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Endocrinology Vol. 148, No. 4 1511-1517
Copyright © 2007 by The Endocrine Society

BRIEF COMMUNICATION

A Direct Effect of Aldosterone on Endothelin-1 Gene Expression in Vivo

Stephen Wong, Francine E. Brennan, Morag J. Young, Peter J. Fuller and Timothy J. Cole

Department of Biochemistry and Molecular Biology (S.W., P.J.F., T.J.C.), Monash University, Clayton, Victoria 3800, Australia; and Prince Henry’s Institute of Medical Research (F.E.B., M.J.Y., P.J.F.), Monash Medical Centre, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Dr. Timothy J. Cole, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: Tim.Cole{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aldosterone regulates sodium reabsorption in epithelial tissues such as the kidney and colon, via a pathway involving the activation of intracellular mineralocorticoid receptors (MR), induction of specific target genes, and a subsequent increase in sodium channel activity. Characterized aldosterone target genes in epithelia include the serum and glucocorticoid-regulated kinase 1 and the corticosteroid hormone-induced factor. Endothelin-1 (ET-1) is a potent vasoconstrictor that alters both sodium transport and hydrogen ion secretion in the kidney. Recent studies in a mouse medullary collecting duct cell line and rat A-10 smooth muscle cells have demonstrated an acute response of ET-1 gene expression to aldosterone. In the present study, we have investigated the ET-1 gene in vivo as a potential direct aldosterone-regulated target gene in the kidney and colon. Adrenalectomized rats given a single dose of aldosterone were found to have a 2-fold increase in ET-1 mRNA levels in the kidney and colon after 1 h. No significant changes in mRNA levels were detected for the related isoforms ET-2 or ET-3. Cotreatment with aldosterone and potassium canrenoate, a MR antagonist, blocked induction of ET-1 mRNA, suggesting that induction was mediated via the MR. In a time course study, ET-1 mRNA levels were induced rapidly by aldosterone, with levels of ET-1 mRNA maximally increased 2- and 2.5-fold after 1 h in the kidney and colon, respectively. These results suggest that ET-1 is a direct aldosterone gene target in the kidney and colon and may play an important role in aldosterone-regulated ion homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALDOSTERONE PROMOTES SODIUM reabsorption in epithelial tissues such as the kidney and colon and plays a critical role in blood pressure homeostasis (1, 2). It also regulates K+ secretion and is implicated in the pathophysiology of cardiac fibrosis and cardiac hypertrophy in end-stage heart failure (3, 4). Aldosterone regulates nuclear gene transcription in target cells by binding to and activating the mineralocorticoid receptor (MR), which functions as a ligand-activated DNA-bound transcription factor (5). Activated MR is able to bind to specific DNA response elements located in the 5' regulatory regions near the promoters of specific target genes. However, activation of MR by aldosterone is complicated by higher circulating levels of glucocorticoids that also bind to MR with the same affinity as aldosterone (6, 7). This is resolved in many epithelial tissues by the presence of the enzyme 11ß-hydroxysteroid dehydrogenase type 2, which rapidly inactivates physiological glucocorticoids allowing aldosterone to bind and activate MR. In nonepithelial tissues lacking 11ß-hydroxysteroid dehydrogenase type 2, the role of aldosterone-mediated effects is not clearly understood and indeed the principal ligand for the MR may, in fact, be cortisol (8).

Only a few aldosterone-induced target genes have been characterized in detail. These include the {alpha}, ß, and {gamma} subunits of the epithelial sodium channel (ENaC) (9). Other characterized targets include the serum and glucocorticoid-regulated kinase-1 (SGK-1), an important mediator of renal sodium homeostasis, corticosteroid hormone-induced factor (CHIF), which regulates the activity of the sodium and potassium-dependent adenosine triphosphatase pump (Na-K-ATPase), the glucocorticoid-induced leucine zipper protein, a transcription factor, and the G protein K-Ras2 (10, 11, 12). SGK-1 is thought to regulate Na+ flux by increasing ENaC activity at the apical surface of epithelial cells. Aldosterone treatment of a rat collecting duct cell line as well as an adrenalectomized rat model demonstrated a rapid induction of SGK-1 mRNA within 30 min of treatment (13, 14). However, SGK-1 null mice only show mild abnormalities in sodium homeostasis, suggesting that other genomic targets are important for overall regulation of sodium transport (15).

