Aldosterone Regulation Research Articles

Abstract 60: Atlas of Distal Nephron Mineralocorticoid Receptor-Dependent Transcriptome Reveals Novel Aldosterone Actions

Aldosterone regulation of Na+ and K+ balance and blood pressure are mediated by the mineralocorticoid receptor (MR, Nr3c2). Although several aldosterone/MR-dependent genes have been identified, their regulation cannot fully explain how aldosterone activates electrogenic Na+- K+ exchange in the aldosterone-sensitive distal nephron (ASDN). Here, we apply RNA-Seq and comprehensive downstream analysis to define the complete inventory of MR-regulated genes. MRfl/fl/Pax8,rtTA/LC1 mice were used to knock out the Nr3c2 gene in an inducible and kidney-specific manner. RNA-Seq analysis was conducted in the micro-dissected connecting tubule and cortical collecting duct tubule segments, followed by multimodal bioinformatic analyses. Direct MR-dependent gene regulation was validated in vitro on immortalized collecting duct cells, ex vivo with isolated distal nephron segments and gene reporter assays. All known aldosterone-response genes, including Sgk1, Scnn1a, Ndrg2, Per3, Tsc22d3, Zbtb16, MLPH, and Atp1a1 were significantly decreased in MR KO mice. 1193 additional genes were down-regulated in the MR KO, and 526 of these have a Mineralocorticoid Responsive Element (MRE), revealing the MR-dependent transcriptome is much larger than previously imagined. MREs were found in most genes with closed chromatin regions, suggesting MR binding might pioneer transcription for most of its regulated genes. The architecture of some MR-regulated genes, highlighted by the co-regulation of 7 Kallikrein genes, points to a novel mechanism of MR-regulation involving epigenetic remodeling in topology-associated domains. Pathway enrichment analysis revealed that MR-regulated genes are highly enriched in mitochondria-associated metabolic processes, and direct MR regulation of Klf15 was identified as a key transcriptional driver. In summary, the inventory of MR-regulated genes in the ASDN is much larger than previously imagined, involving new mechanisms of gene regulation. In addition to pathways that directly up-regulate transport, the data suggest that aldosterone-MR may directly influence metabolism to make energy-consuming transport highly efficient.

Commensal microbiota regulate aldosterone.

The gut microbiome regulates many important host physiological processes associated with cardiovascular health and disease; however, the impact of the gut microbiome on aldosterone is unclear. Investigating whether gut microbiota regulate aldosterone can offer novel insights into how the microbiome affects blood pressure. In this study, we aimed to determine whether gut microbiota regulate host aldosterone. We used enzyme-linked immunosorbent assays (ELISAs) to assess plasma aldosterone and plasma renin activity (PRA) in female and male mice in which gut microbiota are intact, suppressed, or absent. In addition, we examined urinary aldosterone. Our findings demonstrated that when the gut microbiota is suppressed following antibiotic treatment, there is an increase in plasma and urinary aldosterone in both female and male mice. In contrast, an increase in PRA is seen only in males. We also found that when gut microbiota are absent (germ-free mice), plasma aldosterone is significantly increased compared with conventional animals (in both females and males), but PRA is not. Understanding how gut microbiota influence aldosterone levels could provide valuable insights into the development and treatment of hypertension and/or primary aldosteronism. This knowledge may open new avenues for therapeutic interventions, such as probiotics or dietary modifications to help regulate blood pressure via microbiota-based changes to aldosterone.NEW & NOTEWORTHY We explore the role of the gut microbiome in regulating aldosterone, a hormone closely linked to blood pressure and cardiovascular disease. Despite the recognized importance of the gut microbiome in host physiology, the relationship with circulating aldosterone remains largely unexplored. We demonstrate that suppression of gut microbiota leads to increased levels of plasma and urinary aldosterone. These findings underscore the potential of the gut microbiota to influence aldosterone regulation, suggesting new possibilities for treating hypertension.

