Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yin, M.
Right arrow Articles by Thurman, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yin, M.
Right arrow Articles by Thurman, R. G.
J Am Soc Nephrol 12:2691-2700, 2001
© 2001 American Society of Nephrology

Cu/Zn-Superoxide Dismutase Gene Attenuates Ischemia-Reperfusion Injury in the Rat Kidney

Ming Yin*, Michael D. Wheeler*, Henry D. Connor*, Zhi Zhong*, Hartwig Bunzendahl{dagger}, Anna Dikalova||, Richard J. Samulski{ddagger}, Robert Schoonhoven§, Ronald P. Mason||, James A. Swenberg§ and Ronald G. Thurman*

Departments of *Pharmacology and {dagger}Surgery, {ddagger}Gene Therapy Center, and §Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina; and ||Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Science, Research Triangle Park, North Carolina.

Correspondence to Dr. Ronald G. Thurman, Department of Pharmacology, CB 7365, Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599-7365. Phone: 919-966-4745; Fax: 919-966-1893; E-mail: thurman{at}med.unc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Evidence has accumulated for a role of toxic oxygen radicals in the pathogenesis of ischemia-reperfusion injury in the kidney. The aim of this study was to evaluate the hypothesis that reducing postischemic renal injury is possible by delivery of the gene for the antioxidant enzyme superoxide dismutase (SOD). Female Sprague-Dawley rats received intravenous injections of recombinant adenovirus (1 x 109 pfu) containing the transgenes for Escherichia coli ß-galactosidase (Ad-LacZ, as control) or human Cu/Zn-SOD (Ad-SOD). Three days later, renal ischemia was produced by cross-clamping the left renal vessels for 60 min. The right kidney was removed before reperfusion and processed for the transgene. Renal SOD protein and activity in rats given Ad-SOD was 2.5-fold higher than from the animals receiving Ad-LacZ. Urinary lactate dehydrogenase concentrations were elevated by ischemia-reperfusion in the Ad-LacZ group (1403 ± 112 U/L), yet values were 50% lower in Ad-SOD-treated rats. Free radical production was elevated by ischemia-reperfusion but was significantly lower in SOD-treated animals. Importantly, on postischemic day 1, glomerular filtration rates were reduced to 0.21 ml/min per 100 g in the Ad-LacZ group, whereas values remained significantly higher (0.39) in the Ad-SOD group. Two weeks after ischemia-reperfusion, inflammation, interstitial fibrosis, tubular atrophy and tissue levels of tumor necrosis factor alpha and interleukin-1 were significantly higher in the Ad-LacZ-treated than in Ad-SOD-treated rats. In conclusion, these results indicate that SOD expression can be increased by delivery of the sod gene to the kidney by intravenous injection and that sod gene transduction minimized ischemia-reperfusion-induced acute renal failure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute renal failure affects as many as 5% of all hospitalized patients (1) and has an unacceptably high rate of mortality (2). Ischemia-reperfusion-induced renal injury is the most common cause of acute renal failure (3) and is characterized by pronounced depression of the glomerular filtration rates (GFR). After transplantation, decreased GFR due to ischemia-reperfusion may lead to delayed renal graft function, a reversible dysfunction, or even irreversible nonfunction. This problem is almost always observed in transplantation of cadaveric renal allografts that have been subjected to warm or cold ischemia, then reperfusion. Indeed, delayed graft function has adverse effects on long-term survival of allografts (4), hampering efforts to increase the donor pool by using marginal donor organs (5). Therefore, efforts to reduce damage to kidneys exposed to ischemia-reperfusion-induced injury are essential.

Oxygen-derived free radical species have been implicated in the pathogenesis of ischemia-reperfusion-induced renal injury (6,7). During ischemia, increased hypoxanthine and xanthine are produced as a result of ATP degradation (8), and xanthine dehydrogenase is converted into xanthine oxidase (9,10). With the reintroduction of molecular oxygen upon reperfusion, purines are metabolized by xanthine oxidase, leading to the production of superoxide radicals (6,10). Superoxide radicals can be converted into highly reactive hydroxyl radicals, which react with proteins, lipids, or nucleic acids, leading to renal damage. Moreover, the endogenous scavenger superoxide dismutase (SOD) is rapidly depleted during ischemia and reperfusion, especially the cytoplasmic copper-zinc form (11,12). Previously, exogenous SOD has been administered, but the protective effects on tissue injury have been minimal (13), probably because the half-time for plasma elimination of SOD is minutes (14). Therefore, clinical use of SOD as a scavenger for superoxide radicals has been limited, and therapeutic approaches designed to deliver SOD to intracellular sites continuously would be extremely valuable.

