Measurement of serum creatinine.

Kidney function was analyzed by measurement of serum creatinine from sham-operated and experimental animals with a commercially available kit according to manufacturer's directions (Teco Diagnostics, Anaheim, CA). This method is based on Jaffe's reaction, which relies on the reaction of creatinine and picric acid to form a red-orange complex that can be measured biochromatically at 505 nm/573 nm.

Histological scoring for acute tubular necrosis.

Sham-operated and experimental kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4-μm sections, and then stained with either hematoxylin and eosin (H & E) or periodic acid-Schiff reagent (PAS). Indicators of acute tubular necrosis included damaged proximal tubular epithelium, cytoplasmic and basement membrane disruption, and nuclear infiltration in the PAS-stained sections.

We used eosin autofluorescence in necrotic tissue (49) to quantify the severity of tubular injury in the H & E-stained sections. Highly fluorescent cast and necrotic regions were identified with an Olympus BH-2 epifluorescent microscope (Olympus, Center Valley, PA) equipped with a 540-nm excitation filter and a 590-nm emission filter. For each specimen, five randomly chosen outer medullary fields were photographed at ×100 magnification with a Leica DFC 290 digital camera (Leica Microsystems, Bannockburn, IL). After appropriate background thresholding, the fluorescent area containing necrotic tubular epithelium or cast material was quantified with Image-Pro Plus image analysis software (version 6.2, Media Cybernetics, Bethesda, MD). The extent of tubular injury [acute tubular necrosis (ATN) score] for each section was expressed as the mean area occupied by the fluorescent necrotic tubular epithelium or cast material.

Real-time quantitative PCR.

Total RNA was purified from ~100 mg of frozen tissue with the Promega SV RNA total RNA isolation kit (Promega, Madison, WI). Real-time quantitative PCRs were carried out in the Stratagene Mx QPCR system (Stratagene, Cedar Creek, TX) with SYBR Green qPCR SuperMix (Invitrogen). The primer sequences are shown in Table 1. The PCR conditions were as follows: 95°C for 30 s, 60°C for 60 s, 72°C for 30 s for 40 cycles. GADPH mRNA served as the internal control, and the ΔΔC_T method (where C_T is threshold cycle) (30) was used for calculating the relative amount of transcripts.

Western blots assay.

Sham-operated and experimental frozen tissues were homogenized with RIPA buffer and protease inhibitors. Fifty micrograms of the protein was electrophoresed on 7.5%, 10%, 15%, or 4–20% Criterion gels (depending on protein size) and transferred to nitrocellulose membrane. After blocking for 1 h, the blots were incubated with one of the following primary antibodies: cleaved caspase-3 Asp175 (Cell Signaling Technology, Danvers, MA; l:1,000), Bax (Santa Cruz Biotechnology, Santa Cruz, CA; 1:200), Bcl-2 (Santa Cruz Biotechnology; 1:1,000), Fas-L (Santa Cruz Biotechnology; 1:200), xanthine oxidase (Santa Cruz Biotechnology; 1:1,000), GADPH (Chemicon, Temecula, CA; 1:2,000); 4-hydroxy-2-nonenal (HNE) (Calbiochem, San Diego, CA; 1:1,000), nitrotyrosine (Upstate Biotechnology, Lake Placid, NY; 1:1,000), and gp^91phox (Santa Cruz Biotechnology; 1:1,000) overnight at 4°C. After washing, the membranes were probed with secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA) or LI-COR fluorescent (LI-COR Bioscience, Lincoln, NE) antibodies at 1:10,000 for 1 h at room temperature. The blots were either visualized with ECL-Plus or scanned with the LI-COR Odyssey Scanner (LI-COR Bioscience).

SOD activity assay.

The amount of SOD activity from kidney homogenates was measured with a commercially available SOD assay kit (Cayman Chemical, Ann Arbor, MI). This kit utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. To measure Mn SOD activity, 2 mM potassium cyanide was used to inhibit both Cu/Zn SOD and extracellular SOD. Absorbance was read at 450 nm according to manufacturer's instructions and expressed as units per milligram of protein.

Slot blot for nitrotyrosine.

Slot blot immunoassay was used for nitrotyrosine content as described previously (37). Briefly, 3 μg of protein from kidney homogenates was applied to a Bio-Dot apparatus (Bio-Rad) in duplicate on nitrocellulose membranes. The membrane was incubated with 0.8 μg/ml monoclonal anti-nitrotyrosine antibody (Upstate Biotechnology) overnight at 4°C and visualized with enhanced chemiluminescence. Serial dilutions of nitrated BSA standard were used to quantify nitrotyrosine.

