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Keigyou Yoh^1,, Aki Hirayama^2,, Kazusa Ishizaki³, Akiko Yamada³, Masayoshi Takeuchi⁴, Sho-ichi Yamagishi⁵, Naoki Morito¹, Takako Nakano³, Masami Ojima³, Homare Shimohata¹, Ken Itoh⁶, Satoru Takahashi^3,* and Masayuki Yamamoto⁷

¹ Pathophysiology of Renal Diseases, Doctoral Program in Clinical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575, Japan
² Center for Integrative Medicine, Tsukuba University of Technology, Tsukuba 305-8521, Japan
³ Anatomy and Embryology, Doctoral Program in Life System Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575, Japan
⁴ Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa 920-1181, Japan
⁵ Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume 830-0011, Japan
⁶ Department of Stress Response Science, Hirosaki University Graduate School of Medicine, Hirosaki 036-8562, Japan
⁷ Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan

	Abstract

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The transcription factor Nrf2 regulates the expression of antioxidant genes. Hyperglycemia-induced oxidative stress is involved in the pathogenesis of diabetes and its complications. However, little is known about the protective role of Nrf2 in diabetes. To gain insight into the protective role of Nrf2 in diabetes we treated Nrf2 knockout (Nrf2 KO) mice with streptozotocin (STZ). The STZ Nrf2 KO mice did not develop renal hyperfiltration, which was observed in the STZ-treated wild-type (STZ WT) mice, but renal function gradually deteriorated over the 10-week observation period. Urinary excretion of nitric oxide metabolites and the occurrence of 8-nitroguanosine, which was detected in glomerular lesions, were increased in STZ Nrf2 KO mice during the early stages after treatment. In vivo electron paramagnetic resonance analysis revealed an accelerated rate of decay of the 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl spin probe signal in STZ Nrf2 KO mice. The addition of superoxide dismutase prolonged the half-life of the signal, which suggested that increased oxygen radical formation occurred in the STZ Nrf2 KO mice. These results suggested that hyperglycemia increased oxidative and nitrosative stress and accelerated renal injury in the Nrf2 KO mice and that Nrf2 serves as a defense factor against some diabetic complications.

	Introduction

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Oxidative stress plays an important role in the pathogenesis of diabetes and its complications (Giugliano et al. 1996; Ceriello 2003; Wiernsperger 2003). Diabetes introduces multiple sources of oxidative stress, and the stress factors appear early in the disease and worsen as the disease progresses (Nishikawa et al. 2000; Wiernsperger 2003). Hyperglycemia induces oxidative stress by various mechanisms (Wiernsperger 2003; King & Loeken 2004). For instance, glucose can cause the non-enzymatic glycation of proteins, which produces oxidants (Baynes & Thorpe 1999). High glucose concentrations trigger the generation of reactive oxygen species (ROS) (Craven et al. 2001). Free glucose activates aldose reductase and the polyol pathway, which decreases NADPH : NADP ratios (Dunlop 2000). Excess glucose also activates protein kinase C, which increases oxidative stress by activating mitochondrial NADPH oxidase (Xia et al. 1994; Inoguchi et al. 2000). Increased oxidative glucose metabolism itself increases the mitochondrial production of the superoxide anion (), which is converted to the hydroxyl radical (OH^•–) and hydrogen peroxide (H₂O₂) (Nishikawa et al. 2000). Hyperglycemia has also been reported to promote nuclear translocation and activation of NF-B (Yerneni et al. 1999; Nishikawa et al. 2000).

