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Am J Physiol Heart Circ Physiol 285: H1396-H1403, 2003. First published June 5, 2003; doi:10.1152/ajpheart.00096.2003
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Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney

Wei Guo,1 Takeshi Adachi,1 Reiko Matsui,1 Shanqin Xu,1 Bingbing Jiang,1 Ming-Hui Zou,2 Michael Kirber,1 Wilfred Lieberthal,3 and Richard A. Cohen1

1Vascular Biology Unit, Whitaker Cardiovascular Institute, and 3Renal Section, Evans Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118; and 2Department of Surgery, University of Tennessee, Knoxville, Tennessee 37920

Submitted 30 January 2003 ; accepted in final form 4 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV). MnSOD activity decreased ~50% in angiotensin II-infused rat kidneys (24 ± 4.6 vs. 11 ± 5.2 U/mg) without a change in protein expression. Immunohistochemical staining showed 3-nitrotyrosine predominantly in distal tubules and collecting duct cells in the angiotensin II-infused rat kidneys. By two-photon microscopy, 3-nitrotyrosine colocalized with MnSOD. Total 3-nitrotyrosine content in kidney homogenates was increased in angiotensin II-infused rat kidney [3.2 ± 1.9 (sham treated) vs. 9.5 ± 2.3 ng/mg protein by HPLC-UV detection]. With tracer amounts of tyrosine-nitrated recombinant MnSOD, the most sensitive technique to detect tyrosine nitration of MnSOD was immunoprecipitation from tissue with anti-MnSOD antibody, followed by detection of 3-nitrotyrosine by Western blotting or HPLC. By HPLC, 3-nitrotyrosine content of kidney MnSOD increased 13-fold after angiotensin II infusion, representing an increase from approximately one-twentieth to one-fifth of the total 3-nitrotyrosine content in sham-treated and angiotensin II-infused rat kidney, respectively. Angiotensin II-induced hypertension is accompanied by increased tyrosine nitration of MnSOD, which, because it inactivates the enzyme, may contribute to increased oxidant stress in the kidney.

nitrotyrosine; oxidant stress

ANGIOTENSIN II-INDUCED HYPERTENSION is associated with an increase in superoxide production by the activation of NADPH oxidase (23, 24, 30). Superoxide anion readily reacts with nitric oxide (NO) to generate peroxynitrite (ONOO–), which can nitrate the 3-position of tyrosine residues on proteins (5). Therefore, 3-nitrotyrosine is a marker of oxidative stress, which is evoked in various diseases including human atherosclerosis, pulmonary and heart disease, acute and chronic kidney rejection, Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (2, 6, 15–19, 21, 25). Moreover, 3-nitrotyrosine formation itself may change protein function. For example, manganese superoxide dismutase (MnSOD; Refs. 18, 19, 31), neurofilament L (10, 26), the type II isoform of the sarco(endo)plasmic reticulum calcium ATPase (1), and prostacyclin synthase (32) have been reported to be inactivated by 3-nitrotyrosine formation in vivo.

MnSOD, which is the major antioxidant defense within mitochondria, is susceptible to rapid inactivation by relatively low concentrations of ONOO– (19). The overall importance of this enzyme is evident from studies in which mice genetically deficient in MnSOD suffer from a loss of mitochondrial iron-sulfur centers, a modification proving lethal to newborns (18). MnSOD in the kidney is reported to be tyrosine nitrated during aging (28) and transplant graft rejection (18). Therefore, tyrosine nitration of MnSOD may decrease its function and enhance the oxidative damage of cells in disease states.

