Oxidative stress and apoptosis are important factors in the etiology of renal ischemia-reperfusion (I/R) injury. The present study tested the hypothesis that the cell-permeant SOD mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) protects the kidney from I/R-mediated oxidative stress and apoptosis in vivo. Male Sprague-Dawley rats (175–220 g) underwent renal I/R by bilateral clamping of the renal arteries for 45 min followed by reperfusion for 24 h. To examine the role of reactive oxygen species (ROS) in renal I/R injury, a subset of animals were treated with either saline vehicle (I/R Veh) or MnTMPyP (I/R Mn) (5 mg/kg ip) 30 min before and 6 h after surgery. MnTMPyP significantly attenuated the I/R-mediated increase in serum creatinine levels and decreased tubular epithelial cell damage following I/R. MnTMPyP also decreased TNF-α levels, gp91phox, and lipid peroxidation after I/R. Furthermore, MnTMPyP inhibited the I/R-mediated increase in apoptosis and caspase-3 activation. Interestingly, although MnTMPyP did not increase expression of the antiapoptotic protein Bcl-2, it decreased the expression of the proapoptotic genes Bax and FasL. These results suggest that MnTMPyP is effective in reducing apoptosis associated with renal I/R injury and that multiple signaling mechanisms are involved in ROS-mediated cell death following renal I/R injury.
- acute ischemia-reperfusion
- manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin
- reactive oxygen species
renal ischemia-reperfusion (I/R) injury continues to be a significant and persistent problem in kidney transplantation, with serious consequences (8). I/R induces some injury in the cortical proximal tubules and a more severe, generally lethal injury in the outer medullary proximal tubules (8). Apoptosis has been shown to be the major mechanism leading to tubule cell death during the early ischemic renal injury phase (14, 45), and the generation of reactive oxygen species (ROS) plays an important role in this process (26, 27). These free radicals can attack a wide variety of cellular components including DNA, proteins, and lipids, leading to DNA and protein oxidation, protein degradation and lipid peroxidation and thereby enhancing the destruction of the cell structure and loss of cell function (3). ROS also increase the production of proinflammatory cytokines in I/R that can further accelerate apoptotic signaling and cellular damage (51).
Since multiple factors are involved in renal I/R injury cascades, it is important to identify the underlying mechanisms and pathways that ultimately lead to the loss of kidney function. In vivo studies have shown that resistance to renal I/R injury is partly due to a decrease in oxidative stress and preservation of antioxidant proteins (5, 38). Manganese superoxide dismutase (Mn SOD) and copper/zinc superoxide dismutase (Cu/Zn SOD) both protect against global cerebral ischemia by preventing early DNA fragmentation (17, 18). Furthermore, SOD mimetics have also been shown to be protective against cell injury caused by ROS in models of ischemic injury (15, 43), and MnTMPyP attenuates lipopolysaccharide (LPS)-induced production of superoxide (O2•−) and prostaglandin E2 (PGE2) in microglia (50). We showed previously (33) that MnTMPyP, a cell-permeant SOD mimetic, rescues renal proximal tubular epithelial cells from ATP depletion-mediated apoptosis. However, whether MnTMPyP can protect the kidney from acute renal I/R injury in vivo and whether the mechanisms are similar to the in vitro cell culture model are still unclear. The present study tested our hypothesis that MnTMPyP protects the kidney from renal I/R injury-mediated damage in vivo. We measured kidney function, markers of apoptosis, and oxidative stress following renal I/R injury. Finally, to determine the mechanism of action of MnTMPyP we examined the extrinsic and intrinsic pathways of apoptosis following renal I/R injury.
Animal model of renal I/R injury.
