Aldosterone Biosynthesis Research Articles

The differences in brain stem transcriptional profiling in hypertensive ISIAH and normotensive WAG rats

BackgroundThe development of essential hypertension is associated with a wide range of mechanisms. The brain stem neurons are essential for the homeostatic regulation of arterial pressure as they control baroreflex and sympathetic nerve activity. The ISIAH (Inherited Stress Induced Arterial Hypertension) rats reproduce the human stress-sensitive hypertensive disease with predominant activation of the neuroendocrine hypothalamic-pituitary-adrenal and sympathetic adrenal axes. RNA-Seq analysis of the brain stems from the hypertensive ISIAH and normotensive control WAG (Wistar Albino Glaxo) rats was performed to identify the differentially expressed genes (DEGs) and the main central mechanisms (biological processes and metabolic pathways) contributing to the hypertensive state in the ISIAH rats.ResultsThe study revealed 224 DEGs. Their annotation in databases showed that 22 of them were associated with hypertension and blood pressure (BP) regulation, and 61 DEGs were associated with central nervous system diseases. In accordance with the functional annotation of DEGs, the key role of hormonal metabolic processes and, in particular, the enhanced biosynthesis of aldosterone in the brain stem of ISIAH rats was proposed. Multiple DEGs associated with several Gene Ontology (GO) terms essentially related to modulation of BP were identified. Abundant groups of DEGs were related to GO terms associated with responses to different stimuli including response to organic (hormonal) substance, to external stimulus, and to stress. Several DEGs making the most contribution to the inter-strain differences were detected including the Ephx2, which was earlier defined as a major candidate gene in the studies of transcriptional profiles in different tissues/organs (hypothalamus, adrenal gland and kidney) of ISIAH rats.ConclusionsThe results of the study showed that inter-strain differences in ISIAH and WAG brain stem functioning might be a result of the imbalance in processes leading to the pathology development and those, exerting the compensatory effects. The data obtained in this study are useful for a better understanding of the genetic mechanisms underlying the complexity of the brain stem processes in ISIAH rats, which are a model of stress-sensitive form of hypertension.

SAT-356 Differential Effects of Lipophilic and Hydrophilic Statins on Aldosterone Biosynthesis

Statin therapy, which lowers LDL cholesterol, has a profound beneficial cardiovascular (CV) effect. However, some of statins’ CV benefits appear to be independent of decreases in LDL. We showed that statins reduce Aldosterone (ALDO) levels, a key hormone in cardiovascular function, and this effect was greater in lipophilic (e.g., Simvastatin) versus hydrophilic (e.g., Pravastatin) statins. The mechanism by which statins reduce ALDO production is unknown. Thus, the purpose of this study was to determine the effects of statins on the two key regulatory steps of ALDO biosynthesis. We hypothesized that Simvastatin, but not pravastatin, would inhibit the late, but not the early pathway of ALDO biosynthesis. Methods: Wistar rats (n=80, 30% female, 175-250 grams) were fed a 0.03% Na+ diet for 7 days with ad lib access to tap water. After euthanasia Zona Glomerulosa (ZG) cells were isolated from adrenal capsules. ZG cell incubates were studied for 1 hour under the following 8 conditions; Control, Angiotensin II (AngII, 10-7M), Fludrocortisone acetate (Fludro, 3µg), Simvastatin (SIMV, 100µM), Pravastatin (PRAV,100µM), Fludro+AngII, SIMV+AngII, and PRAV+AngII. To separately evaluate the early and late pathways we added Trilostane (TRIL), which inhibits 3β-OH-dehydrogenase enzyme, and a fixed dose of Corticosterone (CORT, 1µM) to the ZG cells. Under these conditions, Pregnenolone levels (PREG reflect early pathway activity and the ALDO/Corticosterone (ALDO/CORT) ratios reflect late pathway activity. Cells in each of the 8 conditions were incubated with and without TRIL (10µM) plus CORT, replicated in duplicate, and repeated 8 times. Results in ALDO and ALDO/CORT Ratio are presented as percent of maximum response (obtained after stimulation with AngII or in control conditions) while other data are in absolute measures, and analyzed by ANOVA and t-test. Results: In comparison to the maximum response caused by AngII (without TRIL); SIMV caused a reduction in aldosterone production while PRAV did not (72.1±26.6% vs 105±32.1%, p < 0.05). In the presence of TRIL, we observed that; 1) SIMV increased PREG in comparison to the control (696.63±98.15ng/dL vs 286.95±167.82ng/dL, p < 0.05) and to PRAV (696.63±98.15ng/dL vs. 291.57±290.39ng/dL, p < 0.05). However, 2) SIMV reduced the Aldo/Cort Ratio when compared to the Control, AngII, and Pravastatin conditions (53.3±5.3% vs 100%, 95.1±8.2%, 81.3±12.0%, p < 0.05 for SIMV vs all other conditions). Conclusions: These data show that SIMV and not PRAV decreases ALDO production. SIMV reduces ALDO by inhibiting the late pathway of ALDO biosynthesis. The SIMV-induced increase in PREG, is insufficient to overcome SIMV’s inhibition of the late pathway. These findings suggest that lipophilic statins have additional benefits (beyond HMG-CoA reductase inhibition) that influence the Renin-Angiotensin-Aldosterone System.