The transcription factor Nrf2 regulates the basal and inducible expression of genes that encode detoxifying and antioxidant enzymes. Nrf2 is essential for the coordinated transcriptional induction of the genes for antioxidant enzymes and phase II drug-metabolizing enzymes through its interaction with antioxidant-responsive elements (Itoh et al. 1997). Antioxidant-responsive elements are usually found in the regulatory sequences of antioxidant enzyme genes, such as those for heme oxygenase-1 (HO-1) (Prestera et al. 1995) and -glutamylcysteine synthetase (Mulcahy et al. 1997), and regulate a wide range of metabolic responses that are activated by ROS or electrophiles (Prestera et al. 1995; Mulcahy et al. 1997; Itoh et al. 1999). Recent studies using Nrf2 knockout (Nrf2 KO) mice suggest that Nrf2 dysfunction is involved in the pathogenesis of human diseases and Nrf2 may play key roles in human disease (Yoh et al. 2001; Yamamoto et al. 2004; Ishii et al. 2005; van Muiswinkel et al. 2005).

Reactive radicals include molecules such as , OH^•–, nitric oxide (NO), and lipid radicals. Although an overproduction of NO has been implicated in the pathogenesis of streptozotocin (STZ) models of diabetes (Flodström et al. 1999; Ishii et al. 2001; Onozato et al. 2002), the NO radical itself does not usually cause severe oxidative damage. STZ-mediated tissue injuries are usually attributable to other detrimental ROS and reactive nitrogen species from radical chain reactions. To detect the responsible ROS/reactive nitrogen species in this model, we employed in vivo electron paramagnetic resonance (EPR) imaging to evaluate oxidative stress. EPR is a technique that is used to detect unpaired electrons, these are free radicals, directly. In vivo EPR measurements that use external nitroxide radicals allow us to evaluate the in vivo redox status in real-time and non-invasively, and this has been a powerful tool for oxidative stress research (Miura & Ozawa 2000; Hirayama et al. 2003, 2005).

Little is known concerning the protective roles of Nrf2 in diabetes. To gain insight into the relationship between Nrf2 and diabetes, we generated hyperglycemia in Nrf2 KO mice. Using this model mouse system and EPR technology, we examined the effect of Nrf2 deficiency on the onset of STZ-induced diabetes and on the early stages of the progression of diabetic nephropathy. We found that hyperglycemia increased oxidative and nitrosative stress to a greater extent, and accelerated early stage renal injury, in Nrf2 KO mice as compared to wild-type (WT) mice. Our results strongly suggested that peroxynitrite is the radical that is responsible for this renal injury.

	Results

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Changes in serum glucose after STZ administration

Plasma glucose concentrations in Nrf2 KO and WT mice before STZ treatment and 2, 6 and 10 weeks after STZ injection are shown in Fig. 1A. The non-fasting serum glucose levels of the control Nrf2 KO and WT mice groups were < 200 mg/dL throughout the observation period. In the STZ-treated Nrf2 KO (STZ Nrf2 KO) and STZ-treated wild-type (STZ WT) mice groups, the serum glucose levels rose markedly after STZ administration and the high glucose levels were maintained throughout the observation period (STZ Nrf2 KO: from 507.4 ± 31.5 mg/dL (2 weeks) to 704.6 ± 58.3 mg/dL (10 weeks); STZ WT: from 591.0 ± 29.9 mg/dL (2 weeks) to 691.2 ± 35.9 mg/dL (10 weeks)). The serum glucose levels in the STZ Nrf2 KO and STZ WT groups after STZ administration were significantly higher than those in the control Nrf2 KO and the WT groups, but there were no significant differences between the STZ Nrf2 KO mice and STZ WT mice.

View larger version (14K): [in this window] [in a new window]   Figure 1  Plasma glucose (A), Creatinine clearance (B), and urinary protein (C) in control and STZ-treated mice. Open squares and closed squares indicate WT mice (n = 10) and STZ-injected WT mice (n = 10), respectively. Open circles and closed circles indicate Nrf2 KO mice (n = 10) and STZ-injected Nrf2 KO mice (n = 10), respectively. Each bar represents the mean ± SEM (*P < 0.01 vs. WT mice, #P < 0.01 vs. Nrf2 KO mice, P < 0.01 vs. STZ WT mice, P < 0.01 vs. STZ Nrf2 KO mice, **P < 0.05 vs. WT mice, ##P < 0.05 vs. Nrf2 KO mice, P < 0.05 vs. STZ Nrf2 KO mice).