Here we report that MnSOD from angiotensin II-infused rat kidney demonstrates both significant increase in tyrosine nitration and decrease in its enzymatic activity. We used immunohistochemistry, Western blot analysis, and HPLC-UV detection with immunoprecipitation techniques to demonstrate 3-nitrotyrosine in the protein. Recombinant MnSOD was tyrosine nitrated with peroxynitrite to track the recovery and sensitivity of detection of the modified native protein.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Surgical procedure. The study was approved by the Boston University Medical Center Institutional Animal Care and Use Committee. Male Wistar rats (Charles River Laboratories, Wilmington, MA) were acclimatized in a temperature- and light-controlled room and allowed access to standard rat chow and water. The rats were randomly divided into sham-treated and angiotensin II-infused groups. Osmotic minipumps (model 2001; Alza, Palo Alto, CA) were implanted under sterile conditions in the midscapular region of rats (250–275 g) anesthetized with inhaled isoflurane. In one group of rats, synthetic angiotensin II (Calbiochem, La Jolla, CA) dissolved in 0.15 mol/l NaCl and 1 mmol/l acetic acid was delivered continuously at a rate of 0.72 mg · kg–1 · day–1 for 7 days. The sham-treated group underwent an identical surgical procedure, except that an osmotic minipump containing 0.15 mmol/l NaCl and 1 mmol/l acetic acid was implanted. Systolic arterial pressures were measured by tail-cuff plethysmography at the end of the drug infusion. Angiotensin II significantly increased systolic blood pressure at day 6 compared with sham-infused animals [190 ± 2(n = 30) vs. 140 ± 3 mmHg (n = 32); P < 0.05].

Immunohistochemistry. The kidneys were cleaned of adherent fat and cut in half, placed in 4% formalin, processed, and embedded in paraffin. After removal of paraffin and rehydration, tissue sections were treated with 10 mmol/l citric acid (pH 6). Sections were microwave heated (2 min, 3 times at 700 W) to recover antigenicity. Nonspecific binding was blocked with 10% normal goat serum in PBS (pH 7.4) for 30 min before incubation with polyclonal anti-nitrotyrosine antibody (1 µg/ml; Upstate Biotechnology) in PBS with 1% BSA overnight at 4°C. Tissue sections were then incubated for 30 min at room temperature with a biotinylated anti-rabbit IgG (1:800) secondary antibody by using the Vectastain ABC kit (Vector). Vector Red alkaline phosphatase substrate (Vector) was used to visualize 3-nitrotyrosine. Specificity of anti-3-nitrotyrosine antibodies was confirmed by preincubation of the antibody with free 3-nitrotyrosine (10 mmol/l) or by using a nonimmune rabbit IgG (Vector) isotypic control.

Fluorescent secondary antibodies were imaged on sections of rat kidney with a two-photon scanning confocal microscope designed and constructed in house in collaboration with the laboratory of Dr. Peter So (Massachusetts Institute of Technology, Cambridge, MA). The light source was a diode laser-pumped titanium-sapphire laser (Coherent, Santa Clara, Ca). The excitation pulses were centered at a wavelength of 800 nm, and the power was reduced using polarizing optics. Fluorescence emission wavelengths were separated by a 565-nm dichroic mirror, and the emission filters were centered at 605 nm with a 55-nm bandwidth and at 535 nm with a 50-nm bandwidth for the long and short emitted wavelengths, respectively (Chroma Technology, Brattleboro, VT). Images were 512 x 512 pixels and were analyzed with NIH Image J and Adobe Photoshop.

Nitration of MnSOD in vitro. Reactions between ONOO– and recombinant human MnSOD (kindly provided by Dr. Lee Ann MacMillan-Crow, Department of Surgery, School of Medicine, University of Alabama at Birmingham, Birmingham, AL) were carried out at room temperature in 0.1 mol/l phosphate buffer at pH 7.4. ONOO– was added to MnSOD while vortex mixing at the final indicated concentrations. Working solutions of peroxynitrite were prepared by diluting stocks in 0.1 mol/l NaOH before use, and the concentration of peroxynitrite was determined by spectrophotometry ({epsilon} = 1,670 M–1cm–1, {lambda} = 302 nm in 0.1 mol/l NaOH). Five micromoles per liter was the minimal concentration of ONOO– to obtain detectable nitrated MnSOD as detected either by Western blot with monoclonal anti-nitrotyrosine antibody or by HPLC. Adding 12.6 µl of ONOO– to 10 µg of MnSOD in a 87.4-µl final volume (50 µmol/l final concentration of ONOO–) resulted in tyrosine nitration of approximately one-half of the MnSOD, as indicated by the nitrotyrosine content determined by HPLC. Thereafter, 50 µmol/l ONOO– was used to nitrate recombinant MnSOD.

