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* School of Medicine and Medical Science, UCD Conway Institute of Biomolecular and Biomedical Research and
Education and Research Center, St. Vincent’s University Hospital, University College Dublin, Ireland;
Nephrology Division, Johns Hopkins University Hospital, Baltimore, Maryland, USA; and
Institute of Molecular and Cell Biology, Singapore, Republic of Singapore
1Correspondence: School of Medicine and Medical Sciences, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: martin.leonard{at}ucd.ie
ABSTRACT
Tissue reoxygenation following hypoxia is associated with ischemia-reperfusion injury (IRI) and may signal the development of ischemic preconditioning, an adaptive state that is protective against subsequent IRI. Here we used microarray RNA analysis of in vivo and in vitro models of IRI to delineate the underlying molecular mechanisms. Microarray analysis of renal tissue after ischemia-reperfusion revealed a number of highly up-regulated antioxidant genes including aldehyde dehydrogenases (ALDH1A1 and ALDH1A7), glutathione S-transferases (GSTM5, GSTA2 and GSTP1), and NAD(P)H quinone oxidoreductase (NQO1). The transcription factor NF-E2-related factor-2 (Nrf2), a master regulator of this antioxidant response, is also elevated in IRI. Furthermore, microarray analysis of renal epithelial cells exposed to hypoxia/reoxygenation identified Nrf2 to be up-regulated on reoxygenation. We also reveal a reoxygenation-specific nuclear accumulation of Nrf2 protein and subsequent activation of a NQO1 promoter reporter construct. Attenuating reactive oxygen species (ROS) in reoxygenation using the antioxidant N-acetyl cysteine results in inhibition of Nrf-2 activation. mRNA levels for Nrf2-dependent genes were detected in human liver biopsy 1 h after transplantation. These results indicate that reoxygenation-dependent Nrf-2 activity facilitates ischemic preconditioning through the induction of antioxidant gene expression and that ROS may be critical in signaling this event.—Leonard, M. O., Kieran, N. E., Howell, K., Burne, M. J., Varadarajan, R., Dhakshinamoorthy, S., Porter, A. G., O’Farrelly, C., Rabb, H., Taylor, C. T. Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia reperfusion injury.
Key Words: antioxidant response element • hypoxia • ischemic preconditioning • NQO1
ISCHEMIC REPERFUSION INJURY, which occurs when blood flow is disrupted to a tissue or organ and subsequently reintroduced, is associated with a diverse array of disease states including myocardial infarction, stroke, acute renal failure, and post-transplantation injury (1
2
3
4)
. In these conditions much of the damage occurs during the reperfusion period, when, for example, thrombolytic therapy has been administered or perfusion has been restored after transplantation. A primary factor in the initiation of the pathological response to reperfusion injury is the generation of high levels of ROS, which can covalently modify protein and lipid macromolecules leading to cell damage, DNA mutation, and initiation of apoptotic and necrotic cascades (5)
. Apart from ROS release from infiltrating cells such as neutrophils, oxygen deprivation (hypoxia) during ischemia and subsequent reoxygenation upon reperfusion are thought to be the major factors contributing to ROS production and the subsequent cellular damage (5)
. It has been well characterized that exposure to ischemia and reperfusion leads to induction of a protective state against subsequent ischemic- reperfusion events—namely, ischemic preconditioning (6)
. Mechanisms underlying the induction of an ischemic preconditioned state remain unclear but likely involve the cells’ ability to maintain sufficient antioxidant and detoxification buffering capacity.
