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Aggravation of ischemia–reperfusion-triggered acute renal failure in xCT-deficient mice
Archives of Biochemistry and Biophysics 490 (2009) 63–69
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Aggravation of ischemia–reperfusion-triggered acute renal failure in xCT-deficient mice Tomohiro Shibasaki a,b,c,d, Yoshihito Iuchi a,b,c, Futoshi Okada a,b,c, Kazuho Kuwata e, Takuya Yamanobe d, Shiro Bannai e, Yoshihiko Tomita d, Hideyo Sato e, Junichi Fujii a,b,c,* a
Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Japan Respiratory and Cardiovascular Diseases Research Center, Research Institute for Advanced Molecular Epidemiology, Yamagata University, Japan Global COE Program for Medical Sciences, Japan Society for the Promotion of Science, Japan d Department of Urology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan e Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan b c
a r t i c l e
i n f o
Article history: Received 19 June 2009 and in revised form 10 August 2009 Available online 18 August 2009 Keywords: Cystine transporter Oxidative stress Ischemia reperfusion Acute renal failure
a b s t r a c t This study examined the question of whether deficiency of xCT, a cystine-transporter gene, exacerbates ischemia–reperfusion-induced acute renal failure (ARF). Two weeks after the right nephrectomy of male mice at 16–18 weeks of age, the left renal vessels were clamped for 45 min to induce renal ischemia. After (24 h) induction of ischemia, xCT/ mice had elevated concentrations of blood urea nitrogen and creatinine indicative of ARF, while in xCT+/ and xCT+/+ mice, these parameters did not differ from the shamoperated mice. Immunohistochemical analyses of kidneys using antibodies against the oxidative stress markers revealed stronger staining in xCT/ mice compared with xCT+/+ mice. Induction of xCT mRNA in the kidneys of xCT+/+ mice was demonstrated using reverse transcriptase (RT)-PCR analysis and was further confirmed using quantitative RT-PCR. These data provide the first in vivo evidence that xCT is induced by oxidative stress and helps prevent ischemia–reperfusion injury to kidneys. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Ischemia induces various pathogenic processes that are involved in acute renal failure (ARF),1 such as endothelial cell injury and invasion of inflammatory cells. Because the mitochondrion is the organelle that is most affected by renal ischemia, depletion of ATP occurs during ischemia reperfusion [1,2]. The resultant purine metabolites become substrates for xanthine oxidase, which produces superoxide anions that initiate a radical chain reaction and generate other reactive oxygen species (ROS) [3]. These ROS cause oxidation of lipids, proteins, and DNA, which consequently leads to cellular dysfunction [4]. In addition, ischemia–reperfusion-induced ARF is associated with an inflammatory response [5]. Upon activation, neutrophils adhere to endothelial cells and participate in this pathogenic process. Because activated neutrophils generate large amounts of ROS via NADPH oxidase and myeloperoxidase, neutrophils that migrate to the inflammatory lesion are another source of
* Corresponding author. Address: Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. Fax: +81 23 628 5230. E-mail address: [email protected] (J. Fujii). 1 Abbreviations used: ARF, acute renal failure; ROS, reactive oxygen species; GPX, glutathione peroxidase; BUN, blood urea nitrogen; Cr, creatinine; HE, hematoxylin and eosin; HNE, anti-4-hydroxy-2-nonenal. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.08.008
ROS [6]. As a result, the kidneys are exposed to severe oxidative stress, which exacerbates the negative consequences of ARF. Recently, we showed that SOD1-deficiency exacerbates ARF in mice [7], demonstrating that detoxification of superoxide plays an important role in renal protection during ARF. In addition to anti-oxidative enzymes, low molecular weight antioxidants, such as glutathione, play pivotal roles in the protection of most tissues against ischemic injury. Besides its direct ROS-scavenging function, glutathione donates electrons to glutathione peroxidase (GPX) and other anti-oxidative systems. Levels of glutathione are maintained by both de novo synthesis from constituent amino acids and recycling by glutathione reductase, which uses NADPH as an electron donor. In cultured cells, cysteine availability determines the rate of glutathione synthesis [8]. In extracellular fluid, cysteine undergoes spontaneous auto-oxidation to cystine. Cystine is then taken up via the cystine/glutamate exchange transport system, which has been designated ‘‘system xc.” Intracellular cystine is simultaneously reduced to cysteine and utilized for glutathione and protein synthesis. Thus, system xc activity indirectly controls intracellular glutathione availability in various cells, such as peritoneal macrophages and neutrophils [9–11]. System xc is composed of two proteins, xCT and 4F2hc; the transport activity of xc has been attributed to the xCT protein [12]. Various stimuli, including electrophilic agents such as diethyl maleate [13], oxygen [14], and bacterial lipopolysaccharide [15],
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increase system xc activity in cultured cells via induction of xCT expression. The induction of xCT by diethyl maleate is regulated by an antioxidant response element located in the 50 -flanking region of xCT, which is also referred to as the electrophile response element [13]. Binding of the transcription factor, Nrf2, to the response element enhances transcription of xCT. On the other hand, cysteine enters cells via neutral amino acid transporters. Because xCT is not expressed in kidneys under normal conditions, cellular cysteine entry via neutral amino acid transporters appears adequate under these conditions. In cultured cells, expression of xCT is induced by various stimuli, including oxidative stress [13–15], while bacterial lipopolysaccharide induces xCT in various mouse tissue [16]. However, whether xCT has a functional role in organs that do not express it in significant amounts, such as kidneys, remains to be determined. Previously, we generated xCT-knockout mice and characterized their phenotype [17]. xCT/ mice appear healthy, but have higher concentrations of cystine in plasma compared with their wild-type littermates. Double-knockout (xCT and SOD1) mice show no exacerbation of the deleterious SOD1-deficient phenotype in the circulatory system [18]. In the present study, the physiological relevance of xCT in kidneys during ischemia–reperfusion injury was examined using xCT-knockout mice. We present the first in vivo evidence that xCT is induced by ischemia reperfusion and protects kidneys against oxidative stress during ARF. Materials and methods Animals xCT-knockout mice of 129/Svj-C57BL/6J mixed genetic backgrounds were created as described previously [17]. Then they were back-crossed with C57BL/6J mice more than 10 times. Genotypes were verified using PCR (Bio-Rad, Tokyo, Japan) with gene-specific primers. The animals were housed under specific pathogen-free conditions at a constant temperature of 20–22 °C with a 12-h alternating light–dark cycle on a regular chow diet. Animal experiments were performed in accordance with the Declaration of Helsinki under a protocol approved by the Animal Research Committee of this institution. Surgery and experimental design The surgical manipulation of kidneys used to induce ARF in the mice was described previously [7]. Briefly, 2 weeks prior to the start of the study, the right kidneys of male mice (14–18 weeks of age) were removed through a small dorsal incision made after administration of pentobarbital anesthesia (40 mg/kg i.p.). After 2 weeks of recovery from the nephrectomy, the mice were anesthetized with pentobarbital (40 mg/kg i.p), and the left kidneys were exposed through a small dorsal incision. To induce ischemia, the left renal artery and vein were clamped for 45 min. At the end of the ischemic period, the clamp was released and blood was allowed to reperfuse the kidneys. For the sham operation, the mice were manipulated in the same manner without clamping of the left renal vessels.
