Presence Of Aldosterone Research Articles

G protein-coupled estrogen receptor: a promising therapeutic target for aldosterone-induced hypertension.

Aldosterone is one of the most essential hormones synthesized by the adrenal gland because it regulates water and electrolyte balance. G protein-coupled estrogen receptor (GPER) is a newly discovered aldosterone receptor, which is proposed to mediate the non-genomic pathways of aldosterone while the hormone simultaneously interacts with mineralocorticoid receptor. In contrast to its cardio-protective role in postmenopausal women via its interaction with estrogen, GPER seems to trigger vasoconstriction effects and can further induce water and sodium retention in the presence of aldosterone, indicating two entirely different binding sites and effects for estrogen and aldosterone. Accumulating evidence also points to a role of aldosterone in mediating hypertension and its risk factors via the interaction with GPER. Therefore, with this review, we aimed to summarize the research on these interactions to help (1) elucidate the role of GPER activated by aldosterone in the blood vessels, heart, and kidney; (2) compare the non-genomic actions between aldosterone and estrogen mediated by GPER; and (3) address the potential of GPER as a new promising therapeutic target for aldosterone-induced hypertension.

Phosphatases Decrease Water and Urea Permeability in Rat Inner Medullary Collecting Ducts.

We previously showed that the phosphatases PP1/PP2A and PP2B dephosphorylate the water channel, AQP2, suggesting their role in water reabsorption. In this study, we investigated whether protein phosphatase 2A (PP2A) and protein phosphatase 2B (PP2B or calcineurin), which are present in the inner medullary collecting duct (IMCD), are regulators of urea and water permeability. Inhibition of calcineurin by tacrolimus increased both basal and vasopressin-stimulated osmotic water permeability in perfused rat IMCDs. However, tacrolimus did not affect osmotic water permeability in the presence of aldosterone. Inhibition of PP2A by calyculin increased both basal and vasopressin-stimulated osmotic water permeability, and aldosterone reversed the increase by calyculin. Previous studies showed that adrenomedullin (ADM) activates PP2A and decreases osmotic water permeability. Inhibition of PP2A by calyculin prevented the ADM-induced decrease in water reabsorption. ADM reduced the phosphorylation of AQP2 at serine 269 (pSer269 AQP2). Urea is linked to water reabsorption by building up hyperosmolality in the inner medullary interstitium. Calyculin increased urea permeability and phosphorylated UT-A1. Our results indicate that phosphatases regulate water reabsorption. Aldosterone and adrenomedullin decrease urea or osmotic water permeability by acting through calcineurin and PP2A, respectively. PP2A may regulate water reabsorption by dephosphorylating pSer269, AQP2, and UT-A1.

OR07-4 Regulation of Mineralocorticoid Receptor Transactivation By Circadian Protein Timeless

Abstract The mineralocorticoid receptor (MR) is a ligand activated transcription factor that plays an important role in cardiovascular physiology and disease. MR transactivation is regulated by ligand-dependent conformational change in the MR and recruitment of coregulator binding partners to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate a candidate MR coregulator, the circadian gene timeless for ligand and cell-selective regulation of MR transactivation. While timeless is not essential for mammalian clock function and does not bind DNA, timeless RNA and protein levels oscillate over 24hr. Co-expression of timeless dose-dependently down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. Timeless also inhibited MR transactivation of synthetic and native gene promoters, and expression of endogenous MR target genes, in H9c2 cardiac myoblasts. Immunofluorescence staining of timeless, and MR showed colocalization to the nucleus in the presence of aldosterone and co-immunoprecipitation which suggested an interaction, although a direct interaction was not confirmed. Taken together, these data suggest timeless is a novel MR coregulatory protein that may contribute to time-of-day-dependent MR transcriptional outputs and suggest new opportunities for the development of MR antagonists with selective actions. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Mineralocorticoid interaction with glycated albumin downregulates NRF – 2 signaling pathway in renal cells: Insights into diabetic nephropathy