Exploring a Role for Gut Microbiota to Modulate Aldosterone

Hypertension is associated with an increased risk of mortality for numerous conditions including heart disease, diabetes, and renal disease (D. Aune, Biomed Res Int, 2021). Hypertension is often viewed as idiopathic and treatment is seldom aimed at addressing an underlying mechanism; however, a recent study indicates that primary aldosteronism is an underappreciated cause of idiopathic hypertension (J. Brown, Ann Intern Med, 2020). Here, we investigate for the first time the influence of gut microbiota on aldosterone regulation. To pursue this, we used C57Bl/6 mice (n=20; males) housed in the Johns Hopkins Animal Care Facility and fed a specific pathogen-free diet. Whole blood from submandibular cheek bleeds were collected into K2EDTA-treated tubes at baseline and mice were allowed to recover. Mice were then treated with antibiotics (ABX; 1g/L ampicillin, 1g/L neomycin, and 0.5g/L vancomycin) in their drinking water ad libitum for a total of 7 days to dramatically suppress gut microbes. Whole blood via submandibular cheek bleeds were collected after 24hrs, and at day 7 on ABXs. Mice were sacrificed on day 7 of ABX. Plasma samples from baseline, 24hrs on ABX and 7 days on ABX were used to determine aldosterone concentration and plasma renin activity (PRA) via ELISA. In addition, 24hr urine samples were collected at baseline and at day 7 on ABXs. Statistics were calculated using paired, one-way ANOVA; data below are mean ± SD. Our results demonstrate that plasma aldosterone and PRA, as well as urine aldosterone, were significantly increased from baseline to 7 days on ABX treatment (plasma aldo (pg/mL): 295.1 ± 164.8 vs 634.9 ± 290.4, p= 0.0197; plasma PRA (ng Ang-1/mL/hr): 191.8 ± 137.6 vs 325.9 ± 141.5, p= 0.0108; urine aldo (pg/mL): 2019 ± 322.1 vs 2668 ± 292.0, p = 0.0047). However, there was no significant difference in plasma aldosterone from baseline to 24hrs on ABX treatment (295.1 ± 164.8 vs 434.5 ± 245.3, p= 0.1047). In contrast, we observed a significant increase in angiotensin I from baseline to 24hrs on ABX treatment (191.8 ± 137.6 vs 355.8 ± 139.6, p=0.0041), and this increase was sustained until day 7 (325.9 ± 141.5 vs baseline, p=0.0108). Preliminary data suggest aldosterone is also increased in the plasma of germ-free mice (without gut microbes) compared to conventionalized controls (750.4 ± 265.0, n= 6, vs 314.4 ± 260.3, n=4, p=0.0667), implying that gut microbiota normally act to suppress plasma aldosterone. Taken together, we demonstrate that in the absence of gut microbiota, plasma aldosterone, urine aldosterone, and PRA are significantly increased. Understanding how gut microbes influence aldosterone levels could provide valuable insights into the development and treatment of hypertension. This knowledge may open new avenues for therapeutic interventions, such as probiotics or dietary modifications, to help regulate blood pressure. William Townsend Porter Predoctoral Fellowship from the American Physiological Society (BNM), Johns Hopkins Post-baccalaureate Research Education Program (PREP; R25GM109441 to ADM), the NIDDK Diabetic Complications Consortium (RRID:SCR_001415m www.diacomp.org, grants DK076169 and DK 115255 (subaward to JLP)) and American Heart Association Established Investigator Award (to JLP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC β- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Systematic review/meta-analysis and a cross-sectional study to assess effects of potassium supplementation on the renin-angiotensin-aldosterone system (RAAS) and blood pressure

Abstract Introduction Nutritional medicine plays a key role in disease prevention and treatment, and we pharmacists must consider our contribution beyond our role in dispensing. Apart from the Dietary Approaches to Stop Hypertension (DASH) diet1, there is a paucity of data on the benefits of nutritional interventions in hypertension with no clear recommendation on potassium in the NICE guidelines. Potassium has been an area of interest in clinical medicine over the decades because of its effect on cardiovascular disease. However, its mechanism of action is poorly understood, and it is unclear it differs between different ethnic groups. Dietary potassium decreases blood pressure (BP) but can also stimulate aldosterone production2. However, the magnitude of potassium-dependent regulation of aldosterone secretion in humans is not fully characterised and it is not clear whether this is mediated by activation of the renin-angiotensin-aldosterone system (RAAS) as a result of a reduction in BP or other mechanisms. Aim To perform a (1) systematic review and meta-analysis of clinical trials assessing effects of potassium on plasma aldosterone and renin in adult individuals alongside a (2) cross-sectional multi-ethnic study to explore the relationship between potassium intake and aldosterone in hypertension. Methods (1) This study was carried out in accordance with PRISMA guidelines3. The full protocol can be found on the PROSPERO database. (2) Self-defined black or white hypertensives were recruited from the hypertension outpatient clinic at St. Thomas’ Hospital and had aldosterone, renin and a 24-hour urinary potassium collected. The systematic review and meta-analysis on already approved clinical trials and did not require ethnical approval. The cross-sectional study was approved by the local Research Ethics Committee in the UK and written informed consent was obtained from all patients. Results 6395 articles were retrieved, and 124 full-text articles were assessed for eligibility. 36 met the prespecified inclusion/exclusion criteria. Potassium supplementation caused a significant decrease in systolic BP (- 3.69 mmHg [-4.91, -2.46], P<0.001) and increase in serum potassium (+0.37 [0.23, 0.52] mmol/L, P<0.001). There was an increase in plasma aldosterone (standardised difference 0.414 [0.291, 0.537], P<0.001) but no effect on plasma renin activity (+0.033 [-0.07, 0.14], P=0.538]. Meta-regression showed a significant positive correlation between change in plasma aldosterone and change in serum potassium (P<0.001). (2) In 469 subjects (72% black), there was a positive association between the measured 24-hour potassium excretion and aldosterone in black (β = 0.200, P = 0.014) but not in white individuals (P = 0.531). Discussion/Conclusion Potassium supplementation increases plasma aldosterone but not renin. The increase in aldosterone correlates with the raise in serum potassium. In subjects with hypertension, potassium intake is related with aldosterone concentration only in black subjects. Overall, the data supports a RAAS-independent potassium regulation of aldosterone which could be particularly relevant in African origin subjects. Limitations: 1. the systematic review results could not be stratified by ethnicity because most of the studies were performed in Caucasian subjects, 2. Use of background therapy in some studies, variable doses of potassium supplementation and duration of supplementation.