Immunohistochemistry for nitrotyrosine.

Immunohistochemistry for nitrotyrosine was performed in paraffin-embedded 4-μm-thick kidney sections. To quench for endogenous background, sections were incubated in 3% H₂O₂ for 5 min and washed twice in phosphate-buffered saline (PBS, pH 7.4). Nonspecific binding of secondary antibody was blocked with 1% BSA for 1 h before incubation with primary antibody. Sections were incubated with 1:50 dilution of monoclonal anti-nitrotyrosine antibody (Upstate Biotechnology) overnight at 4°C. Sections were then incubated with horseradish peroxidase-conjugated secondary IgG (Upstate Biotechnology) and counterstained with methyl green. Negative controls included secondary antibody incubation alone without primary antibody (data not shown).

Lucigenin-enhanced chemiluminescence.

Lucigenin-enhanced chemiluminescence was measured as previously described (24, 38). Briefly, 10 μM lucigenin was added to 0.5 ml of protein homogenate (100 mg/ml) in 50 mM Krebs-HEPES buffer, pH 7.4. Lucigenin-enhanced chemiluminescence was measured for 3 min in a luminometer (Autolomat LB 953, Berthold Instruments, Bad Wilblad, Germany). Krebs-HEPES buffer was read to calculate background luminescence and subtracted from each value. Reactive oxygen production was normalized to protein levels and reported as relative light units (RLU) emitted per minute per milligram protein.

Determination of superoxide formation with dihydroethidium staining.

Kidneys from sham-operated, I/R, and I/R Mn animals were frozen-fixed in liquid nitrogen with optimum cutting temperature (OCT) compound and cut into 12-μm cryosections. Sections were mounted on slides, washed with Dulbecco's PBS (DPBS), and then incubated in 10 μM hydroethidine (HE) in DPBS at 37°C in the dark for 15 min, being washed with DPBS twice. Images were immediately scanned for superoxide with an Olympus IX50 inverted fluorescence microscope equipped with rhodamine filter settings.

Qualitative analysis of superoxide formation.

Dihydroethidium (DHE) intensity was measured with a fluorescence spectrometer. Briefly, 10 μM DHE in 1 ml of prewarmed (37°C) DPBS was added to a 24-well plate containing 20 μl of protein lysate from sham-operated, I/R Veh, and I/R Mn kidney tissues. The plate was immediately read every 2 min for a total of 60 min at excitation/emission filters of 530 nm/620 nm. A blank well was read to calculate background, which was subtracted from each value; DHE alone served as a relative control. DHE values were expressed as intensity per milligram of protein.

Tumor necrosis factor-α ELISA.

Quantitative determination of rat tumor necrosis factor (TNF)-α was performed in serum of all experimental groups with a commercially available TNF-α ELISA kit (R&D Systems, Minneapolis, MN). TNF-α concentration was expressed in picograms per milligram of protein.

TUNEL staining for apoptosis.

Apoptosis was measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay with ApopTag technology (Chemicon International) according to manufacturer's instructions. A minimum of five fields per slide and four slides per group were counted by this method.

Caspase-3 activity.

Caspase-3 activities in tissue homogenates were determined with commercially available colorimetric kits (Chemicon International). Caspase-3 activity was expressed as micromolar per minute per milligram of protein.

Effect of MnTMPyP on expression of O₂^•−-producing enzymes in renal I/R injury.

Xanthine oxidase and gp^91phox, a subunit of NADPH oxidase, are both potentially major sources of O₂^•− radicals in I/R injury. To determine whether the expression of these genes was increased in renal I/R, Western blot analysis was performed on gp^91phox and xanthine oxidase in sham-operated and I/R-, I/R Veh-, and MnTMPyP-treated samples. Although there was only a slight increase in xanthine oxidase protein levels in I/R- or vehicle-treated animals compared with sham-operated animals as shown qualitatively in Fig. 3A and quantitatively in Fig. 3B, the expression of gp^91phox was significantly increased after renal I/R injury (Fig. 3, A and B; P < 0.01, I/R vs. sham operated). MnTMPyP treatment significantly decreased gp^91phox expression (Fig. 3, A and B; P < 0.05, MnTMPyP vs. I/R) but did not affect xanthine oxidase protein levels (Fig. 3, A and B).

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Fig. 3.