Recent studies in cell lines using gene microarray analysis have uncovered a number of novel genes as being potential direct aldosterone gene targets. One such target is endothelin-1 (ET-1), a 21-amino-acid vasoconstrictor peptide that is synthesized by vascular endothelial cells (16). ET-1 has two related isoforms, endothelin-2 (ET-2) and endothelin-3 (ET-3). The three isoforms all bind and activate two cell surface receptors, ETA and ETB. Binding of endothelins to ETA receptors on vascular smooth muscle cells leads to calcium mobilization and smooth muscle contraction. ET-1 is also known to bind to ETB receptors located on vascular endothelial cells to stimulate the formation of nitric oxide. In general, the action of ET-1 is to increase blood pressure and vascular tone (17). Endothelin receptor antagonists such as bosentan have been extensively used in the treatment of pulmonary hypertension (18). Release of ET-1 is stimulated by a number of factors including angiotensin II, antidiuretic hormone, thrombin, cytokines, and reactive oxygen species. ET-1 has other functions apart from vasoconstriction, including roles in the regulation of cellular proliferation (19). A number of studies have shown that ET-1 affects renal Na+ transport and causes increased acidification in the distal nephron (20, 21). Several lines of evidence support a role for ET-1 in the pathogenesis of renal and cardiac interstitial fibrosis (22, 23). For example, ET-1 has been found to activate transcription of the procollagen I promoter via stimulation of ETB receptors; a potential mechanism for aldosterone to increase collagen deposition (24).

It has been reported recently that aldosterone increases ET-1 expression in both the rat A-10 smooth muscle cell line and in a mouse cortical collecting duct cell line (25, 26). The study using A-10 cells also showed stimulation of SGK-1 by ET-1 (26), suggesting that aldosterone and ET-1 may potentially share common intracellular signaling pathways to regulate blood pressure and tissue remodeling. These in vitro studies suggest that ET-1 may represent an aldosterone-mediated target gene. We have investigated the induction of ET-1 mRNA in the kidney and colon in response to a single dose of aldosterone in adrenalectomized rats. We provide evidence that aldosterone stimulates an acute increase in ET-1 mRNA levels in vivo in the kidney and colon, through activation of the MR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Two groups of Sprague Dawley adult rats were bilaterally adrenalectomized, maintained on 0.9% saline to drink, and treated as follows. Group A: rats (200–300 g) received either a single dose of 50 µg/100 g body weight (bw) aldosterone, a dose of 50 µg/100 g bw aldosterone plus 20 mg of the MR antagonist potassium canrenoate, or 20 mg of the MR antagonist potassium canrenoate alone. Rats were killed 60 min later, and tissues were collected for analysis. Group B: rats (100–200 g) received a single dose of 10 µg/100 g bw aldosterone, were coinjected ip with 10 mg of the glucocorticoid receptor (GR) antagonist RU486 to block GRs, and then killed 0, 30, 60, 90, 120, and 180 min later (14).

RNA extraction
Total RNA was prepared from isolated rat kidney and colon by homogenization in TRIzol reagent (Life Technologies, Inc./BRL, Auckland, New Zealand), a guanidinium isothiocyanate/phenol-based homogenization solution. After chloroform extraction, RNA was precipitated from the aqueous phase with isopropanol, washed in 70% ethanol, and redissolved in nuclease-free sterile water.