SAT-LB061 Sex-Specific Role of Retinoic Acid Receptor Alpha Signaling in Adrenal Cortex Development and Functional Zonation

Primary aldosteronism is the major cause of secondary arterial hypertension. Recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1 and ATP2B3 have been identified in aldosterone producing adenoma (APA). Although the role of these mutations in regulating aldosterone biosynthesis has been clearly established, the mechanisms involved in proliferation and APA formation still remain to be elucidated. The aim of our study was to identify mechanisms involved in the development of APA and, in particular, to identify specific signaling pathways responsible for abnormal cell proliferation and nodule formation. To this purpose, we have performed transcriptome analysis on 48 APA and investigated correlations of gene expression with genetic, morphological and functional characteristics of the tumors. Retinoic acid receptor alpha (RARα) signaling was identified as a central molecular network involved in nodule formation. To understand the role of RARα in adrenal cortex structure and function, we have explored the adrenal phenotype of 12 and 52 weeks old Rarα knockout (Rarα-/-) mice. Both male and female mice were investigated. 12 weeks old Rarα-/- mice showed reduced zona glomerulosa thickness and disorganization of the zona fasciculata, with modifications of the extracellular matrix and vessel architecture. This was associated with alterations in Wnt signaling and VegfA expression and an increased proliferative index, as well as a reduced expression of Star, Cyp11a1 and Cyp11b1. No modification of Cyp11b2 expression and aldosterone production were observed. Adrenal cortex disorganization was still present at 52 weeks of age, but without evidence of modification of Wnt signaling and VegfA or steroidogenic gene expression. In female mice, morphological abnormalities of the zona fasciculata were also present in 12 and 52 weeks old Rarα-/- mice. However, even at 12 weeks, no alterations in Wnt signaling and VegfA expression were observed, suggesting that molecular abnormalities may have occurred at an earlier time point in adrenal cortex development. Our results suggest that Rarα contributes in a sex- and time- dependent manner to the normal development and functional zonation of the adrenal cortex, by modulating both Wnt and VegfA signaling. We propose a model in which a homeostatic equilibrium between retinoic acid, Wnt and Vegf signaling pathways is required for normal development of the adrenal cortex. Dysregulation of this equilibrium in adult adrenal cortex may contribute to abnormal cell proliferation and APA development, creating a propitious environment for the emergence of specific driver mutations in APA. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

SAT-LB063 The ZFP36 Family of RNA-Binding Proteins Regulate Human Steroidogenesis

