Urinary Aldosterone Research Articles

Sex differences in the adrenal circadian clock: A role for BMAL1 in the regulation of urinary aldosterone excretion and renal electrolyte balance in mice.

Brain and muscle ARNT-Like 1 (BMAL1) is a circadian clock transcription factor that regulates physiological functions. Male adrenal-specific Bmal1 (ASCre/+::Bmal1) KO mice displayed blunted serum corticosterone rhythms, altered blood pressure rhythm, and altered timing of eating, but there is a lack of knowledge in females. This study investigates the role of adrenal BMAL1 in renal electrolyte handling and urinary aldosterone levels in response to low salt in male and female mice. Mice were placed in metabolic cages to measure 12-hour urinary aldosterone after a standard diet and 7 days low salt diet, as well as daily body weight, 12-hour food and water intake, and renal sodium and potassium balance. Adrenal glands and kidneys were collected at ZT0 or ZT12 to measure expression of aldosterone synthesis genes and clock genes. Compared to littermate controls, ASCre/+::Bmal1 KO male and female mice displayed increased urinary aldosterone in response to a low salt diet, although mRNA expression of aldosterone synthesis genes was decreased. Timing of food intake was altered in ASCre/+::Bmal1 KO male and female mice, with a blunted night/day ratio. ASCre/+::Bmal1 KO female mice displayed decreases in renal sodium excretion in response to low salt, but both male and female KO mice had changes in sodium balance that were time-of-day-dependent. In addition, sex differences were found in adrenal and kidney clock gene expression. Notably, this study highlights sex differences in clock gene expression that could contribute to sex differences in physiological functions.

Rising Prevalence of Obesity and Primary Hyperaldosteronism: Co-incidence or Connected Circumstances Leading to Hypertension? A Narrative Review.

While obesity and its associated complications, mainly diabetes and hypertension, have been the largest public health problems of modern world, the emerging data suggests an increasing prevalence of primary hyperaldosteronism (PA) as one of the most common undiagnosed causes of hypertension. We believe that rising prevalence of PA in the era of high rates of obesity is likely not a chance finding but is deeply intersected with the rising rates of obesity. Higher serum aldosterone concentrations and urinary aldosterone excretion have been observed in patients with increased body mass index or larger waist circumference. The in vitro and pre-clinical studies suggest that adipocytes not only synthesize and secrete aldosterone but also release factors which stimulate production of aldosterone from adrenal glands. Aldosterone excess causing ligand-dependent activation of the mineralocorticoid receptor (MR) has increasingly been recognized as one of the important mechanisms of obesity-related hypertension. The aldosterone excess in these cases can be labelled as acquired hyperaldosteronism to differentiate it from the non-obesity related classical cases of PA. Because of serious consequences, recognizing aldosterone excess in obesity is important, as it gives a more compelling reason for weight loss and guidance to choosing pharmacotherapy wisely. Dietary sodium restriction and mineralocorticoid receptor antagonists play important roles in the management of PA associated with obesity.

ACTH stimulation and oral salt loading tests detect biochemical outcome early after primary aldosteronism surgery.

In primary aldosteronism (PA), the biochemical outcomes of the Primary Aldosteronism Surgical Outcome study are used to assess aldosterone hypersecretion 6-12 months after surgery. However, few studies have investigated whether the outcomes can be predicted in the early postoperative period. In this retrospective study, we evaluated whether the adrenocorticotropin stimulation test (AST) and oral salt loading test (OST) performed immediately after surgery could predict biochemical outcomes 1 year after surgery. We assessed 268 patients with PA who underwent adrenalectomy at our hospital between 2008 and 2020, underwent AST and OST within 15 days of surgery, and were assessed for biochemical outcomes 1 year after surgery. Patients were divided into two groups: biochemical complete success (B-com; n = 219) and incomplete success (B-inc; n = 49). Patients were divided into clinical complete and partial success and absent success groups. The relationships between various AST and OST values and outcomes were analyzed. The B-inc group had significantly higher plasma aldosterone concentration (PAC) and PAC/serum cortisol ratio (PAC/Cort) at baseline and after ACTH loading in AST and 24-hour urine aldosterone in OST than the B-com group. PAC/Cort at 30 min after ACTH loading (area under the curve (AUC) = 0.76) and 24-hour urine aldosterone (AUC = 0.77) were relatively superior predictors of the outcome. Parameters after ACTH loading were better predictors of biochemical and clinical outcomes than baseline. AST and OST immediately after surgery can predict biochemical and clinical outcomes 1 year after surgery in patients with PA.

