Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increase αENaC transcription

Aldosterone is a major regulator of Na+ absorption and acts by activating the mineralocorticoid receptor (MR) to stimulate the epithelial Na+ channel (ENaC). MR −/− mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around postnatal day 10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR −/− mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in inner medullary collecting duct 3 (IMCD3) cells. Real-time RT-quantitative PCR revealed that 5-day-old MR −/− vs. MR +/+ mice had significantly lower αENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (before postnatal day 8) in MR −/− mice.

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Functional tests to guide management in an adult with loss of function of type-1 angiotensin II receptor

Pediatric Nephrology ◽

10.1007/s00467-021-05018-7 ◽

2021 ◽

Author(s):

Daan H. H. M. Viering ◽

Anneke P. Bech ◽

Jeroen H. F. de Baaij ◽

Eric J. Steenbergen ◽

A. H. Jan Danser ◽

...

Keyword(s):

Angiotensin Ii ◽

Collecting Duct ◽

Perinatal Period ◽

Tubular Dysfunction ◽

Loss Of Function ◽

Angiotensin Ii Receptor ◽

Salt Wasting ◽

Truncating Mutation ◽

Urinary Potassium

AbstractBackgroundGenetic loss of function ofAGT(angiotensinogen),REN(renin),ACE(angiotensin-converting enzyme), orAGTR1(type-1 angiotensin II receptor) leads to renal tubular dysgenesis (RTD). This syndrome is almost invariably lethal. Most surviving patients reach stage 5 chronic kidney disease at a young age.MethodsHere, we report a 28-year-old male with a homozygous truncating mutation inAGTR1(p.Arg216*), who survived the perinatal period with a mildly impaired kidney function. In contrast to classic RTD, kidney biopsy showed proximal tubules that were mostly normal. During the subsequent three decades, we observed evidence of both tubular dysfunction (hyperkalemia, metabolic acidosis, salt-wasting and a urinary concentrating defect) and glomerular dysfunction (reduced glomerular filtration rate, currently ~30 mL/min/1.73 m2, accompanied by proteinuria). To investigate the recurrent and severe hyperkalemia, we performed a patient-tailored functional test and showed that high doses of fludrocortisone induced renal potassium excretion by 155%. Furthermore, fludrocortisone lowered renal sodium excretion by 39%, which would have a mitigating effect on salt-wasting. In addition, urinary pH decreased in response to fludrocortisone. Opposite effects on urinary potassium and pH occurred with administration of amiloride, further supporting the notion that a collecting duct is present and able to react to fludrocortisone.ConclusionsThis report provides living proof that even truncating loss-of-function mutations inAGTR1are compatible with life and relatively good GFR and provides evidence for the prescription of fludrocortisone to treat hyperkalemia and salt-wasting in such patients.

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Endothelin receptor mRNA expression in renal medulla identified by in situ RT-PCR

AJP Renal Physiology ◽

10.1152/ajprenal.1995.269.3.f449 ◽

1995 ◽

Vol 269 (3) ◽

pp. F449-F457 ◽

Cited By ~ 19

Author(s):

L. H. Chow ◽

S. Subramanian ◽

G. J. Nuovo ◽

F. Miller ◽

E. P. Nord

Keyword(s):

Mrna Expression ◽

Collecting Duct ◽

Renal Medulla ◽

Receptor Subtypes ◽

Rt Pcr ◽

Receptor Mrna ◽

Pcr Products ◽

Medullary Collecting Duct ◽

Labeled Probe

Three subtypes of endothelin (ET) receptors have been identified by cDNA cloning, namely ET-RA, ET-RB, and ET-RC. In the current study the precise cellular distribution of the ET receptor subtypes in the renal medulla was explored by detecting the corresponding polymerase chain reaction (PCR)-amplified cDNAs by in situ reverse transcription (RT)-PCR. The PCR-amplified cDNAs were detected either by direct incorporation using digoxigenin-dUTP (dig-dUTP) as a nucleotide substrate in the PCR reaction or by in situ hybridization with the dig-dUTP-labeled probe. ET-RB mRNA was detected exclusively in the epithelial cells of the inner and outer medullary collecting duct. In contrast, ET-RA message was observed primarily in interstitial cells and pericytes of the vasae rectae in the outer and inner medulla. Southern blot analysis of PCR-amplified cDNAs reverse transcribed from extracted RNA of rat renal medulla confirmed the specificity of the RT-PCR products. ET-RC mRNA was not detected. We conclude that ET-RB is the major ET receptor found in rat renal medulla and is expressed exclusively on inner medullary collecting duct cells. The pattern of ET receptor mRNA expression described suggests different physiological actions for ET on the diverse cellular structures of the renal medulla.