In diabetic nephropathy, hyperglycemia elevates albumin glycation and also results in increased plasma aldosterone. Both glycation and aldosterone are reported to cause oxidative stress by downregulating the NRF-2 pathway and thereby resulting in reduced levels of antioxidants and glycation detoxifying enzymes. We hypothesize that an interaction between aldosterone and glycated albumin may be responsible for amplified oxidative stress and concomitant renal cell damage. Hence, human serum albumin was glycated by methylglyoxal (MGO) in presence of aldosterone. Different structural modifications of albumin, functional modifications and aldosterone binding were analyzed. HEK-293 T cells were treated with aldosterone+glycated albumin along with inhibitors of receptors for mineralocorticoid (MR) and advanced glycation endproducts (RAGE). Cellular MGO content, antioxidant markers (nitric oxide, glutathione, catalase, superoxide dismutase, glutathione peroxidase), detoxification enzymes (aldose reductase, Glyoxalase I, II), their expression along with NRF-2 and Keap-1 were measured. Aldosterone binds to albumin with high affinity which is static and spontaneous. Cell treatment by aldosterone+glycated albumin increased intracellular MGO, MR and RAGE expression; hampered antioxidant, detoxification enzyme activities and reduced NRF-2, Keap-1 expression. Thus, the glycated albumin-aldosterone interaction and its adverse effect on renal cells were confirmed. The results will help in developing better pharmacotherapeutic strategies for diabetic nephropathy.

Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells.

Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

OR12-02 When the Glucocorticoid Receptor Meets the Mineralocorticoid Receptor in the Nucleus of Human Cells

Adrenal corticosteroids, such as glucocorticoids and mineralocorticoids, are indispensable for mediating response to stress, development, limiting inflammation, and maintaining energy and fluid homeostasis. These hormones exert their actions via binding to two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The GR has low affinity for corticosteroids, but is expressed in nearly every cell. In contrast, the MR shows a higher affinity for corticosteroids and its expression is largely confined to those tissues where electrolyte exchange and fluid balance are required. GR and MR act as ligand-activated transcription factors which, following interaction with co-regulators and DNA responsive elements, either promote or repress gene transcription. The affinity for the same ligands, structural homology, and binding to the same DNA regions suggest GR and MR can compensate for each other’s actions. Yet, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate this interplay, we developed U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (GRMR). Immunofluorescence analysis showed that treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear traslocation of GR and MR. Conversely, treatment with 1 nM of aldosterone (Aldo) promoted nuclear translocation of the MR only. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR dimers were detected in the nucleus of GRMR cells. Surprisingly, nuclear GR-MR dimers were also detected in the presence of Aldo, suggesting that it is necessary to activate at least one receptor to induce nuclear traslocation of the heterocomplex. To decipher the functional contribution of GR-MR dimers in the transcriptional response of GR to Dex and MR to Aldo, we performed RNA-seq in GR, MR, and GRMR cells treated with 1 nM of Dex or Aldo. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a blunted Dex-mediated gene response which affected only 1608 genes, suggesting a functional antagonism of MR. Aldo-activated MR regulated the transcription of 1660 genes. However, co-expression of GR expanded the Aldo-mediated gene response to 3150 genes. Strikingly, 74% of these genes were also regulated by Dex via GR, suggesting that GR-MR dimers in the presence of aldosterone are able to mimic the glucocorticod transcriptional response. Our data suggest that the role of distinct GR and MR homo- and hetero-dimers is relevant for regulating gene expression. Dissecting the mechanism and investigating the cross-talk between GR and MR may be useful to understanding these two receptors in heath and disease.

POTASSIUM INDUCED POLYURIA IN RATS: IS THE ALDOSTERONE PARADOX UP TO DATE?