One such promising therapeutic approach involves gene transfer (15). It is therefore hypothesized that delivery of the sod gene could provide high and long-lasting levels of SOD at the site of free radical generation (i.e., inside the cell). It has been demonstrated that adenovirus (Ad)-mediated transfer of Cu/Zn-SOD cDNAs protects human endothelial cells against oxidant stress in vivo (16). The purpose of this study therefore was to investigate whether levels of SOD in rat kidney can be increased after injection with Ad-SOD gene and to evaluate whether gene delivery could minimize acute renal failure caused by ischemia-reperfusion. Indeed, use of an adenoviral vector encoding Cu/Zn-SOD (Ad-SOD) protected rats against ischemia-reperfusion-induced renal injury. Preliminary accounts of this work have appeared elsewhere (17).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adenoviral Vectors
Recombinant adenovirus containing the transgene for either human Cu/Zn-SOD (Ad-SOD) or Escherichia coli ß-galactosidase (Ad-LacZ) was prepared as described elsewhere (18,19). In short, the plasmid shuttle vectors pAd5.CMV.LacZ and pAd5.CMV.SOD1 were constructed by standard cloning protocols as described by Sambrook et al. (20). The adenoviral shuttle plasmids were transfected into the permissive HEK 293 host cell line to generate recombinant Ad-LacZ. Ad-SOD was obtained as a viral seed stock as a kind gift from Dr. John Engelhardt (University of Iowa). Virus isolates were plaque purified and propagated in HEK 293 cells, isolated, concentrated, and titered by plaque assay. Viral vectors of different titers were diluted in 0.4 ml of saline and injected into the iliac vein via a local incision. The skin incision was closed with two stitches.

Animals and Surgical Procedures
Adult female Sprague-Dawley rats (200 to 250 g) had free access to standard rat chow and tap water and received humane care according to the criteria of the US National Institutes of Health. Three days after viral injections (1 x 109 pfu), animals were anesthetized with ether and placed under a warm lamp to maintain body temperature. A midline laparotomy was performed, and the left kidney was dissected free from the surrounding tissue (21). A nontraumatic vascular clamp was placed across the renal pedicle to induce ischemia for 60 min, and the abdomen was temporarily closed during the ischemic period. Five minutes before the end of the ischemic period, a contralateral nephrectomy was performed. The excised kidney was cut into two pieces; one piece was frozen and one was fixed in formalin for transgene detection and histology. Reperfusion of the left kidney was achieved by releasing the vascular clamp. The abdominal wall was closed with double-layer sutures and the animals were allowed to recover. Rats were killed 14 d after reperfusion, and the kidneys were removed for histology.

Transgene Detection: Western Blot of SOD
Whole kidney tissue was homogenized and 30 µg of protein was resolved by electrophoresis via 16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted with anti-Cu/Zn-SOD antibody (Oxis, Portland, OR), followed by horseradish peroxidase-conjugated secondary antibody. Protein detection was visualized by chemiluminescent detection by ECL Western Detection Reagent (Amersham Life Science, Paisley, England).

Detection of ß-Galactosidase Activity
Whole kidney tissue was homogenized in buffer A containing 40 mmol/L Tris, 140 mmol/L NaCl, and a protease inhibitor cocktail including 1 µg/mL aprotinin, 1 µg/mL leupeptin, 10 mg/ml phenylmethylsulfonyl fluoride (PMSF), and 1 mmol/L dithiothreitol. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C, and the supernatant was collected and diluted with buffer A to a final volume of 150 µl. A 150-µl aliquot of assay buffer B containing 120 mmol/L Na2HPO4, 80 mmol/L NaH2PO4, 2 mmol/L MgCl2, 100 mmol/L 2-mercaptoethanol, and 1.33 mg/ml of O-nitrophenyl-ß-D-galactopyranoside was added to the supernatant and incubated for 30 min at room temperature. The reaction was stopped by addition of 500 µl of a 2.8% sodium carbonate solution. Activity of ß-galactosidase was quantified by nitrophenol formation, which was measured spectrophotometrically at 420 nm. Values were normalized to total protein determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA).

Detection of SOD Activity
Kidney tissue was homogenized in a buffer containing 40 mM Tris, 140 mM NaCl, and the protease inhibitors aprotinin, leupeptin, PMSF, and dithiothreitol, then centrifuged at 10,000 x g for 10 min at 4°C. SOD activity was measured in the supernatant by the reduction of ferricytochrome c as described by McCord and Fridovich (9). Supernatant (10 µl) was added to a solution containing 50 mM K2HPO4, 0.1 mM Na2 ethylenediaminetetraacetate, and 0.5 mg/ml xanthine oxidase (0.4 U/ml). The reduction of cytochrome c was measured spectrophotometrically at 550 nm. SOD activity was calculated on the basis of the millimolar extinction coefficient of 18.5 for reduced cytochrome c.

Immunohistochemical Staining
Because of the endogenous expression of ß-galactosidase and SOD in normal kidney tissue (22,23), localization of recombinant transgenes was difficult via immunohistochemistry. Thus, an adenoviral vector carrying exogenous transgene linked to hemagglutinin (HA) epitope (Ad-HA-tagged I{kappa}B{alpha}) was used as a reporter gene. Rats were injected with Ad-HA-tagged I{kappa}B{alpha} (1 x 109 pfu) intravenously and killed 3 d later. Sections (5 µm) of paraffin-embedded kidneys were stained by use of monoclonal antibody against HA-tagged recombinant protein conjugated with peroxidase (anti-HA-peroxidase, Roche Diagnostics GmbH, Mannheim, Germany) and the Dako Envision System (Dako Corporation, Carpinteria, CA).