To analyze renal function, a comparison of the creatinine clearance rate (Ccr) was performed between the four groups (Fig. 1B). At 2 weeks after STZ administration, the Ccr in the STZ WT mice (6.04 ± 0.86 µL/min/body) was significantly higher than that in the STZ Nrf2 KO mice (4.11 ± 0.65 µL/min/body weight, P < 0.01), or control Nrf2 KO (3.88 ± 0.62 µL/min/body weight, P < 0.05) and WT mice (3.42 ± 0.64 µL/min/body weight, P < 0.01). At 6 weeks, the Ccr in the STZ Nrf2 KO mice (2.60 ± 0.32 µL/min/body weight) was lower compared with that in the STZ WT mice (4.30 ± 0.61 µL/min/body weight, P < 0.01), or control Nrf2 KO (4.03 ± 1.28 µL/min/body weight, P = 0.08), and WT mice (3.89 ± 0.36 µL/min/body weight, P < 0.05). At 10 weeks, the renal function of the STZ WT mice (2.89 ± 0.37 µL/min/body weight) had decreased nearly to that of the STZ Nrf2 KO mice (2.42 ± 0.27 µL/min/body weight). Therefore, the STZ Nrf2 KO mice did not demonstrate renal hyperfiltration, which was seen in the STZ mice, and they developed renal impairment early than the STZ WT mice.

Increased urinary protein in STZ-treated mice

Urinary protein levels were increased after STZ administration in both Nrf2 KO and WT mice (Fig. 1C). At 2 weeks, the urinary protein excretion of the STZ WT mice (3.11 ± 0.66 mg/day) was significantly higher than that of the other groups. The urinary protein output of the STZ Nrf2 KO mice (1.74 ± 0.30 mg/day) was also increased but not significantly more than that of the control Nrf2 KO (0.62 ± 0.26 mg/day) and WT mice (0.66 ± 0.38 mg/day). At 6 weeks, the urinary protein levels of the STZ WT (3.80 ± 0.72 mg/day) and the STZ Nrf2 KO mice (3.23 ± 0.51 mg/day) were both significantly higher than those of the control Nrf2 KO (0.54 ± 0.19 mg/day, P < 0.01) and WT mice (0.80 ± 0.17 mg/day, P < 0.01). At 10 weeks, the urinary protein outputs of the STZ WT mice (3.95 ± 0.20 mg/day) and the STZ Nrf2 KO mice (2.49 ± 0.63 mg/day) were significantly higher than those of the control Nrf2 KO (0.72 ± 0.35 mg/day, P < 0.01, P < 0.05, respectively) and WT mice (0.62 ± 0.11 mg/day, P < 0.01, P < 0.05, respectively). We also analyzed renal histopathology in the STZ-treated mice (Fig. 2). However, the histopathological analysis did not show any significant differences between the STZ Nrf2 KO mice and STZ WT mice. Therefore, although the STZ Nrf2 KO mice developed renal impairment earlier than the STZ WT mice, urinary protein excretion in the STZ Nrf2 KO mice was less severe than that in the STZ WT mice.

View larger version (100K): [in this window] [in a new window]   Figure 2  Histological appearance of the kidney sections obtained pretreatment (A, B) and 2 weeks (C, D, E, F, G, H) after STZ treatment. WT mice (A, C, E, G) and Nrf2 KO mice (B, D, F, H) (x200). Hematoxylin-eosin (A, B, C, D), periodic acid Schiff (E, F) and Masson's trichrome (G, H) staining. Scale bar, 100 µm.