An important mechanism by which cells adapt to oxidant stress is to transcriptionally up-regulate a distinct array of cytoprotective genes responsible for buffering the cells’ antioxidant capacity. These genes act to maintain glutathione content and conjugational activity; they are also responsible for detoxification of damaging electrophilic by-products of oxidant stress and include glutathione S-transferases, aldehyde dehygrogenases, and quinone oxidoreductases (7)
. A master regulator of this specific antioxidant phenotype is the transcription factor Nrf2 (8)
. This transcription factor is held in the cytoplasm by a cytoskeletal-associated specific inhibitory protein KEAP1 under conditions of normal cellular redox state, where Nrf2 is continuously targeted to proteasomal degradation. Under conditions of oxidative stress, cysteine residues within the hinge region of KEAP1 become modified through mechanisms that involve thiol oxidation, resulting in a conformational change in KEAP1 with the loss of Nrf2 binding and proteasomal targeting. Nrf2 then accumulates and localizes to the nucleus, where it heterodimerizes with specific cofactors, including members of the maf protein family, and coordinates up-regulation of cytoprotective genes through the initiation of transactivation at antioxidant response elements (AREs) within the regulatory regions of these genes (9)
. While much of the work to characterize Nrf2 activation and dependent cytoprotective gene expression has been carried out in response to chemical oxidants such as sulforaphane and tBHQ, no studies have been performed to determine the role for Nrf2 in mediating adaptive responses to reperfusion and reoxygenation-mediated cellular injury
In this study we demonstrate the induction of a cohort of Nrf2-dependent antioxidant genes in a murine model of renal IRI. We also demonstrate activation of Nrf2-dependent gene expression in liver biopsy as well as a reoxygenation-specific induction of Nrf2 gene expression and activation of Nrf2-dependent ARE transcriptional activity. These are the first data to demonstrate that reoxygenation-mediated activation of Nrf2 is a likely master regulator for the coordinated adaptive cytoprotective genomic response to oxidative stress during reperfusion injury as a contributory mechanism for the development of ischemic preconditioning.
MATERIALS AND METHODS
Ischemia reperfusion injury
Acute renal failure was induced using an established animal model of renal IRI (10)
. National Institutes of Health (NIH) Swiss mice (25 to 35 g) were anesthetized with 75 mg/kg intraperitoneal sodium pentobarbital. Incisions were made in the abdomen and the renal pedicles were exposed through blunt dissection. A microvascular clamp was placed on both renal pedicles for 30 min. During the procedure, animals were kept hydrated with saline and body temperature was maintained at 36–38°C. After the procedure, clamps were removed and the wounds were sutured. Animals were allowed to recover for 24 h, then the kidneys were dissected and snap frozen before processing for protein and RNA isolation.
Cell culture and experimental conditions
Human proximal tubular epithelial cells (HK-2; American Tissue Type Culture Collection; (11)
were maintained in Dulbecco/Vogt modified Eagle’s medium and Ham’s F-12 medium (DMEM/F-12) containing 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 36 ng/ml hydrocortisone, 4 pg/ml triiodo-L-thyronine, 10 ng/ml epidermal growth factor (EGF), 50U/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine (Sigma-Aldrich, Dublin, Ireland). T84 intestinal epithelial cells were grown in DMEM/F-12 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (Invitrogen, Paisley, UK). Cells were cultured to confluency in 35 mm diameter Petri dishes prior to experimental protocols. Cells were maintained at 37°C in a humidified atmosphere under hypoxic [1% O2, 20 torr (1 torr=133Pa)] or normoxic/reoxygenation [21% O2, 147 torr] conditions with a balance of 95%N2/5%CO2. Hypoxic conditions were maintained using an environmentally controlled hypoxia chamber (Coy Labs, Glass Lake, MI, USA). Cellular PO2 concentrations were measured by fluorescence quenching oxymetry (Oxylite-2000; Oxford Optronix, Oxford, UK) (12)
.
RNA preparation and cDNA synthesis
RNA was isolated from cell line and tissue samples using Trireagent (Sigma-Aldrich). Briefly, after experimental procedure, cells were scraped and lysed in 1 ml of reagent. Tissue samples were homogenized in reagent using a Berthold hand-held homogenizer. Samples were left to stand for 5 min at room temperature before the addition of 0.2 ml chloroform. Samples were then shaken vigorously for 15 s and allowed to stand for 10 min at room temperature before centrifugation at 12,000 g for 15 min at 4°C. The upper aqueous phase was removed to a separate tube and RNA was precipitated using 0.5 ml isopropanol After centrifugation the pellet was washed with 75% ethanol and resuspended in 50 µl of TE buffer. RNA was quantified as absorbance at 260 nM. RNA was reverse transcribed to single-stranded cDNA using the Superscript Choice kit reagents (Invitrogen, Carlsbad, CA, USA). Pooled total RNA (5 µg) was denatured at 65°C for 15 min and added to 1 nM of T7-(dT) primer(5'-(5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGG-CGG-(dT)24-3'). The sample was incubated at 70°C for 10 min, placed on ice, and centrifuged at 14,000 g for 30 s. The sample was then added to 5 x first-strand cDNA buffer, 10 mM DTT, and 500 µM dNTP mix, vortexed, and incubated at 42°C for 2 min. After the addition of SuperScript II reverse transcriptase for 1 h at 42°C, samples were centrifuged briefly and placed on ice. Second-strand cDNA synthesis was carried out through the addition of 5 x second-strand reaction buffer, 200 µM dNTP mix, DNA ligase (10U), DNA polymerase I (40U), and Rnase H (2U). After incubation at 16°C for 2 h, 20U of T4 DNA polymerase was added and subsequently incubated at 16°C for 5 min. Double-stranded cDNA was purified using the GeneChip sample cleanup module per the manufacturer’s instructions and eluted in TE buffer (Qiagen, Valencia, CA, USA).
