Epigenetic Regulation of the Renin-Angiotensin-Aldosterone System in Hypertension.

Activation of the renin-angiotensin-aldosterone system (RAAS) plays an important pathophysiological role in hypertension. Increased mRNA levels of the angiotensinogen angiotensin-converting enzyme, angiotensin type 1 receptor gene, Agtr1a, and the aldosterone synthase gene, CYP11B2, have been reported in the heart, blood vessels, and kidneys in salt-sensitive hypertension. However, the mechanism of gene regulation in each component of the RAAS in cardiovascular and renal tissues is unclear. Epigenetic mechanisms, which are important for regulating gene expression, include DNA methylation, histone post-translational modifications, and microRNA (miRNA) regulation. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in visceral adipose tissue and the heart of salt-sensitive hypertensive rats. Several miRNAs influence AGT expression and are associated with cardiovascular diseases. Expression of both ACE and ACE2 genes is regulated by DNA methylation, histone modifications, and miRNAs. Expression of both angiotensinogen and CYP11B2 is reversibly regulated by epigenetic modifications and is related to salt-sensitive hypertension. The mineralocorticoid receptor (MR) exists in cardiovascular and renal tissues, in which many miRNAs influence expression and contribute to the pathogenesis of hypertension. Expression of the 11beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene is also regulated by methylation and miRNAs. Epigenetic regulation of renal and vascular HSD11B2 is an important pathogenetic mechanism for salt-sensitive hypertension.

Adrenocortical Tumor Associated With Pathogenic Variant in KCNJ5 and DNA Methylation of CYP11B2 in Primary Aldosteronism.

Primary aldosteronism (PA) is a subtype of secondary hypertension categorized as either unilateral PA (eg, aldosterone-producing adenoma [APA]) or bilateral PA. CYP11B2, an aldosterone synthase, is highly expressed in APA. Recent studies have revealed a high prevalence of pathogenic variants in KCNJ5 and the role of DNA methylation on CYP11B2 in APA. We present a case of unilateral PA with pathogenic variants in KCNJ5 and suppressed CYP11B2 expression. A 55-year-old woman with hypertension was referred to our hospital. A high aldosterone-renin ratio was observed; PA was confirmed using the captopril challenge test and the furosemide upright test. Although computed tomography showed no evident tumors in either adrenal gland, adrenal vein sampling revealed left gland dominance. Postoperatively, the aldosterone-renin ratio decreased and captopril challenge test showed negative findings. Pathogenic variants in the KCNJ5 were detected in the adenoma. Although immunohistochemistry for CYP11B2 was negative in adenoma, an aldosterone-producing cell cluster was confirmed in the adjacent left adrenal gland. Furthermore, DNA methylation analysis of the adenoma indicated hypermethylation in the CYP11B2 promoter region. The pathogenic variant in KCNJ5, specific to APA, induces CYP11B2 overexpression, resulting in excess aldosterone. However, these effects can be suppressed by DNA methylation.

Epigenetic alterations of 11beta-hydroxysteroid dehydrogenase 1 gene in the adipose tissue of patients with primary aldosteronism.

11Beta-hydroxysteroid dehydrogenase 1 (11ß-HSD1) is a key enzyme involved in metabolic syndrome. Transcript-specific epigenetic regulation of the gene encoding 11ß-HSD1 (HSD11B1) has been reported. We examined the mRNA level and methylation status of the HSD11B1 promoter region in the adipose tissue of patients with primary aldosteronism (PA). We compared 10 tissue specimens from patients with PA caused by aldosterone-producing adenoma (APA) with 8 adipose tissue specimens from patients with subclinical Cushing's syndrome (SCS) caused by cortisol-producing adenomas, 4 tissue specimens from patients with Cushing's adenoma (Cu), or 7 tissue specimens from patients with non-functioning adrenal adenoma (NFA). PA, SCS, and Cu were diagnosed according to the guideline of the Japan Endocrine Society. The mRNA level of HSD11B1 was quantified using real-time PCR. Isolated DNA was treated with bisulfite and amplified using primers specific to the human HSD11B1 promoter region. The glycohemoglobin level was significantly higher in patients with APA, SCS, or Cu than in those with NFA (p < 0.05). Blood pressure was significantly higher in patients with APA than in those with SCS, Cu, or NFA (p < 0.01). The HSD11B1 mRNA level was significantly increased in the adipose tissues of APA or SCS patients compared with Cu or NFA patients (p < 0.05). The methylation ratio was significantly lower in SCS patients than in APA, Cu, or NFA patients (p < 0.05). HSD11B1 expression is partly controlled by an epigenetic mechanism in human tissues. The pathophysiological role of epigenetic regulation of HSD11B1 expression in adipose tissue requires further study.

Impact of gut microbiome on the renin-aldosterone system: Shika-machi Super Preventive Health Examination results.

