Urinary Aldosterone Excretion Research Articles

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

BMAL1 is a core circadian clock protein that is important for circadian rhythms of physiological function, as well as maintaining physiological homeostasis, including aspects of kidney function. Global Bmal1 knockout (KO) male mice exhibit loss of diurnal rhythm of renal sodium (Na) excretion in response to a normal or high salt diet. However, renal Na excretion rhythm remained intact in kidney-specific Bmal1 KO male mice. The adrenal gland synthesizes hormones, such as aldosterone, which influence renal electrolyte handling. The role of adrenal BMAL1 in the regulation of renal function remains unknown. The study objective was to test the hypothesis that adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na handling. Male and female adrenal-specific aldosterone synthase Cre positive (AS) Bmal1 KO and littermate Cre negative, floxed control mice ( n=5-9/genotype) were acclimated to metabolic cages for 3 days then samples were collected for three days with mice on a normal salt diet followed by 7 days on a low salt diet to challenge the mice to stimulate aldosterone production. Twelve-hour food intake was measured, and urine samples were collected to measure night/day patterns in renal Na balance. Urinary aldosterone excretion was measured by ELISA. Both day and night urinary aldosterone excretion were increased following a low salt diet in control and KO male mice (ANOVA main effect of diet, p<0.05). Male AS Bmal1 KO mice displayed a significant increase in day urinary aldosterone excretion following a normal and low salt diet (ANOVA main effect of genotype, p=0.0230), with a trend for an increase in night urinary aldosterone excretion ( p=0.0571). Interestingly, these genotype effects were not seen in female AS Bmal1 KO mice. Urinary aldosterone was increased following a low salt diet in female mice, but only during the nighttime in control and KO mice ( p=0.0205). Sex differences were also observed in renal Na balance, where Na balance was more positive during the day in male, but not female, AS Bmal1 KO vs. control mice (ANOVA main effect of genotype, p=0.0282). Together, these data suggest adrenal BMAL1 is required for normal rhythms of aldosterone and renal Na balance in a sex-dependent manner. Future work will investigate whether the changes in aldosterone contribute to changes in renal Na balance. This work was supported by the American Heart Association (Postdoctoral FellowshipP0240441 to H.C.; Established Investigator Award 19EIA34660135 to M.G), theRobert and Mary Cade Professorship in Physiology (to M.G.) and the NIH(F32DK121424 to G.R.C.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bmal1 contributes to mean blood pressure rhythms in rats on a low salt diet

Bmal1 is a core component of the molecular clock which is responsible for generating circadian rhythms in many physiological parameters, including blood pressure (BP). Sodium intake can affect circadian rhythms, as high salt consumption can increase BP and alter the expression of the molecular clock. However, the effect of low salt consumption on the circadian rhythm of BP and hormones crucial to sodium homeostasis has not been extensively studied. Using a whole-body Bmal1-knockout (KO) rat, we wanted to determine the contribution of the molecular clock to circadian BP rhythms during low salt diet (LSD, 0.0049%).We hypothesized that Bmal1-KO rats on a LSD would have lower mean BP and higher aldosterone levels in comparison to littermate controls on LSD. Male and female Bmal1-KO rats and littermate controls (WT) had telemeters implanted in the abdominal aorta at 8-10 weeks old. After a 10-day surgical recovery period, BP was continuously recorded while rats were maintained on ad libitum normal salt diet (0.49% NaCl, NSD) for 1 week. Rats were placed in metabolic cages and 12-hour urine samples collected at ZT0 and ZT12 for two days. Next, rats were placed on LSD for one week while maintaining BP measurements. This was again followed by two day, 12-hour urine collections. BP was averaged hourly and analyzed for circadian rhythmicity (cosinor). The MESOR for mean arterial pressure was significantly higher in WT (n=10, 4/10 male) and KO groups (n=8, 4/8 male) on NSD (WT: 108±2 mmHg; KO: 104±2 mmHg) compared to LSD (WT: 104±2 mmHg; KO: 102±2 mmHg; p<0.05 for diet, 2-way ANOVA). BP amplitude was significantly higher in WT rats (NSD: 7.6±0.6 mmHg; LSD: 6.9±0.7 mmHg) compared to KO rats (NSD: 5.2±0.4 mmHg; LSD: 5.3±0.9 mmHg) (p<0.05 for genotype). BP acrophase was similar between groups on both diets. Urinary aldosterone excretion (ELISA) was similar between WT and KO rats on NSD (Daytime: WT 26±5 pg/12hrs, KO 16±1 pg/12hrs; Nighttime: WT 48±8 pg/12hrs, KO 46±4 pg/12hrs) and on LSD (Daytime: WT 95±38 pg/12hrs, KO 109±44 pg/12hrs; Nighttime: WT 174±63 pg/12hrs, KO 155±47 pg/12hrs), with statistically significant overall effects of diet and time of day (p<0.05, 3-way ANOVA). Together, these data support a role for Bmal1 in control of BP amplitude in rats on NSD and LSD. This does not appear to involve changes in aldosterone since urinary excretion followed predicted patterns by time of day and salt diet, but were not impacted by loss of Bmal1 in ad libitum fed rats. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reference intervals for LC-MS /MS measurements of plasma renin activity, aldosterone, angiotensin II, and 24-hour urinary aldosterone in Northern Chinese Han population