Microarray analysis
Sample preparation and microarray analysis were carried out as described previously (13)
. Briefly, after total RNA extraction and cDNA synthesis, complementary biotin-labeled cRNA was prepared using the Bioarray High Yield RNA transcript labeling kit (ENZO Life Sciences Inc., Farmingdale, NY, USA). The labeled cRNA was washed using the GeneChip sample cleanup module per the manufacturer’s instructions (Qiagen, Valencia, CA, USA). cRNA (20 µg) was fragmented by addition of 5 x fragmentation buffer (200 mM Tris acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc [made up fresh in diethyl pyrocarbonate (DEPC) dH2O)] and incubated at 94°C for 35 min, placed on ice, and stored at –20°C until hybridization. After confirmation of cRNA integrity through hybridization to test microarrays, fragmented cRNA was hybridized to human HG U95A or murine U77A microarrays (Affymetrix, Santa Clara, CA, USA) for 16 h, washed, and stained for 2 h with fluorescent detection reagent streptavidin-phycoerythrin. Microarrays were then scanned using an argon-ion laser (excitation 488 nm, detection 570 nm) and results were presented as fluorescent intensities. Microarray Suite 5.0 software (Affymetrix) was used to analyze the relative abundance of each gene from the average difference of fluorescent intensities.
Real-time and semiquantitative RT-polymerase chain reaction (RT-PCR) analysis
Target gene forward and reverse primers for real-time and semiquantitative RT-PCR analysis were designed using the Primer 3 software package (Table 1
) and synthesized by Sigma-Genosys (Haverhill, Suffolk, UK). Real-time polymerase chain reaction (PCR) was carried out using commercially available SYBR green-based detection reagents (Roche, Branchburg, NJ, USA). Each sample contained 50 ng of cDNA, 0.4 mM each of the forward and reverse primers, and 0.1 mM TaqManTM probe (18S rRNA internal standard control only). Temperature conditions consisted of a 5 min step at 95°C, followed by 40 cycles of 60°C for 1 min and 95°C for 15 s performed using a 7900HT sequence detector (Applied Biosystems, Foster City, CA, USA). All measurements were performed in duplicate and water controls were negative in all runs. Data were collected during each extension phase of the PCR reaction and analyzed with the SDS software package (Applied Biosystems). Threshold cycles were determined for each gene and quantification of templates was performed according to the standard curve method. The expression level of each target gene was given as relative amount normalized against 18S standard controls.
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Semiquantitative RT-PCR analysis was carried out on cDNA using a standard reaction mixture for amplification as described previously (14)
. The amplification protocol included an initial 2 min denaturation at 94°C, followed by 26 cycles of 94°C for 40 s, 55°C for 40 s, 72°C for 1 min, with a final extension of 72°C for 5 min. Amplification of 18S RNA (14 cycles) was carried out as a control for equal sample loading. PCR products were analyzed using a 2% agarose gel in 0.5 x TAE buffer, stained with ethidium bromide, and visualized under UV illumination (Uvipro GDS8000, UVItech, Cambridge, UK).
Immunofluorescent staining for Nrf2
Frozen kidney tissue embedded in OCT (optimal cutting temperature compound) was cut to 10 µm sections, dehydrated using graded ethanol steps, rehydrated, and fixed with methanol. After rinsing in PBS, tissue sections were permeabilized in 0.2% Triton-X 100 in PBS, then blocked using 1% goat serum for 1 h at room temperature. Sections were incubated with primary antibody (Ab) (polyclonal rabbit anti-Nrf2 (2 µg/ml), Abcam, Cambridge, UK) overnight at 4°C. Sections were washed with PBS and incubated with secondary Ab (Oregon green-linked goat anti-rabbit (1:500) Invitrogen, Paisley, UK) for 1 h at room temperature. DNA was stained with propidium iodide (Sigma, Poole, Dorset, UK) for 5 min at room temperature. Specific staining was assured using control slides where the primary Ab was omitted. Images were visualized using confocal microscopy with a x20 objective on a UV Zeiss 510 Meta System laser scanning microscope, analyzed using the LSM 5 browser imaging software.
