Download MATERIALS and METHODS

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MATERIALS and METHODS:
Animal preparation
Experiments were performed on groups (n=6) of adult male spontaneously
hypertensive rats (SHR) aged 10 weeks (250-280g), and age-matched male Wistar Kyoto
rats (WKY). Separate groups (n=6 for each) were prepared for the four components of
the study: mRNA expression, protein expression, immunocytochemistry and time course
of p47phox expression. The time-course of changes in protein expression in the kidney
of p47phox were undertaken on groups of 4 week old prehypertensive SHR and WKY
(50-70g), and results contrasted with groups of 10-week old SHR and WKY. Rats were
fed a standard rat chow (Na+ content 0.3g/100g) and were given tap water to drink. They
were anesthetized with pentobarbital sodium (50 mg/kg i.p.). A cannula was inserted into
the abdominal aorta. The kidneys were flushed with ice-cold phosphate-buffered saline
(PBS) and were removed immediately. The kidney cortex was separated by dissection for
extraction of mRNA and protein.
All procedures in animals were performed in
compliance with the Georgetown University Policies and Guidelines Concerning the Use
of Animals in Research and Teaching and the Guide for the Care and Use of Laboratory
Animals (NIH publication No. 93-23, revised 1985).
Immunocytochemical study
The kidneys were perfused with PBS followed by periodate-lysineparaformaldehyde (PLP) solution. Kidney slices for immunohistochemical studies were
immersed
in
PLP
overnight
at
4C.
The
tissue
for
light
microscopic
immunohistochemistry was embedded in wax (Polyethylene glycol 400 distearate;
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Polysciences Inc., Warrington, PA, USA). These methods have been described in detail
previously (1).
Antibodies
Previously characterized monoclonal and polyclonal anti-phox protein antibodies
were used in these studies.
Rabbit anti-p22phox polyclonal antibody (R3179) was
prepared using SDS-polyacrylamide gel-purified human p22phox as the antigen and was
shown to be specific for p22phox (2). Mouse anti-p22phox monoclonal antibody (44.1)
was prepared using affinity purified human flavocytochrome b and was shown to be
specific for p22phox (3). Rabbit polyclonal (R360) and mouse monoclonal (43.27) antip47phox were prepared using recombinant human p47phox, and both antibodies were
shown to be specific for p47phox (4). Rabbit polyclonal (R1497) and mouse monoclonal
(81.1) anti-p67phox were prepared using recombinant human p67phox, and both
antibodies were shown to be specific for p67phox (4).
All of these antibodies have been shown previously to cross-react with analogous
phox proteins in rat vascular tissues (5, 6), and we also performed the western blotting
with human, mouse and rat leukocyte lysate and confirmed cross-reactivity.
Isolation of total RNA
Total RNA was isolated from the kidney cortex using the guanidinium
isothiocyanate method (QIAGEN, Valencia, CA). Briefly, the kidney cortex was first
lysed and homogenized under highly denaturing conditions with guanidinium
isothiocyanate and -mercaptoethanol
applied
to inactivate RNAses. DNase treatment was
to avoid the contamination from genomic DNA.
RNA was quantified
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spectrophotometrically by measuring the absorbency at 260 nm. The RNA integrity was
assessed by comparing the ethdium bromide-stained 18S and 28S ribosomal RNA bands.
Reverse-transcription (RT) and quantitative polymarase chain reaction (PCR)
Quantitative multiplex RT-PCR was used to quantify the expression of mRNA
for gene products of the five principal subunits of phagocyte-type NADPH oxidase:
p22phox, gp91phox, p67phox, p47phox, p40phox, and for MOX-1 and RENOX as
described previously (7). Briefly, two-step RT-PCR reactions were performed using the
SuperScript Preamplification System for the first strand cDNA synthesis (Gibco BRL,
Rockville, MD) and AmpliTaq DNA Polymerase (Applied BioSystems, CA). The first
strand of cDNA was prepared from 1 g total RNA with a random hexomers primer
and reverse transcriptase by incubating for 50 min at 42C, with termination of the
reaction at 70C for 15min. The resulting single-stranded cDNA was amplified using
synthetic oligonucliotide primers (Table 1) based on published sequences for p22phox,
gp91phox, p67phox, p47phox, p40phox, RENOX, and MOX1. PCR amplification was
performed on a 24000 Thermal Cycler (ABI, Model 2400) with 2'-deoxynucleoside5'triphosphates and Taq DNA polymerase. The conditions were 94C for denaturing for
30sec, 58C for annealing for 30sec, 72C for extension for 30sec. Primer sets were used
in multiplex relative quantitative RT-PCR, where 18S was used as internal standard
(Ambion Inc, Austin, TX) and 1l of cDNA amplified from 1g total RNA was used as
a template. Pilot experiments were undertaken for each gene and for the internal control
18S to ensure that quantitative measurements were made only during the exponential
phase of extension. PCR products were separated on a 1.5% (wt/vol) agarose gel
containing ethidium bromide and visualized by ultraviolet transillumination. Band
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intensities were assessed using an Alphaimager 2200 (Alpha Innotech Corporation, San
Leonardo, CA).
