ONLINE APPENDIX
DETAILED MATERIALS AND METHODS
Animals
The investigation was performed in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals and were approved by the local
ethical committee ‘‘Comité regional Nancy-Lorraine/Nord-Est’’ (#B54-547-20) or the
Animal Care and Use Committee of Universidad Complutense de Madrid. Male Wistar rats
(250 g; Harlan Iberica) were treated for 3 weeks with vehicle (sunflower oil, subcutaneous
injection, n=10), Aldo-salt (1 mg/kg/day, subcutaneous injection, n=10 and 1% NaCl as
drinking water), Aldo-salt plus Spironolactone (Spiro) (200 mg/kg/day, subcutaneous
injection, n=10), Aldo-salt plus MCP (100 mg/kg/day, in food, n=9), Spiro (n=7) or MCP
(n=5) alone. After two weeks of treatment, urine was collected in metabolic cages. The model
and the dose of Aldo were chosen from previous studies in which the treatment increased
blood pressure BP (1) and in accordance with previous unpublished dose-response
experiments performed into our laboratory.
Adult male C57BJ6 wild-type mice and Gal-3 KO mice (2) were infused for 3 weeks with
Aldo (1 mg/kg/day) or vehicle (150 mmol/liter NaCl, 5% ethanol) using a subcutaneous
osmotic minipump (Alzet) (n = 7, each group). Tail cuff blood pressure was monitored
throughout the treatment.
Hemodynamic parameters
Rats were anaesthesized with ketamine (Imalgene 1000, Merial; 70 mg/kg, I.P.) plus
xylazine (Rompun 2%, Kup Pharma; 6 mg/kg, I.P.), and a catheter (Sciense FT211B, 1.5mm
diameter) was advanced into the left ventricle through the right carotid artery. The catheter
was connected to a data acquisition system (PowerLab/800, ADInstruments), and signals were
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monitored and digitally stored for analysis with the software Chart for Windows (version 4.2).
The following parameters were evaluated: systolic blood pressure (SBP), diastolic blood
pressure (DBP), left ventricular (LV) end-diastolic pressure (LVEDP), LV systolic pressure
(LVSP), the first derivative of the LV pressure rise over time (+dP/dt), and the first derivative
of the LV pressure decline over time (-dP/dt).
Western Blot analysis
The hearts were dissected and cut transversally in three parts (for histology, RNA and
Western Blot analyses). The kidneys were cut longitudinally, and the left kidneys were used
for histological analyses. In the right kidneys, the medulla was removed and the cortex
employed for RNA and Western Blot analyses. The tissues were immediately frozen in liquid
nitrogen for molecular studies. Aliquots of 40 µg of proteins were electrophoresed onto SDS
polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Amersham
Biosciences). The following specific antibodies were used:
Antibody
Dilution
Manufacturer
Catalog number
1:500 Thermo Scientific
A3A12
Gal-3
1:500
Abcam
ab34710
collagen type I
Santa Cruz
sc-28888
collagen type III 1:500
1:500
Santa Cruz
sc-25440
CTGF
1:500
Santa Cruz
sc-146
TGF-β1
1:500
Abcam
ab6302
β-catenin
1:500
Abcam
ab53033
E-cadherin
1:500
Millipore
MAB1926
fibronectin
1:500
Abcam
A5228
α-SMA
1:1000
Sigma
T6074
tubulin
1:1000
Sigma
A5441
β-actin
Bound antibodies were detected by peroxidase-conjugated secondary antibodies
(Amersham Biosciences) and visualized using the Immun-Star western kit (Bio-rad) or ECLSelect chemiluminescence detection system (Amersham). After densitometric analyses,
optical density values were expressed as arbitrary units and β-actin or tubulin was used to
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normalise protein loading. All Western Blots were performed at least in triplicate for each
experimental condition.