Quantitative real-time PCR
Real-time PCR was performed using the Corbett Research RotorGene 3000 System (Corbett Research, Sydney, Australia) using SYBR Green I. Primer pairs were designed with the Primer 3 RT-PCR program and checked for sequence homology against known genes using the BLAST search program (27). Primers for quantitative RT-PCR (qRT-PCR) were: SGK-1 forward: 5'-TAGCAATCCTCATCGCTTTC-3', reverse: 5'-GAGTTGTTGGCAAGCTTCTG-3'; CHIF forward: 5'-GGGAATAACCTGTGCCTTTC-3', reverse: 5'-AGGGACTGCCTTTATCAACTG-3'; ET-1 forward: 5'-CTCTGCTGTTTGTGGCTTTC-3', reverse: 5'-CCTCTGCCAGTCTGAACAAG-3'; ET-2 forward: 5'-CAACTCCTGGCTTGACAA-3', reverse: 5'-GTCTGTCCTGCAGTGTTC-3'; ET-3 forward: 5'-CCAGTTATTCCAGGAGAGCA-3', reverse: 5'-CTTGACTTCAGCCTTTGACG-3'; ETA forward: 5'-CCCATCAATGTGTTTAAGCTG-3', reverse: 5'-GGAACAGCTTGCAGAGAAAC-3'; ETB forward: 5'-TTTAAGTCGTGTTTGTGCTGC-3', reverse: 5'-AAGCAGGATTGCTTCTCCTC-3'. cDNA templates for real-time PCR were synthesized using the M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant Kit (Promega, Madison, WI) from 2 µg of rat cardiac total RNA. All reactions were optimized to obtain the best amplification kinetics. For the Rotor-Gene, the PCR mixture (20 µl) contained 10 µl of Platinum SYBR Green qPCR SuperMix UDG (SYBR Green I, 60 U/ml Platinum Taq DNA polymerase, 40 mM Tris-HCl (pH 8.4), 100 mM KCl, 6 mM MgCl2, 400 µM dGTP, 400 µM dATP, 400 µM dCTP, 400 µM dUTP, 40 U/ml uracil DNA glycosylase, and stabilizers), 1 µl of forward and reverse primers (1 µM each), and 1 µl cDNA template. Water was used as a no-template control. Cycling conditions were: 1 cycle at 95 C for 2 min, 50 cycles at 95 C for 2 sec, 60 C for 20 sec, and 76 C for 15 sec. DNA amplicons were analyzed for specificity by agarose gel electrophoresis and DNA sequencing of PCR fragments. Analysis was performed with the Rotor-Gene 3000 Software, version 6, with slope correction and reaction efficiency threshold enabled. Samples were performed in triplicate with 18S ribosomal RNA used as an endogenous reference gene. The Ct was determined from a curve generated from a plot of cycle number vs. fluorescence with a manual threshold set above the background fluorescence of the no-template control.

Northern blot analysis
Total RNA (15 µg) was separated in 1.2% agarose/2.2 M formaldehyde gels and blotted onto Genescreen Plus nylon membranes for Northern blot analysis by hybridization with 32P-labeled DNA probes. To make a rat ET-1 probe, 1 µg of total rat RNA was reverse transcribed followed by PCR using the Access Quick RT-PCR kit (Promega). The ET-1 primers were sense, 5'- CTCTGCTGTTTGTGGCTTTC-3', and antisense, 5' CCTCTGCCAGTCTGAACAAG-3', yielding an amplicon of 725 bp. PCR products were then ligated into pGEM-T Easy vector (Promega) and then transformed into Escherichia coli JM109 competent cells. A radioactive DNA probe for ET-1 was then generated using the Prime-a Gene Labeling system (Promega) using isolated insert DNA. All filters were hybridized at 42 C in Ultrahyb buffer (Ambion, Austin, TX) and washed at 45 C in 0.1x SSC, 0.1% SDS two times for 15 min each. Filters were rehybridized with a 32P-labeled 323-bp cDNA fragment of rat 18S rRNA to control for differences in the total RNA loading on agarose gels.