A: representative Western blots of xanthine oxidase and gp^91phox in Sham, IR, IR Veh, and IR Mn groups after renal I/R. B: densitometry of xanthine oxidase and gp^91phox as normalized to GAPDH. Results are means ± SE (n = 3/group). ^¶P < 0.01, IR vs. Sham; *P < 0.05, IR vs. IR Mn. C: changes of Cu/Zn SOD and Mn SOD mRNA level measured by real-time quantitative PCR. *P < 0.05, Sham vs. other groups. D: Western blot for Mn SOD (top) and Cu/Zn SOD (bottom) in Sham, Sham Mn, IR, IR Veh, and IR Mn rats. E: SOD activity in Sham, Sham Mn, IR, IR Veh, and IR Mn rats. Results are means ± SE (n = 3–8/group).

Effect of MnTMPyP on antioxidant protein gene expression after I/R injury.

To determine whether the reported decrease in antioxidant proteins in I/R injury (15, 38) was observed in our model and whether MnTMPyP was able to prevent damage to the kidney by upregulating these genes, we performed real-time quantitative PCR for mRNA levels of Mn SOD and Cu/Zn SOD in sham-operated and I/R-, vehicle-, and MnTMPyP-treated samples. There was a significant decrease in both Mn SOD and Cu/Zn SOD mRNA levels (Fig. 3C; P < 0.05, sham operated vs. I/R and I/R Veh) after the I/R injury. However, MnTMPyP treatment did not upregulate the expression of either of these genes significantly (Fig. 3C; results not significantly different from I/R or I/R Veh), although there was a tendency for Mn SOD RNA levels to be a little higher with MnTMPyP treatment compared with I/R or I/R Veh groups (Fig. 3C). We further examined whether MnTMPyP had any effect on SOD protein levels and SOD activity following I/R injury. As shown in Fig. 3D, there was not much change in Mn SOD protein levels after I/R injury. Cu/Zn SOD protein levels were slightly lower in all I/R groups compared with sham-operated and sham-operated MnTMPyP-treated groups (Fig. 3D). Interestingly, there was a tendency for total SOD activity to be decreased in both I/R- and I/R Veh-treated groups compared with sham-operated and sham-operated MnTMPyP-treated groups (Fig. 3E). MnTMPyP treatment increased SOD activity following I/R but did not reach statistical significance (Fig. 3E). We also evaluated Mn SOD and Cu/Zn SOD activities in sham-operated, I/R Veh, and I/R Mn groups. Mn SOD activity was lower than Cu/Zn SOD activity in sham-operated animals (Mn SOD 4.77 ± 1.34, Cu/Zn SOD 27.69 ± 4.14 U/mg protein). Both Mn SOD and Cu/Zn SOD activity were decreased slightly in the I/R Veh-treated group, although these were not statistically significant from sham-operated groups (Mn SOD 4.09 ± 0.42, Cu/Zn SOD 17.01 ± 2.33 U/mg protein). Treatment with MnTMPyP tended to increase both Mn SOD and Cu/Zn SOD (Mn SOD 4.628 ± 0.23, Cu/Zn SOD 23.35 ± 2.21 U/mg protein; P = not significant, MnTMPyP vs. I/R Veh group).

MnTMPyP attenuates I/R-mediated increase in ROS.

To ensure that MnTMPyP was indeed decreasing ROS, we measured O₂^•− levels by lucigenin-enhanced chemiluminescence and DHE fluorescence. As shown in Fig. 4 A (DHE) and B (lucigenin), there was an expected increase in O₂^•− levels after renal I/R injury. MnTMPyP treatment prevented the increase in ROS by both methods of detection (Fig. 4).

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Fig. 4.

A: top: dihydroethidium (DHE) fluorescent image showing increased superoxide levels after I/R compared with Sham and MnTMPyP-treated animals. Bottom: superoxide levels in Sham and IR and IR Mn animals as measured by a quantitative DHE fluorescence plate assay. Results are means ± SE (n = 3/group). *P < 0.05, IR vs. all other groups. B: superoxide levels as measured by lucigenin-enhanced chemiluminescence in Sham, IR, and IR Mn groups. Results are means ± SE (n = 4–9/group). ^¶P < 0.01, IR vs. Sham and IR Mn. RLU, relative light units.

MnTMPyP partially attenuates nitrotyrosine formation following I/R injury.

Nitrotyrosine is a marker for peroxynitrite damage to proteins and is an indicator of overall nitrative stress. Total protein nitration was evaluated by slot blot analysis in kidney homogenates from sham-operated and experimental I/R injury groups. There was a trend for higher levels of nitrotyrosine in I/R- and I/R Veh-treated animals compared with sham-operated control animals, although this did not reach statistical significance (P = 0.0567, I/R and I/R Veh vs. sham operated) (Fig. 5A). However, when we performed immunohistochemistry for nitrotyrosine, we were able to detect a substantial increase in nitrotyrosine formation in both I/R- and I/R Veh-treated animals compared with sham-operated control animals (Fig. 5B). Some nitrotyrosine was detected in the MnTMPyP-treated animals, but the overall amount was much lower than that of the I/R and I/R-Veh groups (Fig. 5B).

