Endogenous Aldosterone Research Articles

FRI134 Transcriptomic Profiling Of Monocytes To Seek A Potential Biomarker For Primary Aldosteronism

Abstract Disclosure: R. Libianto: None. N. Karthigan: None. J. Morgan: None. D. Powell: None. P.J. Fuller: None. M.J. Young: None. J. Yang: None. Primary aldosteronism (PA), the most common endocrine cause of hypertension, is associated with increased cardiovascular risk compared with essential hypertension. The diagnosis of PA currently relies on measurements of plasma aldosterone and renin levels which can be compromised by patient- or assay-related issues and relies on arbitrary thresholds. A cellular marker that reflects excess endogenous aldosterone activity could enhance the diagnostic process. Mineralocorticoid receptor (MR) activation in macrophages has previously been shown to play a key role in mediating cardiovascular injury. Macrophage precursors, the monocytes, also express MR and can be easily isolated from circulation. We therefore sought to identify unique transcriptomic markers of aldosterone excess in peripheral blood monocytes. Monocytes were isolated from nine male patients with unilateral PA before and 3 months after curative adrenalectomy. Each patient’s post-adrenalectomy samples served as the control for comparison with the pre-adrenalectomy samples. RNA sequencing (RNA-seq) was performed on monocyte RNA samples to identify differentially expressed genes (DEGs), followed by evaluation using a second method by real-time quantitative PCR (RT-qPCR). Distinct clustering of gene expression was observed between the “before” and “after-surgery” samples with 1679 significantly differentially expressed (false discovery rate <0.05). The top 26 DEGs were selected for evaluation by RT-qPCR. Differences were observed in the relative expression of phosphoglycerate mutase 1 (PGAM1), ferrochelatase (FECH), and nuclear enriched abundant transcript 1 (NEAT1) genes pre- and post-surgery. The ratios of the relative expression of these genes accentuated the difference between the before- and after-surgery samples. In conclusion, differential gene expression was found between monocytes of patients with unilateral PA before and after adrenalectomy. Further validation studies are needed in female patients and those with bilateral PA and essential hypertension. These transcriptomic markers or their ratios have the potential to serve as novel biomarkers of aldosterone excess. Presentation: Friday, June 16, 2023

Bisphenol A exposure and reduced male sex hormone levels in a Norwegian population

Background: Bisphenol A (BPA) is used in production of polycarbonate plastics and epoxy resins. Despite endocrine disrupting properties and suspected impact on male reproductive health BPA is still frequently used in a wide variety of consumer products. Few studies have addressed BPA exposure and steroid hormones in a general population sample. We aimed to investigate whether BPA exposure is associated with steroid hormone concentrations (17β-estradiol, estrone, estrone 3-sulfate, progesterone, progesterone 17α-OH-progesterone, testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEAS), aldosterone, cortisone and cortisol) in a healthy Norwegian population. Methods: The study included 205 women and 232 men aged 18-48 years, participating in the RHINESSA study (2014-15) in Norway. Urine concentration of BPA and steroid hormones in serum were measured using liquid chromatography - mass spectrometry. We applied linear mixed effects random intercept models with sex hormone levels (one at a time) as outcome, BPA concentration as exposure and adjusted for age, sex, BMI, smoking and time of day for urine and blood sampling. Results: Bisphenol A was detected in 96% of the urine samples; and median concentration was 1.32 ng/mL (5th-95th percentile: 0.50-5.15, max: 21.9 ng/mL). Increasing exposure to BPA (per ng/mL) was associated with decreasing levels of testosterone (-11.72 pmol/cL, 95% Confidence Interval (CI): -22.93, -0.51) and DHEAS (-6.83 nmol/cL, 95% CI: -12.72, -0.94) for men and women combined. Restricting the analyses to men, increasing exposure to BPA was associated with decreasing levels of 17β-estradiol (-0.88 pmol/L, 95% CI: -1.72, -0.04) and with elevated endogenous aldosterone levels (4.32 pmol/L, 95% CI: 0.91, 7.73). Conclusions: The urinary biomarker concentration of BPA is ubiquitous, but relatively low. BPA was associated with an altered hormone profile, in particular with reduced levels of androgens as well as with 17β-estradiol which is synthesized from androgen precursors. Whether the association is of clinical relevance needs to be elucidated.