Elevated Blood Pressure and Aldosterone Dysregulation in Young Black Women Versus White Women on Controlled Sodium Diets.

Black women have a higher prevalence of hypertension as compared to White women. Differences in dietary sodium intake have been implicated as a contributing factor for the disparities in hypertension. Our objective was to understand whether young Black women would have higher systolic blood pressure (SBP) than White women even on controlled sodium diets and to determine whether SBP differences were due to differences in dietary sodium intake and/or aldosterone regulation. The analyses included 525 hypertensive and normotensive women (ages 18-71) from the International Hypertensive Pathotype consortium, who were maintained on liberal sodium (LIB; >200 mEq sodium/day) and restricted sodium (RES; 10 mEq sodium/day) diets. Multivariate regression analyses (adjusted for age, race, study site, body mass index) found that Black women (ages 18-50) had significantly higher SBP than White women on both sodium diets: +8.7 ± 2.7 mmHg (P-value = .002) on a LIB diet and +8.5 ± 2.5 mmHg (P-value = .001) on a RES diet. Even among 18- to 35-year-olds-who were normotensive and nonobese-Black women had higher SBP: +7.9 ± 2.4 mmHg (P-value = .001) on a LIB diet and +7.6 ± 2.7 mmHg (P-value = .005) on a RES diet. Younger Black women also had higher plasma aldosterone concentration to plasma renin activity ratio (ARR) on both LIB and RES diets as well as a higher sodium-modulated aldosterone suppression-stimulation index-an indicator of aldosterone dysregulation. In younger Black women-but not in White women-there was a significant association between SBP and ARR on both LIB and RES diets. Young Black women had increased SBP and ARR as compared to White women on LIB and RES diets, which offers insights into the possible mechanisms for the increased hypertension and cardiovascular disease risk in an at-risk and understudied population.

Aldosterone activates ERK phosphorylation in the nucleus tractus solitarius

Sodium intake effect of aldosterone has attracted much attention. In our recent study, aldosterone can play a nongenomic regulatory role on rapid sodium intake in the NTS (nucleus tractus solitarius) by activating G protein-coupled estrogen receptor (GPER), and it exhibited an obvious time-dependent and concentration-dependent regulation. However, the molecular mechanism how aldosterone regulated sodium intake rapidly, is unclear. To determine the molecular mechanism of rapid sodium intake regulation of aldosterone, rats with a stainless-steel cannula in the NTS were used (n = 6 each subgroup), and were injected different concentrations of aldosterone/G1 (GPER agonist)/G15 (GPER antagonist) at different time points, then detected ERK1/2 protein expression. The results showed that aldosterone/G1 increased the ERK1/2 protein phosphorylation, and presented a time-dependent and concentration-dependent similar to sodium intake; Meanwhile, G15 partially blocked this effect at least. Taken together, we postulate that ERK1/2 protein may influence nongenomic sodium intake regulated by aldosterone at nucleus tractus solitarius level.

