Increased Plasma Aldosterone Research Articles

Changes in plasma aldosterone and electrolytes levels, kidney epithelial sodium channel (ENaC) and blood pressure in normotensive WKY and hypertensive SHR rats following gonadectomy and chronic testosterone treatment

We hypothesized that testosterone-induced increase in blood pressure involve changes in aldosterone levels and expression of epithelial sodium channel (ENaC) in the kidneys. MethodsOvariectomized female normotensive Wistar Kyoto (WKY) and Spontaneous hypertensive (SHR) rats were given six weeks treatment with testosterone via subcutaneous silastic implant. The rats were anesthetized and mean arterial pressure (MAP) was measured via direct cannulation of the carotid artery. Animals were sacrificed and kidneys were removed and subjected for α, β and γ-ENaC protein and mRNA expression analyses by Western blotting and Real-time polymerase chain reaction (qPCR), respectively. Distributions of α, β and γ-ENaC proteins in kidneys were observed by immunofluorescence. Plasma testosterone, aldosterone, electrolytes, osmolality, urea and creatinine levels were determined by biochemical assays. Analysis were also performed in non-testosterone treated orchidectomized and sham-operated male WKY and SHR rats. ResultsTreatment of ovariectomized female WKY and SHR rats with testosterone causes increased in MAP but decreased in plasma aldosterone, sodium (Na+), osmolality and expression and distribution of α, β and γ-ENaC subunits in the kidneys. Orchidectomy decreased the MAP but increased plasma aldosterone, Na+, osmolality and α, β and γ-ENaC expression and distribution in the kidneys of male WKY and SHR rats. ConclusionsDecreased in plasma aldosterone, Na+ and ENaC levels in kidneys under testosterone influence indicated that testosterone-induced increased in MAP were not due to increased plasma aldosterone and ENaC levels in kidneys, and thus the testosterone effect on MAP likely involve other mechanisms.

Abstract P153: Mineralocorticoid Receptor Inhibition Abolishes Sex-specific, Renin Angiotensin Aldosterone System-associated Salt Sensitivity in Female Mice

Up to half of essential hypertension cases in women are associated with salt sensitive blood pressure (BP) increases, however, the sex-specific mechanisms of salt sensitivity in women are unknown. Our lab has shown that female mice are more sensitive to the hypertensive effects of aldosterone than male mice but it is unknown if aldosterone plays a role in salt sensitivity in female mice. We hypothesized that increasing dietary sodium via a high salt diet would increase blood pressure in female mice which would be abrogated by mineralocorticoid receptor (MR) antagonism. To determine salt sensitivity male and female Balb/C mice were implanted with radiotelemeters for continuous recording of BP. BP was recorded during baseline (7 days) and throughout the administration of a high salt (4% NaCl, HS) diet for 7 days with or without concurrent eplerenone supplementation (daily 200 mg/kg/day). Plasma and kidneys were then harvested. Systolic (SBP) and diastolic (DBP) BP were increased in female mice, but not in males, on HS (7.8±3.3 SBP and 7.8±4.0 DBP Δ change in mmHg in female (P<0.05) vs -3.7±3.1 SBP and 3.1±2.1 DBP in male, respectively, n=7-8). Plasma aldosterone levels were decreased in HS male mice compared to control (224±57 vs 151±19 pg/ml, n=3-5), however, increased modestly in HS females (254±56 vs 394±158 pg/ml, n=3-6). Preliminary data indicated that MR antagonism with eplerenone ablated increases in SBP and DBP in HS female mice, while having no effect on blood pressure in HS males (-22.8 SBP and -23.9 DBP Δ change in mmHg in female vs 1.0 SBP and -1.5 DBP in male, n=2). In addition, and in association with an absence of aldosterone suppression with HS, renal mRNA expression of renin (1.4±0.1-fold, P<0.05, n=5), angiotensinogen (4.4±0.2-fold, P<0.05, n=5), AT1 receptor (52.9±0.9-fold, P<0.05, n=5), MR (1.6±0.2-fold, P<0.05, n=5) and α-ENAC (1.3±0.0-fold, P<0.05, n=5) were increased in HS female mice compared to control females. These data indicate that BP increases in HS female mice are associated with unexpected increases in plasma aldosterone as well as mRNA expression of proteins associated with renin angiotensin aldosterone system activation, which may be novel mechanisms via which females have increased risk for salt sensitive hypertension.