IntroductionAldosterone is a mineralocorticoid produced by the adrenal glands with several physiological functions. The most studied stimulus for aldosterone secretion is the renin‐angiotensin‐aldosterone system (RAAS). Despite the importance of RAAS, the secretion of aldosterone can also be induced independently by an even more powerful stimulus: plasma potassium. It has been shown that a rise of only 0.1 mM of plasma potassium can increase plasma aldosterone by 35%. This rise in aldosterone is important for potassium excretion by the kidney and this does not interfere with sodium handling that happens during RAAS activation. This is known as the aldosterone paradox.ObjectiveIn the present study, we investigated the acute effect of potassium load on the kidney's sodium and potassium handling and its correlation with the aldosterone content in the blood.MethodMale Holtzman rats (280 to 300 g) received a single intragastric load of a solution with high potassium concentration (3.5 mL of 4.6 M KCl) and were immediately returned to metabolic cages for urine collection at each hour to measure sodium and potassium excretion. Groups of rats were decapitated at 3, 6, 9 or 12 hours after the intragastric load for blood collection. Serum sodium, potassium, and aldosterone were analyzed. Data are shown as mean ± SEM, and statistical significance was determined by unpaired t‐test. Experimental protocols were approved by the Ethics Committee for Animal Care and Use (CEUA 05/2018).ResultsWe found that potassium overload induced severe hyperkalemia in the first 9 hours (8.2±0.7; 7.7±0.7 and 8.3±0.1 mEq/l after 3, 6 and 9 h, vs. control: 4.0±0.1 mEq/l). Serum potassium was near the control 12 hours after the load (5.3±0.2 mEq/l). Potassium excretion increased by over 6‐fold reaching the maximum 6 hours after the load (1.2±0.1 μEq/min, vs. control: 0.2±0.2 μEq/min; p<0.05). Interestingly, at the same time, sodium excretion also raised 6‐fold compared to control (2.1±0.5 μEq/min, vs. control: 0.3±0.04 μEq/min, 6 h after the load, p<0.05), along with a 6‐fold increase in urine output with a peak 6 hour after the load (18±2.8 μL/min after 6h, vs. control: 2.8±0.1 μL/min, p<0.05). Aldosterone raised continuously from the 3rd to 12th hour after the load (173±15, 203±18, 208±27, 252±44 ng/dL after 3, 6, 9 and 12 h, vs. control: 10.6±2.8; p<0.05).ConclusionThe present results show that potassium load rapidly (in 3 hours) increases aldosterone in the blood, simultaneously with increases in sodium excretion and urine output. Therefore, potassium alone can promote high urine output even in the presence of aldosterone, which suggests the aldosterone paradox in the presence of hyperkalemia.Support or Funding Information Funding:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Видовые особенности негеномной реакции главных клеток собирательных трубок почек крыс и мышей на альдостерон

We have previously shown the fast nongenomic effect of aldosterone on intracellular sodium ([Na+]i) in the principal cells of rat kidney cortical collecting duct (CCD) in low sodium outer medium (14 mM NaCl). The method of fluorescence microscopy with the intracellular dye Sodium Green was used to study the rate of [Na+]i changes in response to external sodium shift (from 137 to 14 mM ) in Wistar rat and C57bl/6 mouse principal cells. In the presence of aldosterone (10 nM), the level of intracellular sodium in the rat principal cells was higher than in control at the low sodium medium (14 mM NaCl) (р˂0.05). In the principal cells of the mice kidney CCD, the response to a sharp change in the sodium level in the outer medium was significantly faster than we have found in rat, and the nongenomic response to aldosterone (10 nM) was weaker than in rats (р˂0.05). The data obtained for the first time demonstrated the differences in the fast nongenomic aldosterone effects in the principal cells of rat and mice CCD on intracellular sodium balance.

Lipocalin-2 derived from adipose tissue mediates aldosterone-induced renal injury

Increased lipocalin-2 levels are not only the sensitive biomarkers but also causally contribute to the pathogenesis of acute and chronic renal injuries. Mice without lipocalin-2 are protected from the development of chronic kidney diseases. However, the source of lipocalin-2 contributing to the pathological elevation of this molecule in blood and urine during renal injury remain largely unknown. The present study demonstrated that adipose tissue-derived lipocalin-2 represented a major pathological form of this molecule causing inflammatory and fibrotic damages in kidney of mice treated with aldosterone. Compared to sham controls, wild type mice subjected to four-week treatment with aldosterone/uninephrectomy/salt (ANS) showed elevated circulating and urinary lipocalin-2, accompanied by glomerular and tubular injuries in kidney. The same treatment was not able to augment the circulating levels of lipocalin-2 in mice with selective depletion of the Lcn2 alleles in adipose tissues (Adipo-LKO). Despite the increased Lcn2 mRNA expression in kidney and lipocalin-2 protein levels in urine, Adipo-LKO mice were protected from ANS-induced tubular injuries and fibrotic renal damages. Data from acute studies confirmed that aldosterone treatment mainly promoted the urinary excretion of lipocalin-2 originated from adipose tissue; the latter acted to inhibit glomerular expression of Wilms tumor-1 (Wt-1) and enhanced the mRNA levels of clusterin (Clu) and kidney injury marker (KIM)-1. In the presence of aldosterone, acute treatment with recombinant lipocalin-2 variants significantly induced the mRNA expressions of both Clu and kidney injury molecule-1 (Kim-1). Moreover, chronic treatment with R81E, a variant form of lipocain-2, led to renal injuries in mice lacking the Lcn2 alleles, resembling those observed in wild type mice treated with ANS. Collectively, the results demonstrated that lipocalin-2 derived from adipose tissue caused ANS-induced renal injuries.