GFR
Rats were placed in metabolic cages with free access to water and chow, and 24-h urine samples were collected. Blood samples were taken from the tail vein on days 1, 3, 7, and 14 after reperfusion. Creatinine levels in urine and serum were determined with a commercially available kit (Sigma Diagnostic, Inc., St. Louis, MO), and GFR were calculated from the ratio of creatinine in the urine/blood, the volume of urine produced in 24 h, and the body weight (24).

Histopathology
Renal tissue fixed in formalin was processed by dehydration and then embedded in paraffin. Sections were cut at 5 µm and stained with hematoxylin and eosin for histologic assessment in a blinded manner. In the evaluation of kidneys harvested 2 h after reperfusion, the severity of renal damage in terms of tubular epithelial injury and debris accumulation (cast formation) was graded with an arbitrary score of 0 to 3, where 0 was normal, 1 mild, 2 moderate and 3 severe (25). A similar scoring system was also used in the evaluation of tubular atrophy and interstitial inflammation and fibrosis in kidneys 2 wk after reperfusion. The total number of infiltrating leukocytes (e.g., neutrophils and mononuclear cells) in cortical interstitial spaces was counted. The mean values in five high-power fields were used for statistical analyses.

Detection of Free Radicals Adducts and Lactate Dehydrogenase in Urine
In a separate study to detect urinary free radicals and lactate dehydrogenase (LDH), rats were killed after collection of urine samples for 2 h, and kidneys were removed for histology. The spin-trapping reagent 4-POBN (1 g/kg body weight) was dissolved in 2 ml saline and injected slowly into the tail vein 15 min before ischemia. After reperfusion, urine was collected through a catheter placed in the urinary bladder for 2 h and samples were kept at -80°C until electron spin resonance (ESR) analysis. Samples were placed in an aqueous flat cell and bubbled with oxygen for 5 min to eliminate interfering ascorbyl free radical and with nitrogen for 5 min to eliminate line broadening by molecular oxygen. Free radical adducts were detected with a Bruker ESP 300 ESR spectrometer (Bruker Instrument, Billerica, MA). Instrument conditions were as follows: 20-mW microwave power; 1.01-G modulation amplitude, and 80-G scan range (26). LDH assays were performed via standard enzymatic techniques (27).

RNase Protection Assay
Total RNA was isolated from renal tissue by RNA STAT 60 (Tel-Test, Friendswood, TX). RNase protection assays were performed by the RiboQuant multiprobe assay system (Pharmingen, San Diego, CA). Briefly, [32P] RNA probes were transcribed with T7 polymerase with the multiprobe template set at rCK-1, and RNA (10 µg) was hybridized with 4 x 105 cpm of probe overnight at 56°C. Samples were then digested with RNase followed by proteinase K treatment, phenol:chloroform extraction, and ethanol precipitation, and were resolved on a 5% acrylamide-bisacrylamide (19:1) urea gels. After drying, gels were visualized by autoradiography.

Statistical Analyses
ANOVA that used Tukey’s post hoc test, one-way repeated-measures ANOVA, and Wilcoxon signed rank test were used for the determination of statistical significance as appropriate. Data are presented as means ± SEM. P < 0.05 was selected before the study as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of SOD after Gene Transfection
To find an optimal dose of Ad-SOD for this study, serum alanine transaminase and SOD protein expression in kidneys were evaluated in rats receiving 1 x 108, 1 x 109, or 1x 1010 pfu of Ad-SOD and 1 x 109 of Ad-LacZ (n = 4 per group). During the first 2 d after viral injection, animals treated with 1 x 108 or 1 x 109 pfu of adenoviral vectors looked healthy, but most of the rats given 1x 1010 pfu of Ad-SOD were sedentary and feverish. The administration of 1 x 108 or 1 x 109 of Ad-LacZ or Ad-SOD did not affect serum alanine transaminase levels (<40 U/L). However, alanine transaminase levels from rats receiving 1 x 1010 pfu of Ad-SOD were about twofold higher than levels in animals receiving lower titers of virus. This is most likely due to viral-induced toxicity; therefore, a dose of 1 x 109 pfu was chosen for this study because of its lack of side effects.

In kidneys harvested 3 d after Ad-SOD injections, increased levels of human SOD protein were observed in rats receiving 1 x 109 and 1 x 1010 pfu of the Ad-SOD (Figure 1A). Enzyme activity in renal tissue was also measured 3 d after injection of 1 x 109 pfu of either Ad-LacZ or Ad-SOD. ß-Galactosidase (LacZ) activity in kidneys from rats receiving the Ad-LacZ gene was about fourfold higher than animals given saline or the Ad-SOD gene (Figure 1B). Moreover, SOD activity in kidneys from animals receiving Ad-SOD was elevated about 2.5-fold (Figure 1C). Thus, Ad-LacZ and Ad-SOD gene delivery led to protein expression and increased enzyme activity in the kidney.



View larger version (16K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Expression of superoxide dismutase (SOD) in the kidney after gene transfection. (A) Expression of SOD protein was analyzed by Western blot test as described under Materials and Methods. The kidneys used for the measurements of enzyme activity were excised 3 d after injection of either Ad-LacZ or Ad-SOD (1 x 109 pfu). Activity of ß-galactosidase (B) and SOD (C) was determined as described under Materials and Methods. Data represent means ± SEM (n >= 6 per group). *, P < 0.05 versus the other 2 groups by 1-way ANOVA (Tukey’s post hoc test).

