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1 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 4C. The tissue for light microscopic immunohistochemistry was embedded in wax (Polyethylene glycol 400 distearate; 2 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 3 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 42C, with termination of the reaction at 70C 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 94C for denaturing for 30sec, 58C for annealing for 30sec, 72C 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 1l of cDNA amplified from 1g 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 4 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 4C 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 5 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 meanstandard error. Unpaired comparisons using Student’s t test were used to determine significance between specific groups. P<0.05 was considered statistically significant. 6 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.