RAS-challenge as a first-look test for detection of primary aldosteronism in patients with treatment-resistant hypertension

Purpose Primary aldosteronism (PA), characterised by low-renin hypertension, confers a high cardiovascular risk and is the most common cause of secondary hypertension, with an increased prevalence in patients with treatment-resistant hypertension. However, it is estimated that only a small percentage of affected patients are identified in routine clinical practice. Inhibitors of the renin-angiotensin system cause an increase in renin levels in patients with intact aldosterone regulation, and inadequate low renin with concurrent RAS inhibition (RASi) may therefore indicate PA, which could serve as a first look screening test for selection for formal work-up. Methods We analysed patients between 2016–2018 with treatment-resistant hypertension who had inadequate low renin in the presence of RASi (i. e. at risk for PA) and who were offered systematic work-up with adrenal vein sampling (AVS). Results A total of 26 pts were included in the study (age 54.8 ± 11, male 65%). Mean office blood pressure (BP) was 154/95 mmHg on 4.5 antihypertensive drug classes. AVS had a high technical success rate (96%) and demonstrated unilateral disease in the majority of patients (57%), most of which (77%) were undetected by cross-sectional imaging. Conclusion In patients with resistant hypertension, low renin in the presence of RASi is a strong indicator for autonomous aldosterone secretion. It may serve as an on-medication screening test for PA to select for formal PA work up.

Effect of the interaction between the visceral-to-subcutaneous fat ratio and aldosterone on cardiac function in patients with primary aldosteronism.

Primary aldosteronism is the most frequent secondary hypertensive disease and is characterized by an elevated risk for cardiovascular disease. The current standard treatments are adrenalectomy and/or administration of mineralocorticoid receptor blockers, both of which are effective at ameliorating hypertension via intervention for hyperaldosteronism. However, both of these approaches have side effects and contraindications, and mineralocorticoid receptor blockers also have limited preventive efficacy against cardiovascular events. Recently, in vitro experiments have shown that aldosterone regulation is closely related to abdominal fat accumulation and that there is crosstalk between aldosterone and visceral fat tissue accumulation. We previously reported that this interaction was clinically significant in renal dysfunction; however, its effects on the heart remain unclear. Here, we analyzed data from 49 patients with primary aldosteronism and 29 patients with essential hypertension to examine the potential effect of the interaction between the ratio of visceral-to-subcutaneous fat tissue volume and the plasma aldosterone concentration on echocardiographic indices, including the tissue Doppler-derived E/e' ratio. A significant interaction was found in patients with primary aldosteronism (p < 0.05), indicating that patients with the combination of a high plasma aldosterone concentration and high visceral-to-subcutaneous fat ratio show an increased E/e' ratio, which is a well-known risk factor for future cardiovascular events. Our results confirm the clinical importance of the interaction between aldosterone and abdominal fat tissue, suggesting that an improvement in the visceral-to-subcutaneous fat ratio may be synergistically and complementarily effective in reducing the elevated risk of cardiovascular disease in patients with primary aldosteronism when combined with conventional therapies for reducing aldosterone activity. A significant effect of the interaction between plasma aldosterone concentration and the visceral-to-subcutaneous fat ratio on the tissue Doppler-derived E/e' ratio in patients with primary aldosteronism.

LBMON299 Aldosterone Production Is Regulated By Gap Junctions In Human Adrenal Cells

Abstract Aldosterone plays a critical role in blood pressure regulation via the renin-angiotensin-aldosterone system. Autonomous production of aldosterone, primary aldosteronism (PA), is the commonest curable cause of hypertension worldwide. Two cell types in the adrenal cortex produce aldosterone: zona glomerulosa (ZG) cells and aldosterone producing nodules (APN), and in cells of aldosterone producing adenomas (APA). Adrenal cortex cells undergo phenotypic metamorphosis as they migrate from the subcapsular region, centripetally, towards the corticomedullary junction, changing from aldosterone- to cortisol- producing cells as they go. Gap junctions (GJs) are specialised channels of cytoplasmic communication between cells, permitting the regulated transfer of substrates. Loss-of-function mutations in the CADM1 gene, which affects GJ protein CX43, have been found in APAs, implicating CX43 in aldosterone regulation. This study aims to demonstrate the dysregulation of aldosterone production by CX43 inhibition in human adrenal cell line, H295R, and elucidate GJ distribution in human adrenal cortex. Methodology H295R cells were treated with 1×10-8M angiotensin II and connexin mimetic GJ inhibitor, GAP27, at 0µM, 25µM, 83µM and 250µM. Immunofluorescence (IF) was used to identify the abundance of CX43 in different cell types of the adrenal cortex, performed on ex-vivo para-APA adrenal tissue, using antibodies against CX43 (GJA1), colocalised with markers of aldosterone-producing cells (aldosterone synthase, CYP11B2), cells of the zona glomerulosa (ZG) (VSNL1, and DAB2) and zona fasciculata (ZF) (CYP7A1). Results Inhibition of CX43 with Gap27 results in a dose-dependent increase in CYP11B2 mRNA expression and aldosterone production. At the highest concentration of 250µM GAP27, CYP11B2 mRNA expression increased by 133-fold (p=&amp;lt;0. 00001) and aldosterone production by 29-fold (p=0. 0006). IF studies demonstrate a crescendo gradient of CX43 in a centripetal direction from cells of APN to ZF. The abundance was lowest in aldosterone-producing APN cells, moderate in non-aldosterone-producing ZG cells and highest in cortisol-producing ZF cells. GJ distribution was pan-membranous in ZF, but, where present, mostly cytoplasmic in APN. Evidence of recent active communication, in the form of annular gap junctions (AJG), was present in all three cell types. Conclusion The CX43 inhibition-induced upregulation of CYP11B2 expression and aldosterone synthesis indicates that it has an important regulatory role in physiological aldosterone production via CYP11B2 expression. The distribution of CX43 in adrenal tissue suggests its transport substrate not only regulates aldosterone synthesis, but also in adrenal cortex cell phenotypic metamorphosis/migration. Future work will investigate the substrate of CX43 in adrenal cells and its possible effect on adrenal cell phenotype. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Regulation Of Aldosterone On Water And Salt Metabolism During Cooling Therapy On Exertional Heat Stroke

