by Dia-Sorin. The assay begins with samples (zero and incubated) and standards being incubated for 3 hours at room temperature in antibody coated tubes with I125 tracer added. After removing the elluent, antibody coated tubes are counted in a gamma counter and sample values obtained by comparing those of samples to those of the standard curve. The zero sample is then subtracted from the incubated sample to determine the amount of angiotensin I generated (Figsl ,2)(3,4). In mice, due to PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 315 Continuation Format Page Principal Investigator/Program Director (Romero,J.Carlos): Romero, J. Carlos COTG C problems with measuring PRA, we will instead measure PRC. Briefly for PRC generation we will use the methods of Carretero and Beierwaltes (5,6). This involves incubating our mouse samples for 2 hours at 37 ?C with nephrectomized rat substrate in the presence of a peptidase inhibitor. To terminate the reaction, samples are boiled for 10 minutes then centrifuged for 15 minutes at 2000 rpm's. The supernatant is then removed and stored at -20 ?C until assayed using the same kit as used for PRA measurement (Dia-Sorin). Anaiotensin II plasma and tissue: To extract Ang II from the tissue, we will first smash the tissue at-80'|0 C, then homogenize in cold MeOH. Samples are then centrifuged at 20,000 G's, the MeOH supernatant sample removed, lyophilized, and stored at -80?C until reconstituted into assay buffer for extraction. Reconstituted tissue samples and plasma sampleswill use the same methods for extraction and assaying. Basically samples will be extracted on a pre-washed phenyl extraction column washed with dH2O and eluted with MeOH. The MeOH is removed by lyophilization and the samples reconstituted in assay buffer. The Enzyme Immunoassay (EIA) kit used is provided by SPI-Bio and distributed by Cayman Chemical. It involves an incubation of sample and standard with a monoclonal antibody immobilized to the wells of a 96 well plate. After immunological reaction the wells are washed and the trapped ANG II molecule is covalently linked back to the antibody complex with gluteraldehyde. After washing and denaturing the bound ANG II can react with the acetylcholinerase-labelled antibody (tracer). After washing to remove unbound tracer, the tracer bound to the ANG II complex reacts with Ellman's reagent to slowly develop a yellow color that is read on a spectrophotometer plate reader at the wavelength of 405 nm. Readings of samples are compared to those of standards to obtain a value. This value is given as pg per mg protein for the tissue samples and pg/ml for the plasma samples. This assay exhibits a 2-7% intra- and inter-assay coefficient when measuring from 20-100 pg of Ang ll/ml and 2-5% variation when estimating 2-5 pg of Ang II (Fig 2) (4,7,8,9). Nitrotvrosine - tissue: The principle of this assay is based on the fact that NO is the only biological molecule produced in high enough concentration to out-compete SODfor superoxides to form ~OONO. This compound modifies tyrosine in proteins to create nitrotyrosines leaving a marker detectable in vivo. Monoclonal antibodies to nitrotyrosine can then be used to determine immunohistochemically the~OONO- dependent tyrosine nitration. Kidney tissue froze in liquid nitrogen will be homogenized in 20 mM Hepes, pH 7.5, containing protease inhibitors, and will be centrifuged 600 xg to remove cellular debris. Gel electrophoresis analyses for nitrotyrosine residues will be performed on the supernatant as previously described using the anti-nitrotyrosine monoclonal antibody (Cayman,Ann Arbor) as the primary antibody. Following size separation and electrophoretic transfer of kidney proteins to nitrocellulose, Western blot will be performed as described by the vendor for the ECL plus kit (Amersham)for chemiluminescent detection. Bands on exposed films will be quantitated by densitometry. Nitrotyrosine from the vendor will be used as a positive control and GAPDH will be used as the loading control (10). Isoprostane (ISOP)- plasma and tissue: We will measure in plasma samples free levels of ISOP and in tissue samples total levels of ISOP using an enzyme immunoassay kit (EIA, Cayman). Basically all samples are thawed on ice prior to extraction, to reduce the likelihood of further production of ISOP. Tissue samples must first be homogenized in assay buffer with a polytron homogenizer, then undergo an alkaline hydrolysis with 15% potassium hydroxide to release the lipoprotein bound ISOP. Samples are then diluted with absolute ethanol (2X) and centrifuged to remove the precipitate. All samples are then diluted with distilled water, pHed to 3.0 with 1.0 HCI, and added to a Sep Pak C-18 extraction column (Milford, MA). Here they are washed first with distilled water and then Hexane, and eluted off the column with 99/1% Ethyl Acetate/Methanol. The organic solvent is evaporatedfrom the sample, which is reconstituted in assay buffer. For the assay, samples, tracer, and antiserum are added to wells pre-coated with mouse monoclonal secondary antibody and incubated overnight at room temperature. After incubation, plates are washed to remove all unbound ISOP and Ellman's Reagent (containing the substrate to acetylcholinesterase) is added to the wells. Absorbencies produced by this enzymatic reaction will be determined using a spectrophotometer set at 405nm and sample readings will be compared to the standards to obtain values (Fig.3) (4,8,11). Total Anti-oxidant Capacity - plasma This is afast andeasy assayfor measuring the combined actions of all antioxidants in biological fluids and there ability to reduce reactive oxygen species. The assay (AOP-490, Oxis Research) is based on the capacity of the antioxidants in the sample to reduce copper++ to copper +. Basically samples and standards are diluted 1:40 with Bathocuproine Reagent (combines with Copper + to form a compound that absorbs at 490 nm) then added to a microplate with baseline absorbance read at 490 nm. A copper ++ reagent is next added to the samples and standards and jncubated for 3 minutes. Final PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 316 Continuation Format Page Principal Investigator/Program Director (Romero, J.Carlos): Romero, J. Carlos COFG C absorbencies are read at 490mn and net absorbance is recorded. Sample absorbencies are then compared to those of standards with known quantities of uric acid to obtain Total Antioxidant Capacity for each sample. NADPH-oxidase (p47) - tissue NADPH (p47) is assayed using the western blot analysis. Briefly tissue samples are smashed at -80?C and homogenized in lysis buffer. The homogenates are then centrifuged and supernatant removed. For the western blot, 50 ug sample supernatant (as determined by Lowry protein analysis) is loaded onto a 12.5 % Tris-HCI gel, then transferred to a PVDF membrane. Membranes are then blocked with 5 % milk in TBS-T and incubated overnight at 4?C with primary p47-NADPH antibody (1:1000) (Cell Signalling). The next day membranes are incubated for 2 hours at room temperature with secondary (goat anti-rabbit) antibody (1:1000), fluoresced with ECL solution and intensities of the bands recorded on x-ray film. Finally membranes are stripped with guanidine hydrochloride solution, blocked with a 5% milk solution and incubated with B-actin antibody. Upon completion of all western analysis, intensities of p47 NADPH bands are quantified and values are computed relative to the intensities of B-actin(12). GSH/GSSG Assay - tissue (OXIS International. CA) Tissue samples are homogenized in lysis buffer added with 20 ul 1-Methy-2-vinyl-pyridium trifluoromethane sulfonate (M2VP), a thiol-scavenging reagent which rapidly scavenges GSH but does not interfere with glutathione reductase, since GSH oxidation likely occurs rapidly in disrupted tissues. Samples are vortexed with 5% metaphosphoric acid for 20 sec, and then added with assay buffer or GSSG buffer for final measurement. At the time of measurement, 200 pi of standards, blank or samples are added into the cuvettes, mixed with 200 ul Chromogen and 200ul glutathione reductase at room temperature for 5 min. 200 ul of NADPH is added to each cuvettes, the change of absorbance at 412nm recorded for 3 min. A six-point standard curve is constructed, and GSH or GSSG concentration, GSH/GSSG ratio was calculated following the instructions. DHE. In situ production of super oxide anion is measuredin 30-um frozen kidney sections using the oxidative fluorescent dye dihydroethidium (DHE). Cytosolic DHE exhibits blue fluorescence, but once oxidized by super oxide to ethidium bromide it intercalates within the cell's DNA, staining its nucleus a fluorescent red (excitation at 488 nm, emission 610 nm). Serial sections areequilibrated under identical conditions for 30 min at 37?C in Krebs-HEPES buffer. Then, fresh buffer containing DHE (2 umol/l) is applied to each tissue section, cover slips added, and tissues incubatedfor 30 min in a light-protected humidified chamber at 37?C. Quantification of each tissue section for levels of DHE is evaluated using fluorescence microscopy (13). Creatinine will be determined in plasma and urine by means of the Jaffe rate method. Sodium/Potassium will be measured in plasma and urine by flame photometry. StatisticalAnalysis Results will be shown as mean ? SEM. Comparisonswithin groups will be performed by use of paired Student's t test, and among groups by ANOVA, with the Bonferroni correction for multiple comparisons, followed by unpaired Student's t test. Literature cited 1. Schmitz C, Gottthardt M, Hinderlich S, Lehested J-R, Gross V, Vorum H, Christensen El, Luft FC, Takahashi S, Willnow TE. Normal Blood Pressure and Plasma Renin Activity in mice lacking the Renin- binding Protein, a Cellular Renin Inhibitor, J Biol Chem 275: 15357-15362, 2000. 2. Cheng H-F, Wang S-W, Zhang M-Z, McKanna JA, Breyer R, Harris RC. Prostaglandins that increase renin production in response to ACE inhibition are not derived from cyclooxygenase-1. Am J Physiol 288:R638-R646, 2002. 3. Romero JC, Ruilope LM, Bentley MD, Fiksen-Olsen MJ, Lahera V, Vidal MJ . Comparison of the Effects of Calcium Antagonists and converting enzyme inhibitors on renal function under normal and hypertensive conditions. AM J Cardiol 62: 59G-68G, 1988. 4. Bolterman RJ, Manriquez MC, Ruiz MCO, Juncos LA, Romero JC. Effects of captopril on the renin angiotensin system, oxidative stress, and Endothelin in normal and hypertensive rats. Hypertension 46: 943-947, 2005. 5. Harding P, Carretero OA, Beierwaltes, WH. Chronic cyclooxygenase-2 inhibition blunts low sodium- stimulated renin without changing renal hemodynamics. J. Hypertension 18: 1107-1113, 2000. 6. Beierwaltes WH, Schryver S, Olson PS, Romero JC. Interaction of the prostaglandin and renin- angiotensin systems in isolated rat glomeruli. Am J Physiol 239: F602-F608, 1980 PHS 398/2590 (Rev.09/04, Reissued 4/2006) Page 317 Continuation Format Page
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