Circulating Aldosterone Levels Research Articles

Complex equilibrium involving aldosterone underlies the pathophysiology of renovascular hypertension.

The mechanisms underlying the maintenance of hypertension in renovascular hypertension (RVH) are not well understood. To test the current concept of RVH pathophysiology, circulating aldosterone levels in clinical cases were investigated through a literature survey of pediatric cases. Fifty-four patients with documented aldosterone levels were identified. Of these, 42 patients (78%) were assigned to the high renin (HR) group and the rest to the low-normal renin (LR) group. Patients in the HR group were more likely to have unilateral lesions (35/42) than those in the LR group (6/12). In the LR group (corresponding to volume-dependent RVH), 50% (6/12) of patients had elevated aldosterone levels, indicating that the equilibrium between renin and aldosterone shifted towards aldosterone dominance. In the HR group (corresponding to renin-dependent RVH), aldosterone levels were much higher, with 76% (32/42) of patients exceeding the reference range and 14 patients developing hypokalemia. These results are consistent with the notion that pressure natriuresis allows continuous aldosterone action in renin-dependent RVH. In conclusion, the aldosterone status observed in the clinical cases is in agreement with the current understanding of the pathophysiology of RVH, in which a complex equilibrium state involving renin, angiotensin-II, pressure natriuresis, and aldosterone exists.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Kir4.1 deletion prevents salt-sensitive hypertension in early streptozotocin-induced diabetic mice via Na + -Cl - cotransporter in the distal convoluted tubule.

Functional impairment of renal sodium handling and blood pressure (BP) homeostasis is an early characteristic manifestation of type 1 diabetes. However, the underlying mechanisms remain unclear. Metabolic cages, radio-telemetry, immunoblotting, and electrophysiology were utilized to examine effects of high salt (8% NaCl, HS) intake on Na + /K + balance, BP, Na + -Cl - cotransporter (NCC) function, and basolateral K + channel activity in the distal convoluted tubule (DCT) under diabetic conditions. Improper Na + balance, hypernatremia, and a mild but significant increase in BP were found in streptozotocin (STZ)-induced diabetic mice in response to HS intake for 7 days. Compared to the vehicle, STZ mice showed increased Kir4.1 expression and activity in the DCT, a more negative membrane potential, higher NCC abundance, and enhanced hydrochlorothiazide-induced natriuretic effect. However, HS had no significant effect on basolateral Kir4.1 expression/activity and DCT membrane potential, or NCC activity under diabetic conditions, despite a downregulation in phosphorylated NCC abundance. In contrast, HS significantly downregulated the expression of Na + -H + exchanger 3 (NHE3) and cleaved epithelial sodium channel-γ in STZ mice, despite an increase in NHE3 abundance after STZ treatment. Kir4.1 deletion largely abolished STZ-induced upregulation of NCC expression and prevented BP elevation during HS intake. Interestingly, HS causes severe hypokalemia in STZ-treated kidney-specific Kir4.1 knockout (Ks-Kir4.1 KO) mice and lead to death within a few days, which could be attributed to a higher circulating aldosterone level. We concluded that Kir4.1 is required for upregulating NCC activity and may be essential for developing salt-sensitive hypertension in early STZ-induced diabetes.

Lysine specific demethylase 1 deficiency modifies aldosterone synthesis in a sex-specific manner.

Biologic sex influences the development of cardiovascular disease and modifies aldosterone (ALDO) and blood pressure (BP) phenotypes: females secrete more ALDO, and their adrenal glomerulosa cell is more sensitive to stimulation. Lysine-specific demethylase 1 (LSD1) variants in Africans and LSD1 deficiency in mice associate with BP and/or ALDO phenotypes. This study, in 18- and 40-week-old wild type (WT) and LSD1+/- mice, was designed to determine whether: 1) sex modifies ALDO biosynthetic enzymes; 2) LSD1 deficiency disrupts the effect of sex on these enzymes; 3) within each genotype, there is a positive relationship between ALDO biosynthesis (proximate phenotype), plasma ALDO (intermediate phenotype) and BP levels (distant phenotype); and 4) sex and LSD1 genotype interact on these phenotypes. In WT mice, female sex increases the expression of early enzymes in ALDO biosynthesis, but not ALDO levels or SBP. However, enzyme expressions are shifted downward in LSD1+/- females vs. males, so that early enzyme levels are similar but the late enzymes are substantially lower. In both age groups, LSD1 deficiency modifies the adrenal enzyme expressions, circulating ALDO levels and SBP in a sex specific manner. Finally, significant sex/LSD1 genotype interactions modulate the three phenotypes in mice. In conclusion, biologic sex in mice interacts with LSD1 deficiency to modify several phenotypes: 1) proximal (ALDO biosynthetic enzymes); 2) intermediate (circulating ALDO); and 3) distant (SBP). These results provide entrée to better understand the roles of biologic sex and LSD1 in: 1) hypertension heterogeneity and 2) in providing more personalized treatment.