Whole-cell and nuclear protein extract preparation and Western blot analysis
Whole-cell extracts were prepared in radio-immuno-precipitation assay (RIPA) lysis buffer (20 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 5 mmol/L ethylene diaminetetraacetic acid, 1% Nonidet P-40, 0.1% SDS, 5 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 1 µmol/L leupeptin, 0.3 µmol/L aprotinin). Nuclear extracts were prepared from cells scraped into hypotonic buffer (10 mM HEPES-NaOH buffer, pH7.9, containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF.), incubated for 10 min, and centrifuged for 10 min at 21,000 g at 4°C. Samples were then lysed for 10 min on ice in hypotonic buffer containing 0.1% (v/v) Nonidet P-40, followed by centrifugation at 21,000 g for 6 min. The resulting pellets were resuspended in high-salt buffer [20 mM HEPES-NaOH buffer pH7.9 containing 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (w/v) glycerol, and 0.5 mM PMSF] for 15 min on ice followed by centrifugation at 21,000 g for 6 min at 4°C. Supernatants were removed as the nuclear extracts. Protein content was quantified and normalized using the Bradford method (Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK) and electrophoresed on 10% SDS-PAGE gels. Expression levels for Nrf1 and Nrf2 were measured using specific antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by Western blot analysis as described previously (15)
. Phosphorylation-specific (Ser-473) Akt Ab was obtained from Abcam (Cambridge, UK).
Transient transfection
Cells were transfected with 2 µg of an ARE promoter-luciferase reporter construct [NQO1 wild-type (WT)] kindly provided by Prof. Alan Porter (Institute of Molecular and Cell Biology, Proteos, Singapore; ref. 16
). Transfection was carried out using Fugene 6 transfection reagent (Roche Applied Science, Lewes, East Sussex, UK) according to the manufacturer’s guidelines. After transfection for 24 h and subsequent experimental procedures, cells were washed with 2 ml of 1 x PBS (ice-cold) and lysed in 200 µl 1 x luciferase reporter whole-cell lysis buffer (Promega, Southampton, UK). Luciferase activity was quantified using a luciferin substrate (Promega) and luminometry (Junior LB 9509, Berthold Technologies, Redbourn, Hertfordshire, UK). All readings were normalized to protein using the Bradford method (Bio-Rad).
Liver biopsy extraction and preparation
Liver wedge biopsy was obtained from donor liver at the beginning of multiorgan donor retrieval before cold perfusion and stored at –80°C (retrieval biopsy) as described previously (17)
. After a period of cold ischemia and reimplantation, a second wedge biopsy was performed 1 h after portal reperfusion and stored at –80°C (reperfusion biopsy). RNA was isolated as described above.
Statistical analysis
All data are presented as mean ± SEM for n independent experiments. Statistical significance was evaluated using ANOVA carried out using software package InStat (GraphPad, San Diego, CA, USA).
RESULTS
Ischemia-reperfusion injury induced Nrf2-regulated antioxidant gene expression
Gene expression profiling of disease models has permitted clearer insight into the molecular mechanisms governing the adaptive response to disease conditions. In the context of IRI, a preconditioning event can protect against subsequent ischemic events. To understand the implications of altered gene expression patterns responsible for this phenomenon, we analyzed global gene expression mRNA levels in a murine model of IRI after a single event (30 min ischemic time followed by 24 h reperfusion) using the Affymetrix U77A microarray system. Initial analysis revealed a cluster of genes up-regulated compared with sham-operated control animals. Within this cluster of the 20 most highly up-regulated genes were 7 genes involved in cytoprotection against oxidant stress including ALDH1A1, ALDH1A7, NQO1, GSTM5, GSTA2, and GSTP1 (Fig. 1
A). These enzymes are involved in phase II detoxi-fication of xenobiotics and metabolites created during oxidative stress such as those produced on ischemia reperfusion, and therefore are likely candidates as mediators of protection observed with ischemic preconditioning. We confirmed alterations in gene expression using real-time PCR analysis, where we observed a statistically significant increase in mRNA levels of all genes analyzed (Fig. 1B
). Levels of increase were comparable to those levels observed on microarray analysis. Since a high percentage of genes with similar cytoprotective function were up-regulated under these conditions, it is likely they have a similar signaling mechanism of induction. One candidate master regulator of this response is the transcription factor Nrf2. This transcription factor has been observed in other model systems as a regulator of expression of 6 of the 7 cytoprotective genes induced in this model of IRI (18
19
20
21)
(Fig. 1A
). As activation of Nrf2 involves stabilization and accumulation of the protein before nuclear translocation and activation of gene expression, we next assessed protein levels for Nrf2. Specific immunofluorescent staining for Nrf2 was observed in sham-operated and IRI-treated kidney sections (Fig. 2
A) compared with sections where the primary Ab was omitted (data not shown). Staining of sham-operated kidneys revealed a pattern specific for the cortex, with little or no staining observed in the medulla. IRI resulted in a substantial increase in staining for Nrf2 localized mainly within the medulla (Fig. 2A
). We also observed a substantial increase in levels of Nrf2 protein on ischemia-reperfusion compared with sham-operated control (Fig. 2B
).