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Fig. 5.

A: nitrotyrosine slot blot analysis in Sham, IR, IR Veh, and IR Mn groups. Results are means ± SE (n = 4–9/group). Results not significantly different from each other. B: nitrotyrosine immunostaining (brown stain) in Sham, IR, IR Veh, and IR Mn groups. C: top: Western blot showing effect of MnTMPyP on 4-hydroxy-2-nonenal (HNE) levels after renal I/R. Bottom: densitometry analysis of Western blot of HNE. Results are means ± SE (n = 3–4/group). ^¶P < 0.01, IR and IR Veh vs. Sham and IR Mn.

MnTMPyP decreases lipid peroxidation following renal I/R injury.

Oxidation of ω-6 unsaturated fatty acids by superoxide releases HNE, a highly reactive and cytotoxic aldehyde. HNE-protein adducts were measured to determine the extent of lipid peroxidation in kidney homogenates from sham-operated and I/R-, vehicle-, and MnTMPyP-treated rats. As shown in Fig. 5C, here was an increase in HNE adducts in both I/R- and I/R Veh-treated animals compared with sham-operated control animals. Treatment with MnTMPyP decreased HNE adduct formation after I/R, indicating that this compound was effective in preventing I/R-induced lipid peroxidation (Fig. 5C).

MnTMPyP treatment reduces TNF-α protein levels after I/R injury.

We next wanted to determine whether proinflammatory cytokines such as TNF-α were increased in our model and whether MnTMPyP treatment attenuated the increase in TNF-α. Serum TNF-α protein levels were significantly increased after I/R (P < 0.05, I/R and I/R Veh vs. sham operated). Treatment with MnTMPyP significantly attenuated the increase in I/R-induced increase in TNF-α (P < 0.05, MnTMPyP vs. I/R and I/R Veh) (Fig. 6).

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Fig. 6.

Changes of tumor necrosis factor (TNF)-α protein after renal I/R measured by ELISA. *P < 0.05, IR and IR Veh vs. Sham and IR Mn.

MnTMPyP protects against I/R-induced apoptosis.

To determine whether the decreased histological damage and oxidative stress in MnTMPyP-treated rats were related to decreased apoptosis after I/R injury, we measured apoptosis by TUNEL assay in kidney tissue slices from all four groups. Figure 7A, top, is a representative image of TUNEL staining in sham-operated and I/R-, I/R Veh-, and MnTMPyP-treated rats, and Fig. 7A, bottom, shows the number of apoptotic nuclei per field in each group. There was increased apoptosis in I/R (Fig. 7A; P = 0.054, I/R vs. sham operated) and I/R Veh (Fig. 7A; P = 0.027, I/R Veh vs. sham operated) groups. Treatment with MnTMPyP partially attenuated the increase in apoptotic nuclei (Fig. 7A).

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Fig. 7.

A: top: TUNEL staining depicting apoptotic nuclei (indicated by arrows) in Sham, IR, IR Veh, and IR Mn kidney. Bottom: apoptotic nuclei/high-power field in each group. Results are means ± SE (n = 4–9/group). *P < 0.05, IR Veh vs. Sham. B: effect of MnTMPyP on caspase-3 activation in Sham, Sham Mn, IR, IR Veh, and IR Mn rats after renal I/R. Increased caspase-3 activity and protein levels were significantly reduced by MnTMPyP treatment. *P < 0.05 IR vs. Sham, Sham Mn, and IR Mn. C: Western blot showing effect of MnTMPyP on Bax, FasL, and Bcl-2 expression after renal I/R. D: densitometry of Western blot of pro- and antiapoptotic gene expression. Results are means ± SE (n = 3/group). *P < 0.05, IR and IR Veh vs. Sham and IR Mn.

To further examine the effect of MnTMPyP on apoptosis after I/R injury, we measured activation of the effector protease of apoptosis, caspase-3, in kidney homogenates from sham-operated and experimental animals. Caspase-3 activity was significantly increased in the I/R group compared with control animals (Fig. 7B, top; P < 0.05, I/R vs. sham operated). MnTMPyP treatment attenuated the I/R-mediated increase in caspase-3 by ~50% (Fig. 7B, top; P < 0.05 MnTMPyP vs. I/R, P = 0.11 MnTMPyP vs. I/R Veh). We also performed Western blotting in all four groups to confirm our results. There was an increase in cleaved caspase-3 after I/R injury that was significantly attenuated by MnTMPyP treatment (Fig. 7B, bottom).