Abstract
To ascertain the possible roles of nuclear erythroid 2 p45-related factor 2 (Nrf2), a key transcription factor of phase 2 drug-metabolizing enzymes, in renal cellular defense against oxidative stress, wild-type and Nrf2-knockout (–/–) mice were treated with ferric nitrilotriacetate (Fe-NTA) at doses of 3 or 6 mg iron/kg body weight. After Fe-NTA treatment, Nrf2 (–/–) mice consistently showed lower levels of glutathione (GSH) in the kidney at the low dose and the liver at the high dose than the wild-type mice. Gamma-glutamylcysteine ligase (GCL) activity in the kidney and liver of Nrf2 (–/–) mice was also consistently lower than in wild-type mice after the Fe-NTA treatment. Histopathological examination revealed that nephrotoxicity of Fe-NTA, reflected in necrosis of renal tubule epithelial cells following nuclear damage, was more severe in the Nrf2 (–/–) mice than in their wild-type counterparts. Overall, the data suggest that Nrf2 (–/–) mice are unable to compensate for depletion of renal GSH because of oxidative stress, being more susceptible to Fe-NTA-induced nephrotoxicity. In conclusion, the present study showed that Nrf2 might play an important role in protecting cells from oxidative stress in the kidney through its regulation of antioxidant enzymes.
Keywords
Introduction
Molecular mechanisms underlying detoxification and removal of reactive oxygen species (ROS) are of major interest regarding intracellular defense against oxidative stress (Willcox et al., 2004). Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a key transcription factor that controls the constitutional and inducible expression of phase 2 drug-metabolizing enzymes through interaction with the antioxidant-response element (ARE) in their promoter regions (Itoh et al., 1997). Oxidative signals trigger release of Nrf2 from its cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1) and subsequent translocation to the nucleus for transcription of ARE-response genes (Itoh et al., 1999; McMahon et al., 2003). Since phase 2 drug-metabolizing enzymes catalyze various detoxification reactions and conjugation of ROS (Sheweita, 2000), Nrf2 is an important intracellular mediator between oxidative stress and antioxidant responses. Roles of Nrf2 in preventing toxicity caused by oxidative stress can be investigated in detail using Nrf2-null (–/–) mice, which have already been shown to be more susceptible to the hepatotoxicity of the common analgesic acetaminophen than are their wild-type counterparts (Enomoto et al., 2001; Chan et al., 2001). We also have demonstrated that liver DNA of Nrf2-null mice is vulnerable to oxidative stress derived from pentachlorophenol, a mouse liver carcinogen (Umemura et al., 2006). These reports point to a functional deficiency of detoxifying systems regulated by Nrf2, including glutathione (GSH) conjugation, glucuronidation, and quinone reduction. Similar mechanisms responsible for decrease in antioxidant responses have been demonstrated in the lungs and brains of Nrf2 (–/–) mice, in which oxidative damage is also prone to occur (Cho et al., 2002; Rangasamy et al., 2005; Lee et al., 2003). However, it remains unclear how Nrf2 works in the kidney, one major organ for xenobiotic metabolism.
The kidney plays important roles in blood filtration, concentration, and excretion of waste products and electrolytes and reabsorption of nutrients, thus serving as a main organ for maintaining homeostatic condition (Robertson, 1998). Active transport of such metabolites through the renal tubules increases the opportunities for tubule cells to produce or come in contact with harmful agents such as ROS, suggesting that the kidney is at high risk of oxidative stress in its metabolic action, as are the liver and lung. In particular, the kidney, like the liver, is a main organ for GSH (γ-glutamyl-cysteinyl-glycine) biosynthesis catalyzed by γ-glutamyl-cycle enzymes (Lash, 2005), and 2 pivotal enzymes for GSH synthesis, γ-glutamylcysteine ligase (GCL) and GSH synthetase (GSS), are under Nrf2-ARE regulation (Wild et al., 1999; Lee et al., 2005). In fact, it has already been reported that the kidney shows a high level of Nrf2 expression (Itoh et al., 1997).