Male Sprague-Dawley rats (175–220 g) were purchased from Harlan. All experiments were approved by the Animal Care and Use Committee at the Medical College of Wisconsin. The animals were anesthetized with pentobarbital sodium (50 mg/kg ip). Renal I/R was induced by bilateral clamping of the renal arteries for 45 min followed by revascularization for 24 h. A subset of animals were treated with either MnTMPyP (5 mg/kg ip, 30 min before surgery and 6 h after reperfusion) or saline vehicle. Sham-operated control animals underwent surgery without renal artery clamping. Animals were killed at the end of 24 h, and kidneys were isolated, quick frozen in liquid nitrogen, and stored at −80°C until further analysis. Blood was sampled with heparinized syringes and centrifuged for 1 min at 8,500 rpm, and serum was stored in heparinized tubes at −80°C until further analysis.
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 ΔΔCT method (where CT 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 gp91phox (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 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 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.
Results are expressed as means ± SE. Statistical analysis was done by analysis of variance (ANOVA) followed by Newman-Keuls multiple-comparison tests. Statistical significance was set at P < 0.05.
MnTMPyP improves kidney function and reduces histological damage after I/R injury.
Serum creatinine level was used for assessment of kidney function after renal I/R. Preoperative baseline serum creatinine levels in the rats were 0.74 ± 0.07 mg/dl. Serum creatinine levels were significantly increased in all I/R groups compared with sham-operated animals (P <0.001, I/R and I/R Veh vs. sham operated). MnTMPyP treatment significantly decreased serum creatinine levels (P < 0.001, MnTMPyP vs. I/R, I/R Veh), although it did not decrease creatinine to levels seen in sham-operated animals (P < 0.05, MnTMPyP vs. sham operated) (Fig. 1).
Histological analysis of the kidney also revealed more significant damage in the I/R- and I/R Veh-treated rats compared with sham-operated animals. We took advantage of the increased intensity of eosin autofluorescence in necrotic tissue to quantify ATN. A representative eosin fluorescent image and a corresponding light image in an H & E-stained section are shown in Fig. 2A. There was good correlation between necrotic tissue and increased fluorescence (arrow, Fig. 2A) and normal proximal tubules and decreased fluorescence (asterisk, Fig. 2A). As shown in qualitatively in Fig. 2B, top, and quantitatively in Fig. 2C, there was a massive increase in eosin autofluorescence in both the I/R and I/R Veh groups compared with sham-operated animals. MnTMPyP-treated sections displayed significantly less eosin autofluorescence, indicating lower tubular damage (Fig. 2, B, top, and C). PAS staining of the sections from I/R- and vehicle-treated rats also revealed increased tubular basement membrane disruption, infiltration of nuclei, and increased vacuolization of tubular epithelium (Fig. 2B, bottom) compared with sham-operated rats. Similar to the finding with serum creatinine and H & E staining, MnTMPyP treatment decreased the tubular damage following I/R injury (Fig. 2B).
Effect of MnTMPyP on expression of O2•−-producing enzymes in renal I/R injury.
Xanthine oxidase and gp91phox, 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 gp91phox 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 gp91phox 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 gp91phox 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).
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).
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).
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).
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).
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).
Effect of MnTMPyP on pro- and antiapoptotic gene expression following renal I/R injury.
To further elucidate the apoptotic signaling pathways that could lead to the activation of caspase-3, we performed a gene expression profile of pro- and antiapoptotic genes in sham-operated and I/R-, vehicle-, and MnTMPyP-treated animals. Surprisingly, the antiapoptotic protein Bcl-2 was increased by I/R injury (Fig. 7, C and D) and MnTMPyP treatment did not affect Bcl-2 expression significantly (Fig. 7, C and D). Nevertheless, the expression of the proapoptotic genes Bax and FasL was also dramatically increased after I/R injury (Fig. 7, C and D). Both of these were significantly downregulated by MnTMPyP.