5042 The Prevalence of Primary Aldosteronism in Incidentally Discovered Adrenocortical Adenomas Irrespective of Blood Pressure: A Prospective Cohort Study

Abstract Disclosure: T. Uslar: None. A. Burnier: None. B. Sanfuentes: None. P. Böhm: None. M. Orellana: None. F.J. Guarda: None. R.I. Olmos: None. Á. Huete: None. N. Mertens: None. C. Besa: None. A. Majerson: None. J. Cartes: None. C.E. Fardella: None. F. Allende: None. S. Solari: None. A. Vaidya: Consulting Fee; Self; Corcept Therapeutics, HRA Pharmaceuticals. R. Baudrand: None. Background: Incidental adrenocortical adenomas are commonly found on imaging. Guidelines recommend screening for primary aldosteronism (PA) only when adrenocortical adenomas are accompanied by hypertension or unexplained hypokalemia. Objective: To prospectively evaluate the prevalence of PA in consecutive patients with incidentally discovered adrenocortical adenomas. Design: Prospective, longitudinal, single-center study. Methods: 254 consecutive patients with incidentally discovered adrenocortical adenomas were studied, without any evidence of autonomous cortisol secretion, excluded via 1mg dexamethasone suppression test. A structured protocol was followed, which involved measurement of plasma aldosterone (PAC), 24h-urinary aldosterone and sodium, plasma renin activity (PRA) and/or direct renin (DRC). Samples were collected in the morning, in a seated posture, after withdrawing interfering medications. Possible PA was defined across a continuum by having a low renin (DRC <10 uIU/mL or PRA < 1ng/mL/hr) in addition to one of the following 3 criteria reflecting the severity of PA: 1) overt PA: aldosterone/renin ratio (ARR) >30, PA >15 ng/dL, and/or 24h urinary aldosterone >10 ug/24h; 2) moderate PA: ARR 20-30 and PAC 10-15 ng/dL; 3) mild PA: ARR <20 and PAC 5-10ng/dL. Adjusted linear regressions were performed. Patients were followed longitudinally to evaluate treatment outcomes with paired T-test. Results: Among patients with incidentally discovered adrenocortical adenomas, 35% (n=89/254) had possible PA. Overt, moderate, and mild PA were identified in 10%, 12%, and 13% of the overall sample, respectively. Notably, one in five of these individuals (20%) were normotensive. There were trends across the 3 phenotypes of PA indicating a continuum of severity, including: systolic blood pressure (152 ±19, 144 ±13, 142 ±16 mmHg, p-trend=0.02), resistant hypertension (56%, 38%, 33% p-trend=0.034), daily defined dose of antihypertensives (DDD) (2.9 ±1.8, 2.1 ±1.5, 1.0 ±0.8 p-trend=0.001), higher PAC (30.7 ±29.4, 10.3 ± 2.7, 7.9 ±2.5ng/dL p-trend<0.001) and urinary aldosterone (18.2 ±12, 5.6 ±3.6, 5.5 ±3.9 ug/24h p-trend<0.001). In longitudinal treatment follow-up (mean 28-months), 83% had a favorable response to treatment (84% medical, 16% adrenalectomy). After treatment, there was a decrease in SBP of -31.3 ±23 mmHg in overt PA, -13.2 ±19 mmHg in moderate PA, and -9.4 ±18 mmHg in mild PA (p<0.001 in all groups). Similar trends were observed for DDD, with a significant increase in renin in all groups. Conclusions: Overt PA, as well as a continuum of PA, was frequently seen in up to 35% of incidentally discovered adrenocortical adenomas. These findings underscore the importance of screening for PA in all patients with adrenocortical adenomas, irrespective of blood pressure and/or hypokalemia. Presentation: 6/1/2024

8399 Adherens Junctions Regulate Aldosterone Production and Zona Glomerulosa Morphogenesis

Abstract Disclosure: M. Berber: None. N.A. Guagliardo: None. P.Q. Barrett: None. B. Haykir: None. V. Chortis: None. K.S. Borges: None. D.L. Carlone: None. D.T. Breault: None. Primary aldosteronism (PA) caused by the autonomous production of aldosterone by zona Glomerulosa (zG) cells of the adrenal gland is the most common cause of endocrine hypertension, which increases the risk of cardiovascular and renal disease, independent of blood pressure. Current treatments with mineralocorticoid receptor antagonists do not fully mitigate these risks. Thus, a better understanding of the mechanisms underlying PA pathogenesis is important for the development of new therapeutic strategies. zG cells are organized in multicellular rosette structures, which are the functional aldosterone-producing units. Rosettes are stabilized by intercellular adherens junctions (AJs) comprised of cadherins, beta-catenin (βCat) and alpha-catenin (αCat), which link to the actin non-muscle myosin II (NMII) network. Recent data implicate AJs and rosettes in aldosterone-producing cell clusters and an unbiased proteomic analysis of aldosterone-producing adenomas detected an upregulation of the GTPase RhoC, an important regulator of AJ stability.We hypothesized that AJs are potent modulators of aldosterone production and zG morphogenesis with important implications for PA. Using the NCI-H295R cell line with confocal microscopic and co-immunoprecipitation assays, we found that potassium (K+) and angiotensin II (AngII) stimulation markedly enhanced the formation and maintenance of AJs. In addition, treatment with inhibitors of Rho-associated kinase (ROCK) (Y-27632) and NMII (blebbistatin) largely abrogated the effects of K+ and AngII stimulation on AJs, suggesting the Rho-ROCK-NMII pathway regulates AJ stability in the zG. Moreover, disruption of AJs, using either blebbistatin or deletion of αCat using CRISPR, blunted the aldosterone response to K+ stimulation (100% and 50%, respectively). Furthermore, zG-specific deletion of αCat (αCat-LOF) in mice led to disruption of AJs, impaired rosette formation and decreased 24-hour urinary aldosterone excretion on standard (Control 5.6±1.7 ng/mg; αCat-LOF 2.9±0.7 ng/mg (aldosterone/creatinine)) or high K+ (Control: 34.7±13.7 ng/mg; αCat-LOF 19.4±9.5 ng/mg (aldosterone/creatinine)) diets. Finally, mice with zG-specific βcat-GOF demonstrated enhanced AJ stability, increased rosette size and zG hyperplasia, which was abrogated in zG-specific bigenic βCat-GOF::αCat-LOF mice, without affecting gene expression of canonical βcat targets. These data indicate that AJs modulate aldosterone production and zG morphology, revealing AJs as a novel molecular target for the treatment of high-risk patients with PA. Presentation: 6/3/2024