Corresponding Author: Bing Zhang. The pathophysiological mechanism of exertional heat stroke involves the imbalance of water and salt metabolism, but the effect of cooling therapy on serum aldosterone and the further regulation mechanism of water and salt metabolism is not clear. PURPOSE: To determine the changes of serum aldosterone and its regulation on water and salt metabolism in rats with exertional heat stroke under cooling therapy. METHODS: In our study, there are six groups of male SD rats, including the Normal environment Control group(NC n = 8), the Onset group of EHS(EHSO,n = 9), the Rest group 1 after EHS(EHSR n = 9), the Cooling group 1 after EHS(EHSC1,n = 8), the Rest group 2 after EHS(EHSR2 n = 9), the Cooling group 2 after EHS(EHSC2,n = 8). In hot and humid conditions(36 °C,75%RH), the rat model of exertional heat stroke was induced by exercising to exhaustion and raising their rectal temperature to about 42 °C. Cold water immersion for 5 min at 19 °C was used as rapid cooling therapy. Blood was harvested. Aldosterone in serum was measured by ELISA. Enzolife company in the United States provided ALD ELISA kit for rats. Mann-Whitney U test in nonparametric test was used for statistical analysis. RESULTS: With the onset of exertional heat stroke, serum ALD regulating water and salt metabolism was significantly up-regulated(EHSO vs. NC) (ALD: 122.51 ± 15.52 vs. 63.97 ± 16.28 pg/ml, p < 0.01). There was no significant change in serum ALD immediately after cooling therapy for 5 minutes(EHSC1 vs. EHSO or EHSR1) (ALD: 133.12 ± 17.62 vs. 122.51 ± 15.52 or 115.39 ± 13.90 pg/ml, p > 0.05). After cooling therapy for 5 minutes and resting for 60 minutes, serum ALD still had no significant change(EHSC2 vs. EHSO or EHSR2) (ALD: 131.49 ± 18.79 vs. 122.51 ± 15.52 or 115.70 ± 17.88 pg/ml, p > 0.05). CONCLUSIONS: When exertional heat stroke occurs, serum aldosterone increases significantly, which proves that the body has seriously unbalanced water and salt metabolism. Cooling therapy has no significant effect on serum aldosterone, which indicate that the regulation of cooling therapy on water and salt metabolism through aldosterone is not obvious or lagging. The research of this thesis has sponsored by the key fund project of philosophy and social science planning project of Anhui Province in 2020(Project Number: AHSKZ2020D25).

MANP Activation Of The cGMP Inhibits Aldosterone Via PDE2 And CYP11B2 In H295R Cells And In Mice.

Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear. In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice. We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased. Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.

A Role for the Adrenal Clock in Aldosterone Regulation and Renal Excretory Function

