neutrophil gelatinase-associated lipocalin is a novel mineralocorticoid target in the cardiovascular...
TRANSCRIPT
Rossignol, Faiez Zannad, Nicolette Farman and Frederic JaisserHernandez-Diaz, Diego Alvarez de la Rosa, Claudine Perret, Natalia López Andrés, Patrick Celine Latouche, Soumaya El Moghrabi, Smail Messaoudi, Aurélie Nguyen Dinh Cat, Ivan
Cardiovascular SystemNeutrophil Gelatinase-Associated Lipocalin Is a Novel Mineralocorticoid Target in the
Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2012 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.111.187872
2012;59:966-972; originally published online April 2, 2012;Hypertension.
http://hyper.ahajournals.org/content/59/5/966World Wide Web at:
The online version of this article, along with updated information and services, is located on the
http://hyper.ahajournals.org/content/suppl/2012/04/02/HYPERTENSIONAHA.111.187872.DC1.htmlData Supplement (unedited) at:
http://hyper.ahajournals.org//subscriptions/
is online at: Hypertension Information about subscribing to Subscriptions:
http://www.lww.com/reprints Information about reprints can be found online at: Reprints:
document. Permissions and Rights Question and Answer this process is available in the
click Request Permissions in the middle column of the Web page under Services. Further information aboutOffice. Once the online version of the published article for which permission is being requested is located,
can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions:
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
Neutrophil Gelatinase-Associated Lipocalin Is a NovelMineralocorticoid Target in the Cardiovascular System
Celine Latouche, Soumaya El Moghrabi, Smail Messaoudi, Aurélie Nguyen Dinh Cat,Ivan Hernandez-Diaz, Diego Alvarez de la Rosa, Claudine Perret, Natalia López Andrés,
Patrick Rossignol, Faiez Zannad, Nicolette Farman, Frederic Jaisser
Abstract—Mineralocorticoid receptor (MR) activation may be deleterious to the cardiovascular system, and MRantagonists improve morbidity and mortality of patients with heart failure. However, mineralocorticoid signaling in theheart remains largely unknown. Using a pan-genomic transcriptomic analysis, we identified neutrophil gelatinase-associatedlipocalin (NGAL or lipocalin 2) as a strongly induced gene in the heart of mice with conditional and targeted MRoverexpression in cardiomyocytes (whereas induction was low in glucocorticoid receptor–overexpressing mice). NGALmRNA levels were enhanced after hormonal stimulation by the MR ligand aldosterone in cultured cardiac cells and in theheart of wild-type mice. Mineralocorticoid pathological challenge induced by nephrectomy/aldosterone/salt treatmentupregulated NGAL expression in the heart and aorta and its plasma levels. We show evidence for MR binding to an NGALpromoter, providing a mechanism for NGAL regulation. We propose that NGAL may be a marker of mineralocorticoid-dependent injury in the cardiovascular system in mice. (Hypertension. 2012;59:966-972.) ● Online Data Supplement
Key Words: transgenic mice � cardiomyocyte � cardiac cell lines � chromatin immunoprecipitation� mineralocorticoid receptor
Inappropriate mineralocorticoid signaling has been shownto play an important role in the progression of cardiovas-
cular (CV) disease. Aldosterone (Aldo) is a main regulator ofrenal sodium reabsorption, with an overall effect on volemiaand blood pressure. Aldo binds to the mineralocorticoidreceptor (MR), a transcription factor of the nuclear receptorfamily present in the kidney.1 Extrarenal pathophysiologicaleffects of this hormone have been characterized, extending itsactions to the CV system, the brain, the adipose tissue, theskin, and the eye.2–5 Inappropriate MR activation has beenshown to promote cardiac fibrosis in experimental models6,7
The Randomized Aldactone Evaluation Study,8 EplerenonePost-Acute Myocardial Infarction Heart Failure Efficacy andSurvival Study,9 and Eplerenone in Mild Patients Hospital-ization and Survival Study in Heart Failure10 clinical trialshave demonstrated that the addition of the MR antagonistsspironolactone or eplerenone to standard care markedlyreduced the overall and CV mortality in patients with leftventricular systolic dysfunction and mild or severe symptomsof chronic heart failure (HF) or with HF signs after acutemyocardial infarction.
The molecular mechanisms of Aldo and MR activation inthe CV system are not yet fully established. A better under-standing of these mechanisms may unveil novel biotargets forpharmacological modulation of the signaling cascades in-duced by mineralocorticoids in CV diseases.
In this study we identified neutrophil gelatinase-associatedlipocalin (NGAL) in a pan-genomic transcriptomic analysisperformed on hearts of transgenic mice that overexpress theMR in cardiomyocytes. NGAL (lipocalin 2 or 24p3) is a25-kDa glycoprotein belonging to the lipocalin superfam-ily.11,12 The cell-specific roles of NGAL remain elusive, butenhanced NGAL in tissues, plasma, or urine has beenreported in several pathological states, such as kidney fail-ure13 and obesity,14,15 and many other situations. Increasedsystemic and myocardial expressions of NGAL have beenobserved in clinical and experimental HF.16,17
Hormonal regulation of NGAL expression has not beeninvestigated. Here we document NGAL induction after min-eralocorticoid challenge, in vivo in the mouse heart and exvivo in cultured cells.
Received November 19, 2011; first decision December 9, 2011; revision accepted February 29, 2012.From the Institut National de la Santé et de la Recherche Médicale UMR 872 Team 1 (C.L., S.E.M., S.M., A.N.D.C., C.P., N.F., F.J.), Centre de
Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France; Department of Physiology and Institute of Biomedical Technologies (I.H.-D.,D.A.d.l.R.), University of La Laguna, Tenerife, Spain; Institut National de la Santé et de la Recherche Médicale (N.L.A., P.R., F.Z.), Centred’Investigations Cliniques-9501, Nancy, France; Nancy-Université (P.R., F.Z.), Nancy, France; Institut National de la Santé et de la Recherche MédicaleU961 (N.L.A., P.R., F.Z.), Nancy, France.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.187872/-/DC1.
