Urinary Aldosterone Concentration Research Articles

PS-C26-8: UNIQUE PROPERTIES OF DEPRESSOR, RENAL, AND ALDOSTERONE RESPONSES TO ALISKIREN THERAPY IN HYPERTENSIVE PATIENTS WITH NEPHROPATHY

Objective: Blood pressure (BP) control using renin-angiotensin system (RAS) inhibitor as a first choice is recommended in hypertension accompanied by chronic kidney disease (CKD), and RAS localized in the kidney is known to exist independent of circulating RAS. Aliskiren, direct renin inhibitor (DRI), is most excellent in kidney tissue migration among RAS inhibitors despite it has been reported to cause aldosterone breakthrough like angiotensin II receptor blocker (ARB) in essential hypertension. Therefore, DRI-induced antihypertensive, renoprotective and aldosterone-suppressing effects were investigated as compared to those of ARB in hypertensive CKD patients. Design and method: 168 hypertensive patients with CKD (stage G1–4) were employed in this study. Home BP in the morning, estimated glomerular filtration rate (eGFR), urinary protein/creatinine ratio (UPCR), urinary liver-type fatty-acid-binding protein (U-LFABP) as an indicator of tubular dysfunction, plasma and urinary aldosterone concentration (PAC and UAC) were measured before and 0.5 to 5 years after administration of ARB or DRI and assessed retrospectively. Aldosterone breakthrough was defined as any increase in PAC from the baseline level following treatment with a RAS inhibitor. Results: In ARB-treated group, BP and UPCR decreased significantly at 6 months, but sufficient declines were not observed 1st year onward. In DRI-treated group, a significant reduction in BP was sustained until 3 years and BP unchanged decreasing tendency even 4 years later, meanwhile UPCR decreased significantly for 2 years and was below the pretreatment value 3 years after. ARB raised BP and UPCR again in patients with CKD G3–4 prior to those with CKD G1–2, however, DRI expressed no difference between stages. Although PAC diminished up to 6 months in ARB group and 1 year in DRI group, it turned up 1 year and 2 years after, respectively. PAC-ascensive rate and maximum elevation in PAC were higher and greater in ARB group than in DRI group (48.7 vs 22.6%, 56.1 vs 29.3pg/ml). All significant differences in rising PAC cases disappeared by 5 years, whereas in non-rising PAC cases, only DRI reduced U-LFABP without deterioration in eGFR and UAC further lowered while PAC was maintained. Conclusions: In comparison with ARB, aliskiren prolongs hypotensive and proteinuria-lowering effects with retained eGFR regardless of CKD severity possibly via improvement of tubular disorders and less PAC reascent in hypertensive nephropathy. Aliskiren given to CKD patients might elicit mild and delayed aldosterone breakthrough rather than ARB due to a direct action against aldosterone derived from the kidney.

PS-C21-9: REVISITING ORAL SALT LOADING TEST: A NEW CHEMILUMINESCENT IMMUNOASSAY OF 24-HOUR URINE ALDOSTERONE MEASUREMENT

Primary aldosteronism (PA) is widely recognized as the most frequent cause of secondary hypertension, and its prevalence ranges from 5% to 10% in hypertensive patients. The number of patients diagnosed with PA has increased due to more frequent screening in JAPAN. PA is diagnosed by a screening test, followed by a confirmatory or exclusionary test and a subtype test using computed tomography and adrenal vein sampling. Measuring of the urinary aldosterone concentration is important in oral sodium loading test (OSLT), one of the confirmatory tests. Therefore, an accurate aldosterone measurement method is required for clinical practice. Since April 2021, the plasma aldosterone concentration has been measured by chemiluminescent enzyme immunoassay (CLEIA) in Japan. In the present study, we developed a new CLEIA using a two step sandwich method to measure the 24 hour urine aldosterone level. We collected urine samples and measured 24 hour urine aldosterone levels employing radioimmunoassay (RIA), CLEIA, and liquid chromatography tandem mass spectrometry (LC MS/MS). The results showed that the 24-hour urine aldosterone levels measured using CLEIA and LC MS/MS were significantly correlated. The 24 hour urine aldosterone levels measured using CLEIA and RIA were also significantly correlated. However, the aldosterone level measured by CLEIA was lower than that measured by RIA (slope, 0.729; intercept, 120.9). In Japan, a new guideline for primary aldosteronism has been announced, with changes in the aldosterone measurement method, and the cutoff values for oral sodium loading test (OSLT) were changed. Therefore, we examined the cut off value of the 24 hour urine aldosterone level after the OSLT. Receiver operating characteristic analysis revealed a cut off value for primary aldosteronism of 3 mcg/day.