Purpose of studyIt is well documented that most physiological functions, including hormone secretion, renal electrolyte excretion and blood pressure (BP), exhibit circadian rhythms. Disruption in these rhythms is associated with adverse cardiorenal outcomes. BMAL1 is a core circadian clock protein. Global BMAL1 knockout (KO) male mice exhibit lower and non‐dipping BP which is associated with inappropriate renal sodium (Na) retention (Zhang et al.). Kidney‐specific BMAL1 KO male mice displayed lower BP, but BP and renal Na excretion rhythm remained intact. The adrenal gland synthesizes the hormones, cortisol (corticosterone in rodents) and aldosterone, which exert a powerful influence on BP rhythm and renal electrolyte handling. Others have shown that the BMAL1 in the adrenal gland is necessary for proper regulation of corticosterone but the role of the adrenal clock in the regulation of aldosterone and renal function remains unknown.HypothesisWith kidney BMAL1 not playing a role in the inappropriate renal Na retention seen in global BMAL1 KO mice under basal conditions and unpublished data from our lab suggesting a crosstalk between the adrenal gland and kidney clock, we hypothesized a role for adrenal BMAL1 in maintenance of aldosterone and renal Na excretion rhythm.MethodsTo test this, adrenal‐specific aldosterone synthase Cre positive (AS)‐BMAL1 KO mice and Cre negative control mice were generated, and efficiency of KO was evaluated using immunohistochemistry and recombination PCR. AS‐BMAL1 male mice and littermate controls were placed in metabolic cages to assess urinary 12‐hour aldosterone excretion and renal Na and potassium (K) rhythm during normal Na diet and 7 days Na depletion. Furthermore, body weight, and 12‐hour food intake and water intake were measured (n=5‐6).ResultsHere, we show that efficiency of KO of adrenal BMAL1 was &gt;80%, localized to the zona glomerulosa (ZG) of the adrenal gland. Body weight and water intake were similar between genotypes under normal Na or 7 days low Na conditions. There was an increase in food intake consumed during the inactive phase on both diets in AS‐BMAL1 KO mice compared with controls (ANOVA genotype effect p=0.0087), with no significant difference in 24‐hour food intake between genotypes. Urinary aldosterone levels were increased with blunted night/day differences in AS‐BMAL1 KO male mice under normal salt and low Na conditions compared with controls (ANOVA interaction p=0.0407). Na balance was calculated, showing a trend for a significant interaction between genotype and time (p=0.05), appearing more positive during their inactive period. K levels were not manipulated in the diet and there was no significant difference in K balance in AS‐BMAL1 KO mice under normal Na or 7 days low Na conditions (ANOVA interaction p=0.3808).ConclusionsConsistent with our hypothesis, loss of the core clock protein BMAL1 in the ZG of the adrenal gland resulted in blunted night/day aldosterone levels and disturbance in renal Na handling. This work is the first to investigate the role of adrenal BMAL1 on aldosterone regulation and renal function, suggesting a link between the adrenal clock and the kidney. Future studies will assess whether BP rhythm in AS‐BMAL1 KO mice is disrupted and determine if there are sex‐specific differences in the role of adrenal BMAL1.

Early Renin Recovery After Adrenalectomy in Aldosterone-Producing Adenomas: A Prospective Study.

The aim of the study was to clarify the relationship and the time of aldosterone and renin recoveries at immediate and long-term follow-up in aldosterone-producing adenoma (APA) patients who underwent adrenalectomy. Prospective and longitudinal protocol in a cohort of APA patients was followed in a single center. Among 43 patients with primary aldosteronism (PA), thirteen APA patients were enrolled in this study. Blood was collected for aldosterone, renin, potassium, creatinine, cortisol, and ACTH before and 1, 3, 5, 7, 15, 30, 60, 90, 120, 180, 270, 360 days after adrenalectomy. At diagnosis, most patients (84%) had hypokalemia and high median aldosterone levels (54.8; 24.0-103 ng/dl) that decreased to undetectable (<2.2) or very low (<3.0) levels between fifth to seventh days after surgery; then, between 3-12 months, its levels gradually increased to the lower normal range. The suppressed renin (2.3; 2.3-2.3 mU/l) became detectable between the fifteen and thirty days after surgery, remaining normal throughout the study. The aldosterone took longer than renin to recover (60 vs.15 days; p<0.002) and patients with higher aldosterone had later recovery (p=0.03). The cortisol/ACTH levels remained normal despite the presence of a post-operative hypoaldosteronism. Blood pressure and antihypertensive requirement decreased after adrenalectomy. In conclusion, our prospective study shows the borderline persistent post-operative hypoaldosteronism in the presence of early renin recovery indicating incapability of the zona glomerulosa of the remaining adrenal gland to produce aldosterone. These findings contribute to the comprehension of differences in renin and aldosterone regulation in APA patients, although both are part of the same interconnected system.

Salt Sensitivity of Blood Pressure and Aldosterone: Interaction Between the Lysine-specific Demethylase 1 Gene, Sex, and Age.