Statistical analysis
For all Northern blot analyses, RNA levels were quantified by densitometric analysis on a Storm Phosphoimager from Molecular Dynamics (Sunnyvale, CA) using ImageQuant software, and standardized to the expression of 18S rRNA, with statistical significance set at P < 0.05. Statistical analyses for real-time PCR were performed using a Student’s t test. P < 0.05 was considered to denote statistically significant differences between treatment groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aldosterone increases ET-1 mRNA levels in vivo in the kidney and colon
Total RNA from rat kidney and distal colon was first examined for induction of two known aldosterone target genes, SGK-1 and CHIF, after treatment with aldosterone. Using qRT-PCR, SGK-1 and CHIF mRNA levels in the distal colon increased 4.5- and 4.8-fold 1 h after aldosterone treatment, respectively (Fig. 1Go, A and B). The levels of induction corresponded to those previously published and demonstrate that aldosterone-mediated effects on target genes were present in the treated animals (10, 14). To investigate the effect of aldosterone on ET-1 gene expression, qRT-PCR was used to measure ET-1 mRNA levels in rats 1 h after treatment with aldosterone. Figure 1CGo shows a statistically significant 1.8-fold increase in ET-1 mRNA levels in the distal colon 1 h after aldosterone treatment, whereas in the kidney from the same animals, mRNA levels for ET-1 increased by approximately 1.7-fold (Fig. 1DGo).


Figure 1
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  		FIG. 1. Analysis by qRT-PCR of SGK-1, CHIF, and ET-1 mRNA levels in kidney and colon 1 h after aldosterone treatment. A, SGK-1 mRNA levels in colon total RNA. Levels were standardized to 18S rRNA mRNA as a housekeeping control. B, CHIF mRNA levels in colon total RNA 1 h after aldosterone standardized as in A. C, Analysis of ET-1 mRNA levels in colon total RNA from 1-h vehicle and aldosterone-treated rats standardized to levels of 18S rRNA. D, Analysis of ET-1 mRNA levels in kidney total RNA from 1-h vehicle and aldosterone-treated rats standardized to levels of 18S rRNA. E, Agarose gel analysis of real-time PCR amplicons. Ethidium bromide stained 1.5% agarose gel showing ET-1, ET-2, ET-3, ETA receptor, ETB receptor, and 18S rRNA amplicon DNA bands. All values are the mean ± SEM (n = 4); *, P < 0.05; **, P < 0.005. Veh, Vehicle; Aldo, aldosterone.

 
Analysis of the endothelin peptide family and endothelin receptor gene expression in the kidney and colon after treatment with aldosterone
We next assessed whether the other isoforms of ET-1 and the receptors ETA and ETB were also induced by aldosterone in the kidney and distal colon, using real-time PCR. Each PCR amplicon was analyzed by agarose gel electrophoresis (Fig. 1EGo), and their identities were verified by DNA sequencing (data not shown). The mRNA levels of ET-2 (Fig. 2AGo), ET-3 (Fig. 2BGo), and ETB (Fig. 2DGo) did not change significantly in either the kidney or the distal colon after treatment with aldosterone. ETA mRNA levels increased slightly (1.5-fold) in colon but not in kidney, although this increase did not achieve statistical significance (P = 0.055) (Fig. 2CGo). To confirm the induction of ET-1 by aldosterone, Northern blot analysis was performed with total RNA samples isolated from kidney and colon 1 h after aldosterone treatment. ET-1 mRNA levels in distal colon were observed to increase 1.5-fold compared with control while aldosterone increased ET-1 mRNA levels by 2-fold in total RNA from the kidney (data not shown). These changes were consistent with those measured using quantitative real-time PCR shown in Fig. 1Go.


Figure 2
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  		FIG. 2. Analysis by qRT-PCR of mRNA levels for ET-2, ET-3, and the endothelin receptors ETA and ETB in the kidney and colon 1 h after treatment with aldosterone. ET-2 (A), ET-3 (B), ETA (C), and ETB (D) mRNA levels in kidney and colon, from vehicle and aldosterone-treated rats standardized to the level of 18S rRNA. Values are the mean ± SEM (n = 4). Veh, Vehicle; Aldo, aldosterone.