It is considered that acute nephrotoxicity and renal carcino-genesis in rodents induced by iron chelate ferric nitrilotriacetate (Fe-NTA) resulted from iron-catalyzed oxidative injury (Li et al., 1987; Ebina et al., 1986). Actually, Fe-NTA is known to reduce renal GSH levels by generating hydroxyl radicals via the iron-catalyzed Fenton reaction (Athar and Iqbal, 1998), with an increase of oxidative stress markers such as 8-OHdG and thiobarbituric acid-reactive substances (TBARS; Umemura et al., 1990a, 1990b; Li et al., 1988). In the present study, to cast light on roles of Nrf2 in antioxidant defense in the kidney, the effects of Nrf2-deficiency on development of renal lesions in mice exposed to Fe-NTA were evaluated with reference to levels of GSH, enzymatic GCL activity, histopathological parameters, and lipid peroxidation.
Materials and Methods
Reagent Preparation
Fe-NTA solution was prepared as previously described (Li et al., 1988). Briefly, iron (III) nitrate enneahydrate (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and nitrilotriacetic acid disodium salt (STREM Chemicals, Newburyport, Massachusetts) were separately dissolved in deionized water to form 20- and 40-mmol/L solutions, respectively. These solutions were then mixed immediately before use at the volume ratio of 1:2 (molar ratio, 1:4), and the pH was adjusted to 7.4 by adding sodium bicarbonate.
Animals
Nrf2 (–/–) mice of the ICR/129SV background were produced at University of Tsukuba (Itoh et al., 1997). Wild-type ICR mice were purchased from Charles River Japan Inc. (Kanagawa, Japan). Nrf2 (+/–) mice were mated to yield Nrf2 (–/–) mice, and the genotypes of offspring were determined by the polymerase chain reaction (PCR) with genomic DNA from tail tips. PCR amplification was carried out using 3 different primers: 5′-TGGACGGGACTATTGAAGGCTG-3′ (sense for both genotypes), 5′-GCCGCCTTTTCAGTAGATGGAGG-3′ (antisense for wild type), and 5′-GCGGATTGACCGTAATGGGATAGG-3′ (antisense for LacZ). They were housed in a room with a barrier system and maintained under the following constant conditions: temperature of 24 ± 1°C, relative humidity of 55 ± 5%, ventilation frequency of 18 times/hour, and a 12-hour light–dark cycle with free access to commercial mouse diet CRF-1 (Charles River Japan) and tap water.
Experimental Protocol
Groups of 45 male Nrf2 (–/–) and wild-type mice at 6 weeks of age were used in this study. Males were chosen because they are more susceptible to the oxidative stress and nephrotoxicity of Fe-NTA than females (Li et al., 1988; Ma et al., 1998). In a previous study, the renal TBARS levels of mice treated with 3 mg iron of Fe-NTA peaked at 90 min after treatment and then gradually decreased to the base-line level (Li et al., 1988). Therefore, the animals were given a single intraperitoneal injection of Fe-NTA at the doses of 3 mg (low dose) or 6 mg (high dose) iron/kg body weight and killed at 1, 2, 4, or 24 hours thereafter (n = 5). Five animals of each genotype were killed without injection of Fe-NTA as untreated controls (0 hours). The kidneys and liver of each animal were immediately removed and weighed. A part of each organ was fixed in 10% buffered for-malin and then routinely processed for embedding in paraffin, and 4-μm sections were stained with hematoxylin and eosin (H&E) stain for histopathological study. The remainder was frozen in liquid nitrogen and stored at –80°C for subsequent biochemical analyses.
