Orchiectomy Attenuates Post-ischemic Oxidative Stress and Ischemia/Reperfusion Injury in Mice

Males are much more susceptible to ischemia/reperfusion (I/R)-induced kidney injury when compared with females. Recently we reported that the presence of testosterone, rather than the absence of estrogen, plays a critical role in gender differences in kidney susceptibility to I/R injury in mice. Although reactive oxygen species and antioxidant defenses have been implicated in I/R injury, their roles remain to be defined. Here we report that the orchiectomized animal had significantly less lipid peroxidation and lower hydrogen peroxide levels in the kidney 4 and 24 h after 30 min of bilateral renal ischemia when compared with intact or dihydrotestosterone-treated orchiectomized males. The post-ischemic kidney expression and activity of manganese superoxide dismutase (MnSOD) in orchiectomized mice was much greater than in intact or dihydrotestosterone-administered orchiectomized mice. Four hours after 30 min of bilateral ischemia, superoxide formation was significantly lower in orchiectomized mice than in intact mice. In Madin-Darby canine kidney cells, a kidney epithelial cell line, 1 mm H2O2 decreased MnSOD activity, an effect that was potentiated by pretreatment with dihydrotestosterone. Orchiectomy prevented the post-ischemic decrease of catalase activity. Treatment of male mice with manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a SOD mimetic, reduced the post-ischemic increase of plasma creatinine, lipid peroxidation, and tissue hydrogen peroxide. These results suggest that orchiectomy accelerates the post-ischemic activation of MnSOD and reduces reactive oxygen species and lipid peroxidation, resulting in reduced kidney susceptibility to I/R injury.

Gender differences in disease susceptibility have been well characterized in many cardiovascular diseases (1)(2)(3). The increased risk for cardiovascular diseases in men and postmenopausal women (4) has been attributed primarily to lack of estrogen-mediated protection (5)(6)(7). Recently we demonstrated that male mice are much more susceptible to kidney ischemia/reperfusion (I/R) 5 injury when compared with female mice (8), and that orchiectomy decreases kidney susceptibility to I/R injury (9), whereas ovariectomy has no effect on kidney susceptibility to I/R injury (8). Testosterone administration to orchiectomized male or female mice reverses the protective phenotype, increasing susceptibility to kidney I/R injury. We concluded that the presence of testosterone, rather than the absence of estrogen, plays a critical role in the gender differences in kidney susceptibility to I/R injury. The detailed molecular mechanisms responsible for this testosterone effect remain to be defined.
Ischemia/reperfusion markedly increases the production of reactive oxygen species (ROS) including superoxide anions, hydroxyl radicals, hypochlorous acid, hydrogen peroxide, and peroxynitrite (10). The abnormal excessive production of ROS results in lipid peroxidation, leukocyte activation, endothelial cell damage, and cytokine production, all of which contribute to tissue damage (11)(12)(13). Reactive oxygen species disrupt the cellular cytoskeleton and cellular integrity and break down DNA (11)(12)(13). A number of studies have demonstrated that the activation of antioxidant enzymes or treatment with antioxidant agents ameliorates I/R injury (11, 14 -17).
There are gender differences in the oxidant-antioxidant systems (18 -21). Base-line oxidation is greater in males than in females (22). Estrogen has antioxidant properties at pharmacological concentrations, whereas testosterone does not (23). We hypothesized that testosterone may inhibit antioxidant defense systems, resulting in increased susceptibility to kidney I/R injury.
Here we report that orchiectomy accelerates the post-ischemic activation and expression of MnSOD in the kidney, leading to reduced superoxide radical levels and lipid peroxidation, effects that can explain the attenuated susceptibility of the kidney of the orchiectomized mouse to ischemic injury. Consistent with this is the finding that manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a mimic of SOD, reduces kidney susceptibility to I/R injury. Thus, the increase in antioxidant defenses associated with orchiectomy may account for reduced susceptibility to ischemic injury in orchiectomized mice.

MATERIALS AND METHODS
Animal Preparation-Experiments were performed in 12-14week-old BALB/c male mice. In all the cases, studies were done according to animal experimental procedures approved by the Animal Care and Use Committee of Kyungpook National University. Each animal group consisted of more than four mice. Blood was collected to determine base line plasma creatinine concentrations.