BackgroundExamination of aldosterone to Renin Ratio (ARR) and plasma aldosterone concentration (PAC) or 24-h urinary aldosterone excretion (24-h UALD) was the necessary tests in confirmatory tests for primary aldosteronism (PA). We developed a combined liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for plasma renin activity (PRA), PAC, and angiotensin II (Ang II) and investigated their reference intervals (RIs) in northern Chinese Han population. The RIs of 24-h UALD excretion were also studied using LC-MS/MS. MethodsA total of 309 healthy volunteers were recruited in 3 cities in China. PRA, PAC, Ang II, and 24-h UALD were measured using the laboratory-developed LC-MS/MS. Multiple linear regression and the variance component model were applied to determine if the RI needed to be split. The RIs of PRA, PAC, and Ang II were determined using the nonparametric percentile method. ResultsThe laboratory-developed LC-MS/MS method was verified and showed good performance. Standard deviation ratio (SDR) sex for PAC and SDR region for Ang II are 0.466 and 0.407, respectively, indicating that the RIs of PAC and Ang II must be divided by sex and region, respectively. In addition, the SDR 24hUK for 24-h UALD is 0.579, indicating that the RI of 24-h UALD must be partitioned by urine potassium. ConclusionRIs were established for tests related to the renin-angiotensin-aldosterone system in the apparently healthy northern Chinese Han population by the LC-MS/MS method.

S-55-1: PRIMARY ALDOSTERONISM – WHERE TO FROM HER E?

Over the past decade there have been remarkable advances in the pathophysiology of primary aldosteronism (PA); in contrast, there has been little if any change in the clinical management of PA for over 20 years. A patient suspected of PA is referred to an expert center, and their medications withdrawn for 4–6 weeks, with CCBs/alpha blockers used if needed to control blood pressure (BP).They undergo a single blood draw for plasma aldosterone concentration (PAC) and plasma renin activity or concentration (PRA/PRC), to establish the aldosterone to renin ratio (ARR). If the ARR 'suggests’ PA, the patient then undergoes confirmatory testing by one of at least five different procedures – to ensure no patient who does not have PA ever slips through the net. There are multiple reasons for change. First, in no country are more than 1% of hypertensives ever screened for PA. Secondly, a single blood draw for PAC is a very imperfect index of a patient’s actual aldosterone status; among those referred many are excluded on the basis of their PAC who in fact have PA. Third, the current work up – in addition to being exclusionary - is demanding and clunky. Fourth, the prevailing attitude among those treating hypertension is that if the BP is controlled all will be well, when in fact patients with PA have a three-fold higher risk profile than matched essential hypertensives with the same BP. What we are thus facing is a major public health crisis. There is finally increasing recognition that the current estimate of PA prevalence (5% - 10% of hypertensives) is far too low, and the actual prevalence in hypertensives is of the order of at least 30% (and of 10% in normotensives). The current protocols are useless facing figures such as these; what is needed are much simpler and inclusive measures to diagnose probable PA – rather than excluding all but the most obvious cases. This will require input not only from endocrinologists, but also from hypertension specialists, internists, primary care and public health physicians. It is important to distinguish newly presenting and established hypertension. When a patient is newly diagnosed with an elevated BP eschew immediate antihypertensive(s) and order a PRA/PRC assay. If renin is not suppressed, onto antihypertensives. If the renin is suppressed coach the patient to provide a 24 h urine collection for Na, K, and urinary excretion of aldosterone (UEA). If the UEA is < 6 mcg/24 h the patient does not have PA; if the UEA is 6–12, the patient may have PA; if the UEA is > 12 the patient has PA. For these two latter categories order 25 mg/d spironolactone and review at four weeks. If BP falls by < 10mmHg onto antihypertensives; if BP falls by > 10 mmHg continue spironolactone. Established hypertension is rather simpler. Add spironolactone 25 mg/d to current medication and review at 4 weeks. If BP falls < 10mmHg, discontinue; if BP falls > 10mmHg order a UEA. If UEA and BP are moderately elevated continue spironolactone, as such patients very probably have bilateral hyperaldosteonism; if UEA and BP are high, refer to an expert center. What this is calling for is a radical reassessment of how physicians should address hypertensive patients. The rationale is to save them from the double jeopardy of hypertension and PA, and the attendant increased morbidity and premature mortality. The time is now.

Autonomous Aldosterone Secretion in Patients with Adrenal Incidentaloma.

In recent years, research has emphasized the significance of mild clinical and biochemical presentations of primary aldosteronism (PA) that do not meet current diagnostic criteria of the syndrome. In this study, we assessed the prevalence of autonomous aldosterone (Ald) secretion (AAS), defined as a positive (>1.2 ng/dL/mIU/L) Ald-to-renin ratio (ADRR) combined with unsuppressed Ald (>4 ng/dL), and its associations with blood pressure (BP), cardiac function, and common carotid artery (CCA) intima-media thickness (IMT) in patients with incidentally discovered adrenal adenomas (AI), who were either normo- or hypertensive but had no other cardiovascular disease. Among 332 AI patients hospitalized between November 2018 and December 2019, 63 study participants were recruited (26 normo- and 37 hypertensive), who underwent hormonal examinations, 24 h ambulatory BP measurement, transthoracic echocardiography, and CCA IMT assessment without altering chronic medications. AAS was found in approximately 25% of subjects (seven normo- and nine hypertensive); urinary aldosterone excretion (UAldE) exceeded 10 ug/day in none of the subjects. The left ventricular mass index correlated positively with UAldE in non-diabetic patients (n = 50), and negatively with renin in those without beta blocker therapy (n = 38). The study shows that a pragmatic approach to hormonal assessment (no chronic therapy modification) may reveal patients with AAS. Screening for this subclinical PA presentation is probably more effective with a permissive ADRR than UAldE in such a setting.