istry. Kidneys were excised and bisected. One half was frozen in liquid nitrogen, and stored at 80 °C for determination of protein. The other half was fixed in Bouin’s solution overnight, immersed sequentially in 50%, 75% and 99% ethanol for 24 h in each solution, embedded in paraffin, and cut into 4-lm sections for histochemical analysis and hematoxylin and eosin (HE) staining. The tissue sections were incubated with 1% hydrogen peroxide in methanol. After rinsing, the sections were incubated for 60 min with blocking solution A (Histofine mouse stain kit, Nichirei, Tokyo) at room temperature, followed by incubation with a mouse anti-8-hydroxy guanine (8-OHdG) monoclonal antibody (MOG-100 at a concentration of 5.0 lg/ml; Nikken Foods Co. Inc., Shizuoka, Japan), an anti4-hydroxy-2-nonenal (HNE) monoclonal antibody (MHN-100; Nikken Seil, Shizuoka), and anti-nitrotyrosine (NT) polyclonal antibody (Millipore, Bedford, MA) overnight in a humidified chamber at 4 °C. After rinsing, the sections were incubated for 10 min at room temperature with blocking solution B (Histofine mouse stain kit, Nichirei, Tokyo). Blocking solutions A and B were intended to inhibit non-specific binding of the antibody to endogenous mouse immunoglobulin. A MAX-PO complex, in which the Fab’ portion of the secondary antibody was conjugated with an amino acid polymer and peroxidase (Histofine mouse stain kit, Nichirei, Tokyo) was placed on the tissue for 10 min. After a final rinse, the tissue sections were exposed to the chromogen, 3,30 -diaminobenzidine (Nichirei, Tokyo), for detection of specific immunolabeling. The reaction of 3,30 -diaminobenzidine with peroxidase was stopped by washing the tissues with distilled water. The sections were then dehydrated, mounted, and photographed. SDS–PAGE and immunoblot analysis Kidneys were homogenized in PBS and centrifuged at 10,000 rpm in a microcentrifuge. Soluble proteins were assayed using a BCA kit (Pierce) with bovine serum albumin as the standard. SDS–PAGE and immunoblot analyses of kidney proteins were also performed. Protein samples were subjected to 10–15% SDS– PAGE and then transferred to a HybondÒ nitrocellulose membrane (Amersham Pharmacia) under semi-dry conditions by means of a Transfer-blot SD semi-dry transfer cell (Bio-Rad). Non-specific binding sites on the membrane were then blocked by incubation with 5% non-fat milk in 150 mM NaCl and 20 mM Tris/HCl, pH 7.6 (TBS) for 1 h at room temperature. The membranes were then incubated with antibodies specific for the gene products of SOD1, SOD2 [7], aldose reductase (AKRB1) [19], aldehyde reductase (AKRA1) [20], cytosolic glutathione peroxidase (GPX1) [21], and glutathione reductase (GSR) [7] overnight at 4 °C or for 2 h at room temperature. After washing with TBS containing 0.1% Tween-20 (TBST), the membrane was incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After washing with TBST, the peroxidase activity on the membranes was detected using a chemiluminescent method with an ECL PlusÒ kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), followed by membrane exposure to X-ray film (Kodak, Rochester, USA). Detection of xCT mRNA by RT-PCR and quantification by real-time PCR
Blood measurements Blood urea nitrogen (BUN) and creatinine (Cr) concentrations in plasma were determined using commercially available test systems (Wako Pure Chemicals, Osaka, Japan) as described previously [7]. Immunohistochemistry Mice were euthanized under ether anesthesia and kidneys were harvested for subsequent immunoblotting and immunohistochem-
Total cellular RNA was prepared from the cells in triplicate using Isogen (Wako). First strand cDNA was synthesized from total RNA (1 lg) using oligo-dT12–18 as a primer. A pair of primers, 50 -TT GCAAGCTCACAGCAATTCTG-30 in exon 4 and 50 -CAGGGTTGTCTACT TCTTCAGT-30 in exon 6 that were synthesized based on mouse xCT exon sequences (GenBank/European Molecular Biology Laboratory/ DNA Databank of Japan Accession No. AB022345) were used to amplify 200 bp of the xCT cDNA in a conventional PCR (PCR Thermal cycler, Takara, Tokyo). As an internal standard, a 104-bp b-actin
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cDNA was amplified using a pair of primers: 50 -TGAGGAGCACCCT GTGCT-30 in exon 3 and 50 -ACATGGCTGGGGTGTTGAAG-30 in exon 4. RT-PCR products of the xCT cDNA and b-actin cDNA were separated on a 1% agarose gel and visualized by staining with ethidium bromide. The same primers were used for quantification of the xCT cDNA and b-actin cDNA by a real-time PCR (Light Cycler, Roche). The abundance of xCT mRNA was calculated relative to b-actin mRNA. Measurement of total glutathione Total glutathione was extracted with 5% trichloroacetic acid and then measured using an enzymatic method [22] that is based on the reduction of 5,50 -dithiobis(2-nitrobenzoic acid) by the glutathione reductase system. We measured only total glutathione content because most of the glutathione extracted from the cells was in a reduced form, and there is no reliable standard method to accurately measure the content of oxidized glutathione [23]. Assay for lipid peroxidation products Thiobarbituric acid-reactive substances (TBARS) in renal tissues were determined as described previously [18]. Precipitates from kidneys, prepared as described for the immunoblot analysis, were used for the assay. A suspension of the precipitated materials in PBS was added to and then mixed thoroughly with 0.6 ml of a solution containing 15% trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, 0.25 M HCl, and 1.8 mM butylhydroxytoluene. The solution was heated for 15 min in boiling water, cooled in ice-cold water, and centrifuged at 10,000g for 10 min. The absorbance of the sample was measured at 535 nm. We used a solution of 1,1,3,3-tetraethoxypropane to standardize the experiment. TBARS content was estimated using an extinction coefficient of 1.56 105 M1 cm1. Statistical analysis Statistical analyses of the data were carried out using a Mann– Whitney U-test. The number of samples is shown in parentheses in each figure. A P-value of P < 0.05 was considered significant (*P < 0.05; **P < 0.01). Results Comparison of renal function of mice under normal conditions We first examined how xCT deficiency affects kidney function under normal conditions. Body weight, kidney weight, and serum levels of BUN and Cr did not differ significantly among xCT+/+, xCT+/, and xCT/ mice at 16–18 weeks of age (Fig. 1). Histological examination of kidneys showed no differences among groups (data not shown). This observation is consistent with the absence of xCT expression in kidneys under normal conditions [17]. Comparison of kidney function after ischemia-reperfusion injury We then examined the effects of ischemia reperfusion on kidney function in 16–18-week old xCT+/+, xCT+/, and xCT/ male mice. Mice were right-nephrectomized 2 weeks prior to the start of the study. Ischemic ARF was induced by clamping the left renal blood vessels for 45 min followed by reperfusion. We examined renal function 24 h after the induction of ischemia. Ischemic AFR was examined at the acute stage at 24 h after ischemia reperfusion, because, based on our preceding study using SOD1-knockout mice, renal damage was evident at this period and was reversed thereafter [7]. According to the results, the xCT/ mice had greater serum
Fig. 1. Renal function of xCT-deficient mice under normal conditions. Male mice (16–18-week old) with the indicated genotypes were euthanized and body (A) and kidney (B) weights were obtained. Plasma concentrations of BUN (C) and Cr (D) were assayed. Means + SEM are shown.