Abstract P039: The Endogenous Ouabain And Changes In Blood Pressure And Sodium Excretion Following An Acute Saline Load In Essential Hypertension

Among ~25% of patients with essential hypertension (EH), circulating endogenous ouabain (EO) and aldosterone (Aldo) are typically coelevated and their BP is especially salt-sensitive (SS). We probed for exaggerated natriuresis in a large cohort (712) of hypertensive patients that underwent an acute 2hr (T 120 ) load with saline (0.9%/2 L). Results: Using a quartiles analysis of sodium excretion, the 4th quartile subgroups showed higher SBP and DBP at T 120 , and at 2 hrs recovery (T 240 ). Fractional sodium excretion (FE Na) was significantly higher during loading and recovery in the 4 th quartile subgroup. Plasma Aldo was suppressed after saline in all subgroups as expected. SS patients showed an increased EO. In the 4 th quartiles EO was elevated at baseline and after saline (see figure), p Manova > 0.001 and the urinary Na/K ratio output was higher. Further, increases in plasma Na + were greater in the 4 th quartile group (1.91 ± 0.15 vs 0.83 ± 0.13 mM, p<0.001). These data suggest circulating EO is a potential mediator of the exaggerated natriuresis in SS EH, and that this phenomenon is reminiscent of an “aldosterone escape”.

Neuronal aldosterone elicits a distinct genomic response in pain signaling molecules contributing to inflammatory pain

BackgroundRecently, mineralocorticoid receptors (MR) were identified in peripheral nociceptive neurons, and their acute antagonism was responsible for immediate and short-lasting (non-genomic) antinociceptive effects. The same neurons were shown to produce the endogenous ligand aldosterone by the enzyme aldosterone synthase.MethodsHere, we investigate whether endogenous aldosterone contributes to inflammation-induced hyperalgesia via the distinct genomic regulation of specific pain signaling molecules in an animal model of Freund’s complete adjuvant (FCA)-induced hindpaw inflammation.ResultsChronic intrathecal application of MR antagonist canrenoate-K (over 4 days) attenuated nociceptive behavior in rats with FCA hindpaw inflammation suggesting a tonic activation of neuronal MR by endogenous aldosterone. Consistently, double immunofluorescence confocal microscopy showed abundant co-localization of MR with several pain signaling molecules such as TRPV1, CGRP, Nav1.8, and trkA whose enhanced expression of mRNA and proteins during inflammation was downregulated following i.t. canrenoate-K. More importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by continuous intrathecal delivery of a specific aldosterone synthase inhibitor prevented the inflammation-induced enhanced transcriptional expression of TRPV1, CGRP, Nav1.8, and trkA and subsequently attenuated nociceptive behavior. Evidence for such a genomic effect of endogenous aldosterone was supported by the demonstration of an enhanced nuclear translocation of MR in peripheral sensory dorsal root ganglia (DRG) neurons.ConclusionTaken together, chronic inhibition of local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons may contribute to long-lasting downregulation of specific pain signaling molecules and may, thus, persistently reduce inflammation-induced hyperalgesia.

Aldosterone Synthase in Peripheral Sensory Neurons Contributes to Mechanical Hypersensitivity during Local Inflammation in Rats.

Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.

Enabling insights into the maturation of the renin–angiotensin–aldosterone system in children—Development of a low-volume LC–MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite

IntroductionAs part of the renin–angiotensin–aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. MethodsA HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight–mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. ResultsUsing the Sciex TripleTOF 6600, a reliable determination in 50 µL serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between −1.21 and −6.99 % and 2.07 and −10.22 %, respectively, confirming compliance with international guidelines. ConclusionA simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 µL serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.

Salt-sensitive transcriptome of isolated kidney distal tubule cells.