ODP270 Prevalence of Primary Hyperaldosteronism in Patients with Resistant Hypertension - An Outpatient Cross-Sectional Clinic-Based Study

Abstract Background Primary hyperaldosteronism is the non-suppressible over secretion of aldosterone caused either by an adenoma or bilateral adrenal hyperplasia. Although its reported prevalence is 8-10% in recent studies, intraindividual variability for aldosterone levels poses diagnostic challenges during the screening of these patients. In this study, we aim to assess the prevalence of primary hyperaldosteronism in patients presenting with drug-resistant hypertension, with or without hypokalemia, at a general endocrinology practice with an emphasis on the variability of renin and aldosterone levels in the screening tests used to detect primary hyperaldosteronism. Method: We included participants presenting at our outpatient clinic from June 2020 until January 2022 with blood pressure>140/90mm Hg despite the use of three antihypertensive medications including a diuretic. The screening tests used were morning (8-9 AM) plasma aldosterone concentration (PAC) and plasma renin (PR) level. Aldosterone was measured using L iquid Chromatography-Tandem Mass Spectrometry assay determined by Mayo Clinic. The screening was considered positive when PAC was>10 ng/dL (277 pmol/l) and PR was <1 ng/ml/hr. The tests were repeated if PAC was between 5-10 ng/dL and PR was between 1-2 ng/ml/hr without any medication changes. If the repeat PR was >2 ng/ml/hr, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers were stopped and changed to calcium channel blockers, and the patient was tested again after 2 weeks. Positive results were further tested with 24-hour urinary aldosterone excretion during high dietary salt ingestion with 24-hour urine sodium and creatinine. Primary hyperaldosteronism was confirmed if plasma renin activity was <1. 0 ng/mL per hour and urinary aldosterone was >12 mcg/24-hour during high urinary sodium excretion (>200 mEq/24-hour). Results We studied a total of 138 patients during this study period. Mean age was 58.7 years and BMI 33.7 kg/m 2, out of which 95 participants had type 2 diabetes mellitus, 7 had obstructive sleep apnea, and 20 had chronic kidney disease stage 3 or higher. We found 3 patients were positive on screening tests and two patients were confirmed to have primary hyperaldosteronism, making prevalence 2/138(1.45%). Conclusion Hyperaldosteronism is the most common cause of secondary hypertension, especially resistant hypertension. Repeat testing of aldosterone levels with intraindividual variability can be very challenging, and time-consuming resulting in repeated follow-up visits and multiple communications with providers posing challenges for effective care coordination, and financial burden on patients. Further large-scale studies are recommended to re-analyze the protocols for screening hyperaldosteronism and treatment initiation without extensive confirmatorytesting. We also suggest health care providers emphasize on effective management of other secondary causes of hypertension such as obesity and Metabolic Syndrome with diabetes for a greater impact on blood pressure control on these patients. Presentation: No date and time listed

RENIN ANGIOTENSIN SYSTEM BLOCKADE AT BEDTIME VERSUS ON AWAKENING TO PREVENT ALDOSTERONE BREAKTHROUGH

Objective: Aldosterone breakthrough (AB) is defined by an increase of serum aldosterone levels over baseline values before starting blockers of renine angiotensine system (RAS) treatment by angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARB). AB may limit the efficacy of these drugs. We hypothesized that short-acting blockers taken on awakening may not inhibit the aldosterone peak occuring at the awakening. To address this hypothesis, we compared the incidence of AB in patients randomized to take their medications either on awakening or at bedtime. Design and method: participants had chronic kidney disease stage 3 or 4 and a urinary protein over creatinine ratio above than 0.5 g/g. Blood pressure, renal function, urinary sodium excretion, levels of aldosterone and renin were measured at baseline and after 1 year exposure to RAS blockers. Incidence of AB was tested with Chi-square test and the variables associated with AB were searched with Mann-Whitney-Wilcoxon tests. Results: among 105 participants, 97 were randomized and 77 completed the study. The incidence of AB was not significantly different between subjects having their treatment on awakening or at bedtime (43% versus 41%, p = 0.83). AB was associated with the decrease of systolic blood pressure (-1 ± 14 vs 5 ± 19 mm Hg, p = 0.05), the decrease of urinary sodium excretion (-31 ± 52 vs 1 ± 59 meq/d, p = 0.007), or the increase of serum creatinine levels (-0.2 ± 0.36 vs -0.10 ± 0.30 mg/dl, p = 0.018). As expected, AB was also associated with the increase of plasma levels of renin (42 ± 125 vs -1 ± 65 ng/L, p = 0.04) and the increase of urinary aldosterone excretion (4 ± 13 vs -1 ± 3, p = 0.0003). The independent factors associated with AB were systolic blood pressure (p = 0.03) and urinary aldosterone excretion (0.03). Conclusions: the intake of a short half-life RAS blockers at bedtime does not prevent AB in CKD patients with macroproteinuria. Mineralocorticoid receptor antagonists may be required to prevent the deleterious consequences of AB.

