Download Materials and Methods

SUPPLEMENTAL DATA
Materials and Methods
Animals. Male Sprague-Dawley rats (150g-180g) were housed in an
environmentally controlled room with a 12 h light/dark cycle, where they had free
access to tap water and special rat chow. The Ethics Committee of the Faculty of
Medicine of the University Los Andes approved all protocols for animal
experimentation, according to the National Institutes of Health Guide for the Care
and Use of Laboratory Animals.
Chronic uremia was induced in the animals by 5/6 nephrectomy in one stage
procedure. Under anesthesia (intraperitoneal ketamine 80 mg/kg, Troy
Laboratories PTY Limited, Australia; Xilacine 2.9 mg/kg, Agroland-Alfasan,
Holland), approximately 60% of one kidney was removed. When hemostasis was
achieved, the contra lateral kidney was removed. Control animals underwent
sham operation. After surgery, the rats were allowed to recover for 7 days.
During the following 5 weeks the animals had access to a 20 gr pellet daily
(AIN76 Rodent purified diet, Dyets Inc., Bethlehem, Pennsylvania, USA) that
allowed, when pertinent, to include spironolactone (Spironolactone 15 mg/kg
body weight/day). For the last 2 days of the 5-week period, a group of rats were
held in metabolic cages, and 24-h urine samples were collected for biochemical
analysis. At the end of the study period, blood samples were drawn for
biochemical analysis and immediately the heart was removed. Creatinine
clearance was calculated using the following standard formula: Urinary creatinine
(mg/dL) x Urinary volume (mL/24h)/1440min x plasma creatinine (mg/dL).
Blood Pressure Measurement by the Direct Method: We performed direct BP
measurement in a group of rats, for the accurate BP evaluation, in accordance
with the recommendation of the Subcommittee of Professional and Public
Education of the American Heart Association Council on High Blood Pressure
Research. Under light ether anaesthesia, a polyethylene catheter was implanted
through the left femoral artery into the lower aorta. The distal end of catheter was
threaded through the subcutaneous space, and exteriorized from the
subscapular region, according to Blouin et al.1. Animals have free movements
and access to water and food. Blood pressure was measured daily during the
last experimental week.
RNA isolation and Reverse Transcription. Left ventricles were homogenized in
the presence of the RNA extraction reagent TRIzol (Invitrogen- Life
Technologies, Carlsbad, CA, USA), and total RNA extraction was performed on
the tissue homogenate as described by Michea et al. 2. Total RNA samples were
all treated with DNAse I to remove contaminating genomic DNA from RNA
preparations (DNA-free
TM,
Ambion, Austin, TX, USA), as per manufacturer
instructions. RNA (0.5 μg) was reverse transcribed with random hexamers
(ImProm-IITM Reverse Transcription System, Promega Corporation, Madison,
WI, USA).
Real time semi quantitative PCR analysis. Real-time PCR reactions were
carried out to determine rat tissue ANP, BNP, 11-βHSD2, CYP11B2 and MR
transcripts, using a PTC-200 thermal cycler coupled to a Chromo4 detector (MJ
Research, Alameda, CA, USA). PCR amplification of the 18S ribosomal RNA
served as internal control. Primers for all target genes are listed in Table S1.
Reaction volumes were 20 μl and each contained 0.5 μmol/L of primers, 2.0 U
Taq polymerase (MBI Fermentas, Lithuania), PCR buffer, 0.2 mmol/L dNTPs, 1.5
mmol/L MgCl2, 1/200000 dilution of SYBR Green (Molecular Probes-Invitrogen,
Carlsbad, CA, USA), 2 μL of template cDNA. To confirm amplification specificity
the PCR product was subjected to a melting curve program. Abundance of target
genes mRNA was calculated from standard curves (correlation coefficient ≥
0.98). The results are expressed as the ratio of the target gene amount to 18S
amount for each sample, measured in triplicate. Table S1 includes the primers
used for each measured gene.