 
Aldosterone acts via MR to increase ET-1 mRNA levels in the kidney and colon
To explore the signaling pathway responsible for the aldosterone-induced increase in ET-1 mRNA, we examined the role of the MR using potassium canrenoate, a MR antagonist. Adrenalectomized rats were treated with aldosterone with and without potassium canrenoate and expression levels determined after 1 h by densitometric analysis of hybridized Northern blots. As shown in Fig. 3Go, A and B, the observed aldosterone-induced increase in ET-1 mRNA in both the kidney and colon was attenuated by coadministration of potassium canrenoate. This demonstrates that MR is involved in mediating the induction of ET-1 mRNA by aldosterone in both kidney and colon.


Figure 3
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  		FIG. 3. Northern blot analysis of ET-1 mRNA levels in kidney and colon after aldosterone treatment and in the presence of the MR antagonist potassium canrenoate. Tissue samples from kidney (A) and colon (B) were from rats treated with aldosterone alone (Aldo), potassium canrenoate alone (KC), aldosterone plus potassium canrenoate (Aldo + KC), or vehicle (Veh). Total RNA (15 µg) was analyzed in a 1.2% agarose gel, and after blotting, the filter was hybridized with a probe specific for rat ET-1. Filters were reprobed for 18S rRNA, scanned and quantified as described in Materials and Methods. Statistical analysis was performed using Student’s t test. Values are the mean ± SEM (n = 3); *, P < 0.05.

 
Time course of induction of ET-1 mRNA in the kidney and colon by aldosterone
ET-1 mRNA levels were analyzed in a time course extending up to 3 h after treatment with aldosterone by Northern blot analysis. In the kidney, there was a rapid increase in ET-1 mRNA after only 30 min with levels reaching a maximal 2-fold increase after 1 h (Fig. 4AGo). In the colon, induction of ET-1 mRNA by aldosterone was also maximal at 60 min postinjection of aldosterone; with the ET-1 mRNA levels approximately 2.5 those of control rats at time 0. After this time, the levels of ET-1 mRNA began to steadily decrease (Fig. 4BGo). Overall, this demonstrates a rapid and significant induction of ET-1 mRNA by aldosterone in vivo in both the kidney and colon.


Figure 4
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  		FIG. 4. Time course of the response to aldosterone of ET-1 mRNA levels in kidney and colon. Total RNA (15 µg) was prepared from kidney (A) and colon (B) at 0, 30, 60, 90, 120, and 180 min after rats had been treated with a single dose of aldosterone (10 µg/100 g bw). Total RNA was analyzed by Northern blot analysis as described in Materials and Methods. Levels of ET-1 mRNA were quantified and normalized to levels of 18S rRNA. Statistical analysis was performed using Student’s t test. Values are the mean ± SEM (n = 4 or 5); *, P < 0.05; **, P < 0.005; #, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The effects of aldosterone action on ion channels and transporters such as ENaC and the Na-K-ATPase in the kidney have been well established (2). The actual gene targets that are directly regulated by aldosterone have been less well defined, particularly in the context of sodium transport and blood pressure regulation. In general, the search for aldosterone-induced genes has been a challenge due to the relatively small changes in either mRNA or protein levels of genes involved in the ion transporting pathways. Most characterized aldosterone gene targets such as SGK-1, CHIF, the ENaC subunits, and K-Ras2A have only shown modest 2- to 3-fold changes shortly after treatment with aldosterone (1, 2). Other targets such as the cardiac AT1 receptor have shown a modest 3-fold change over a longer 3- to 4-wk time period after treatment with aldosterone, although this may not reflect a direct transcriptional effect (28).