G Protein‐coupled Estrogen Receptor 1 and Pregnancy Confer Protection against Renal Injury in Aged Female Mice

Chronic kidney disease, which is a leading risk factor impacting 1 in 7 American women, is more frequent in aged compared to younger women. Along with the natural aging process, aged women undergo a decline in circulating estrogen level, which is a predisposing factor for renal disease. Estrogen activates the G protein‐coupled estrogen receptor 1 (GPER1), which elicits cardiovascular and renal protective actions. Additionally, pregnancy augments GPER1‐induced vasodilation in rats. However, there is limited information on the potential interaction between GPER1 and pregnancy in the renal health later in life. We hypothesize that GPER1 signaling, and pregnancy confer protection against renal injury in aged female mice. In our study, 14‐16 months‐old GPER1 knock‐out (KO) and wild‐type (WT) female mice with and without a history of former pregnancies were placed into metabolic cages for collection of 24h urine samples. Then, animals were euthanized, plasma and kidneys were harvested for assessment of renal injury. No significant differences in body weight were observed between groups. GPER1 deletion in virgin mice did not impact food intake or urinary Na+ excretion (UNaV). Former pregnancies reduced food intake (p=0.0003) and UNaV (p<0.0001) in GPER1 KO mice. To assess renal function, we measured urinary albumin excretion and plasma creatinine (Cr). GPER1 deletion increased urinary albumin excretion in virgin mice (KO: 16.47±2.08; WT: 6.06±1.04 μg/day; n=6 & 7 respectively; p=0.0005). Interestingly, prior pregnancies eliminated this genotypic difference in albuminuria (KO: 6.12±1.44; WT: 5.79±1.66 μg/day; n=6 & 8 respectively; p=0.986). GPER1 deletion also resulted in higher plasma Cr level in virgin mice (KO: 1.36±0.08; WT: 0.97±0.05 mg/mL; n=6 & 7 respectively; p=0.0018). Prior pregnancies decreased plasma Cr levels in GPER1 KO mice (0.84±0.10 mg/dL, n= 5; p= 0.0012). We also measured urinary excretion of endothelin (ET‐1) and aldosterone which are causally linked to renal injury. GPER1 KO elicited greater levels of urinary ET‐1 compared to WT virgin mice (0.37±0.06 vs.0.17±0.01 pg/day; n=6 & 7, respectively; p=0.0063). Previous pregnancies lowered urinary levels of ET‐1 and aldosterone in GPER1 KO mice (ET‐1: p=0.0049; aldosterone: p=0.0099). Furthermore, histological assessment of kidney sections revealed diffuse proliferative glomerulonephritis (GN) with intracapillary hyaline/protein thrombi, in GPER1 KO virgin mice only. No significant acute tubular injury was observed in any group. Further experiments will determine blood pressure and characterize the glomerular lesions evident in aged GPER1 KO virgin mice. Overall, our data demonstrate significant glomerular injury (GN), greater plasma Cr and greater urinary levels of albumin and ET‐1 in GPER1 KO virgin mice compared to WT virgin and previously‐pregnant GPER1 KO aged mice. The current data suggest that GPER1 and former pregnancies provide protection against glomerular injury in aged females.

Hepatocellular carcinoma induces body mass loss in parallel with osmolyte and water retention in rats

AimsThe number of cancer survivors with cardiovascular disease is increasing. However, the effect of cancer on body fluid regulation remains to be clarified. In this study, we evaluated body osmolyte and water imbalance in rats with hepatocellular carcinoma. Main methodsWistar rats were administered diethylnitrosamine, a carcinogenic drug, to establish liver cancer. We analyzed tissue osmolyte and water content, and their associations with aldosterone secretion. Key findingsHepatocellular carcinoma rats had significantly reduced body mass and the amount of total body sodium, potassium, and water. However, these rats had significantly increased relative tissue sodium, potassium, and water content per tissue dry weight. Furthermore, these changes in sodium and water balance in hepatocellular carcinoma rats were significantly associated with increased 24-h urinary aldosterone excretion. Supplementation with 0.25% salt in drinking water improved body weight reduction associated with sodium and water retention in hepatocellular carcinoma rats, which was suppressed by treatment with spironolactone, a mineralocorticoid receptor antagonist. Additionally, the urea-driven water conservation system was activated in hepatocellular carcinoma rats. SignificanceThese findings suggest that hepatocellular carcinoma induces body mass loss in parallel with activation of the water conservation system including aldosterone secretion and urea accumulation to retain osmolyte and water. The osmolyte and water retention at the tissue level may be a causative factor for ascites and edema formation in liver failure rats.

Abstract 23: Body Fluid And Sodium Loss Leads To Aldosterone Hypersecretion And Muscle Catabolism In Hepatocellular Carcinoma Rats.

Background: The number of cancer survivors coincides with cardiovascular diseases is increasing, therefore, we are promoting the concept of “Onco-Hypertension” to clarify the mechanism linking cancer and blood pressure. In this study, we evaluated body osmolyte and water imbalance in hepatocellular cancer (HCC) model rats. Methods: Wistar rats were administered diethylnitrosamine (DEN) (1.5 μg/day, p.o.), a carcinogenic drug, for 8 weeks to establish liver cancer. Three weeks after the completion of DEN administration, we investigated blood pressure, tissue osmolyte and water content, and its association with aldosterone secretion. Results: HCC rats significantly reduced blood pressure, skin sodium, potassium, and water content. In the carcass (muscle + bone), dry weight, sodium, potassium, and water content were dramatically reduced without changing bone mass in HCC rats, suggesting that HCC causes muscle wasting to supply osmolyte and water for the dehydrated organs. These osmolytes and water loss were significantly associated with increased urinary aldosterone excretion. Supplementation of 0.25% salt water to drink improved body sodium and water loss and muscle wasting in HCC rats, which were completely suppressed by treatment with spironolactone (75 mg/kg/day, p.o.), a mineralocorticoid receptor (MR) blocker. Conclusion: These findings suggest that HCC causes body osmolyte and water loss, which leads to aldosterone hypersecretion and muscle catabolism to compensate for dehydration. A relatively small amount of salt supplement ameliorates the HCC-induced dehydration and muscle wasting via aldosterone/MR-mediated sodium and water restoration.