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Reoxygenation of renal epithelial cells after hypoxia induces Nrf2 gene expression
Having observed the induction of phase II detoxification antioxidative gene expression in a murine model of IRI with the likely involvement of Nrf2 as a master regulator of this transcriptional response, we wanted to delineate specific cellular events responsible for altered gene expression that may be responsible for the phenomenon of ischemic preconditioning. One component of reperfusion overlooked in previous studies has been that of reoxygenation. To address this we used an in vitro model of human renal epithelial (HK-2) cells exposed to hypoxia and reoxygenation. Characterization of altered oxygen tension within our system was established using fluorescence-quenching oxymetry. Oxygen tension at the cell-liquid interface fell rapidly upon hypoxic incubation (1% atmospheric O2) and reached detectable 0 torr within 40 min (Fig. 3
). Measured oxygen tension fell more rapidly in plates with cells than plates with medium alone, reflecting cellular oxygen consumption. Reintroduction of oxygen caused a more rapid increase in measured oxygen tension than the decline observed during hypoxic exposure (Fig. 3)
. Having established a defined model of reoxygenation, we carried out a microarray experiment to determine the contribution to global gene expression of this reoxygenation response. After exposure of cells to various time points (8–36 h) of hypoxia and a 6 h reoxygenation time point, we identified a cluster of 17 genes up-regulated specifically on reoxygenation. This cluster contained a number of genes, including Nrf2 (2.1-fold compared with normoxia) (Fig. 4
A). Confirmation of Nrf2 mRNA induction by reoxygenation was carried out using semiquantitative (Fig. 4B
) and real-time (Fig. 4C
) PCR analysis. Both methods revealed a significant increase in expression compared with 18S control.
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Reoxygenation results in nuclear accumulation and activation of Nrf2
We have so far observed the induction of Nrf2-dependent genes on reperfusion and induction of the Nrf2 gene itself on reoxygenation. We next wanted to examine whether the accumulation of Nrf2 protein on reperfusion is correlative with a response observed on reoxygenation. Incubation of HK-2 cells in hypoxia for 16 h followed by reoxygenation for various time points resulted in nuclear accumulation of Nrf2 protein as early as 2 h as assessed by Western blot analysis (Fig. 5
A). We also observed a pronounced nuclear accumulation of Nrf2 in colonic epithelial T84 cells exposed to hypoxia/reoxygenation in a pattern reflective of that seen in HK-2 cells (Fig. 5B
). There were no significant alterations in nuclear accumulation of the NF-E2-like family member Nrf1, indicating a signaling pathway specific for Nrf2 activation (Fig. 5A, B
). We next analyzed whether this accumulation results in the activation of Nrf2-dependent gene expression. Cells were transfected with a NQO1 promoter reporter construct containing 1.1kbp of promoter sequence upstream from the transcription start site (NQO1 WT). Reoxygenation but not hypoxia caused a significant increase in reporter activity (2.45-fold compared with empty vector control) (Fig. 5C
). Mutation of the Nrf2 binding ARE within the NQO1 promoter resulted in a loss of reoxygenation-specific activation of reporter activity (Fig. 5C
). In addition to NQO1, we analyzed the expression of another Nrf2-dependent gene that was up-regulated on IRI, GSTP1. Reoxygenation but not hypoxia resulted in a substantial increase in GSTP1 after 8 h, assessed using real-time PCR analysis (Fig. 5D
).