Human steroid hormones produced by the adrenal cortex control important physiology including metabolism, inflammation, blood pressure and volume, and sexual characteristics. While the signaling components, transcriptional regulators, and steroidogenic enzymes necessary for adrenocortical production of hormones have been identified, little to nothing is known about post-transcriptional regulation by RNA-binding proteins (RBPs). Recently technological advances have revolutionized our ability to investigate RBP-driven RNA regulation making it possible for the first time to investigate how this mechanism controls steroidogenesis. We have recently carried out an siRNA screen of RBPs regulating human aldosterone production that revealed a critical role for the tristetraprolin (ZFP36) family of RNA-binding protein. The ZFP36 family of RBPs binds to AU-rich elements in 3’UTRs and consequently destabilizes and/or translationally represses these ARE-containing mRNAs. Remarkably, depletion of either ZFP36L2 or ZFP36L1 significantly increased aldosterone levels. In addition, we have data demonstrating that: 1) mRNA stability controls the temporal pattern of RNA expression during steroidogenesis; 2) mRNAs with AU-rich elements (AREs) in their 3’ UTR are rapidly induced and cleared out in response to steroidogenic stimulation; 3) The ZFP36 family of RBPs are induced during steroidogenesis. We propose a model in which the ZFP36 family of proteins operate a negative feedback loop that prevent overproduction of aldosterone by destabilizing and/or translationally repressing ARE-containing mRNAs encoding steroidogenic proteins. Notably, over-production of aldosterone is a major cause of hypertension, suggesting that failure of this negative feedback loop could have important implications for human health. Our ongoing work will elucidate the mechanism underlying this negative feedback loop that controls aldosterone biosynthesis post-transcriptionally through the action of ZFP36 RNA binding proteins and yet to be discovered factors that they interact with. The adrenal cortex is amenable to the delivery of modified antisense oligonucleotides. Thus, our discoveries can facilitate the design of oligonucleotide therapeutics that can be used to precisely and specifically modulate human steroidogenesis. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.

Abstract 095: Renal Transcriptomic Analysis of Transgenic Mice Containing Human Cyp11b2 Gene

The hCYP11B2 gene encodes aldosterone synthase, the rate-limiting enzyme in the biosynthesis of aldosterone. Inappropriate excess levels of aldosterone induce positive sodium balance and predisposes to hypertension and other cardiovascular problems. Epidemiological studies have suggested that the variant -344T of the Cyp11B2 gene is associated with hypertension and myocardial hypertrophy. We have found that hCyp11B2 gene has three SNPs in 1 Kb of its promoter that are in complete linkage disequilibirium. These SNPs are T/C at -344, C/T at -470 and C/A at -663. Thus variant -344T always occurs with variants -470C, -663A (Hap-I), and variant -344C almost always occurs with variants, -470T, -663T (Hap-II). We have generated novel transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype I or II variants. We observed increased baseline blood pressure in Hap I TG mice (Hap-I 117±2.5 vs. Hap-II 109±1.9 mm Hg, p&lt;0.05), an effect accentuated by high-salt diet (Hap-I 135±2.6 vs. Hap-II 122±2.2 mm Hg, p&lt;0.05). In order to identify the downstream genes that are involved in Cyp11B2 induced hypertension and kidney damage, we have performed transcriptome analysis in kidneys of TG animals using RNAseq. Our results indicate that the highly significant differentially expressed genes are involved in the neuro-inflammation signaling pathway (p=2.71E-10), complement system (p=2.83E-07) and IL-7 signaling pathway (p=1.43E-04) with Z scores 2.333, 0.816 and 2.714 respectively. Using Ingenuity pathway analysis, we have identified genes that are associated with renal fibrosis. We have also found that expression of CTSW, CCL5, CCR2, CX3CR1, IRF1, and IRF7 are increased by 12.27 fold (p=3.49E-05), 4.23 fold (p= 3.78E-08), 2.9 fold (p= 3.45E-11), 3.37 fold (p=2.60E-12), 2.1 fold (p= 3.20E-06) and 2.0 fold (p= 1.21E-04) respectively in kidneys of hypertensive animals. Our RNA seq results delineate the regulatory molecules involved in the aldosterone mediated nephrotoxicity. These findings not only provide the valuable information on the pathophysiology of kidney damage but also can be used to identify novel therapeutic targets to reduce high blood pressure and end organ damage.

Tumor Cell Subtypes Based on the Intracellular Hormonal Activity in KCNJ5-Mutated Aldosterone-Producing Adenoma.