AGEs contribute to diabetic nephropathy (Forbes et al. 2003). We analyzed the levels of serum glucose-derived AGEs (Glc-AGE) and found that they were increased after STZ administration in both the Nrf2 KO and WT mice (Fig. 3A). At 2 weeks, Glc-AGE levels in the STZ WT mice (59.5 ± 5.0 U/mL) and STZ Nrf2 KO mice (46.9 ± 8.4 U/mL) were also higher than those in the control groups (WT mice, 39.8 ± 5.6 U/mL; Nrf2 KO, 34.2 ± 13.2 U/mL), but there was only a significant difference between the STZ WT mice and control Nrf2 KO mice. At 6 weeks, the Glc-AGE level in the STZ Nrf2 KO mice (76.8 ± 8.1 U/mL)) was significantly higher than those in the control Nrf2 KO (39.8 ± 12.9 U/mL, P < 0.01) and WT mice (49.2 ± 0.8 U/mL, P < 0.05). The Glc-AGE level in the STZ WT mice (67.3 ± 6.7 U/mL) was only significantly higher than that in the control Nrf2 KO mice (P < 0.05). At 10 weeks, the Glc-AGE levels in the STZ WT (81.0 ± 12.4 U/mL) and STZ Nrf2 KO mice (81.5 ± 11.7 U/mL) were both significantly higher than those in the control Nrf2 KO (28.7 ± 2.4 U/mL, P < 0.01) and WT mice (32.1 ± 4.0 U/mL, P < 0.01). We could not detect any differences between the STZ Nrf2 KO mice and STZ WT mice. This result showed that the deficiency in Nrf2 did not induce higher levels of Glc-AGE production during the 10-week observation period.

View larger version (15K): [in this window] [in a new window]   Figure 3  Serum Glc-AGE levels (A) and daily urinary NOx excretion (B). Open and closed squares indicate WT mice (n = 5) and STZ WT mice (n = 5), respectively. Open and closed circles indicate Nrf2 KO mice (n = 5) and STZ Nrf2 KO mice (n = 5), respectively. Each bar represents the mean ± SEM (*P < 0.01 vs. WT mice, #P < 0.01 vs. Nrf2 KO mice, P < 0.05 vs. STZ WT mice, **P < 0.05 vs. WT mice, ##P < 0.05 vs. Nrf2 KO mice).

Due to the fact that NO plays a key role in STZ-induced diabetes (Flodström et al. 1999; Ishii et al. 2001; Onozato et al. 2002), we evaluated the changes in NO production by measuring daily urinary nitrite and nitrate (NOx) excretion. Because NOx excretion is affected by renal function, the data were corrected for creatinine clearance (Fig. 3B). Before STZ treatment, there was no significant difference in urinary NOx excretion between the four groups (Nrf2 KO, 12.2 ± 1.1 nmol/day/Ccr; WT mice, 15.2 ± 2.3 nmol/day/Ccr; STZ Nrf2 KO, 10.7 ± 0.83 nmol/day/Ccr; STZ WT mice, 11.0 ± 2.7 nmol/day/Ccr). Two weeks after STZ treatment, however, NOx excretion in the STZ Nrf2 KO mice (100.2 ± 40.6 nmol/day/Ccr) was significantly increased compared to that in the other groups (WT mice, 14.0 ± 2.9 nmol/day/Ccr, P < 0.05; Nrf2 KO mice, 17.4 ± 6.8 nmol/day/Ccr, P < 0.05; STZ WT mice, 35.9 ± 12.4 nmol/day/Ccr, P < 0.05). At 6 weeks after STZ treatment, NOx excretion in the STZ WT mice (82.7 ± 37.9 nmol/day/Ccr) was also increased but a significant difference was only seen between the STZ Nrf2 KO mice (117.0 ± 28.2 nmol/day/Ccr) and the control mice groups (WT mice, 13.0 ± 2.7 nmol/day/Ccr, P < 0.05; Nrf2 KO mice, 19.5 ± 7.3 nmol/day/Ccr, P < 0.05). At 10 weeks, NOx excretion in the STZ Nrf2 KO mice had further increased to a level of 145.3 ± 77.2 nmol/day/Ccr, whereas in the STZ WT mice the level of excretion had fallen to 50.9 ± 7.7 nmol/day/Ccr at this point. The NOx analysis showed that the STZ Nrf2 KO mice developed NO overproduction.