8401 Genetic Characterization of a Case of Primary Aldosteronism in an 11-Year-Old Girl carrying a Germline VUS of CACNA1H and a Somatic Mutation of CTNNB1

Abstract Disclosure: S. Parisien-La Salle: None. G. Van Vliet: None. G. Corbeil: None. M. Labrecque: None. M. Tétreault: None. I. Bourdeau: None. Introduction: Primary aldosteronism is a frequent cause of secondary hypertension in adults but is rare in children. We report the case and genetic studies of an 11 yo girl presenting with primary aldosteronism at puberty. Case presentation: An 11-year-old female patient was referred to the pediatric endocrinology clinic for hypertension (166/118). Her past medical history and family history were unremarkable. She reported polyuria, polydipsia and occasional headaches and abdominal pain. Her Tanner stage indicated that puberty was initiated (P2M2). An abdominal CT revealed a 9x10x11cm right adrenal mass with calcifications and a necrotic zone. On [1]8F-FDG PET/CT, the mass had an uptake of 2.9 SUVmax. Laboratory results revealed hypokalemia (K: 2.6 mmol/L [N: 3.5-4.5]) and elevated aldosterone (1712.8 pmol/L [N: 110-1330]) with suppressed renin (0.2 ng/ml/h [N: <5.3]). Her urinary aldosterone level was elevated (114 nmol/day [N: <67]). The rest of the biochemical adrenal panel was normal. Patient underwent a right adrenalectomy by laparotomy. The pathology report showed an adrenal tumor without capsular or vascular invasion. The only malignancy criterion was an area of necrosis and the ki-67% proliferation index was < 1%. Genetic investigations: After informed consent, the patient underwent germline analysis for the chimeric gene CYP11B2/CYP11B1 (Ruhr University Bochum, Germany) and a multigene panel including KCNJ5, CACNA1D, CACNA1H and CLCN2 genes (Fulgent, CA), which were both negative except for a variant of unknown significance (VUS) that was identified in CACNA1H (c.3868G>A, p.Val1290Ile) with a minor allele frequency of 3.291E-5 (GnomAD). Tumor DNA was extracted after adrenalectomy from fresh frozen tissues. Somatic genetic analysis of GNAS, CTNNB1, KCNJ5, ATP1A1, ATP2B3 and CACNA1D was performed using direct Sanger Sequencing (Genome Quebec Innovation Centre) and revealed a heterozygous missence pathogenic variant in the CTNNB1 gene (c.121 A>G p.Thr41Ala). This mutation was absent in leukocyte DNA of the patient. Immunohistochemistry of the adrenal tumor disclosed strong cytoplasmic β-catenin staining in most tumor cells (monoclonal anti-β-catenin antibody; BD Transduction Laboratories). RNA-sequencing of patient tumor RNA was performed (Research Center CHU Québec) and compared to human adrenal total RNA (Clontech, Mountain View, CA). Pathway enrichment analysis showed activation of the Wnt signaling pathway. No variant was identified in GNAQ or GNA11 genes. However, GNRHR was upregulated compared to the control sample with a log2 fold change of 3.96 (p=0.000074). Conclusion: We present a rare pediatric case of an aldosterone producing adenoma harbouring a somatic β-catenin pathogenic variant with upregulation of GNRHR. The implication of the germline CACNA1H VUS still needs to be clarified. Presentation: 6/3/2024