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Investigation into the signaling mechanisms involved in reoxygenation-mediated activation of Nrf2
Having demonstrated a role for Nrf2 in the transcriptional response to reoxygenation as a possible contributor to gene expression patterns in IRI, we then characterized the possible signaling events involved in this activation. It has been well characterized that hypoxia reoxygenation result in the creation of ROS such as superoxide and hydrogen peroxide (5)
. Not only have ROS been implicated in mediating protein and lipid membrane damage, they have also been suggested as signaling effectors (5)
. During insults where elevated ROS are observed and subsequent oxidative stress ensues, the cell uses the cytoprotective glutathione conjugation buffering system to eliminate toxic oxidizing intermediates. Depletion of glutathione can lead to further oxidative stress and cellular damage. To investigate whether oxidative stress through glutathione depletion is a contributor to Nrf2 activation on reoxygenation, we used the GSH synthesis inhibitor butathione sulfoxamine (BSO). Treatment of cells with BSO resulted in a moderate increase in Nrf2 accumulation after 3 h reoxygenation at a concentration of 1 mM. This increase was not observed at any concentration used after 6 h reoxygenation (Fig. 6
A). To complement these data, we set out to increase the buffering capacity of the cells’ glutathione system through use of the antioxidant N-acetyl cysteine (NAC). After treatment of cells with NAC, we observed an inhibition of reoxygenation-specific nuclear accumulation of Nrf2 (Fig. 6B
). Through indirect alteration of the cells’ glutathione content, these results implicate the involvement of ROS as a trigger for the reoxygenation-specific activation of Nrf2. Finally, we used various pharmacological protein kinase inhibitors to delineate putative signaling pathways involved in Nrf2 activation (Fig. 7
A). Pretreatment of cells with the p38 and c-Jun NH2-terminal kinase (JNK) MAPK pathway inhibitors SB203580 and SP600125, respectively, resulted in a moderate inhibition of reoxygenation-mediated activation of Nrf2. Inhibition of ERK MAPK and PKC using the specific inhibitors PD98059 and GF020848X resulted in significant attenuation of Nrf2 activation, whereas treatment with the PI3K inhibitor LY294002 resulted in a near ablation of Nrf2 nuclear accumulation. We also observed a similar pattern of inhibition of Nrf2 nuclear accumulation when these compounds were used under normoxic conditions. Our results indicate that the PI3K-Akt signaling pathway may play a role in the reoxygenation-specific response. To address this, we looked for activation of Akt using a phosphorylation-specific Ab that detects activation of this protein. Hypoxia resulted in a significant increase in Akt phosphorylation, which rapidly returned to normoxic levels on reoxygenation (Fig. 7B
).
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Nrf2-dependent gene expression from transplanted human liver
Since we observed Nrf2 activation and the induction of Nrf2-dependent gene expression in a murine model of IRI, we wanted to know whether this observation is translated into the human disease condition. Analysis of mRNA levels by real-time PCR analysis of liver biopsy taken postcold ischemia and portal reperfusion (reperfusion biopsy) for 1 h revealed a significant increase in Nrf2, ALDH1A7, and GSTP1 gene expression compared with donor retrieval biopsy (Fig. 8
).
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DISCUSSION
The phenomenon known as ischemic preconditioning (IPC) is a continuing area of important research for potential treatment development programs for IRI. The protective effect of IPC against subsequent ischemic injury was first described in the heart (22)
and is now well established as a universal phenomenon among tissues and organs, including brain, liver, small intestine, and kidney (23
24
25
, 6)
. Much of the work on the mechanisms involved in IPC has been carried out using myocardial models (26)
and has defined a well-characterized biphasic response. These studies have defined an initial acute-phase or classical immediate phase (within 1–4 h postinitial ischemic event) and a second, delayed phase (27)
24–48 h after the first preconditioning ischemic event, where protection against subsequent ischemic events is seen.