Increased oxygen radical formation in STZ-treated Nrf2 KO mice detected by in vivo EPR

Based upon the above results, we speculated that hyperglycemia induced an increased oxidative stress in STZ Nrf2 KO mice, which occurred primarily in the early period following STZ treatment. We then used in vivo EPR to analyze oxidative stress during the first 2 weeks after treatment (Fig. 4). Because free radical-related reactions have high rate constants, oxidative stress is usually estimated indirectly by its end-products. However, such methods potentially introduce problems, which are similar to those seen in vivo. To avoid these issues, we employed in vivo EPR for oxidative stress evaluation. The murine in vivo redox status was evaluated by in vivo EPR, which measured the elimination half-life of a nitroxyl radical spin-probe, carbamoyl-PROXYL. The disappearance of the EPR signal from nitroxyl spin probes is determined by two major factors; reduction by intracellular antioxidants and reaction with locally-produced free radicals, which lead to accelerated disappearance of the EPR signal.

View larger version (40K): [in this window] [in a new window]   Figure 4  Elimination half-lives of carbamoyl-PROXYL. Open, filled, dotted, and hatched bars indicate WT mice (n = 5), Nrf2 KO mice (n = 5), WT mice with SOD injection (n = 5) and Nrf2 KO mice with SOD injection (n = 5), respectively. W, WT mice; K, Nrf2 KO mice; S, STZ-treated mice. 0 weeks, pretreatment. Each bar represents the mean + SEM (*P < 0.01, **P < 0.05).

Changes in glutathione and HO-1 levels after STZ treatment

In addition, we measured total glutathione (GSH) and HO-1 levels in the kidney before STZ treatment and at 1 and 2 weeks after STZ treatment (Table 1). The GSH levels in the STZ WT mice did not change significantly over the period of observation. However, the GSH levels in the STZ Nrf2 KO mice were significantly lower at 1 and 2 weeks after STZ treatment than before treatment. HO-1 levels in the STZ WT and STZ Nrf2 KO mice at 2 weeks after treatment were all higher than those of before treatment, but the difference was only significant in the WT mice (Nrf2 KO mice, P = 0.056).

In order to measure nitrosative stress, we evaluated 8-nitroguanosine formation in the kidneys of the mice by immunohistochemical analysis (Fig. 5). 8-Nitroguanosine is a biomarker for nitrosative stress (Akuta et al. 2006). Very little immunostaining for 8-nitroguanosine was observed in WT and Nrf2 KO kidneys before STZ administration. However, immunostaining became obvious in glomerular mesangial lesions at 1 week after STZ treatment in both WT and Nrf2 KO mice. The immunostaining then declined at 2 weeks in the STZ WT mouse. However, the immunostaining became more obvious at 2 weeks in the STZ Nrf2 KO mouse.

View larger version (51K): [in this window] [in a new window]   Figure 5  (A) 8-Nitroguanosine immunohistochemistry in control and STZ-treated mice. Immunostaining in kidneys obtained 0 (pretreatment), 1 or 2 weeks after STZ treatment (x200). Scale bar, 100 µm. (B) Quantification of 8-nitroguanosine. Data are shown as the mean ± SEM of the relative optical densities in arbitrary units (AU) (*P < 0.01 vs. WT 0, 1, and 2 weeks, and Nrf2 KO 0, and 1 weeks).