Sequencing of PCR products
Reamplified PCR products for p22phox,
gp91phox,
p67phox,
p47phox,
p40phox, MOX1,and RENOX were sequenced and compared to published data for the
mouse and human counterparts. Direct sequencing of PCR products was performed after
gel purification of the PCR products according to the DyePrimer and DyeTerminator
system (Applied BioSystems, CA). The labeled extension products were analyzed on an
Applied BioSystems Model 373A DNA sequencer.
Western Blotting
As described in detail previously (1), the kidneys were removed immediately after
perfusion with ice-cold PBS. They were homogenized on ice with a Teflon-glass tissue
homogenizer (Iwaki, Chiba, Japan), in 2 ml of buffer containing 20mM Tris, at pH 7.2,
0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.5mM ethylene glycol-bis (aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), 20 µM leupeptin, 10 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 0.1 mM Pefabloc SC.
Homogenates were centrifuged at 4C and 12.000 rpm for 15 min. The supernatants were
diluted in the same volume of sodium dodecyl sulfate (SDS) sample buffer (0.125 M Tris
HCl, 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue in final
concentration). Samples containing 25 µg of protein were applied to an 8.5% gel and
electroblotted to nitrocellulose membranes. The membranes were incubated with 5%
nonfat dried milk in TBST for 30 min, following overnight incubation with previously
characterized monoclonal and polyclonal antibodies at a 1:1000 dilution. After rinsing
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in TBST, membranes were incubated for 1 h with anti-rabbit IgG antibody conjugated
HRP at a 1:1000 dilution., and rinsed with TBST followed by 0.8 mM diaminobenzidine
with 0.01% H2O2 and 3 mM NiCl2 for the detection of blots .
Light microscopic immunohistochemistry
Kidney slices were processed for immunohistochemistry using the labeled
streptavidin biotin method as described previously (1). Wax sections (2µm) were
dewaxed, incubated first with 3% H2O2 for 10 min to eliminate endogenous peroxidase
activity and thereafter with above indicated polyclonal antibodies directed against
p22phox at a dilution of 1:200 or p47phox and p67phox at a dilution of 1:400 for 2 h,
after exposure to blocking serum. The sections were rinsed with Tris buffered saline
containing 0.1% Tween 20 (TBST) and a biotinylated secondary antibody against rabbit
immunoglobulin (Dako, Glostrup, Denmark) for 1h. After rinsing with TBST, the
sections were incubated for 1 h with horseradish peroxidase (HRP)-conjugated
streptavidin solution. HRP labeling was detected using a peroxide substrate solution with
diaminobenzidine (0.8 mM; Dojindo Laboratories, Kumamoto, Japan) and 0.01% H2O2.
The sections were counterstained with hematoxylin before being examined under a light
microscope.
STATISTICAL ANALYSIS:
All values shown are meanstandard error. Unpaired comparisons using Student’s
t test were used to determine significance between specific groups. P<0.05 was
considered statistically significant.
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Referencies
1. Tojo A, Bredt DS, Wilcox CS. Distribution of postsynaptic density proteins in rat
kidney: relationship to neuronal nitric oxide synthase. Kidney Int. 1999;55:1384-94.
2. Jesaitis, A.J., Buescher, E., Harrison, D., Quinn, M.T., Parkos, C.A., Livesey, S., and
Linner, J. Ultrastructural Localization of Cytochrome b in Resting and
Phagocytosing Human Granulocytes. J Clin Invest. 1990;85: 821-835.
3. Burritt, J.B., Quinn, M.T., Jutila, M.A., Bond, C.W., and Jesaitis, A.J. Topological
Mapping of Neutrophil Cytochrome b Epitopes with Phage-display Libraries. J Biol
Chem. 1995;270: 16974-16980.
4. DeLeo, F.R., Ulman, K.V., Davis, A.R., Jutila, K.A., and Quinn, M.T. Assembly of
the Human Neutrophil NADPH Oxidase Involves Binding of p67-phox and
Flavocytochrome b to a Common Functional Domain in p47-phox. J Biol. Chem.
1996;271: 17013-17020.
5. Wang, H.D., Pagano, P.J., Du, Y., Cayatte, A.J., Quinn, M.T., Brecher, P., and
Cohen, R.A. Superoxide anion from the adventitia of the rat thoracic aorta
inactivates nitric oxide. Circulation Research. 1998;82:810-818.
6. Wang, H.D., Hope, S., Du, Y., Quinn, M.T., Cayatte, A., Pagano, P.J., and Cohen,
R.A. Paracrine role of adventitial superoxide anion in mediating spontaneous tone of
the isolated rat aorta in angiotensin II-induced hypertension. Hypertension
1999;33:1225-1232.
7. Kitiyakara C, Chabrashvili T, Jose P, Welch WJ, Wilcox CS. Effects of dietary salt
intake on plasma arginine. Am J Physiol. 2001;280:R1069-75.