Aldosterone Hypothesis for Cognitive Impairment in Diabetes Mellitus

Increased plasma aldosterone concentration is significantly associated with dementia, which is accentuated by diabetes mellitus (DM). Angiotensin II (AngII) deteriorates cognitive function through neuronal degradation. Lipoproteins, a major source of cholesterol for aldosterone biosynthesis, undergo glycoxidative modifications in the presence of hyperglycemia. We hypothesize that there would be a pathophysiological link between diabetically-modified lipoproteins, angiotensin II, and increased plasma aldosterone concentration for induction of cognitive impairment. Glycoxidized lipoproteins produce significantly more aldosterone from AngII-sensitized adrenocortical cells compared to their native counterparts. The elucidation of signaling mechanisms revealed that modified lipoproteins follow the similar signaling mechanism like AngII for adrenocortical aldosterone release via ERK1/2 and Janus kinase-2 (Jak-2)-mediated pathways. The enhanced aldosterone release from AngII-sensitized adrenocortical cells induced by glycoxidatively modified lipoproteins may play a crucial role in cognitive dysfunction in diabetic individuals along with AngII via a prevailing mode of signaling cascade involving ERK1/2- and Jak-2-dependent pathways.

Salt wasting in two neonates with posterior urethral valves: secondary pseudohypoaldosteronism

Secondary pseudohypoaldosteronism (PHA) is a rare condition that presents with hyperkalemia, hyponatremia, and metabolic acidosis, which develops due to renal tubular unresponsiveness to aldosterone. It may be seen in infants with obstructive uropathy and acute pyelonephritis. Three important reasons of the aldosterone unresponsiveness of the tubules are early infancy kidney, obstructive uropathy, and urinary tract infection. Besides other findings, diagnosis is established with highly increased plasma aldosterone and renin levels. In infants who present with hyperkalemia, hyponatremia, and metabolic acidosis, PHA should be considered in the differential diagnosis of congenital adrenal hyperplasia. Urinary ultrasonography and urine analysis should be performed in these patients. Herein, we present two patients with secondary PHA caused by posterior urethral valves.

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.

Stimulatory Effect of Food Restriction on the Steroidogenesis of Aldosterone in Ovariectomized Rats.

Food or calorie restriction (FR or CR) induces several physiological changes including weight loss, metabolic adaptations, mineral and hormonal changes. However, the effects of FR on aldosterone steroidogenesis in zona glomerulosa (ZG) cells have not been elucidated. Therefore, the present study was designed to investigate the effects of FR on aldosterone secretion and the involved mechanisms in ovariectomized (Ovx) rats. Ovx rats were divided into ad libitum fed (control) and FR groups. The FR rats exhibited decreased body weight, water intake, urine flow, sodium excretion and increased plasma aldosterone in comparison with control rats. FR elevated the basal and angiotensin II-stimulated aldosterone secretion from ZG cells. The conversions of 25-hydroxy-cholesterol to pregnenolone or corticosterone to aldosterone in ZG cells of FR group were greater than that in control group. FR group had a higher protein expression of steroidogenic acute regulatory (StAR) protein in ZG cells. However, there was no different protein expression of cytochrome P450 sidechain cleavage enzyme (P450scc) in ZG cells between control and FR groups. In summary, the increased activities of P450scc and aldosterone synthase as well as the protein expression of StAR protein in ZG cells are involved in the effects of FR on aldosterone steroidogenesis in Ovx rats. We also suggest that the increase of aldosterone might be associated with anti-diuresis and antinatriuresis in FR group. These results are helpful for understanding the role of aldosterone in physiological adaptation and renal sodium conservation during FR.

The Aldosterone Synthase Inhibitor FAD286 is Suitable for Lowering Aldosterone Levels in ZDF Rats but not in db/db Mice.