5165 Cushing’s Disease And Primary Aldosteronism Confounded By A Non-functioning Solitary Adrenal Incidentaloma

Abstract Disclosure: J.G. Friedman: None. W. Huang: None. Background: Adrenal incidentalomas are common, affecting ∼2% of the general population. The presence of adrenal incidentaloma necessitates biochemical testing for hypersecretion, however hypercortisolism and/or hyperaldosteronism may not necessarily originate from the nodule that prompted the initial workup. Clinical Case: A 53-year-old man with a history of obesity and hypertension was referred for workup of a 2.4 x 1.4 cm R adrenal nodule that was incidentally discovered on an abdominal CT to rule out pancreatitis. He was noted on examination to have some Cushingoid features. He was on three antihypertensives (amlodipine 10 mg, carvedilol 50 mg, and spironolactone 50 mg) with adequate blood pressure control and no hypokalemia. He denied spells nor exogenous steroid exposure. Initial testing for hypersecretion showed an elevated cortisol 8.7 ug/dL (<1.8) after 1 mg dexamethasone with adequate dexamethasone level, aldosterone concentration (PAC) 9 ng/dL (3-16), plasma renin activity (PRA) 0.7 ng/mL/h (0.25-5.82), potassium of 3.8 mmol/L (3.5-5.1), and normal plasma metanephrines. Further testing revealed elevated 24-hour urinary free cortisol 81.3 mcg/24h (4-50) and elevated late night salivary cortisol 0.10 and 0.13 mg/dL (<0.09). Baseline 8 AM ACTH was not suppressed 16.3 pg/mL (7.2-63.6). DDAVP stimulation resulted in an increase in cortisol and ACTH suggestive of Cushing’s disease, which was confirmed by inferior petrosal sinus sampling (IPSS). Pituitary MRI showed a 3.6 mm L-sided microadenoma. He underwent transsphenoidal resection with pathology consistent with an ACTH-staining pituitary adenoma. Immediate post-op cortisol nadir was 4.9 ug/dL. A few weeks after surgery, he underwent retesting for primary aldosteronism notable for PAC 14 ng/dL, PRA 0.58 ng/mL/h, and K 4.0 mmol/L. After oral salt loading, 24-hour urine aldosterone was 14.4 mcg/24h (<12) with urine sodium 247 mmol/L/24h (>200). He completed AVS which lateralized to the L adrenal gland despite a R adrenal nodule. He underwent L adrenalectomy with significant improvement in blood pressure which is now well-controlled on carvedilol monotherapy with normalization of PRA to 1.94 ng/ml/h. The R adrenal nodule remains stable in size and appearance on follow up imaging. Conclusion: The presence of an adrenal adenoma with concomitant biochemical evidence of hormonal excess cannot inherently be attributed to nodular autonomous function without further workup. Other hormonal assessment including ACTH level and localization studies are necessary prior to any surgical procedure to avoid missed diagnoses of Cushing’s disease and contralateral primary aldosteronism in the presence of a non-functioning adrenal nodule. Presentation: 6/1/2024

O79 DAILY URINARY POTASSIUM EXCRETION IS NOT A FIXED PERCENTAGE OF DAILY POTASSIUM INTAKE

Background and Objective: High potassium intake is associated with a lower blood pressure and lower incidence of cardiovascular events. For estimation of potassium intake, the World Health Organization advices to multiply the 24-hour renal potassium excretion by 1.3, considering the substantial fecal potassium loss. We examined the variation in the required conversion factor in healthy volunteers and evaluated the effect of factors associated with tissue cation storage on the renal potassium excretion. Methods: We performed a post-hoc analysis of the Mars long-term sodium balance studies. Healthy male participants consumed a fixed amount of salt (6, 9 and 12 grams/day for >29 days) and potassium (4 grams/day) for 105 (n=4) and 205 days (n=6). All urine was collected in 24-hour aliquots. We calculated the percentage of potassium intake that was excreted in the corresponding 24-hour urine collection during steady-state. A multivariable mixed-effects model was used to assess associations of this percentage with sodium intake, potassium intake, urinary aldosterone, urinary cortisol and urine volume. Results: The median renal potassium excretion was 80% of potassium intake (IQR 65-97%, range 24-304%), corresponding to a conversion factor of 1.25 (IQR 1.03-1.54). Within individual subjects we also observed a marked day-to-day variation in the percentage of renal potassium excretion: the average intraperson width of the IQR was 25%. Sodium intake (figure A), urinary aldosterone (figure B), urinary cortisol (figure C) and urine volume (figure D) were positively associated with the percentage of renal potassium excretion, whereas potassium intake showed a negative association (figure A). A significant positive interaction between the effects of sodium and potassium intake was present (P<0.001). Conclusions: The percentage of dietary potassium that is excreted via urine varies considerably. During steady-state, this percentage is independently affected by sodium intake, potassium intake, urinary aldosterone, urinary cortisol, and urine volume. Our results suggest that the WHO's fixed 1.3 conversion factor may lead to inaccurate estimations of potassium intake.