Kidney homogenization. Animals were anesthetized with pentobarbital sodium, and both kidneys were collected and immediately frozen in liquid N2 and stored at –80°C until further processing. Frozen tissues were homogenized manually in a buffer containing 50 mmol/l Tris · HCl (pH 7.4), 150 mmol/l NaCl, 0.1 mmol/l diethylenetriamine pentaacetic acid (DTPA), 1 mmol/l NaF, 1% NP-40, and 0.25% deoxycholic acid, with the addition of 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 2 µg/ml aprotinin, and 10 mg/ml PMSF. Each sample was sonicated on ice with 10 pulses at 40% duty cycle and output 3 in a Branson sonifier (model 450) and then centrifuged at 16,000 g for 30 min at 4°C. The protein concentration of the supernatant was determined with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).

MnSOD activity. Horse heart cytochrome c (type III), xanthine, and xanthine oxidase were purchased from Sigma (St. Louis, MO). After thawing, rat kidney samples were washed with 0.9% NaCl solution. The tissue was then homogenized in 2 ml of 50 mmol/l Tris · HCl buffer containing 0.1 mmol/l EDTA at pH 7.0. After centrifugation at 15,000 g for 30 min, the supernatant was removed and total protein concentration was measured. SOD activity was measured by inhibition of the reduction of cytochrome c by xanthine and xanthine oxidase as described previously (20). Ten microliters of the supernatant was used in the assay mixture consisting of 50 mmol/l Tris · HCl, 0.1 mmol/l EDTA, 50 µmol/l xanthine, 10 µmol/l cytochrome c, and enough xanthine oxidase (~6 nmol/l) to cause {Delta}A550 = 0.025 min–1, at pH 7.0, in a total reaction volume of 300 µl. Under these conditions, one unit of SOD activity is defined as the amount that causes 50% inhibition of the initial rate of reduction of cytochrome c. MnSOD activity was measured by inhibiting extracellular and cytosolic Cu/Zn SOD activity with KCN (1 mmol/l). In a separate series of angiotensin II- or sham-infused rat kidneys we found no substantial difference in the results reported here when we made measurements at pH 7.8 and with 5 mmol/l NaCN, as suggested by Okado-Matsumoto and Fridovich (22) to slow the reaction of cyanide with cytochrome c.

Western blots. Kidney protein samples, 50 µg/lane, were electrophoretically size fractionated on 15% SDS-polyacrylamide gels at 120 V for 90–120 min and transferred to Immun-Blot cellulose membranes (Bio-Rad) at 200 mA for 90 min. Membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk for 1 h. For detection of MnSOD, membranes were incubated with the primary antibody (polyclonal rabbit anti-MnSOD Ig-G, dilution 1:2,000; Upstate Biotechnology) at 4°C overnight. Nitrotyrosine in MnSOD was detected by incubating membranes at 4°C overnight with a mouse monoclonal IgG anti-nitrotyrosine antibody (dilution 1:1,000; Upstate Biotechnology). The membranes were washed for 30 min, with the wash buffer changed every 10 min before incubation with the Fc fragment-specific, horseradish peroxidase-labeled goat anti-rabbit IgG (1:10,000; Pierce, Rockford, IL) in Tris-buffered saline with 5% milk for 45 min at room temperature for MnSOD detection, using Fc fragment-specific, horseradish peroxidase-labeled goat anti-mouse IgG (1:10,000, Pierce, Rockford, IL) for nitrotyrosine in MnSOD detection. The washes were again repeated for 30 min before the membrane was scanned for bound antibody by chemiluminescence with enhanced chemiluminescence detection reagents (Super Signal Family; Pierce). The staining of blots by anti-nitrotyrosine antibody was blocked by preincubation of the antibody with 10 mmol/l nitrotyrosine (data not shown). The films were scanned with a densitometer to determine the integrated density values. Data were normalized to the density of the average control value.