Animals were anesthetized with pentobarbital sodium (60 mg/kg body weight; intraperitoneally) prior to surgery. Kidney ischemia was carried out as described previously (24,25). In some animals, either orchiectomy or sham orchiectomy was carried out 15 days before either 30 min of bilateral renal ischemia or sham surgery. Some animals were administered dihydroxytestosterone (DHT; 500 g/kg body weight, Sigma), a non-aromatizible analogue of testosterone or vehicle (sesame oil, Sigma) by subcutaneous injection every day for 14 days prior to ischemia. Some animals were administered intraperitoneally either MnTMPyP (5 mg/kg body weight), a SOD mimetic, or vehicle (saline) for 12 days and then subjected to either 30 min of bilateral renal ischemia or sham operation. Kidneys were harvested for Western blot analysis and biochemical studies and frozen using liquid nitrogen. For the determination of superoxide formation, kidneys were frozen-fixed using oxytetracyline compound (Sakura FineTek, Torrance, CA).
Plasma Creatinine Concentration-Seventy microliters of blood was taken from the retrobulbar vein plexus at the times indicated in Figs. 1 and 8. Plasma creatinine concentrations were measured using a Beckman Creatinine Analyzer II (Beckman).
Measurement of Kidney Lipid Peroxidation and Hydrogen Peroxide-Thiobarbituric acid-reactive substances were determined as a measure of lipid peroxidation. Samples were evaluated for malondialdehyde (MDA) production using a spectrophotometric assay for thiobarbituric acid-reactive substances (26). Hydrogen peroxide concentrations were determined using a ferric oxide-sensitive dye, xylenol orange, as described (26). H 2 O 2 oxidizes iron(II) to iron(III) in the presence of sorbitol, which acts as a catalyst. Iron(III) then forms a purple complex with xylenol orange.
Preparation of Kidney Cytosolic and Mitochondrial Fractions-Kidneys in sucrose buffer (0.32 M sucrose, 10 mM Tris-HCl, pH 7.4) on ice were homogenized with a Dounce homogenizer. The homogenate was centrifuged at 1,000 ϫ g for 5 min, and the supernatant was centrifuged at 15,000 ϫ g for 30 min. The supernatant was used to measure the activity of catalase or copper-zinc superoxide dismutase (CuZnSOD). The precipitate was washed twice with sucrose buffer to collect mitochondrial pellets. The mitochondrial pellets were sus-pended in phosphate-buffered saline containing 0.1% Triton X-100, disrupted twice using a sonicator (4710 series; Cole-Palmer, Chicago, IL) at 40% of maximum setting for 10 s, and centrifuged at 15,000 ϫ g for 30 min. The supernatant was used to measure the activities of manganese superoxide dismutase (MnSOD).
Measurement of Catalase, CuZnSOD, and MnSOD Activities in Kidney and MDCK Cells-Catalase activity was measured by the decomposition of hydrogen peroxide, determined by a decrease in absorbance at 240 nm. SOD activity was assayed spectrophotometrically using a pyrogallol assay as described previously (27). MnSOD or CuZnSOD activity was measured using the mitochondrial or cytosolic fraction, respectively. One unit of SOD activity was defined as the quantity of enzyme that reduces the superoxide-dependent color change by 50%. MDCK cells were grown in the phenol red-free Dulbecco's modified Eagle's medium containing 10% charcoal-deprived fetal bovine serum. Cells were pretreated in serum-free medium for 24 h, incubated with serum-free medium containing various concentrations of DHT for 24 h, and treated with either 1 mM H 2 O 2 or vehicle.
Determination of Superoxide Formation Using Nitro Blue Tetrazolium (NBT)-Kidneys were harvested either at 4 h after sham operation or 30 min of bilateral renal ischemia. The kidneys were frozen-fixed in liquid nitrogen and cut into 4-m sections using a cryomicrotome (Cryotome). Sections were incubated in 1 mg/ml NBT (Sigma) in phosphate-buffered saline, pH 7.4, for 2 h at 37°C and washed with phosphatebuffered saline. Signals were observed using a light microscope. The area of nitro blue tetrazolium positivity was analyzed using the image acquisition and analysis software, LabWorks (Ultra-Violet Products Ltd.).
Statistics-The results were expressed as mean Ϯ S.E. Statistical differences among groups were calculated using analysis of variance followed by least significance difference post hoc comparison using the SPSS 12.0 program. Differences between groups were considered statistically significant at a p value of Ͻ0.05.