concentrations of BUN and Cr than both the xCT+/+and xCT+/ mice (Fig. 2), suggesting that xCT/ mice had an increased severity of ARF. Thus, xCT deficiency exacerbated the symptoms of ischemic ARF, despite the fact that xCT is not expressed in kidneys under normal conditions. Histological examination of kidneys after ischemia reperfusion Renal injury, characterized by proximal tubular expansion, vacuolation of proximal tubular epithelial cells, and defluvium of the proximal tubule epithelium, was more severe in xCT/ mice than that in xCT+/+ mice (Fig. 3). However, glomerular injury was mild compared with the damage observed in epithelial cells. This result is consistent with the previous observation that endothelial damage is a classic symptom of ischemia-induced ARF [24]. Results of immunohistochemical examination showed that after renal ischemia–reperfusion 8-OHdG immunoreactivity was detected in both the glomerulus and the proximal tubules in xCT+/+ mice, and it was enhanced in xCT/ mice. Staining for HNE, a lipid peroxidation product, in the proximal tubules was more pronounced in xCT/ mice compared with xCT+/+ mice. There was an increase in immunostaining by an anti-nitrotyrosine (NT) antibody, especially in the kidneys of xCT/ mice, during ischemia reperfusion. These findings suggest that ROS were produced as a consequence of renal ischemia reperfusion and that the reactive species are involved in the pathogenesis of ARF, especially in xCT/ mice. Levels of anti-oxidative/detoxificating enzymes in kidneys after ischemia reperfusion To examine the role of anti-oxidative-detoxifying enzymes, we examined the amount of CuZn-superoxide dismutase (SOD1), Mnsuperoxide dismutase (SOD2), cytosolic glutathione peroxidase (GPX1), glutathione reductase (GSR), aldehyde reductase (AKR1A), and aldose reductase (AKR1B) protein after ischemia–reperfusioninduced ARF in the different genotypes of mice (Fig. 4). The superoxide dismutases, SOD1 and SOD2, scavenge superoxide, which is
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Fig. 2. Renal function parameters of male mice 24 h after ischemia reperfusion. After 45 min of ischemia, kidneys were reperfused. Plasma concentrations of BUN (A) and Cr (B) were measured in xCT+/+, xCT+/, and xCT/ mice (16–18-week old) 24 h after either ischemia reperfusion (I/R) or sham operation (sham). Means + SEM are shown.
the primary oxygen radical produced by donation of one electron. GPX1 and GSR are glutathione-related anti-oxidative/redox enzymes that are abundant in kidneys. Aldo–keto reductase 1A and 1B (AKR1A and AKR1B) are the predominant aldo–keto reductases in kidneys [25], which protect against oxidative stress by reducing toxic carbonyl compounds present in urine. Levels of each of these
enzymes in xCT+/+, xCT+/ and xCT/ mice were not altered by ischemia reperfusion compared with the sham operation. However, we could not examine levels of the xCT protein because no specific antibody against the xCT protein was available. Thus, xCT deficiency appears to have little or no effect on renal expression of these genes during ischemia reperfusion.
Fig. 3. Immunohistochemical analyses of renal oxidative stress markers after ischemia reperfusion. Renal sections obtained 24 h after sham operation or ischemia reperfusion were allowed to react with an anti-8-OHdG, an anti-HNE antibody, and an anti-NT and were stained with HE. Typical data from several experiments are shown.
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Fig. 4. Immunoblot analysis of renal anti-oxidative/redox proteins. Soluble proteins were isolated from the kidneys of mice 24 h after either ischemia reperfusion (I/R) or sham operation (sham), and were subjected to immunoblot analysis using antibodies against SOD1, SOD2, GPX1, GSR, AKR1A, and AKR1B. GAPDH was used as a loading control. Typical data from several experiments are shown.
Induction of xCT gene expression in kidneys after ischemia reperfusion Although xCT expression is normally at a level that is not detectable in kidneys by Northern blotting [12,16], ischemia–reperfusion-induced AFR in xCT/ mice was more severe than that in xCT+/+ and xCT+/ mice. This observation can be reasonably explained if xCT expression is induced in mice with a wild-type allele. Therefore, we performed RT-PCR analysis of total RNA isolated from the kidneys. As expected from the results showing exacerbation of kidney function in xCT/ mice, xCT expression was elevated in xCT+/+ mice after ischemia reperfusion (Fig. 5A). We then performed quantitative PCR to assure the induction of xCT in xCT+/+ mice. The induction of xCT mRNA was further supported by quantitative PCR, which showed a 20-fold increase in renal expression in xCT+/+ mice after ischemia reperfusion (Fig. 5B). Thus, xCT was induced by ischemia reperfusion and appeared to play a protective role during AFR. Because the cystine that is transported into cells via system xc is used to synthesize glutathione, which protects against oxidative stress, we measured levels of both total glutathione and lipid peroxidation products in the kidneys of xCT+/+ and xCT/ mice. However, there was no significant difference in the levels of either total glutathione (Fig. 6) or lipid peroxidation products (data not shown) between the two groups.
Fig. 5. Induction of xCT expression in kidneys after ischemia reperfusion. RT-PCR analysis was performed for total RNA isolated from kidneys 24 h after either ischemia reperfusion (I/R) or sham operation (sham) (A). Quantitative xCT mRNA data were obtained by quantitative RT-PCR using a Light Cycler. The relative abundance of xCT mRNA to b-actin mRNA was presented. Means + SD are shown (B).
that radical scavengers, SOD [4] and edaravone [29], and an antioxidant, N-acetyl cysteine [30], effectively ameliorate the consequences of ischemic injury. Moreover, attenuated expression of SOD1, glutathione peroxidase, and catalase has been reported in ischemia–reperfusion-induced ARF [31,32]. The results of the present study indicate that xCT deficiency exacerbates ischemia–reper-
Discussion In this study, we examined the role of system xc in renal ischemia–reperfusion injury using xCT-deficient mice. After ischemia– reperfusion injury, renal oxidative damage in xCT/ mice was more severe than that observed in either the xCT+/+ or xCT+/ mice (Fig. 3). Although xCT is not expressed in the kidneys of xCT+/+ mice under normal conditions, its expression was induced by ischemia– reperfusion injury (Fig. 5). This result suggests that xCT is induced by ischemia–reperfusion injury to protect against the cellular damage caused by ARF and by other pathological conditions associated with oxidative stress. Oxidative and nitrosative stress have been implicated in the pathogenesis of ischemic ARF [26,27], and the production of ROS during ischemic injury has been demonstrated [28]. Involvement of ROS in the pathogenesis of ARF is supported by the observation
Fig. 6. Total glutathione and lipid peroxidation products. Levels of total glutathione in kidneys were measured in xCT+/+ and xCT/ mice (16–18-weeks old) 24 h after either ischemia reperfusion (I/R) or sham operation (sham). Means + SEM are shown.