S1. Primers used for Real-time
Target
Sense
Antisense
MR
AACTTCAGGCTGCTCAGAGG
TGAAGAACGCTCCAAGGTCT
11 ß HSD-2
CCCGTTGTAGATGCCATCA
AGCTGATACTGTGGGGGAAG
CYP11B-2
GAAAGTGGCCCAAAGCATAA
CAACTGCTTTCCTCGGCTAC
ANP
ATCTGATGGATTTCAAGAACC
CTCTGAGACGGGTTGACTTC
BNP
ACAATCCACGATGCAGAAGCT
GGGCCTTGGTCCTTTGAGA
α-GR
CCTAAGGAAGGTCTGAAGAGC
GCCAAGTCTTGGCCCTCTAT
SGK1
GCTGCTCGAAGTACCCTCAC
TTCAACAGAACATTGCGCTC
NOX-2
CCA TTC GGA GGT CTT ACT TTG
CTG GGC ACT CCT TTA TTT TTC
NOX-4
GAACCTCAACTGCAGCCTGATC
CCTTTGTCCAACAATCTTCTTGTTCC
18S
CGACGACCCATTCGAACGTCT
GCTATTGGAGCATGGAATTACCG
PCR reactions were carried out using the following primer sets (all 5'
MR,
mineralocorticoid
receptor;
11βHSD-2,
11
beta
3').
hydroxysteroid
dehydrogenase type 2; CYP11B-2, aldosterone synthase; ANP, atrial natriuretic
peptide; BNP, brain natriuretic peptide; αGR, glucorticoid receptor; SGK1, serum
glucocorticoid kinase -1; NADPH oxidases: NOX-2 and
NOX-4, 18S, 18S
ribosomal RNA.
Western Bot Analysis: Equal protein amounts were electrophoresed on a SDSPAGE 10% polyacrilamide gel and processed for Western blotting as previously
described.3 Proteins were then transferred to polyvinylidene difluoride membrane
and blocked with 5% non fat milk in Tris buffered saline (20mmol/L Tris/HCl,
137mmol/L NaCl) plus 0.1% Tween-20. Membrane was incubated with
corresponding primary antibody. Blots were developed using the enhanced
chemiluminescent method (Perkin Elmer Life Sciences Inc, Boston, MA, U.S.A.)
with horseradish peroxidase-linked secondary antibody. The membrane was
finally exposed to Biomax MR Kodak scientific imaging film. The following
primary antibodies were used: SGK1, serum and glucocorticoid-regulated protein
kinase (United States Biological, MA. U.S.A.) and p47-phox (Santa Cruz
Biotechnology, Santa Cruz, CA, U.S.A.).
Histology. Equatorial ventricular sections were stained with hematoxylin-eosin
to determine cardiomyocyte cross-sectional area (CSA).4 Images of sections
were analyzed in their entirety (at least 300 cells/image, 20 images/ventricle).
Images were acquired in a Zeiss Axioskop (Zeiss, Germany), coupled to a digital
camera (Nikon Coolpix995, Nikon Corp. Japan). CSA was quantified by a single
investigator who was unaware of the nature of the experimental groups by
computer assisted morphometry (IPLab Spectrum V 3.7, Scanalitycs, Fairfax,
VA, USA).
Biochemical analysis. Plasma and urine electrolytes were determined using the
Roche 9180 Electrolyte Analyzer (Manhein, Germany). Serum and urine
creatinine were measured with a Beckman analyzer. Plasma aldosterone
concentrations were determined by radioimmunoassay (Diagnostic Products
Corporation, Los Angeles, CA, USA). Cardiac aldosterone was extracted from
hearts as described by Chai et al.4 Briefly, the excised hearts were washed
thoroughly with ice-cold PBS to remove blood and then homogenized in 1 mL of
methanol with the use of a Polytron homogenizer. After centrifugation at 3000g
for 15 minutes, the supernatant was dried under vacuum (Savant Speed Vac SC100 system) and then mixed with 1 mL PBS. Aldosterone concentrations were
determined by radioimmunoassay system. Tissue aldosterone levels were
expressed in pg/mg protein.