RESULTS
Orchiectomy Reduces Kidney Susceptibility to Ischemia/ Reperfusion Injury in Mice-Thirty minutes of bilateral renal ischemia in intact mice markedly increased the plasma creati-nine level at 4 and 24 h after reperfusion ( Fig. 1). By contrast, this period of ischemia in orchiectomized mice did not result in the increase of plasma creatinine 4 and 24 h after ischemia (Fig. 1). To determine whether the reduced susceptibility of orchiectomized mice to ischemia is associated with testosterone, we induced ischemia in orchiectomized mice that had been pretreated with DHT. In these mice, post-ischemic plasma creatinine levels were similar to those in intact males ( Fig. 1).

Ischemia/Reperfusion-induced Kidney Lipid Peroxidation and Production of Hydrogen Peroxide
Were Reduced in Orchiectomized Mice-Thirty minutes of bilateral renal ischemia followed by 4 or 24 h reperfusion significantly increased lipid peroxidation and tissue hydrogen peroxide levels ( Fig. 2A). The lipid peroxidation in intact males was much greater than in orchiectomized males 4 and 24 h after I/R ( Fig. 2A). Tissue hydrogen peroxide levels were significantly lower in orchiectomized mice when compared with that in intact mice 4 and 24 h after I/R (Fig. 2B). This suggests that the reduced susceptibility to I/R injury in orchiectomized mice may be related to a reduction of ROS.
Activity of Catalase in Kidneys Subjected to Ischemia/ Reperfusion-In intact males, 30 min of bilateral renal ischemia significantly decreased the kidney catalase activity 24 h after reperfusion, whereas in orchiectomized mice it did not (Fig. 3). To examine whether the reduced activity of catalase in intact males is associated with the levels of androgen, we determined the post-ischemic catalase activity in orchiectomized mice administered 500 g/kg DHT. Twenty-four hours after ischemia, catalase activities in DHT-treated orchiectomized mice were significantly lower than sham-operated control (Fig. 3). We reported previously that orchiectomy decreases plasma testosterone, and DHT treatment increases plasma testosterone levels to those of the intact male (8).
Activity of CuZnSOD and MnSOD in Kidneys Subjected to Ischemia-Four and 24 h after ischemia, the activity of CuZn-SOD in the cytosolic fraction of kidney tissues was not significantly changed in any of the three experimental animal groups when compared with their respective sham-operated control mice (Fig. 4A). Because MnSOD is a major antioxidant in mitochondria and is essential for life (28,29), we determined the activity of MnSOD in the mitochondrial fraction of the kidneys. In intact males, ischemia significantly decreased MnSOD activity 24 h after ischemia (Fig. 4B). In orchiectomized males, however, ischemia did not attenuate the activity of MnSOD 24 h after reperfusion (Fig. 4B). In fact, MnSOD activity was increased 4 h after ischemia in orchiectomized males (Fig. 4B). Administration of DHT to orchiectomized mice prevented the increase (Fig. 4B). The increased MnSOD activity in orchiectomized mice was associated with an increase in total MnSOD protein in kidneys 4 h after reperfusion (Fig. 5). By contrast,   JULY 21, 2006 • VOLUME 281 • NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20351 ischemia in intact mice and in orchiectomized mice treated with DHT did not result in the change of MnSOD expression (Fig. 5).

Increased Post-ischemic MnSOD Activity in Orchiectomized Mice
Activity and Expression of MnSOD in MDCK Cells after Treatment with DHT or H 2 O 2 -Because epithelial cell injury is an important component of ischemia/reperfusion-induced injury in vivo, we determined the effect of DHT on the activity and expression of MnSOD in MDCK cells, an established kidney epithelial cell line. Treatment with DHT alone did not alter MnSOD expression and activity (Fig. 6A). One mM H 2 O 2 treatment significantly decreased MnSOD activity in the mitochondrial fraction of the cells (Fig. 6B). Pretreatment with DHT resulted in significantly greater reductions in MnSOD activity after treatment with 1 mM H 2 O 2 (Fig. 6B).
Orchiectomy Reduces Post-ischemic Superoxide Formation in the Kidneys Subjected to Ischemia-Because superoxide produces blue formazan after reaction with nitro blue tetrazolium, we examined the deposition of blue formazan in kidney sections. Four hours after I/R, the formation of blue formazan dramatically increased in the tubular cells of the kidneys in intact mice (Fig. 7A). Superoxide formation was less in orchiectomized mice than it is in intact mice 4 h after I/R (Fig. 7A). The production of formazan was quantitated, and data are presented in Fig. 7B. This suggests that the higher activity of antioxidant enzymes such as MnSOD in orchiectomized mice reduces the amount of superoxide radicals leading to less susceptibility of the kidney to I/R injury.