Salt sensitivity of blood pressure (SSBP) is associated with increased cardiovascular risk, especially in individuals of African descent, although the underlying mechanisms remain obscure. Lysine-specific demethylase 1 (LSD1) is a salt-sensitive epigenetic regulator associated with SSBP and aldosterone dysfunction. An LSD1 risk allele in humans is associated with SSBP and lower aldosterone levels in hypertensive individuals of African but not European descent. Heterozygous knockout LSD1 mice display SSBP and aldosterone dysregulation, but this effect is modified by age and biological sex. This might explain differences in cardiovascular risk with aging and biological sex in humans. This work aims to determine if LSD1 risk allele (rs587618) carriers of African descent display a sex-by-age interaction with SSBP and aldosterone regulation. We analyzed 297 individuals of African and European descent from the HyperPATH cohort. We performed multiple regression analyses for outcome variables related to SSBP and aldosterone. LSD1 risk allele carriers of African (but not European) descent had greater SSBP than nonrisk homozygotes. Female LSD1 risk allele carriers of African descent had greater SSBP, mainly relationship-driven by women with low estrogen (postmenopausal). There was a statistically significant LSD1 genotype-sex interaction in aldosterone response to angiotensin II stimulation in individuals aged 50 years or younger, with female carriers displaying decreased aldosterone responsiveness. SSBP associated with LSD1 risk allele status is driven by women with a depleted estrogen state. Mechanisms related to a resistance to develop SSBP in females are uncertain but may relate to an estrogen-modulating effect on mineralocorticoid receptor (MR) activation and/or LSD1 epigenetic regulation of the MR.

Abstract 10: MANP Activation of the Cyclic GMP/PDE2/CYP11B2 Pathway Inhibits Aldosterone In Vitro and In Vivo

Aldosterone is a critical driver for cardiovascular disease (CVD). We recently discovered that MANP, a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP. MANP is currently entering clinical trials for hypertension and thus understanding its aldosterone suppressing mechanism is important. The mechanism of aldosterone inhibition by natriuretic peptides (NPs) remains to be clearly defined. Conflicting results were reported on the roles of particulate guanylyl cyclase A receptor (pGC-A) and NP clearance receptor (NPRC) in aldosterone inhibition. Furthermore, the functions of protein kinase G (PKG) and phosphodiesterases (PDE) on aldosterone regulation are not clear. Herein, we investigated the molecular mechanism of aldosterone regulation in the human adrenocortical cell line H295R and in mice. We firstly showed that pGC-A mediates aldosterone inhibition. In contrast, with NPRC agonist and antagonist, we showed that NPRC did not inhibit aldosterone. Next, we confirmed that MANP inhibits aldosterone via PDE2, not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer (FRET) experiments. Specifically, MANP suppressed ANGII mediated activation of aldosterone (fold change) MANP+ANGII 3.2±0.1* vs. ANGII 3.8±0.1 (*p&lt;0.05) with IBMX, a PDEs inhibitor and the PDE2 antagonist Bay 60-7550 reversed MANP-mediated aldosterone suppression (IBMX+MANP+ANGII 3.9±0.2 and Bay+MANP+ANGII 4.1±0.1). With PKG agonists and inhibitors, aldosterone levels were not changed. In PDE2 activity FRET studies, aldosterone control was 3.7±0.4 and with MANP 0.9±0.2* supporting PDE2 activation by MANP. Further, the inhibitory effect of PDE2 is mediated by a reduction of intracellular Ca2+ concentration (~22%). We then showed that MANP directly reduced aldosterone synthase CYP11B2 expression in vitro. Lastly, in PDE2 knockout mice (embryonic lethal), embryonic adrenal CYP11B2 expression is markedly increased (wild type: 1±0.2, KO: 2.8±0.5*). Our findings innovatively elucidate the pGC-A/cGMP/PDE2 pathway in aldosterone inhibition by MANP in vitro and in vivo. Additionally, our data also support the development of MANP as a novel ANP analog drug for CVD.

The potential role of RAAS-related hsa_circ_0122153 and hsa_circ_0025088 in essential hypertension

ABSTRACT Background: The dysregulation of renin-angiotensin-aldosterone system (RAAS) is closely related to the development of essential hypertension (EH). MicroRNAs (miRNAs) are an important regulator of RAAS. The sponge effect of circular RNAs (circRNAs) on miRNAs makes the circRNA-miRNA-mRNA axis in EH possible, however, there is currently a lack of relevant evidence. Material and Methods: A circRNA-miRNA network was constructed based on the previous circRNAs microarray results. The expression of RAAS-related miRNAs and circRNAs were verified by qRT-PCR. Peripheral blood samples of 106 EH patients and 106 healthy volunteers were included in this study. GO and KEGG enrichment were performed to predict the role of candidate circRNAs in EH. Results: In EH patients, RAAS-related hsa-miR-483-3p and hsa-miR-27a-3p were down-regulated, and hsa_circ_0122153 and hsa_circ_0025088 were up-regulated. The relative expression of RAAS-related circRNAs and target miRNAs showed a negative correlation (hsa_circ_0122153-hsa-miR-483-3p and hsa_circ_0025088-hsa-miR-27a-3p). Hsa_circ_0122153 or hsa_circ_0025088 combined with corresponding miRNAs and environmental factors may support the early diagnosis of EH. Hsa_circ_0122153 and hsa_circ_0025088 may participate in the regulation of aldosterone and the secretion of renin through the circRNA-miRNA-mRNA network, respectively. Conclusion: Highly expressed hsa_circ_0122153 and hsa_circ_0025088 increase the risk of EH. The hsa_circ_0122153/hsa-miR-483-3p and hsa_circ_0025088/hsa-miR-27a-3p axis involving RAAS were potential EH pathways.