A safety and pharmacodynamic study of the highly selective aldosterone synthase inhibitor PB6440 in the cynomolgus monkey

Abstract Background Aldosterone is an important mediator of hypertension, particularly resistant hypertension, heart failure, and chronic kidney disease. PB6440 is a potent inhibitor of aldosterone synthase (CYP11B2) with high selectivity over the closely related enzyme CYP11B1. In previous studies in cynomolgus monkeys, PB6440 exhibited excellent oral bioavailability and a marked suppression of aldosterone synthesis at doses as low as 1 mg/kg/day. Importantly, no effect was observed on cortisol production nor significant changes noted in plasma concentrations of the steroid precursors 11-deoxycortisol or deoxycorticosterone (DOC), which are dependent on CYP11B1 activity. The purpose of the current study was to assess the safety and pharmacodynamics of higher doses of PB6440 in the cynomolgus monkey in order to determine a therapeutic index prior to initiation of studies in humans. Methods Male and female cynomolgus monkeys (2/sex/dose group) were administered 0, 10, 30 or 100 mg/kg PB6440 once-daily for 14 days by oral gavage. Assessment of safety was based on mortality, clinical observations, body weights, and clinical and anatomic pathology. Blood samples were also collected for pharmacodynamic analysis, including aldosterone, cortisol, 11-deoxycortisol, DOC, and ACTH. Results PB6440 was well tolerated at all doses. There were no deaths and no reports of clinical observations at any dose level. Mild decreases in body weight were observed in PB6440-treated animals, which were likely due to the diuretic effects of the compound. PB6440 caused an increase in adrenal weights which was associated with hypertrophy of the adrenal zona fasciculata, a finding which has previously been observed with other aldosterone synthase inhibitors. PB6440 led to marked declines in basal aldosterone levels of −97%, −97% and −98% from baseline at 10, 30 and 100 mg/kg/day, respectively, at 24-hours following the final dose. Despite these clear declines in circulating aldosterone levels, no meaningful changes in cortisol, 11-deoxycortisol, DOC, or ACTH levels were observed, supporting lack of CYP11B1 inhibition. PB6440 led to expected decreases in plasma sodium (up to 7 nmol/L at the high dose). No effect on plasma potassium levels were observed at any dose level. Conclusions PB6440 was well tolerated in the cynomolgus monkey at doses that are much higher than needed for potent aldosterone suppression based on previous studies. At the highest dose level tested (100 mg/kg), which led to a 98% reduction in basal aldosterone levels, no evidence of significant CYP11B1 inhibition was observed. The absence of an increase in plasma potassium in the presence of a plasma sodium decline merits further study. Thus, PB6440 is a highly selective novel aldosterone synthase inhibitor for the potential treatment of hypertension, heart failure, and chronic kidney disease in humans. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): PhaseBio Pharmaceuticals Inc.

Mineralocorticoid Receptor Antagonists Cause Natriuresis in the Absence of Aldosterone.

MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. We studied AS (aldosterone synthase)-deficient (AS-/-) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR-/-) mice. KS-MR-/- mice were used to compare MR expression and ENaC localization and cleavage with AS-/- mice. MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR-/- mice but were fully preserved along DCT2 and were partially reduced along CCD in AS-/- mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS-/- mice and were not increased along CCD after low salt. AS-/- mice exhibited transient Na+ wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS-/- mice led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR-/- mice. Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS-/- mice.