Utility of Urinary Ratio of Cortisol to Aldosterone as Inflammatory and Metabolic Parameters

Corticosteroids are important bioactive substances in the body that regulate inflammation, metabolism, immunity, and circulatory dynamics. Cortisol and aldosterone are two major naturally occurring steroids, which synthesized in the adrenal fasciculata and in the adrenal glomerulosa under the regulation of adrenocorticotropin (ACTH) and renin-angiotensin system (RAS), respectively. The conventional strategy for evaluating adrenocortical function has been to measure random serum cortisol or 24-hour stored levels of urine free cortisol (UFC). UFC is a more appropriate marker to avoid the effects of diurnal fluctuation of cortisol, serum cortisol-binding proteins, and cortisol clearance in the kidney. Thus, measurement of UFC has been a reliable test for diagnosis of Cushing’s disease or adrenal insufficiency. However, since the normal range of UFC varies widely, it is often difficult to evaluate whether the UFC level is optimal or not for each patient. That is because UFC is greatly affected by the amount of fluid intake or urine volume, and an immunoassay for UFC, which is the usual method for measuring UFC, is susceptible to interference from other steroid metabolites and synthetic glucocorticoids. To explore an alternative indicator, we tried to standardize the levels of UFC by the levels of urinary aldosterone concentration (UAC) in the same urinary sample. Medical records of all 246 patients in whom daily excretions of UFC and UAC had been measured between 2015 and 2018 at our department were reviewed. 142 patients (including 93 females) were included after exclusion of 104 patients because of corticosteroid replacement therapy. UFC/UAC ratio showed significant positive and negative correlations with the levels of serum cortisol (R=0.287) and aldosterone (R=-0.762), respectively. UFC/UAC ratio increased with aging in female patients, while the ratio was not altered by the levels of BMI in either gender. Markers for metabolic and inflammatory status including hemoglobin A1c (R=0.327), albumin (R=-0.331), chorine esterase (R=-0.248), C-reactive protein (R=0.317), ferritin (R=0.473), and D-dimer (R=0.569) showed correlations to the ratio of UFC/UAC that were more significant than the correlations to the serum level of cortisol or UFC alone. Of note, the UFC/UAC ratio was shown as an indicator for risks of diabetes (AUC: 0.765), hypoalbuminemia (0.839), hyper-CRPemia (0.748), and thrombophilia (0.824), in which the cut-off levels of UFC/UAC ratios were found to be around 12. These results indicate that the UFC/UAC ratio is a suitable variable for the detection of metabolic and inflammatory complications related to adrenocortical dysfunction.

SAT-552 Epigenetic Regulation of 11beta-Hydroxysteroid Dehydrogenase 1 and 2 Gene in Salt-Sensitive Hypertensive Rats

ENDO 2020Epigenetic regulation of 11beta-hydroxysteroid dehydrogenase 1 and 2 gene in salt-sensitive hypertensive rats[Objective]11Beta-hydroxysteroid dehydrogenase type1 (11-HSD1) is the modulator of glucocorticoid hormone and type2 (11-HSD2) is the modulator of mineralocorticoid hormone. We investigated the effect of high salt diet on the methylation of both enzyme gene in salt-sensitive hypertensive (SSH) rats [Methods]SSH rats were fed a high (7% NaCl) or normal (0.45%) salt chow for 4 weeks. Body weight, blood pressure, plasma and urinary aldosterone concentration and PRA were measured. DNA was extracted from kidneys and visceral fats. Bisulfite sequencing and Pyrosequencing were done for the analysis of methylation status of 11-HSD1 and 2 gene. [Results] High salt diet significantly decreased methylation ratio of 11-HSD1 gene in the visceral fats of SSH rats compared with controls (p<0.05). The methylation ratio of 11-HSD2 gene in the kidney of SSH rats was not influenced by high salt diet. [Discussion and Conclusion]11-HSD1 overexpression in visceral fats in mice was reported to show SSH. We reported decreased 11-HSD2 activity in the artery in SSH rats. In this study high salt diet affected methylation status of 11-HSD1 in the adipose tissue but not 11-HSD2 gene in the kidney in SSH. Food intake such as salt may influence the epigenesis of 11-HSD and induce hypertension.