Correspondence to Frederic Jaisser, Institut National de la Santé et de la Recherche Médicale UMR 872 Team 1, Centre de Recherche des Cordeliers,Université Pierre et Marie Curie, 15 rue de l’Ecole de Médecine, 75006 Paris, France. E-mail [email protected]
© 2012 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.111.187872
966 by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
MethodsAnimalsThe use of mice was in accordance with the guidelines of theEuropean Community and approved by our institutional animal careand use committees. For conditional MR or glucocorticoid receptor(GR) overexpression in cardiomyocytes, the previously describeddouble-transgenic MRcardio (�-myosin heavy chain-tetracyclintransactivator/tetracyclin response element [tTA/tetO]-human MR)18
and GRcardio mice (�-myosin heavy chain-tTA/tetO-human GR)19
were used and compared with their control littermates.Wild-type mice were treated with Aldo alone (low or high doses)
or submitted to nephrectomy/Aldo/salt treatment (NAS) as describedin the supplementary Methods section (please see the online-onlyData Supplement). MR antagonists used in vivo were either potas-sium canrenoate (30 mg/kg of body weight [BW] per day in thedrinking water) for MRcardio or NAS mice or spironolactone (200mg/kg of BW per day in the drinking water) for mice with chronicAldo infusion.
Cell CultureReagents are from Sigma-Aldrich (Saint-Quentin Fallavier, France)unless specified. H9C2/MR� cells, a clonal cell line of cardiomyo-cytes derived from the embryonic rat ventricle and stably expressingthe MR (kindly provided by A. Naray-Fejes-Toth, Dartmouth Schoolof Medicine, Lebanon, NH) were grown as described.20 Cells weretreated with Aldo, corticosterone, the GR antagonist RU38486, orthe MR antagonist RU28318 (Tocris Biosciences, Bristol, UnitedKingdom), as indicated in the legend of Figure 3.
HL-1 cells (kindly provided by W.C. Claycomb, Louisiana StateUniversity Health Sciences Center, New Orleans, LA), an immor-talized cell line derived from neonatal mouse cardiac myocytes, werecultured as described21 and transiently transfected with a mouseMR-expressing plasmid.22 Cell treatments are indicated in legends ofFigure 4. Cell transfection and chromatin immunoprecipitation aredescribed in the supplementary Methods section.
Primary mouse aortic endothelial cells and primary mouse vascu-lar smooth muscle cells derived from 8- to 10-week–old mice wereisolated under sterile conditions as described.23,24 Endothelial cellswere used at passages 2 to 3 and smooth muscle cells at passages 4to 7. After 24 hours in serum-free medium, cells were incubated asindicated in legends of Figure 2.
Cell BiologyReal-time PCR, Western blot, immunolocalization, and measure-ments of plasma levels of NGAL are indicated in the supplementaryMethods section.
Statistical AnalysesResults are provided as mean�SEM. The nonparametric Mann-Whitney U test was used to assess statistical differences between 2experimental groups. Differences among �2 experimental condi-tions were tested by the ANOVA 1-way analysis followed by theNewman-Keuls test; P�0.05 was considered significant.
ResultsCardiac NGAL Expression Is Enhanced After MROverexpression in CardiomyocytesA pan-genomic transcriptomic analysis on hearts of double-transgenic mice that overexpress the MR in cardiomyocytesonly (MRcardio mice) identified NGAL as a gene highlyinduced in the heart of MRcardio mice; the induction isessentially MR dependent and not GR dependent, becausecardiac NGAL mRNA was strongly enhanced in MRcardiomice (�150 fold compared with the control littermates),whereas GR overexpression (in GRcardio mice) led to amuch smaller (�5-fold) increase in NGAL mRNA (Figure1A). Treatment of MRcardio mice with the MR antagonist
NGALControl MRcardio200 ***A B
(25kD)
GAPDH(39kD)150
mR
NA C
30
40
50
**
Car
diac
NG
AL
m
+ MRA
0
10
20
+ Dox***N 15 16 10 12 6 6 6 6
ControlMRcardio GRcardio
Control
N 15 16 10 12 6 6 6 6
Figure 1. Neutrophil gelatinase-associated lipocalin (NGAL) expression in the heart of mineralocorticoid receptor (MR)- versus gluco-corticoid receptor (GR)-overexpressing mice. A, NGAL mRNA expression in the heart of mice overexpressing MR (MRcardio) or GR(GRcardio) in cardiomyocytes and their littermate controls. MRcardio mice were treated for 8 weeks with the MR antagonist canrenoate(�MRA) or with doxycycline (�Dox) to switch off the human MR expression. NGAL mRNA levels (quantitative PCR) were normalized forubiquitin C expression as an internal control. Values in control mice were set as 1, and fold changes are shown on the Figure. Numberof mice per condition is indicated below the bars. ***P�0.001, **P�0.01, transgenic mice vs their littermate controls (control-MR orcontrol-GR), Mann-Whitney U test. B, NGAL protein expression (Western blot) in the heart of mice overexpressing MR in cardiomyo-cytes (MRcardio) vs control mice. GAPDH was used as internal control for protein loading. C, NGAL immunohistochemistry in the heartof MRcardio mice (panel b) vs littermate control (panel a). Negative control indicates omission of primary antibody (panel c). MRcardiomice treated with the MR antagonist canrenoate (panel d). Bar, 100 �m.