Loss of Female Sex Hormones Does Not Further Elevate Blood Pressure in Obese Female Mice but Preserves the Contribution of Leptin to Hypertension

Obesity, which is a major risk factor for hypertension and cardiovascular disease, abolishes the cardioprotective effects of female sex hormones and predisposes women to hypertension. Although 40% of postmenopausal women are obese and 75% are hypertensive, the mechanisms whereby obesity and menopause interact to elevate blood pressure (BP) remains unknown. Formerly, our laboratory demonstrated that hypertension involves leptin‐mediated increases in aldosterone production in obese female mice of reproductive age. Herein, we hypothesized that loss of female sex hormones with ovariectomy (OVX) will further elevate BP and impairs vascular function in obese female mice. Obese agouti yellow mice (Ay) on a KK background and lean controls underwent OVX or sham surgery at 12‐week of age. At 15 weeks, mice were implanted with radiotelemeters to record BP under baseline conditions and in response to leptin receptor blockade (Allo‐Aca, 0.05mg/kg/day s.c. osmotic minipump). To ascertain the effects of OVX on autonomic control of BP, we recorded BP and heart rate (HR) responses to ganglionic blockade (hexamethonium), atropine, and propranolol. At 18 weeks, mice were euthanized and mesenteric arteries isolated to measure endothelial function via wire myography. Obesity significantly increased mean arterial pressure (MAP), systolic BP, diastolic BP, and HR but OVX did not further elevate BP. Interestingly, obesity also disrupted circadian rhythm. Unexpectedly, OVX significantly reduced MAP response to ganglionic blockade (2‐way ANOVA, P<0.05) in obese mice but preserved HR responses to propranolol and atropine respectively suggesting lower neurogenic control of BP. Allo‐Aca treatment significantly decreased (2‐way ANOVA, P<0.05) MAP in both sham and OVX obese mice, indicating BP elevation remains leptin‐dependent. Obesity significantly impaired relaxation to acetylcholine (2‐way ANOVA, P<0.05) but OVX did not further impair relaxation. Compared to lean controls, LNAME further reduced (2‐way ANOVA, P=0.054) relaxation responses to acetylcholine in obese OVX mice suggesting greater contribution of nitric oxide synthase activity to vascular relaxation. Quantitative real‐time PCR showed a trend for decreased adrenal aldosterone synthase (CYP11B2) expression in obese OVX mice. Interestingly, menopause induced a similar decrease in CYP11B2 expression in human adrenal tissue collected from kidney donors with elevated BMIs (postmenopausal 56 yo female: BMI 27.4, vs. premenopausal 43 yo female, BMI 32). Quantification of mouse plasma aldosterone levels via LC‐MS revealed no significant difference in circulating aldosterone levels, suggesting that tissue(s) other than the adrenals could potentially produce aldosterone in obese OVX mice. Plasma analysis also revealed Allo‐Aca treatment increased ANG II, ANG I and ANG 1‐7 in obese mice. In conclusion, these data suggest that obesity‐associated hypertension remains leptin dependent in the absence of female sex hormone and that female sex steroids play limited role in the control of the development of hypertension associated with obesity.

Eplerenone‐induced natriuresis and hyperkalemia in mice lacking aldosterone results from aldosterone‐independent mineralocorticoid receptor occupancy

BackgroundMineralocorticoid receptor (MR) antagonists are recommended for patients with resistant hypertension independent of circulating aldosterone levels. Aldosterone, secreted in response to salt depletion or hyperkalemia, activates MR to increase epithelial sodium channel (ENaC) activity. While glucocorticoids can also activate MR, they are metabolized by 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), protecting MR from illicit glucocorticoid occupation. 11β‐HSD2 is expressed at increasing levels from the DCT through the collecting duct. Here, we hypothesized that glucocorticoid occupies MR to stimulate ENaC in the DCT2 and early connecting tubule.MethodsWe studied aldosterone synthase knockout (AS−/−) mice provided by Dr. JM Luther. Kidney–specific MR knockout (MR−/−) mice were used for validation of Western blot for ENaC and immunofluorescence for MR. We measured plasma and urinary electrolytes at baseline, and following dietary salt restriction with or without administration of the MR antagonist eplerenone.ResultsAS−/− mice displayed higher plasma K+ levels than AS+/+ mice but not salt wasting under baseline. Under low salt (LS) diet, AS−/− mice exhibited higher urine Na+ excretion, but preserved body weight. Cleaved αENaC and γENaC abundances were lower in AS−/− than AS+/+ mice, and were not increased in AS−/− mice after LS diet. Notably, we found nuclear localization of MR was preserved in DCT2 of AS−/− mice. Administration of the MR blocker eplerenone to AS−/− mice fed LS diet led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice.ConclusionsOur results provide evidence that MR is occupied in the absence of aldosterone, suggesting glucocorticoid‐binding to MR preserves sodium homeostasis in the DCT2 of AS−/− mice. This likely explains the milder phenotype of AS−/− mice compared with MR−/− mice.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

The Herbicide Atrazine Potentiates Angiotensin II-Induced Aldosterone Synthesis and Release From Adrenal Cells.