Inhibition of aldosterone synthase is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone actions. FAD286 is one of the best characterized aldosterone synthase inhibitors to date. FAD286 improves glucose tolerance and increases glucose-stimulated insulin secretion in obese and diabetic ZDF rats. However, there is limited knowledge about the dose-dependent effects of FAD286 on plasma aldosterone, corticosterone, and 11-deoxycorticosterone in ZDF rats and in db/db mice, a second important rodent model of obesity and type 2 diabetes. In addition, effects of FAD286 on plasma steroids in mice and rats are controversial. Therefore, obese Zucker diabetic fatty (ZDF) rats and db/db mice were treated with FAD286 for up to 15 weeks and plasma steroids were evaluated using highly sensitive liquid chromatography-tandem mass spectrometry. In ZDF rats, FAD286 (10 mg/kg/d) treatment resulted in nearly complete disappearance of plasma aldosterone while corticosterone levels remained unaffected and those of 11-deoxycorticosterone were increased ~4-fold compared to vehicle control. A lower dose of FAD286 (3 mg/kg/d) showed no effect on plasma aldosterone or corticosterone, but 11-deoxycorticosterone was again increased ~4-fold compared to control. In contrast to ZDF rats, a high dose of FAD286 (40 mg/kg/d) did not affect plasma aldosterone levels in db/db mice although 11-deoxycorticosterone increased ~2.5-fold. A low dose of FAD286 (10 mg/kg/d) increased plasma aldosterone without affecting corticosterone or 11-deoxycorticosterone. In conclusion, the aldosterone synthase inhibitor, FAD286, lowers plasma aldosterone in obese ZDF rats, but not in obese db/db mice.

Metoclopramide unmasks potentially misleading contralateral suppression in patients undergoing adrenal vein sampling for primary aldosteronism.

As metoclopramide stimulates aldosterone secretion, we tested its usefulness in the assessment of lateralization of primary aldosteronism by adrenal vein sampling (AVS). Prospective within-patient study in consecutive patients undergoing AVS for primary aldosteronism subtyping. We compared the diagnostic accuracy of baseline and postmetoclopramide lateralization index and relative (to cortisol) aldosterone secretion indices (RASI) for each adrenal gland with aldosterone-producing adenoma (APA) determined by the four corners criteria as the reference diagnosis. We recruited 93 consecutive patients (mean age: 52 years; women 31%). Metoclopramide increased plasma aldosterone in the inferior vena cava and in both adrenal veins. The postmetoclopramide lateralization index was accurate in identifying APA, but did not increase diagnostic accuracy over baseline lateralization index, because the RASI increased similarly in both sides. Conversely, metoclopramide raised RASI to values more than 0.90 bilaterally in non-APA patients allowing accurate identification of factitious aldosterone suppression. In contrast, RASI was 0.90 or less in 48% contralateral to the tumor in APA patients. Regression analysis showed the APA patients with persistent suppression of RASI contralaterally showed a more florid primary aldosteronism phenotype. Metoclopramide does not enhance lateralization of aldosterone excess in APA, but consistently increased the value of RASI in non-APA cases, thus unmasking potentially misleading suppression of aldosterone. Postmetoclopramide RASI may therefore allow a more precise diagnosis when AVS can be achieved only unilaterally.

Prenatal corticosterone exposure programs sex-specific adrenal adaptations in mouse offspring.

Maternal stress can impair foetal development and program sex-specific disease outcomes in offspring through the actions of maternally produced glucocorticoids, predominantly corticosterone (Cort) in rodents. We have demonstrated in mice that male but not female offspring prenatally exposed to Cort (33 µg/kg/h for 60 h beginning at E12.5) develop cardiovascular/renal dysfunction at 12 months. At 6 months of age, renal function was normal but male offspring had increased plasma aldosterone concentrations, suggesting that altered adrenal function may precede disease. This study investigated the long-term impact of prenatal exposure to Cort on adrenal growth, morphology and steroidogenic capacity as well as plasma Cort concentrations in offspring at postnatal day 30 (PN30), 6 months and 12 months of age. Prenatal Cort exposure decreased adrenal volume, particularly of the zona fasciculata, in male offspring at PN30 but increased both relative and absolute adrenal weight at 6 months of age. By 12 months of age, male Cort-exposed offspring had reduced absolute adrenal weight in association with increased adrenal plaque deposition (lipogenic pigmentation). Plasma Cort concentrations were elevated in male 6-month offspring but not at other ages. mRNA expression of Mc2r (ACTH receptor) was increased in males at PN30, and Cyp11a1 expression was decreased at 6 and 12 months of age. There were no changes in the adrenals of female Cort-exposed offspring. This study demonstrates that prenatal Cort exposure induces offspring adrenal gland dysfunction in an age- and sex-specific manner, which may contribute to long-term programmed disease in male offspring after maternal stress.