 
Localization of Gene Transfection
Three days after injection of an Ad-HA-tagged reporter gene, the tagged gene products could be detected almost everywhere in the kidney (Figure 2). More than 80% of proximal tubules in outer cortical areas showed strong positive staining (Figure 2B), and nearly all of the proximal tubules in the inner cortex were positive (Figure 2D). In glomeruli, capillary endothelial cells were positive for gene transfection, whereas mesangial cells were mostly negative. Nearly all of the epithelial cells of the tubules and loop of Henle in the outer medulla were transfected (Figure 2F). Moreover, the majority of cells in the inner medulla were also positive (Figure 2H), and nearly all of the peritubular endothelial cells were stained positive, although to varying extents. These results indicate that most tubular epithelial cells and vascular endothelial cells are transfected by intravenous administration of adenoviral vectors.



View larger version (164K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Representative photomicrographs of kidneys from rats receiving saline or hemagglutinin (HA)-tagged adenoviral reporter gene. Animals were injected with saline (0.4 ml) or HA-tagged adenovirus (Ad)-reporter gene (1 x 109 pfu) intravenously and killed 3 d later. Immunohistochemical staining was performed to locate the sites of gene delivery in kidneys as described under Materials and Methods. The left column is the kidney from rat given saline; the right column is the one receiving adenovirus. (A and B) Outer cortex. (C and D) Inner cortex. (E and F) Inner medulla. Magnification, x200.

 
Effects of Ad-SOD on Ischemia-Reperfusion-Induced Renal Injury
Morphology of kidneys that were excised 3 d after injection of 1 x 109 pfu of Ad-LacZ or Ad-SOD is depicted in Figure 3, A and B. These photomicrographs demonstrate normal renal histology with intact integrity of tubules and interstitial space, indicating that viral vectors at the dose used here did not cause morphologic damage. Representative photomicrographs of kidneys 2 h after ischemia-reperfusion are shown in Figure 3, C and D. Kidneys from rats receiving Ad-LacZ exhibited dramatic renal injury (Figure 3C), with tubular cell swelling, loss of brush border, and cast formation. On the other hand, only mild tubular injury was observed in kidneys from rats treated with Ad-SOD (Figure 3D). Pathology scores of these kidneys 2 h after ischemia-reperfusion are shown in Figure 4. Nonischemic kidneys scored zero, whereas the extent of tubular epithelial injury in the kidneys of rats receiving Ad-LacZ was increased dramatically. Ad-SOD-treated kidneys, however, exhibited much less injury than kidneys receiving Ad-LacZ (P < 0.05). Furthermore, cast formation in kidneys from rats given Ad-LacZ was more than fourfold higher than values from Ad-SOD-treated kidneys (P < 0.05). As a result, the sum of pathology scores from Ad-LacZ-treated kidneys was 3.1 ± 0.4, a value about twofold higher than that of Ad-SOD-treated kidneys. This indicates that early ischemia-reperfusion-induced renal injury was largely reduced by the successful delivery of the Ad-SOD gene and expression of SOD protein.



View larger version (121K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Representative photomicrographs of kidneys from rats receiving Ad-LacZ or Ad-SOD before and after ischemia and reperfusion. Animals were treated as described under Materials and Methods. (A) Kidney from rat 3 d after receiving Ad-LacZ. (B) Kidney 3 d after receiving Ad-SOD. (C) Kidney of Ad-LacZ-treated rat 2 h after ischemia and reperfusion. (D) Ad-SOD-treated kidney 2 h after ischemia and reperfusion. (E) Ad-LacZ-treated kidney 2 wk after ischemia and reperfusion. (F) Ad-SOD-treated kidney 2 wk after ischemia and reperfusion. (G) Ad-LacZ-treated kidney 2 wk after ischemia and reperfusion. Blue is due to fibrosis. (H) Ad-SOD-treated kidney 2 wk after ischemia and reperfusion. Stain, hematoxylin and eosin (A to F) and trichome (G and H). Magnification, x200.

 


View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Effects of delivery of Ad-LacZ or Ad-SOD genes on renal histology 2 h after ischemia and reperfusion. Pathology was scored as described under Materials and Methods. N, nontreated normal kidneys. Data represent means ± SEM (n >= 6 per group). *, P < 0.05 versus the Ad-LacZ-treated groups by Wilcoxon signed rank test.

 
Representative photomicrographs of kidneys treated with either Ad-LacZ or Ad-SOD 2 wk after ischemia and reperfusion are shown in Figure 3E and Figure 3F, respectively. Increased inflammation, interstitial fibrosis, and tubular atrophy were observed in kidneys from rats receiving the Ad-LacZ gene (Figure 3E); however, Ad-SOD-treated kidneys exhibited almost normal renal architecture (Figure 3F). Pathology scores quantitating the extent of tubular atrophy, interstitial infiltration, and fibrosis were significantly two- to threefold higher in the Ad-LacZ-treated kidneys than in kidneys from Ad-SOD-treated rats (Figure 5A). Quantitation of infiltrating leukocytes showed that there were only a minimal number of leukocytes in the nonischemic kidneys (Figure 5B); however, a large number of infiltrating leukocytes were observed in the renal cortex of rats receiving the Ad-LacZ gene (Figure 2E). sod gene transduction significantly reduced inflammatory cell infiltration about two- to threefold (Figure 5B). Figure 3G shows dramatic interstitial fibrosis around atrophic tubules in kidneys from Ad-LacZ-treated rats, whereas changes were minimal after Ad-SOD transfection (Figure 3H).