The renin-angiotensin-aldosterone system (RAAS) is a regulatory mechanism of the endocrine system and is associated with various diseases, including hypertension and renal and cardiovascular diseases. The gut microbiota (GM) have been associated with various diseases, mainly in animal models. However, to our knowledge, no studies have examined the relationship between the RAAS and GM in humans. The present study aimed to assess the association between the systemic RAAS and GM genera and their causal relationships. The study participants were 377 members of the general population aged 40 years or older in Shika-machi, Japan. Plasma renin activity (PRA), plasma aldosterone concentration (PAC), aldosterone-renin ratio (ARR), and GM composition were analyzed using the 16S rRNA method. The participants were divided into high and low groups according to the PRA, PAC, and ARR values. U-tests, one-way analysis of covariance, and linear discriminant analysis of effect size were used to identify the important bacterial genera between the two groups, and binary classification modeling using Random Forest was used to calculate the importance of the features. The results showed that Blautia, Bacteroides, Akkermansia, and Bifidobacterium were associated with the RAAS parameters. Causal inference analysis using the linear non-Gaussian acyclic model revealed a causal effect of Blautia on PAC via SBP. These results strengthen the association between the systemic RAAS and GM in humans, and interventions targeting the GM may provide new preventive measures and treatments for hypertension and renal disease.

Molecular and Epigenetic Control of Aldosterone Synthase, CYP11B2 and 11-Hydroxylase, CYP11B1.

Aldosterone and cortisol serve important roles in the pathogenesis of cardiovascular diseases and metabolic disorders. Epigenetics is a mechanism to control enzyme expression by genes without changing the gene sequence. Steroid hormone synthase gene expression is regulated by transcription factors specific to each gene, and methylation has been reported to be involved in steroid hormone production and disease. Angiotensin II or potassium regulates the aldosterone synthase gene, CYP11B2. The adrenocorticotropic hormone controls the 11b-hydroxylase, CYP11B1. DNA methylation negatively controls the CYP11B2 and CYP11B1 expression and dynamically changes the expression responsive to continuous stimulation of the promoter gene. Hypomethylation status of the CYP11B2 promoter region is seen in aldosterone-producing adenomas. Methylation of recognition sites of transcription factors, including cyclic AMP responsive element binding protein 1 or nerve growth factor-induced clone B, diminish their DNA-binding activity. A methyl-CpG-binding protein 2 cooperates directly with the methylated CpG dinucleotides of CYP11B2. A low-salt diet, treatment with angiotensin II, and potassium increase the CYP11B2 mRNA levels and induce DNA hypomethylation in the adrenal gland. A close association between a low DNA methylation ratio and an increased CYP11B1 expression is seen in Cushing's adenoma and aldosterone-producing adenoma with autonomous cortisol secretion. Epigenetic control of CYP11B2 or CYP11B1 plays an important role in autonomic aldosterone or cortisol synthesis.

Impact of gut microbiome on dyslipidemia in japanese adults: Assessment of the Shika-machi super preventive health examination results for causal inference.

Dyslipidemia (DL) is one of the most common lifestyle-related diseases. There are few reports showing the causal relationship between gut microbiota (GM) and DL. In the present study, we used a linear non-Gaussian acyclic model (LiNGAM) to evaluate the causal relationship between GM and DL. A total of 79 men and 82 women aged 40 years or older living in Shika-machi, Ishikawa Prefecture, Japan were included in the analysis, and their clinical information was investigated. DNA extracted from the GM was processed to sequence the 16S rRNA gene using next-generation sequencing. Participants were divided into four groups based on sex and lipid profile information. The results of one-way analysis of covariance, linear discriminant analysis effect size, and least absolute value reduction and selection operator logistic regression model indicated that several bacteria between men and women may be associated with DL. The LiNGAM showed a presumed causal relationship between different bacteria and lipid profiles in men and women. In men, Prevotella 9 and Bacteroides were shown to be potentially associated with changes in low- and high-density lipoprotein cholesterol levels. In women, the LiNGAM results showed two bacteria, Akkermansia and Escherichia/Shigella, had a presumptive causal relationship with lipid profiles. These results may provide a new sex-based strategy to reduce the risk of developing DL and to treat DL through the regulation of the intestinal environment using specific GM.

Explainable Machine Learning for Atrial Fibrillation in the General Population Using a Generalized Additive Model　- A Cross-Sectional Study.

Background: Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased thromboembolic stroke risk and heart failure. Although various prediction models for AF risk have been developed using machine learning, their output cannot be accurately explained to doctors and patients. Therefore, we developed an explainable model with high interpretability and accuracy accounting for the non-linear effects of clinical characteristics on AF incidence. Methods and Results: Of the 489,073 residents who underwent specific health checkups between 2009 and 2018 and were registered in the Kanazawa Medical Association database, data were used for 5,378 subjects with AF and 167,950 subjects with normal electrocardiogram readings. Forty-seven clinical parameters were combined using a generalized additive model algorithm. We validated the model and found that the area under the curve, sensitivity, and specificity were 0.964, 0.879, and 0.920, respectively. The 9 most important variables were the physical examination of arrhythmia, a medical history of coronary artery disease, age, hematocrit, γ-glutamyl transpeptidase, creatinine, hemoglobin, systolic blood pressure, and HbA1c. Further, non-linear relationships of clinical variables to the probability of AF diagnosis were visualized. Conclusions: We established a novel AF risk explanation model with high interpretability and accuracy accounting for non-linear information obtained at general health checkups. This model contributes not only to more accurate AF risk prediction, but also to a greater understanding of the effects of each characteristic.