Abstract P295: Endothelial Sodium Channel Activation Promotes Cardiac Stiffness in Obese Female Mice

Cardiac diastolic dysfunction (DD) and diastolic heart failure is increasing in concert with obesity and aging population in the United States. In obese and diabetic women, DD is more common than in their male counterparts. This disproportionate increase in DD in obese females may partly explain their loss of sex-related cardiovascular (CV) disease protection. Recent studies have suggested a role for endothelial sodium channel (ENaC) activation in promotion of endothelial stiffness and suppression of flow- (nitric oxide) mediated vasodilation. Moreover, increased mineralocorticoid receptor (MR) activation mediated endothelial stiffness is promoted, in part, by ENaC activation. In this regard, we have recently reported increased plasma aldosterone levels, aortic and cardiac stiffness, and cardiac and vascular MR expression in female mice fed a high fat and high fructose diet (western diet [WD]). This increase in CV stiffness was prevented by very low dose MR antagonism. Accordingly, we hypothesized that inhibition of MR-mediated ENaC activation by using a very low dose of the ENaC inhibitor, amiloride would prevent cardiac stiffening (DD) in WD-fed female mice. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1mg/kg/day) for 16 weeks. Amiloride significantly attenuated WD-induced impairment of cardiac relaxation in vivo as measured by high resolution magnetic resonance imaging (MRI) as well as cardiac interstitial fibrosis as measured by immunohistochemistry by picrosirius red staining. Moreover, amiloride prevented the development of DD in obese female mice without having effects on blood pressure. These observations support a role for ENaC activation in diet-induced cardiac stiffening (DD) in obese females.

OS 15-03 A NOVEL MECHANISM FOR ALDOSTERONE PRODUCTION THROUGH BETA 3 ADRENERGIC RECEPTOR (β3-AR) IN HEART FAILURE

Objective: Numerous clinical trials demonstrated that renin-angiotensin-aldosterone system (RAAS) was involved in the pathogenesis of hypertension-induced heart failure. While RAS is a major machinery required for aldosterone production, multiple minor systems, including catecholamine, could give rise to the aberrant aldosterone production beyond the RAS activation. We investigated the mechanism underlying the RAS-independent aldosterone production in heart failure. Design and Method: Dahl-salt sensitive rats fed high salt diet developed malignant hypertension, resulting in heart failure with inappropriately increased plasma aldosterone level. The gene expression pattern of the adrenal gland from heart failure rats showed strikingly upregulated b3-Adrenergic receptor (b3-AR) expression. Using Dahl-salt sensitive rats and H295R human adrenocortical carcinoma cells, we conducted immunohistochemical and molecular biological studies to assess whether b3-AR contribute to aldosterone production in heart failure. Results: Immunohistochemical staining confirmed that heart failure increased b3-AR expression in zona glomerulosa cells in adrenal gland of Dahl-salt sensitive rats. And the staining of pimonidazole, a hypoxia probe, revealed that the outer layer of adrenal cortex in the heart failure rat was under hypoxic condition. Hypoxia and DMOG, a chemical stabilizer of hypoxia-inducible factor, strongly induced b3-AR mRNA expression in H295R cells. Hypoxia-reoxygenation stimuli induced aldosterone production from H295R cells, which was blocked by SR59230A, a selective b3-AR antagonist, but not by losartan, an angiotensin II receptor blocker (ARB). Hypoxia endowed catecholamine responsiveness in H295R cells by increased b3-AR expression, wherein b3-AR agonist increased the phosphorylation levels of hormone-sensitive lipase (HSL) through activation of ERK pathway. Conclusions: In the present study, we demonstrated that the b3-AR induction in adrenal gland by hypoxia played an important role in aberrant aldosterone production in heart failure. The b3-AR-mediated catecholamine system could regulate the utilization of cholesterol, a substrate of aldosterone, via the ERK-HSL pathway.

Abstract P632: Nlrp3/inflammasome Activation Contributes To Aldosterone-induced Vascular Dysfunction In Type 2 Diabetes.