Latouche et al NGAL as Cardiac Mineralocorticoid Target 967
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
potassium canrenoate reduced noticeably the NGAL cardiaclevels, which were fully suppressed when mice were treatedwith doxycycline, that prevents transgene (MR) expression(Figure 1A). The MR-dependent induction of NGAL in theheart of MRcardio mice was also evident at the protein level,as measured by Western blot (Figure 1B). Immunolocaliza-tion of NGAL was negative in the heart from control miceand showed signal over cardiomyocytes of MRcardio mice(Figure 1C, panel b versus panel a) that was reduced by invivo MR antagonism (Figure 1C, panel d).
Increase in NGAL Expression After In VivoAldo AdministrationAcute administration of Aldo (1 mg/kg of BW) inducedcardiac NGAL expression 6 hours later (Figure 2A). Chronicinfusion of Aldo (1 mg/kg of BW per day for 4 weeks)induced a 2-fold increase in cardiac NGAL expression, aneffect prevented by coadministration of the MR antagonistspironolactone (Figure 2B). Of note, in the mouse, infusionwith Aldo alone at low concentrations (60 �g/kg of BW per
day) had no effect on cardiac NGAL expression (Figure 2C).The NAS model is a classic model of mineralocorticoid stressthat associates mineralocorticoid administration on top of saltexcess and reduced renal mass. We found that NGAL mRNAexpression was increased in the heart of mice with NASchallenge, whereas nephrectomy alone, Aldo alone, or excesssalt intake alone had no effect (Figure 2C). Because NGALmay be secreted, its plasma concentration was estimated inmice challenged with NAS treatment followed by MR antag-onism. Four weeks of NAS challenge enhanced plasmaNGAL levels, which returned to low values when mice weretreated by an MR antagonist (Figure 2D).
NAS challenge also enhanced by 2.5-fold NGAL mRNAexpression in the aorta (data not shown). Immunolocalizationexperiments showed that NAS challenge led to clear NGALsignal over large cardiac vessels, whereas cardiomyocyteswere barely positive; small arterioles and capillaries werenegative (Figure 2E). Mineralocorticoid sensitivity of vascu-lar NGAL expression was evidenced on primary cultures ofaortic endothelial and smooth muscle cells: Aldo upregulated
A4N
A
* NA *
B5
**NA
C
1
2
3
4
ardi
acN
GA
L m
R1
2
ardi
acN
GA
L m
R
1
2
3
4
ardi
acN
GA
L m
R
0V 0.1 1.0
Aldo (mg/kg)
C
0V Aldo Aldo
+ MRA
C
C
evel
(µg/
l) ** **
400
500
600
D ControlE NAS
Plas
ma
NG
AL
l
NAS0
100
200
300 F MAEC
2
4
6**
AL
mR
NA
VSMCs
234 **
Basal NAS NAS + MRA
V Aldo Aldo+ MRA
0
NG
01
V Aldo Aldo+ MRA
Figure 2. Regulation of neutrophil gelatinase-associated lipocalin (NGAL) mRNA by mineralocorticoids in mice. A and B, Wild-typemice were treated with vehicle (V), with a single injection of aldosterone (0.1 or 1 mg/kg of body weight [BW]) and euthanized 6 hourslater (A) or treated for 4 weeks with high-dose aldosterone (1 mg/kg of BW per day using osmotic minipumps) alone or with the miner-alocorticoid receptor (MR) antagonist spironolactone 200 mg/kg of BW per day (B). N�6 to 10 mice per group. * P�0.05 vs vehicle(ANOVA analysis, Newman-Keuls test). C, Mice have been subjected to the nephrectomy/aldosterone/salt challenge (NAS) for 4 weeksor to each component of NAS: uninephrectomy alone (nephrect), aldosterone (60 �g/kg of BW per day) alone, high salt intake alone(salt). Values of cardiac NGAL mRNA expression (quantitative PCR) were normalized for ubiquitin C or GAPDH expression as internalcontrol in panels A–C. N�5 to 9 mice per group; **P�0.01 treatment vs control (ANOVA analysis, Newman-Keuls test). D, Follow-up ofindividual plasma NGAL levels in 5 mice before (basal) or after 4 weeks of NAS challenge (NAS), followed by 4 weeks of treatment withthe MR antagonist canrenoate 30 mg/kg of BW per day (NAS�MR agonist [MRA]). **P�0.01 (repeated-measures ANOVA analysis,paired t test). E, NGAL immunolocalization in the heart of control and NAS mice (bar, 50 �m). F, NGAL mRNA expression (quantitativePCR) in mouse aortic endothelial cells (MAECs) and smooth muscle cells (VSMCs) in primary culture. Cells were treated for 24 hourswith vehicle (V), with 10�8 M aldosterone alone (aldo), or with the MR antagonist spironolactone 1 �m (aldo�MRA). Data are means of4 to 6 independent experiments, each performed in duplicate. **P�0.01 vs vehicle (ANOVA analysis, Newman-Keuls test). Values ofNGAL mRNA (quantitative PCR) were normalized to ubiquitin C expression. Values in control mice (sham) or vehicle-treated cells ormice (V) were set as 1, and fold changes are shown on the Figures.
968 Hypertension May 2012
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
NGAL transcripts in both cell types, and MR antagonist fullyprevented the induction (Figure 2F).