Urinary Angiotensinogen is Essential for Renal Potassium Handling

Apart from the well‐known systemic renin‐angiotensin system (RAS), the urinary RAS acts as a local autocrine/paracrine system along the nephron. The regulation of renal K+ handling by systemic vs urinary RAS remains unclear. Liver‐specific (Alb‐Cre) angiotensinogen knockout (LKO) and liver + proximal tubule (Alb‐Cre + KAP‐Cre) angiotensinogen knockout (DKO) were utilized in this study. WT, LKO, and DKO mice were kept on either a regular diet (RD) or a high K diet (HK; 5% K+) for 10 days. Mice were acclimated to metabolic cages twice before collecting urine for 24 hours. Mice were then euthanized to collect blood and kidneys. Plasma and urinary [Na+] and [K+] were measured by flame photometry. Plasma and urinary angiotensin II (Ang II) concentrations were measured by ELISA (Enzo Life Sciences). Plasma and urinary aldosterone (Aldo) concentrations were measured by ELISA (DRG Diagnostics). Urinary creatinine (Cr) concentrations were measured by colorimetric assays (Cayman Chemical). Plasma [Ang II] was significantly lower in LKO, compared to WT, and the value in DKO was not significantly different from zero. Urinary [Ang II] was significantly lower in DKO compared to WT and LKO. Plasma [K+] was significantly higher in DKO but not LKO compared to WT on HK. Urinary K:Cr ratio was significantly lower in DKO compared to WT on HK. However, plasma and urinary [Aldo] were not different between DKO and WT on HK. Overall, our results showed that urinary RAS but not systemic RAS plays an essential role in renal K handling.Support or Funding InformationThis project was funded by F30 DK108456.

PP.23.04] RESPONSES OF THE RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM TO PLASMA VOLUME DEPLETION IN PRE-ECLAMPSIA &amp; SUPERIMPOSED PRE-ECLAMPSIA

Objective: Women with chronic kidney disease (CKD) are at increased risk of superimposed pre-eclampsia (SPE) and associated adverse pregnancy outcomes. Diagnosis of SPE using blood pressure and proteinuria is of limited use because these may develop in women with CKD even without the condition. Inaccurate diagnosis may result in unnecessary iatrogenic preterm delivery. Plasma and urinary aldosterone concentrations are elevated in patients with CKD, but suppressed in established pre-eclampsia (PE). The aim of the study was to determine plasma active renin, total plasma and urinary angiotensinogen (AGT) and aldosterone availability in women with CKD with and without SPE compared with normotensive controls (NC) and those with PE without CKD. Design and method: NC women (n = 19), women with PE (n = 14), CKD without SPE (n = 6) and with SPE (n = 11) were recruited from two tertiary antenatal clinics (mean ± SD 38 ± 3 weeks). Active renin and total AGT concentrations were measured by ELISA; urinary tetrahydroaldosterone was measured by gas chromatography-mass spectrometry; creatinine was measured using a standard clinical laboratory assay for normalisation. Results: Aldosterone and plasma active renin (Kruskal Wallis, both P < 0.05) were significantly different between groups. Aldosterone: creatinine ratios were significantly lower in both PE and SPE compared to the NC and women with CKD (median [IQR], PE: 6.5 [3, 14.4]; SPE: 10.8 [5.4, 16.4]; NC: 13.8 [9.1, 29.8]; CKD: 24.2 [16.3, 37.9] μg/mmol creatinine; P < 0.05 for all). Plasma active renin concentrations were also lower in PE, SPE and CKD women compared to NC women (PE: 5.3 [9.7, 21.8]; SPE: 3.4 [2.4, 12]; CKD: 3.9 [3.2, 6.2]; NC: 5.3 [0.2, 8.7] pg/ml; P < 0.05 for all). No differences in either plasma or urinary total AGT were observed between the groups. Conclusions: Women with SPE have lower urinary aldosterone and plasma active renin than women with CKD in keeping with women with PE without pre-existing kidney disease, suggestive of similar pathophysiology. Further exploration of the predictive value of low urinary aldosterone and plasma renin for adverse pregnancy events in women with CKD and its role in cases of diagnostic uncertainty is required.

Determination of urinary aldosterone using a plasma aldosterone 2D ID LC-MS/MS method.