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Mediators of mineralocorticoid receptor-induced profibrotic inflammatory responses in the heart

Clinical Science ◽

10.1042/cs20080247 ◽

2009 ◽

Vol 116 (9) ◽

pp. 731-739 ◽

Cited By ~ 30

Author(s):

Peter Wilson ◽

James Morgan ◽

John W. Funder ◽

Peter J. Fuller ◽

Morag J. Young

Keyword(s):

Oxidative Stress ◽

Mrna Expression ◽

Mineralocorticoid Receptor ◽

Time Course ◽

Cardiac Fibrosis ◽

Inflammatory Responses ◽

Receptor Activation ◽

Mrna Levels ◽

Tissue Remodelling ◽

Perivascular Inflammation

Coronary, vascular and perivascular inflammation in rats following MR (mineralocorticoid receptor) activation plus salt are well-characterized precursors for the appearance of cardiac fibrosis. Endogenous corticosterone, in the presence of the 11βHSD2 (11β hydroxysteroid dehydrogenase type 2) inhibitor CBX (carbenoxolone) plus salt, produces similar inflammatory responses and tissue remodelling via activation of MR. MR-mediated oxidative stress has previously been suggested to account for these responses. In the present study we thus postulated that when 11βHSD2 is inhibited, endogenous corticosterone bound to unprotected MR in the vessel wall may similarly increase early biomarkers of oxidative stress. Uninephrectomized rats received either DOC (deoxycorticosterone), CBX or CBX plus the MR antagonist EPL (eplerenone) together with 0.9% saline to drink for 4, 8 or 16 days. Uninephrectomized rats maintained on 0.9% saline for 8 days served as controls. After 4 days, both DOC and CBX increased both macrophage infiltration and mRNA expression of the p22phox subunit of NADPH oxidase, whereas CBX, but not DOC, increased expression of the NOX2 (gp91phox) subunit. eNOS [endothelial NOS (NO synthase)] mRNA expression significantly decreased from 4 days for both treatments, and iNOS (inducible NOS) mRNA levels increased after 16 days of DOC or CBX; co-administration of EPL inhibited all responses to CBX. The responses characterized over this time course occurred before measurable increases in cardiac hypertrophy or fibrosis. The findings of the present study support the hypothesis that endogenous corticosterone in the presence of CBX can activate vascular MR to produce both inflammatory and oxidative tissue responses well before the onset of fibrosis, that the two MR ligands induce differential but overlapping patterns of gene expression, and that elevation of NOX2 subunit levels does not appear necessary for full expression of MR-mediated inflammatory and fibrogenic responses.

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Activation of Bone Marrow-Derived Cells Angiotensin (Ang) II Type 1 Receptor by Ang II Promotes Atherosclerotic Plaque Vulnerability

International Journal of Molecular Sciences ◽

10.3390/ijms19092621 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2621

Author(s):

Maxime Pellegrin ◽

Karima Bouzourène ◽

Jean-François Aubert ◽

Aimable Nahimana ◽

Michel Duchosal ◽

...

Keyword(s):