View larger version (11K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Effects of delivery of Ad-LacZ or Ad-SOD on renal histology 2 wk after ischemia and reperfusion. Pathology was scored (A) and the number of infiltrating leukocytes was quantitated (B) as described under Materials and Methods. N, nontreated normal kidneys. Data represent means ± SEM (n = 10 per group). *, P < 0.05 versus the Ad-LacZ-treated groups by Wilcoxon signed rank test (pathology score) or 1-way ANOVA (number of leukocytes).

 
Effects of Ad-SOD on GFR
GFR during the first 2 wk after ischemia-reperfusion are shown in Figure 6. The values at day 0 (i.e., normal GFR measured before ischemia) were about 5.5 ml/min per 100 g. In rats receiving the Ad-LacZ gene, GFR was reduced to about 40% of normal values 1 d after ischemia-reperfusion and did not recover fully until 2 wk later. This was comparable to values of rats given saline (data not shown); however, GFR was reduced significantly less and increased much faster in rats receiving Ad-SOD.



View larger version (15K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. Effects of delivery of Ad-SOD on glomerular filtration rates (GFR) after ischemia and reperfusion. Urine and blood samples were collected and GFR was determined as described under Materials and Methods. Data represent means ± SEM (n >= 8 per group). *, P < 0.05 versus the Ad-LacZ-treated group by 1-way repeated-measures ANOVA.

 
Effects of Ad-SOD on Urinary Release of LDH and Free Radical Adduct Formation after Ischemia-Reperfusion
LDH release in urine from normal rats was minimal (Figure 7); however, 2 h after reperfusion, LDH levels were increased dramatically in Ad-LacZ-treated animals. Importantly, values were increased significantly less in rats receiving Ad-SOD (P < 0.05). Reperfusion subsequent to renal ischemia could lead to free radical formation. Accordingly, free radicals were trapped with the spin-trapping reagent 4-POBN and detected with ESR. Figure 8 shows representative ESR spectra due to free radical adducts in urine collected during the first 2 h of reperfusion. A six-line ESR spectrum due to radical adducts was detected in urine samples from both groups. However, free radical signals from urine of a rat receiving Ad-LacZ were almost threefold larger than Ad-SOD-treated rats, indicating that transfer of sod gene reduced free radicals.



View larger version (13K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Effects of delivery of Ad-SOD on urinary lactate dehydrogenase (LDH) release after ischemia and reperfusion. Urine samples were collected via a catheter placed in the urinary bladder during the first 2 h after ischemia, and activity of LDH was determined as described under Materials and Methods. Data represent means ± SEM (n >= 6 per group). *, P < 0.05 versus the other 2 groups by 1-way ANOVA (Tukey’s post hoc test). Typical spectra are shown.

 


View larger version (22K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 8. Effects of delivery of Ad-SOD on electron spin resonance (ESR) spectrum of free radical adducts in urine. The spin trapping reagent 4-POBN (1 g/kg) was injected intravenously 10 min before ischemia. Urine samples were collected through a catheter placed in the urinary bladder during the first 2 h of reperfusion. Free radical adducts in urine were detected by ESR as described under Materials and Methods. Typical spectra are shown. (A) Rat received saline. (B) Rat received Ad-LacZ (1 x 109 pfu). (C) Computer simulation of the radical adduct spectrum from Ad-LacZ-treated rat. (D) Rat received Ad-SOD (1 x 109 pfu). n >= 3 per group.

 
Effects of Ad-SOD on Tissue Levels of Inflammatory Cytokines
Tissue levels of the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-{alpha}) could be detected in ischemically injured kidneys from rats receiving Ad-LacZ 2 wk after ischemia-reperfusion (Figure 9). However, in tissue from rats treated with Ad-SOD, IL-1 was reduced dramatically and TNF-{alpha} could not be detected.



View larger version (45K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 9. Effects of delivery of Ad-SOD on inflammatory cytokines in postischemic kidneys. Tumor necrosis factor alpha (TNF-{alpha}) and interleukin-1 (IL-1) mRNA were determined in renal tissue after 2 wk of ischemia-reperfusion by RNase protection assay as detailed under Materials and Methods. Representative data are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene Delivery of SOD Reduces Ischemia-Reperfusion Injury in the Kidney
Although ischemia will ultimately destroy living tissues, oxidant stress caused by reperfusion increases injury (6,7). The pathogenesis of ischemia-reperfusion-related changes involves reactive oxygen species (6,8,10), and the results of the study reported here support the harmful role of oxidants. It was observed that increased levels of intracellular SOD in kidney minimized ischemia-reperfusion-induced tubular injury and improved postischemic renal function (Figures 3 to 7). Importantly, S OD levels in kidney were elevated by delivering the Cu/Zn sod gene in vivo with an adenoviral vector as evidenced by increased tissue SOD protein expression and elevated enzyme activity (Figure 1). Moreover, attenuated postischemic renal failure in animals with overexpression of SOD was accompanied by reduced free radical adducts in urine (Figure 8). To our knowledge, this report is the first to demonstrate protection against kidney ischemia-reperfusion injury by antioxidant gene products by successful gene delivery.