In the diagnosis for primary aldosteronism, the measurement of urinary aldosterone is part of the confirmation test but diagnostic accuracy may be blunted due to poor immunoassay performance for urinary aldosterone. Nowadays, plasma aldosterone concentrations are measured preferably by LC-MS/MS yet such methods for urinary aldosterone are lacking. We show that plasma and urinary aldosterone can be measured with the same 2D isotope dilution LC-MS/MS method. The accuracy of the method was tested against a certified reference material. The reference values for plasma and urinary aldosterone were established. With this method, urinary aldosterone concentrations can be measured precisely, simply and accurately together with plasma samples with one set of calibration standards.

Effect of benazepril and robenacoxib and their combination on glomerular filtration rate in dogs.

Combined use of angiotensin-converting enzyme inhibitors and nonsteroidal anti-inflammatory drugs may induce acute kidney injury, especially when combined with diuretics. The objective of this investigation was to evaluate the effect of benazepril, robenacoxib and their combination in healthy dogs. In each of two studies (studies 1 and 2), 32 beagle dogs were randomized into one of four groups in a parallel-group design. Groups received once-daily oral treatment for 7days with placebo, benazepril, robenacoxib or benazepril plus robenacoxib. In study 2, all dogs received additionally 2mg/kg furosemide orally twice daily. The primary endpoint was the glomerular filtration rate (GFR) estimated from the plasma clearance of iohexol. Secondary endpoints included standard clinical monitoring and, in study 2, plasma renin activity, urine volume, specific gravity and aldosterone concentration and water intake. Administration of furosemide induced diuresis, reduced GFR and activated the renin-aldosterone-angiotensin system. Benazepril and robenacoxib, administered alone or in combination, were tolerated well, did not decrease GFR with or without co-administration of furosemide and significantly reduced urinary aldosterone concentrations. No increased risk of acute kidney injury was identified with the combination of benazepril and robenacoxib in healthy dogs. Different effects might occur in dogs with heart or renal disease.

Effect of Cyclooxygenase(COX)-1 and COX-2 inhibition on furosemide-induced renal responses and isoform immunolocalization in the healthy cat kidney

BackgroundThe role of cyclooxygenase(COX)-1 and COX-2 in the saluretic and renin-angiotensin responses to loop diuretics in the cat is unknown. We propose in vivo characterisation of isoform roles in a furosemide model by administering non-steroidal anti-inflammatory drugs (NSAIDs) with differing selectivity profiles: robenacoxib (COX-2 selective) and ketoprofen (COX-1 selective).ResultsIn this four period crossover study, we compared the effect of four treatments: placebo, robenacoxib once or twice daily and ketoprofen once daily concomitantly with furosemide in seven healthy cats. For each period, urine and blood samples were collected at baseline and within 48 h of treatment starting. Plasma renin activity (PRA), plasma and urinary aldosterone concentrations, glomerular filtration rate (GFR) and 24 h urinary volumes, electrolytes and eicosanoids (PGE2, 6-keto-PGF1α, TxB2), renal injury biomarker excretions [N-acetyl-beta-D-glucosaminidase (NAG) and Gamma-Glutamyltransferase] were measured. Urine volume (24 h) and urinary sodium, chloride and calcium excretions increased from baseline with all treatments. Plasma creatinine increased with all treatments except placebo, whereas GFR was significantly decreased from baseline only with ketoprofen. PRA increased significantly with placebo and once daily robenacoxib and the increase was significantly higher with placebo compared to ketoprofen (10.5 ± 4.4 vs 4.9 ± 5.0 ng ml−1 h−1). Urinary aldosterone excretion increased with all treatments but this increase was inhibited by 75 % with ketoprofen and 65 % with once daily robenacoxib compared to placebo. Urinary PGE2 excretion decreased with all treatments and excretion was significantly lower with ketoprofen compared to placebo. Urinary TxB2 excretion was significantly increased from baseline only with placebo. NAG increased from baseline with all treatments. Immunohistochemistry on post-mortem renal specimens, obtained from a different group of cats that died naturally of non-renal causes, suggested constitutive COX-1 and COX-2 co-localization in many renal structures including the macula densa (MD).ConclusionsThese data suggest that both COX-1 and COX-2 could generate the signal from the MD to the renin secreting cells in cats exposed to furosemide. Co-localization of COX isoenzymes in MD cells supports the functional data reported here.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0598-z) contains supplementary material, which is available to authorized users.