Bone Marrow ◽

Mrna Expression ◽

Atherosclerotic Plaque ◽

Cholesterol Metabolism ◽

Mrna Levels ◽

Ang Ii ◽

Plaque Vulnerability ◽

Plaque Development ◽

Pro Inflammatory Cytokines

Angiotensin (Ang) II triggers vulnerable atherosclerotic plaque development. Bone marrow (BM)-derived cells are key players in atherogenesis but whether Ang II induces plaque vulnerability directly through Ang II type 1 receptor (AT1R) activation on these cells remains to be clarified. In the present study, we investigated whether a lack of AT1R on BM-derived cells might affect Ang II-mediated vulnerable plaque development. The 2-kidney, 1-clip (2K1C) model (Ang II-dependent mouse model of advanced atherosclerosis and vulnerable plaques) was generated in ApoE−/− mice transplanted with AT1aR−/− or AT1aR+/+ BM. Plasma cholesterol as well as hepatic mRNA expression levels of genes involved in cholesterol metabolism were significantly lower in 2K1C mice transplanted with AT1aR−/− BM than in controls. Atherosclerotic lesions were significantly smaller in AT1aR−/− BM 2K1C mice (−79% in the aortic sinus and −71% in whole aorta compared to controls). Plaques from AT1aR−/− BM 2K1C mice exhibited reduced lipid core/fibrous cap and macrophage/smooth muscle cells ratios (−82% and −88%, respectively), and increased collagen content (+70%), indicating a more stable phenotype. Moreover, aortic mRNA levels of pro-inflammatory cytokines IL-12p35, IL-1β, and TNF-α were significantly reduced in AT1aR−/− BM 2K1C mice. No significant differences in either the number of circulating Ly6Chigh inflammatory monocytes and Ly6Clow resident anti-inflammatory monocyte subsets, or in mRNA levels of aortic M1 or M2 macrophage markers were observed between the two groups. No significant differences were observed in splenic mRNA levels of T cell subsets (Th1, Th2, Th17 and Treg) markers between the two groups. In conclusion, direct AT1R activation by Ang II on BM-derived cells promotes hepatic mRNA expression of cholesterol-metabolism-related genes and vascular mRNA expression of pro-inflammatory cytokines that may lead to plaque instability.

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Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.3.f596 ◽

1998 ◽

Vol 274 (3) ◽

pp. F596-F601 ◽

Cited By ~ 2

Author(s):

Géza Fejes-Tóth ◽

Erzsébet Rusvai ◽

Emily S. Cleaveland ◽

Anikó Náray-Fejes-Tóth

Keyword(s):

Mrna Expression ◽

Collecting Duct ◽

Cell Types ◽

Alkaline Media ◽

Mrna Levels ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Acid Base Balance ◽

Acid Base ◽

Base Balance

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3 (base) loading of rabbits for ∼18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for β-actin levels. We found that CCD cells express high levels of AE2 mRNA (∼500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 ± 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, α-intercalated cells, and β-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+ concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of β-intercalated cells.

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Type 1 cannabinoid receptor modulates water deprivation-induced homeostatic responses

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00536.2014 ◽

2015 ◽

Vol 309 (11) ◽

pp. R1358-R1368 ◽

Cited By ~ 7

Author(s):

Silvia G. Ruginsk ◽

Fernanda M. V. Vechiato ◽

Ernane T. Uchoa ◽

Lucila L. K. Elias ◽

Jose Antunes-Rodrigues

Keyword(s):

Mrna Expression ◽

Water Deprivation ◽

Energy Homeostasis ◽

Cannabinoid Receptor ◽

Plasma Concentrations ◽

Hydration Status ◽

Potential Candidate ◽

Mrna Levels ◽

Type 1 Cannabinoid Receptor

The present study investigated the type 1 cannabinoid receptor (CB1R) as a potential candidate to mediate the homeostatic responses triggered by 24 h of water deprivation, which constitutes primarily a hydroelectrolytic challenge and also significantly impacts energy homeostasis. The present results demonstrated for the first time that CB1R mRNA expression is increased in the hypothalamus of water-deprived (WD) rats. Furthermore, the administration of ACEA, a CB1R selective agonist, potentiated WD-induced dipsogenic effect, whereas AM251, a CB1R antagonist, attenuated not only water but also salt intake in response to WD. In parallel with the modulation of thirst and salt appetite, we confirmed that CB1Rs are essential for the development of appropriated neuroendocrine responses. Although the administration of ACEA or AM251 did not produce any effects on WD-induced arginine vasopressin (AVP) secretion, oxytocin (OXT) plasma concentrations were significantly decreased in WD rats treated with ACEA. At the genomic level, ACEA significantly decreased AVP and OXT mRNA expression in the hypothalamus of WD rats, whereas AM251 potentiated both basal and WD-induced stimulatory effects on the transcription of AVP and OXT genes. In addition, we showed that water deprivation alone upregulated proopiomelanocortin, Agouti-related peptide, melanin-concentrating hormone, and orexin A mRNA levels in the hypothalamus, and that CB1Rs regulate main central peptidergic pathways controlling food intake, being that most of these effects were also significantly influenced by the hydration status. In conclusion, the present study demonstrated that CB1Rs participate in the homeostatic responses regulating fluid balance and energy homeostasis during water deprivation.