Diabetic patients and animal models of type 2 diabetes (DM2) display increased plasma aldosterone (aldo) levels. Aldo induces vascular inflammation and endothelial dysfunction. NOD-like receptors, which are pattern recognition receptors involved in a variety of host innate immune responses, promote vascular inflammation. We hypothesized that aldo via mineralocorticoid receptors (MR) activates the inflammasome platform in the vasculature of DM2 mice. Control (db/+) and diabetic (db/db) mice were treated with vehicle or spironolactone (spiro - MR antagonist, 50 mg/Kg/day). Mesenteric resistance arteries (MA) from db/db mice exhibited reduced acetylcholine (ACh) dilation, which was reversed by spiro [Emax (% of relaxation): db/+: 78.5±4.1; db/db: 40.5±6.4; db/+spiro: 77.0±3.8; db/db+spiro: 62.8±5.9 n=3-6 p<0.05]. Spiro treatment reduced caspase-1 and mature IL-1β content in MA from db/db mice. Spiro also reduced caspase-1 activity in macrophages from peritoneal lavage of db/db mice [% of activity: db/+: 33.9±2.5; db/db: 51.8±7.4; db/+spiro: 31.1±1.9; db/db+spiro: 34.8±3.8 n=4-7, p<0.05]. In vitro, aldo increased mature IL-1β in vascular smooth muscle cells (VSMC) (cont: 0.9±0.01 ; LPS+Nigericine: 6.1±2.1 ; Aldo 4h: 9.7±2.6; LPS+Aldo 4h: 12.8±1.9 n=3-5, p<0.05). To determine whether aldo in vivo directly activates NLRP3/inflammasome in the vasculature and whether NLRP3 activation contributes to aldo-induced vascular injury, aldo was infused (600 ug/Kg/day for 14 days) in wild type (WT) and NLRP3 knockout mice ( NLRP3-/- ) after bone marrow transplantation from WT donor. The groups were constituted: WT->WT, WT->WT+aldo and WT-> NLRP3 -/-+aldo. NLRP3 -/- mice were protected against aldo-induced endothelial dysfunction [Emax: WT: 89.3±2.9; WT+aldo: 39.8±1.8; NLRP3-/- +aldo: 87.7±4.2, p<0.05]. Aldo treatment leaded to endothelial dysfunction in WT ->WT mice, but WT-> NLRP3 -/- mice were protected from aldo-induced endothelial dysfunction [Emax: WT->WT: 95.1±3.1; WT->WT+aldo: 57.1±4.7; WT->NLRP3-/-+aldo: 85.3±3.1 p<0.05]. These results suggest that NLRP3/inflammasome in the vasculature plays a crucial role on aldo/MR-induced vascular damage and on DM2-associated vascular dysfunction. Financial Support: FAPESP, CAPES, CNPq.

Abstract 254: A Novel Mechanism for Aberrant Aldosterone Production in Heart Failure

Objective: Aldosterone production by the adrenal gland is mainly regulated by the renin-angiotensin system (RAS), which is normally suppressed by a high-salt diet. However, plasma aldosterone levels are elevated in proportion to the severity of heart failure even if caused by high-salt intake. The inappropriate increase in plasma aldosterone level could not be diminished by pharmacological blockade of the RAS. We investigated the cellular mechanism underlying the aberrant aldosterone production in heart failure beyond the activation of RAS. Methods and Results: Dahl-salt sensitive rats fed high salt diet developed malignant hypertension, resulting in heart failure with increased plasma aldosterone level. The gene expression pattern of the adrenal gland from heart failure rats showed strikingly upregulated β3-adrenergic receptor (β3-AR) expression. Immunohistochemical staining confirmed that heart failure increased β3-AR expression in zona glomerulosa cells in adrenal gland of Dahl-salt sensitive rats. And the staining of pimonidazole, a hypoxia probe, revealed that the outer layer of adrenal cortex in the heart failure rat was under hypoxic condition. Hypoxia and DMOG, a chemical stabilizer of hypoxia-inducible factor, strongly induced β3-AR mRNA expression in H295R human adrenocortical carcinoma cells. Hypoxia-reoxygenation stimuli induced aldosterone production from H295R cells, which was blocked by SR59230A, a selective β3-AR antagonist, but not by losartan, an angiotensin II receptor blocker (ARB). Hypoxia endowed catecholamine responsiveness in H295R cells by increased β3-AR expression, wherein β3-AR agonist increased the phosphorylation levels of hormone-sensitive lipase (HSL) through activation of ERK pathway. Conclusions: In the present study, we demonstrated that the β3-AR induction in adrenal gland by hypoxia played an important role in aberrant aldosterone production in heart failure. The catecholamine stimuli through β3-AR in adrenocortical cells caused the ERK-mediated phosphorylation of HSL, suggesting that the HSL promotes lipolysis to supply substrates for the aldosterone biosynthesis.