Reduced urinary aldosterone in preeclampsia, superimposed pre-eclampsia compared to standard- and high-risk pregnant women

Women with chronic kidney disease (CKD) and chronic hypertension (CHT) are at increased risk of superimposed pre-eclampsia (SPE) and associated adverse pregnancy outcomes. Diagnosis of SPE using blood pressure and proteinuria is of limited use because they may develop in women with CKD or CHT without the condition. Inaccurate diagnosis may result in unnecessary iatrogenic preterm delivery. In established pre-clampsia (PE), aldosterone levels are supressed. The aim of the study was to compare the urinary aldosterone concentrations between normotensive controls (NC), PE and CKD/CHT women with and without SPE at term.

Cardiovascular Complications Associated With Primary Aldosteronism

A higher risk of cardiovascular events has been reported in patients with primary aldosteronism (PA) than in otherwise similar patients with essential hypertension (EH). However, the evidence is limited by small sample size and potential confounding factors. We, therefore, compared the prevalence of cardiovascular events in 459 patients with PA diagnosed in our hypertension unit from 2001 to 2006 and 1290 controls with EH. PA cases and EH controls were individually matched for sex, age (± 2 years), and office systolic blood pressure (± 10 mm Hg). Patients with PA and EH differed significantly in duration of hypertension, serum potassium, plasma aldosterone and plasma renin concentrations, aldosterone-to-renin ratio, and urinary aldosterone concentration (P<0.001 for all comparisons). The prevalence of electrocardiographic and echocardiographic left ventricular hypertrophy was about twice higher in patients with PA even after adjustment for hypertension duration. PA patients also had a significantly higher prevalence of coronary artery disease (adjusted odds ratio, 1.9), nonfatal myocardial infarction (adjusted odds ratio, 2.6), heart failure (adjusted odds ratio, 2.9), and atrial fibrillation (adjusted odds ratio, 5.0). The risks associated with PA were similar across levels of serum potassium and plasma aldosterone. To conclude, patients with PA are more likely to have had a cardiovascular complication at diagnosis than otherwise similar patients with EH. Target organ damage and complications disproportionate to blood pressure should be considered as an additional argument for suspecting PA in a given individual and possibly for broadening the scope of screening at the population level.

Associations among serum N-terminal procollagen type III concentration, urinary aldosterone-to-creatinine ratio, and ventricular remodeling in dogs with myxomatous mitral valve disease

To assess relationships among serum N-terminal procollagen type III concentration, urinary aldosterone-to-creatinine concentration ratio (UAC), and clinical variables in dogs with myxomatous mitral valve disease (MMVD) and healthy dogs. 162 dogs with MMVD and 24 healthy control dogs of comparable age and body weight. Blood and urine samples were collected from each dog. Dogs with MMVD underwent echocardiography and ECG. Ventricular diameter measurements were normalized for body weight. Serum N-terminal procollagen type III and urinary aldosterone concentrations were measured via radioimmunoassay. Each dog was examined on 1 to 3 occasions. Examinations were repeated at approximately 6-month intervals. Serum N-terminal procollagen type III concentration decreased with increasing severity of MMVD and was negatively associated with age and left ventricular end-diastolic and end-systolic diameters. The UAC increased with prior percentage change in left ventricular end-diastolic diameter per month, subsequent percentage change in left ventricular end-systolic diameter per month, and treatment with diuretics and was negatively associated with age. Both UAC and serum N-terminal procollagen type III concentration were higher in Cavalier King Charles Spaniels than in other breeds when other measured variables were controlled for. In dogs with MMVD, echocardiographic indicators of left ventricular remodeling appeared to be associated with a decrease in serum concentration of a marker of collagen type III turnover and an increase in urinary aldosterone concentration.