Measurement of GSH Content and GCL Activity
The GSH content and GCL activity in the kidney and liver were determined according to the method of White et al. (2003). Briefly, tissue was homogenized in TES/SB buffer consisting of 20 mM Tris, 1 mM EDTA, 250 mM sucrose, 20 mM sodium borate, and 2 mM serine. The homogenates were centrifuged at 4°C for 10 minutes at 10,000 g, and then the supernatants were taken. The samples were adjusted to 1 mg protein/ml, and 50 μl of them were mixed with an equal volume of GCL reaction cocktail consisting of 400 mM Tris, 40 mM ATP, 20 mM L-glutamic acid, 2 mM EDTA, 20 mM sodium borate, 2 mM serine, and 40 mM MgCl2 on separate 96-well plates. The GCL reaction was initiated by adding 50 μl of 2 mM cysteine (dissolved in TES/SB buffer) to each GCL activity well (cysteine was not added to the GSH-baseline wells at this time) and the plates were incubated at 37°C for 10 minutes. The GCL reaction was stopped by adding 50 μl of 200 mM 5-sulfosalicylic acid to all wells, and then 50 μl of cysteine was added to the GSH-baseline wells. Following protein precipitation by centrifugation, 20 7mu;l of supernatants from each well were transferred to a 96-well plate designed for fluorescence detection. Next, 180 μl of 2,3-naphthalenedicarboxyaldehyde (NDA; Aldrich Chemical, St. Louis, Missouri) in derivatization solution (50 mM Tris, pH 10, 0.5N NaOH, and 10 mM NDA in Me2SO, v/v/v 1.4/0.2/0.2) was added to all wells, and the plate was incubated in the dark at room temperature for 30 minutes. NDA-gamma-GC or NDA-GSH fluorescence intensity was finally measured (472 ex/528 em) with a Fluoroskan AscentFL fluorescence plate reader (Thermo Labsystems Oy, Vantaa, Finland).
Measurement of TBARS
The levels of lipid peroxidation in the kidneys were determined by the method of Uchiyama and Mihara (1978). Briefly, the kidneys were homogenized with a 9-fold volume of ice-cold KCl solution (1.15%) to form 10% homogenates and were centrifuged at 3,000 rpm for 10 minutes. Fifty-μl aliquots of supernatant were incubated with a reaction mixture consisting of 0.2 ml of 8.1% sodium lauryl sulfate, 3 ml of 0.4% thiobarbituric acid (pH 3.5), and 0.75 ml of distilled water at 95°C for 60 minutes. A standard series of malondialdehyde (MDA) was prepared by hydrolysis of 1,1,3,3, -tetramethoxypropane (Wako). The reaction products were extracted with a mixture of n-butanol and pyridine (15:1, v/v) and separated by centrifugation. The butanol phase was then taken, and its absorbance was measured at 553 nm. Protein assays were performed with BCA protein assay kit (PIERCE Rockford, Illinois) to allow expression of renal TBARS content as nmol MDA/mg protein.
Statistical Evaluation
For statistical analysis, the Student’s t-test was used to compare quantitative data for GSH content, GCL activity, and TBARS between groups.
Results
GSH Content and GCL Activity in the Kidney
Basal GSH levels in the kidneys of Nrf2 (–/–) mice (0 hours) were slightly low but not significantly different from those of wild-type mice. The GSH levels of wild-type mice showed slight decrease after the low-dose treatment of Fe-NTA, followed by gradual recovery up to 24 hours. However, the same dose treatment induced significant decrease of GSH levels in Nrf2 (–/–) mice at 1, 2, and 4 hours after the injection (p < .05 vs. 0 hours), and at 4 and 24 hours, levels were lower than in wild-type mice (p < .05; Figure 1A). The high-dose treatment caused similar and statistically significant reduction of renal GSH levels in both genotypes without recovery to baseline. Basal activity of GCL in the kidneys of Nrf2 (–/–) mice was significantly lower (p < .01) than in wild-type mice (Figure 1B), and this was also the case after the Fe-NTA treatment throughout the experimental period. After the low-dose treatment, GCL activity with both genotypes decreased and thereafter increased gradually to basal levels by 24 hours. On the other hand, the high-dose treatment caused rapid decrease of GCL activity in both genotypes, and the low levels were maintained up to 24 hours.