	Discussion

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The hyperfiltration phenomenon was not detected at 2 weeks in the STZ Nrf2 KO mice, therefore we surmised that oxidative stress occurred before 2 weeks and aggravated renal function. In addition, we examined oxidative stress and nitrosative stress in the period from pre-administration to 2 weeks after STZ administration. Previous reports showed that oxidative stress was enhanced and nitric oxide was increased during the early stages after STZ treatment (Kakkar et al. 1997; Cosenzi et al. 2002). Kakkar et al. showed that oxidative stress begins quite early in STZ-induced diabetes and they proposed that oxidative stress plays a role in the initiation and development of complications in the STZ model. Cosenzi et al. showed that increased production of NO occurs immediately after the STZ treatment. Furthermore, NO and its derivative S-nitrosothiols are known to cause accumulation of Nrf2 in the nucleus and up-regulation of antioxidant-responsive element-regulated genes (Dhakshinamoorthy & Porter 2004; Buckley et al. 2008). These observations suggest that the generation of ROS and NO in Nrf2 KO mice could contribute to the renal impairment that is observed during the early phases of STZ treatment. We also measured GSH levels and found that GSH levels in the Nrf2 KO mice at 1 and 2 weeks after STZ treatment were significantly lower than those of the Nrf2 KO mice before treatment and the STZ WT mice at 1 and 2 weeks. From the NOx, in vivo EPR, GSH and 8-nitroguanosine analyses, we propose that NO overproduction and increased oxidative stress might accelerate renal injury in Nrf2-deficient mice in the early stages after STZ treatment.

EPR and spin probe kinetic analysis showed that the defense mechanism against oxidative stress in the liver and kidney is impaired in aged Nrf2 KO mice and this substantially decreases their ability to eliminate ROS (Hirayama et al. 2003). Therefore in this study we used young Nrf2 KO mice, which do not show a decreased ability to eliminate ROS as do aged Nrf2 KO mice. EPR signal decay of nitroxyl radicals, which include carbamoyl-PROXYL, is determined by two antithetical phenomena: radical-reducing activity in an organ or tissue and a direct reaction between ROS and the nitroxyl radicals. Carbamoyl-PROXYL is reduced to a hydroxylamine by antioxidants or reducing molecules and loses its EPR signal in organs. Carbamoyl-PROXYL molecules also lose their paramagnetic properties when they react with hydroxyl radicals (Miura & Ozawa 2000; Han et al. 2001; Hirayama et al. 2005). Therefore, the balance between the reducing activity of a tissue and the local production of free radicals determines the signal decay rate of carbamoyl-PROXYL. To distinguish between these opposing factors, we added SOD to eliminate the production of ROS. If the carbamoyl-PROXYL half-life is prolonged by the addition of SOD, this means that an increase in local ROS production is primarily responsible for the accelerated rate of carbamoyl-PROXYL signal decay. We found that SOD prolonged the carbamoyl-PROXYL half-life in the SZT Nrf2 KO mice at 1 week and in the SZT WT mice at 1 and 2 weeks (Fig. 4), which suggested that ROS production was increased. Carbamoyl-PROXYL is known to react almost exclusively with hydroxyl radicals, and this leads to a loss of the EPR signal (Takeshita et al. 2002). Therefore, our finding that the half-life is prolonged by SOD treatment means that the amount of hydroxyl radicals, whose origin is superoxide, has been decreased by the addition of SOD. On the other hand, our results also revealed an increase in NO, which leads to the overproduction of peroxynitrite. The reaction between NO and yields peroxynitrite with a second order rate constant, which has been determined for the reaction, that is near the diffusion-controlled limit (Huie & Padmaja 1993). Thus, this result predicts an increase in peroxynitrite, which will lead to ^•OH formation (Fig. 6). 8-Nitroguanosine is formed in DNA and RNA from guanine by peroxynitrite-induced nitrosative stress (Akuta et al. 2006). The immunohistochemical results revealed remarkable deposition of 8-nitroguanosine in the glomeruli of STZ Nrf2 KO mice, which agreed with the increase of peroxynitrite (Fig. 5). Mesangial damage affects the glomerular filtration rate. Therefore, we conclude that peroxynitrite is the radical that is responsible for the early stage renal damage in STZ Nrf2 KO mice. At 2 weeks after STZ treatment, the carbamoyl-PROXYL half-life was prolonged in Nrf2 KO mice, and this increase persisted throughout the later phase (data not shown). Because Nrf2 regulates several antioxidative enzymes including glutathione S-transferase (Itoh et al. 1997), this result suggested that the intracellular antioxidative molecules that would reduce carbamoyl-PROXYL were exhausted by the increased amount of free radicals that were present during these phases. The immunohistochemical result that showed enhanced 8-nitroguanosine staining in the glomeruli of Nrf2 KO mice at 2 weeks after the STZ treatment agreed with this hypothesis. Thus, free radical-related tissue damage plays a key role in the early stages of STZ-induced renal damage. The decreased antioxidative response that is due to the deficiency in Nrf2 emphasizes this damage.