Aldosterone synthase inhibition in humans

Aldosterone synthase (CYP11B2) inhibition has emerged as a new option for the treatment of hypertension, heart failure and renal disorders, in addition to mineralocorticoid receptor (MR) blockade. The aim is to decrease aldosterone concentrations in both plasma and tissues, thereby decreasing MR-dependent and MR-independent effects in the cardiac, vascular and renal target organs. LCI699 was the first orally active aldosterone-synthase inhibitor to be developed for human use. Its structure is similar to that of FAD286, the dextroenantiomer of the aromatase inhibitor, fadrozole. It dose-dependently decreases plasma and urine aldosterone concentrations by up to 70 or 80% and increases plasma renin activity in healthy male subjects on a low-sodium diet. LCI699 does not decrease basal plasma cortisol concentrations at doses of 0.5-3 mg q.d., but it blocks the cortisol response to adrenocorticotropic hormone (ACTH) at doses ≥ 3 mg q.d. In a proof-of-concept study in patients with primary aldosteronism (PA), LCI699 (0.5-1 mg b.i.d.) induced a dose-dependent and reversible 70-80% decrease in plasma and urinary aldosterone concentration accompanied by a massive dose-dependent accumulation of deoxycorticosterone (>+700%), the aldosterone precursor, in the plasma, thereby confirming the inhibition of the CYP11B2 gene product. This effect was associated with a rapid correction of hypokalaemia, a modest decrease in blood pressure (BP) and a mild increase in plasma renin concentration in patients with PA. LCI699 administration induced biological signs of partial inhibition of the glucocorticoid axis, such as dose-dependent increases in both plasma ACTH and 11-deoxycortisol (the precursor of cortisol) concentrations, consistent with the inhibition of the CYP11B1 gene product. An 8-week placebo-controlled dose-response study on patients with Stage 1 and 2 essential hypertension reported an optimal decrease in BP with a dose of 1 mg LCI699 q.d., which had an antihypertensive effect similar to that of 50 mg b.i.d. eplerenone. A blunted cortisol response to ACTH was observed in 20% of patients, but the clinical and biological safety and tolerability of LCI699 were similar to those of placebo and eplerenone. The discovery of this first orally active aldosterone synthase inhibitor, LCI699, has provided new opportunities to assess the feasibility and the haemodynamic, biological and safety consequences as well as the limitations of this new approach to block the aldosterone pathway in hypertensive patients. However, as the effects of LCI699 on the glucocorticoid axis limit the use of higher doses range because of the loss of selectivity for CYP11B2, this aldosterone synthase inhibitor cannot replace the MR blockade in patients with hypertension, other cardiovascular or renal disorders. The development of second-generation aldosterone synthase inhibitors with a higher selectivity index for CYP11B2 than LCI699 should make it possible to test this approach at much higher doses in these patients, after the necessary toxicology and Phase I studies.

A new transgenic rat model overexpressing the angiotensin II type 2 receptor provides evidence for inhibition of cell proliferation in the outer adrenal cortex

This study aimed to elucidate the role of the AT(2) receptor (AT(2)R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT(2)R) that overexpresses the AT(2)R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT(2)R number amounted to 82-98% of total Ang II binding sites. In the ZG of TGRCXmAT(2)R, the AT(2)R density was elevated threefold relative to wild-type (WT) littermates, whereas AT(1)R density remained unchanged. TGRCXmAT(2)R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT(2)R at baseline. Three and 14 days of Ang II infusion (300 ng·min(-1)·kg(-1)) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT(1)R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT(2)R. In conclusion, AT(2)R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

β2-Adrenergic receptor genotype affects the renin-angiotensin-aldosterone system response to the Dietary Approaches to Stop Hypertension (DASH) dietary pattern

Beta(2)-adrenergic receptor (beta2-AR) is a susceptibility locus for hypertension, and polymorphisms at this site relate to salt sensitivity and low plasma renin activity (PRA). The Dietary Approaches to Stop Hypertension (DASH) dietary pattern lowers blood pressure and appears to interact with the renin-angiotensin-aldosterone system (RAAS). We hypothesized that the DASH diet associates with increased RAAS activity, and genotype status at beta2-AR G46A modifies this response. We genotyped participants in the DASH-Sodium study (n = 372) at beta2-AR G46A to determine the association with blood pressure, RAAS components, and consumption of the DASH diet. We used 2-way mixed linear regression and an additive model for all primary analyses. Mean (+/-SEM) PRA was significantly higher in participants in the DASH group than in participants in the control group (0.68 +/- 0.03 compared with 0.54 +/- 0.03 ng x mL(-1) x h(-1), P = 0.002). Serum aldosterone, urinary aldosterone, and urinary potassium concentrations were also significantly higher in the DASH group (P < 0.01 for all). We observed significant gene-diet interactions for changes in systolic blood pressure (SBP) and concentrations of aldosterone and urinary potassium (P for interaction = 0.048, 0.017, and 0.001 for SBP and aldosterone and urinary potassium concentrations, respectively). There was an association between the A allele of beta2-AR G46A and greater blood pressure reduction and blunted aldosterone and PRA responses to the DASH diet. Our results indicate that the DASH diet lowers blood pressure and increases PRA and aldosterone concentrations. There is an association between the G46A polymorphism of beta2-AR and blood pressure and RAAS responses to the DASH diet, which suggests that beta2-AR may be a genetic modifier of DASH-diet responsiveness. This trial was registered at clinicaltrials.gov as NCT00000608.