In the distal kidney tubule, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel (ENaC). Most studies seeking to identify ENaC-regulating aldosterone-induced proteins have used transcriptional profiling of cultured cells. To identify salt-sensitive transcripts in an in vivo model, we used low-NaCl or high-NaCl diet to stimulate or suppress endogenous aldosterone, in combination with magnetic- and fluorescence-activated cell sorting to isolate distal tubule cells from mouse kidney for transcriptional profiling. Of the differentially expressed transcripts, 162 were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, and 161 were more abundant in distal tubule cells isolated from mice fed high-NaCl diet. Enrichment analysis of Gene Ontology biological process terms identified multiple statistically overrepresented pathways among the differentially expressed transcripts that were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, including ion transmembrane transport, regulation of growth, and negative regulation of apoptosis. Analysis of Gene Ontology molecular function terms identified differentially expressed transcription factors, transmembrane transporters, kinases, and G protein-coupled receptors. Finally, comparison with a recently published study of gene expression changes in distal tubule cells in response to administration of aldosterone identified 18 differentially expressed genes in common between the two experiments. When expression of these genes was measured in cortical collecting ducts microdissected from mice fed low-NaCl or high-NaCl diet, eight were differentially expressed. These genes are likely to be regulated directly by aldosterone and may provide insight into aldosterone signaling to ENaC in the distal tubule.

Canine hypoadrenocorticism - an update on pathogenesis, diagnosis and treatment

Canine hypoadrenocorticism (HoAC) results from a loss of functional adrenal cortex, the most common etiology of which is an immune-mediated destruction leading to an inadequate production of glucocorticoids and mineralocorticoids. The term "atypical" HoAC is used for a subgroup of dogs with either an isolated glucocorticoid deficiency or a combined glucocorticoid and mineralocorticoid deficiency but normal electrolytes. Dogs with HoAC can present with a large variety of clinical signs, ranging from shaking, weakness, and mild gastrointestinal signs to seizures, hypovolemic shock, and collapse. Routine clinicopathologic and diagnostic imaging findings are usually nonspecific and frequently mimic those of other common diseases. However, the absence of a stress leukogram, eosinophilia, hyponatremia, hyperkalemia, and azotemia and small adrenal glands on abdominal ultrasound are characteristic findings in dogs with HoAC. The ACTH stimulation test is currently the gold standard method for diagnosing HoAC. Other endocrine laboratory diagnostics, including the quantification of endogenous ACTH, basal and ACTH-stimulated aldosterone levels, cortisol:ACTH ratio, and aldosterone:renin ratio, may further aid in differentiating between primary, secondary, and "atypical" HoAC. Aggressive intravenous fluid therapy is the cornerstone of treatment in paients with an acute Addisonian crisis because it restores normovolemia and normal blood electrolytes. Maintenance therapy consists of glucocorticoid (e.g., prednisolone) and mineralocorticoid (e.g., des- oxycortone pivalate) supplementation and aims for stable electrolyte concentrations and a clinically well dog. The optimal dose of desoxy- cortone pivalate for a specific dog is determined based on blood so- dium and potassium concentrations by using a standardized protocol. Regular reevaluation of blood electrolytes is required for early identifi- cation of a mineralocorticoid deficiency in dogs with "atypical" HoAC. The long-term prognosis for dogs with HoAC is excellent provided that patients receive adequate treatment and there is good owner com- pliance.

Aldosterone‐Induced Transcripts Identified from Rapidly Isolated Collecting Duct Cells