View larger version (8K): [in this window] [in a new window]   Figure 6  Model for early renal damage in STZ-treated Nrf2 KO mice. CP T half, half-life of carbamoyl-PROXYL.

	Experimental procedures

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Treatment of mice

The generation of female Nrf2 KO mice has been described previously (Itoh et al. 1997). All the mice that were used in the present study were from the same litter, had an ICR background, and were under 10 weeks old. Diabetes was induced by a single intraperitoneal injection of STZ (150 mg/kg body weight: Sigma Chemical Co., St. Louis, MO) that had been diluted in citrate buffer, pH 4.5. Control mice were injected with an equal volume of citrate buffer alone. After 7 days, the induction of diabetes was confirmed by the measurement of blood glucose concentrations. Fed animals with blood glucose levels that were greater than 300 mg/dL were considered to be diabetic. Only mice that reached a stable hyperglycemia of more than 300 mg/dL were included in the study. Mice were maintained in the Laboratory Animal Resource Center. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the University of Tsukuba.

Serum glucose, creatinine, urinary protein and creatinine clearance measurements

Serum glucose and creatinine were measured by a Dry-Chem 3500 automated analyzer for routine laboratory tests (Fuji Film, Inc., Tokyo, Japan). The urine of each mouse was collected in an individual metabolic cage over a 24-h period. The methods for urinary protein determination and measurement of urinary creatinine were described previously (Yoh et al. 2001).

Histopathological of renal tissues

Each mouse was bled while under ether anesthesia. At autopsy, organs were fixed with 10% formalin in 0.01 M phosphate buffer, pH 7.2, and embedded in paraffin. Sections (3 µm) were stained by hematoxylin-eosin, periodic acid Schiff and Masson's trichrome staining for histopathological examination under the light microscope.

Determination of AGE concentration

Serum levels of Glc-AGE were measured with a competitive enzyme-linked immunosorbent assay (ELISA), as described previously (Takeuchi et al. 1999). 1 U corresponds to 1 µg of the Glc-AGE bovine serum albumin standard as described previously (Takeuchi et al. 1999).

Measurement of NOx

NOx were measured by the Griess reaction. Details of this method are described elsewhere (Nagase et al. 1997).