ELISA
Protein concentrations were measured by ELISA according to the manufacturer's
instructions. List of the ELISA kit used:
Rat
Mouse
Gal-3
R&D Systems
NGAL Biosensis R&D Systems
Albumin Abcam
Abcam
Creatinine dosage
Creatinine concentrations were measured according to the manufacturer's instructions
(Abcam).
Reverse-transcription PCR
Total RNA from hearts and kidneys was extracted with Trizol Reagent (Euromedex) and
purified using the RNeasy kit, according to the manufacturer’s instructions (Qiagen). First
strand cDNA was synthesized according to the manufacturer’s instructions (Fermentas).
Quantitative PCR analysis was then performed with SYBR green PCR technology (Biorad).
List of the primers (Invitrogen) used in rat:
Gene
Gal-3
Col1a1
Col3a1
Col4a1
CTGF
TGF-β
β-catenin
E-cadherin
Primers
Sequence (5' to 3')
Forward
AGCCCAACGCAAACAGTATC
Reverse
GGCTTCAACCAGGACCTGTA
Forward
GCCTCCCAGAACATCACCTA
Reverse
ATGTCTGTCTTGCCCCAAGT
Forward CTCACCCTGCTTCACCCCTCTC
Reverse TGGACATATTGCACAACATTCTC
Forward GCCAAGTGTGCATGAGAAGA
Reverse
AGCGGGGTGTGTTAGTTACG
Forward
GAGTCGTCTCTGCATGGTCA
Reverse
CCACAGAACTTAGCCCGGTA
Forward
TGCTTCAGCTCCACAGAGAA
Reverse
CAGAAGTTGGCATGGTAGCC
Forward
GCCAGTGGATTCCGTACTGT
Reverse
GAGCTTGCTTTCCTGATTGC
Forward
GGGTTGTCTCAGCCAATGTT
Reverse
CACCAACACACCCAGCATAG
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Fibronectin Forward
Reverse
α-SMA
Forward
Reverse
GAPDH
Forward
Reverse
GGGGTCACGTACCTCTTCAA
TGGAGGTTAGTGGGAGCATC
TGTGCTGGACTCTGGAGATG
GAAGGAATAGCCACGCTCAG
AGATCCACAACGGATACATT
TCCCTCAAGATTGTCAGCAA
Relative quantification was achieved with MyiQ (Bio-rad) software according to the
manufacturer's instructions. GAPDH was used as the endogenous control. All PCRs were
performed at least in triplicate for each experimental condition.
Morphological and histological evaluation
Hearts (arrested in diastole using KCl before being harvested) and kidneys were fixed and
included in paraffin. Sections (5µm-thick) were stained with Sirius red for collagen content.
Quantitative measurement of the area of perivascular fibrosis was calculated from at least 5
arterial cross sections per slide. The ratio of the stained surface to the vessel area was
measured to evaluate the percentage of staining. The area of interstitial collagen was
identified after excluding the vessel (and the glomeruli for kidney slides) area from the region
of interest. The ratio of the stained surface to the total tissue area was measured to evaluate
the percentage of staining. For analyses of the glomerular collagen 10 glomeruli were
randomly selected from each kidney section. The ratio of the stained surface to the glomerular
area was measured to evaluate the percentage of staining. For cell width, cross sectional area
and length, at least 50 cardiomyocytes per section were measured. Cardiomyocyte length was
measured only in cardiomyocytes where intercalated disks were visible. All measurements
were performed blind using an automated image-analysis system (Nikon). Images were
calibrated with known standards.
Immunochemistry
Paraffin-embedded myocardial and renal sections of 5 µm were used. Slides were treated
with H2O2 for 10 min to block peroxidase activity. All sections were blocked with 5% normal
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goat serum in PBS for 1 h and incubated overnight with antibodies described in the table
below, washed three times, and then incubated for 30 min with the horseradish peroxidaselabeled polymer conjugated to secondary antibodies (Dako Cytomation, Carpenteria, CA).
