HYPERTENSION/2006/078121.R2

MATERIALS AND METHODS

Animal Treatment

The investigation was approved by the Vertebrate Animal Review Committee of the Medical University of South Carolina. Male Dahl salt-sensitive (DSS) rats (Sprague-Dawley Harlan, Indianapolis, IN) were housed at a constant room temperature with a 12-hr light/dark cycle and had free access to tap water and rat chow. All procedures complied with the standards for care and use of animal subjects as stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Resources, National Academy of Sciences, Bethesda, MD). DSS rats (5 weeks-old) were fed either a normal salt (NS; 0.4% NaCl, n=5) or a high salt (HS; 4% NaCl, n=12) diet for 5 weeks. After 2 weeks of salt loading, six DSS rats on a HS diet were infused with bradykinin (BK) at a dose of 500 ng/hr via osmotic minipumps (ALZET, Cupertino, CA) implanted subcutaneously in the back. All rats were anaesthetized with pentobarbital (50 mg/kg body weight i.p.) 3 weeks after minipump infusion.

Blood Pressure Measurement and Blood and Urine Collection

Systolic blood pressure was measured with DASYlab 5.5 software (Kent Scientific Corporation, Turnington, CT) by the tail-cuff method. Blood was collected from the heart at the time of sacrifice. Twenty-four-hour urine was collected from rats in metabolic cages 1 day before sacrifice.

Renal Function Assays

Blood urea nitrogen (BUN) levels were determined using a modified urease-indophenol method1 and serum creatinine levels were measured by a commercial kit (BioAssay Systems, Hayward, CA). Urinary protein levels were measured by Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA).

Morphological and Histological Analysis

Kidneys were fixed in 4% formaldehyde, dehydrated and paraffin-embedded. Four mm thick sections were stained with periodic acid-Schiff (PAS) for morphological analysis and determination of the extent of renal injury; hematoxylin and eosin (H&E) for measuring blood vessel width; and Masson’s trichrome or Sirius red to determine the extent of fibrosis. Collagen fraction volume was determined from tissue sections stained with Sirius red. Briefly, ten fields from each tissue section were photographed and analyzed by Adobe Photoshop software.2 Sirius red-stained material from each field was selected and the number of red pixels was determined. The sum of stained extracellular material was then divided by the total amount of tissue (number of total pixels in the field, including the extracellular material) for each section to calculate collagen fraction volume. Immunohistochemistry was performed using the Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlingame, CA). Kidney sections were incubated at 4oC overnight with primary antibodies against the monocyte/macrophage marker ED-1 (1:100; Chemicon, Temecula, CA), collagen I and III (1:2000, Sigma, St Louis, MO), and collagen IV (1:10, Southern Biotech, Birmingham, AL). The number of monocytes/macrophages was counted in a double-blind fashion from 20 different fields of each section (n=5 or 6).

Histological Evaluation of Interlobular Artery Diameter and Structure

H&E stained slides of kidney cortex from each animal were scanned at medium magnification and two randomly chosen interlobular arteries with complete luminal cross-sections were evaluated in each slide. The arteries were analyzed for the following features: (1) total blood-vessel diameter (outer adventitia width), (2) media width (outer media width including lumen), and (3) average smooth muscle cell layers in the media (estimated by counting nuclei in the media on each side of the lumen and averaging). The numerical values for widths of arterial layers were obtained using a 100 mm diameter grid placed in the eyepiece of the microscope.

TUNEL Staining

Apoptotic cells were determined using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and co-stained with propidium iodide (PI).3 The TUNEL procedure was performed using an in situ cell death detection kit (Roche, Indianapolis, IN). TUNEL-positive renal cells were counted under a microscope under double-blinded conditions. The ratio of TUNEL-positive renal cells to the total number of renal cells was then calculated.

Measurement of Renal NO Levels, NADH/NADPH Oxidase Activity and Superoxide Formation

Nitrate/nitrite (NOx) levels (indicative of NO formation) were measured by a fluorometric assay.4 NADH and NADPH oxidase activities in renal extracts were measured in the presence of NADH or NADPH substrate (0.1 mM) and lucigenin (0.25 mM) (Sigma) at a wavelength of 425 nm using a TD-20/20 luminometer as previously described.5,6 Superoxide levels were quantified by a spectrophotometric assay based on the rapid reduction of ferricytochrome c (Sigma) to ferrocytochrome c as previously described.5 Non-superoxide-dependent reduction of ferricytochrome c was corrected for by deducting the activity not inhibited by superoxide dismutase (Sigma). An SOD baseline was run to normalize the SOD-inhibitable reduction of ferricytochrome c.

Western Blot Analysis

Western blot analysis was performed as previously described5 using the cytosolic fraction of kidney extracts to detect the total and/or phosphorylated forms of extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun N-terminal kinase (JNK), (Cell Signaling Tech., Beverly, MA), TGF-b1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and GAPDH (Advanced Immunochemical Inc., Long Beach, CA). Membranes were incubated with secondary antibody conjugated to LumiGLO chemiluminescent reagent. Chemiluminescence was detected using an ECL-Plus kit (Perkin Elmer Life Science, Boston, MA).

Quantitative Real-Time PCR

Total RNA was extracted from kidney using Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was transcribed using a cDNA Archive Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR reactions were carried out using 10 ml of 2X TaqMan Universal PCR Master Mix, 1 ml of 20X TaqMan Gene Expression Assay Mix (Rn01475962_mL for TGF-b1, Rn00801649_gl for collagen I, Rn01437650_gl for collagen III, Rn00578261_mL for B1 receptor, and Rn00597384_mL for B2 receptor), and 9 ml of cDNA in duplicate. Reactions were run on a 7300 real-time PCR system (Applied Biosystems) with the following thermal cycler conditions: 95oC for 10 min, followed by 40 cycles of 95oC for 15 sec and 60oC for 1 min. Transcription of the housekeeping gene GAPDH was determined by specific primer/probe mix.

Statistical Analysis

Data were analyzed using standard statistical methods and ANOVA followed by Fisher’s PLSD and Bonferroni post hoc tests where appropriate. Group data were expressed as mean ± SEM. Values of all parameters were considered significantly different at a value of P<0.05.

References

1. Bauer JD. Carbohydrates and nitrogen compounds. In: Bauer JD, ed. Clinical Laboratory Methods. St. Louis, MO: The CV Mosby Co.; 1982:472–505.

2. Lehr HA, van der Loos CM, Teeling P, Gown AM. Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem. 1999;47:119-125.

3. Yin H, Chao L, Chao J. Kallikrein/kinin protects against myocardial apoptosis after ischemia/reperfusion via Akt-glycogen synthase kinase-3 and Akt-Bad.14-3-3 signaling pathways. J Biol Chem. 2005;280:8022-8030.

4. Misko TP, Schilling RJ, Salvemini D, Moore W, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993;214:11-16.

5. Zhang JJ, Bledsoe G, Kato K, Chao L, Chao J. Tissue kallikrein attenuates salt-induced renal fibrosis by inhibition of oxidative stress. Kidney Int. 2004;66:722-732.

6. Griendling KK, Minineri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994;74:1141-1148.

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