P175 ESTIMATION OF SODIUM INTAKE CAN BE IMPROVED BY CORRECTION OF 24H SODIUM EXCRETION FOR 24H URINE VOLUME

Background and Objective: Long-term sodium (Na) balance studies demonstrated that 24 h sodium excretion does not reflect 24 h sodium intake, which complicates the use of 24 h urine sodium measurements for sodium intake estimation. This discrepancy has been linked to infradian rhythms of aldosterone and cortisol, which are responsible for adaptive responses targeted to (renal) water conservation. We hypothesize that correction for these parameters will improve the accuracy of sodium intake estimates. Methods: We used data from two sodium balance studies that controlled dietary salt intake for 105 (4 men) and 205 days (6 men) at levels of 6, 9 and 12 g/day. We tested whether correction of 24 h urine sodium for 24 h urine volume, 24 h urine potassium, 24 h urine urea, 24 h urine aldosterone, 24 h urine cortisol, 24 h urine cortisol/cortisone and/or urine osmolality, increased the accuracy of 24 h sodium intake estimates using forward selection linear mixed-effects analysis with a random intercept per participant. Results: The median difference between 24 h sodium intake and excretion was 26 mmol (IQR 13 - 45 mmol). 24 h urine sodium explained only 49% of the variance in 24 h sodium intake. In 7 out of 10 subjects, 24 h urine volume was of most added value to the model, after 24 h urine sodium, increasing R2 with 0.08±0.03. Including 24 h urine volume in the model significantly improved the model fit (P<0.001, marginal R2 0.55). The addition of urine urea, urine aldosterone and urine osmolality also contributed significantly but the marginal R2 only further increased to 0.56. After correction of 24 h urine sodium for 24 h urine volume, sodium intake could be estimated more accurately: the 50-mmol discrepancy between 24 h sodium intake and excretion was reduced from 18% to 10% and the 25-mmol discrepancy from 52% to 43% (P<0.001, Figure 1). Conclusions: In healthy young males, estimation of sodium intake can be improved by correcting 24 h urine sodium for 24 h urine volume.

Factors associated with water consumption measured using the stable isotope techniques among Japanese adults: a cross-sectional study.

While some dietary guidelines have established daily water requirements for adults, those for older adults are not well defined. Factors associated with water turnover (WT), indicating water requirements and preformed water (PW), referring to fluids consumed from food and beverages, remain poorly understood. Therefore, we aimed to investigate these factors in Japanese adults. This cross-sectional study included 57 participants aged 54-85 years, living in Fukuoka City, Japan. The WT and PW were measured using the doubly labelled water (DLW) methods from August 2015 to September 2015. Dietary intake and physical activity were evaluated using a 3-day dietary record (DR) and a validated triaxial accelerometer, respectively. Fasting blood and urine samples, as well as environmental conditions, were assessed using a validated technique. We evaluated the variables for predicting water consumption by using a multivariate generalised linear model, with forward stepwise selection using these covariates. The median WT and PW were 2842 ml/day and 2227 ml/day, respectively. In the multivariate model, WT (R2 = 0.629) and PW (R2 = 0.621) were moderately predicted by variables such as sex, body weight, moderate-to-vigorous physical activity, water consumption estimated by DR, urinary aldosterone and osmolality, creatinine clearance and wet-bulb globe temperature. The PW estimates from DR were ~40% lower than those from DLW method, despite a significant correlation between the values. WT and PW are associated not only with lifestyle and environmental factors but also with urinary markers and kidney function. These findings provide useful insights into the differences in each individual's water requirement.

Effects of sodium intake, age, gender, blood sampling time on distribution of plasma aldosterone, renin activity, deoxycorticosterone, cortisol, cortisone, and 24 h urinary aldosterone levels in normotensive individuals based on LC-MS/MS.

This study aims to analyze the distribution of plasma aldosterone, renin activity, deoxycorticosterone (DOC), cortisol, cortisone, and 24 h urinary aldosterone (24 h-uAld) levels based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Plasma and 24 h urine were collected from 129 healthy volunteers in Northeast China. The effect of sodium intake, age, gender, blood sampling time on plasma aldosterone concentration (PAC), plasma renin activity (PRA), PAC to PRA ratio (ARR), DOC, cortisol, cortisone, cortisol to cortisone ratio, and 24 h-uAld were investigated by nonparametric test, multiple linear regression and Harris-Boyd's standard deviate test. There was no significant difference observed in 24 h-uAld, PAC (AM), PRA(AM), ARR (AM), DOC (AM), cortisol (AM), cortisone (AM), and cortisol to cortisone (AM) between high and low sodium intake group. Significant differences were observed between morning and afternoon sampling groups in terms of PAC, ARR, DOC, cortisol, and cortisone. Reference intervals (RIs) of 24 h-uAld, PAC (AM) were recommended to be partitioned by gender. RI of PRA was recommended age stratification. We recommend that the same reference interval could be used regardless of sodium intake. Gender is the main influence factor for 24 h-uAld, PAC, and ARR. Age is key influence factor for PRA.

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.