Immunoprecipitation of MnSOD and nitrotyrosine. Solubilized protein (1 mg) was diluted in 1 ml of buffer containing (in mmol/l) 150 NaCl, 50 Tris · HCl, and 5 EDTA with 0.5% NP-40 and precleared with 50 µl of protein G Sepharose (Amersham Pharmacia) for 45 min at 4°C, and the supernatant was incubated with 10 µg of monoclonal anti-nitrotyrosine antibody (Upstate Biotechnology) or 10 µg of polyclonal anti-MnSOD antibody (StressGen Biotechnologies) with mixing by end-over-end inversion overnight at 4°C. Immune complexes were precipitated with 50 µl of protein G Sepharose for 2 h at 4°C, washed in SNNTE buffer [0.5 mol/l NaCl, 50 mmol/l Tris · HCl (pH 7.4), 5 mmol/l EDTA, 5% sucrose, and 4% NP-40] three times, resuspended in Laemmli sample buffer (Bio-Rad) supplemented with 0.1 mmol/l DTPA and 5% {beta}-mercaptoethanol, and heated at 95°C for 5 min. After centrifugation at 16,000 g for 1 min, samples were immediately loaded and fractionated by reducing SDS-PAGE in 15% gels. Repeat addition of either anti-nitrotyrosine or anti-MnSOD antibody and protein G Sepharose showed no further immunoprecipitated protein, indicating that each procedure was quantitative.

HPLC analysis. Whole homogenate protein (1 mg) or immunoprecipitates obtained with anti-MnSOD antibody were hydrolyzed for 24 h at 110°C under vacuum in 6 N HCl. After drying by evaporation under vacuum, hydrolyzed samples were suspended in 120 µl of buffer A (50 mmol/l sodium acetate, pH 4.7) and centrifuged at 12,000 g for 10 min to pellet any particulates. Clarified samples (100 µl) were injected onto an octadecyl silica gel reverse-phase column and eluted at 1 ml/min with 90% buffer A-10% methanol. Peaks were monitored with a photodiode array detector at 365 nm.

Data analysis. Statistical comparisons were made by one- or two-way ANOVA and a post hoc Student's t-test. The null hypothesis was rejected when P was <0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Immunohistochemistry. Immunohistochemical staining performed with a polyclonal anti-nitrotyrosine antibody was far more intense in cortex, outer medulla, and papilla from kidneys of angiotensin II-infused rats compared with that in kidneys from sham-operated animals (Fig. 1; A–D). Nitrotyrosine was localized predominantly to collecting ducts of cortex (Fig. 1, A and B), outer medulla (Fig. 1E) and tubules within the inner medulla (papilla) of angiotensin II-infused rats (Fig. 1, B and C). When cortex of kidney from angiotensin II-treated rats was immunostained for nitrotyrosine and MnSOD and examined by two-photon microscopy, substantial colocalization of MnSOD and nitrotyrosine was evident within cells of the collecting ducts (Fig. 1F).



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  		Fig. 1. Immunohistochemical staining with a polyclonal antibody for 3-nitrotyrosine in kidney of sham-treated and angiotensin (ANG) II-infused rats. A and B: cortex from control and angiotensin II-infused rats, respectively (x10 magnification). Intense nitrotyrosine staining is present in kidneys from angiotensin II-infused rats, whereas staining is far less prominent in control kidneys. Nitrotyrosine is localized predominantly to cortical collecting ducts. C and D: papilla from control and angiotensin II-infused rats, respectively (x10 magnification). Although there is some nitrotyrosine staining of tubules within the papilla of kidneys obtained from control animals, the staining is far more intense in papilla obtained from angiotensin II-infused rats. E: outer medulla of kidneys from angiotensin II-infused rat (magnification x40). Prominent staining of collecting ducts is evident in the outer medulla. Also, collecting ducts appear somewhat dilated. F: cortex of kidney from angiotensin II-treated rats was stained with fluorescent secondary antibodies against primary antibodies directed toward nitrotyrosine (green) or manganese superoxide dismutase (MnSOD; red) and examined by 2-photon microscopy. Substantial colocalization of staining (yellow) for MnSOD and nitrotyrosine was present in the cells of the collecting ducts.