11ß-HSDActivityAssay. 11-Dehydrogenase activity of both 11ß-HSD isoforms
was determined by measuring the rate of conversion of corticosterone (B) to 11dehydrocorticosterone (A) as described by Alzamora5. In brief, heart was
homogenized in ice-cold KCl buffer (0.154 mol/L, pH 7.6) using a Polytron
homogenizer (Kinematica). Homogenates were centrifuged for 10 minutes at
1000g, and protein concentration in the supernatant was determined (Bradford,
Bio-Rad). Homogenates (0.5 mg protein/mL) were incubated in 0.5 mL of
phosphate buffer (0.1 mol/L, pH 7.6), which contained 50000 cpm of 1,2,6,7-
[3H]corticosterone (specific activity 88 Ci/mmol; DuPont-New England Nuclear),
0.1 or 2.5 µmol/L B, and 400 µmol/L oxidized nicotinamide adenine dinucleotide
(NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+), for 20 minutes
at 37°C. On the basis of previous studies, assays with 0.1 µmol/L B and NAD+
were designed to detect 11ß-HSD2 activity and assays with 2.5 µmol/L B with
NADP+, to measure 11ß-HSD1. Aliquots were extracted into 1:10 chloroform
(vol/vol), and steroids were separated by thin-layer chromatography using
acetone/chloroform (18:82) as a mobile phase. Areas corresponding to steroids
were identified under UV light and scraped off, and radioactivity was counted in a
liquid scintillation analyzer (Packard Instrument Co.). Activity was expressed as
picomoles of product per minute per 100 mg of protein for each tissue
Measurement
of
O2.-Production.
We
used
lucigenin-enhanced
chemiluminescence to estimate O2.- levels in left ventricle tissue from SHAM,
NPX, and NPXspiro rats (n=4 each group).6 Briefly, ventricular tissue (1 mm x 5
mm segments of the left ventricle) was transferred into scintillation vials
containing Krebs-Hepes buffer (pH 7.4 after 30 min aeration with 95%O2 and
5%CO2)
and
5
µmol/L
lucigenin
(bis-N-methylacridinium
nitrate).
Chemiluminescence was measured in duplicate by a luminometer (Berthold
FB12 Luminometer; Pforzheim, Germany) recording relative light units (RLU)
emitted over 1-min intervals (10 consecutive measurements). 5 mmol/L of 4,5dihydroxy-1,3-benzene disulfonic acid (Tiron), a superoxide dismutase mimetic
and cell membrane permeable scavenger of superoxide anion, was added to
each sample after the measurements. The data were averaged and
chemiluminescence
of
lucigenin-containing
buffer
with
tissue
minus
chemiluminescence in the presence of Tiron was calculated, and the decreased
values were reported as net superoxide anion production. Similar data were
obtained when chemiluminescence of lucigenin-containing buffer with tissue
minus background chemiluminescence (without tissue) were calculated. After the
assay, the tissues were removed from the buffer and dried at 90ºC for 4 hours,
and the RLU were normalized per milligram of dry tissue weight.
Results
Figure S1.
Effect of Spironolactone on cardiac hypertrophy induced by 5/6 nephrectomy. A,
Heart weight/ body weight ratio and B, left ventricle/ body weight ratio, of control
rats (SHAM), 5/6 nephrectomy (NPX) and NPX plus spironolactone (NPXspiro).
Values are mean ± SE; n = 9 for each group of rats. *P<0.05 compared with
SHAM, #P<0.05 with respect to NPX.
Figure S2.
11ß-Hydroxysteroid Dehydrogenase (11ß-HSD) activity and expression in left
ventricle of SHAM, NPX and NPXspiro rats. A, 11ß-HSD type 1 activity in left
ventricle protein homogenates (nmol/mg protein x min-1). B, 11ß-HSD type 2
activity (pmol/mg protein x min-1). C, 11ß-HSD2 mRNA abundance normalized to
the abundance of 18S rRNA (18S). Data are expressed as mean ± SE. n= 6 in
each group.
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