GSH Content and GCL Activity in the Liver
Basal GSH levels in the livers of wild-type mice were not different from those of Nrf2 (–/–) mice (Figure 2A). Wild-type mice treated with the low dose of Fe-NTA retained almost the basal level of hepatic GSH up to 24 hours. However, the same dose treatment induced temporary but significant decrease at 2 hours in the Nrf2 (–/–) mice (p < .05). Hepatic GSH levels of wild-type mice treated with the high dose of Fe-NTA showed temporary decrease at 2 hours and then returned to the basal level up to 24 hours, while dramatic decrease was evident in the Nrf2 (–/–) mice, with significantly low values at 2, 4, and 24 hours as compared to 0 hours. The lowest hepatic GSH level of the Nrf2 (–/–) mice was observed at 24 hours after the high-dose treatment, being 52% less than that of wild-type mice at this time point. Basal activity of GCL in the livers of Nrf2 (–/–) mice was 41% less than that of wild-type mice (Figure 2B). Hepatic GCL activity of Nrf2 (–/–) mice given the low dose of Fe-NTA increased gradually and, consequently, showed no significant difference from the wild type at 2 and 24 hours. However, GCL activity of the Nrf2 (–/–) mice given the high-dose treatment was markedly decreased at 24 hours, being 51% less than that of wild-type mice.
Histopathology of Kidney
Histopathological changes in the kidneys of mice treated with the low and high doses of Fe-NTA are illustrated in Figures 3 and 4, respectively. After the low-dose treatment with Fe-NTA, nephrotoxicity was observed more severely in the Nrf2 (–/–) mice as compared with the wild-type mice (Figure 3). Pyknosis in proximal tubular epithelial cells was found as an initial lesion, which was much more evident in Nrf2 (–/–) mice than in wild-type mice at 1 hour after the treatment (A, E). The lesion in Nrf2 (–/–) was exacerbated time-dependently from karyorrhexis to karyolysis; extensive tubular necrosis consequently became apparent (F, G, H). However, the magnitude of these lesions was relatively smaller in the wild-type mice (B, C, D). The incidences of histopathological lesions are summarized in Table 1. Pyknosis was also found in the distal straight tubular cells, the downstream target exposed to redox-active iron (Kawabata et al., 1997), of the Nrf2 (–/–) mice.
With the high dose of Fe-NTA, the same histopathological lesions as at the low dose were extensively observed in the kidneys of both genotypes (Figure 4). Despite differences’ being less clear than at the low dose, the levels of the lesions were relatively milder in the wild-type mice (A, B, C) as compared with the Nrf2 (–/–) mice (E, F, G). Especially at 24 hours, while tubules with normal appearance were still found among necrotic tubules in wild-type mice (D), the necrotic areas in Nrf2 (–/–) mice consisted almost of necrotic tubules filled with debris and degenerative tubules with pyknosis (H). The incidences of histopathological lesions are summarized in Table 2. In the distal straight tubules, pyknosis and karyorrhexis were more frequent in the Nrf2 (–/–) mice than in wild-type mice.
Oxidative Damage
The levels of lipid peroxidation in the kidney were determined by measuring amounts of TBARS (Figure 5). Fe-NTA treatments caused dose-dependent accumulation of renal TBARS, and both the low-dose and high-dose treatments caused peaks in TBARS levels in both genotypes at 1 hour after the injection. With the low-dose treatment, significant TBARS accumulation was observed at 1, 2, and 4 hours (p < .01 vs. 0 hours) in the Nrf2 (–/–) mice and 1 hour (p < .01) and 2 hours (p < .05) in the wild-type mice. The levels of TBARS accumulation were not significantly different between the two genotypes. With the high-dose treatment, TBARS levels were significantly elevated (p < .01) at 1, 2, and 4 hours after the treatment in both genotypes. Nrf2 (–/–) mice showed significantly higher TBARS levels at 24 hours than the wild-type mice.