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fusion-induced ARF. A protective role of xCT against the oxidative stress induced by renal ischemia–reperfusion injury was supported by the increase in oxidative stress markers, 8-OHdG and HNE, in the xCT/ mice compared with xCT+/+ and xCT+/ mice (Fig. 3). We found severe proximal tubule damage in xCT/ mice. Ischemic ARF results in various types of kidney damage, and the mitochondrion appears to be the organelle most affected by renal ischemia reperfusion [1,2]. Glutathione is present at high concentrations in mitochondria where it protects against oxidative damage. In addition to mitochondrial production, ROS are generated by the action of xanthine oxidase during purine metabolism [3] and by neutrophils attached to endothelial cells, as seen during most inflammatory processes [5]. Activated neutrophils produce ROS via two major enzymatic systems: NADPH oxidase, which produces superoxide by transfer of electrons from NADPH to molecular oxygen; and, myeloperoxidase, which produces HOCl [6]. While the resulting ROS protect against infection at sites of inflammation, they also damage nearby cells. Thus, endothelial cells can be damaged by the ROS released from activated neutrophils. Transplantation of functional endothelial cells into kidneys has a renoprotective effect [33]. Oxidative stress also elevates production of nitric oxide (NO) by inducing transcription of the nitric oxide synthase gene (NOSII). Regarding reactive nitrogen oxide species, the harmful molecule that is directly responsible for kidney damage during ARF appears to be peroxynitrite [24], which results from the reaction between NO and superoxide. Because SOD scavenges superoxide and, hence, suppresses peroxynitrite formation, the levels of nitrotyrosine, which is produced by the reaction between tyrosine and peroxynitrite [34], are elevated in the kidneys of SOD1/ mice [7]. In xCT-deficient kidneys, nitrotyrosine levels increased slightly (Fig. 3) and may also be involved in deterioration of ARF. We recently generated SOD1/; xCT/ double-knockout mice, and found no significant exacerbation of the SOD-deficiency in any of the organs examined, including kidneys [18]. This result suggests that the oxidative stress caused by deficiency of both SOD1 and xCT is insufficient to cause renal damage, and that ischemia–reperfusion injury is responsible for induction of ARF. Glutathione plays multiple functions. It acts directly as an antioxidant to neutralize deleterious ROS, and it is also a cofactor for antioxidant enzymes, including glutathione peroxidase, glutathione S-transferase, and glutaredoxin. Moreover, cell proliferation and apoptosis are also regulated by glutathione [35]. Oxidative stress perturbs cellular glutathione levels either by affecting glutathione biosynthesis or by altering the intracellular ratio of reduced and oxidized forms, thereby affecting multiple physiological responses. Because xCT is required for the provision of cysteine for glutathione synthesis, xCT deficiency could limit glutathione availability. In addition, genes that are involved in either glutathione synthesis (GCLM, GCLC, and xCT) or in glutathione recycling via reduction (GSR) are induced during oxidative stress. Furthermore, Nrf2 is known to play an essential role in the induction of these genes [13,36–39]. When we measured total glutathione levels in kidneys, there was no significant difference in total glutathione levels (Fig. 6). The damage caused by ARF was limited to specific regions of the kidney. Most of the damage was localized to the proximal tubules, which constitute only a small part of kidneys. Thus, changes in the glutathione levels in the damaged cells may not affect either total glutathione content in the entire kidney. It is also possible that glutathione levels had recovered to physiological levels via induction of xCT by the time they were assayed. Here we have measured only total glutathione levels in the kidneys. An increase in the oxidized form of glutathione is observed in many diseases [40,41], and, hence, the significance of individual measurement of both the reduced and the oxidized forms of glutathione is recognized. However, standard values for these measures
in biological samples are lacking because there is no consensus or standardization of assay methods [23]. Other than glutathione synthesis, xCT is involved in the maintenance of extracellular redox balance, which is defined by the ratio of cysteine to cystine, i.e. [cysteine/cystine]. Recently, we demonstrated that xCT overexpression maintained integrity of Burkitt’s lymphoma cells that were depleted of bulk glutathione as a result of incubation with L-buthionine sulfoximine [42]. This finding shows that xCT protects against oxidative stress by driving a highly efficient cystine/cysteine redox cycle. Moreover, this redox cycle by itself, i.e., without participation of the intracellular glutathione system, is regarded as one of the most effective anti-oxidative systems in culture. Because renal ischemia reperfusion would shift the extracellular redox balance to the oxidized state, the exacerbation of ARF observed in xCT-deficient mice may have resulted from a failure to restore the redox state. The observation that N-acetyl cysteine improves ischemia–reperfusion-induced renal damage [30] supports the hypothesis that an xCT-mediated redox cycle protects against ARF. Additional studies are required to determine if the xCT-driven redox cycle is functional in vivo. In conclusion, while xCT may not be essential for renal function under normal conditions, xCT expression is induced during severe oxidative stress, such as that induced by ischemia–reperfusion injury, and exerts an anti-oxidative function. Thus, xCT belongs to a collection of genes that are induced in kidneys during oxidative stress and function in a coordinated manner to protect cells against oxidative injury during AFR. Acknowledgments This work was supported, in part, by the Global COE Program (F03) from the Japan Society for the Promotion of Science. References [1] D.L. Cruthirds, L. Novak, K.M. Akhi, P.W. Sanders, J.A. Thompson, L.A. MacMillan-Crow, Arch. Biochem. Biophys. 412 (2003) 27–33. [2] D.L. Cruthirds, H. Saba, L.A. MacMillan-Crow, Arch. Biochem. Biophys. 437 (2005) 96–105. [3] R. Harrison, Free Radic. Biol. Med. 33 (2002) 774–797. [4] M.S. Paller, J.R. Hoidal, T.F. Ferris, J. Clin. Invest. 74 (1984) 1156–1164. [5] J.V. Bonventre, A. Zuk, Kidney Int. 66 (2004) 480–485. [6] M.B. Hampton, A.J. Kettle, C.C. Winterbourn, Blood 92 (1998) 3007–3017. [7] T. Yamanobe, F. Okada, Y. Iuchi, K. Onuma, Y. Tomita, J. Fujii, Free Radic. Res. 41 (2007) 200–207. [8] S. Bannai, N. Tateishi, J. Membr. Biol. 89 (1986) 1–8. [9] S. Bannai, E. Kitamura, J. Biol. Chem. 255 (1980) 2372–2376. [10] H. Watanabe, S. Bannai, J. Exp. Med. 165 (1987) 628–640. [11] Y. Sakakura, H. Sato, A. Shiiya, M. Tamba, J. Sagara, M. Matsuda, N. Okamura, N. Makino, S. Bannai, J. Leukoc. Biol. 81 (2007) 974–982. [12] H. Sato, M. Tamba, T. Ishii, S. Bannai, J. Biol. Chem. 274 (1999) 11455–11458. [13] H. Sasaki, H. Sato, K. Kuriyama-Matsumura, K. Sato, K. Maebara, H. Wang, M. Tamba, K. Itoh, M. Yamamoto, S. Bannai, J. Biol. Chem. 277 (2004) 44765– 44771. [14] S. Bannai, H. Sato, T. Ishii, Y. Sugita, J. Biol. Chem. 264 (1989) 18480–18484. [15] H. Sato, K. Fujiwara, J. Sagara, S. Bannai, Biochem. J. 310 (1995) 547–551. [16] K. Taguchi, M. Tamba, S. Bannai, H. Sato, J. Inflamm. 4 (2007) 20. [17] H. Sato, A. Shiiya, M. Kimata, K. Maebara, M. Tamba, Y. Sakakura, N. Makino, F. Sugiyama, K. Yagami, T. Moriguchi, S. Takahashi, S. Bannai, J. Biol. Chem. 280 (2005) 37423–37429. [18] Y. Iuchi, N. Kibe, S. Tsunoda, F. Okada, S. Bannai, H. Sato, J. Fujii, Mol. Cell. Biochem. 319 (2008) 125–132. [19] M. Takahashi, J. Fujii, E. Miyoshi, A. Hoshi, N. Taniguchi, Int. J. Cancer 62 (1995) 749–754. [20] M. Takahashi, J. Fujii, T. Teshima, K. Suzuki, T. Shiba, N. Taniguchi, Gene 127 (1993) 249–253. [21] T. Fujii, T. Endo, J. Fujii, N. Taniguchi, Free Radic. Res. 36 (2002) 1041–1049. [22] F. Tietze, Anal. Biochem. 27 (1969) 502–522. [23] P. Monostori, G. Wittmann, E. Karg, S. Túri, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. (2009) [Epub ahead of print]. [24] M.S. Goligorsky, S.V. Brodsky, E. Noiri, Kidney Int. 61 (2002) 855–861. [25] J.Y. Jung, Y.H. Kim, J.H. Cha, K.H. Han, M.K. Kim, K.M. Madsen, J. Kim, Am. J. Physiol. Renal Physiol. 283 (2002) F481–F491. [26] E. Noiri, A. Nakao, K. Uchida, H. Tsukahara, M. Ohno, T. Fujita, S. Brodsky, M.S. Goligorsky, Am. J. Physiol. Renal Physiol. 281 (2001) F948–F957.