NGAL Is an Aldo/MR Target Gene in CulturedCardiac CellsTo distinguish consequences of enhanced MR signaling fromlong-term cardiac adaptation to chronic MR activation, wemeasured NGAL expression in a rat cardiomyocyte cell linestably transfected with the MR (H9C2/MR� cells). Aldoinduced NGAL mRNA expression after 24-hour incubation,in a dose-dependent manner (Figure 3A and 3B). Cells treatedby corticosterone (Figure 3C) or Aldo (Figure 3D) exhibitedMR-specific enhanced NGAL mRNA (Figure 3D) and pro-tein (Figure 3E) expression that was prevented by coincuba-tion with the MR antagonist RU28318, whereas the GRantagonist RU28486 was ineffective (Figure 3C through 3E).Thus, NGAL is upregulated by both MR-Aldo and MR-corticosterone complexes in this cell context.
To evaluate the mechanism of NGAL regulation by MRactivation, we used the mouse cardiomyocyte cell line HL-1cells that do not express detectable levels of endogenous MRactivity22 and that were transiently transfected with mouseMR. Aldo produced a 6-fold increase in NGAL mRNA levelsin HL-1 cells transfected with mouse MR but not in HL-1cells transfected with empty vector (Figure 4A). Cotransfec-tion of mouse MR and NGAL-luciferase reporter construct
(using the �794 to �36 NGAL promoter region) thatcontains a hormone response element (Figure 4B), followedby Aldo treatment, resulted in a 4-fold increase in luciferaseactivity (Figure 4C) that was blocked by the MR antagonistspironolactone (Figure 4C). When the hormone responseelement was deleted, no significant increase in luciferaseactivity on Aldo treatment was detected (Figure 4C). Thissuggests that MR binds to the hormone response elementpresent in the �794 to �36 NGAL promoter and that NGALis an Aldo/MR responsive gene. Chromatin immunoprecipi-tation showed that the hormone response element–mediatedAldo/MR effect is a direct consequence of MR binding to thispromoter element (Figure 4D). When MR was transfected,the NGAL promoter was enriched in the chromatin immuno-precipitation product by 4-fold. Aldo treatment producedfurther enrichment of the NGAL promoter (�11-fold), indi-cating increased binding of MR to the promoter. These datashow that the MR directly controls NGAL expression incardiomyocytes by binding to its promoter.
DiscussionInappropriate or excessive MR activation is a hallmark of anumber of CV diseases that may benefit from treatments withMR antagonists. In this study we have provided evidence thatNGAL is a mineralocorticoid-target gene in the CV system.We have shown that NGAL is a cardiac gene with high
BA
L m
RN
AA
3
4
5
6
7 **
****
A(fo
ldch
ange
)
** **
4
6
8
Control AldoAldo+MRA
E
NG
A
Aldosterone in medium (nM)
0
1
2
3
0 1 10Incubation time (hours)
NG
AL
mR
NA
0 2 6 10 48240
2
2
nge)
NGALGAPDH
6** **
hang
e) 7
D
6
7
**hang
e)
C
Aldosterone in medium (nM) Incubation time (hours)
1
prot
ein
(fold
chan
*
1
2
3
4
5
AL
mR
NA
(fold
c
2
3
4
5 **
AL
mR
NA
(fold
ch
0Control Aldo Aldo+
MRA
NG
AL
p
0
1
AldoMRAGRA
---
+--
++-
+-+
NG
A
CorticoMRAGRA
0
1
---
+--
++-
+-+
NG
A
Figure 3. Expression of neutrophil gelatinase-associated lipocalin (NGAL) mRNA in a cellular model of rat cardiomyocyte overexpress-ing the mineralocorticoid receptor (MR; H9C2/MR� cells). H9C2/MR� cells were treated with aldosterone (added to the culture medium)at different doses (24 hours; A) or during 0 to 48 hours (10�8 M; B). Specificity of induction of NGAL mRNA was assessed in cellstreated for 24 hours with 10�8 M aldosterone (Aldo; D) or 10�8 M corticosterone (Cortico; C) alone or in association with the 10�6 M MRantagonist RU28318 (MRA) or 10�6 M GR antagonist RU38486 (GRA). Values of NGAL mRNA levels (quantitative PCR) are normalizedfor �-actin expression (fold change vs control, nontreated cells set as 1); n�4 to 8 wells per condition, from 2 independent experi-ments. Western blot of cells treated for 48 hours with 10�8 M aldosterone in the absence/presence of MRA (E) show specificity ofNGAL (25 kDa) expression at the protein level (n�3–5 wells per condition); GAPDH (39 kDa) was used for normalization. *P�0.05,**P�0.01 treatment vs control (ANOVA analysis, Newman-Keuls test).
Latouche et al NGAL as Cardiac Mineralocorticoid Target 969
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
differential expression between mice with conditional MRoverexpression restricted to the cardiomyocytes and theirlittermate controls. MRcardio mice have 5-fold more cardiacMR than control mice,20 an experimental situation reminis-cent of the increase in cardiac MR expression reported inrodent HF and myocardial infarction25,26; cardiac NGALexpression is specifically increased by MR activation ratherthan by GR activation; NGAL appears to be a direct target ofMR, because chromatin immunoprecipitation experimentsdemonstrated that the MR binds on the NGAL promoter, atleast in the cardiomyocyte cellular context NGAL is alsodependent on MR activation in vascular cells; and plasmaNGAL is modulated by Aldo/salt and MR antagonism. Takentogether, our results show that NGAL is a novel actor of theAldo/MR signaling cascade. In the literature, induction ofNGAL has been reported in the hearts of rats with autoim-mune myocarditis27 or myocardial infarction17; NGAL im-munoreactivity was also evidenced in the cardiomyocytes andin the aortic atherosclerotic plaque in a mouse model ofatherosclerosis, whereas it was undetectable in normalmice.28 NGAL may bind covalently to the matrix metallo-proteinase 9, leading to its stabilization and increased activ-ity.29 Although it remains difficult to attribute a specificfunction to the different forms of NGAL, it is tempting topropose that the NGAL-matrix metalloproteinase 9 complexmay be particularly relevant in the context of matrix remod-eling, observed in CV diseases and also on experimental MRactivation.