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.

Mineralocorticoid receptor as a therapeutic target in chronic kidney disease and hypertension.

The kidney has a central role in long-term control of blood pressure, and decreased kidney function is a common but difficult-to-treat cause of hypertension. Conversely, elevated blood pressure contributes to the progression of chronic kidney disease. Steroid hormone aldosterone and its receptor mineralocorticoid receptor (MR) contribute to hypertension by increasing renal salt reabsorption and promote kidney dysfunction through direct effects on renal parenchymal cells. Accumulating data indicate that various mechanisms affect aldosterone-MR signaling. Using a genetically engineered mouse model, we identified crosstalk between small GTPase Rac1 and MR. This crosstalk pathway promotes glomerular podocyte injury, and is also involved in the pathogenesis of hypertension. Notably, salt loading increases renal Rac1 activity in several models of salt-sensitive hypertension, which, in the presence of aldosterone, synergistically activates MR signaling, causing hypertension and kidney injury. There is also a mechanism regulating MR in a cell-selective manner. In the principal cells of the collecting duct, aldosterone directly binds and activate MR. In neighboring intercalated cells, however, binding of aldosterone to MR is regulated by phosphorylation at the ligand-binding domain. This mechanism serves as a switch to turn on electrolyte flux pathways in intercalated cells, allowing aldosterone to exert distinct effects in different physiological contexts. Given the potential benefit of MR blockade in hypertensive kidney disease, the delineation of these pathways may lead to the identification of alternative therapeutic targets. In this review, we discuss the roles of MR in mediating kidney disease and hypertension, with a focus on the crosstalk among related signaling pathways.

The effect of aldosterone on Na+/K+/ATPase expression and development of embryos derived from vitrified-warmed sheep oocytes

Media supplementation with various compounds in order to increase the oocyte developmental competence is of particular importance. This experiment was conducted to evaluate the effect(s) of media supplementation with aldosterone on embryo development and Na+/K+/ATPase expression following sheep oocyte vitrification. The abattoir-derived sheep COCs (cumulus oocyte complexes) were randomly allocated into six experimental groups: IVM of fresh and vitrified COCs in the presence of aldosterone followed by IVF/IVC (F-IVM and Vit-IVM groups, respectively); IVM/IVF of fresh and vitrified COCs followed by IVC wherein the embryos were exposed to aldosterone on Day 4 of IVC (F-D4 and Vit-D4 groups, respectively); and IVM/IVF and IVC of fresh and vitrified COCs in the absence of aldosterone (F-Cont and Vit-Cont groups, respectively). The expression of Na+/K+/ATPase α1 and β1 subunits in embryos were assessed at morula and blastocyst stages with related primary antibodies. The hatching rate was greater in aldosterone supplemented groups in both fresh and vitrified COCs compared to their controls. The expression of Na+/K+/ATPase β1 subunit was significantly greater in F-D4 and Vit-D4 groups compared to other groups. The ICM/Total ratio in F-IVM was greater compared to the other groups. In conclusion, addition of aldosterone to the IVM and IVC media could improve the hatching rate of blastocysts derived from both fresh and vitrified COCs. These improvements, in D4 supplemented groups, might be related to the greater expression of Na+/K+/ATPase β1 subunit induced by aldosterone supplementation.

