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Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia-reperfusion injury

Martin O. Leonard^*,1, Niamh E. Kieran^*, Katherine Howell^*, Melissa J. Burne, Raghu Varadarajan, Saravanakumar Dhakshinamoorthy, Alan G. Porter, Cliona O’Farrelly, Hamid Rabb and Cormac T. Taylor^*

^* 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, 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

SPECIFIC AIMS

In this study we investigated the underlying transcriptional response to ischemia-reperfusion injury (IRI), with particular emphasis on the role for NF-E2-related factor-2 (Nrf2) as a reoxygenation-specific activated transcription factor protective against oxidant-mediated injury on reperfusion.

PRINCIPAL FINDINGS

1. Ischemia reperfusion injury induces Nrf2 activation and antioxidant gene expression
To better understand the underlying adaptive transcriptional responses to IRI, we analyzed mRNA levels in a murine model of renal pedicle clamping for 30 min of ischemia followed by 24 h reperfusion. Microarray analysis revealed that within a cluster of the 20 most highly up-regulated genes were 7 genes with antioxidant and detoxification function, including aldehyde dehydrogenases 1A7 and 1A1, NAD(P)H:quinone oxidoreductase (NQO1), and glutathione S-transferases 5, 2, and 1 (Fig 1 A). Results were confirmed by real-time polymerase chain reaction (PCR) analysis (Fig 1B ). The antioxidant transcription factor Nrf2 has been noted in other model systems as a regulator of the expression of 6 of the 7 identified cytoprotective genes. We therefore hypothesized Nrf2 as a likely master regulator of this antioxidant gene expression on reperfusion injury. As activation of Nrf2 involves stabilization and accumulation of the protein prior to nuclear translocation and gene transactivation, evidence to support its role in mediating gene expression in ischemia-reperfusion was demonstrated as a significant increase in renal Nrf2 protein levels. We also observed increased localized staining for Nrf2 protein in the medullar region of ischemia-reperfused kidney compared with sham-operated control tissue.

View larger version (27K): [in this window] [in a new window]   Figure 1. Ischemia-reperfusion injury induced Nrf2-regulated gene expression. Ischemia reperfusion injury was induced in NIH Swiss mice by clamping renal pedicles for 30 min, followed by reperfusion for 24 h, whereupon kidneys were removed and processed for mRNA or protein isolation. A) mRNA from 4 IRI-treated and 4 sham-operated animals were pooled separately and processed for microarray analysis. Analysis was performed using the U77A Affymetrix chip set. Relative gene expression is depicted as fold over control (F.O.C.) sham-operated mRNA levels. Shown are 7 antioxidant genes expressed in the top 20 most highly up-regulated genes on IRI. B) Levels of mRNA expression were confirmed using real-time RT-polymerase chain reaction (RT-PCR) analysis. Results are expressed as IRI (n=8) F.O.C. sham (n=5) -operated levels and deemed statistically significant at a P value <0.05 (*) or <0.01 (**).

2. Reoxygenation of renal epithelial cells after hypoxia results in nuclear accumulation and activation of Nrf2
To identify specific mechanisms of Nrf2 induction and activation of antioxidant gene expression in IRI, we investigated a role for reoxygenation after hypoxia. Exposure of human renal epithelial HK-2 cells to hypoxia for 16 h followed by reoxygenation resulted in nuclear accumulation of Nrf2 protein as early as 2 h (Fig 2 A). We also observed reoxygenation-specific nuclear accumulation of Nrf2 in colonic epithelial T84 cells, indicating a response specific not only to renal epithelial cells (Fig 2B ). There were no significant alterations in nuclear accumulation of the NF-E2-like family member Nrf1 indicating a signaling pathway specific for Nrf2 activation. We next analyzed whether this accumulation results in activation of Nrf2-dependent gene expression. Reoxygenation of cells transfected with a NQO1 promoter reporter construct resulted in a significant increase in reporter activity (2.45-fold compared with empty vector control). Mutation of the Nrf2 binding antioxidant response element (ARE) within the NQO1 promoter resulted in a loss of reoxygenation-specific activation (Fig 2C ). Further evidence for a role for Nrf2 in reoxygenation-specific gene activation was revealed as the presence of Nrf2 in a reoxygenation-specific cluster of genes identified by microarray analysis and through induction of the Nrf2-dependent gene GSTP1 (Fig 2D ).

View larger version (13K): [in this window] [in a new window]   Figure 2. Reoxygenation-specific activation of Nrf2 and Nrf2-dependent gene expression. A) HK-2 cells were exposed to hypoxia (1% atmospheric O2) for 16 h before reoxygenation (21% atmospheric O2) for 1–6 h. Protein nuclear extracts were prepared and analyzed for Nrf2 and Nrf1 expression by SDS-PAGE and Western blot analysis. B) T84 cells exposed to hypoxia and reoxygenation were also analyzed for nuclear Nrf1 and Nrf2 protein content by SDS-PAGE and Western blot analysis. C) HK-2 cells were transfected with either human NQO1 promoter wild-type (WT) (NQO1 WT), antioxidant response element mutated (NQO1 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 F.O.C. normoxic levels ± SEM for n = 4 independent experiments.