It is highly plausible that several factors contribute todetermine NGAL expression levels, in physiological situa-tions and in pathology. Of interest, it has been shown thatnuclear factor �B (NF-�B) can directly enhance NGALexpression, as demonstrated in vascular smooth muscle cells.Interleukin 1� triggers binding of NF-�B to the NGALpromoter.30 Further analysis of induction of NGAL throughthe NF-�B pathway was provided recently.31 Cross-talkbetween nuclear receptors and NF-�B has been reported.32 Inthe literature, there are data that show transcriptional antag-onism between the MR and NF-�B.33 Aldo also inhibitedNF-�B activation (leading to production of tumor necrosisfactor-�) in neutrophils, and the authors concluded that MRmediates anti-inflammatory effects in these cells when theyinteract with the endothelium.34 Conversely, activation ofNF-�B by Aldo has been reported in renal collecting ductcells; however, this may not be a direct effect but rather anadaptative phenomenon to limit excessive Aldo signaling.35
In cardiac cells, balance between MR and NF-�B mayregulate NGAL expression; this balance may be differentafter short-term and long-term situations and even more in apathological context. Further experiments are needed to testsuch hypotheses, to understand how and why NGAL levelsvary. Altogether, these data illustrate the complexity ofNGAL regulation that is far from being elucidated.
NGAL/lipocalin 2 is expressed at low levels in normaltissues and highly in a number of situations with tissue injury,where its roles are multiple and not fully understood.36
BA
Exon 1HRE
-794 +36
l2
4
6
8
L m
RN
Ale
vels *
luc
se *NGAL(-794/+36)-luc
4
5C 15D
*
0
2
HL-1 HL-1 + MRN
GA
Lrm
aliz
edlu
cife
raac
tivity NGAL(-794/+36)∆HRE-luc
1
2
3
4
5
10
Fold
enr
ichm
ent
Control Aldo Aldo+MRA
Nor 0
0HL-1 HL-1-MR HL-1-MR
+ Aldo
Figure 4. Neutrophil gelatinase-associated lipocalin (NGAL) is a direct target of mineralocorticoid receptor (MR). A, NGAL mRNA abun-dance (quantitative PCR) in cardiac HL-1 cells transfected with mouse MR (HL-1�MR) or with empty vector (HL-1) and treated or notwith 10�8 M aldosterone for 16 hours. B, Schematic representation of mouse NGAL promoter. HRE indicates hormone response ele-ment. Arrowheads indicate the approximate location of primers used to analyze chromatin immunoprecipitation products. The linebelow represents the fragment of NGAL promoter fused to luciferase (NGAL-luc construct). Numbers refer to the transcription startsites. C, Luciferase expression mediated by the wild-type (NGAL[�794/�36]-luc) or mutated (NGAL[�794/�36]�HRE-luc) fragment ofmouse NGAL promoter in transfected HL-1 cells, treated or not with 10�8 M aldosterone alone or with 10�6 M spironolactone (MRA) for16 hours. Renilla luciferase was used as internal control. Luciferase values are normalized to control conditions. D, Quantitative PCRanalysis of MR binding to the HRE in mouse NGAL promoter in HL-1 cells transfected or not with mouse MR and treated or not with10�8 M aldosterone. Bars represent the fold enrichment of the 184-bp fragment surrounding the HRE over the value obtained with theirrelevant IgG. *P�0.05, treatment vs control (ANOVA analysis, Newman-Keuls test). Data are means of 3 to 4 independent experi-ments, each performed in triplicate.
970 Hypertension May 2012
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
NGAL/lipocalin 2 was initially identified as a critical com-ponent of innate immunity, limiting bacterial infection,through sequestration of iron.37 Later on, this protein wasshown to be a modulator of cell proliferation, differentiation,apoptosis, and more generally in the reaction to various stressfactors.38,39 Its expression is strongly enhanced in response torenal tubular injury. It has been shown that NGAL mayprotect the kidney from failure after ischemia/reperfusion40;conversely, it was reported that it may belong to a criticalpathway leading to progression toward renal failure.41 Exten-sive literature data have been provided to designate plasmaand/or urinary NGAL levels as sensitive markers of earlyprediction of kidney injury and its outcomes in humans.13
NGAL, as a biomarker of renal tubular damage, may alsoreflect renal consequences of cardiac impairment after car-diac surgery, myocardial infarction, or cardiorenal syndromeassociated with HF. However, recent data suggest that cardiacNGAL and circulating NGAL levels may be altered bycardiac dysfunction, independent of renal dysfunction.42
Plasma NGAL is high in patients with acute HF aftermyocardial infarction (Optimal Trial in Myocardial InfarctionWith the Angiotensin II Antagonist Losartan),17 as well as inpatients with chronic HF17 or coronary heart disease43 and inthe atherosclerotic plaque.28 We found that NGAL plasmaconcentration is enhanced on mineralocorticoid stress in mice(although it is difficult to determine the origin of plasmaNGAL in the NAS model). Human trials should determinewhether plasma NGAL correlates with circulating Aldolevels and MR antagonism in patients with HF.
PerspectivesWe demonstrate in the present study that NGAL is upregu-lated by mineralocorticoids, through MR activation, in theCV system, where its cellular functions remain to be eluci-dated. Future studies should also determine whether the riseof plasma NGAL may serve as a biomarker of excessive MRactivation in patients with HF, to select those who couldbenefit from treatment with MR antagonists.