Long-Term Modulation of Ion Channels by Aldosterone in Adult Rat Atrial Myocytes

In recent years, both aldosterone and mineralocorticoid receptor (MR) have drawn attention as important factors that promote structural remodeling of the atrium. Here, we investigated effects of chronic aldosterone treatment on both intracellular Ca2+ and ion channels. Atrial myocytes were cultured in either the absence or presence of aldosterone, and then the activity of ion channels was studied, under whole-cell patch-clamp conditions. Aldosterone increased both the cell membrane capacitance (Cm) and the maximal conductance of ion channels that give rise to IKsus, INa, ICaT, and ICaL (30-100%). Except for inactivation curves of INa, which were shifted by −10 mV, aldosterone produced no major alterations in the biophysical properties of the channels. Interestingly, at resting membrane potentials the increase in INa was cancelled by a greater fraction of inactivation. The onset and recovery of the changes -in ICaT, ICaL, and Cm- were also assessed. In general, they required 2d to be noticeable, reached their maximal value in 6d, and returned to basal values after 1-3d of aldosterone removal. The effects on both Cm and ICaL were further studied -to explore both a potential dose-response relationship and a possible implication of the MR. In fact, co-incubating with 10 µM of spironolactone (an MR antagonist) abolished both effects. Furthermore, their corresponding magnitudes fitted well with the Hill equation, being the EC50 values for Cm and ICaL 20 and 130 (nM), respectively. Interestingly, aldosterone did not alter expression levels of Cav1.2, suggesting that the action on ICaL arises from a stimulus in open probability. The hormone also produced a 40% increase in the amplitude of Ca2+ transients along with a higher proportion of arrhythmic cells (2.5-fold increase). These results contribute to understanding the role of the MR and aldosterone in atrial electrophysiology.

Identification and characterization of a ligand-selective mineralocorticoid receptor coactivator.

The mineralocorticoid receptor (MR) is unique in responding to 2 physiological ligands: aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11β-hydroxysteroid dehydrogenase type 2. In other tissues, cortisol is the primary ligand. To understand the structural determinants of ligand-specific MR activation, we sought to identify coregulatory molecules that interact with the ligand-binding domain (LBD) of the MR. A yeast-2-hybrid (Y2H) kidney cDNA library was screened with the human MR-LBD in the presence of aldosterone and cortisol. One clone, identified as aldosterone-specific in the Y2H assay, exhibited a 7-fold greater response, aldosterone vs. cortisol, in a mammalian-2-hybrid (M2H) assay. This clone encodes the region of the tesmin gene that has 2 leucine-x-x-leucine-leucine (LxxLL) motifs. Full-length tesmin coactivates (>2-fold) MR-mediated transactivation in the presence of aldosterone, but not of cortisol; this specificity is observed with a range of promoters. GST pulldown and coimmunoprecipitation of the MR by tesmin supports a direct interaction, mediated by the 2 LxxLL motifs. Tesmin thus represents a novel MR coregulator that exhibits a differential interaction, providing further evidence of the adoption of ligand-dependent conformations by the MR-LBD.

Aberrant Rac1–mineralocorticoid receptor pathways in salt‐sensitive hypertension

According to Guyton's model, impaired renal sodium excretion plays a key role in the increased salt sensitivity of blood pressure (BP). Several factors contribute to impaired renal sodium excretion, including the sympathetic nervous system, the renin-angiotensin system and aldosterone. Accumulating evidence suggests that abnormalities in aldosterone and its receptor (i.e. the mineralocorticoid receptor (MR)) are involved in the development of salt-sensitive (SS) hypertension. Patients with metabolic syndrome often exhibit hyperaldosteronism and are susceptible to SS hypertension. Aldosterone secretion from the adrenal glands is not suppressed in obese hypertensive rats fed a high-salt diet because of the abundant production of adipocyte-derived aldosterone-releasing factors, which are independent of the negative feedback regulation of aldosterone secretion by the renin-angiotensin-aldosterone system. Increased plasma aldosterone levels lead to SS hypertension via MR activation in the kidney. Renal MR activity is increased in Dahl salt-sensitive rats fed a high-salt diet, despite the appropriate suppression of plasma aldosterone levels. In this rat strain, activation of MR in the distal nephron causes salt-induced hypertension. This paradoxical response of the MR to salt loading can be attributed to activation of Rac1, a small GTPase. In the presence of aldosterone, activated Rac1 synergistically and directly activates MR in a ligand-independent manner. Thus, Rac1 activation in the kidney determines the salt sensitivity of BP. Together, the available evidence suggests that the aberrant Rac1-MR pathway plays a key role in the development of SS hypertension.