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Aggravation of ischemia–reperfusion-triggered acute renal failure in xCT-deficient mice Tomohiro Shibasaki a,b,c,d, Yoshihito Iuchi a,b,c, Futoshi Okada a,b,c, Kazuho Kuwata e, Takuya Yamanobe d, Shiro Bannai e, Yoshihiko Tomita d, Hideyo Sato e, Junichi Fujii a,b,c,* a
Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, Japan Respiratory and Cardiovascular Diseases Research Center, Research Institute for Advanced Molecular Epidemiology, Yamagata University, Japan Global COE Program for Medical Sciences, Japan Society for the Promotion of Science, Japan d Department of Urology, Yamagata University School of Medicine, 2-2-2 Iidanishi, Yamagata 990-9585, Japan e Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan b c
a r t i c l e
i n f o
Article history: Received 19 June 2009 and in revised form 10 August 2009 Available online 18 August 2009 Keywords: Cystine transporter Oxidative stress Ischemia reperfusion Acute renal failure
a b s t r a c t This study examined the question of whether deficiency of xCT, a cystine-transporter gene, exacerbates ischemia–reperfusion-induced acute renal failure (ARF). Two weeks after the right nephrectomy of male mice at 16–18 weeks of age, the left renal vessels were clamped for 45 min to induce renal ischemia. After (24 h) induction of ischemia, xCT/ mice had elevated concentrations of blood urea nitrogen and creatinine indicative of ARF, while in xCT+/ and xCT+/+ mice, these parameters did not differ from the shamoperated mice. Immunohistochemical analyses of kidneys using antibodies against the oxidative stress markers revealed stronger staining in xCT/ mice compared with xCT+/+ mice. Induction of xCT mRNA in the kidneys of xCT+/+ mice was demonstrated using reverse transcriptase (RT)-PCR analysis and was further confirmed using quantitative RT-PCR. These data provide the first in vivo evidence that xCT is induced by oxidative stress and helps prevent ischemia–reperfusion injury to kidneys. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Ischemia induces various pathogenic processes that are involved in acute renal failure (ARF),1 such as endothelial cell injury and invasion of inflammatory cells. Because the mitochondrion is the organelle that is most affected by renal ischemia, depletion of ATP occurs during ischemia reperfusion [1,2]. The resultant purine metabolites become substrates for xanthine oxidase, which produces superoxide anions that initiate a radical chain reaction and generate other reactive oxygen species (ROS) [3]. These ROS cause oxidation of lipids, proteins, and DNA, which consequently leads to cellular dysfunction [4]. In addition, ischemia–reperfusion-induced ARF is associated with an inflammatory response [5]. Upon activation, neutrophils adhere to endothelial cells and participate in this pathogenic process. Because activated neutrophils generate large amounts of ROS via NADPH oxidase and myeloperoxidase, neutrophils that migrate to the inflammatory lesion are another source of
* Corresponding author. Address: Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. Fax: +81 23 628 5230. E-mail address: [email protected] (J. Fujii). 1 Abbreviations used: ARF, acute renal failure; ROS, reactive oxygen species; GPX, glutathione peroxidase; BUN, blood urea nitrogen; Cr, creatinine; HE, hematoxylin and eosin; HNE, anti-4-hydroxy-2-nonenal. 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.08.008
ROS [6]. As a result, the kidneys are exposed to severe oxidative stress, which exacerbates the negative consequences of ARF. Recently, we showed that SOD1-deficiency exacerbates ARF in mice [7], demonstrating that detoxification of superoxide plays an important role in renal protection during ARF. In addition to anti-oxidative enzymes, low molecular weight antioxidants, such as glutathione, play pivotal roles in the protection of most tissues against ischemic injury. Besides its direct ROS-scavenging function, glutathione donates electrons to glutathione peroxidase (GPX) and other anti-oxidative systems. Levels of glutathione are maintained by both de novo synthesis from constituent amino acids and recycling by glutathione reductase, which uses NADPH as an electron donor. In cultured cells, cysteine availability determines the rate of glutathione synthesis [8]. In extracellular fluid, cysteine undergoes spontaneous auto-oxidation to cystine. Cystine is then taken up via the cystine/glutamate exchange transport system, which has been designated ‘‘system xc.” Intracellular cystine is simultaneously reduced to cysteine and utilized for glutathione and protein synthesis. Thus, system xc activity indirectly controls intracellular glutathione availability in various cells, such as peritoneal macrophages and neutrophils [9–11]. System xc is composed of two proteins, xCT and 4F2hc; the transport activity of xc has been attributed to the xCT protein [12]. Various stimuli, including electrophilic agents such as diethyl maleate [13], oxygen [14], and bacterial lipopolysaccharide [15],
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increase system xc activity in cultured cells via induction of xCT expression. The induction of xCT by diethyl maleate is regulated by an antioxidant response element located in the 50 -flanking region of xCT, which is also referred to as the electrophile response element [13]. Binding of the transcription factor, Nrf2, to the response element enhances transcription of xCT. On the other hand, cysteine enters cells via neutral amino acid transporters. Because xCT is not expressed in kidneys under normal conditions, cellular cysteine entry via neutral amino acid transporters appears adequate under these conditions. In cultured cells, expression of xCT is induced by various stimuli, including oxidative stress [13–15], while bacterial lipopolysaccharide induces xCT in various mouse tissue [16]. However, whether xCT has a functional role in organs that do not express it in significant amounts, such as kidneys, remains to be determined. Previously, we generated xCT-knockout mice and characterized their phenotype [17]. xCT/ mice appear healthy, but have higher concentrations of cystine in plasma compared with their wild-type littermates. Double-knockout (xCT and SOD1) mice show no exacerbation of the deleterious SOD1-deficient phenotype in the circulatory system [18]. In the present study, the physiological relevance of xCT in kidneys during ischemia–reperfusion injury was examined using xCT-knockout mice. We present the first in vivo evidence that xCT is induced by ischemia reperfusion and protects kidneys against oxidative stress during ARF. Materials and methods Animals xCT-knockout mice of 129/Svj-C57BL/6J mixed genetic backgrounds were created as described previously [17]. Then they were back-crossed with C57BL/6J mice more than 10 times. Genotypes were verified using PCR (Bio-Rad, Tokyo, Japan) with gene-specific primers. The animals were housed under specific pathogen-free conditions at a constant temperature of 20–22 °C with a 12-h alternating light–dark cycle on a regular chow diet. Animal experiments were performed in accordance with the Declaration of Helsinki under a protocol approved by the Animal Research Committee of this institution. Surgery and experimental design The surgical manipulation of kidneys used to induce ARF in the mice was described previously [7]. Briefly, 2 weeks prior to the start of the study, the right kidneys of male mice (14–18 weeks of age) were removed through a small dorsal incision made after administration of pentobarbital anesthesia (40 mg/kg i.p.). After 2 weeks of recovery from the nephrectomy, the mice were anesthetized with pentobarbital (40 mg/kg i.p), and the left kidneys were exposed through a small dorsal incision. To induce ischemia, the left renal artery and vein were clamped for 45 min. At the end of the ischemic period, the clamp was released and blood was allowed to reperfuse the kidneys. For the sham operation, the mice were manipulated in the same manner without clamping of the left renal vessels.