Sources of FundingC.L. was recipient of PhD grant from the Ministere de la Recherche.Fellowships from Région Ile-de-France (CODDIM) supported S.M.and A.N.D.C. This work was supported by grants from InstitutNational de la Santé et de la Recherche Médicale (CoPoc), theAgence Nationale pour la Recherche (ANR-09-BLAN-0156-01 andANR-09-EBIO-024-01), the Programme National de RechercheCardiovasculaire, the Institut National de la Santé et de la RechercheMédicale, the Société Française d’Hypertension artérielle 2007 toF.J., and the Ministerio de Ciencia e Innovación (Spain) grantsBFU2010-16265 and Consolider SICI-CSD2008-000005 (toD.A.d.l.R).
DisclosuresNone.
References1. Farman N, Rafestin-Oblin ME. Multiple aspects of mineralocorticoid
selectivity. Am J Physiol Renal Physiol. 2001;280:F181–F192.2. Messaoudi S, Azibani F, Delcayre C, Jaisser F. Aldosterone, mineralo-
corticoid receptor, and heart failure. Mol Cell Endocrinol. 2012;350:266–272.
3. Zennaro MC, Caprio M, Feve B. Mineralocorticoid receptors in themetabolic syndrome. Trends Endocrinol Metab. 2009;20:444–451.
4. Sainte Marie Y, Toulon A, Paus R, Maubec E, Cherfa A, Grossin M,Descamps V, Clemessy M, Gasc JM, Peuchmaur M, Glick A, Farman N,Jaisser F. Targeted skin overexpression of the mineralocorticoid receptorin mice causes epidermal atrophy, premature skin barrier formation, eyeabnormalities, and alopecia. Am J Pathol. 2007;171:846–860.
5. Zhao M, Valamanesh F, Celerier I, Savoldelli M, Jonet L, Jeanny JC,Jaisser F, Farman N, Behar-Cohen F. The neuroretina is a novel miner-alocorticoid target: aldosterone up-regulates ion and water channels inmuller glial cells. FASEB J. 2010;24:3405–3415.
6. Robert V, Silvestre JS, Charlemagne D, Sabri A, Trouve P, Wassef M,Swynghedauw B, Delcayre C. Biological determinants of aldosterone-induced cardiac fibrosis in rats. Hypertension. 1995;26:971–978.
7. Brilla CG. Aldosterone and myocardial fibrosis in heart failure. Herz.2000;25:299–306.
8. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, PalenskyJ, Wittes J. The effect of spironolactone on morbidity and mortality inpatients with severe heart failure: Randomized Aldactone EvaluationStudy investigators. N Engl J Med. 1999;341:709–717.
9. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, BittmanR, Hurley S, Kleiman J, Gatlin M. Eplerenone, a selective aldosteroneblocker, in patients with left ventricular dysfunction after myocardialinfarction. N Engl J Med. 2003;348:1309–1321.
10. Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, ShiH, Vincent J, Pocock SJ, Pitt B. Eplerenone in patients with systolic heartfailure and mild symptoms. N Engl J Med. 2011;364:11–21.
11. Flower DR, North AC, Sansom CE. The lipocalin protein family:structural and sequence overview. Biochim Biophys Acta. 2000;1482:9–24.
12. Grzyb J, Latowski D, Strzalka K. Lipocalins: a family portrait. J PlantPhysiol. 2006;163:895–915.
13. Devarajan P. Emerging biomarkers of acute kidney injury. ContribNephrol. 2007;156:203–212.
14. Wang Y, Lam KS, Kraegen EW, Sweeney G, Zhang J, Tso AW, ChowWS, Wat NM, Xu JY, Hoo RL, Xu A. Lipocalin-2 is an inflammatorymarker closely associated with obesity, insulin resistance, and hyper-glycemia in humans. Clin Chem. 2007;53:34–41.
15. Catalan V, Gomez-Ambrosi J, Rodriguez A, Ramirez B, Silva C, RotellarF, Gil MJ, Cienfuegos JA, Salvador J, Fruhbeck G. Increased adiposetissue expression of lipocalin-2 in obesity is related to inflammation andmatrix metalloproteinase-2 and metalloproteinase-9 activities in humans.J Mol Med. 2009;87:803–813.
16. Bolignano D, Basile G, Parisi P, Coppolino G, Nicocia G, Buemi M.Increased plasma neutrophil gelatinase-associated lipocalin levels predictmortality in elderly patients with chronic heart failure. Rejuvenation Res.2009;12:7–14.
17. Yndestad A, Landro L, Ueland T, Dahl CP, Flo TH, Vinge LE, EspevikT, Froland SS, Husberg C, Christensen G, Dickstein K, Kjekshus J, OieE, Gullestad L, Aukrust P. Increased systemic and myocardial expressionof neutrophil gelatinase-associated lipocalin in clinical and experimentalheart failure. Eur Heart J. 2009;30:1229–1236.
18. Ouvrard-Pascaud A, Sainte-Marie Y, Benitah JP, Perrier R, SoukaseumC, Cat AN, Royer A, Le Quang K, Charpentier F, Demolombe S,Mechta-Grigoriou F, Beggah AT, Maison-Blanche P, Oblin ME,Delcayre C, Fishman GI, Farman N, Escoubet B, Jaisser F. Conditionalmineralocorticoid receptor expression in the heart leads to life-threateningarrhythmias. Circulation. 2005;111:3025–3033.
19. Sainte-Marie Y, Nguyen Dinh Cat A, Perrier R, Mangin L, Soukaseum C,Peuchmaur M, Tronche F, Farman N, Escoubet B, Benitah JP, Jaisser F.Conditional glucocorticoid receptor expression in the heart induces atrio-ventricular block. Faseb J. 2007;21:3133–3141.