3. Investigation into intracellular signaling pathways 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 characterized possible signaling events involved in its activation. Reactive oxygen species (ROS) generation after hypoxia and reoxygenation has been implicated as a trigger for gene expression in reperfusion injury. To address whether such signals are responsible for Nrf2 activation on reoxygenation, we altered glutathione content to adjust the cells’ response to ROS and oxidative stress. Using the glutathione synthesis inhibitor butathione sulfoxamine (BSO), we observed a moderate increase in Nrf2 accumulation after 3 h reoxygenation. Treatment of cells with the antioxidant N-acetyl cysteine (NAC) resulted in inhibition of reoxygenation-specific induction of Nrf2 nuclear accumulation.

We used pharmacological protein kinase inhibitors to delineate signaling cascade involvement in Nrf2 activation. Pretreatment of HK-2 cells with the p38 and c-Jun NH2-terminal kinase (JNK) MAPK pathway inhibitors SB203580 and SP600125 resulted in a slight inhibition of reoxygenation-mediated activation of Nrf2. Inhibition of ERK MAPK and PKC using specific inhibitors PD98059 and GF020848X resulted in significant attenuation of Nrf2 activation, and treatment with the PI3K inhibitor LY294002 resulted in near ablation of Nrf2 nuclear accumulation. A similar pattern of inhibition of Nrf2 nuclear accumulation was observed with these compounds under normoxic conditions.

4. Induction of Nrf2-dependent gene expression in transplanted ischemic reperfused human liver
We wanted to know whether Nrf2 activation and induction of Nrf2-dependent gene expression in a murine model of IRI were translated into human disease. Analysis of mRNA levels by real-time PCR analysis of liver biopsy taken after cold 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.

CONCLUSIONS AND SIGNIFICANCE

In this study microarray analysis revealed induction of a group of 7 phase II detoxification and antioxidant genes all within the top 20 most highly up-regulated on renal IRI. This coordinated response likely involves Nrf2-regulated transactivation of cis-acting AREs within the regulatory regions of these genes, as Nrf2 has been identified as a master regulator of these genes in other model systems. We demonstrate an increased staining for and accumulation of Nrf2 protein in ischemia-reperfused kidney, implicating activation of this transcription factor as a mechanism of induction of cytoprotective antioxidant gene expression. From our in vitro studies, we demonstrate a reoxygenation-specific induction of Nrf2 protein nuclear accumulation. We also demonstrate the reoxygenation-specific activation of NQO1 promoter reporter activity, which is inhibited upon deletion of the ARE through which Nrf2 binds. RNA microarray analysis identified Nrf2 specifically within a cluster of genes induced on reoxygenation alone.

Investigation into the intracellular signaling mechanisms in reoxygenation-specific activation of Nrf2 revealed a role for ROS by use of the antioxidant NAC and the glutathione synthesis inhibitor BSO. We noted a role for kinase signaling pathways in Nrf2 expression that likely contributes to basal but not reoxygenation-triggered expression of Nrf2. Evidence points to activation of a pathway specific for cytoprotection against oxidative damage in IRI induced by reoxygenation after hypoxia/ischemia.

We have demonstrated for the first time induction of Nrf2 activation and Nrf2-dependent antioxidant gene expression in an in vivo model of IRI, as well as reoxygenation-specific activation of Nrf2 and Nrf2-dependent antioxidant gene expression in a renal epithelial cell in vitro model. We postulate that reoxygenation-specific activation of the Nrf2 antioxidant pathway is a contributory mechanism to the adaptive cytoprotective response to ongoing and subsequent oxidant damage in ischemia-reperfusion. Further delineation of the signaling mechanisms involved in this response—specifically the involvement of ROS signaling pathways—may enhance our understanding of ischemic preconditioning in protecting against oxidative damage.

View larger version (10K): [in this window] [in a new window]   Figure 3. Schematic illustrating proposed mechanism of induction of Nrf2-dependent antioxidant cytoprotective gene expression in ischemia reperfusion injury. Ischemia reperfusion induces expression of phase II detoxification/antioxidant gene expression and Nrf2 activation, which protects the cell from oxidant damage and cellular injury. We propose that hypoxia/reoxygenation as a part of ischemia reperfusion is a trigger for Nrf2 activation through ROS generation. Nrf2 then binds to antioxidant response elements in the promoters of these genes and increases gene expression, causing a reduction in oxidant-mediated tissue injury.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5097fje

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