MnTMPyP, a SOD Mimetic, Reduces Post-ischemic Lipid Peroxidation and Protects the Kidney from Ischemia-To determine whether the increased activation of MnSOD mediates the lower susceptibility of orchiectomized males to ischemia, we examined the effect of MnTMPyP, a mimetic of SOD, on I/R injury. Treatment with MnTMPyP for 12 days did not change body weight (data not shown). At 24 h after ischemia, plasma creatinine concentration significantly increased in vehicletreated mice, whereas ischemia resulted in only modest increases in MnTMPyP-treated mice (Fig. 8). To examine whether the protection afforded by MnTMPyP was associated with reduced oxidative stress, we determined the lipid peroxidation and the tissue hydrogen peroxide levels in the kidney 4 and 24 h after reperfusion. Prior administration of MnTMPyP resulted in significant reductions in post-ischemic lipid peroxidation and tissue hydrogen peroxide levels when compared with vehicle-treated animals (Fig. 9). Catalase activity was not affected  by MnTMPyP administration and decreases 24 h after ischemia in both vehicle-and MnTMPyP-treated mice (Fig. 10A). Pretreatment with MnTMPyP did not affect the activities of CuZnSOD 4 and 24 h after ischemia (Fig. 10B). MnTMPyP prevented the postischemic reduction of MnSOD activity (Fig. 10C).

DISCUSSION
Our findings build on our previous data that removal of the testes in males reduces kidney susceptibility to ischemia/reperfusion injury and that the reduced susceptibility associated with orchiectomy is reversed by addition of exogenous testosterone. We have now found a mechanism of this important gender difference in susceptibility to ischemia. The protection afforded by orchiectomy is related to a reduction of oxidative stress as reflected by reduced lipid peroxidation and reduced tissue levels of hydrogen peroxide and superoxide formation with greater MnSOD activity in the kidneys of orchiectomized mice. We also demonstrate that MnTMPyP, a SOD mimetic, reduces the post-ischemic lipid peroxidation and renal functional impairment in mice. Although many studies have emphasized the beneficial effect of estrogen in gender differences in ischemia/reperfusion injury, this study, together with our prior observations, demonstrates that male sex hormones play an important role in the gender difference in kidney susceptibility to I/R injury (8,30).
Excessive ROS production is an important cause of I/Rinduced injury (11,32). ROS have been implicated in gender FIGURE 7. Effect of orchiectomy on superoxide formation in kidneys subjected to ischemia/reperfusion injury. Some mice were subjected to orchiectomy on day 0. The mice were subjected to either sham operation or 30 min of bilateral renal ischemia on day 15. Four hours after reperfusion, the kidneys were harvested for the analysis of superoxide formation by conversion of NBT to formazan (dark brown) as described under "Materials and Methods." A, sections were stained with NBT. B, formazan production was quantified using the image acquisition and analysis software LabWorks (Ultra-Violet Products Ltd.) (n ϭ 3). a, p Ͻ 0.05 versus their respective sham-operated controls. b, p Ͻ 0.05 orchiectomy in ischemia.

Increased Post-ischemic MnSOD Activity in Orchiectomized Mice
differences in diseases (33,34). Borras et al. (18) reported that females have higher antioxidant enzymes resulting in the lower production of ROS than in males and that ovariectomy increases the production of ROS. In postmenopausal women with coronary heart disease, testosterone concentration and MDA production, an indicator of lipid peroxidation, are elevated in serum when compared with values obtained in normal healthy women (34). By contrast, SOD and estradiol levels are significantly lower than in normal healthy women (34). Strehlow et al. (33) reported that 17␤-estradiol up-regulates MnSOD and extracellular superoxide dismutase expression and enzyme activity in vascular smooth muscle cells.
Lipid peroxidation and production of ROS are regulated by various extracellular and intracellular antioxidant enzymes. A contributing factor to the increase of ROS levels after I/R is the reduction of antioxidant enzyme activity. It has been reported that catalase activity is decreased after I/R (11). In the present study, the activity of catalase, which transforms H 2 O 2 to H 2 O, was significantly decreased after ischemia in the intact mice, whereas orchiectomy prevented the post-ischemic decreases of catalase activity. In addition, DHT administration to orchiectomized mice prevented the effects of orchiectomy on the post-ischemic decrease of catalase activity, indicating that testosterone plays an important role in the regulation of antioxidant enzyme activity. H 2 O 2 production was greater in intact males than in orchiectomized mice.