Aldosterone-producing adenomas (APAs) harbor marked intratumoral heterogeneity in terms of morphology, steroidogenesis, and genetics. However, an association of biological significance of morphologically identified tumor cell subtypes and genotypes is virtually unknown. KCNJ5 mutation is most frequently detected and generally considered a curable phenotype by adrenalectomy. Therefore, to explore the biological significance of KCNJ5 mutation in APA based on intracellular hormonal activities, 35 consecutively selected APAs (n=18; KCNJ5 mutated, n=17; wild type) were quantitatively examined in the whole tumor areas by newly developed digital image analysis incorporating their histological and ultrastructural features (14 cells from 2 KCNJ5-mutated APAs and 15 cells from 1 wild type) and CYP11B2 immunoreactivity. Results demonstrated that KCNJ5-mutated APAs had significantly lower nuclear/cytoplasm ratio and more abundant clear cells than wild type. CYP11B2 immunoreactivity was not significantly different between these genotypes, but a significant correlation was detected between the proportion of clear cells and CYP11B2 immunoreactivity in all of the APAs examined. CYP11B2 was predominantly immunolocalized in clear cells in KCNJ5-mutated APAs. Quantitative ultrastructural analysis revealed that KCNJ5-mutated APAs had significantly more abundant and smaller-sized mitochondria with well-developed cristae than wild type, whereas wild type had more abundant lipid droplets per unit area despite the small number of the cases examined. Our results did provide the novel insights into the morphological features of APA based on their biological significance. KCNJ5-mutated APAs were characterized by predominance of enlarged lipid-rich clear cells possibly resulting in increased neoplastic aldosterone biosynthesis.

Aldosterone Suppression by Dexamethasone in Patients With KCNJ5-Mutated Aldosterone-Producing Adenoma.

Aldosterone biosynthesis is regulated principally by ACTH and gene mutations as well as by angiotensin II and serum potassium. In addition, previous studies have reported the potential effects of KCNJ5 mutations in aldosterone-producing adenoma (APA) on cardiovascular diseases. However, responsiveness to ACTH in APAs according to potassium inwardly rectifying channel, subfamily J, member 5 (KCNJ5) mutations remains unknown. To investigate KCNJ5 genotype-specific differences in aldosterone biosynthesis in response to ACTH stimulation. A cross-sectional study through retrieval of clinical records. One hundred forty-one patients aged ≥20 years with APA were examined. Associations between KCNJ5 mutations and clinical parameters reflecting the renin-angiotensin system [saline infusion test (SIT)] and ACTH pathways [dexamethasone suppression test (DST)]. KCNJ5 mutations were detected in 107 cases. In the crude comparison, patients with mutations in KCNJ5 had higher plasma aldosterone concentrations (PACs) both at baseline and after the SIT. PAC after the DST showed a significant inverse association with KCNJ5 genotypes after controlling for age, sex, tumor size, and PAC after the SIT. Immunohistochemical analysis of 101 cases revealed more abundant immunoreactivity of CYP11B1 and CYP17 in the KCNJ5-mutated group than in the KCNJ5 wild-type group. This report of marked suppression of PAC by dexamethasone in patients with KCNJ5-mutated APAs indicates that such APAs respond to endogenous ACTH more readily than APAs in nonmutated cases. Further molecular and epidemiologic studies are required to validate our results and clarify the clinical effectiveness of the DST for predicting KCNJ5 mutations before adrenalectomy.

Epigenetic Regulation of Aldosterone Synthase Gene by Sodium and Angiotensin II.