istry. Kidneys were excised and bisected. One half was frozen in liquid nitrogen, and stored at 80 °C for determination of protein. The other half was fixed in Bouin’s solution overnight, immersed sequentially in 50%, 75% and 99% ethanol for 24 h in each solution, embedded in paraffin, and cut into 4-lm sections for histochemical analysis and hematoxylin and eosin (HE) staining. The tissue sections were incubated with 1% hydrogen peroxide in methanol. After rinsing, the sections were incubated for 60 min with blocking solution A (Histofine mouse stain kit, Nichirei, Tokyo) at room temperature, followed by incubation with a mouse anti-8-hydroxy guanine (8-OHdG) monoclonal antibody (MOG-100 at a concentration of 5.0 lg/ml; Nikken Foods Co. Inc., Shizuoka, Japan), an anti4-hydroxy-2-nonenal (HNE) monoclonal antibody (MHN-100; Nikken Seil, Shizuoka), and anti-nitrotyrosine (NT) polyclonal antibody (Millipore, Bedford, MA) overnight in a humidified chamber at 4 °C. After rinsing, the sections were incubated for 10 min at room temperature with blocking solution B (Histofine mouse stain kit, Nichirei, Tokyo). Blocking solutions A and B were intended to inhibit non-specific binding of the antibody to endogenous mouse immunoglobulin. A MAX-PO complex, in which the Fab’ portion of the secondary antibody was conjugated with an amino acid polymer and peroxidase (Histofine mouse stain kit, Nichirei, Tokyo) was placed on the tissue for 10 min. After a final rinse, the tissue sections were exposed to the chromogen, 3,30 -diaminobenzidine (Nichirei, Tokyo), for detection of specific immunolabeling. The reaction of 3,30 -diaminobenzidine with peroxidase was stopped by washing the tissues with distilled water. The sections were then dehydrated, mounted, and photographed. SDS–PAGE and immunoblot analysis Kidneys were homogenized in PBS and centrifuged at 10,000 rpm in a microcentrifuge. Soluble proteins were assayed using a BCA kit (Pierce) with bovine serum albumin as the standard. SDS–PAGE and immunoblot analyses of kidney proteins were also performed. Protein samples were subjected to 10–15% SDS– PAGE and then transferred to a HybondÒ nitrocellulose membrane (Amersham Pharmacia) under semi-dry conditions by means of a Transfer-blot SD semi-dry transfer cell (Bio-Rad). Non-specific binding sites on the membrane were then blocked by incubation with 5% non-fat milk in 150 mM NaCl and 20 mM Tris/HCl, pH 7.6 (TBS) for 1 h at room temperature. The membranes were then incubated with antibodies specific for the gene products of SOD1, SOD2 [7], aldose reductase (AKRB1) [19], aldehyde reductase (AKRA1) [20], cytosolic glutathione peroxidase (GPX1) [21], and glutathione reductase (GSR) [7] overnight at 4 °C or for 2 h at room temperature. After washing with TBS containing 0.1% Tween-20 (TBST), the membrane was incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After washing with TBST, the peroxidase activity on the membranes was detected using a chemiluminescent method with an ECL PlusÒ kit (Amersham Pharmacia Biotech, Buckinghamshire, UK), followed by membrane exposure to X-ray film (Kodak, Rochester, USA). Detection of xCT mRNA by RT-PCR and quantification by real-time PCR
Blood measurements Blood urea nitrogen (BUN) and creatinine (Cr) concentrations in plasma were determined using commercially available test systems (Wako Pure Chemicals, Osaka, Japan) as described previously [7]. Immunohistochemistry Mice were euthanized under ether anesthesia and kidneys were harvested for subsequent immunoblotting and immunohistochem-
Total cellular RNA was prepared from the cells in triplicate using Isogen (Wako). First strand cDNA was synthesized from total RNA (1 lg) using oligo-dT12–18 as a primer. A pair of primers, 50 -TT GCAAGCTCACAGCAATTCTG-30 in exon 4 and 50 -CAGGGTTGTCTACT TCTTCAGT-30 in exon 6 that were synthesized based on mouse xCT exon sequences (GenBank/European Molecular Biology Laboratory/ DNA Databank of Japan Accession No. AB022345) were used to amplify 200 bp of the xCT cDNA in a conventional PCR (PCR Thermal cycler, Takara, Tokyo). As an internal standard, a 104-bp b-actin
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cDNA was amplified using a pair of primers: 50 -TGAGGAGCACCCT GTGCT-30 in exon 3 and 50 -ACATGGCTGGGGTGTTGAAG-30 in exon 4. RT-PCR products of the xCT cDNA and b-actin cDNA were separated on a 1% agarose gel and visualized by staining with ethidium bromide. The same primers were used for quantification of the xCT cDNA and b-actin cDNA by a real-time PCR (Light Cycler, Roche). The abundance of xCT mRNA was calculated relative to b-actin mRNA. Measurement of total glutathione Total glutathione was extracted with 5% trichloroacetic acid and then measured using an enzymatic method [22] that is based on the reduction of 5,50 -dithiobis(2-nitrobenzoic acid) by the glutathione reductase system. We measured only total glutathione content because most of the glutathione extracted from the cells was in a reduced form, and there is no reliable standard method to accurately measure the content of oxidized glutathione [23]. Assay for lipid peroxidation products Thiobarbituric acid-reactive substances (TBARS) in renal tissues were determined as described previously [18]. Precipitates from kidneys, prepared as described for the immunoblot analysis, were used for the assay. A suspension of the precipitated materials in PBS was added to and then mixed thoroughly with 0.6 ml of a solution containing 15% trichloroacetic acid, 0.375% (w/v) thiobarbituric acid, 0.25 M HCl, and 1.8 mM butylhydroxytoluene. The solution was heated for 15 min in boiling water, cooled in ice-cold water, and centrifuged at 10,000g for 10 min. The absorbance of the sample was measured at 535 nm. We used a solution of 1,1,3,3-tetraethoxypropane to standardize the experiment. TBARS content was estimated using an extinction coefficient of 1.56 105 M1 cm1. Statistical analysis Statistical analyses of the data were carried out using a Mann– Whitney U-test. The number of samples is shown in parentheses in each figure. A P-value of P < 0.05 was considered significant (*P < 0.05; **P < 0.01). Results Comparison of renal function of mice under normal conditions We first examined how xCT deficiency affects kidney function under normal conditions. Body weight, kidney weight, and serum levels of BUN and Cr did not differ significantly among xCT+/+, xCT+/, and xCT/ mice at 16–18 weeks of age (Fig. 1). Histological examination of kidneys showed no differences among groups (data not shown). This observation is consistent with the absence of xCT expression in kidneys under normal conditions [17]. Comparison of kidney function after ischemia-reperfusion injury We then examined the effects of ischemia reperfusion on kidney function in 16–18-week old xCT+/+, xCT+/, and xCT/ male mice. Mice were right-nephrectomized 2 weeks prior to the start of the study. Ischemic ARF was induced by clamping the left renal blood vessels for 45 min followed by reperfusion. We examined renal function 24 h after the induction of ischemia. Ischemic AFR was examined at the acute stage at 24 h after ischemia reperfusion, because, based on our preceding study using SOD1-knockout mice, renal damage was evident at this period and was reversed thereafter [7]. According to the results, the xCT/ mice had greater serum
Fig. 1. Renal function of xCT-deficient mice under normal conditions. Male mice (16–18-week old) with the indicated genotypes were euthanized and body (A) and kidney (B) weights were obtained. Plasma concentrations of BUN (C) and Cr (D) were assayed. Means + SEM are shown.