In the distal nephron, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel ENaC. Previous attempts to identify the aldosterone‐induced proteins that mediate the action of aldosterone on ENaC have primarily used transcriptional profiling of cultured cells. Here, we present a method to rapidly isolate collecting duct cells from the mouse kidney for transcriptional profiling. Our objective is to use this method, in combination with dietary sodium manipulation, to identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone.An antibody against the cell adhesion molecule L1‐CAM was previously used for fluorescence activated cell sorting (FACS) of collecting duct cells from human kidney. We verified L1‐CAM is also expressed in the collecting ducts of mouse kidney using immunofluorescence. We next developed a protocol for physical and enzymatic separation of mouse kidney into a single cell suspension. The kidney cells were labeled with L1‐CAM magnetic microbeads as well as antibodies against markers for endothelial cells (CD31) and immune cells (CD45). This allowed isolation of an L1‐CAM+ cell population using magnetic activated cell sorting (MACS), followed by separation of collecting duct cells from L1‐CAM+ endothelial and immune cells using FACS. RNA was purified from the FACS‐isolated collecting duct cells and, following poly(A) selection, used to generate Illumina TruSeq libraries. The libraries were sequenced on the Illumina HiSeq platform and the reads were aligned to the mouse genome using the Subread aligner. The aligned reads were mapped to transcripts using feature Counts and differentially expressed transcripts are identified using EdgeR.To identify transcripts that are differentially expressed in the collecting ducts of mouse kidney under conditions of high versus low plasma aldosterone, C57BL/6J mice were fed sodium‐deficient diet (to stimulate endogenous aldosterone) or high‐sodium diet (to suppress endogenous aldosterone). After 5 days of dietary sodium manipulation, the kidneys were harvested, collecting duct cells were isolated, and RNA was sequenced and analyzed according to the above protocol. Of the differentially expressed transcripts, 234 were up‐regulated by aldosterone (more highly expressed in the sodium‐deficient diet samples) while 276 were down‐regulated by aldosterone (more highly expressed in the high‐sodium diet samples); false discovery rate < 0.05, no log2FC or log2CPM threshold. The transcriptome data were then compared with recently published single‐cell RNA‐seq data for the major collecting duct cell types, specifically intercalated cells and principal cells. It was found that 16 of the up‐regulated transcripts and 32 of the down‐regulated transcripts were highly expressed in principal cells, thereby limiting the number of candidate transcripts for subsequent validation. Of the transcripts that were highly expressed in principal cells, Sgk1 was the most differentially expressed transcript induced by aldosterone.Support or Funding InformationU.S. Department of Veterans Affairs: I01BX002228This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dietary salt blunts vasodilation by stimulating epithelial sodium channels in endothelial cells from salt-sensitive Dahl rats.

Our recent studies show that the reduced activity of epithelial sodium channels (ENaC) in endothelial cells accounts for the adaptation of vasculature to salt in Sprague-Dawley rats. The present study examines a hypothesis that enhanced ENaC activity mediates the loss of vasorelaxation in Dahl salt-sensitive (SS) rats. We used the cell-attached patch-clamp technique to record ENaC activity in split-open mesenteric arteries. Western blot and immunofluorescence staining were used to evaluate the levels of aldosterone, ENaC, eNOS and NO. Blood pressure was measured with the tail-cuff method and the artery relaxation was measured with the wire myograph assay. High-salt (HS) diet significantly increased plasma aldosterone and ENaC activity in the endothelial cells of Dahl SS rats. The endothelium-dependent artery relaxation was blunted by HS challenge in these rats. Amiloride, a potent blocker of ENaC, increased both phosphorylated eNOS and NO and therefore prevented the HS-induced loss of vasorelaxation. As, in SS rats, endogenous aldosterone was already elevated by HS challenge, exogenous aldosterone did not further elevate ENaC activity in the rats fed with HS. Eplerenone, a mineralocorticoid receptor antagonist, attenuated the effects of HS on both ENaC activity and artery relaxation. These data suggest that HS diet blunts artery relaxation and causes hypertension via a pathway associated with aldosterone-dependent activation of ENaC in endothelial cells. This pathway provides one of the mechanisms by which HS causes hypertension in Dahl SS rats. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Aldosterone regulates a 5' variant sgk1 transcript via a shared hormone response element in the sgk1 5' regulatory region.