Background DNA methylation is believed to be maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not stable. We demonstrate that stimulatory signals can change the DNA methylation status around transcription factor binding sites and a transcription start site and activate expression of the aldosterone synthase gene (CYP11B2).Methods and Results DNA methylation of CYP11B2 was analyzed in aldosterone‐producing adenomas, nonfunctioning adrenal adenomas, and adrenal glands and compared with the gene expression levels. CpG dinucleotides in the CYP11B2 promoter were found to be hypormethylated in tissues with high expression, but not in those with low expression, of CYP11B2. Methylation of the CYP11B2 promoter fused to a reporter gene decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including CREB1, NGFIB (NR4A1), and NURR1 (NR4A2) diminished their DNA‐binding activity. A methylated‐CpG‐binding protein MECP2 interacted directly with the methylated CYP11B2 promoter. In rats, low salt intake led to upregulation of CYP11B2 expression and DNA hypomethylation in the adrenal gland. Treatment with angiotensin II type 1 receptor antagonist decreased CYP11B2 expression and led to DNA hypermethylation.Conclusions DNA demethylation may switch the phenotype of CYP11B2 expression from an inactive to an active state and regulate aldosterone biosynthesis.

Pharmacophore Modeling and in Silico/in Vitro Screening for Human Cytochrome P450 11B1 and Cytochrome P450 11B2 Inhibitors.

Cortisol synthase (CYP11B1) is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing's syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2) is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8–10), one selective CYP11B1 inhibitor (Compound 11, IC50 = 2.5 μM), and one selective CYP11B2 inhibitor (compound 12, IC50 = 1.1 μM), respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

11β-Hydroxylase deficiency detected by urine steroid metabolome profiling using gas chromatography-mass spectrometry

Introduction11β-hydroxylase deficiency is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5–8% of all cases. It is an autosomal recessive enzyme defect that impairs the biosynthesis of cortisol and aldosterone. Mutation of the CYP11B1 gene on chromosome 8q22 causes partial or total reduction of enzyme activity. Clinical manifestations of 11β-hydroxylase deficiency include hypertension, and other signs related to overproduction of mineralocorticoids, and virilisation. Here, we report on a case of 11β-hydroxylase deficiency detected by urine steroid metabolome profiling. Case SubjectThe patient, a 3-month-old male, suffered from truncus arteriosus type I (congenital cardiovascular anomaly) and also presented with hyperpigmentation. An endocrinology consultation was sought and biochemical and molecular testing was conducted. ResultsThe patient’s urine steroid metabolome, as analysed by GC-MS, showed high excretion of tetrahydrodeoxycortisol (THS) and a THS/(THE + THF + 5αTHF) ratio of 2.3, which was higher than normal. Diagnosis of 11β-hydroxylase deficiency was confirmed by mutation analysis of the CYP11B1 gene. ConclusionAnalysis of the urine steroid metabolome by GC-MS can be used to assist in diagnosis of 11β-hydroxylase deficiency. We recommend consideration of urine steroid analysis as a first-line test in the diagnosis of CAH.

Disordered CYP11B2 Expression in Primary Aldosteronism.

Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations.

Abstract 054: Visinin Like Protein 1 Regulation of Aldosterone Biosynthesis

Visinin-Like Protein-1(Vsnl1) is a member of the EF-hand calcium sensor proteins. Its mRNA was found to be upregulated in aldosterone-producing adenomas and to be expressed in the zona glomerulosa (ZG) of the rat and human adrenal. We studied the expression pattern of Vsnl1 and its co-localization with the CYP11B2 enzyme and ZG marker Dlk1 (ZOG) using double and triple immunofluorescence in adrenal glands of rats on normal, high and low sodium diets and the effect of Vsnl1 on calcium mobilization and aldosterone biosynthesis in the human adrenocarcinoma cell line, the HAC15 cell. In rat adrenals, Vsnl1 was expressed only in ZG, in the same cells as Dlk1, in both functional and undifferentiated ZG cells. CYP11B2 co-localized with Vsnl1 in nearly all ZG cells of rats on a low sodium diet. Co-expression of CYP11B2 with Vsnl1 in adrenals of rats on normal and high sodium diets was significantly less. HAC15 were transduced with a lentivirus carrying Vsnl1 or an empty virus. Three days later cells were incubated with vehicle, A-II 10 nM, Forskolin 10μM, or potassium 16 mM. Aldosterone and cortisol were measured after 24 hr. Basal secretion of aldosterone was not increased; cortisol was. Aldosterone and cortisol secretion were greater after A-II, forskolin or potassium stimulation in cells overexpressing Vsnl1. Intracellular calcium measured by Fluo-4 AM dye and proliferation were increased by Vsnl1 overexpression. HAC15 cells were transduced with 2 different shRNA for Vsnl1. Aldosterone synthesis was reduced in HAC15 cells transduced with the shRNA for Vsnl1 stimulated with A-II. Vsnl1 has a myristoylation consensus sequence at the N-terminal domain. Site directed mutagenesis of Vsnl1 at the 2 position (G2A) resulted in a significant decrease in the aldosterone response to A-II. In summary, the calcium sensor protein Vsnl1 is ZG specific and detected in both steroidogenically active and inactive cells, as well as undifferentiated cells in the subcapsular area of the rat adrenal. Vsnl1 expression and activity in HAC15 cell correlate positively with calcium mobilization, proliferation and aldosterone synthesis.