A link between mineralocorticoids and ET-1 was first suggested by studies with deoxycorticosterone acetate-salt hypertensive rats, where increased ET-1 immunoreactivity and mRNA levels were detected in aortic and mesenteric arteries 2–3 wk after the development of hypertension (29, 30). More recently, a gene microarray study using a cortical collecting duct cell line has described several potential aldosterone-regulated genes that included ET-1 (25). This study demonstrated up-regulation of ET-1 mRNA by a modest 2.1-fold at 1 h after treatment with aldosterone, which was blocked by concurrent treatment with the MR antagonist spironolactone. Despite numerous studies involving both aldosterone and ET-1, a direct link between them in vivo has remained uncertain. In this current study, we present data that extends the previous in vitro studies (25, 26) and demonstrates acute induction of ET-1 mRNA by aldosterone in vivo in the kidney and colon, two important aldosterone target tissues. We show through quantitative real-time PCR that of the three members of the endothelin family, ET-1, -2, and -3, only ET-1 is induced by aldosterone. Endothelins mediate their physiological effects via two receptors, ETA and ETB. The ETA receptor has been linked to pathophysiological roles in cardiovascular and renal disease, whereas the ETB receptor has been linked to regulation of sodium balance and arterial blood pressure (16). However, neither ETA nor ETB mRNAs were induced by aldosterone in the kidney or colon in the animal model used here, whereas antagonism of the ETA receptor has been shown to block the vascular remodeling effects seen in chronically aldosterone-treated rats (31, 32).

First, we show that ET-1 mRNA is increased by aldosterone in a rapid time-dependent manner, and second, via the use of a coadministered antagonist potassium canrenoate, we show that the MR is involved in aldosterone-mediated induction of ET-1 mRNA. Previous in vitro studies have used high concentrations of aldosterone (up to 10–6 M) with the consequence that the induction could be mediated via GR, for which aldosterone has a low but appreciable affinity. An earlier study performed in amphibian kidney-derived A6 cells showed that aldosterone induces SGK-1 activity via GR (13). Moreover, there have been claims that GR and MR can heterodimerize in vitro and in vivo with resultant GR/MR-mediated effects (33). In the present study, we have investigated the induction of ET-1 mRNA by aldosterone using a relatively low physiological dose of aldosterone (10 µg/100 g bw) and also included RU486 with aldosterone treatment to prevent the possibility of GR occupancy and activation by aldosterone.

Our results strongly suggest that ET-1 mRNA levels in the kidney and colon are increased via the classic MR-mediated genomic actions of aldosterone. The time-course studies described in this paper demonstrate that ET-1 is induced by aldosterone within 1 h in both kidney and colon, with kidney levels peaking at 3-fold after 2 h. The difference observed in the temporal response for ET-1 between kidney and colon may reflect cell-type-specific differences in expression within these tissues. The ligand-activated MR regulates gene expression via hormone response elements (HREs) normally found in upstream promoter regions of target genes. Analysis of the promoter region of the rat ET-1 gene using the TRANSFAC program (34) revealed a putative HRE 1.4 kb upstream of the transcription start site for the ET-1 gene (data not shown). Sequence comparisons between the rat, mouse, and human ET-1 genes showed that this putative HRE is highly conserved across the three species. Whether this HRE functions to mediate the direct effect of aldosterone-activated MR to increase ET-1 gene transcription remains to be demonstrated.

Induction of ET-1 by aldosterone in A-10 smooth muscle cells was described by Wolf et al. (26), who examined the relationship between SGK-1 and ET-1. Their findings suggested that, as with aldosterone, the blood pressure elevating effects of ET-1 may be mediated via SGK-1. Interestingly, experiments with ET-1 and aldosterone together had no additive effects on SGK-1 mRNA or protein levels. Therefore, it was proposed that aldosterone and ET-1 might stimulate SGK-1 through similar signal transduction pathways. We have found in our studies that ET-1 induction by aldosterone is correlated with induction of SGK-1 in the kidney and colon. However, further analysis is required to define cell-specific localization of these induced proteins in the kidney and colon, which may provide evidence for functional interactions in specific subcompartments. Stimulation of ET-1 by aldosterone in the kidney may provide an additional level of induction or alteration in phosphorylation status of other regulators, such as SGK-1, further promoting renal sodium reabsorption in times of need. Alternatively increased renal ET-1 may directly promote vasoconstriction to exert positive effects on blood pressure. In contrast, however, tissue-specific gene-targeted mice lacking ET-1 expression specifically in kidney collecting ducts are hypertensive and on a high-salt diet, and have reduced sodium excretion (35). This may reflect other endocrine actions of collecting duct-derived ET-1 or that ET-1 may play a role in the development of the collecting duct that differs from its acute role, and indicates that dissecting the nephron vs. vascular actions of ET-1 in the kidney will not be straightforward.