We previously identified a 5ʹ variant alternate transcript of Sgk1 (Sgk1_v3) encoding an NH 2‐terminal variant Sgk1 isoform, Sgk1_i3 that, like Sgk1, is expressed in the distal convoluted tubule, connecting tubule and collecting duct and can stimulate epithelial Na+ transport (Am J Physiol Renal Physiol 303: F1527–F1533, 2012). We now demonstrate that, similar to Sgk1, aldosterone and glucocorticoids stimulate Sgk1_v3 expression in cell lines from the collecting duct and airway epithelia. In mice, short term aldosterone infusion and maneuvers that increase endogenous aldosterone secretion including dietary Na+ deprivation and K+ loading increases distal nephron Sgk1_v3 expression in vivo. Although Sgk1_v3 has a different 5ʹ proximal regulatory region from Sgk1, the transcription start sites are less than 1000 bp apart. We cloned the 5ʹ regulatory region for Sgk1 and Sgk_v3 upstream of a luciferase gene and by deletion and reporter gene analysis we localized the corticosteroid regulatory region for Sgk1_v3 to a glucocorticoid response element (GRE) that had previously been identified for Sgk1 (Am J Physiol Endo Metab 283: E971–E979, 2002). We tested this element with MR in an MR‐null cell line and demonstrate that aldosterone stimulates Sgk1 and Sgk1_v3 via this GRE. We conclude that corticosteroids stimulate Sgk1 and Sgk1_v3 expression in epithelial cells via activation of a common conserved GRE in the 5ʹ flanking region of Sgk1.

Dietary sodium restriction decreases insulin secretion without affecting insulin sensitivity in humans.

Interruption of the renin-angiotensin-aldosterone system prevents incident diabetes in high-risk individuals, although the mechanism remains unclear. To test the hypothesis that activation of the endogenous renin-angiotensin-aldosterone system or exogenous aldosterone impairs insulin secretion in humans. We conducted a randomized, blinded crossover study of aldosterone vs vehicle and compared the effects of a low-sodium versus a high-sodium diet. Academic clinical research center. Healthy, nondiabetic, normotensive volunteers. Infusion of exogenous aldosterone (0.7 μg/kg/h for 12.5 h) or vehicle during low or high sodium intake. Low sodium (20 mmol/d; n = 12) vs high sodium (160 mmol/d; n = 17) intake for 5-7 days. Change in acute insulin secretory response assessed during hyperglycemic clamps while in sodium balance during a low-sodium vs high-sodium diet during aldosterone vs vehicle. A low-sodium diet increased endogenous aldosterone and plasma renin activity, and acute glucose-stimulated insulin (-16.0 ± 5.6%; P = .007) and C-peptide responses (-21.8 ± 8.4%; P = .014) were decreased, whereas the insulin sensitivity index was unchanged (-1.0 ± 10.7%; P = .98). Aldosterone infusion did not affect the acute insulin response (+1.8 ± 4.8%; P = .72) or insulin sensitivity index (+2.0 ± 8.8%; P = .78). Systolic blood pressure and serum potassium were similar during low and high sodium intake and during aldosterone infusion. Low dietary sodium intake reduces insulin secretion in humans, independent of insulin sensitivity.

Feedforward activation of endothelial ENaC by high sodium.

Kidney epithelial sodium channels (ENaCs) are known to be inactivated by high sodium concentrations (feedback inhibition). Recently, the endothelial sodium channel (EnNaC) was identified to control the nanomechanical properties of the endothelium. EnNaC-dependent endothelial stiffening reduces the release of nitric oxide, the hallmark of endothelial dysfunction. To study the regulatory impact of sodium on EnNaC, endothelial cells (EA.hy926 and ex vivo mouse endothelium) were incubated in aldosterone-free solutions containing either low (130 mM) or high (150 mM) sodium concentrations. By applying atomic force microscopy-based nanoindentation, an unexpected positive correlation between increasing sodium concentrations and cortical endothelial stiffness was observed, which can be attributed to functional EnNaC. In particular, an acute rise in sodium concentration (+20 mM) was sufficient to increase EnNaC membrane abundance by 90% and stiffening of the endothelial cortex by 18%. Despite the absence of exogenous aldosterone, these effects were prevented by the aldosterone synthase inhibitor FAD286 (100 nM) or the mineralocorticoid receptor (MR)-antagonist spironolactone (100 nM), indicating endogenous aldosterone synthesis and MR-dependent signaling. Interestingly, in the presence of high-sodium concentrations, FAD286 increased the transcription of the MR by 69%. Taken together, a novel feedforward activation of EnNaC by sodium is proposed that contrasts ENaC feedback inhibition in kidney.

Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L, CCR7, and CXCR4 expression

The role of mineralocorticoid receptors (MRs) in human T-cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR-agonist fludrocortisone or placebo and numbers of eight T-cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4+, central memory CD4+, and naïve CD8+ T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T-cell migration and suggest a role for endogenous aldosterone in the redistribution of T-cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T-cell homing following sleep-dependent aldosterone release might thus essentially contribute to sleep's well-known role in supporting adaptive immunity.

An Individualized Exercise Prescription to Attenuate Symptoms Associated With Postural Orthostatic Tachycardia Syndrome

An Individualized Exercise Prescription to Attenuate Symptoms Associated With Postural Orthostatic Tachycardia Syndrome

Expression of mineralocorticoid and glucocorticoid receptors in preautonomic neurons of the rat paraventricular nucleus.

Activation of mineralocorticoid receptors (MR) of the hypothalamic paraventricular nucleus (PVN) increases sympathetic excitation. To determine whether MR and glucocorticoid receptors (GR) are expressed in preautonomic neurons of the PVN and how they relate to endogenous aldosterone levels in healthy rats, retrograde tracer was injected into the intermediolateral cell column at T4 to identify preautonomic neurons in the PVN. Expression of MR, GR, 11-β hydroxysteroid dehydrogenase1 and 2 (11β-HSD1, 2), and hexose-6-phosphate dehydrogenase (H6PD) required for 11β-HSD1 reductase activity was assessed by immunohistochemistry. RT-PCR and Western blot analysis were used to determine MR gene and protein expression. Most preautonomic neurons were in the caudal mediocellular region of PVN, and most expressed MR; none expressed GR. 11β-HSD1, but not 11β-HSD2 nor H6PD immunoreactivity, was detected in the PVN. In rats with chronic low or high sodium intakes, the low-sodium diet was associated with significantly higher plasma aldosterone, MR mRNA and protein expression, and c-Fos immunoreactivity within labeled preautonomic neurons. Plasma corticosterone and sodium and expression of tonicity-responsive enhancer binding protein in the PVN did not differ between groups, suggesting osmotic adaptation to the altered sodium intake. These results suggest that MR within preautonomic neurons in the PVN directly participate in the regulation of sympathetic nervous system drive, and aldosterone may be a relevant ligand for MR in preautonomic neurons of the PVN under physiological conditions. Dehydrogenase activity of 11β-HSD1 occurs in the absence of H6PD, which regenerates NADP(+) from NADPH and may increase MR gene expression under physiological conditions.

Chronic hyperaldosteronism in Cryptochrome-null mice induces high-salt- and blood pressure-independent kidney damage in mice

Although aldosterone has an essential role in controlling electrolyte and body fluid homeostasis, aldosterone also exerts certain pathological effects on the kidney. Several previous studies have attempted to examine these deleterious effects. However, the majority of these studies were performed using various injury models, including high-salt treatment and/or mineralocorticoid administration, by which the kidney changes observed were not only due to aldosterone but also due to prior injury caused by salt and hypertension. In the present study, we investigated aldosterone's pathological effect on the kidney using a mouse model with a high level of endogenous aldosterone. We used cryptochrome-null (Cry 1, 2 DKO) mice characterized by high aldosterone levels and low plasma renin activity and observed that even under normal salt exposure conditions, these mice showed increased albumin excretion and kidney tubular injury, decreased nephrin expression and increased reactive oxygen species production in the absence of hypertension. Exposure to high salt levels exacerbated the kidney damage observed in these mice. Moreover, we noted that decreasing blood pressure without blocking aldosterone action did not provide beneficial effects to the kidney in high-salt-treated Cry 1, 2 DKO mice. Thus, our findings support the hypothesis that aldosterone has deleterious effects on the kidney independent of high-salt exposure and high blood pressure.