A novel haplotype of low-frequency variants in the aldosterone synthase gene among northern Han Chinese with essential hypertension.

Low-frequency variants showed that there is more power to detect risk variants than to detect protective variants in complex diseases. Aldosterone plays an important role in the renin–angiotensin–aldosterone system, and aldosterone synthase catalyzes the speed-controlled steps of aldosterone biosynthesis. Polymorphisms of the aldosterone synthase gene (CYP11B2) have been reported to be associated with essential hypertension (EH). CYP11B2 polymorphisms such as –344T/C, have been extensively reported, but others are less well known. This study aimed to assess the association between human CYP11B2 and EH using a haplotype-based case–control study. A total of 1024 EH patients and 956 normotensive controls, which consist of north Han population peasants, were enrolled. Seven single nucleotide polymorphisms (SNPs) (rs28659182, rs10087214, rs73715282, rs542092383, rs4543, rs28491316, and rs7463212) covering the entire human CYP11B2 gene were genotyped as markers using the MassARRAY system. The major allele G frequency of rs542092383 was found to be risk against hypertension [odds ratio (OR) 3.478, 95% confidence interval (95% CI) 1.407–8.597, P = .004]. The AG genotype frequency of SNP rs542092383 was significantly associated with an increased risk of hypertension (OR 4.513, 95% CI 1.426–14.287, P = .010). In the haplotype-based case–control analysis, the frequency of the T-G-T haplotype was higher for EH patients than for controls (OR 5.729, 95% CI 1.889–17.371, P = .000495). All |D′| values of the seven SNPs were >0.9, and r2 values for rs28659182- rs10087214-rs28491316-rs7463212 SNPs were >0.8 and showed strong linkage intensity. Haplotype T-G-T may therefore be a useful genetic marker for EH.

Aldosterone Hypothesis for Cognitive Impairment in Diabetes Mellitus

Increased plasma aldosterone concentration is significantly associated with dementia, which is accentuated by diabetes mellitus (DM). Angiotensin II (AngII) deteriorates cognitive function through neuronal degradation. Lipoproteins, a major source of cholesterol for aldosterone biosynthesis, undergo glycoxidative modifications in the presence of hyperglycemia. We hypothesize that there would be a pathophysiological link between diabetically-modified lipoproteins, angiotensin II, and increased plasma aldosterone concentration for induction of cognitive impairment. Glycoxidized lipoproteins produce significantly more aldosterone from AngII-sensitized adrenocortical cells compared to their native counterparts. The elucidation of signaling mechanisms revealed that modified lipoproteins follow the similar signaling mechanism like AngII for adrenocortical aldosterone release via ERK1/2 and Janus kinase-2 (Jak-2)-mediated pathways. The enhanced aldosterone release from AngII-sensitized adrenocortical cells induced by glycoxidatively modified lipoproteins may play a crucial role in cognitive dysfunction in diabetic individuals along with AngII via a prevailing mode of signaling cascade involving ERK1/2- and Jak-2-dependent pathways.

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

Expression and Histopathological Significance of Disabled-2 in Aldosterone-Producing Adenoma.