concentrations of BUN and Cr than both the xCT+/+and xCT+/ mice (Fig. 2), suggesting that xCT/ mice had an increased severity of ARF. Thus, xCT deficiency exacerbated the symptoms of ischemic ARF, despite the fact that xCT is not expressed in kidneys under normal conditions. Histological examination of kidneys after ischemia reperfusion Renal injury, characterized by proximal tubular expansion, vacuolation of proximal tubular epithelial cells, and defluvium of the proximal tubule epithelium, was more severe in xCT/ mice than that in xCT+/+ mice (Fig. 3). However, glomerular injury was mild compared with the damage observed in epithelial cells. This result is consistent with the previous observation that endothelial damage is a classic symptom of ischemia-induced ARF [24]. Results of immunohistochemical examination showed that after renal ischemia–reperfusion 8-OHdG immunoreactivity was detected in both the glomerulus and the proximal tubules in xCT+/+ mice, and it was enhanced in xCT/ mice. Staining for HNE, a lipid peroxidation product, in the proximal tubules was more pronounced in xCT/ mice compared with xCT+/+ mice. There was an increase in immunostaining by an anti-nitrotyrosine (NT) antibody, especially in the kidneys of xCT/ mice, during ischemia reperfusion. These findings suggest that ROS were produced as a consequence of renal ischemia reperfusion and that the reactive species are involved in the pathogenesis of ARF, especially in xCT/ mice. Levels of anti-oxidative/detoxificating enzymes in kidneys after ischemia reperfusion To examine the role of anti-oxidative-detoxifying enzymes, we examined the amount of CuZn-superoxide dismutase (SOD1), Mnsuperoxide dismutase (SOD2), cytosolic glutathione peroxidase (GPX1), glutathione reductase (GSR), aldehyde reductase (AKR1A), and aldose reductase (AKR1B) protein after ischemia–reperfusioninduced ARF in the different genotypes of mice (Fig. 4). The superoxide dismutases, SOD1 and SOD2, scavenge superoxide, which is
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Fig. 2. Renal function parameters of male mice 24 h after ischemia reperfusion. After 45 min of ischemia, kidneys were reperfused. Plasma concentrations of BUN (A) and Cr (B) were measured in xCT+/+, xCT+/, and xCT/ mice (16–18-week old) 24 h after either ischemia reperfusion (I/R) or sham operation (sham). Means + SEM are shown.
the primary oxygen radical produced by donation of one electron. GPX1 and GSR are glutathione-related anti-oxidative/redox enzymes that are abundant in kidneys. Aldo–keto reductase 1A and 1B (AKR1A and AKR1B) are the predominant aldo–keto reductases in kidneys [25], which protect against oxidative stress by reducing toxic carbonyl compounds present in urine. Levels of each of these
enzymes in xCT+/+, xCT+/ and xCT/ mice were not altered by ischemia reperfusion compared with the sham operation. However, we could not examine levels of the xCT protein because no specific antibody against the xCT protein was available. Thus, xCT deficiency appears to have little or no effect on renal expression of these genes during ischemia reperfusion.
Fig. 3. Immunohistochemical analyses of renal oxidative stress markers after ischemia reperfusion. Renal sections obtained 24 h after sham operation or ischemia reperfusion were allowed to react with an anti-8-OHdG, an anti-HNE antibody, and an anti-NT and were stained with HE. Typical data from several experiments are shown.
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Fig. 4. Immunoblot analysis of renal anti-oxidative/redox proteins. Soluble proteins were isolated from the kidneys of mice 24 h after either ischemia reperfusion (I/R) or sham operation (sham), and were subjected to immunoblot analysis using antibodies against SOD1, SOD2, GPX1, GSR, AKR1A, and AKR1B. GAPDH was used as a loading control. Typical data from several experiments are shown.
Induction of xCT gene expression in kidneys after ischemia reperfusion Although xCT expression is normally at a level that is not detectable in kidneys by Northern blotting [12,16], ischemia–reperfusion-induced AFR in xCT/ mice was more severe than that in xCT+/+ and xCT+/ mice. This observation can be reasonably explained if xCT expression is induced in mice with a wild-type allele. Therefore, we performed RT-PCR analysis of total RNA isolated from the kidneys. As expected from the results showing exacerbation of kidney function in xCT/ mice, xCT expression was elevated in xCT+/+ mice after ischemia reperfusion (Fig. 5A). We then performed quantitative PCR to assure the induction of xCT in xCT+/+ mice. The induction of xCT mRNA was further supported by quantitative PCR, which showed a 20-fold increase in renal expression in xCT+/+ mice after ischemia reperfusion (Fig. 5B). Thus, xCT was induced by ischemia reperfusion and appeared to play a protective role during AFR. Because the cystine that is transported into cells via system xc is used to synthesize glutathione, which protects against oxidative stress, we measured levels of both total glutathione and lipid peroxidation products in the kidneys of xCT+/+ and xCT/ mice. However, there was no significant difference in the levels of either total glutathione (Fig. 6) or lipid peroxidation products (data not shown) between the two groups.
Fig. 5. Induction of xCT expression in kidneys after ischemia reperfusion. RT-PCR analysis was performed for total RNA isolated from kidneys 24 h after either ischemia reperfusion (I/R) or sham operation (sham) (A). Quantitative xCT mRNA data were obtained by quantitative RT-PCR using a Light Cycler. The relative abundance of xCT mRNA to b-actin mRNA was presented. Means + SD are shown (B).
that radical scavengers, SOD [4] and edaravone [29], and an antioxidant, N-acetyl cysteine [30], effectively ameliorate the consequences of ischemic injury. Moreover, attenuated expression of SOD1, glutathione peroxidase, and catalase has been reported in ischemia–reperfusion-induced ARF [31,32]. The results of the present study indicate that xCT deficiency exacerbates ischemia–reper-
Discussion In this study, we examined the role of system xc in renal ischemia–reperfusion injury using xCT-deficient mice. After ischemia– reperfusion injury, renal oxidative damage in xCT/ mice was more severe than that observed in either the xCT+/+ or xCT+/ mice (Fig. 3). Although xCT is not expressed in the kidneys of xCT+/+ mice under normal conditions, its expression was induced by ischemia– reperfusion injury (Fig. 5). This result suggests that xCT is induced by ischemia–reperfusion injury to protect against the cellular damage caused by ARF and by other pathological conditions associated with oxidative stress. Oxidative and nitrosative stress have been implicated in the pathogenesis of ischemic ARF [26,27], and the production of ROS during ischemic injury has been demonstrated [28]. Involvement of ROS in the pathogenesis of ARF is supported by the observation
Fig. 6. Total glutathione and lipid peroxidation products. Levels of total glutathione in kidneys were measured in xCT+/+ and xCT/ mice (16–18-weeks old) 24 h after either ischemia reperfusion (I/R) or sham operation (sham). Means + SEM are shown.