Atrazine is one of the most commonly used pre-emergence and early post-emergence herbicides in the world. We have shown previously that atrazine does not directly stimulate the pituitary or adrenal to trigger hormone release but acts centrally to activate a stress-like activation of the hypothalamic-pituitary-adrenal axis. In doing so, atrazine treatment has been shown to cause adrenal morphology changes characteristic of repeated stress. In this study, adrenals from atrazine treated and stressed animals were directly compared after 4 days of atrazine treatment or restraint stress. Both atrazine and stressed animals displayed reduced adrenocortical zona glomerulosa thickness and aldosterone synthase (CYP11B2) expression, indicative of repeated adrenal stimulation by adrenocorticotropic hormone. To determine if reduced CYP11B2 expression resulted in attenuated aldosterone synthesis, stressed and atrazine treated animals were challenged with angiotensin II (Ang II). As predicted, stressed animals produced less aldosterone compared to control animals when stimulated. However, atrazine treated animals had higher circulating aldosterone concentrations compared to both stressed and control groups. Ang II-induced aldosterone release was also potentiated in atrazine pretreated human adrenocortical carcinoma cells (H295R). Atrazine pretreated did not alter the expression of the rate limiting steroidogenic StAR protein or angiotensin II receptor 1. Atrazine treated animals also presented with higher basal blood pressure than vehicle treated control animals suggesting sustained elevations in circulating aldosterone levels. Our results demonstrate that treatment with the widely used herbicide, atrazine, directly increases stimulated production of aldosterone in adrenocortical cells independent of expression changes to rate limiting steroidogenic enzymes.

Acquired hypoaldosteronism as classified by circulating aldosterone levels: characteristics

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Mineralocorticoid Receptor Antagonists Eplerenone and Spironolactone Modify Adrenal Cortex Morphology and Physiology.

Mineralocorticoid receptor antagonists (MRAs) are a class of anti-hypertensive drugs that act by blocking aldosterone action. The aim of this study was to evaluate whether the MRAs spironolactone and eplerenone influence adrenal cortical physiology and morphology. Spontaneous hypertensive rats (SHR, n = 18) and normotensive rats (WKY, n = 18) were randomly exposed to a daily dose of spironolactone (n = 6), eplerenone (n = 6), or no drug (n = 6) over 28 days. After that, aldosterone, corticosterone, and 11-deoxycorticosterone plasma concentrations were quantified. Adrenal glands were subjected to morphological analysis to assess lipid droplets content, capsular width, cell proliferation, and steroidogenic proteins expression. The adrenal cortex in untreated SHR showed higher lipid droplet content as than in WKY. In SHR, MRA treatment was associated with higher circulating aldosterone levels and Ki-67 expression in aldosterone-secreting cells. In WKY, the only difference observed after MRA spironolactone treatment was a narrower capsule. There was no difference in abundance of steroidogenic enzyme between groups. In conclusion, MRAs modify adrenal gland function and morphology in SHR. The effects observed within the adrenal glomerulosa with aldosterone-secreting cell proliferation and higher circulating aldosterone levels suggests that MRA treatment provokes activation of the renin angiotensin system. The prognostic value of hyperaldosteronism secondary to MRAs blockade requires further investigation.

Variability of Aldosterone Measurements During Adrenal Venous Sampling for Primary Aldosteronism.

Variability of aldosterone concentrations has been described in patients with primary aldosteronism. We performed a retrospective cohort study of 340 patients with primary aldosteronism who underwent adrenal venous sampling (AVS) at a tertiary referral center, 116 of whom also had a peripheral venous aldosterone measured hours before the procedure. AVS was performed by the same interventional radiologist using bilateral, simultaneous sampling, under unstimulated and then stimulated conditions, and each sample was obtained in triplicate. Main outcome measures were: (i) change in day of AVS venous aldosterone from pre-AVS to intra-AVS and (ii) variability of triplicate adrenal venous aldosterone concentrations during AVS. Within an average duration of 131 minutes, 81% of patients had a decline in circulating aldosterone concentrations (relative decrease of 51% and median decrease of 7.0 ng/dl). More than a quarter (26%) of all patients had an inferior vena cava aldosterone of ≤5 ng/dl at AVS initiation. The mean coefficient of variation of triplicate adrenal aldosterone concentrations was 30% and 39%, in the left and right veins, respectively (corresponding to a percentage difference of 57% and 73%), resulting in lateralization discordance in up to 17% of patients if the lateralization index were calculated using only one unstimulated aldosterone-to-cortisol ratio rather than the average of triplicate measures. Circulating aldosterone levels can reach nadirs conventionally considered incompatible with the primary aldosteronism diagnosis, and adrenal venous aldosterone concentrations exhibit acute variability that can confound AVS interpretation. A single venous aldosterone measurement lacks precision and reproducibility in primary aldosteronism.