Many studies of the kidney document the early phase of IPC, when 3–10 min of ischemia is followed by a short reperfusion period of up to 60 min that protects against damage due to a subsequent severe ischemic event (30–45 min) (6)
. This early protection has been attributed to the release of soluble mediators such as adenosine and bradykinin (6)
. Much less work has been devoted to documenting the late delayed phase of IPC in the kidney, although Bonventre and others (28)
have demonstrated that 30 min of bilateral ischemia protects against ischemic injury 8 to 15 days later. The mechanisms responsible for triggering and affecting this delayed IPC phase are suggested to involve new protein synthesis because of the length of time it takes to develop (12–24 h). Proteins thought to be involved include heat shock proteins, inducible NOS (iNOS), COX-2, aldosereductase, and antioxidant enzymes such as MnSOD (29
30
31)
. In our present study we examined the pattern of gene expression after 30 min bilateral renal pedicle clamping and 24 h reperfusion to characterize putative effectors of this preconditioning effect observed at later time points.
Analysis of microarray data revealed the induction of a group of 7 phase II detoxification and antioxidant genes all within the top 20 most highly up-regulated, including aldehyde dehydrogenases, glutathione S transferases, and NQO1. The high incidence of antioxidant gene expression suggests a highly specific and important coordinated response to protect the cell against ongoing or future oxidative stress. This coordinated response likely involves Nrf2, as this transcription factor has been identified as an activator of these antioxidant genes in other model systems. Evidence to support this was demonstrated through the accumulation of Nrf2 protein in ischemia-reperfused kidney compared with sham-operated controls. We also observed specific medullary staining for Nrf2 within the kidney on IRI. Further analysis of the specific cell type or types within the medulla responsible for this staining will reveal greater insight into the mechanisms of Nrf2 activation and its importance in protecting against oxidant stress within the kidney.
Though much work has established the basic pathological characteristics of IRI and delayed ischemic preconditioning, the precise signaling mechanisms responsible remain unclear. It was recently demonstrated that ROS are generated during IRI (5)
. During ischemia-reperfusion, increased intracellular calcium levels are considered to contribute to cellular injury and cell death through an alteration in mitochondrial respiratory function causing an increase in damaging ROS, including superoxide anion radical (O2–), the hydroxyl radical (OH·), hydrogen peroxide (H2O2), and peroxynitrate (ONOO–). These ROS have also been postulated as cytoprotective signaling intermediates for preconditioning, although downstream targets have not been extensively investigated to date. The rise in levels of ROS on ischemia/reperfusion appears to be essential for the development of delayed IPC (32
33
34)
. An important overlooked source of ROS on ischemia reperfusion involves those produced upon reoxygenation. Hypoxia causes accumulation of hypoxanthine, which on reoxygenation forms O2– through the action of xanthine oxidase. The spontaneous production of hydroxyl radical forms from H2O2 can also occur through the action of free Fe2+ (Fenton reaction) (35
36)
. The generation of ROS on reoxygenation has been demonstrated in various model systems, including HUVECs through mitochondrial sources (37)
.
It has been widely demonstrated that Nrf2 activation occurs due to an alteration in the redox state of the cell due to the presence of increased amounts of electrophiles or ROS. In the context of our current work, we identified Nrf2 in a model of hypoxia/reoxygenation of renal epithelial cells within a cluster genes up-regulated specifically on reoxygenation through microarray analysis. We postulate that this reoxygenation-specific up-regulation is a mechanism through which cells activate an antioxidant response to protect themselves from future oxidant damage. It has been demonstrated that Nrf2 activation induces the expression of the Nrf2 gene itself (38)
. We also demonstrated a rapid nuclear accumulation of Nrf2 on reoxygenation and the induction of promoter reporter activity of one of the identified antioxidant genes, NQO1, which was abolished on deletion of the ARE sequence. We also observed a reoxygenation-specific up-regulation of the Nrf2-dependent gene GSTP1. All these data suggest that reoxygenation-specific activation of Nrf2 results in accumulation and transactivation of Nrf2-dependent Phase II detoxification and antioxidant gene expression, including Nrf2 itself, and this is a vital mechanism through which these genes are up-regulated in IRI to protect the cell from further damage. We also suggest this as a contributory mechanism through which the development of ischemic preconditioning occurs. Activation of Nrf2 has been observed in response to different ROS, and in our current study we present evidence to support that reoxygenation-specific activation of Nrf2 is mediated through ROS generation. This was evidenced through the use of the antioxidant NAC, which resulted in an inhibition of reoxygenation-specific activation of Nrf2. Additional evidence was generated through modification of the cells redox status using the glutathione synthesis inhibitor BSO, resulting in further activation of Nrf2 on reoxygenation.