Ischemia-reperfusion injury of the kidney involves components of a typical inflammatory reaction (28,29). Oxygen free radicals can directly trigger the activation of leukocytes and expression of adhesion molecules (30). Enhancement of inflammatory cytokine production (IL-1 and TNF-{alpha}), which increases expression of cell adhesion molecules (ICAM-1), allows neutrophils to adhere and accumulate in peritubular vessels causing capillary plugging and obstruction (31–33), leading to tissue destruction and reduced GFR. Indeed, the inflammatory reaction in ischemically injured kidney could still be detected 2 wk after the initial insult in kidneys from Ad-LacZ-treated rats (Figures 3E and 5B) and was accompanied by expression of the proinflammatory cytokines, TNF-{alpha} and IL-1 (Figure 9). Importantly, these phenomena were attenuated by SOD overexpression, which may be attributed both to extra enzyme in the kidney upon reperfusion and its continuous production during the recovery period.

Advantages of Antioxidant Gene Delivery in Protection of Renal Ischemia-Reperfusion Injury
SOD is a naturally occurring intracellular enzyme that catalyzes the breakdown of superoxide radicals (34). Ischemia and reperfusion leads not only to an increase in superoxide production, but also to a rapid depletion of SOD (11,12). Numerous studies have assessed the potential benefits of exogenous SOD in preventing reperfusion injury, but data are conflicting (8,35). As a result, an efficient way to inhibit oxidative injury has not been established. One important factor responsible for unfavorable effects of exogenous SOD administration is most likely its short half-life in plasma (14). Gene transfer technology has the theoretical advantage of introducing SOD genes into kidney tissue, leading to continuous production of the gene product. Indeed, effective gene delivery was established by intravenous injection of the gene vectors (1 x 109 pfu) 3 d before the ischemic insult without toxic side effects (Figure 1). Moreover, nearly all components of renal tissue were transfected 3 d after intravenous administration of the adenoviral vector (Figure 2), most likely making vulnerable renal regions more resistant to the ischemia-reperfusion insults.

The use of adenovirus as a vector for kidney-directed gene therapy has made significant progress in the area of kidney biology, in particular in hereditary kidney disease and inflammatory and fibrotic disease (36). Adenoviral vectors have distinct advantages of high titers and high expression of the transgene. Because the adenovirus can infect both dividing and nondividing cells (37), the vector has a significant advantage in delivering genes into quiescent or terminally differentiated cells and is thus suitable for gene transfer into complex organs such as the kidney. Adenoviral vectors also have disadvantages. First, the expression of the transfected gene is limited to weeks or months because the adenovirus does not integrate into the host genome. Second, the adenovirus can elicit immunological responses; therefore, the vector cannot be administered repeatedly. Nonetheless, ischemia-reperfusion-induced renal injury usually occurs in emergency situations, with harmful effects maximal within a week (e.g., posttransplant acute renal failure). This makes the adenoviral vector especially suitable for antioxidant gene delivery in prevention of acute renal failure.

Previously, Heikkila and colleagues (38) tried to use ex vivo kidney perfusion system in adenoviral gene delivery. They found that about 75% of glomerular cells were transfected and no tubular cells expressed transgene. Apparently, this is a good model for gene therapy in kidney transplantation and allows renal allografts to be treated before implantation. Kidneys in a perfusion system may behave differently as a result of surgical procedures, temperature, and use of perfusion media. Moreover, Moullier and colleagues (39) achieved adenoviral gene transfer to tubular cells by infusion through renal artery. Although this technique enables the vector to go through kidney, then into bloodstream, it appears to be associated with trauma. In this study, virus circulates in the blood until it enters cells and has minimal side effects.

Clinical Implications
The study reported here demonstrates the feasibility of delivering exogenous SOD with an adenoviral gene transfer approach, with successful expression of the gene product. Moreover, overexpression of SOD in kidney by gene delivery provides protection against ischemia-reperfusion-induced renal injury. This study raises the intriguing possibility of the potential therapeutic use of SOD, not only in hypoxia-reoxygenation injury, but also in other inflammatory renal disease states. It has been reported that initial postischemic injury and free radicals are involved in progression of acute and chronic rejection (35,40,41). The protection of free radical-mediated early injury by SOD gene transfection shown in this study may have beneficial effects on the long-term outcome of kidney allografts.