20. Latouche C, Sainte-Marie Y, Steenman M, Castro Chaves P, Naray-Fejes-Toth A, Fejes-Toth G, Farman N, Jaisser F. Molecular signature ofmineralocorticoid receptor signaling in cardiomyocytes: from culturedcells to mouse heart. Endocrinology. 2010;151:4467–4476.
21. Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB,Bahinski A, Izzo NJ Jr. Hl-1 cells: a cardiac muscle cell line that contractsand retains phenotypic characteristics of the adult cardiomyocyte. ProcNatl Acad Sci U S A. 1998;95:2979–2984.
22. Hernandez-Diaz I, Giraldez T, Arnau MR, Smits VA, Jaisser F, FarmanN, Alvarez de la Rosa D. The mineralocorticoid receptor is a constitutivenuclear factor in cardiomyocytes due to hyperactive nuclear localizationsignals. Endocrinology. 2010;151:3888–3899.
Latouche et al NGAL as Cardiac Mineralocorticoid Target 971
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
23. Kobayashi M, Inoue K, Warabi E, Minami T, Kodama T. A simplemethod of isolating mouse aortic endothelial cells. J Atheroscler Thromb.2005;12:138–142.
24. Callera GE, Touyz RM, Tostes RC, Yogi A, He Y, Malkinson S, SchiffrinEL. Aldosterone activates vascular p38MAP kinase and NADPH oxidasevia c-Src. Hypertension. 2005;45:773–779.
25. Nagata K, Obata K, Xu J, Ichihara S, Noda A, Kimata H, Kato T, IzawaH, Murohara T, Yokota M. Mineralocorticoid receptor antagonismattenuates cardiac hypertrophy and failure in low-aldosterone hyper-tensive rats. Hypertension. 2006;47:656–664.
26. Ohtani T, Ohta M, Yamamoto K, Mano T, Sakata Y, Nishio M, TakedaY, Yoshida J, Miwa T, Okamoto M, Masuyama T, Nonaka Y, Hori M.Elevated cardiac tissue level of aldosterone and mineralocorticoidreceptor in diastolic heart failure: Beneficial effects of mineralocorticoidreceptor blocker. Am J Physiol Regul Integr Comp Physiol. 2007;292:R946–R954.
27. Ding L, Hanawa H, Ota Y, Hasegawa G, Hao K, Asami F, Watanabe R,Yoshida T, Toba K, Yoshida K, Ogura M, Kodama M, Aizawa Y.Lipocalin-2/neutrophil gelatinase-B associated lipocalin is stronglyinduced in hearts of rats with autoimmune myocarditis and in humanmyocarditis. Circ J. 2010;74:523–530.
28. Hemdahl AL, Gabrielsen A, Zhu C, Eriksson P, Hedin U, Kastrup J,Thoren P, Hansson GK. Expression of neutrophil gelatinase-associatedlipocalin in atherosclerosis and myocardial infarction. ArteriosclerThromb Vasc Biol. 2006;26:136–142.
29. Yan L, Borregaard N, Kjeldsen L, Moses MA. The high molecular weighturinary matrix metalloproteinase (MMP) activity is a complex of gela-tinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL):modulation of MMP-9 activity by NGAL. J Biol Chem. 2001;276:37258–37265.
30. Bu DX, Hemdahl AL, Gabrielsen A, Fuxe J, Zhu C, Eriksson P, Yan ZQ.Induction of neutrophil gelatinase-associated lipocalin in vascular injuryvia activation of nuclear factor-�B. Am J Pathol. 2006;169:2245–2253.
31. Karlsen JR, Borregaard N, Cowland JB. Induction of neutrophilgelatinase-associated lipocalin expression by co-stimulation withinterleukin-17 and tumor necrosis factor-� is controlled by I�B-zeta butneither by c/EBP-� nor c/EBP-�. J Biol Chem. 2010;285:14088–14100.
32. De Bosscher K, Vanden Berghe W, Haegeman G. Cross-talk betweennuclear receptors and nuclear factor �b. Oncogene. 2006;25:6868–6886.
33. Kolla V, Litwack G. Inhibition of mineralocorticoid-mediated tran-scription by NF-�B. Arch Biochem Biophys. 2000;383:38–45.
34. Bergmann A, Eulenberg C, Wellner M, Rolle S, Luft F, Kettritz R.Aldosterone abrogates nuclear factor �B-mediated tumor necrosis factor� production in human neutrophils via the mineralocorticoid receptor.Hypertension. 2010;55:370–379.
35. Leroy V, De Seigneux S, Agassiz V, Hasler U, Rafestin-Oblin ME,Vinciguerra M, Martin PY, Feraille E. Aldosterone activates NF-�B inthe collecting duct. J Am Soc Nephrol. 2009;20:131–144.
36. Devarajan P. Neutrophil gelatinase-associated lipocalin: new paths for anold shuttle. Cancer Ther. 2007;5:463–470.
37. Goetz DH, Holmes MA, Borregaard N, Bluhm ME, Raymond KN, StrongRK. The neutrophil lipocalin NGAL is a bacteriostatic agent thatinterferes with siderophore-mediated iron acquisition. Mol Cell. 2002;10:1033–1043.
38. Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P,Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J AmSoc Nephrol. 2007;18:407–413.
39. Leng X, Ding T, Lin H, Wang Y, Hu L, Hu J, Feig B, Zhang W, PusztaiL, Symmans WF, Wu Y, Arlinghaus RB. Inhibition of lipocalin 2 impairsbreast tumorigenesis and metastasis. Cancer Res. 2009;69:8579–8584.
40. Mori K, Lee HT, Rapoport D, Drexler IR, Foster K, Yang J, Schmidt-OttKM, Chen X, Li JY, Weiss S, Mishra J, Cheema FH, Markowitz G,Suganami T, Sawai K, Mukoyama M, Kunis C, D’Agati V, Devarajan P,Barasch J. Endocytic delivery of lipocalin-siderophore-iron complexrescues the kidney from ischemia-reperfusion injury. J Clin Invest. 2005;115:610–621.