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

BackgroundCombined use of angiotensin-converting enzyme inhibitors and nonsteroidal anti-inflammatory drugs may induce acute kidney injury in humans, especially when combined with diuretics. The objective of this investigation was to evaluate the effects of benazepril, robenacoxib and their combination in healthy cats.In each of two studies (study 1 followed by study 2), 32 healthy cats were randomised to one of four groups (n = 4 male and 4 female cats per group) in a parallel-group design. The groups received orally once daily for 7 days either placebo (control group), benazepril, robenacoxib or benazepril plus robenacoxib. In study 2, all groups received in addition 0.5 mg/kg furosemide twice daily by subcutaneous injection for 7 days.ResultsBenazepril, robenacoxib and their combination were well tolerated as evidenced from lack of clinical signs and no negative effects on body weight, feed consumption and clinical chemistry, haematology and urinalysis variables.The primary endpoint of the study was the glomerular filtration rate (GFR), which was estimated from the plasma clearance of iohexol. In the absence of furosemide, GFR was significantly higher in cats receiving the combination of benazepril plus robenacoxib compared to the other three groups, and was also significantly higher in females receiving only benazepril compared to the control.Administration of furosemide induced diuresis, reduced GFR and activated the renin-aldosterone-angiotensin system, evidenced from increased plasma renin activity and plasma aldosterone concentrations. Compared to the control group in cats treated with furosemide, GFR was increased by benazepril (females only) but decreased by robenacoxib (males only). Benazepril, robenacoxib and their combination significantly inhibited the increase in plasma aldosterone induced by furosemide.ConclusionsThe combination of benazepril and robenacoxib was well tolerated and either increased or had a neutral effect on GFR in healthy cats without or with concomitant furosemide. The combination of benazepril and robenacoxib reduced plasma aldosterone concentrations increased by furosemide. It is recommended to test the efficacy and safety of the combined use of benazepril and robenacoxib in cats with clinical disease, notably proteinuric chronic kidney disease.

MTORC2 critically regulates renal potassium handling.

The mTOR pathway orchestrates cellular homeostasis. The rapamycin-sensitive mTOR complex (mTORC1) in the kidney has been widely studied; however, mTORC2 function in renal tubules is poorly characterized. Here, we generated mice lacking mTORC2 in the distal tubule (Rictorfl/fl Ksp-Cre mice), which were viable and had no obvious phenotype, except for a 2.5-fold increase in plasma aldosterone. Challenged with a low-Na+ diet, these mice adequately reduced Na+ excretion; however, Rictorfl/fl Ksp-Cre mice rapidly developed hyperkalemia on a high-K+ diet, despite a 10-fold increase in serum aldosterone levels, implying that mTORC2 regulates kaliuresis. Phosphorylation of serum- and glucocorticoid-inducible kinase 1 (SGK1) and PKC-α was absent in Rictorfl/fl Ksp-Cre mice, indicating a functional block in K+ secretion activation via ROMK channels. Indeed, patch-clamp experiments on split-open tubular segments from the transition zone of the late connecting tubule and early cortical collecting duct demonstrated that Ba2+-sensitive apical K+ currents were barely detectable in the majority of Rictorfl/fl Ksp-Cre mice. Conversely, epithelial sodium channel (ENaC) activity was largely preserved, suggesting that the reduced ability to maintain K+ homeostasis is the result of impaired apical K+ conductance and not a reduced electrical driving force for K+ secretion. Thus, these data unravel a vital and nonredundant role of mTORC2 for distal tubular K+ handling.

Genetic and potential autoimmune triggers of primary aldosteronism.