#1429 Daily urinary potassium excretion is not a fixed percentage of daily potassium intake

Abstract Background and Aims High potassium intake is associated with a lower blood pressure and lower incidence of chronic kidney disease. Since a significant amount of potassium is lost via stool, the World Health Organization advices to multiply 24-hour renal potassium excretion by 1.3 for estimation of dietary intake. We analyzed whether this 1.3 factor for potassium intake varies in healthy volunteers and evaluated whether factors associated with tissue cation storage, namely sodium intake, aldosterone, cortisol and urine volume, affect the renal potassium excretion. Method We performed a post-hoc analysis of the long-term sodium balance studies Mars105 and Mars520. Ten healthy male participants consumed a fixed amount of salt (6, 9 and 12 grams/day for >29 days) and potassium (4 grams/day) for 105 (n = 4) and 205 days (n = 6). During the entire study period all urine was collected in 24-hour aliquots. We calculated the percentage of potassium intake that was excreted in the corresponding 24-hour urine collection during steady-state. We assessed whether this percentage was associated with sodium intake, potassium intake, urinary aldosterone, urinary cortisol and urine volume using a multivariable mixed-effects model. Results Overall, the median renal potassium excretion was 80% of potassium intake (IQR 65-97%; range 24-304%). This corresponds to a conversion factor of 1.25 (IQR 1.03-1.54). Within individual subjects we also observed a marked day-to-day variation in the percentage of renal potassium excretion: the average intraperson width of the IQR was 25%. A higher sodium intake was associated with an increased percentage of renal potassium excretion, whereas a higher potassium intake was associated with a decreased percentage of renal potassium excretion. A significant positive interaction between the effects of sodium and potassium intake was present (P < .001; Fig. A). Furthermore, urinary aldosterone (Fig. B), urinary cortisol (Fig. C) and urine volume (Fig. D) were positively associated with the percentage of renal potassium excretion. Conclusion The percentage of consumed potassium that is excreted via urine shows considerable intra- and inter-subject variability. During steady-state this percentage is independently affected by sodium intake, potassium intake, urinary aldosterone, urinary cortisol, and urine volume. Our results suggest that the WHO's fixed 1.3 conversion factor may lead to inaccurate estimations of potassium intake.

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.

Adrenal circadian clock protein BMAL1 regulates aldosterone and renal excretory function in a sex- and time-dependent manner

BMAL1 is a core circadian clock protein that is important for circadian rhythms of physiological function, as well as maintaining physiological homeostasis, including aspects of kidney function. Global Bmal1 knockout (KO) male mice exhibit loss of diurnal rhythm of renal sodium (Na) excretion in response to a normal or high salt diet. However, renal Na excretion rhythm remained intact in kidney-specific Bmal1 KO male mice. The adrenal gland synthesizes hormones, such as aldosterone, which influence renal electrolyte handling. The role of adrenal BMAL1 in the regulation of renal function remains unknown. The study objective was to test the hypothesis that adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na handling. Male and female adrenal-specific aldosterone synthase Cre positive (AS) Bmal1 KO and littermate Cre negative, floxed control mice ( n=5-9/genotype) were acclimated to metabolic cages for 3 days then samples were collected for three days with mice on a normal salt diet followed by 7 days on a low salt diet to challenge the mice to stimulate aldosterone production. Twelve-hour food intake was measured, and urine samples were collected to measure night/day patterns in renal Na balance. Urinary aldosterone excretion was measured by ELISA. Both day and night urinary aldosterone excretion were increased following a low salt diet in control and KO male mice (ANOVA main effect of diet, p<0.05). Male AS Bmal1 KO mice displayed a significant increase in day urinary aldosterone excretion following a normal and low salt diet (ANOVA main effect of genotype, p=0.0230), with a trend for an increase in night urinary aldosterone excretion ( p=0.0571). Interestingly, these genotype effects were not seen in female AS Bmal1 KO mice. Urinary aldosterone was increased following a low salt diet in female mice, but only during the nighttime in control and KO mice ( p=0.0205). Sex differences were also observed in renal Na balance, where Na balance was more positive during the day in male, but not female, AS Bmal1 KO vs. control mice (ANOVA main effect of genotype, p=0.0282). Together, these data suggest adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na balance in a sex-dependent manner. Future work will investigate whether the changes in aldosterone contribute to changes in renal Na balance. This work was supported by the American Heart Association (Postdoctoral FellowshipP0240441 to H.C.; Established Investigator Award 19EIA34660135 to M.G), theRobert and Mary Cade Professorship in Physiology (to M.G.) and the NIH(F32DK121424 to G.R.C.). 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.

DIAGNOSTIC ACCURACY OF 24-HOUR URINARY ALDOSTERONE TO CREATININE RATIO AND 24-HOUR URINARY ALDOSTERONE FOR PRIMARY ALDOSTERONISM IN DIFFERENT AGE GROUP

Objective: The aim of this study was to explore and evaluate the diagnostic accuracy of 24-hour urinary aldosterone to creatinine ratio (UACR) and 24-hour urinary aldosterone and in different age group. Design and method: There were 423 patients eligible and consecutively enrolled in this study. After 1:2 propensity score matching (PSM) analysis, 100 patients were in primary aldosterone (PA) group and 194 patients were in essential hypertension (EH) group. The information of clinical characteristic and biochemistry measurement were collected and analysis. Receiver operator characteristic curve (ROC) was introduced and calculated area under the curve (AUC) to explore the diagnostic accuracy and the optimal cut-off value. Results: There was no significant difference for the average age in PA group (53.38±11.31) and in EH group (52.77±11.25) (P>0.05). The 24-hour UACR and urinary aldosterone was higher in PA group (1.1 ug/(mmol/L) vs. 0.7 ug/(mmol/L), 6.4 ug/24h vs. 4.8 ug/24h, P<0.05) among all age group. The optimal cut-off value of 24-hour UACR was 1.2 ug/(mmol/L) with 0.702 for AUC and 0.300 for the Youden index, in contrast with 11.4 ug/24h, 0.652 and 0.257 for 24-hour UACR. For those 55 years old or below, it was the same for both the two index, while the optimal cut-off value was declined to 0.8 ug/(mmol/L) for 24-hour UACR and 2.7 ug/24h for 24-hour urinary aldosterone among those over 55 years old. Conclusions: 24-hour UACR was a promising index and was better than 24-hour urinary aldosterone on PA diagnosing. More research is needed to explore the underlying influence factors.