A non-invasive left ventricular pressure-strain loop study on myocardial work in primary aldosteronism

We investigated the myocardial work derived from left ventricular pressure-strain loop in patients with primary aldosteronism or primary hypertension. We enrolled 50 patients with primary aldosteronism, 50 age- and sex-matched patients with primary hypertension, and 25 normotensive control subjects. We performed transthoracic echocardiography and speckle-tracking echocardiography-based left ventricular pressure-strain loop analysis to evaluate cardiac structure and function. Patients with primary aldosteronism and those with primary hypertension had similar clinic and ambulatory blood pressures, except that the former had a significantly (P = 0.03) higher nighttime systolic blood pressure. All subjects had normal left ventricular ejection fraction (66.4 ± 4.7%). Patients with primary aldosteronism had a greater left ventricular mass index than those with primary hypertension and the normal controls (111.0 ± 21.6 g/m2 versus 95.7 ± 17.7 and 77.9 ± 13.5 g/m2, respectively, P < 0.001). The global myocardial work index (GWI, 2336 ± 333, 2366 ± 288, and 2292 ± 249 mmHg%, respectively), and global constructive work (GCW, 2494 ± 325, 2524 ± 301, and 2391 ± 193 mmHg%, respectively), were comparable in the three groups (P ≥ 0.18). However, the global work efficiency (GWE) differed significantly (P < 0.001), being lowest in primary aldosteronism (91.1 ± 2.7%), intermediate in primary hypertension (93.5 ± 2.5%) and highest in controls (95.3 ± 1.5%). The opposite was true for the global wasted work (GWW) (205.6 ± 74.6, 142.0 ± 56.4 and 99.4 ± 33.7 mmHg%, respectively, P < 0.001). GWE was significantly correlated with the logarithmically transformed plasma concentration and the urinary excretion of aldosterone in patients with primary aldosteronism or primary hypertension (r = -0.43 for both, P < 0.001). The associations remained statistically significant (P ≤ 0.04) after further adjustment for several factors, including left ventricular mass index and clinic or nighttime blood pressure. In conclusion, GWE decreased and GWW increased in primary hypertension and further in primary aldosteronism, probably because of the adrenal aldosterone hypersecretion and the left ventricular mass index increase, while GWI and GCW were similar, indicating that similar and normalized total myocardial work might be a compensation in hypertension at the expense of work efficiency.

G Protein‐Coupled Estrogen Receptor 1 is Required for Greater Endothelin‐1 Excretion in Female Mice

Estrogen signaling via G protein-coupled estrogen receptor 1 (GPER1) can elicit cardiovascular and renal protective actions. Endothelin-1 (ET-1) is a pro-natriuretic peptide that contributes to sex differences in blood pressure regulation and Na+ homeostasis. We recently showed that activation of GPER1 in the renal medulla promotes ET-1-dependent natriuresis in female but not male rats. Also, renal GPER1 expression is significantly higher in female rats than in male rats. In the current study, we hypothesized that genetic deletion of GPER1 would elevate blood pressure and impair renal ET-1 production/release in a sex-specific manner. Twelve-week-old male and female GPER1-knockout (KO) and wild-type (WT) littermate mice were implanted with telemeters to monitor blood pressure. Parallel groups of mice were placed into metabolic cages, and 24-h urine samples were collected while food and water intake were monitored. Then, animals were euthanized to obtain plasma. Urinary levels of electrolytes and ET-1 were measured. GPER1 deletion did not significantly affect mean arterial blood pressure in female (KO: 102±1 mmHg; WT: 102±2 mmHg; n=7-8/group, p=0.9) or male mice (KO: 106±3 mmHg; WT: 106±2 mmHg; n=6-10/group, p=1). No genotypic differences were observed in food intake, water intake, or urine flow rate. GPER1 deletion did not significantly affect urinary Na+excretion in female (KO: 0.25±0.05 μg/24 h; WT: 0.17±0.05 μg/24 h; n=9-12/group, p=0.52) or male mice (KO: 0.34±0.06 μg/24 h; WT: 0.35±0.04 μg/24 h; n=10-12/group, p=1.00). Similarly, GPER1 deletion did not affect urinary K+ excretion in either sex. However, GPER1 deletion decreased urinary ET-1 excretion in females (KO: 75.16±16.35 ng/24 h; WT: 172.83±24.67 ng/24 h; n=6-10/group, p<0.005) but not males (KO: 95.18±17.52 ng/24 h; WT: 76.03± 9.90 ng/24 h; n=10-12/group, p=0.69). Of note, WT females had significantly greater urinary ET-1 excretion than WT males (p=0.001), whereas KO mice had no sex differences in ET-1 excretion. Importantly, urinary excretion of ET-1 reflects intrarenal ET-1 production/release. GPER1 deletion did not significantly change plasma ET-1 concentration in either sex. Finally, GPER1 deletion did not affect urinary aldosterone excretion in females (KO: 315.98±73.13 pg/24 h; WT: 306.79±81.45 pg/24 h; n=7-8, p=1.00) or males (KO: 544.09±130.20 pg/24 h; WT: 481.84±68.30 pg/24 h; n=8/group, p=0.87). However, male mice had significantly greater urinary aldosterone excretion than female mice (p=0.04), regardless of genotype. Overall, we demonstrate that WT female mice have higher urinary ET-1 excretion than WT males, and that GPER1 deletion eliminates this sex-related difference in intrarenal ET-1 production/release. Our data suggest that GPER1 may contribute to the enhanced capacity of the female kidney to maintain Na+ homeostasis and have led us to hypothesize this as a mechanism for protecting females against salt-sensitive hypertension.