The current pathological diagnosis of aldosterone-producing adenoma (APA) is challenging because no histological markers of aldosterone production are available in routine practice. A previous study demonstrated that Disabled-2 (DAB2) is a specific marker of the zona glomerulosa (ZG) in rodents. The aim of the present study was to investigate the significance of immunohistochemical staining to detect DAB2 in the adrenal tissue of patients with APA. We investigated the expression of DAB2 in 36 adrenal glands with APA, 23 adrenal glands with cortisol-producing adenoma (CPA), and 33 adrenal glands with non-functioning adenoma (NFA). Immunohistochemical staining was performed using anti-DAB2 antibodies on paraffin-embedded sections. We analysed the expression of DAB2 semi-quantitatively by scoring staining intensity, and assessed the correlation of this information with the clinical findings. DAB2 mRNA expression in adenoma tissues was evaluated by RT-PCR. DAB2 was highly expressed in the ZG in normal human adrenal glands. DAB2 expression was heterogeneous in APA, with spotted, strong staining noted in most samples (25 of 36 APA). CPA and NFA also exhibited extensive low or moderate DAB2 expression. DAB2 mRNA was significantly increased and positively correlated with CYP11B2 in APA (p<0.05). In APA, the DAB2 score adjusted for tumour volume was positively correlated with plasma aldosterone (p<0.05). Patients with low or moderate DAB2 staining more frequently exhibited high blood pressure and were diagnosed at a younger age compared with patients with high DAB2 staining. The present study clearly demonstrates that DAB2 is a specific marker of the ZG in normal human adrenal glands but that DAB2 immunostaining is not sufficiently powerful for histopathological diagnosis of APA. DAB2 might be involved in excessive aldosterone biosynthesis and correlate with specific clinical characteristics of APA patients.

Visinin-Like Protein-1 in the regulation of aldosterone biosynthesis

Visinin-Like Protein-1 in the regulation of aldosterone biosynthesis

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron.

A key role of aldosterone and mineralocorticoid receptor is to regulate fluid volume and K+ homeostasis in the body by acting on the renal distal nephron. Global responses of the kidney to elevated aldosterone levels are determined by the coordinate action of different constituent tubule cells, including principal cells, intercalated cells and distal convoluted tubule cells. Recent studies on genetic mutations causing aldosterone overproduction have identified the molecules involved in aldosterone biosynthesis in the adrenal gland, and there is also increasing evidence for mechanisms and signaling pathways regulating the balance between renal NaCl reabsorption and K+ secretion, the two major effects of aldosterone. In particular, recent studies have demonstrated that mineralocorticoid receptor in intercalated cells is selectively regulated by phosphorylation, which prevents ligand binding and activation. Moreover, the ubiquitin ligase complex composed of Kelch-like 3 and Cullin 3 acts downstream of angiotensin II and plasma K+ alterations, regulating Na-Cl cotransporter independently of aldosterone in distal convoluted tubule cells. These and other effects are integrated to produce appropriate kidney responses in a high-aldosterone state, and are implicated in fluid and electrolyte disorders in humans. This review summarizes the current knowledge on mechanisms modulating mineralocorticoid receptor and its downstream effectors in the distal nephron.

ACTH and Polymorphisms at Steroidogenic Loci as Determinants of Aldosterone Secretion and Blood Pressure.

The majority of genes contributing to the heritable component of blood pressure remain unidentified, but there is substantial evidence to suggest that common polymorphisms at loci involved in the biosynthesis of the corticosteroids aldosterone and cortisol are important. This view is supported by data from genome-wide association studies that consistently link the CYP17A1 locus to blood pressure. In this review article, we describe common polymorphisms at three steroidogenic loci (CYP11B2, CYP11B1 and CYP17A1) that alter gene transcription efficiency and levels of key steroids, including aldosterone. However, the mechanism by which this occurs remains unclear. While the renin angiotensin system is rightly regarded as the major driver of aldosterone secretion, there is increasing evidence that the contribution of corticotropin (ACTH) is also significant. In light of this, we propose that the differential response of variant CYP11B2, CYP11B1 and CYP17A1 genes to ACTH is an important determinant of blood pressure, tending to predispose individuals with an unfavourable genotype to hypertension.