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Functional upregulation of H+-ATPase by lethal acid stress in cultured inner medullary collecting duct cells

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.4.c1194 ◽

1997 ◽

Vol 273 (4) ◽

pp. C1194-C1205 ◽

Cited By ~ 25

Author(s):

Hassane Amlal ◽

Zhaohui Wang ◽

Manoocher Soleimani

Keyword(s):

Collecting Duct ◽

Acid Stress ◽

Northern Hybridization ◽

Mrna Levels ◽

Inner Medullary Collecting Duct ◽

Duct Cells ◽

Acid Load ◽

Collecting Duct Cells ◽

Medullary Collecting Duct ◽

Mutant Cells

The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells ( P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N, N′-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.

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Characterization of Na+/ HCO 3 − cotransporter isoform NBC-3

AJP Renal Physiology ◽

10.1152/ajprenal.1999.276.6.f903 ◽

1999 ◽

Vol 276 (6) ◽

pp. F903-F913 ◽

Cited By ~ 21

Author(s):

Hassane Amlal ◽

Charles E. Burnham ◽

Manoocher Soleimani

Keyword(s):

Mrna Expression ◽

Ph Regulation ◽

Collecting Duct ◽

Cdna Probe ◽

Spinal Column ◽

Acid Stress ◽

Kidney Cortex ◽

Mrna Levels ◽

Coding Region ◽

Low Levels

Na+-[Formula: see text]cotransporters mediate the transport of[Formula: see text] into or out of the cell. Two Na+-[Formula: see text]cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by ∼90% but increased the Na+-dependent[Formula: see text] cotransport activity by ∼7-fold (as assayed by DIDS-sensitive, Na+-dependent,[Formula: see text]-mediated intracellular pH recovery). This increase was associated with ∼5.5-fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li+ in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting [Formula: see text] from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of [Formula: see text] into the cells.

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Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis

AJP Renal Physiology ◽

10.1152/ajprenal.1998.274.2.f259 ◽

1998 ◽

Vol 274 (2) ◽

pp. F259-F267 ◽

Cited By ~ 8

Author(s):

Shuichi Tsuruoka ◽

Ann M. Kittelberger ◽

George J. Schwartz

Keyword(s):

Metabolic Acidosis ◽

Carbonic Anhydrase ◽

Mrna Expression ◽

Collecting Duct ◽

Proximal Tubules ◽

Rabbit Kidney ◽

Rt Pcr ◽

Chronic Metabolic Acidosis ◽

Nephron Segments ◽

Medullary Collecting Duct

Carbonic anhydrase (CA) facilitates renal bicarbonate reabsorption and acid excretion. Cytosolic CA II catalyzes the buffering of intracellular hydroxyl ions by CO2, whereas membrane-bound CA IV catalyzes the dehydration of carbonic acid generated from the secretion of protons. Although CA II and IV are expressed in rabbit kidney, it is not entirely clear which segments express which isoforms. It was the purpose of this study to characterize the expression of CA II and CA IV mRNAs by specific segments of the nephron using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and to determine the effect of chronic metabolic acidosis on CA expression by those segments. Individual nephron segments (usually 1–2 mm) were isolated by microdissection and subjected to RT-PCR. Amplification was performed simultaneously for CA IV, CA II, and malate dehydrogenase (MDH), a housekeeping gene. The intensities of the PCR products were quantitated by densitometry. CA IV mRNA was expressed by S1 and S2 proximal tubules and by outer medullary collecting duct from inner stripe (OMCDi) and outer stripe and initial inner medullary collecting duct (IMCDi). CA II mRNA was expressed by S1, S2, and S3 proximal tubules, thin descending limb, connecting segment (CNT), and all collecting duct segments. Acid loading induced CA IV mRNA expression in S1 and S2 proximal tubules and in OMCDi and IMCDi. CA II mRNA was induced by acidosis in all three proximal segments and nearly all distal segments beginning with CNT. No upregulation of MDH mRNA expression occurred. These adaptive increases in CA II and IV mRNAs are potentially important in the kidney’s adaptation to chronic metabolic acidosis.

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