Paradoxical Increase of 24-Hour Urinary Aldosterone Levels in Obese Patients With Resistant Hypertension on a High Salt Diet

Having previously reported that aldosterone levels increase progressively with body mass index (BMI), the current analysis was done to determine to what extent this association is related to dietary high salt intake. We anticipated that aldosterone levels would decrease with higher sodium status consistent with classical suppression of aldosterone release secondary to progressive fluid retention induced by high dietary sodium intake. Cross-sectional analysis of a large diverse cohort of 2,705 patients with resistant hypertension (HTN) seen in a referral HTN Clinic. Dietary sodium intake was indexed by 24-hour (h) urinary sodium (UNa), aldosterone status was determined by plasma aldosterone concentration, plasma renin activity, and 24 h urinary aldosterone (UAldo). Patients with normal weight served as control. In this study, 1,572 individuals with complete 24 h urine collections were analyzed. Mean BMI was 32.5 ± 7.1 kg/m2 and ranged from 24.6 ± 2.4 kg/m2 (first quartile) to 41.0 ± 4.2 kg/m2 (fourth quartile). BMI was positively associated with 24 h UNa and UAldo levels (P < 0.0001), 24 h UNa and UAldo. There was a positively stronger correlation in obese (r = 0.273, P < 0.0001) compared with normal weight individuals (r = 0.108, P = 0.0342) independent of number and classes of antihypertensive medications. Our analysis shows that there is an altered regulation of aldosterone in obese patients in the setting of high dietary salt intake.

Aldosterone is Aberrantly Regulated by Various Stimuli in a High Proportion of Patients with Primary Aldosteronism

In primary aldosteronism (PA), aldosterone secretion is relatively independent of the renin-angiotensin system, but can be regulated by several other stimuli. To evaluate aldosterone response to several stimuli in a series of patients with PA secondary either to bilateral adrenal hyperplasia (BAH) or unilateral aldosterone-producing adenoma (APA). Prospective cohort study conducted in a university teaching hospital research center. Forty-three patients with confirmed PA and subtyped by adrenal vein sampling (n = 39) were studied, including 11 with BAH, 28 with APA, and 4 with undefined etiology. We also studied 4 other patients with aldosterone and cortisol cosecretion. We systematically explored aberrant regulation of aldosterone using an in vivo protocol that included the following stimulation tests performed over 3 days under dexamethasone suppression: upright posture, mixed meal, adrenocorticotropin (ACTH) 1-24, gonadotropin-releasing hormone (GnRH), vasopressin, and serotonin R4 agonist. Positive response was defined as >50% renin or ACTH-independent increase in plasma aldosterone/cortisol concentration following the various stimulation tests. Renin-independent aldosterone secretion increased in response to several aberrant stimuli (upright posture, GnRH) in up to 83% of patients with APA or BAH in whom ACTH 1-24 and HT4R agonists also produced aldosterone oversecretion in all patients. The mean significant aberrant responses per patient was similar in BAH (4.6) and in APA (4.0). Aldosterone secretion in PA is relatively autonomous from the renin-angiotensin system, but is highly regulated by several other stimuli, which contributes to the large variability of aldosterone levels in PA patients.

OR34-02 Somatic Transmembrane Domain Mutations of a Cell Adhesion Molecule, CADM1, Cause Primary Aldosteronism by Preventing Gap Junction Communication Between Adrenocortical Cells

Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich.Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1.All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p<0.01) and 10-20-fold increase in CYP11B2 expression (p<0.001) compared to WT. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. CX43 inhibition increased aldosterone secretion 2-fold (p<0.01), and CYP11B2 expression 3 to 8-fold (<0.001). Knock-down of GJ proteins increased aldosterone secretion 1.5-fold (p<0.01) and CYP11B2 expression 1.7-fold (p<0.001).Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p<0.001].The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.