 

MnSOD expression and activity in angiotensin II-infused rat kidney. MnSOD protein expression was not significantly different between sham-treated and angiotensin II-infused groups (Fig. 2, A and B). The activity of total SOD in kidney was also unchanged between these two groups [901 ± 69 U/mg (n = 4) in sham-treated group vs. 833 ± 43 U/mg (n = 4) in angiotensin-II infused group; Fig. 2C]. However, MnSOD activity decreased significantly by ~50% in the angiotensin II-infused group compared with the sham-treated group (24 ± 4.6 vs. 11 ± 5.1 U/mg, P < 0.01; Fig. 2C).



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  		Fig. 2. MnSOD expression and activity in angiotensin II infused rat kidney. A: Western blots of samples of sham-treated (n = 6) and angiotensin II-infused (n = 6) rat kidney homogenates, stained with polyclonal anti-MnSOD antibody. There was no change in MnSOD expression between sham-treated and angiotensin II-infused rat samples. B: summary of MnSOD expression as determined by densitometry of the blot shown in A. C: total SOD and MnSOD activity were measured with the cytochrome c method by spectrophotometry and expressed as units per milligram of protein. Angiotensin II had no effect on total SOD activity but significantly decreased MnSOD activity in rat kidney. **P < 0.01 vs. sham treated. S1–S4, sham-treated rats 1–4; A1–A4, angiotensin II-treated rats 1–4.

 

Detection of 3-nitrotyrosine containing recombinant MnSOD added to homogenates of rat kidney with monoclonal anti-nitrotyrosine antibody and HPLC. We first determined the sensitivity of Western blotting and HPLC to detect tyrosine-nitrated recombinant MnSOD added to kidney homogenate. When increasing amounts of tyrosine-nitrated MnSOD were added to 50 µg of normal rat kidney homogenate and then subjected to SDS-PAGE and Western blotting, the detection limit with monoclonal anti-nitrotyrosine antibody was 0.3 µg of nitrated MnSOD (containing 2.8 ng of nitrotyrosine, data not shown). The detection limit by HPLC was reached by adding 0.5 µg tyrosine-nitrated MnSOD (containing 4.7 ng nitrotyrosine) to 1 mg of kidney homogenate (data not shown).

Sensitivity of detection of tyrosine-nitrated MnSOD by immunoprecipitation from rat kidney homogenate. Different amounts of tyrosine-nitrated MnSOD were added to normal rat kidney homogenate. MnSOD was then immunoprecipitated from the mixture with polyclonal anti-MnSOD antibody. The immunoprecipitated proteins were separated by SDS-PAGE and subsequently blotted and stained with monoclonal anti-nitrotyrosine antibody. The threshold for detection of tyrosine-nitrated MnSOD was reached by adding 0.1 µg of tyrosine-nitrated MnSOD (containing 0.9 ng of nitrotyrosine) per milligram of tissue homogenate (Fig. 3A). Thus the immunoprecipitation improved the detection limit threefold over directly immunostaining the blot of homogenate proteins with anti-nitrotyrosine antibody.