Effect of sodium bicarbonate supplementation on the renin-angiotensin system in patients with chronic kidney disease and acidosis: a randomized clinical trial

BackgroundAcidosis-induced kidney injury is mediated by the intrarenal renin-angiotensin system, for which urinary renin is a potential marker. Therefore, we hypothesized that sodium bicarbonate supplementation reduces urinary renin excretion in patients with chronic kidney disease (CKD) and metabolic acidosis.MethodsPatients with CKD stage G4 and plasma bicarbonate 15–24 mmol/l were randomized to receive sodium bicarbonate (3 × 1000 mg/day, ~ 0.5 mEq/kg), sodium chloride (2 × 1,00 mg/day), or no treatment for 4 weeks (n = 15/arm). The effects on urinary renin excretion (primary outcome), other plasma and urine parameters of the renin-angiotensin system, endothelin-1, and proteinuria were analyzed.ResultsForty-five patients were included (62 ± 15 years, eGFR 21 ± 5 ml/min/1.73m2, plasma bicarbonate 21.7 ± 3.3 mmol/l). Sodium bicarbonate supplementation increased plasma bicarbonate (20.8 to 23.8 mmol/l) and reduced urinary ammonium excretion (15 to 8 mmol/day, both P < 0.05). Furthermore, a trend towards lower plasma aldosterone (291 to 204 ng/L, P = 0.07) and potassium (5.1 to 4.8 mmol/l, P = 0.06) was observed in patients receiving sodium bicarbonate. Sodium bicarbonate did not significantly change the urinary excretion of renin, angiotensinogen, aldosterone, endothelin-1, albumin, or α1-microglobulin. Sodium chloride supplementation reduced plasma renin (166 to 122 ng/L), and increased the urinary excretions of angiotensinogen, albumin, and α1-microglobulin (all P < 0.05).ConclusionsDespite correction of acidosis and reduction in urinary ammonium excretion, sodium bicarbonate supplementation did not improve urinary markers of the renin-angiotensin system, endothelin-1, or proteinuria. Possible explanations include bicarbonate dose, short treatment time, or the inability of urinary renin to reflect intrarenal renin-angiotensin system activity.Graphic abstract

Adrenal (Pro)renin Receptor Expression and Serum Soluble (Pro)renin Receptor Concentration in Primary Aldosteronism

The (pro)renin receptor [(P)RR] is a multifunctioning protein playing roles in various pathological conditions. A soluble form of (P)RR [s(P)RR] has been considered a biomarker for (P)RR expression in tissues. Expression of (P)RR has been described in aldosterone-producing adenoma (APA), but the roles of (P)RR have yet to be fully determined. This study investigated the significance of (P)RR and serum s(P)RR concentrations in patients with APA. We evaluated associations between (P)RR expression and expression of CYP11B2, an aldosterone synthase, and aldosterone production by the adrenal glands and assessed the relationships between serum s(P)RR concentration and background factors. (P)RR colocalized with CYP11B2 and expression levels of (P)RR were positively associated with those of CYP11B2 in APA tissues. (P)RR immunoreactivity in these tissues correlated positively with plasma aldosterone concentrations (PAC) and urinary aldosterone excretion. Also, in APA, (P)RR mRNA abundance was positively correlated with β-catenin mRNA abundance. Significant positive correlations were identified between serum s(P)RR concentration and plasma glucose, hemoglobin A1c, and serum creatinine levels, but not with PAC (in either peripheral vein or adrenal vein) or adrenal (P)RR expression level. This study showed that (P)RR expression level correlates with CYP11B2 expression in APA tissues and PAC and urinary aldosterone excretion, suggesting that (P)RR expression may contribute to aldosterone synthesis via CYP11B2 activation in APAs, although serum s(P)RR concentration failed to show any significant relationship with adrenal (P)RR expression. Adrenal (P)RR activity might offer a therapeutic target in the treatment of PA, although this issue needs to be investigated in future studies.

A speckle tracking echocardiographic study on right ventricular function in primary aldosteronism.

We investigated right ventricular function using speckle tracking echocardiography (STE) in patients with primary aldosteronism. Our study included 51 primary aldosteronism patients and 50 age and sex-matched primary hypertensive patients. We performed two-dimensional echocardiography to measure cardiac structure and function. We performed STE offline analysis on right ventricular four-chamber (RV4CLS) and free wall longitudinal strains (RVFWLS). Primary aldosteronism patients, compared with primary hypertensive patients, had a significantly (P ≤ 0.045) greater left ventricular mass index (112.0 ± 22.6 vs. 95.8 ± 18.5 g/m) and left atrial volume index (26.9 ± 6.0 vs. 24.7 ± 5.6 ml/m) and higher prevalence of left ventricular concentric hypertrophy (35.3 vs. 12.0%), although they had similarly normal left ventricular ejection fraction (55-77%). Primary aldosteronism patients also had a significantly (P ≤ 0.047) larger right atrium and ventricle, lower tricuspid annular plane systolic excursion, and higher E/E't (the peak early filling velocity of trans-tricuspid flow to the peak early filling velocity of lateral tricuspid annulus ratio), estimated pulmonary arterial systolic pressure and right ventricular index of myocardial performance. On the right ventricular strain analysis, primary aldosteronism patients had a significantly (P < 0.001) lower RV4CLS (-18.1 ± 2.5 vs. -23.3 ± 3.4%) and RVFWLS (-21.7 ± 3.7 vs. -27.9 ± 4.5%) than primary hypertensive patients. Overall, RV4CLS and RVFWLS were significantly (r = -0.58 to -0.41, P < 0.001) correlated with plasma aldosterone concentration and 24-h urinary aldosterone excretion. After adjustment for confounding factors, the associations for RV4CLS and RVFWLS with 24-h urinary aldosterone excretion remained significant (P < 0.001), with a standardized coefficient of -0.48 and -0.55, respectively. In addition to left ventricular abnormalities, primary aldosteronism patients also show impaired right ventricular function, probably because of hyperaldosteronism.