To investigate other possible signaling events involved in the activation of Nrf2 on reoxygenation, we used an array of protein kinase inhibitors to the MAPKs p38, JNK, ERK, PI3kinase, and PKC. We observed substantial inhibition of reoxygenation-stimulated Nrf2 accumulation using the PKC and ERK MAPK pathway inhibitors and an abolition of activation using the PI3kinase pathway inhibitor LY294002. We observed a modest inhibition using p38 and JNK MAPK inhibitors. These results are interesting in the context that all of these pathways are activated in various models of IRI and hypoxia/reoxygenation. Indeed, the PI3K-Akt and ERK MAPK pathways are recruited in the setting of IPC (39
40)
. Inhibition of either PI3K or ERK kinase cascades abolished the protective effect of preconditioning to a subsequent ischemic event in these models. In the heart, inhibition of PKC prevented the protection seen with ischemic preconditioning (41)
. The involvement of PKC may not be all that clear; for example, PKC activation during ischemia is reported to be harmful and the specific actions of PKC-epsilon are reported to be inhibited by potent inhibitors of Ca2+-dependent PKC inhibitors such as GF109203X. Therefore, inhibition of Nrf2 accumulation observed in our model system using GF109203X could be due to multiple PKC isoforms, not just PKC
. There is compelling evidence for the involvement of these signaling cascades as specific triggers or mediators for reoxygenation and reperfusion-stimulated activation of Nrf2. However, treatment of cells with these kinase inhibitors under normoxic conditions resulted in a nearly identical pattern of inhibition of Nrf2 expression, as was observed under reoxygenation. This result indicates that these signaling pathways are likely necessary for basal expression of Nrf2 but are not the reoxygenation-specific trigger. This is further emphasized by the lack of reoxygenation-specific activation of Akt in these cells. It is therefore likely that some as yet undefined signaling cascade specifically activated on reoxygenation likely involving ROS is responsible for Nrf2 activation.
It has been put forward as to whether IPC actually occurs in humans. Some studies have been carried out, for example, using a human forearm model, where preconditioning 3 x 5 min ischemia before subsequent 20 min ischemia protects against endothelial dysfunction and neutrophil activation (42)
. There has also been evidence in the heart for the existence of IPC. It has been observed that patients with preinfarction angina have smaller infarct sizes, better functional recovery, and better prognosis, which the authors have suggested as a functional readout of ischemic preconditioning. We have demonstrated an up-regulation of Nrf2 and Nrf2-dependent antioxidant gene expression in ischemic reperfused human liver biopsies pre- and post-transplant, which we suggest as a mechanism of protection against current and subsequent oxidant damage as would occur with future ischemic events.
In summary we have demonstrated for the first time the induction of Nrf2 activation and Nrf2-dependent antioxidant gene expression in an in vivo model of IRI. We also demonstrate for the first time a reoxygenation-specific activation of Nrf2 and Nrf2-dependent antioxidant gene expression in a renal epithelial cell in vitro model. We therefore postulate that reoxygenation-specific activation of the Nrf2 antioxidant pathway as a contributory mechanism to the adaptive cytoprotective response to ongoing and subsequent oxidant damage in IRI. We also present evidence for the involvement of ROS in the reoxygenation-specific activation of Nrf2. Further investigation into the signaling pathways involved in this response may help our understanding of ischemic preconditioning toward future therapeutic potential.
ACKNOWLEDGMENTS
We acknowledge technical assistance from Annemarie Griffin. This work was supported by grants from the Health Research Board of Ireland, the Wellcome Trust (to C.T.T.), the Science Foundation of Ireland (to C.T.T.), and from the National Institutes of Health (M.J.B. and H.R.: N100K R01 and NH/LBI Lung Injury SCCOR).
Received for publication November 29, 2005. Accepted for publication August 7, 2006.
REFERENCES
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ARE), or empty vector (pGL3) luciferase-reporter constructs for 24 h prior to incubation under normoxic (40 h), hypoxic (40 h), or reoxygenation (hypoxia 16 h+24 h reoxygenation) conditions. Cells were then lysed and assayed for luciferase activity using luminometry. Results are expressed as mean fold over normoxia (F.O.N.) ± SEM for n = 4 independent experiments and deemed statistically significant at a P value <0.05 (*). D) HK-2 cells were exposed to hypoxia for 16 h, followed by various periods of reoxygenation (2–8 h). mRNA expression levels for GSTP1 were assessed using real-time PCR analysis. Results are expressed as mean fold over control (F.O.C.) normoxic levels ± SEM for n = 4 independent experiments.