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  		Fig. 3. Sensitivity of detecting 3-nitrotyrosine in MnSOD in kidney homogenate. A: different amounts of tyrosine-nitrated recombinant MnSOD were added to 1 mg of protein homogenate of normal rat kidney. After immunoprecipitation (IP) with polyclonal anti-MnSOD antibody (p-MnSOD), the protein was separated by SDS-PAGE and Western blotted (WB) for nitrotyrosine with monoclonal antibody (m-NY). The detection limit was 0.1 µg of tyrosine-nitrated MnSOD (NY-MnSOD). Note the detection of the rat IgG light chain with the anti-mouse secondary antibody at a slightly higher molecular weight than MnSOD. B: different amounts of tyrosine-nitrated MnSOD were added to 1 mg of protein homogenate of normal rat kidney. After immunoprecipitation with monoclonal anti-nitrotyrosine antibody, the protein was blotted for MnSOD with polyclonal antibody. The detection limit was 0.6 µg of tyrosine-nitrated MnSOD.

 

Kidney homogenates prepared in the same way and subjected to immunoprecipitation with monoclonal anti-nitrotyrosine antibody followed by blotting with polyclonal anti-MnSOD antibody revealed that the detection threshold by this method was 0.6 µg of nitrated MnSOD per milligram of homogenate (containing 5.65 ng nitrotyrosine; Fig. 3B). Thus immunoprecipitation with MnSOD antibody and staining with anti-nitrotyrosine antibody offered a sixfold higher sensitivity over the immunoprecipitation with nitrotyrosine antibody and staining with anti-MnSOD antibody.

MnSOD is tyrosine-nitrated in angiotensin II-infused rat kidney. MnSOD immunoprecipitated from both sham-treated and angiotensin II-infused rat kidney stained positively with the monoclonal anti-nitrotyrosine antibody, although the staining was consistently seen only in the angiotensin II-infused rats (Fig. 4A). The nitrotyrosine immunoreactivity of MnSOD immunoprecipitates was increased in angiotensin II-infused rat kidneys, indicating a 100-fold increase in nitrotyrosine immunoreactivity of the protein [0.23 ± 0.23 U (n = 3) in sham-treated vs. 18 ± 3.2 U (n = 4) in angiotensin II-infused rat kidneys, P < 0.01; Fig. 4A]. When 0.15 µg of tyrosine-nitrated MnSOD (containing 1.5 ng nitrotyrosine) was added to both sham-treated and angiotensin II-infused rat kidney homogenate, the nitrotyrosine staining of the MnSOD immunoprecipitate was increased (Fig. 4A).



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  		Fig. 4. 3-Nitrotyrosine in MnSOD from angiotensin II-infused rat kidney. A: proteins were immunoprecipitated with polyclonal anti-MnSOD antibody from 1 mg of protein homogenate of both sham-treated (S) and angiotensin II-infused (A) rat kidneys and blotted for nitrotyrosine with monoclonal antibody. Staining was observed in 2 of the 3 S animals, in all 6 of the A animals, and in all samples to which tyrosine-nitrated MnSOD (0.15 µg containing 1.5 ng nitrotyrosine) was added to enhance the signal (+NY). Note that the rat IgG was not detected as it was in Fig. 3A, because an anti-mouse Fc fragment-specific secondary antibody was used. No staining was observed if antibody (no anti) or homogenate (no homo) was omitted from the immunoprecipitation. B: proteins were first immunoprecipitated with polyclonal anti-MnSOD antibody from 1 mg of protein homogenates of both sham-treated (n = 4) and angiotensin II-infused (n = 6) rat kidneys, and the immunoprecipitate was then hydrolyzed in 6 N HCl, and nitrotyrosine was detected with HPLC-UV (left). Hatched bars indicate the values obtained in samples to which tyrosine-nitrated MnSOD (0.15 µg containing 1.5 ng nitrotyrosine) was added to enhance the signal. Total protein homogenates (1 mg) of both sham-treated (n = 8) and angiotensin II-infused (n = 10) rat kidneys were hydrolyzed by 6 N HCl, and then total homogenate 3-nitrotyrosine was detected by HPLC-UV (right). Note the different ordinate values for right and left sides of B. *P < 0.01 vs. sham-treated group. **P < 0.01 vs. with nitrotyrosine added.