Dietary potassium effects on renal calcium transport at single nucleus resolution

Calcium homeostasis is maintained by the coordination of the intestines, bones, and kidneys. Dietary calcium is absorbed in the intestines, enters the bloodstream, and eventually finds its place in the bone reservoir or undergoes filtration by the glomerulus before being reabsorbed throughout the nephron. The late distal convoluted tubule (DCT2) and connecting tubule (CNT) determine the final rate of urinary calcium excretion, since no calcium reabsorption takes place beyond the connecting tubule (CNT). When excess calcium is excreted in the urine, also known as hypercalciuria, it contributes to the development of osteoporosis and the formation of kidney stones. High dietary potassium intake is strongly associated with a lower risk of kidney stone formation. Yet, the precise mechanism by which potassium intake modulates calcium excretion is not clear. To examine the transcriptional changes linking high dietary potassium intake and calcium transport, we applied single-nucleus RNA-sequencing (snRNA-Seq) in enriched distal nephron cells. To achieve the enrichment, we crossed a Calbindin 1 (Calb1)-driven Cre mouse line with the INTACT (for Isolation of Nuclei TAgged in Specific Cell Types) reporter line, which allows the GFP reporter to be expressed at the nuclear envelope of all calbindin 1-expressing cells. As the Calb1-Cre is constitutively expressed, all cells that have calbindin 1 expression at any time point during development will express the reporter. We examined the GFP expression by immunofluorescence, and found that it is expressed along the entire distal nephron from the distal convoluted tubule (DCT), CNT, to the collecting duct (CD). Male Calb1-Cre-INTACT mice were provided either a normal (NK, 1.05%) or a high (HK, 2%) potassium diet for 4 days and kidneys were snap-frozen for targeted snRNA-Seq using 10X Chromium (n=3 mice per group, targeting 10,000 nuclei per mouse). Our snRNA-seq dataset showed 6 major clusters, including DCT (DCT1 and DCT2), CNT (3 subclusters), CD principal cells (3 subclusters), ɑ-intercalated cells (2 subclusters), β-intercalated cells (2 subclusters), and proliferating cells. We curated a calcium score from the expression of known calciotropic genes ( Slc8a1, Vdr, Trpv5, Calb1, S100g, Ryr2, Trpv6) and used it as an index of calcium handling capacity. The calcium score is the highest in DCT2 and CNT along the distal nephron. In all CNT subclusters of the HK-treated animals, the calcium score is higher compared to the NK-treated animals, suggesting more calcium transport in CNT. Given that HK stimulates aldosterone and causes CNT hypertrophy, we compared the results with mice subjected to low dietary potassium (LK) treatment and metolazone (thiazide) treatment. The aim was to delineate the effects of dietary potassium and aldosterone A comprehensive summary of plasma potassium levels, urinary calcium excretion, aldosterone levels, calcium scores, and CNT morphology has been presented in the table below. Notably, the comparative analysis reveals a significant correlation between aldosterone levels and CNT size, both of which exhibit an inverse relationship with urinary calcium excretion: [Formula: see text] Our results suggest that DCT2 and CNT are the major sites for calcium transport. Yet, CNT exhibit more heightened adaptability in response to physiological or pharmacological perturbations at both transcriptional and morphological levels. Calcium homeostasis coincides with the regulation of aldosterone levels and CNT remodeling. DK51496 and DK133220 to DHE; K01DK121737 and AHA 20CDA35320169 to JWN. 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.

Mineralocorticoid receptor and aldosterone: Interaction between NR3C2 genetic variants, sex and age in a mixed cohort.