SUN-220 High Salt Intake May Paradoxically Drive Autonomous Aldosterone Production

Many modifiable factors contribute to the high prevalence rates of hypertension, among them is the consumption of too much salt (sodium). Another curable cause of hypertension is the excess of the hormone aldosterone. Aldosterone is normally produced by the zona glomerulosa (ZG) of the adrenal glands in response to a lack of salt and conversely suppressed by salt excess. We hypothesize that [i] suppression of aldosterone production induces apoptosis of ZG cells, as occurs following genetic deletion1; [ii] this sets up a maladaptive response to chronic salt overload by conferring a survival advantage to cells in which mutations drive autonomous aldosterone production. To address [i], we measured apoptosis of cells in which aldosterone synthesis was inhibited; to address [ii] we undertook a cross-sectional clinical study of aldosterone and sodium excretion, hypothesising that aldosterone excretion will be highest in the outside quartiles of sodium excretion. Aldosterone was inhibited by modification, in human adrenocortical H295R cells, of either CADM1 expression (mutated in aldosterone-producing adenomas2) or intracellular calcium concentration3. Apoptosis was measured by flow cytometric analysis of annexin V conjugates. 24-hour urinary aldosterone excretion (24h-Ualdo) was correlated with 24-hour urinary sodium (24h-Usodium) in 24h-urine samples collected for a Malaysian population-based salt intake study (MyCoSS). The prevalence of autonomous aldosterone production was estimated from the proportion of subjects with serum measurement whose “SUSSPUP” ratio (= serum sodium to urinary sodium)/(serum potassium2 to urinary potassium) was >5.34. Modification of CADM1 in human adrenocortical H295R cells decreased aldosterone production by half compared to vector control, and this was associated with a 3 to 5-fold increase of apoptotic cells (p<0.05; n>3). Pilot investigation of a Cav1.3 inhibitor decreased aldosterone production by 70%, and increased apoptosis by 7-fold (p<0.10; n=2). In 767 subjects, 24h-urine samples from the high urinary sodium quartile (>150mmol/d) had higher urinary aldosterone (4+0.18 ug/d) than other quartiles (p=0.00001). Overall, the estimated prevalence of autonomous aldossterone production using SUSSPUP ratio was 4.5% (8 of 179 subjects). In 63 subjects with 24h-Usodium>200 mmol/day, autonomous aldosterone secretion (conventionally >10µg/d) was found in 9.5%. Our results support the hypothesis that initial suppression of aldosterone production by salt excess may create a selective advantage for cells which autonomously produce aldosterone, and hence an inappropriate long-term increase in aldosterone production. 1Lee et al., Endocrinology. 2005;146:2650-6. 2Wu et al., 21st European Congress of Endocrinology. Vol. 63. BioScientifica, 2019. 3Xie et al., Sci Rep. 2016;6:24697. 4Willenberg et al., Eur J Clin Invest. 2009;39:43-50.

PD31-02 WHAT TIMING IS APPROPRIATE FOR A BIOCHEMICAL EVALUATION AFTER LAPAROSCOPIC ADRENALECTOMY FOR UNILATERAL ALDOSTERONE-PRODUCING ADENOMA?

INTRODUCTION AND OBJECTIVE: The over-secretion of plasma aldosterone by unilateral aldosterone-producing adenoma (APA) can be cured by adrenalectomy. However, the time needed for the endocrine environment to normalize remains unclear. The aim of this study is to clarify adequate timing for a biochemical evaluation in unilateral APA patients after adrenalectomy. METHODS: A total of 166 unilateral APA patients were retrospectively reviewed. We evaluated renin-angiotensin axis agents: the plasma aldosterone concentration (PAC) (pg/ml), active renin concentration (ARC) (pg/ml) and aldosterone-renin ratio (ARR: PAC/ARC), serum potassium concentration, and estimated glomerular filtration rate (eGFR) at 1, 3, and 6 post operation months (POM). By applying the Primary Aldosteronism Surgical Outcome criteria, which is based with ARR, PAC and serum potassium levels, biochemical outcomes were also evaluated. RESULTS: PAC was significantly lower at 1POM than at pre-surgery (pre-surgery; 407.2, 1 POM; 90.0 pg/mL, p<0.001) (Fig. A). ARC did not increase from baseline at 1POM, but significantly increased at 3POM (pre-surgery; 4.43, 1POM; 4.87, 3POM; 11.3 pg/mL, p<0.001) (Fig. B). The patients who didn’t achieve the ARC normalization at 1POM had lower pre-surgery ARC (p<0.001) and higher pre-surgery 24-hour urinary aldosterone excretion (p=0.042). Although ARC did not significantly increase at 1POM, ARR significantly decreased at 1POM (pre-surgery; 146.9, 1 POM; 26.3, p<0.001) (Fig. C). Among the 34 patients who had hypokalemia pre-surgery, it was resolved in 28 (82%) at 1POM and in all (100%) at 3POM (Fig. D). The biochemical outcomes at 1POM were 131 (79%) complete, 20 (12%) partial, and 15 (9%) absent successes, while at 3POM, 147 (89%) were complete, 9 (5%) partial, and 10 (6%) absent. Twenty-three (14%) patients were changed biochemical outcomes between 1 and 3POM, whereas only 5 (3%) changed between 3 and 6POM. CONCLUSIONS: The appropriate timing for a biochemical evaluation of unilateral APA patients treated with laparoscopic adrenalectomy appears to be 3 months or more after surgery.Source of Funding: None

P-glycoprotein influences urinary excretion of aldosterone in healthy individuals.