In conclusion, our study shows for the first time in vivo that the ET-1 gene is a direct aldosterone-regulated target gene in the kidney and colon in a time-dependent manner. We also demonstrate that induction of ET-1 mRNA by aldosterone is mediated via the MR, most likely via direct interaction with a HRE upstream of the ET-1 gene promoter, although this awaits formal demonstration.


   Footnotes
 
Disclosure Statement: S.W., F.E.B., and T.J.C. have nothing to declare. M.J.Y. and P.J.F. have been the recipients of a previous research grant from Pfizer Inc., and M.J.Y. has been the recipient of a previous research grant from Merck. The present study does not relate to these activities.

First Published Online January 11, 2007

Abbreviations: bw, Body weight; CHIF, corticosteroid hormone-induced factor; ET-1, endothelin-1; GR, glucocorticoid receptor; HRE, hormone response element; MR, mineralocorticoid receptor; qRT-PCR, quantitative RT-PCR; SGK-1, serum and glucocorticoid-regulated kinase-1.

Received July 19, 2006.

Accepted for publication January 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cole TJ, Pearce D 2001 Mineralocorticoid target genes. Curr Opin Endocrinol Diabetes 8:118–123
  2. Fuller PJ, Young MJ 2005 Mechanisms of mineralocorticoid action. Hypertension 46:1227–1235[Abstract/Free Full Text]
  3. Pearce D, Bhargava A, Cole TJ 2003 Aldosterone: its receptor, target genes, and actions. Vitam Horm 66:29–76[Medline]
  4. Young MJ, Funder JW 1996 Mineralocorticoids, salt, hypertension: effects on the heart. Steroids 61:233–235[CrossRef][Medline]
  5. Rogerson FM, Brennan FE, Fuller PJ 2003 Dissecting mineralocorticoid receptor structure and function. J Steroid Biochem Mol Biol 85:389–396[CrossRef][Medline]
  6. Krozowski ZS, Funder JW 1983 Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc Natl Acad Sci USA 80:6056–6060[Abstract/Free Full Text]
  7. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268–275[Abstract/Free Full Text]
  8. Funder JW 2004 Aldosterone, mineralocorticoid receptors and vascular inflammation. Mol Cell Endocrinol 217:263–269[CrossRef][Medline]
  9. Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA 1999 Aldosterone-mediated regulation of ENaC {alpha}, ß, and {gamma} subunit proteins in rat kidney. J Clin Invest 104:R19–R23
  10. Brennan FE, Fuller PJ 1999 Acute regulation by corticosteroids of channel-inducing factor gene messenger ribonucleic acid in the distal colon. Endocrinology 140:1213–1218[Abstract/Free Full Text]
  11. Brennan FE, Fuller PJ 2006 Mammalian K-ras2 is a corticosteroid-induced gene in vivo. Endocrinology 147:2809–2816[Abstract/Free Full Text]
  12. Soundararajan R, Zhang TT, Wang J, Vandewalle A, Pearce D 2005 A novel role for glucocorticoid-induced leucine zipper protein in epithelial sodium channel-mediated sodium transport. J Biol Chem 280:39970–39981[Abstract/Free Full Text]
  13. Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D 1999 Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96:2514–2519[Abstract/Free Full Text]
  14. Bhargava A, Fullerton MJ, Myles K, Purdy TM, Funder JW, Pearce D, Cole TJ 2001 The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action. Endocrinology 142:1587–1594[Abstract/Free Full Text]
  15. Wulff P, Vallon V, Huang DY, Volkl H, Yu F, Richter K, Jansen M, Schlunz M, Klingel K, Loffing J, Kauselmann G, Bosl MR, Lang F, Kuhl D 2002 Impaired renal Na+ retention in the sgk1-knockout mouse. J Clin Invest 110:1263–1268[CrossRef][Medline]