HORMONAL EFFECTS OF THE NEW DIRECT ALDOSTERONE SYNTHASE INHIBITOR, LCI699, IN PATIENTS WITH PRIMARY ALDOSTERONISM: 5C.06

Objective: Aldosterone synthase inhibition with LCI699 is a new therapeutic option aimed at decreasing hormone concentrations in both plasma and tissues. We report the hormonal effects of LCI699 to patients with primary aldosteronism (PA). Design and Methods: After a two-week placebo run-in, patients with PA received oral LCI699 (0.5 mg bid) for two weeks, LCI699 (1 mg bid) for two weeks, and placebo for one week. We assessed changes in plasma and urinary aldosterone (PAC/UAldo), renin (PRC), cortisol and ACTH concentrations associated with correction of hypokalemia and decrease in BP. From the screening visit onwards, all patients received oral KCl (3–6 g/day) and 10/14 patients received a calcium channel blocker alone or in combination with prazosin to ensure that home BP remained ≤170/105 mmHg. Results: 14 patients (13 men, age: 50.3 ± 6.7yrs) with hypertension (office SBP/DBP: 151 ± 17/91 ± 12mmHg) and low plasma K (3.0mmol/L [min:2.5-max:3.5]) were included in the study. They had high plasma aldosterone (630pmol/L [359–997]), low plasma renin (4.5mU/L [1.0–9.5]) concentrations, and high aldosterone/renin ratio (123pmol/pg [84–553]). Baseline PAC decreased from 540pmol/L (95%CI:394;739) to 171pmol/L (95%CI:128;230) after 0.5 mg LCI699 (−68% [95%CI:−77;−55], P < 0.001 vs. baseline) and to 133pmol/L (95%CI:100;177) after 1 mg LCI699 (−75%, [95%CI:−84;−63]; P < 0.001 vs. baseline). UAldo was 82nmol/24 h (95%CI:61;109) at baseline and decreased by −78% ([95%CI:−82;−73]; P < 0.0001) after 0.5 mg LCI699 and by −88% ([95%CI:−92;−84]; P=0.0003) after 1 mg LCI699. PRC increased mildly from baseline by 26% to 39%; (P = 0.0007). Basal plasma cortisol remained unchanged, whereas plasma ACTH concentration increased by 35% after 0.5 mg LCI699 (P = 0.08) and by 113% after 1 mg LCI699 (P < 0.001). The PAC and the plasma cortisol response to an ACTH test (250 μg IV) after 1 mg LCI699 was blunted. All variables returned to initial levels after the placebo. LCI699 administration was well tolerated. Conclusion: The administration of LCI699, up to 1 mg bid, effectively and safely inhibits aldosterone synthase in patients with PA. The effects on the glucocorticoid axis were consistent with a latent inhibition of cortisol synthesis.

Physiological Partial Aldosterone Resistance in Human Newborns

In the neonatal period, the human kidney is characterized by an impaired ability to regulate water and sodium homeostasis, resembling partial aldosterone resistance. The aim of our study was to assess this hormonal insensitivity in newborn infants and to determine its relationship with neonatal sodium handling. We conducted a prospective study in 48 healthy newborns and their mothers. Aldosterone, renin, and electrolyte concentrations were measured in umbilical cords and in maternal plasma. Urinary aldosterone concentrations and sodium excretion were determined at urination within 24 h after birth. A significant difference was observed between aldosterone and renin levels in newborn infants compared with their mothers (817 +/- 73 versus 575 +/- 55 pg/mL and 79 +/- 10 versus 15 +/- 2 pg/mL, respectively, p < 0.001). This hyperactivation of the renin-angiotensin-aldosterone system was associated with hyponatremia and hyperkalemia in the newborn infants, and high urinary sodium loss, consistent with a partial aldosterone resistance at birth. Unlike plasma aldosterone, urinary aldosterone concentration was found highly correlated with plasma potassium concentrations, thus representing the best index for accurate evaluation of mineralocorticoid sensitivity. Our study represents a comprehensive characterization of the renin-aldosterone axis in newborn infants and provides evidence for physiologic partial aldosterone resistance in the neonatal period.