Hypertension is a major life-threatening disease of high morbidity and mortality, which affects between 10% to 40% of the general population in an age-dependent manner. The pathogenesis of essential hypertension is multifactorial, with various underlying contributory mechanisms. Primary aldosteronism (PA) is the leading cause of secondary hypertension that affects 4.3% of patients in the general hypertensive population and 9.5% of patients referred to hypertension units.1 The 2 predominant causes of the constitutive aldosterone secretion that define PA is either aldosterone-producing adenomas (APA) or bilateral adrenal hyperplasia (BAH, also called idiopathic hyperaldosteronism). Exome sequencing has unraveled the pathogenetic basis of around half of APA, but the underlying cause of the aldosterone excess in BAH remains an enigma. The diagnosis of PA is made by a multistep process, recommended by the Endocrine Society guidelines,2 that ends with the invasive protocol of adrenal venous sampling. Once diagnosed, it is imperative to distinguish the surgically correctable forms (APA) from those that should be treated pharmacologically with mineralocorticoid receptor antagonists (BAH). With the application of wider screening for PA, >70% of cases of aldosterone excess have been defined as caused by BAH, with APA making up most of the remainder. APA and BAH are sporadic forms of PA, but 3 rarer familial forms of PA also exist called familial hyperaldosteronism (FH) types I, II, and III, described in more detail below. Three familial forms of PA with distinct clinical characteristics have been described.3,4 FH type I or glucocorticoid remediable aldosteronism is caused by a hybrid gene resulting from the unequal crossing-over between the adjacent genes CYP11B1 and CYP11B2 .5,6 Only few families (≈50) have been identified worldwide. FH type II seems to be inherited as an autosomal dominant trait. A locus has been mapped on chromosome 7p22 …

Reduced Renal K+ Excretion With Compensatory Hyperaldosteronism In KCa1.1 Channel β2‐subunit KO Mice

The kidneys are the primary organs responsible for excreting K+, ensuring whole body K+ homeostasis by precisely matching K+ excretion to dietary K+ intake. K+ is secreted into the urine in the collecting ducts, and two distinct mechanisms for K+ secretion exist; a constitutive mechanism mediated by ROMK (Kir1.1) in principal cells and a flow‐induced mechanism mediated by BK channels (KCa1.1) in intercalated cells. Both mechanisms are up‐regulated by aldosterone. Here we studied renal K+ excretion in KO mice for the β2‐subunit of the BK channel.The β2 KO mice have increased plasma aldosterone, low renin expression and normal plasma [K+]. The low renin in β2 KO mice indicates that hyperaldosteronism was triggered by a K+ handling deficiency, rather than hypotension and activation of the renin‐angiotensin‐aldosterone system. We hypothesize that β2 KO mice have decreased BK channel‐mediated renal K+ secretion, which is compensated by hyperaldosteronism and up‐regulation of ROMK‐mediated K+ secretion, allowing β2 KO mice to maintain normal plasma [K+]. In fact, when treated with eplerenone (mineralocorticoid receptor antagonist) for 4 days, β2 KO mice develop slight hyperkalemia (4.15 mM ± 0.13 in WT vs. 4.60 mM ± 0.10 in KO, P = 0.013).Urinary K+ excretion following oral K+ load (20% of normal daily intake) was not different between WT and KO mice under control conditions. However, when treated with eplerenone, β2 KO mice had a significantly lower urinary K+ excretion rate (P=0.044) and significantly higher plasma [K+] 3 hours after oral K+ load (10.0 mM ± 0.4 in WT vs. 11.4 mM ± 0.5 in KO, P = 0.044). Our data support that hyperaldosteronism in β2 KO mice is part of a chronic compensation to a decreased BK channel‐mediated renal K+ secretion.Supported by the Lundbeck foundation.

Mast cell hyperplasia is associated with aldosterone hypersecretion in a subset of aldosterone-producing adenomas.

Adrenal mast cells can stimulate aldosterone secretion through the local release of serotonin (5-HT) and activation of the 5-HT4 receptor (5-HT4). In aldosterone-producing adenomas (APAs), 5-HT4 receptor is overexpressed and the administration of 5-HT4 receptor agonists to patients with APA increases plasma aldosterone levels. These data and the well-documented role of mast cells in tumorigenesis suggest that mast cells may be involved in the pathophysiology of APA. The study aimed at investigating the occurrence of mast cells in a series of APA tissues and to examine the influence of mast cells on aldosterone secretion. The occurrence of mast cells in APAs was investigated by immunohistochemistry. Mast cell densities were compared with clinical data. The influence of mast cells on aldosterone production was studied by using cultures of human mast cell and adrenocortical cell lines. In APA tissues, the density of mast cells was found to be increased in comparison with normal adrenals. Mast cells were primarily observed in adrenal cortex adjacent to adenomas or in the adenomas themselves, distinguishing two groups of APAs. A subset of adenomas was found to contain a high density of intratumoral mast cells, which was correlated with aldosterone synthase expression and in vivo aldosterone secretory parameters. Administration of conditioned medium from cultures of human mast cell lines to human adrenocortical cells induced a significant increase in aldosterone synthase (CYP11B2) mRNA expression and aldosterone production. APA tissues commonly contain numerous mast cells that may influence aldosterone secretion through the local release of regulatory factors.