The present study is a follow-up of our previous in vitro study (33) in which we showed that MnTMPyP is effective in protecting ATP-depleted proximal tubular epithelial cells against apoptosis. In the present study, we wanted to examine whether MnTMPyP is effective in protection against renal I/R injury in vivo. The dose of MnTMPyP that we used has been used by us before in a model of cardiac allograft rejection without any cytotoxic effect (40). Furthermore, sham-operated animals treated with MnTMPyP did not have any significant changes in kidney function compared with sham-operated animals alone. Here the main findings are that 1) MnTMPyP treatment significantly improved kidney function after renal I/R injury, 2) MnTMPyP reduced the I/R-mediated increase in TNF-α protein levels, 3) MnTMPyP was partially effective in decreasing nitrotyrosine formation and it decreased lipid peroxidation after I/R injury, and 4) MnTMPyP treatment partially reversed I/R-mediated apoptosis, mostly by decreasing proapoptotic gene expression.
The ischemic phase of I/R is characterized by a rapid depletion of ATP followed by a burst of ROS including superoxide anions, hydroxyl radicals, and peroxynitrite (4, 11, 39) in the reperfusion phase. The abnormal excessive production of ROS during reperfusion can induce lipid peroxidation, leukocyte activation, endothelial cell damage, and cytokine production, all of which contribute to tissue damage. ROS also disrupt the cellular cytoskeleton and cellular integrity or break down DNA, inducing apoptosis or necrosis (8). Recent compelling evidence has shown the generation of ROS not only during the reperfusion period but, in fact, during the ischemic period as well (41, 42). Thus pharmacological interventions that scavenge ROS are attractive as a therapeutic strategy to diminish the damage in I/R injury.
In this study, we used a small cell-permeant SOD mimetic, MnTMPyP, that has been shown by us previously to be effective in attenuating ATP depletion-mediated apoptosis in proximal tubular epithelial cells (33). MnTMPyP treatment significantly reduced serum creatinine and improved the renal histology structure after I/R injury in our model. This is consistent with other reports in the literature that have demonstrated that activation of antioxidant enzymes or treatment with antioxidant agents ameliorates I/R injury (9, 10, 28).
Xanthine oxidase is thought to be one of the main oxygen radical-generating systems after I/R injury in rat kidney (1). Surprisingly, in our study there was a very small increase in xanthine oxidase expression after I/R injury that was not affected by MnTMPyP treatment. On the other hand, there was a more pronounced increase in the expression of gp91phox, a subunit of NADPH oxidase, after I/R injury that was attenuated by MnTMPyP. This indicated that the loss of kidney function in our model of renal I/R injury was linked more to superoxide production through the actions of gp91phox. Although studies examining the role of this protein in renal I/R injury are limited, there is a report demonstrating that inhibition of NADPH oxidase via apocynin could reverse the damage in streptomycin-induced diabetic kidneys (44). Furthermore, using gp91phox-knockout mice (23) and inhibitors of NADPH oxidase in models of stroke and focal cerebral ischemia (47), studies have shown that this protein plays a critical role in ROS formation and disruption of the blood-brain barrier. Thus it is likely that activation of the NADPH oxidase subunit is at least partially responsible for the increase in O2•− observed in our model and the tissue damage following I/R injury.
Overall oxidative stress is, however, more dependent on both oxidant production and removal by antioxidant proteins. In our model, I/R injury significantly reduced Mn SOD and Cu/Zn SOD mRNA expression, resulting in increased superoxide, and caused oxidative stress. This corresponded well with previous reports that there is a loss of antioxidant proteins that contributes to overall oxidative stress in I/R injury (15, 38). Treatment with MnTMPyP was insufficient to restore the expression of either of these two genes, indicating that this compound does not act by upregulation of antioxidant proteins.