 

To confirm the increase in nitrotyrosine in MnSOD caused by angiotensin II infusion, the immunoprecipitated protein from 1 mg of sham-treated or angiotensin II-infused rat kidney homogenate, with or without the addition of recombinant tyrosine-nitrated MnSOD containing 1.5 ng of nitrotyrosine, was hydrolyzed and nitrotyrosine was measured by HPLC. In sham-treated rat kidney samples, there was 0.15 ± 0.08 ng nitrotyrosine/mg tissue protein. The 3-nitrotyrosine content in MnSOD immunoprecipitated from angiotensin II-infused rat kidneys was increased 13-fold to 2 ± 0.4 ng nitrotyrosine/mg tissue protein (P < 0.01). The recovery of the added MnSOD containing 1.5 ng of nitrotyrosine was nearly quantitative. The amount of 3-nitrotyrosine in the total homogenate from angiotensin II-infused rat kidneys was approximately three times more than that of sham-treated rats (Fig. 4B). Thus 3-nitrotyrosine-containing MnSOD increased from one-twentieth to one-fifth of the total tissue 3-nitrotyrosine content after angiotensin II infusion.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
Protein tyrosine nitration in the kidney has been detected in a number of pathological conditions, such as human renal allograft rejection (18), experimental glomerulonephritis (11), diabetic nephropathy (23, 27), endotoxin-induced kidney injury (7), transgenic sickle cell mice (4), IL-1{beta}-treated mesangial cells in culture (32), and the Goldblatt rat, an angiotensin II-dependent model of hypertension (8). However, MnSOD (19) and prostacyclin synthase (32) are the only specific proteins previously reported to be tyrosine nitrated in renal cells. Tyrosine nitration at the active site of MnSOD is associated with inactivation of enzymatic activity (19, 31).

In the present study, tyrosine nitration of MnSOD in angiotensin II-infused rat kidney was accompanied by a decrease in superoxide anion scavenging activity of the enzyme of ~50% without a change in expression of the protein. This suggests that oxidative posttranslational modification of the enzyme can explain the reduced function. Indeed, we found 2 ng nitrotyrosine/mg tissue protein in MnSOD immunoprecipitated from angiotensin II-infused rat kidney. This represents ~9 pmol of nitrotyrosine in the MnSOD immunoprecipitated per milligram of tissue protein. The control MnSOD activity that we measured was 24 U/mg tissue protein. With a specific activity of MnSOD of 4,000 U/mg, the control activity measured corresponds to 0.6% tissue protein, or ~25 pmol/mg protein, assuming that the average molecular mass of tissue proteins equals that of MnSOD (24 kDa). Thus an estimated 9/25th or 36% of MnSOD was found to be tyrosine nitrated in angiotensin II-infused rat kidney. This is near the measured 50% reduction in activity and consistent with stoichiometric inactivation of MnSOD by nitration of its active site tyrosine.