Nicotine-induced adrenal beta-arrestin1 upregulation mediates tobacco-related hyperaldosteronism leading to cardiac dysfunction.

BACKGROUNDTobacco-related products, containing the highly addictive nicotine together with numerous other harmful toxicants and carcinogens, have been clearly associated with coronary artery disease, heart failure, stroke, and other heart diseases. Among the mechanisms by which nicotine contributes to heart disease is elevation of the renin-angiotensin-aldosterone system (RAAS) activity. Nicotine, and its major metabolite in humans cotinine, have been reported to induce RAAS activation, resulting in aldosterone elevation in smokers. Aldosterone has various direct and indirect adverse cardiac effects. It is produced by the adrenal cortex in response to angiotensin II (AngII) activating AngII type 1 receptors. RAAS activity increases in chronic smokers, causing raised aldosterone levels (nicotine exposure causes the same in rats). AngII receptors exert their cellular effects via either G proteins or the two βarrestins (βarrestin1 and-2).AIMSince adrenal ßarrestin1 is essential for adrenal aldosterone production and nicotine/cotinine elevate circulating aldosterone levels in humans, we hypothesized that nicotine activates adrenal ßarrestin1, which contributes to RAAS activation and heart disease development.METHODSWe studied human adrenocortical zona glomerulosa H295R cells and found that nicotine and cotinine upregulate βarrestin1 mRNA and protein levels, thereby enhancing AngII-dependent aldosterone synthesis and secretion.RESULTSIn contrast, siRNA-mediated βarrestin1 knockdown reversed the effects of nicotine on AngII-induced aldosterone production in H295R cells. Importantly, nicotine promotes hyperaldosteronism via adrenal βarrestin1, thereby precipitating cardiac dysfunction, also in vivo, since nicotine-exposed experimental rats with adrenal-specific βarrestin1 knockdown display lower circulating aldosterone levels and better cardiac function than nicotine-exposed control animals with normal adrenal βarrestin1 expression.CONCLUSIONAdrenal βarrestin1 upregulation is one of the mechanisms by which tobacco compounds, like nicotine, promote cardio-toxic hyperaldosteronism in vitro and in vivo. Thus, adrenal βarrestin1 represents a novel therapeutic target for tobacco-related heart disease prevention or mitigation.

Allopurinol and valproic acid improve cardiac triglyceride and Na+-K+-ATPase activity independent of circulating aldosterone in female rats with glucose intolerance

Context: Studies have shown that cardiac triglyceride accumulation and impaired Na+-K+-ATPase activity are linked to diabetes- related cardiovascular disease, particularly in women.Objectives: We hypothesised that allopurinol (ALL) and valproic acid (VPA) treatment would improve cardiac triglyceride and Na+-K+-ATPase activity independent of circulating aldosterone in Combined Oral Contraceptive (COC)-induced dysglycemiaMaterials and methods: Rats received COC (1.0 μg ethinylestradiol and 5.0 μg levonorgestrel; po) with or without ALL (1 mg; po) and VPA (20 mg; po) for 6 weeks.Results: COC-treatment led to impaired glucose tolerance, accumulated abdominal fat, dyslipidemia, elevated plasma MDA, PAI-1 and aldosterone levels and also reduced plasma nitric oxide bioavailability and cardiac Na+-K+-ATPase activity. However, either ALL or VPA treatment ameliorated these alterations comparably independent of elevated aldosterone levelDiscussion and conclusion: Our results suggest that either ALL or VPA would improve cardiac TG and Na+-K+-ATPase activity comparably in COC-treated rats, regardless of circulating aldosterone level.