Role of Circadian Rhythms in Potassium Homeostasis

It has been known for decades that urinary potassium excretion varies with a circadian pattern. In this review, we consider the historical evidence for this phenomenon and present an overview of recent developments in the field. Extensive evidence from the latter part of the past century clearly shows that circadian potassium excretion does not depend on endogenous aldosterone. Of note is the recent discovery that the expression of several renal potassium transporters varies with a circadian pattern that appears to be consistent with substantial clinical data regarding daily fluctuations in urinary potassium levels. We propose the circadian clock mechanism as a key regulator of renal potassium transporters, and consequently renal potassium excretion. Further investigation into the regulation mechanism of renal potassium transport by the circadian clock is warranted to increase our understanding of the clinical relevance of circadian rhythms to potassium homeostasis.

Effects of mineralocorticoid receptor signaling during sleep on the expression of CD62L and CCR7 on naïve T cells

Sleep supports adaptive immune functions. However, the underlying mechanisms are still obscure. Animal studies point to a supporting effect of sleep on the migration of T cells to lymph nodes, where adaptive immunity is generated. In line with this assumption, we were able to show in humans that sleep, compared to nocturnal wakefulness, acutely reduces the number of naı¨ve T cells in blood. This effect is likely mediated via mineralocorticoid receptors (MR), the blockade of which mimics the effect of nocturnal wakefulness on cell numbers. To elucidate the mechanism by which MR signaling affects naı¨ve T cell distribution, we focused on the expression of the ‘homing receptor’ L-selectin (CD62L) and the chemokine receptor CCR7 that are both essential in the migratory cascade that directs these cells to lymph nodes. To this end, blood from 13 sleeping healthy men was incubated either with an MR agonist (fludrocortisone), an MR antagonist (spironolactone) or PBS as control. After two and four hours of incubation, the expression of CD62L and CCR7 on CD4 + and CD8 + naı¨ve T cells was examined via flow cytometry. Preliminary results show that the MR antagonist reduced the expression of both CD62L and CCR7. Accordingly, the agonist slightly increased the expression of CD62L whereas CCR7 levels were unaffected, probably reflecting a ceiling effect due to already high endogenous aldosterone levels. We propose that sleep-dependent activation of MR enhances the migration of naı¨ve T cells from blood to lymph nodes by increasing the expression of CD62L and CCR7 on these cells.

Aldosterone deficiency prevents high-fat-feeding-induced hyperglycaemia and adipocyte dysfunction in mice.

Obesity is associated with aldosterone excess, hypertension and the metabolic syndrome, but the relative contribution of aldosterone to obesity-related complications is debated. We previously demonstrated that aldosterone impairs insulin secretion, and that genetic aldosterone deficiency increases glucose-stimulated insulin secretion in vivo. We hypothesised that elimination of endogenous aldosterone would prevent obesity-induced insulin resistance and hyperglycaemia. Wild-type and aldosterone synthase-deficient (As (-/-)) mice were fed a high-fat (HF) or normal chow diet for 12weeks. We assessed insulin sensitivity and insulin secretion using clamp methodology and circulating plasma adipokines, and examined adipose tissue via histology. HF diet induced weight gain similarly in the two groups, but As (-/-) mice were protected from blood glucose elevation. HF diet impaired insulin sensitivity similarly in As (-/-) and wild-type mice, assessed by hyperinsulinaemic-euglycaemic clamps. Fasting and glucose-stimulated insulin were higher in HF-fed As (-/-) mice than in wild-type controls. Although there was no difference in insulin sensitivity during HF feeding in As (-/-) mice compared with wild-type controls, fat mass, adipocyte size and adiponectin increased, while adipose macrophage infiltration decreased. HF feeding significantly increased hepatic steatosis and triacylglycerol content in wild-type mice, which was attenuated in aldosterone-deficient mice. These studies demonstrate that obesity induces insulin resistance independently of aldosterone and adipose tissue inflammation, and suggest a novel role for aldosterone in promoting obesity-induced beta cell dysfunction, hepatic steatosis and adipose tissue inflammation.