It is well established that superoxide and nitric oxide can react rapidly at rates that are diffusion limited to form peroxynitrite, a potent tyrosine-nitrating and damaging radical (6, 7, 21, 22). It has been shown that there is increased nitration of several proteins including Mn SOD and cytochrome c in both heart and kidney allograft rejection and during renal I/R injury (13, 31, 32, 37). In our studies, we found an increase in nitrotyrosine formation by qualitative immunohistochemistry techniques. Most of the nitrotyrosine staining was evident in the proximal tubular region of the kidney, an area that is most susceptible to damage in renal I/R injury. Although there was no attenuation of nitrotyrosine by the slot blot method, immunohistochemistry data indicated that nitrotyrosine was decreased in the proximal tubular region of MnTMPyP-treated rats. Other studies have demonstrated that MnTMPyP can significantly attenuate nitrotyrosine generation in cyclosporine A-treated endothelial cells (36) and can prevent nitrotyrosine immunoreactivity in glial cells subject to glucose deprivation (12). The reason for the variability of our results and this disparity with the literature is not really clear, although it could be related to the amount of nitrotyrosine formed by the particular model of injury and different susceptibility of various cell types to oxidative stress and injury. Increased oxidative stress also results in an escalation in lipid peroxidation, which plays an important role in the etiology of reperfusion injury. HNE-modified proteins have been used as indicators of lipid peroxidation and are increased after monolateral renal I/R (2). Furthermore, modified HNE can induce apoptosis by activation of JNK and p38 MAPK pathways in neuronal cells (46). In conjunction with previous reports in the literature (25), MnTMPyP treatment was effective in partially attenuating HNE-protein adducts, demonstrating that MnTMPyP could prevent downstream effects of increased oxidative stress.
There is growing evidence that indicates that inflammatory mechanisms contribute to I/R injury-induced acute renal failure. Studies have shown that increased TNF-α is one of the earliest indicators of I/R injury and mediated neutrophil infiltration (16). Furthermore, there is evidence that TNF-α mediates apoptosis (35) through the TNF receptor-associated protein TRADD and Fas activation (20) and p38 MAPK pathways (34). In our study also, there was a massive increase in TNF-α production indicative of inflammation that was significantly attenuated by MnTMPyP. This decrease suggested that MnTMPyP could also function as an anti-inflammatory agent and prevent apoptosis. To test this, we examined apoptosis by TUNEL and caspase-3 activation in all groups. These results showed that MnTMPyP was effective in partially attenuating both caspase-3 activity and TUNEL-positive nuclei, suggesting that the increase in TNF-α is responsible for increased cell death in I/R injury.
Apoptosis is largely mediated by either extrinsic mechanisms or intrinsic mechanisms. Fas and FasL are members of the “extrinsic” cell death pathway and are known to transduce apoptotic signals emanating from cell surface receptors, resulting in activation of caspase-8 that can either directly activate caspase-3 or cleave BID, a BH3 domain-containing proapoptotic Bcl-2 family member, to activate apoptosis via the mitochondrial or intrinsic pathway (29). Proapoptotic proteins such as Bax also promote the release of cytochrome c from the mitochondria, whereas antiapoptotic members of the Bcl-2 family prevent the release of cytochrome c from the mitochondria.
To further delineate the pathways through which cells undergo apoptosis after I/R injury, we examined the expression of pro- and antiapoptotic genes of the extrinsic and intrinsic pathways. As expected, there was an increase in protein levels for the proapoptotic genes Bax and FasL after I/R injury, although there was also a corresponding increase in the expression of the antiapoptotic protein Bcl-2. Although unexpected, this was not totally surprising to us since other investigators have shown that Bcl-2 is upregulated in distal tubules as a compensatory protective mechanism after renal ischemic injury (19, 48). MnTMPyP treatment dramatically decreased Bax and FasL expression. However, MnTMPyP did not affect Bcl-2 protein levels, although in our previous in vitro study (33) using ATP-depleted proximal tubular epithelial cells we found that MnTMPyP was effective in increasing Bcl-2 protein levels in the early time points of ischemia. Thus whether Bcl-2 expression is increased in the distal or proximal tubular region may impact on the resistance/susceptibility of that particular tubular segment of the kidney to ischemic damage.
In summary, the present study suggests that MnTMPyP plays an important role in reducing the oxidative stress and apoptosis associated with renal I/R injury. However, more work needs to be done to completely define the signaling pathways that lead to oxidant-mediated programmed cell death in renal ischemic injury.
This work was supported in part by the P&F project in National Institute of Diabetes and Digestive and Kidney Diseases Grant NIH-P50-1DK-079306-01 and by departmental funds to V. Nilakantan.
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.
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