OR34-03 Variable and Pulsatile Circulating Aldosterone Levels in Primary Aldosteronism: Implications for Diagnosis and Sub-Type Differentiation

Introduction: Considerable intra-individual variability in circulating aldosterone levels has been observed in patients with primary aldosteronism (PA). The magnitude and implications of this phenomenon are not well characterized. Objective: To evaluate the acute variability in aldosterone in patients with confirmed PA. Methods: 373 patients with confirmed PA underwent adrenal venous sampling (AVS) after appropriate catheterization of bilateral adrenal veins. Peripheral venous aldosterone levels were measured 2 hours prior to AVS while in supine posture. After anesthesia induction with fentanyl and midazolam, AVS was performed while in the same supine posture, and aldosterone levels were drawn from the inferior vena cava (IVC) and in triplicate from the bilateral adrenal veins over 10 minutes. Differences between the pre-AVS and intra-AVS IVC aldosterone levels were analyzed, and regression models used to identify independent predictors of change. Coefficients of variation (COV) between triplicate aldosterone levels in each adrenal vein were calculated. Results: 81% of patients demonstrated a decrease in aldosterone concentration from pre-AVS to the intra-AVS IVC measurement. The mean decrease in aldosterone was 10.5 ng/dL (95% CI: 7.6–13.3) and the mean relative decrease in aldosterone was 39% (95% CI: 27–51%, P<0.0001). The absolute decrease in aldosterone was striking, with 48% of patients who had a decrease in aldosterone exhibiting an IVC aldosterone of less than or equal to 5 ng/dL. The absolute decrease in aldosterone was significantly associated with a higher aldosterone level (p<0.001) and lower systolic blood pressure at diagnosis (p=0.02). A wide variation in triplicate aldosterone values was seen in the span of 10-minute sampling, ranging from 1–300%, with COV of 21.0% in the left adrenal vein and 25.0% in the right adrenal vein. If the lowest of three aldosterone-to-cortisol (A/C) ratios on the dominant side and highest of three A/C ratios on the contralateral side were used instead of the average of the three values, the interpretation of the AVS would have changed from unilateral PA to bilateral PA in 15.9% of cases. Conclusions: These findings underscore the pulsatile and variable nature of circulating aldosterone levels in patients with bona fide PA. Aldosterone levels substantially declined in 81% of patients within a period of 2 hours while maintaining a fixed and supine posture. In half of these patients, aldosterone levels declined to 5 ng/dL or below, a threshold typically considered incompatible with PA. Further, adrenal venous aldosterone levels exhibited large variations on repeated sampling within a 10-minute span that could have influenced the interpretation of sub-type differentiation in nearly 16% of cases. Single circulating aldosterone values lack precision and reproducibility and may result in incorrect diagnosis and sub-type differentiation.

Upregulation of Renal ENaC Activity in Spontaneously Hypertensive Rats Is Independent of Aldosterone

Spontaneously hypertensive rat (SHR) has been widely used as a model to interrogate the contribution of the kidney to the pathogenesis of essential hypertension in humans. Elevation of blood pressure in SHR has been associated with altered renal function, since predisposition to hypertension can be transferred with a transplanted kidney into a normotensive Wistar‐Kyoto (WKY) strain. Renal defects result in excessive water and sodium retention leading to increased circulating volume and blood pressure. Abnormally elevated Na+ reabsorption in SHR is associated with the overactive renin‐angiotensin‐aldosterone system (RAAS) and with the increased abundance of the renal epithelial Na+ channel (ENaC) responsible for final regulation of sodium reuptake in the kidney and pivotal for long‐term blood pressure control. The existing data on regulation of ENaC by RAAS components in SHR is inconsistent and molecular determinants of excessive ENaC activation remain largely obscure. In the present study, we tested the efficacy of aldosterone antagonism to inhibit ENaC activity, decrease renal Na+ reabsorption and reduce blood pressure in SHR of both sexes. Where necessary, age‐matched (12–15 week‐old) WKY rats were used as controls. Consistent with previously published data, enzyme immunoassay analysis revealed elevated circulating aldosterone levels and semi‐quantitative immunoblotting – higher expression of renal ENaC protein in SHR when compared to WKY controls. Patch‐clamp experiments in split‐opened collecting ducts showed that ENaC activity was moderately increased in SHR when compared to WKY. Systemic administration of aldosterone antagonist, spironolactone (30 mg/kgBW·day) did not affect renal ENaC activity or protein abundance in SHR. Aldosterone antagonism did not alter the expression of sodium‐chloride co‐transporter (NCC) in the kidneys of SHR. Tail‐cuff plethysmography showed that treatment with spironolactone at doses 30–200 mg/kgBW·day for 2 weeks failed to decrease blood pressure in SHR and did not result in any detectable changes in urinary sodium excretion. We did not observe any sex‐specific differences pertaining to ENaC activity or responsiveness to spironolactone in SHR males and females. Thus, we conclude that aldosterone is not a primary driver of excessive ENaC‐dependent renal Na+ reabsorption in SHR.