Redox measurement using in vivo EPR

The murine in vivo redox status was evaluated by in vivo EPR using carbamoyl-PROXYL as a spin probe. The disappearance of the EPR signal from nitroxyl spin probes is determined by two major factors: reduction by intracellular antioxidants and reaction with locally-produced free radicals, which lead to accelerated disappearance of the EPR signal. First, the carbamoyl-PROXYL (300 mM, 2 mL/kg) was injected into the mice through the tail vein 15 min after pentobarbital anesthesia. Each mouse was then placed in a plastic holder and put into the EPR system with the upper abdominal area in the center and the bladder outside the resonator. Rate constants (k) were calculated from the EPR signal intensities, which were measured every 20 s from 6 to 15 min after the carbamoyl-PROXYL injection. The peak-to-peak height of the lowest magnetic field signal in the triplet spectrum was defined as the signal intensity. The carbamoyl-PROXYL signal intensity was plotted semilogarithmically against time and the first order spin reduction rate constant was estimated from the slope value of the observed clearance curve which was obtained by best fit. The half-life was calculated using the equation: t_1/2 = ln 2/k. To distinguish the effect on the carbamoyl-PROXYL half-life of direct reaction with ROS from that of intracellular antioxidants, the effects of the addition of a free radical specific scavenger on the carbamoyl-PROXYL half-life were measured. SOD (4 U/g) was injected intraperitoneally into the mice and, soon after the injection, the half-life of carbamoyl-PROXYL was measured. The L-band in vivo EPR system was used for this measurement. L-band EPR measurements were performed according to the previously-reported method, with minor modifications (Hirayama et al. 2003). The L-band in vivo EPR system manufactured by JEOL, Tokyo, Japan, which consists of a 1 GHz microwave unit and a bridged four-gap loop-gap resonator (38 mm diameter and 28 mm long), was used in this study. The signal intensity was measured using ESR-NT software (JEOL). EPR conditions for these in vivo measurements were: magnetic field, 37.0 ± 5.0 mT; modulation width, 0.69 mT; time constant, 0.03 s; microwave power, 0.25 mW; and scanning time, 30 s.

Measurement of GSH level

GSH quantification was performed with the Total Glutathione Quantification Kit (Dojindo Laboratories, Kumamoto, Japan). Kidney tissue (100 mg/kidney) was homogenized in 1 mL of 5% 5-sulfosalicylic acid, and the particulate cellular debris was removed by centrifugation (8000 g) for 10 min at 4 °C. The supernatant was used for the assay. GSH levels in the supernatant were determined according to the manufacturer's protocol by measuring absorbance at 405 nm with a microplate reader.

Measurement of HO-1 level

HO-1 quantification was performed with the Mouse HO-1 EIA Kit (Takara Bio Inc., Shiga, Japan). Kidney tissue (100 mg/kidney) was homogenized in 0.75 mL of phosphate buffered saline containing 1% Nonidet P-40, and the particulate cellular debris was removed by centrifugation (8 000 g) for 5 min at 4 °C. The supernatant was used for the assay. HO-1 levels in the supernatant were determined according to the manufacturer's protocol by measuring absorbance at 450 nm with a microplate reader.

Immunohistochemical staining of 8-nitroguanosine

Kidneys were removed from individual mice either before or 1 or 2 weeks after the induction of diabetes by STZ injection, snap-frozen in acetone/dry ice, and embedded in optimal cutting temperature compound (Sakura Finetechical Co. Ltd., Tokyo, Japan). Sections (3 µm) were immunostained with a monoclonal antibody against 8-nitroguanosine (Dojindo Laboratories) as the primary antibody and detected using the Histofine Kit (Nichirei, Tokyo, Japan). The amount of 8-nitroguanosine immunostaining was quantified using the ImageJ imaging software from NIH. The proportional glomerular area that showed positive 8-nitroguanosine immunostaining was measured from sections (n = 4/group) that contained in excess of 10 glomeruli.

Statistical analysis

All data are expressed as means ± SEM. Multiple data comparisons were performed by using one-way analysis of variance (ANOVA). Significant differences between the groups of mice were analyzed using a t-test for paired samples. P values < 0.05 were considered statistically significant.

	Acknowledgements

 
Authors thank Drs Y. Shimizu and H. Shimamo (University of Tsukuba) for help and discussion. Authors also thank Dr Burton D. Cohen for his warm support for our study. This work was supported by the Grants-in-Aid for Encouragement of Young Scientists, Grant-in-Aid for Scientific Research (C), and Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by ERATO-JST.

	Footnotes

 
Communicated by: Kohei Miyazono

These authors contributed equally to this work.

^* Correspondence: satoruta{at}md.tsukuba.ac.jp

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Accepted: 18 August 2008

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