DNA Methylation of the Angiotensinogen Gene, AGT, and the Aldosterone Synthase Gene, CYP11B2 in Cardiovascular Diseases.

Angiotensinogen (AGT) and aldosterone play key roles in the regulation of blood pressure and are implicated in the pathogenesis of cardiovascular diseases. DNA methylation typically acts to repress gene transcription. The aldosterone synthase gene CYP11B2 is regulated by angiotensin II and potassium. DNA methylation negatively regulates AGT and CYP11B2 expression and dynamically changes in response to continuous promoter stimulation of each gene. High salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT-enhancer-binding-protein (CEBP) sites of the CYP11B2 promoter region, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue and heart. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in salt-sensitive hypertensive rats. Salt-dependent hypertension may be partially affected by increased cardiac AGT expression. CpG dinucleotides in the CYP11B2 promoter are hypomethylated in aldosterone-producing adenomas. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminish their DNA-binding activity. The methylated CpG-binding protein MECP2 interacts directly with the methylated CYP11B2 promoter. Low salt intake and angiotensin II infusion lead to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with the angiotensin II type 1 receptor antagonist decreases CYP11B2 expression and leads to DNA hypermethylation. A close association between low DNA methylation and increased CYP11B2 expression are seen in the hearts of patients with hypertrophic cardiomyopathy. These results indicate that epigenetic regulation of both AGT and CYP11B2 contribute to the pathogenesis of cardiovascular diseases.

Pregnancy-associated diabetes insipidus in Japan-a review based on quoting from the literatures reported during the period from 1982 to 2019.

This Review Article overviews the literature on diabetes insipidus (DI) associated with pregnancy and labor in Japan published from 1982 to 2019. The total number of patients collected was 361, however, only one-third of these cases had detailed pathophysiologic information enabling us to identify the respective etiology and subtype. Pregnancy-associated DI can be divided into 3 etiologies, central (neurogenic) DI, nephrogenic DI, and excess vasopressinase-associated DI. Neurogenic DI has various causes: for example, DI associated with tumoral lesions in the pituitary and neighboring area, DI associated with Sheehan's syndrome and/or pituitary apoplexy, and DI associated with lymphocytic infundibuloneurohypophysitis (LINH, stalkitis). Nephrogenic DI results from defective response of the kidney to normal levels of vasopressin. However, the most interesting causal factor of pregnancy-associated DI is excess vasopressinase, caused either by excess production of vasopressinase by the placenta or defective clearance of vasopressinase by the liver. Hepatic complications resulting in pregnancy-associated DI include acute fatty liver of pregnancy (AFLP) and HELLP syndrome (syndrome of hemolysis, elevated liver enzymes, low platelets), as well as pre-existing or co-incidental hepatic diseases. A possible role of glucose uptake in putative stress-induced DI and the importance of correct diagnosis and treatment of pregnancy-associated DI, including use of 1-deamino 8-D arginine vasopressin, are also discussed.

Effect of potassium on DNA methylation of aldosterone synthase gene.

BACKGROUND: Aldosterone synthase gene, CYP11B2 is regulated by potassium and angiotensin II (Ang II). We have reported that Ang II could change the DNA methylation status around transcription factor-binding sites and a transcription start site (TSS) and activate expression of CYP11B2. Similar to Ang II, small increases in extracellular potassium levels also increase CYP11B2 mRNA levels. METHODS AND RESULTS: Adrenocortical H295R cells were treated with different doses of potassium. Methylation analysis of CYP11B2 promoter region was done by bisulfite sequencing. CYP11B2 mRNA and protein levels, chromatin accessibility, methylation and demethylation activity were estimated. The transcriptional ability of CYP11B2 promoter with or without methylation was assessed. Potassium stimulation caused DNA demethylation around cyclic AMP responsive element binding protein 1 (CREB1) and nuclear receptor subfamily 4 group A (NR4A) family-binding sites and a TSS; demethylation was accompanied by recruitment of CREB1 and NR4A1 and increased chromatin accessibility of the CYP11B2 promoter. DNA methylation activity decreased in the nucleus. DNA demethylation at CpG1 (Ad1), CpG2 (Ad5) and CpG3 were detected within 2 to 4 days after potassium (16 mmol/l) stimulation. The changes reached a maximum level by day 7. DNA at CpG2 (Ad5) and CpG3 was re-methylated to levels that were similar to those of nontreated cells at day 9. Potassium treatment significantly reduced DNA methylation activity at days 7 and 9. DNA demethylation activity was not changed by potassium. CONCLUSION: : Potassium induced reversibly DNA demethylation, which switches the phenotype of CYP11B2 expression from an inactive to an active state.