The adrenal cortex after estradiol or daidzein application in a rat model of the andropause: Structural and hormonal study

Introduction and AimDaidzein application may represent an effective and less harmful alternative to indicated, classical estrogenization of ageing men. The aim of this study was to perform structural and hormonal analysis of the adrenal cortex, after estradiol or daidzein supplementation in a rat model of the andropause. Material and methodsMiddle-aged Wistar rats were divided into sham operated (SO; n = 8), orchidectomized (Orx; n = 8), estradiol treated orchidectomized (Orx + E; n = 8) and daidzein treated orchidectomized (Orx + D; n = 8) groups. Estradiol (0.625 mg/kg b.m./day) or daidzein (30 mg/kg b.m./day) were administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using stereology, histochemistry/immunohistochemistry, immunoassays and ultrastructural analysis. ResultsBoth estradiol and daidzein treatment significantly increased volumes of the zona glomerulosa cell and nuclei, but decreased circulating aldosterone levels. Estradiol markedly increased volumes of the zona fasciculata cell and nuclei in parallel with significant decrease of the adrenal tissue level of corticosterone, while daidzein significantly decreased both the adrenal and circulating levels of corticosterone. Serum DHEA level and volumes of the zona reticularis cell and nuclei significantly increased upon estradiol treatment, whereas daidzein even stronger increased the circulating level of DHEA. Shunting of the corticosteroidogenesis pathways towards adrenal androgens production, after the treatments, corresponded to the ultrastructural findings and zonal capillary network rearrangements. ConclusionsGiven the coherence of its effects and relative safety, daidzein could be the remedy of choice for the treatment of ageing-caused androgen deprivation and the hypothalamo-pituitary-adrenal axis hyperfunction/related metabolic issues in males.

GRK2-Mediated Crosstalk Between β-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein βarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. β-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the βAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the βAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol’s effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the β-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a βAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

SHORT-TERM CHANGES IN DIETARY SODIUM INTAKE INFLUENCE SWEAT SODIUM CONCENTRATION AND MUSCLE SODIUM CONTENT IN HEALTHY SUBJECTS

Objective: There is increasing evidence that sodium can be stored in the skin and muscles without being osmotically active, yet whether acute changes in dietary sodium intake alter sweat and muscle sodium content has not been investigated previously. Design and method: In a cross-over design, we assessed muscle sodium content by 23Na magnetic resonance imaging (MRI) in 38 healthy normotensive volunteers (aged 33.5 ± 11.1 years, 76.3% female) after 5 days of high sodium diet (HS) (6 g of salt added to their normal diet) and 5 days of a low-sodium diet (LS). Women performed each study visit during the same phase of the menstrual cycle. In a sub-group of 18 participants (72.2% female) we conducted quantitative pilocarpine iontophoretic sweat collections and measured the sodium concentration in sweat. Office blood pressure, electrolytes, creatinine and circulating aldosterone levels were measured in all participants, and 24 h urine was collected Results: Under HS conditions urinary sodium excretion, muscle sodium content and sweat sodium concentration all increased significantly (see Figure). Muscle sodium content by MRI (rm = 0.47, p = 0.03) and sodium sweat concentration (rm = 0.72, p < 0.001) correlated positively with salt intake as estimated by 24-hour urine sodium excretion. Age, gender or the phase of the menstrual cycle did not influence muscle or sweat sodium concentrations or their changes. In contrast, plasma aldosterone levels were strongly and negatively associated with both muscle sodium content (rs = −0.42, p = 0.0001) and sweat sodium (rs = −0.52, p = 0.002). Sweat and muscle sodium content were not different in participants with a salt-sensitive blood pressure compared to non-sensitives. Conclusions: Muscle and sweat sodium concentrations are significantly higher under high salt conditions in healthy male and female subjects, suggesting that muscle and sweat play a role in the dietary sodium balance in humans.