The Effect of Aldosterone on the miRNA Content of a Murine Inner Medullary Collecting Duct Cell

MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension‐related renal disease.Aldosterone is the major known hormone responsible for sodium retention in the kidney collecting duct. Our working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption.Here we report the miRNA landscape in mIMCD‐3 cells using Toray 3D‐GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 35 highly expressed miRNAs, as well as several miRNAs whose levels appear to be affected by the presence of aldosterone. Two aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1and sodium/potassium‐transporting ATPase subunit beta‐1 mRNA. Argonaute pull‐down experiments also demonstrated that endothelin‐1 mRNA is a target of the RNA‐induced silencing complex indicating miRNA action on this transcript.This work was supported by NIH NIDDK 1RO1DK082680 to B.D.C and C.S.W. and a 5T32DK076541–04 pre‐doctoral fellowship to M.E.J. Additional funding support and miRNA microarrays were provided by Toray Industries, Inc.

The Migratory Capacity of Human Trophoblastic BeWo Cells: Effects of Aldosterone and the Epithelial Sodium Channel

Aldosterone is a key regulator of the epithelial sodium channel (ENaC) and stimulates protein methylation on the β-subunit of the ENaC. We found that aldosterone (100 nM) promotes cellular migration in a wound-healing model in trophoblastic BeWo cells. Here, we tested if the positive influence of aldosterone on wound healing is related to methylation reactions. Cell migration and proliferation were measured in BeWo cells at 6h, when mitosis is still scarce. Cell migration covered 12.4, 25.3, 19.6 and 45.1% of the wound when cultivated under control, aldosterone (12h), 8Br-cAMP and aldosterone plus 8Br-cAMP, respectively. Amiloride blocked the effects of aldosterone alone or in the presence of 8Br-cAMP on wound healing. Wound healing decreased in aldosterone (plus 8Br-cAMP) coexposed with the methylation inhibitor 3-deaza-adenosine (3-DZA, 12.9% reinvasion of the wound). There was an increase in wound healing in aldosterone-, 8Br-cAMP- and 3-DZA-treated cells in the presence of AdoMet, a methyl donor, compared to cells in the absence of AdoMet (27.3 and 12.9% reinvasion of the wound, respectively). Cell proliferation assessed with the reagent MTT was not changed in any of these treatments, suggesting that cellular migration is the main factor for reinvasion of wound healing. Electrophysiological studies showed an increase in ENaC current in the presence of aldosterone. This effect was higher with 8Br-cAMP, and there was a decrease when 3-DZA was present. AdoMet treatment partially reversed this phenomenon. We suggest that aldosterone positively influences wound healing in BeWo cells, at least in part through methylation of the ENaC.

Role of Per1 and the mineralocorticoid receptor in the coordinate regulation of αENaC in renal cortical collecting duct cells

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.

Activation of an apoptotic signal transduction pathway involved in the upregulation of calpain and apoptosis-inducing factor in aldosterone-induced primary cultured cardiomyocytes

In this study, aldosterone (ALD)-induced apoptosis of cardiomyocyte was evaluated based on the previous studies, and the roles of calpain signaling were clarified. Primary cultured rat cardiomyocytes were injured by ALD (0.01-10 μM) for varying time periods. Then, the effects of ethylene glycol tetraacetic acid (EGTA) (0.5 mM), calpeptin (2.5 μM), and spironoclactone (10 μM) were evaluated on cardiomyocytes activated by ALD. Cardiomyocytes that were injured by ALD were assayed by the MTT and LDH leakage ratio. Apoptosis was evaluated by a TUNEL assay, annexin V/PI staining, and caspase-3 activity. The expression of cleavage of Bid (tBid), calpain and apoptosis-inducing factor (AIF) was evaluated by western blot analysis. ALD increased calpain expression and caspase-3 activity and promoted Bid cleavage. It also induced the release of AIF from mitochondria into the cytosol. The upregulation of calpain, tBid and caspase-3 activity were further inhibited by treatment with EGTA in the presence of ALD. Additionally, AIF levels in the cytosol decreased due to EGTA but not due to calpeptin. This was also accompanied by a significant decrease in apoptosis. Furthermore, treatment with spironoclactone not only attenuated the pro-apoptotic effect of ALD but reversed the ALD-induced increase of calpain and AIF levels.

The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na+ transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,β,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.