P-glycoprotein (P-gp), the product of the ABCB1 gene, is involved in the transport of aldosterone and cortisol in adrenal cells in vitro but its physiological role in humans remains controversial. Our objective was to test the influence of P-gp polymorphisms on aldosterone. We evaluated plasma aldosterone concentration (PAC), urinary aldosterone, and blood pressure in a cohort of white normotensive men at baseline on diets unrestricted for sodium and potassium and after a 5-day treatment with 500 mg b.i.d. clarithromycin, a P-gp inhibitor. Included were 20 homozygous wild-type (P-gp0), 20 heterozygous (P-gp1), and 20 individuals with combined 2677G>T/A-3435C>T loss-of-function polymorphism of the ABCB1 gene (P-gp2). At baseline, PAC, urinary aldosterone, urinary free cortisol to urine creatinine ratios, and blood pressure did not differ in the three genotypes. After clarithromycin administration, the urinary aldosterone to creatinine ratio increased by an average of 30% in the entire cohort (P < 0.001, n = 60). Increases were pronounced in P-gp1 (+40%; P = 0.014) and P-gp2 individuals (+50%; P = 0.020) but lesser and were NS in P-gp0 individuals (+10%; P = 0.259). PAC also increased from baseline after clarithromycin treatment in all individuals (+19%, P = 0.050); however, the increase in PAC was NS when the three genotypes were analyzed separately. In our experimental conditions, the interaction between P-gp inhibition and the ABCB1 genotype, suggests that aldosterone is indeed a physiological endogenous substrate of P-gp in humans and that P-gp interferes with the net equilibrium between aldosterone secretion and elimination processes in humans.Clinical Trial Registration - URL: http://www.clinicaltrials.gov. Unique identifier: NCT01627665.

Left atrial myocardial dysfunction in patients with primary aldosteronism as assessed by speckle-tracking echocardiography.

We investigated the left atrial myocardial deformation in patients with primary aldosteronism using the speckle-tracking echocardiographic (STE) strain imaging technique. Our study included 107 primary aldosteronism patients [52 aldosterone-producing adenoma (APA) and 55 idiopathic hyperaldosteronism (IHA)] and 50 primary hypertensive patients. We performed conventional echocardiography to measure left atrial volume and ejection fraction, and STE to estimate left atrial myocardial deformation including peak velocity, strain and strain rate and calculate the ratio of E/e' to left atrial strain during left ventricular systole as the left atrial stiffness index. Patients with APA, compared with those with IHA and primary hypertension had a significantly (P < 0.001) lower serum potassium concentration and higher 24-h urinary aldosterone excretion and plasma aldosterone-to-renin ratio. Patients with APA had a significantly (P < 0.01) larger maximal, precontraction, and minimal left atrial volumes and lower total, active and passive left atrial emptying fractions than those with IHA and primary hypertension. Among the three groups, patients with APA showed lowest left atrial velocity, strain, and strain rate during ventricular systole, early diastole and late diastole (P < 0.05) and highest left atrial stiffness index (P < 0.001). In unadjusted analysis, the left atrial strain, strain rate and stiffness index were significantly (P < 0.05) associated with plasma aldosterone concentration and urinary aldosterone excretion. After adjustment for various confounding factors, these associations remained statistically significant for urinary aldosterone excretion (P < 0.05) but not plasma aldosterone concentration (P ≥ 0.05). Patients with primary aldosteronism, especially APA, had impaired left atrial deformation mechanics and increased left atrial stiffness, providing a promising insight into early detection of subclinical left atrial dysfunction by strain echocardiography.

Primary Aldosteronism: Does Underlying Pathology Impact Clinical Presentation and Outcomes Following Unilateral Adrenalectomy?

Patients undergoing unilateral adrenalectomy for primary aldosteronism (PA) may have a solitary adenoma, unilateral hyperplasia, or multiple adenomas on final pathology. This study investigated whether the underlying pathological diagnosis was associated with differences in clinical presentation and postoperative outcomes. A retrospective cohort study of patients undergoing unilateral adrenalectomy for PA from 2004 to 2015 at our institution was performed. Baseline clinical and laboratory parameters, as well as postoperative biochemical and hypertension cure rates, were compared across the three aforementioned pathological groups. Of 206 patients who met criteria for inclusion, 152 (73.8%) had a single adenoma, 33 (16%) had unilateral hyperplasia, and 21 (10.2%) had multiple unilateral adenomas. Patients with unilateral hyperplasia were more likely to be male (81.2% vs 57.9%, P = .03), undergo left-sided adrenalectomy (78.8% vs 47.4%, P < .01), and had a lower median adrenal venous sampling lateralization index (9.8 vs 19.8, P = .04) compared to those with solitary, but not multiple unilateral adenomas. No differences were seen in age, duration of hypertension, preoperative plasma aldosterone levels, plasma renin activities, 24-h urinary aldosterone excretion, serum potassium concentrations, and the number of preoperative antihypertensive medications across all three pathological groups. All patients achieved biochemical cure following adrenalectomy, and no significant differences in the rates of hypertension cure or improvement were observed in comparisons across pathological subtype. Clinical presentation and postoperative outcomes are similar regardless of underlying pathology in patients with PA. Because one in four patients may harbor unilateral hyperplasia or multiple adenomas, total unilateral adrenalectomy should be performed as the operation of choice over adrenal-sparing approaches.