Antibody Dilution
Manufacturer
Catalog number
1:50
Thermo Scientific
A3A12
Gal-3
1:50
Abcam
ab6302
β-catenin
1:50
Abcam
ab53033
E-cadherin
1:50
Millipore
MAB1926
fibronectin
1:50
Abcam
A5228
α-SMA
The signal was revealed by using DAB Substrate Kit (BD Pharmingen). The ratio of the
stained surface to the total tissue area was measured in 10 different regions. All measurements
were performed blind using an automated image-analysis system (Nikon). Images were
calibrated with known standards.
Sodium and potassium dosage
Urinary Na+ and K+ concentrations were measured by flame photometry (IL943; Instruments
Laboratory).
Statistics
Results are presented as meanSEM, computed from the average measurements obtained
from each group or animals. Normal distribution of data was checked by means of the Shapiro
Wilks test and a Levene statistic test was performed to check the homogeneity of variances.
Differences among more than 2 experimental conditions were tested by the ANOVA one way
test, followed by the Scheffé test to analyze differences between groups. The unpaired
Student’s t test or the Mann-Whitney U tests were used to assess statistical differences
between two experimental conditions. P values lower than 0.05 were considered significant.
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REFERENCES
1. López-Andrés N, Martin-Fernandez B, Rossignol P, et al. A role for cardiotrophin-1 in
myocardial remodeling induced by aldosterone. Am. J. Physiol. Heart Circ. Physiol.
2011;301:H2372–2382.
2. Colnot C, Fowlis D, Ripoche MA, Bouchaert I, Poirier F. Embryonic implantation in
galectin 1/galectin 3 double mutant mice. Dev. Dyn. 1998;211:306–313.
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Online Figure 1
Online Figure 1. In a model of Aldo-salt-treated rats, representative pictures and
quantifications of renal sections stained for Gal-3 are presented. Aldo-salt increases Gal-3
expression in tubular cells in kidney but not in glomeruli. Spironolactone (Spiro) and
modified citrus pectin (MCP) co-treatment reversed both Aldo-salt-induced tubular Gal-3
expression. Representative pictures and quantifications of renal sections stained for Gal-3
show that Gal-3 is expressed in the tubules but not in the glomeruli. Histogram bars represent
the mean ± SEM of each group of animals (n≥9 per group). *, p<0.05 vs. control; †, p<0.05
vs. Aldo-salt.
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Online Table 1. Cardiac phenotype of Aldo-salt-treated rats after spironolactone and MCP
treatment.
Control
Spiro
MCP
10
7
5
322 ± 2.3
310 ± 3.1
341 ± 7.7
2.50 ± 0.01
2.35 ± 0.01
2.28 ± 0.01
SBP (mmHg)
123 ± 0.6
124 ± 1.9
124 ± 2.8
DBP (mmHg)
88 ± 0.6
89 ± 1.3
84 ± 0.7
PP (mmHg)
35 ± 0.4
35 ± 1.0
36.1 ± 6.4
HR (beat/min)
322 ± 4.5
330 ± 13.5
324 ± 11
LVSP (mmHg)
132 ± 3.5
126 ± 5.6
127 ± 4.7
LVEDP (mmHg)
3.8 ± 0.3
4.4 ± 0.3
3.9 ± 0.2