There are several methods to detect 3-nitrotyrosine in tissues including immunohistochemistry, Western blot, HPLC-UV, electrochemical detection, and GC-MS analysis. Immunodetection is sensitive and conventional; however, it is not quantitative and requires the careful consideration of the specificity of the antibodies used. HPLC-UV detection is quantitative; however, artifacts have also been reported with this method (13). There are many reports of tyrosine nitration that use only one method, at the risk of one or more of these shortcomings. Therefore, we used both immunochemical and HPLC methods to detect tyrosine nitration on a specific protein in an animal model of disease. This study benefited from the fact that we had specific antibodies to MnSOD, enabling immunoprecipitation of the protein and increasing sensitivity of its detection. We also were able to tyrosine nitrate the recombinant MnSOD to test sensitivity and specificity of the methods used. Our data indicate that Western blot of whole kidney protein with anti-nitrotyrosine antibody has nearly twice the sensitivity of HPLC-UV when detecting tyrosine-nitrated recombinant MnSOD added to kidney homogenate (2.8 compared with 4.7 ng nitrotyrosine/mg protein). However, if the goal is to detect low amounts of nitrotyrosine on unknown proteins in tissue homogenates, Western blot of tissue homogenates with anti-nitrotyrosine antibody is not as sensitive as HPLC. In the case of whole tissue homogenates of angiotensin II-infused rat kidney, we could not detect any protein bands by Western blot with either polyclonal or monoclonal anti-nitrotyrosine antibodies. In contrast, with HPLC of hydrolyzed MnSOD immunoprecipitate, we detected 0.15 ng nitrotyrosine/mg tissue in sham-treated rat kidney homogenates, which was increased 13-fold by angiotensin II infusion. Finally, we estimate that ~5% of nitrotyrosine in sham-treated rat kidneys is accounted for by tyrosine-nitrated MnSOD, which increased to ~20% of the total nitrotyrosine content in angiotensin II-infused rat kidney. The reason for the greater relative susceptibility of MnSOD to nitration is not known but may be due to its ability to catalyze tyrosine nitration of itself by peroxynitrite (12).

Immunoprecipitation with an anti-nitrotyrosine antibody is the preferred technique for detecting unknown tyrosine-nitrated proteins in tissues. This technique has been used to identify tyrosine-nitrated proteins including MnSOD in lung and liver of endotoxemic rats (3). However, immunoprecipitation with anti-nitrotyrosine antibody followed by Western blotting for nitrotyrosine failed to show any detectable proteins in our samples, except for the IgG present in the immunoprecipitate (data not shown). Comparing "nonspecific" immunoprecipitation with anti-nitrotyrosine antibody followed by staining with anti-MnSOD antibody with "specific" immunoprecipitation of MnSOD followed by staining for nitrotyrosine, we found the latter to be sixfold more sensitive.

Nitrotyrosine, as detected by immunohistochemistry, was more intense in kidneys of angiotensin II-treated rats compared with sham-treated rats. These findings are consistent with the quantitative data obtained from kidney homogenate. In addition, we were able to use two-photon microscopy to substantiate the colocalization of 3-nitrotyrosine and MnSOD in renal tubular cells. Interestingly, nitrotyrosine staining was most intense in distal tubules (predominantly collecting ducts) within the cortex and outer medulla and in the tubules within the renal papilla. These cells are reported to have increased expression of MnSOD and, presumably for that reason, to resist damage during ischemia-reperfusion (14). In addition, distal tubular cells have recently been reported to be those in which NADPH oxidase is induced in response to angiotensin II (9), and in preliminary studies (S. Xu and B. Jiang, unpublished observations) we have confirmed the presence in these cells of p47phox. It is possible that oxidants generated by this enzyme are the cause of tyrosine nitration of MnSOD, although this conclusion awaits further studies.

The study of the role of NO and oxidants in vivo is hindered by technical difficulties inherent in the noninvasive measurement and short half-life of NO, superoxide anion, peroxynitrite, and related species. In an effort to circumvent this difficulty, biomarkers that specifically indicate tissue nitration have been sought. 3-Nitrotyrosine was the first candidate for such a biomarker (5), and early attempts to detect nitrotyrosine used antibodies to provide some qualitative indication of protein nitration (29). Unfortunately, quantitative immunochemical analysis is subject to variability in both antibody specificity and affinity. We therefore used different methods to quantify nitrotyrosine in vivo, and the sensitivity of each method was compared. This approach suggests appropriate methods for identifying and quantifying novel proteins that are tyrosine nitrated in tissues.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
These studies were supported by National Institutes of Health (NIH) Grants R01-HL-55620-07 and R01-HL-31607-20 (R. A. Cohen) and by the NIH Boston University Cardiovascular Proteomics Center (N01-HV-28178; T. Adachi, M. Kirber, and R. A. Cohen).


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Cohen, Vascular Biology Unit, Boston Medical Center X708, 650 Albany St., Boston, MA 02118 (Email: racohen{at}bu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 

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