PPARγ Agonist-Induced Fluid Retention Depends on αENaC Expression in Connecting Tubules

Background/Aims: Thiazolidinediones (TZDs, like rosiglitazone (RGZ)) are peroxisome proliferator-activated receptor γ (PPARγ) agonists used to treat type 2 diabetes. Clinical limitations include TZD-induced fluid retention and body weight (BW) increase, which are inhibited by amiloride, an epithelial-sodium channel (ENaC) blocker. RGZ-induced fluid retention is maintained in mice with αENaC knockdown in the collecting duct (CD). Since ENaC in the connecting tubule (CNT) rather than in CD appears to be critical for normal NaCl retention, we aimed to further explore the role of ENaC in CNT in RGZ-induced fluid retention. Methods: Mice with conditional inactivation of αENaC in both CNT and CD were used (αENaC lox/lox AQP2-Cre; ‘αENaC-CNT/CD-KO') and compared with littermate controls (αENaC lox/lox mice; ‘WT'). BW was monitored and total body water (TBW) and extracellular fluid volume (ECF) were determined by bioelectrical impedance spectroscopy (BIS) before and after RGZ (320 mg/kg diet for 10 days). Results: On regular NaCl diet, αENaC-CNT/CD-KO had normal BW, TBW, ECF, hematocrit, and plasma Na⁺, K⁺, and creatinine, associated with an increase in plasma aldosterone compared with WT. Challenging αENaC-CNT/CD-KO with a low NaCl diet unmasked impaired NaCl and K homeostasis, consistent with effective knockdown of αENaC. In WT, RGZ increased BW (+6.1%), TBW (+8.4%) and ECF (+10%), consistent with fluid retention. These changes were significantly attenuated in αENaC-CNT/CD-KO (+3.4, 1.3, and 4.3%). Conclusion: Together with the previous studies, the current results are consistent with a role of αENaC in CNT in RGZ-induced fluid retention, which dovetails with the physiological relevance of ENaC in this segment.

Abstract 18390: Ang II activates the RhoA Exchange Factor Arhgef1 in Humans

Introduction: Although a causative role for RhoA-Rho kinase signaling has been recognized in the development of human hypertension, the molecular mechanism(s) as well as the RhoA exchange factors (GEFs) responsible for the over activation of RhoA remain unknown. Arhgef1 has been recently identified as a RhoA GEF involved in Ang II-mediated regulation of vascular tone and hypertension in mice. Hypothesis: Here we assessed the hypothesis that Arhgef1 is activated and involved in the activation of RhoA-Rho kinase signaling by Ang II in humans. Methods: We used in vitro stimulation of human coronary artery smooth muscle cells and human peripheral blood cells (PBMC) by Ang II (0.1 μmol/L), and PBMC isolated from normotensive subjects before and after activation of the renin-angiotensin system by a low-salt diet for 7 days (checked by the increase in plasma aldosterone and active renine). Activation of Arhgef1 was monitored by measuring its tyrosine phosphorylation by western blot and phosphorylation of the Rho kinase target MYPT was used to measure the activity of RhoA/Rho kinase signaling. Results: In vitro, Ang II induced activation of Arhgef1 in human vascular smooth muscle cells and PBMC. Silencing of Arhgef1 expression by siRNA in human vascular smooth muscle cells inhibited Ang II-induced activation of RhoA-Rho kinase signaling (0.67±0.12 vs 3.21±0.91relative to unstimulated condition, n=4, P<0.05). In normotensive subjects, activation of the renin-angiotensin system by a low-salt diet stimulated Arhgef1 activity in PBMC (1.26±0.12-fold over basal level, n=39, P<0.05). This activation was associated with an increase activity of RhoA-Rho kinase signaling (1.33±0.14-fold over basal level, n=39, P<0.05). Conclusions: Our results show that Ang II stimulates Arhgef1 activity and strongly suggest that Arhgef1 mediates Ang II-induced RhoA activation in humans. Moreover, we show for the first time that measurement of RhoA GEF activity in PBMC might be a useful method to evaluate RhoA GEF activity in humans.