dP/dt+ (mmHg/s)
9697 ± 430
9196 ± 519
9252 ± 168
dP/dt- (mmHg/s)
-6651 ± 200 -7073 ± 748
-7310 ± 520
LVWT (mm)
2.27 ± 0.08
2.36 ± 0.5
2.39 ± 0.04
LVCSA (mm²)
35.9 ± 1.4
37.7 ± 1.9
33.4 ± 0.8
Diameter (µm)
20.5 ± 1.3
22.2 ± 0.3
21.9 ± 1.0
Cross sectional area (µm²)
343 ± 30
396 ± 10
379 ± 32
n
BW (g)
Cardiac and hemodynamic parameters
HW/BW (mg/g)
Cardiac morphometry
Cardiomyocyte morphometry
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Length (µm)
96.1 ± 2.2
95.7 ± 2.3
95.0 ± 0.9
Gal-3 (mRNA)
1.00 ± 0.11
0.70 ± 0.16
1.00 ± 0.64
Protein (protein)
1.00 ± 0.04
1.38 ± 0.21
1.42 ± 0.29
Col1a1 (mRNA)
1.00 ± 0.29
1.48 ± 0.04
1.10 ± 0.28
CTGF (mRNA)
1.00 ± 0.11
1.66 ± 0.31
1.13 ± 0.08
TGF-β (mRNA)
1.00 ± 0.10
0,62 ± 0.09
1.71 ± 0.08
Interstitial collagen (%)
2.25 ± 0.60
2.39 ± 0.56
2.63± 0.41
Perivascular collagen (%)
16.60 ± 3.57 20.34 ± 2.15 17.98 ± 2.01
Cardiac Gal-3
Cardiac Fibrosis
Spiro, spironolactone; MCP, modified citrus pectin; BW, body weight; HW, heart weight;
SBP, systolic blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; HR, heart
rate; LVSP, left ventricular (LV) systolic pressure; LVEDP, LV end-diastolic pressure;
dP/dt+, first derivative of the LV pressure rise over time; dPdt-, first derivative of the LV
pressure decline over time; LVWT, LV wall thickness; LVCSA, LV cross-sectional area
(CSA); Col1a1, collagen type I α1; CTGF, connective tissue growth factor; TGF-β,
transforming growth factor β. Values are mean ± SEM.
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Online Table 2. Renal phenotype of Aldo-salt-treated rats after spironolactone and MCP
treatment.
n
Control
Spiro
MCP
10
7
5
Renal parameters
KW/BW (mg/g)
0.26 ± 0.02 0.28 ± 0.02 0.27 ± 0.01
Urine Na+/ K+
0.58 ± 0.04 0.56 ± 0.03 0.48 ± 0.03
Renal Gal-3
Gal-3 (mRNA)
1.00 ± 0.17 1.05 ± 0.09 1.41 ± 0.37
Protein (protein)
1.00 ± 0.17 0.60 ± 0.20 1.11 ± 0.53
Renal Fibrosis
Col1a1 (mRNA)
1.00 ± 0.17 1.41 ± 0.46 1.21 ± 0.40
Col4a1 (mRNA)
1.00 ± 0.15 1.17 ± 0.34 1.44 ± 0.36
CTGF (mRNA)
1.00 ± 0.20 1.32 ± 0.54 1.37 ± 0.12
TGF-β (mRNA)
1.00 ± 0.16 0.97 ± 0.27 1.41 ± 0.16
Interstitial collagen (%)
1.96 ± 0.34 1.80 ± 0.19 2.19 ± 0.13
Glomerular collagen (%)
8.49 ± 0.77 9.97 ± 0.84 9.92 ± 0.46
Perivascular collagen (%)
7.77 ± 0.47 8.27 ± 0.57 7.33 ± 0.13
Renal epithelial-mesenchymal transition
β-catenin (%)
5.55 ± 0.62 4.85 ± 0.12 5.32 ± 0.26
E-cadherin (%)
6.87 ± 0.65 6.39 ± 2.23 6.93 ± 0.87
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Fibronectin (%)
3.19 ± 0.40 1.82 ± 0.02 1.71 ± 0.18
α-SMA (%)
1.95 ± 0.32 1.96 ± 0.31 1.30 ± 0.40
Spiro, spironolactone; MCP, modified citrus pectin; BW, body weight; KW, kidney weight;
Col1a1, collagen type I α1; Col4a1, collagen type IV α1; CTGF, connective tissue growth
factor; TGF-β, transforming growth factor β. Values are mean ± SEM.
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