Conditional Transgenic Mice for Studying the Role of the Glucocorticoid Receptor in the Renal Collecting Duct

The mineralocorticoid receptor (MR) is a major regulator of renal sodium reabsorption and body fluid homeostasis. However, little is known about glucocorticoid receptor (GR)-dependent renal effects. Glucocorticoids may activate both receptors, so it is difficult to distinguish between MR- and GR-mediated effects in vivo. To overcome this complexity, we used a transgenic mouse model allowing conditional GR overexpression (doxycycline inducible TetON system, Hoxb7 promoter) in the renal collecting duct (CD) to identify GR-regulated genes involved in sodium transport in the CD. In microdissected cortical CD, induction of GR expression led (after 2 d of doxycycline) to increased alpha-epithelial sodium channel and glucocorticoid-induced leucine zipper and decreased abundance of with-no-lysine kinase 4 transcripts, without modification of Na,K-ATPase, serum- and glucocorticoid-kinase-1, or MR expression. No changes occurred in the upstream distal and connecting tubules [distal convoluted tubule (DCT), connecting tubule (CNT)]. Sodium excretion was unaltered, but the urinary aldosterone concentration was reduced, suggesting compensation of transitory extracellular volume expansion that subsequently disappeared. At steady state, i.e. after 15 d of doxycycline administration, transcript abundance remained altered in the CD, whereas mirror changes appeared in the DCT and CNT. Plasma aldosterone or glucocorticoids and blood pressure were all unaffected. These experiments show that: 1) GR, in addition to MR, controls epithelial sodium channel- and glucocorticoid-induced leucine zipper expression in vivo in the CD; 2) with-no-lysine kinase 4 is negatively controlled by GR; and 3) the DCT and CNT compensate for these alterations to maintain normal sodium reabsorption and blood pressure. These results suggest that enhanced GR expression may contribute to enhanced sodium retention in some pathological situations.

Estimation of 24-h aldosterone secretion in the dog using the urine aldosterone:Creatinine ratio

Objectives One potential method of evaluating renin–angiotensin–aldosterone system (RAAS) activation involves the quantification of urinary aldosterone excretion. While blood concentrations of aldosterone are easily obtained, results may be misleading because of minute-to-minute variation in aldosterone secretion and subsequent blood concentrations. Urinary aldosterone concentration measurement represents a more consistent “pooled” index of aldosterone secretion, but obtaining 24-h urine samples is time-consuming, difficult, and fraught with potential error. We postulated that the urinary aldosterone:creatinine ratio, measured from spot urine samples, would correlate well with 24-h urinary aldosterone excretion, and would provide a simple index of aldosterone excretion that would eliminate the need for 24-h urine collection. Animals, materials and methods After validating an assay for aldosterone in canine urine, 24-h urinary aldosterone excretion was determined by radioimmunoassay from 8 normal, male beagle dogs under control conditions, after RAAS stimulation with amlodipine administration, and after RAAS attenuation with the addition of enalapril to amlodipine administration. Spot urine samples, each obtained at the same time of day, were used to determine the aldosterone:creatinine ratio during control conditions, RAAS stimulation, and RAAS attenuation. Results The aldosterone:creatinine ratio from spot-checked urine samples correlated well with 24-h urinary aldosterone excretion ( r = 0.77, P < 0.0001). Conclusions A spot urinary aldosterone:creatinine ratio might be substituted for 24-h urinary aldosterone determination.

Measurement of aldosterone in feline, canine and human urine

Systemic hypertension is an important problem in older cats associated with kidney disease and hypokalaemia, suggesting that excessive activity of the renin-angiotensin-aldosterone system might contribute to the hypertensive state. Fluctuations in plasma renin activity and plasma aldosterone concentrations complicate the interpretation of these assays. The aim of this study was to determine whether measurement of urinary aldosterone excretion in cats aided the investigation of hypertension. Urine concentrations of free (ethyl acetate extract) and 18-glucuronidated aldosterone (acid hydrolysis before extraction) were measured by radioimmunoassay in normal, normotensive and hypertensive azotaemic cats (n=11 per group). Urine samples from 11 healthy human volunteers and eight normal dogs were also analysed for comparison. Urinary aldosterone concentration was corrected for the urinary creatinine concentration. Cats excreted 7.3 times less free aldosterone than human beings, and no free aldosterone was detected in dog urine. Acid hydrolysis led to large increases in aldosterone recovery from both human beings and dog but not feline urine. No significant effect of hypertension or azotaemia on feline urinary aldosterone concentration was found. Measurement of aldosterone in feline urine using the available methodology has limited or no utility in investigating feline hypertension.