Discussion
GSH homeostasis is maintained in organs intensely exposed to exogenous toxins and oxidative stress such as the liver, kidneys, lungs, and intestine (Kretzschmar, 1996). Reduction of electrophiles and conjugation of hydrophobic compounds are important roles of GSH in protecting cells against deleterious agents. GCL, whose gene expression is controlled through the Nrf2-ARE interaction in its promoter region, is a rate-limiting enzyme of GSH biosynthesis (Chan and Kwong, 2000). The present data demonstrated that there were no differences between GSH levels in the kidneys of the no-treated Nrf2 (–/–) mice and their wild-type counterparts. However, from 4 hours after Fe-NTA treatment at the low dose, GSH levels in Nrf2 (–/–) mice were significantly lower than those in the wild-type mice. There was no significant induction of GCL activity and GSH accumulation in the wild-type mice after the Fe-NTA treatment. The possible explanation is that the acute renal injury caused by the treatment might obstruct the normal induction response in the wild-type mice. However, the constant levels of GCL activity in the wild-type mice might play a role in protecting GSH levels from decreasing after the Fe-NTA treatment. Taking into consideration sustained lower levels of renal GCL in Nrf2 (–/–) mice as compared with those in the wild-type mice, the results suggest that Nrf2 (–/–) mice lack the ability to compensate for GSH consumption because of oxidative stress in the kidney. At the high dose, GSH levels were reduced in the wild-type mice with the same profile as in the Nrf2 (–/–) mice, albeit with significant differences in GCL levels between the two through the experimental period except at 2 hours. It might be assumed that GSH depletion caused by the high dose might exceed the supply from the Nrf2-GCL system.
In contrast, in the livers at the high dose but not the low dose, remarkable differences in GSH levels between the Nrf2 (–/–) and the wild-type mice were found. Fe-NTA also causes a certain level of oxidative stress in the liver as well as kidney (Li et al., 1988; Iqbal et al., 1995). However, hepatic GSH is not only used in hepatic metabolism but also is exported to other tissues for delivering reduced cysteinyl residues via the blood circulation (Ookhtens and Kaplowitz, 1998). Accordingly, the supply of GSH from the livers to the kidneys might occur. These results may imply the possibility that Nrf2 works for prevention of systemic oxidative stress.
The present histopathological survey of renal tissue provided clear evidence that Nrf2 (–/–) mice are more susceptible than wild-type counterparts to Fe-NTA-induced nephrotoxicity mediated by oxidative stress from the iron-catalyzed Fenton reaction (Toyokuni, 2002). This was supported by the observation of consistent deficiency of GSH synthesis in the kidneys of Nrf2 (–/–) mice. Although GSH metabolites cysteine and cysteinyl-glycine are required for the expression of nephrotoxicity of Fe-NTA (Okada et al., 1993), tissue susceptibility to oxidative stress largely depends on the internal levels of antioxidants. For instance, several antioxidants have been reported to decrease oxidative damage and ameliorate the nephrotoxicity of Fe-NTA (Umemura et al., 1991; Umemura et al., 1996; Zhang et al., 1997). It has also been reported that the artificial depletion of endogenous GSH by L-buthionine-sulfoximine (BSO), a potent inhibitor of GSH synthesis, leads to increase in susceptibility to oxidative stress (Chen et al., 2005; Chung et al., 2005; Seckin et al., 2001). Based on the low level of GCL activity, therefore, it is suggested that constitutional decline of GSH synthesis is involved in the enhanced susceptibility to oxidative damages in Nrf2 (–/–) mice.
The levels of TBARS were not significantly different between genotypes, being highest at 1 hour after the Fe-NTA treatment. A previous report revealed significant increase of TBARS as early as 30 minutes after intraperitoneal injection of Fe-NTA to A/J mice (Li et al., 1988). Therefore, in the light of our finding that morphological alterations were already apparent at 1 hour in Nrf2 (–/–) mice, the maximum level of TBARS in the Nrf2 (–/–) mice might be reached earlier than 1 hour after treatment. Judging from the histopathological features, the higher level of TBARS in the Nrf2 (–/–) mice observed at 24 hours might be linked to the debris filling proximal tubule cells.
In conclusion, our results suggest that Nrf2 plays a pivotal role in cell defenses against oxidative stress in the kidney by controlling the intracellular antioxidant states. In addition, the possibility of Nrf2 participation in prevention of systemic oxidative stress can be proposed.
Footnotes
Acknowledgment
The authors thank M. Maeda, A. Kaneko, and F. Takaki for their technical help. This work was supported in part by a grant for the Research Fellow of the Japan Society for the Promotion of Science and a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.