  16. Granger JP 2003 Endothelin. Am J Physiol Regul Integr Comp Physiol 285:R298–R301
  17. Agapitov AV, Haynes WG 2002 Role of endothelin in cardiovascular disease. J Renin Angiotensin Aldosterone Syst 3:1–15[Medline]
  18. Iglarz M, Schiffrin EL 2003 Role of endothelin-1 in hypertension. Curr Hypertens Rep 5:144–148[Medline]
  19. Kohan DE 1997 Endothelins in the normal and diseased kidney. Am J Kidney Dis 29:2–26[Medline]
  20. Gallego MS, Ling BN 1996 Regulation of amiloride-sensitive Na+ channels by endothelin-1 in distal nephron cells. Am J Physiol 271:F451–F460
  21. Wesson DE, Dolson GM 1997 Endothelin-1 increases rat distal tubule acidification in vivo. Am J Physiol 273:F586–F594
  22. Ammarguellat FZ, Gannon PO, Amiri F, Schiffrin EL 2002 Fibrosis, matrix metalloproteinases, and inflammation in the heart of DOCA-salt hypertensive rats: role of ET(A) receptors. Hypertension 39:679–684[Abstract/Free Full Text]
  23. Park JB, Schiffrin EL 2002 Cardiac and vascular fibrosis and hypertrophy in aldosterone-infused rats: role of endothelin-1. Am J Hypertens 15:164–169[CrossRef][Medline]
  24. Seccia TM, Belloni AS, Kreutz R, Paul M, Nussdorfer GG, Pessina AC, Rossi GP 2003 Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II. J Am Coll Cardiol 41:666–673[Abstract/Free Full Text]
  25. Gumz ML, Popp MP, Wingo CS, Cain BD 2003 Early transcriptional effects of aldosterone in a mouse inner medullary collecting duct cell line. Am J Physiol Renal Physiol 285:F664–F673
  26. Wolf SC, Schultze M, Risler T, Rieg T, Lang F, Schulze-Osthoff K, Brehm BR 2006 Stimulation of serum- and glucocorticoid-regulated kinase-1 gene expression by endothelin-1. Biochem Pharmacol 71:1175–1183[CrossRef][Medline]
  27. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402[Abstract/Free Full Text]
  28. Robert V, Heymes C, Silvestre JS, Sabri A, Swynghedauw B, Delcayre C 1999 Angiotensin AT1 receptor subtype as a cardiac target of aldosterone: role in aldosterone-salt-induced fibrosis. Hypertension 33:981–986[Abstract/Free Full Text]
  29. Lariviere R, Day R, Schiffrin EL 1993 Increased expression of endothelin-1 gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats. Hypertension 21:916–920[Abstract/Free Full Text]
  30. Lariviere R, Thibault G, Schiffrin EL 1993 Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension 21:294–300[Abstract/Free Full Text]
  31. Park JB, Schiffrin EL 2001 ET(A) receptor antagonist prevents blood pressure elevation and vascular remodeling in aldosterone-infused rats. Hypertension 37:1444–1449[Abstract/Free Full Text]
  32. Pu Q, Neves MF, Virdis A, Touyz RM, Schiffrin EL 2003 Endothelin antagonism on aldosterone-induced oxidative stress and vascular remodeling. Hypertension 42:49–55[Abstract/Free Full Text]
  33. Liu W, Wang J, Sauter NK, Pearce D 1995 Steroid receptor heterodimerization demonstrated in vitro and in vivo. Proc Natl Acad Sci USA 92:12480–12484[Abstract/Free Full Text]
  34. Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV, Ignatieva EV, Ananko EA, Podkolodnaya OA, Kolpakov FA, Podkolodny NL, Kolchanov NA 1998 Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26:362–367[Abstract/Free Full Text]
  35. Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE 2004 Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 114:504–511[CrossRef][Medline]



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N. J. Brown
Aldosterone and Vascular Inflammation
Hypertension, February 1, 2008; 51(2): 161 - 167.
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