Superoxide is an important free radical mediating cellular damage. It is scavenged by CuZnSOD located in cytoplasm and MnSOD in mitochondria. MnSOD is the major antioxidant enzyme located in mitochondria and is essential for life (28,29). Chien et al. (37) reported that intravenous treatment with SOD in rats results in reduced apoptotic cell death and ROS production after kidney ischemia/reperfusion. Macmillan-Crow and Cruthirds (35) reported that mice expressing only 50% of the normal complement of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of ROS (35). On the other hand, overexpression of MnSOD enzyme renders the cell and tissues resistant to ischemia/reperfusion injury (36). In the present study, orchiectomy resulted in enhanced post-ischemic MnSOD, but not CuZnSOD, activity and expression, resulting in the reduction of post-ischemia superoxide radical formation. Thus the higher ROS stress in intact males may be directly related to lower MnSOD activity (37).
To test whether testosterone alone suppresses the expression and activity of MnSOD by a direct effect on the epithelial cell,  we evaluated whether DHT modulates MnSOD activity and expression in MDCK cells, a well established tubular epithelial cell line. DHT treatment alone did not affect the expression and activity of MnSOD in these cells. Thirty or sixty minutes after treatment with 1 mM H 2 O 2 , however, MnSOD activity was significantly lower in DHT-pretreated MDCK cells than that in vehicle-pretreated cells (Fig. 6B). This indicates that the postischemic suppression of MnSOD activity in DHT-treated orchiectomized mice may reflect, at least in part, a direct effect on the epithelial cell.
MnSOD is susceptible to nitrotyrosylation, which resulted in enzyme inactivation (38,39). In the present study, ischemia in intact male mice decreases MnSOD activity without a significant decrease of MnSOD expression. We speculate therefore that MnSOD is inactivated by nitration. MacMillan-Crow et al. (38,39) demonstrated in chronic renal allograft rejection that MnSOD is endogenously tyrosine nitrated and inactivated. Cruthirds et al. (40) reported that I/R in kidney decreases MnSOD activity without a reduction in MnSOD expression, and this decrease of MnSOD activity is associated with an increase in MnSOD nitration. Sam et al. (41) reported that in human hearts there can be a dissociation between mRNA expression and protein abundance or activity of catalase and MnSOD.
ROS production by mitochondria from females is much lower than that from males (18), indicating that levels of sexual hormones affect SOD activity. In the present study, although the treatment of orchiectomized mice with DHT restored kidney susceptibility from a resistant to susceptible phenotype, the post-ischemic reduction of MnSOD activity was not restored. This may be caused by subtle differences of hormone levels between intact and DHT-treated orchiectomized mice.
Thirty minutes of bilateral ischemia in non-orchiectomized mice results in the disruption of most of tubular cells in the outer medulla leading to kidney dysfunction (8,24,25,42), whereas the same period of ischemia in orchiectomized mice does not induce extensive cellular death. In the present study, 30 min of ischemia in orchiectomized mice produced small amounts of free radicals as seen in Fig. 7 without extensive tubular cell death, lipid peroxidation, and kidney functional impairment (8). Orchiectomy alone did not modulate superoxide formation. Differences in oxidants and antioxidant enzymes related to orchiectomy only emerged after an ischemic challenge.
MnTMPyP is a SOD mimetic that protects the heart from ischemia/reperfusion injury (31). In the present study, MnT-MPyP reduced the post-ischemic lipid peroxidation and the tissue levels of hydrogen peroxide in the kidney. These results support the hypothesis that the increased MnSOD activity in orchiectomized mice may account for the reduced susceptibility associated with orchiectomy. Recently we also reported that MnTMPyP protects cells from oxidative stress associated with radiation (26).
In summary, our data indicate that removal of testes reduces kidney susceptibility to ischemia/reperfusion injury and results in decreased lipid peroxidation, increased post-ischemic catalase, and MnSOD activities. The higher activity of antioxidant enzymes may account for the lower susceptibility of orchiectomized mice to ischemic injury. Our findings may have impor-tant implications for understanding the pathophysiology of gender differences in kidney ischemia/reperfusion injury and should lead to consideration of androgens and the ROS-antioxidant systems as targets for protective strategies to avoid acute kidney injury.