aldosterone concentration or hypokalemia. We assessed the PA database established by the multicenter JPAS (Japan Primary Aldosteronism Study) and compared the prevalence of CVD among patients with PA with that among age-, sex-, and blood pressure-matched essential hypertension patients and participants with hypertension in a general population cohort. We also performed binary logistic regression analysis to determine which parameters significantly increased the odds ratio for CVD. Of the 2582 patients with PA studied, the prevalence of CVD, including stroke (cerebral infarction, cerebral hemorrhage, or subarachnoid hemorrhage), ischemic heart disease (myocardial infarction or angina pectoris), and heart failure, was 9.4% (stroke, 7.4%; ischemic heart disease, 2.1%; and heart failure, 0.6%). The prevalence of CVD, especially stroke, was higher among the patients with PA than those with essential hypertension/hypertension. Hypokalemia (K+ ≤3.5 mEq/L) and the unilateral subtype significantly increased adjusted odds ratios for CVD. Although aldosterone levels were not linearly related to the adjusted odds ratio for CVD, patients with plasma aldosterone concentrations ≥125 pg/mL had significantly higher adjusted odds ratios for CVD than those with plasma aldosterone concentrations <125 pg/mL. Thus, patients with PA seem to be at a higher risk of developing CVD than patients with essential hypertension. Moreover, patients with PA presenting with hypokalemia, the unilateral subtype, or plasma aldosterone concentration ≥125 pg/mL are at a greater risk of CVD and have a greater need for PA-specific treatments than others.

Significance of dopamine D1 receptor signalling for steroidogenic differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are expected to be both a revolutionary cell source for regenerative medicine and a powerful tool to investigate the molecular mechanisms underlying human cell development in vitro. In the present study, we tried to elucidate the steroidogenic differentiation processes using hiPSC-derived intermediate mesoderm (IM) that is known to be the origin of the human adrenal cortex and gonads. We first performed chemical screening to identify small molecules that induce steroidogenic differentiation of IM cells expressing Odd-skipped related 1 (OSR1), an early IM marker. We identified cabergoline as an inducer of 3ß-hydroxysteroid dehydrogenase, an essential enzyme for adrenogonadal steroidogenesis. Although cabergoline is a potent dopamine D2 receptor agonist, additional experiments showed that cabergoline exerted effects as a low-affinity agonist of D1 receptors by increasing intracellular cyclic AMP. Further analysis of OSR1+ cells transfected with steroidogenic factor-1/adrenal 4 binding protein revealed that D1 receptor agonist upregulated expression of various steroidogenic enzymes and increased secretion of steroid hormones synergistically with adrenocorticotropic hormone. These results suggest the importance of dopamine D1 receptor signalling in steroidogenic differentiation, which contributes to effective induction of steroidogenic cells from hiPSCs.

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells.

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.