   Acknowledgments
 
This study was supported in part by a grant from the US National Institutes of Health.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DuBose TD, Warnock DG, Mehta RL, Bonventre JV, Hammerman MR, Molitoris BA, Paller MS, Siegel NJ, Scherbenske J, Striker GE: Acute renal failure in the 21st century: Recommendations for management and outcomes assessment. Am J Kidney Dis 29: 793–799, 1997[Medline]
  2. Smithies NN, Cameron JS: Can we predict outcome in acute renal failure? Nephron 51: 287–300, 1989[Medline]
  3. Padanilam BJ, Lewington AJ: Molecular mechanisms of cell death and regeneration in acute ischemic renal injury. Curr Opin Nephrol Hypertens 8: 15–19, 1999[Medline]
  4. Shoskes DA, Halloran PF: Delayed graft function in renal transplantation: Etiology, management and long-term significance. J Urol 155: 1831–1840, 1999
  5. Kootstra G: The asystolic, or non-heartbeating, donor. Transplantation 63: 917–921, 1997[Medline]
  6. McCord JM: Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159–163, 1985[Abstract]
  7. Paller MS: Free radicals-mediated postischemic injury in renal transplantation. Ren Fail 14: 257–260, 1992[Medline]
  8. Paller MS, Hoidal JR, Ferris TF: Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 74: 1156–1164, 1984
  9. McCord JM, Fridovich I: The utility of superoxide dismutase in studying free radical reactions. II. The mechanism of the mediation of cytochrome c reduction by a variety of electron carriers. J Biol Chem 245: 1374–1377, 1970[Abstract/Free Full Text]
  10. Roy RS, McCord JM: Superoxide and ischemia: Conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy Radicals and Their Scavenger Systems, Vol. 2, Cellular and Medical Aspects, edited by Greenwald RA and Cohen G, New York, Elsevier, 1983, pp 145–153
  11. Singh I, Gulati S, Orak JK, Singh AK: Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury. Mol Cell Biochem 125: 97–104, 1993[Medline]
  12. Davies SJ, Reichardt-Pascal SY, Vaughan D, Russell GI: Differential effect of ischemia-reperfusion injury on anti-oxidant enzyme activity in the rat kidney. Exp Nephrol 3: 348–354, 1995[Medline]
  13. Johnson KJ, Weinberg JM: Postischemic renal injury due to oxygen radicals. Curr Opin Nephrol Hypertens 2: 625–635, 1993[Medline]
  14. Bayati A, Kallskog O, Odlind B, Wolgast M: Plasma elimination kinetics and renal handling of copper/zinc superoxide dismutase in the rat. Acta Physiol Scand 134: 65–74, 1988[Medline]
  15. Lehmann TG, Wheeler MD, Schoonhoven R, Bunzendahl H, Samulski RJ, Thurman RG: Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat. Transplantation 69: 1051–1057, 2000[Medline]
  16. Crawford LE, Milliken EE, Irani K, Zweier JL, Becker LC, Johnson TM, Eissa NT, Crystal RG, Finkel T, Goldschmidt-Clermon PJ: Superoxide-mediated actin response in post-hypoxic endothelial cells. J Biol Chem 271: 26863–26867, 1996[Abstract/Free Full Text]
  17. Yin M, Wheeler MD, Bunzendahl H, Samulski RJ, Thurman RG: Adenoviral gene delivery of Cu/Zn-superoxide dismutase attenuates ischemia/reperfusion-induced acute renal failure in the rat [Abstract]. J Am Soc Nephrol 10: A642–A643, 1999
  18. Rigby PW: Cloning vectors derived from animal viruses. J Gen Virol 64: 255–266, 1983[Abstract/Free Full Text]
  19. Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD: Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 252: 431–434, 1991[Abstract/Free Full Text]
  20. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1989
  21. Yin M, Kurvers HAJM, Tangelder GJ, Booster MH, Buurman WA, Kootstra G: Ischemia-reperfusion injury of rat kidney relates more to cortical tubular than to microcirculatory disturbances. Ren Fail 18: 211–223, 1996[Medline]
  22. Guder WG, Ross BD: Enzyme distribution along the nephron. Kidney Int 26: 101–111, 1984[Medline]
  23. Muse KE, Oberley TD, Sempf JM, Oberley LW: Immunolocalization of antioxidant enzymes in adult hamster. Histochem J 26: 734–753, 1994[Medline]
  24. Laiken ND, Fanestil DD: Body fluids and renal function.In: Physiological Basis of Medical Practice, 11th ed., edited by West JB, Baltimore, Williams & Wilkins, 1985,pp 438–543
  25. Linas SL, Shanley PF, Whittenburg D, Berger E, Repine JE: Neutrophils accentuate ischemia-reperfusion injury in isolated perfused rat kidney. Am J Physiol 255: F728–F735, 1988[Abstract/Free Full Text]
  26. Knecht KT, Bradford BU, Mason RP, Thurman RG: In vivo formation of a free radical metabolite of ethanol. Mol Pharmacol 38: 26–30, 1990[Abstract]
  27. Bergmeyer HU: Methods of Enzymatic Analysis. New York, Academic Press, 1988
  28. Willinger CC, Schramek H, Pfaller K, Pfaller W: Tissue distribution of neutrophils in postischemic acute renal failure. Virchows Arch B Cell Pathol Incl Mol Pathol 62: 237–243, 1992[Medline]
  29. Rabb H, O’Meara YM, Maderna P, Coleman P, Brady HR: Leukocytes, cell adhesion molecules and ischemic acute renal failure. Kidney Int 51: 1463–1468, 1997[Medline]
  30. Fraticelli A, Serrano CV,Jr Bochner BS, Capogrossi MCZJL: Hydrogen peroxide and superoxide modulate leukocyte adhesion molecule expression and leukocyte endothelial adhesion. Biochim Biophys Acta 1310: 251–259, 1996[Medline]
  31. Kelly KJ, Williams WW, Colvin RB, Meehan SM, Springer TA, Gutierrez-Ramos J, Bonventre JV: Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 97: 1056–1063, 1996[Medline]
  32. Humes HD, Liu S: Cellular and molecular basis of renal repair in acute renal failure. J Lab Clin Med 124: 749–754, 1994[Medline]
  33. Yin M, Buurman WA, Daemen JHC, Janssen MA, Kootstra G: PAF antagonist TCV-309 reduces PMN infiltration and enhances early function of 24-h preserved rat kidneys with long warm ischemia. Transplantation 61: 1443–1446, 1996[Medline]
  34. McCord JM: Human disease, free radicals and the oxidant/antioxidant balance. Clin Biochem 26: 351–357, 1993[Medline]
  35. Land W, Zweler JL: Prevention of reperfusion-induced, free radical-mediated acute endothelial injury by superoxide dismutase as an effective tool to delay/prevent chronic renal allograft failure: A review. Transplant Proc 29: 2567–2568, 1997[Medline]
  36. Moullier P, Salvetti A, Champion-Arnaud P, Ronco PM: Gene transfer into the kidney: Current status and limitations. Nephron 77: 139–151, 1997[Medline]
  37. Imai E, Isaka Y: Strategies of gene transfer to the kidney. Kidney Int 53: 264–272, 1997
  38. Heikkila P, Parpala T, Lukkarinen O, Weber M, Tryggvason K: Adenovirus-mediated gene transfer into kidney glomeruli using an ex vivo and in vivo kidney perfusion system—First steps towards gene therapy of Alport syndrome. Gene Ther 3: 21–27, 1996[Medline]
  39. Moullier P, Friedlander G, Galise D, Ronco P, Perricaudet M, Ferry N: Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int 45: 1220–1225, 1994[Medline]
  40. Schneeberger H, Schleibner S, Illner WD, Messmer K, Land W: The impact of free radical-mediated reperfusion injury on acute and chronic rejection events following cadaveric renal transplantation. Clin Transpl 219: 232, 1993
  41. Shiraishi T, Kuroiwa A, Shirakusa T, Kawahara K, Yoneda S, Kitano K, Okabayashi K, Iwasaki A: Free radical-mediated tissue injury in acute lung allograft rejection and the effect of superoxide dismutase. Ann Thorac Surg 64: 821–825, 1997[Abstract/Free Full Text]
Received for publication June 16, 2000. Accepted for publication May 30, 2001.