Hypertension, a prevalent cardiovascular risk, often involves dysregulated aldosterone and its interaction with the mineralocorticoid receptor (MR). Experimental designs in animal models and human cohorts have demonstrated a sex and age dependency of aldosterone secretion that expands our pathophysiologic understanding. This study explores the genetic variation of NR3C2, which encodes MR, in relation to aldosterone, considering age, sex, and race. Incorporating 720 Caucasians and 145 Africans from the HyperPATH cohort, we investigated the impact of rs4835490, a single nucleotide risk allele variant, on aldosterone levels and vasculature. Notably, a significant association between rs4835490 and plasma aldosterone under liberal salt conditions emerged in individuals of European ancestry (P=0.0002). Homozygous carriers of the risk A allele exhibited elevated plasma aldosterone levels (AA=8.1±0.9 vs GG=4.9±0.5 ng/dl). Additionally, aldosterone activation through posture (P=0.025) and urinary excretion (P=0.0122) showed notable associations. Moreover, genetic interactions with race, sex, and age were observed. Caucasian females under 50 years displayed higher plasma aldosterone, urine aldosterone, and posture aldosterone with the AA genotype compared to females over 50 years, suggesting a potential connection with menopausal or estrogen influences. Interestingly, such age-dependent interactions were absent in the African cohort. our study highlights the significance of NR3C2 genetic variation and its interplay with age, sex, and race in aldosterone activation. The findings point towards an estrogen-modulating effect on MR activation, particularly in women underlining the role of aldosterone dysregulation in hypertension development. This insight advances our comprehension of hypertension's complexities and opens avenues for personalized interventions.

Spironolactone improves left atrial function and atrioventricular coupling in patients with resistant hypertension.

To determine the blood pressure independent effects of spironolactone on left atrial (LA) size and function in patients with resistant hypertension (RHTN). Patients with RHTN (n = 36, mean age 55 ± 7) were prospectively recruited. Spironolactone was initiated at 25mg/day and increased to 50mg/day after 4 weeks. Other antihypertensives were withdrawn to maintain constant blood pressure. Cardiac magnetic resonance imaging was performed at baseline and after 6 months of spironolactone treatment and changes in LA functional metrics were assessed. LA size and function parameters were improved (p < 0.05) from baseline to month-6: LA volumes indexed to body surface area (LAVI) were reduced (LAVImaximum 41.4±12 vs. 33.2±9.7 mL/m2; LAVIpre-A 32.6±9.8 vs. 25.6±8.1 mL/m2; median LAVIminimum 18.5 [13.9-24.8] vs. 14.1 [10.9-19.2] mL/m2); left atrioventricular coupling index was reduced (28.2±11.5 vs. 22.7±9.2%); LA emptying fractions (LAEF) were increased (median total LAEF 52.4 [48.7-60.3] vs. 55.9 [50.3-61.1] %; active LAEF 40.2±8.6 vs. 43.1±7.8%). LA global longitudinal strain in the active phase was increased (16.3±4.1 vs. 17.8±4.2%). The effect of spironolactone was similar in patients with high (N = 18) and normal (N = 18) aldosterone status (defined by plasma renin activity and 24-h urine aldosterone). Treatment of RHTN with spironolactone is associated with improvements in LA size and function, and atrioventricular coupling, regardless of whether aldosterone levels were normal or high at baseline. This study suggests the need for larger prospective studies examining effects of mineralocorticoid receptor antagonists on atrial function and atrioventricular coupling.

Associations of maternal exposure to 2,4-dichlorophenoxyacetic acid during early pregnancy with steroid hormones among one-month-old infants

BackgroundExposure to 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used hormonal herbicide, may disrupt steroid hormone homeostasis. However, evidence from population-based studies is limited, especially for one-month-old infants whose steroid hormones are in a state of adjustment to extrauterine life and can be important indicators of endocrine development. This study aimed to explore the associations between maternal 2,4-D exposure during early pregnancy and infant steroid hormone levels. MethodsThe 885 mother-infant pairs were from a birth cohort in Wuhan, China. Maternal exposure to 2,4-D was determined in urine samples from early pregnancy, and nine steroid hormones were determined in infant urine. The associations of maternal 2,4-D exposure with infant steroid hormones and their product-to-precursor ratios were estimated based on generalized linear models, and bioinformatic analysis was conducted with public databases to explore the potential mechanisms involved. ResultsThe detection frequency of 2,4-D was 99.32 %, and the detection frequency of steroid hormones ranged from 98.42 % to 100.00 %. After adjusting for covariates, an interquartile range increase in 2,4-D concentrations was associated with a 7.84 % decrease in 11-deoxycortisol (95 % confidence interval, CI: −14.12 %, −1.10 %), an 8.09 % decrease in corticosterone (95 % CI: −14.56 %, −1.14 %), an 8.67 % decrease in cortisol (95 % CI: −14.43 %, −2.52 %), a 13.00 % decrease in cortisone (95 % CI: −20.64 %, −4.62 %), and an 11.17 % decrease in aldosterone (95 % CI: −19.62 %, −1.83 %). Maternal 2,4-D was also associated with lower infant cortisol/17α-OH-progesterone, cortisol/pregnenolone, and aldosterone/pregnenolone ratios. In bioinformatic analysis, pathways/biological processes related to steroid hormone synthesis and secretion were enriched from target genes of 2,4-D exposure. ConclusionsMaternal urinary 2,4-D during early pregnancy was associated with lower infant urinary 11-deoxycortisol, corticosterone, cortisol, cortisone, and aldosterone, reflecting that 2,4-D exposure may interfere with infant steroid hormone homeostasis. Further efforts are still needed to study the relevant health effects of exposure to 2,4-D, particularly for vulnerable populations.