41. Viau A, El Karoui K, Laouari D, Burtin M, Nguyen C, Mori K, PilleboutE, Berger T, Mak TW, Knebelmann B, Friedlander G, Barasch J, Terzi F.Lipocalin 2 is essential for chronic kidney disease progression in miceand humans. J Clin Invest. 2010;120:4065–4076.
42. Bolignano D, Coppolino G, Lacquaniti A, Buemi M. From kidney tocardiovascular diseases: NGAL as a biomarker beyond the confines ofnephrology. Eur J Clin Investig. 2010;40:273–276.
43. Choi KM, Lee JS, Kim EJ, Baik SH, Seo HS, Choi DS, Oh DJ, Park CG.Implication of lipocalin-2 and visfatin levels in patients with coronaryheart disease. Eur J Endocrinol. 2008;158:203–207.
972 Hypertension May 2012
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN IS A NOVEL MINERALOCORTICOID TARGET IN THE CARDIOVASCULAR SYSTEM SUPPLEMENTARY METHODS Celine Latouche*, Soumaya El Moghrabi*, Smail Messaoudi*, Aurélie Nguyen Dinh Cat *, Ivan Hernandez-Diaz**, Diego Alvarez de la Rosa**, Claudine Perret*, Natalia López Andrés***, Patrick Rossignol***, Faiez Zannad***, Nicolette Farman*, Frederic Jaisser* *INSERM UMR 872 Team 1, Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, 15 rue de l’Ecole de Médecine, 75006 Paris, France; ** Department of Physiology and Institute of Biomedical Technologies, University of La Laguna, Tenerife, Spain; ***INSERM, Centre d’Investigations Cliniques- 9501, Nancy, France (PR, FZ) ; Nancy-Université, France (PR, FZ), INSERM U961, Nancy, France (NL, PR, FZ).
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from
Animal treatments To switch off the transgene expression in 3 months old MRcardio mice, doxycycline (dox, 2mg/ml in drinking water) was administered for 4 weeks before sacrifice. Adult mice were treated with aldosterone alone either acutely (bolus of 0.1 or 1 mg/kg BW six hours before sacrifice) or for 4 weeks (1 mg/kg BW/day delivered subcutaneously via ALZET® osmotic mini pumps (Charles River Laboratories, L’Arbresle, France). In other series, mice with nephrectomy/aldosterone/salt treatment (NAS) were uninephrectomized (xylazine/ketamine anesthesia) and received 60 µg/kg BW/day aldosterone, delivered subcutaneously via ALZET® osmotic mini pumps, and 1% sodium chloride to drink, for 4 weeks. The control mice were sham-operated and maintained on standard chow and tap water. In some series, mice were subjected to only one of the 3 components of the NAS challenge (uninephrectomy alone or aldosterone alone or 1% sodium chloride to drink). Cell transfection and chromatin immunoprecipitation HL-1 cells were transfected using Lipofectamine 2000. A NGAL promoter fragment including -794 to +36 relative to the transcription start site was cloned in pGL4.1-luc (Promega, Madison, WI). Deletion of a hormone responsive element (HRE) in the NGAL promoter (position -551 to -501) was performed by site-directed mutagenesis using a commercial kit (Quickchange, Agilent, Santa Clara, CA). The effect of MR activation on NGAL promoter activity was tested by co-transfecting these plasmids with mouse MR 22. Steroid treatments were performed after 24 h in charcoal-stripped medium. Luciferase activity was determined with a kit (DualGlo, Promega). For chromatin immunoprecipitation (ChIP) cells were cross-linked with formaldehyde. Chromatin was purified, sonicated and used for ChIP as recommended by the manufacturer (ChIP-IT Express, Active Motif, Carlsbad, CA). Three reactions were performed: positive control with an anti-RNApolII antibody; a negative control with an irrelevant IgG; a test reaction using an anti-MR antibody (H-300, Santa Cruz Biotechnology, Santa Cruz, CA). ChIP products were then purified and the enrichment of NGAL promoter area containing the HRE was tested by qPCR. Cell biology RNA extraction and qPCR analyses were performed as described 20, 22. Primers for NGAL were 5’-GGACCAGGGCTGTCGCTACT-3’ and 5’-GGTGGCCACTTGCACATTGT-3’ in mouse samples, and 5’-TCACCCTGTACGGAAGAACC-3’ and 5’-GGTGGGAACAGAGAAAACGA-3’ for H9C2 rat cells (forward and reverse primers); UBC mouse primers were 5’-AGCCCAGTGTTACCACCAAG-3’ and 5’-ACCCAAGAACAAGCACAAGG-3’; rat β actin primers were 5’-TTCTACAATGAGCTGCGTGTG-3’ and 5’-CAGGTCCAGACGCAGGAT-3’; GAPDH primers were 5'-AACTTTGGCATTGTGGAAGG-3' and 5'-GGATGCAGGGATGATGTTCT-3'. Western blots were performed using goat polyclonal anti-NGAL antibody (1:1000, R&D Systems Europe, Lille, France). GAPDH was used as internal control for protein loading. For immunohistochemical detection of NGAL on paraformaldehyde-fixed tissues, the NGAL antibody was diluted 1:100. Mouse plasma samples were collected using lithium-heparin Microvette® CB300 (Sarstedt, Marnay, France) and plasma NGAL levels were measured by ELISA (Bioporto Diagnostics, Gentofte, Denmark).
by guest on July 22, 2013http://hyper.ahajournals.org/Downloaded from