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fusion-induced ARF. A protective role of xCT against the oxidative stress induced by renal ischemia–reperfusion injury was supported by the increase in oxidative stress markers, 8-OHdG and HNE, in the xCT/ mice compared with xCT+/+ and xCT+/ mice (Fig. 3). We found severe proximal tubule damage in xCT/ mice. Ischemic ARF results in various types of kidney damage, and the mitochondrion appears to be the organelle most affected by renal ischemia reperfusion [1,2]. Glutathione is present at high concentrations in mitochondria where it protects against oxidative damage. In addition to mitochondrial production, ROS are generated by the action of xanthine oxidase during purine metabolism [3] and by neutrophils attached to endothelial cells, as seen during most inflammatory processes [5]. Activated neutrophils produce ROS via two major enzymatic systems: NADPH oxidase, which produces superoxide by transfer of electrons from NADPH to molecular oxygen; and, myeloperoxidase, which produces HOCl [6]. While the resulting ROS protect against infection at sites of inflammation, they also damage nearby cells. Thus, endothelial cells can be damaged by the ROS released from activated neutrophils. Transplantation of functional endothelial cells into kidneys has a renoprotective effect [33]. Oxidative stress also elevates production of nitric oxide (NO) by inducing transcription of the nitric oxide synthase gene (NOSII). Regarding reactive nitrogen oxide species, the harmful molecule that is directly responsible for kidney damage during ARF appears to be peroxynitrite [24], which results from the reaction between NO and superoxide. Because SOD scavenges superoxide and, hence, suppresses peroxynitrite formation, the levels of nitrotyrosine, which is produced by the reaction between tyrosine and peroxynitrite [34], are elevated in the kidneys of SOD1/ mice [7]. In xCT-deficient kidneys, nitrotyrosine levels increased slightly (Fig. 3) and may also be involved in deterioration of ARF. We recently generated SOD1/; xCT/ double-knockout mice, and found no significant exacerbation of the SOD-deficiency in any of the organs examined, including kidneys [18]. This result suggests that the oxidative stress caused by deficiency of both SOD1 and xCT is insufficient to cause renal damage, and that ischemia–reperfusion injury is responsible for induction of ARF. Glutathione plays multiple functions. It acts directly as an antioxidant to neutralize deleterious ROS, and it is also a cofactor for antioxidant enzymes, including glutathione peroxidase, glutathione S-transferase, and glutaredoxin. Moreover, cell proliferation and apoptosis are also regulated by glutathione [35]. Oxidative stress perturbs cellular glutathione levels either by affecting glutathione biosynthesis or by altering the intracellular ratio of reduced and oxidized forms, thereby affecting multiple physiological responses. Because xCT is required for the provision of cysteine for glutathione synthesis, xCT deficiency could limit glutathione availability. In addition, genes that are involved in either glutathione synthesis (GCLM, GCLC, and xCT) or in glutathione recycling via reduction (GSR) are induced during oxidative stress. Furthermore, Nrf2 is known to play an essential role in the induction of these genes [13,36–39]. When we measured total glutathione levels in kidneys, there was no significant difference in total glutathione levels (Fig. 6). The damage caused by ARF was limited to specific regions of the kidney. Most of the damage was localized to the proximal tubules, which constitute only a small part of kidneys. Thus, changes in the glutathione levels in the damaged cells may not affect either total glutathione content in the entire kidney. It is also possible that glutathione levels had recovered to physiological levels via induction of xCT by the time they were assayed. Here we have measured only total glutathione levels in the kidneys. An increase in the oxidized form of glutathione is observed in many diseases [40,41], and, hence, the significance of individual measurement of both the reduced and the oxidized forms of glutathione is recognized. However, standard values for these measures
in biological samples are lacking because there is no consensus or standardization of assay methods [23]. Other than glutathione synthesis, xCT is involved in the maintenance of extracellular redox balance, which is defined by the ratio of cysteine to cystine, i.e. [cysteine/cystine]. Recently, we demonstrated that xCT overexpression maintained integrity of Burkitt’s lymphoma cells that were depleted of bulk glutathione as a result of incubation with L-buthionine sulfoximine [42]. This finding shows that xCT protects against oxidative stress by driving a highly efficient cystine/cysteine redox cycle. Moreover, this redox cycle by itself, i.e., without participation of the intracellular glutathione system, is regarded as one of the most effective anti-oxidative systems in culture. Because renal ischemia reperfusion would shift the extracellular redox balance to the oxidized state, the exacerbation of ARF observed in xCT-deficient mice may have resulted from a failure to restore the redox state. The observation that N-acetyl cysteine improves ischemia–reperfusion-induced renal damage [30] supports the hypothesis that an xCT-mediated redox cycle protects against ARF. Additional studies are required to determine if the xCT-driven redox cycle is functional in vivo. In conclusion, while xCT may not be essential for renal function under normal conditions, xCT expression is induced during severe oxidative stress, such as that induced by ischemia–reperfusion injury, and exerts an anti-oxidative function. Thus, xCT belongs to a collection of genes that are induced in kidneys during oxidative stress and function in a coordinated manner to protect cells against oxidative injury during AFR. Acknowledgments This work was supported, in part, by the Global COE Program (F03) from the Japan Society for the Promotion of Science. References [1] D.L. Cruthirds, L. Novak, K.M. Akhi, P.W. Sanders, J.A. Thompson, L.A. MacMillan-Crow, Arch. Biochem. Biophys. 412 (2003) 27–33. [2] D.L. Cruthirds, H. Saba, L.A. MacMillan-Crow, Arch. Biochem. Biophys. 437 (2005) 96–105. [3] R. Harrison, Free Radic. Biol. Med. 33 (2002) 774–797. [4] M.S. Paller, J.R. Hoidal, T.F. Ferris, J. Clin. Invest. 74 (1984) 1156–1164. [5] J.V. Bonventre, A. Zuk, Kidney Int. 66 (2004) 480–485. [6] M.B. Hampton, A.J. Kettle, C.C. Winterbourn, Blood 92 (1998) 3007–3017. [7] T. Yamanobe, F. Okada, Y. Iuchi, K. Onuma, Y. Tomita, J. Fujii, Free Radic. Res. 41 (2007) 200–207. [8] S. Bannai, N. Tateishi, J. Membr. Biol. 89 (1986) 1–8. [9] S. Bannai, E. Kitamura, J. Biol. Chem. 255 (1980) 2372–2376. [10] H. Watanabe, S. Bannai, J. Exp. Med. 165 (1987) 628–640. [11] Y. Sakakura, H. Sato, A. Shiiya, M. Tamba, J. Sagara, M. Matsuda, N. Okamura, N. Makino, S. Bannai, J. Leukoc. Biol. 81 (2007) 974–982. [12] H. Sato, M. Tamba, T. Ishii, S. Bannai, J. Biol. Chem. 274 (1999) 11455–11458. [13] H. Sasaki, H. Sato, K. Kuriyama-Matsumura, K. Sato, K. Maebara, H. Wang, M. Tamba, K. Itoh, M. Yamamoto, S. Bannai, J. Biol. Chem. 277 (2004) 44765– 44771. [14] S. Bannai, H. Sato, T. Ishii, Y. Sugita, J. Biol. Chem. 264 (1989) 18480–18484. [15] H. Sato, K. Fujiwara, J. Sagara, S. Bannai, Biochem. J. 310 (1995) 547–551. [16] K. Taguchi, M. Tamba, S. Bannai, H. Sato, J. Inflamm. 4 (2007) 20. [17] H. Sato, A. Shiiya, M. Kimata, K. Maebara, M. Tamba, Y. Sakakura, N. Makino, F. Sugiyama, K. Yagami, T. Moriguchi, S. Takahashi, S. Bannai, J. Biol. Chem. 280 (2005) 37423–37429. [18] Y. Iuchi, N. Kibe, S. Tsunoda, F. Okada, S. Bannai, H. Sato, J. Fujii, Mol. Cell. Biochem. 319 (2008) 125–132. [19] M. Takahashi, J. Fujii, E. Miyoshi, A. Hoshi, N. Taniguchi, Int. J. Cancer 62 (1995) 749–754. [20] M. Takahashi, J. Fujii, T. Teshima, K. Suzuki, T. Shiba, N. Taniguchi, Gene 127 (1993) 249–253. [21] T. Fujii, T. Endo, J. Fujii, N. Taniguchi, Free Radic. Res. 36 (2002) 1041–1049. [22] F. Tietze, Anal. Biochem. 27 (1969) 502–522. [23] P. Monostori, G. Wittmann, E. Karg, S. Túri, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. (2009) [Epub ahead of print]. [24] M.S. Goligorsky, S.V. Brodsky, E. Noiri, Kidney Int. 61 (2002) 855–861. [25] J.Y. Jung, Y.H. Kim, J.H. Cha, K.H. Han, M.K. Kim, K.M. Madsen, J. Kim, Am. J. Physiol. Renal Physiol. 283 (2002) F481–F491. [26] E. Noiri, A. Nakao, K. Uchida, H. Tsukahara, M. Ohno, T. Fujita, S. Brodsky, M.S. Goligorsky, Am. J. Physiol. Renal Physiol. 281 (2001) F948–F957.
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