This article has been cited by other articles:


Home page
 Nephrol Dial TransplantHome page
S. Efrati, S. Berman, G. B. Aharon, Y. Siman-Tov, Z. Averbukh, and J. Weissgarten
Application of normobaric hyperoxia therapy for amelioration of haemorrhagic shock-induced acute renal failure
Nephrol. Dial. Transplant., July 1, 2008; 23(7): 2213 - 2222.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
H. Zhu, L. Zhang, A. R. Amin, and Y. Li
Coordinated Upregulation of a Series of Endogenous Antioxidants and Phase 2 Enzymes as a Novel Strategy for Protecting Renal Tubular Cells from Oxidative and Electrophilic Stress
Experimental Biology and Medicine, June 1, 2008; 233(6): 753 - 765.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
D. Son, I. Kojima, R. Inagi, M. Matsumoto, T. Fujita, and M. Nangaku
Chronic hypoxia aggravates renal injury via suppression of Cu/Zn-SOD: a proteomic analysis
Am J Physiol Renal Physiol, January 1, 2008; 294(1): F62 - F72.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
A. Hirayama, S. Nagase, A. Ueda, T. Oteki, K. Takada, M. Obara, M. Inoue, K. Yoh, K. Hirayama, and A. Koyama
In vivo imaging of oxidative stress in ischemia-reperfusion renal injury using electron paramagnetic resonance
Am J Physiol Renal Physiol, March 1, 2005; 288(3): F597 - F603.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. Liu, B. Xu, T. A. Cavalieri, and C. E. Hock
Attenuation of antioxidative capacity enhances reperfusion injury in aged rat myocardium after MI/R
Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2719 - H2727.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
C. Thiemermann, N. S.A. Patel, E. O. Kvale, G. W. Cockerill, P. A.J. Brown, K. N. Stewart, S. Cuzzocrea, D. Britti, H. Mota-Filipe, and P. K. Chatterjee
High Density Lipoprotein (HDL) Reduces Renal Ischemia/Reperfusion Injury
J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1833 - 1843.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
H. Shimizu, S. Maruyama, Y. Yuzawa, T. Kato, Y. Miki, S. Suzuki, W. Sato, Y. Morita, H. Maruyama, K. Egashira, et al.
Anti-Monocyte Chemoattractant Protein-1 Gene Therapy Attenuates Renal Injury Induced by Protein-Overload Proteinuria
J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1496 - 1505.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yin, M.
Right arrow Articles by Thurman, R. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yin, M.
Right arrow Articles by Thurman, R. G.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP