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Am J Physiol Renal Physiol 294: F491-F498, 2008. First published December 26, 2007; doi:10.1152/ajprenal.00263.2007
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Amelioration of oxidative mitochondrial DNA damage and deletion after renal ischemic injury by the KATP channel opener diazoxide

Zhaoli Sun,1 Xiuying Zhang,1,2 Kazushige Ito,1 Yulin Li,2 Robert A. Montgomery,1 Shingo Tachibana,1 and George Melville Williams1

1Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2The Key Laboratory of Pathobiology, China's Ministry of Education and Department of Pathology, Norman Bethune School of Medicine, Jilin University, Changchun, China

Submitted 6 June 2007 ; accepted in final form 24 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal ischemia was induced in the rat by constriction of the renal artery for 45 min, and the ability of the ATP-sensitive K+ (KATP) channel opener diazoxide (DZ) to ameliorate renal ischemia-reperfusion (I/R) injury was evaluated. In this model, blood urea nitrogen and creatinine were elevated 2 days after I/R injury but returned closer to normal levels by 7 days after reperfusion. Histological staining for reactive oxygen species (ROS) was clearly positive and oxidized DNA, detected by the presence of the stable adduct 8-hydroxy-2'-deoxyguanosine, was clearly present in the cytoplasm of tubular cells after 1 h of reperfusion and declined 7 days after reperfusion. This finding was confirmed by ELISA, which detected 8-hydroxy-2'-deoxyguanosine in the mitochondrial fraction of kidney homogenates. Despite evidence of improved function measured by blood urea nitrogen and creatinine 7 days after reperfusion, the early changes in tubules were alarming. Mitochondrial DNA showed the common deletion, and the number of TdT-mediated dUTP nick-end label-positive tubular cells increased. Activation of caspase-3 continued, and abnormal levels of ROS were found in the mitochondrial fraction of cellular homogenates. Treatment with DZ before ischemia reduced or prevented the acute and subacute deleterious effects associated with renal I/R injury. We conclude that excess production of ROS by mitochondria on reperfusion is a major upstream event in renal reperfusion injury and that DZ functioned by preventing ROS accumulation in the mitochondria after I/R injury, thereby reducing oxidative stress as measured by the presence of oxidized mitochondrial DNA and features of apoptosis.

transplantation; reactive oxygen species; inflammatory response

ALL TRANSPLANTED ORGANS SUFFER some degree of ischemia, and, on reperfusion, additional cellular injury may occur. When ischemia is prolonged, the injury is severe and may lead to primary nonfunction, defeating attempts to recover badly needed organs for transplantation. The destructive role of reactive oxygen species (ROS) in ischemia-reperfusion (I/R) injury is widely accepted (23, 29, 35). Free radicals (ROS) originate from several sources, including NADPH, xanthine oxidase-hypoxanthine, inflammatory cells, and mitochondria of parenchymal cells, as the result of ischemia-provoked derangement of the electron transport chain. Excessive amounts of ROS cause damage to proteins, lipids, and DNA and stimulate components of the inflammatory response (40, 28), making the organs more prone to rejection (21).

Experimental attempts in swine to reduce I/R injury by intra-arterial injections of SOD were successful, as were experiments providing allopurinol to the transplant to block the xanthine oxidase-hypoxanthine reaction (18). However, neither treatment reduced the incidence of delayed graft function in human kidney transplants. Interestingly, kidneys treated with SOD fared much better in the long run (22). It was somewhat surprising that agents opening the ATP-sensitive K+ (KATP) channel, which has been found to be effective in ameliorating cardiac and neural I/R injury (7, 9, 31, 34, 39), have been reported to be ineffective in kidney I/R injury and, possibly, injurious (8, 33, 36). Glibenclamide, which closes KATP channels, was reported to be effective in reducing renal I/R injury (33). Struck by our findings that the KATP channel opener diazoxide (DZ) prevented the dramatic early accumulation of ROS in the mitochondrial fraction of the ischemic spinal cord neurons, thereby arresting apoptosis (39), we questioned whether the kidney was unique in managing the mitochondrial output of ROS associated with I/R injury. Our hypothesis was that the kidney was not unique and that a significant I/R injury would be ameliorated by administration of DZ before ischemia. We tested this idea in rats subjected to a significant I/R injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN; 8–10 wk of age) were maintained in a pathogen-free facility at Johns Hopkins Medical Institutions and cared for according to National Institutes of Health guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee.

Renal I/R. The abdomen was opened in the midline, and small clips were placed on the renal arteries for 45 min. After 1 h of reperfusion, the left kidney was excised, and portions were immediately frozen, selected for formalin fixation, or used to separate mitochondria. The right kidney remained in situ for various periods, after which it was studied in similar fashion. Blood samples were taken from the tail vein at 2 and 7 days after reperfusion and tested for blood urea nitrogen (BUN) and creatinine (Cr). The rats were divided into six groups: 1) sham-operated animals with unconstricted renal artery, 2) control-operated animals treated with saline, 3) animals treated with DZ (5 mg/kg im; Sigma, St. Louis, MO) dissolved in DMSO, 4) animals treated with DZ (5 mg/kg im) and then with 5-hydroxydecanoate (5-HD, 5 mg/kg im; Sigma) to block DZ, 5) animals treated with DMSO alone to control for the antioxidative effects of DMSO, and 6) animals treated with 5-HD alone to control for the effects of 5-HD.

Preparation of mitochondria. Mitochondrial fractions were isolated from kidneys using mitochondria isolation kits (Sigma) in accordance with the manufacturer's instructions. All procedures were carried out at 0–2°C. For testing, the protein concentration was adjusted to 15–20 mg/ml.

ROS release measurements. ROS production in isolated mitochondria was measured using the Amplex Red H2O2/peroxidase assay kit (catalog no. A22188, Molecular Probes) according to the manufacturer's instruction. Mitochondrial suspensions were incubated in the presence of 50 µM Amplex Red and 0.1 U/ml horseradish peroxidase, and fluorescence was monitored over time using a temperature-controlled (37°C) fluorescence microplate reader (FlexStation II, Molecular Devices) operating at excitation and emission wavelengths of 544 and 590 nm, respectively, with gentle continuous stirring. The change of mitochondrial ROS production in different groups was calculated as the percentage of ROS production in the sham-operated animals.

In situ detection of ROS production by histochemical staining. Unfixed cryostat sections of kidney were subjected to cytochemical procedures based on the 3,3'-diaminobenzidine (DAB) deposition technique, as described elsewhere (37). Briefly, renal tissue was immediately frozen in liquid nitrogen, and cryostat sections (8 µm) were cut immediately in a cabinet maintained at –25°C. The sections are placed on Star-Frost adhesive slides and immediately air dried for 3 min at room temperature. The incubation medium contained 10% (wt/vol) polyvinyl alcohol dissolved in 100 mM Tris maleate buffer (pH 8.0). Sodium azide (5 mM) was added to inhibit endogenous myeloperoxidase activity. DAB (12.5 mM) and MnCl2 (2.5 mM) were added shortly before incubation of the cryostat sections. All the compounds were added in strict order and thoroughly mixed from stock solutions into the polyvinyl alcohol-containing medium. After incubation for 60 min at 37°C, sections were washed in distilled water to stop the reaction and then mounted in glycerol for light microscopy.

Histopathological analysis. Cut sections (4 µm) were prepared from frozen tissue for 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining as described elsewhere (32). Each representative section was stained with hematoxylin and eosin. The slides were incubated with anti-8-OHdG antibody (Institute for the Control of Aging, Shizuoka, Japan; 1:100 dilution) at 4°C overnight and stained with diaminobenzamide tetrahydrochloride (DAB) and counterstained with hematoxylin. TdT-mediated dUTP nick-end labeling (TUNEL) was carried out using dUTP-FITC according to the manufacturer's instructions (In Situ Cell Death Detection Kit, Roche). For Kir6.2 staining, the slides were incubated with anti-Kir6.2 antibody (Lifespan Biosciences; 7.5 µg/ml) at room temperature for 1.5 h and then with Texas red-labeled donkey anti-rabbit IgG (Santa Cruz Biotechnology; 1:75 dilution) for 30 min.

ELISA assay for measurement of 8-OHdG levels in mitochondrial DNA. The levels of 8-OHdG in mitochondrial DNA (mtDNA) were measured by ELISA (32). The nucleoside samples were used for the determination of 8-OHdG by a competitive ELISA kit (8-OHdG Check, Institute for the Control of Aging). The determination range was 0.125–10 ng/ml or 0.5–200 ng/ml. The levels of 8-OHdG were expressed as amounts of 8-OHdG (ng) per milligram of mtDNA.

Detection of mtDNA deletion by PCR. The primer sets for amplification of common mtDNA deletion of 4,834 bp, which was reported to be one of the most frequent deletions (12, 30, 48), were 5'-TTTCTTCCCAAA CCTTTCCT-3' (base pair 7837–7856) and 5'-AAGCCTGCTAGGATGCTTC-3' (base pair 13108–13126). The primer sets for control amplification of wild-type mtDNA were 5'-GGT TCTTACTTCAGGGGCCATC-3' (base pair 15782–15892) and 5'-GTGGAATTTTCTGAG GGTAGGC-3' (base pair 16279–16300) (32). Sequence and numbering are based on the rat complete mitochondrial genome (GenBank accession no. AJ 428514). PCRs contained 0.2 mmol/l of dNTP, 0.2 µmol/l of each primer, 1.25 U of Taq DNA polymerase (Qiagen), and 0.05 µg of total DNA as template in a 50-µl reaction solution. The thermal cycling condition was started with 1 cycle at 94°C for 3 min and 6 cycles at 94°C for 1 min, 64°C for 1 min (–1°C/cycle), and 72°C for 1.5 min followed by 29 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1.5 min and final extension at 72°C for 5 min. PCR products were electrophoresed on 1.3% agarose gel and visualized with ethidium bromide staining.

Western blot analysis. Caspase-3 antibody (catalog no. sc-7148, Santa Cruz Biotechnology; 1:1,000 dilution) was used for Western blotting to quantitate active caspase-3. Monoclonal antibody against β-actin (Ab-6, Oncogene Research Products) was used as control for equal protein loading. Anti-Kir6.2 and anti-voltage-dependent anion channel (VDAC) antibodies (1:1,000 dilution; Cell Signaling) were used to quantitate Kir6.2 and VDAC expression in mitochondrial fractions. After reacting with the primary and secondary antibodies, the membrane was subjected to the colorimetric detection system with use of a stabilized solution of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Promega, Madison, WI).

Statistics. Values are means ± SE of n independent experiments. Statistical significance was determined by ANOVA; P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Renal function after I/R. In survival experiments, 2 of 40 rats died during the 1st wk after I/R (Fig. 1A); both of these rats were in the untreated control group. At 2 days after I/R injury and left nephrectomy, mean BUN and Cr levels were 150 and 3.6 mg/dl, respectively, in the untreated animals and 41 and 1.0 mg/dl, respectively, in animals treated with DZ before ischemia (P < 0.05). DMSO, the carrier for DZ, had little effect, and 5-HD blunted the action of DZ. BUN and Cr levels were closer to normal 7 days after I/R (Fig. 1, B and C).


Figure 1
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  		Fig. 1. Renal function after ischemia-reperfusion (I/R) injury. A: rat survival. B and C: renal injury evaluated by blood urea nitrogen (BUN) and creatinine (Cr). DZ, diazoxide; 5-HD, 5-hydroxydecanoate; sham-op, sham-operated. Values are means ± SE (n = 4 or 8 animals per group). *P < 0.05.

 
Free radical generation. A few DAB-positive cells were present in tissue sections from normal kidneys. After 1 h of reperfusion, tissue sections from control ischemic kidneys and all treatment groups, except DZ-treated animals, demonstrated tubular cell staining (Fig. 2A). DZ dramatically decreased ROS production in tubules to nearly nonischemic control levels at all time periods. The amount of DAB staining in tubular cells declined over time but was present in greater amounts than in nonischemic control kidneys (Fig. 2A). Glomeruli, interstitium, and inflammatory cells did not react with DAB, indicating that tubular epithelial cells, rather than inflammatory cells, produced ROS.


Figure 2
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  		Fig. 2. Free radical generation in ischemic kidneys after reperfusion. A: in situ detection of reactive oxygen species (ROS) generation by histochemical procedures. ROS-positive cells show brown stain. At 1 h after reperfusion, a large number of tubular cells were strongly diaminobenzidine (DAB) positive; at 7 days, DAB staining in tubular cells diminished. DZ dramatically decreased ROS production in tubules at 1 h and 7 days. Glomeruli, interstitium, and inflammatory cells reacted negatively to DAB. Original magnification x40. Renal tissue sections from 1 of 4 animals in each group are shown. B: mitochondrial ROS production shown as percentage of H2O2 in sham-operated group. Note significant increase in mitochondrial H2O2 in DMSO- compared with DZ-treated animals 1 h and 7 days after reperfusion. Data represent 1 of 3 samples in each group. Values are means ± SD. *P < 0.05 vs. DZ.

 
We used the Amplex Red detection kit to measure H2O2 production in intact mitochondria. After 1 h of reperfusion, significantly increased levels of H2O2 were detected in the mitochondrial fraction of ischemic kidneys recovered from animals pretreated with DMSO, the carrier for DZ, compared with sham-operated animals. H2O2 in the mitochondrial fraction of ischemic kidneys was reduced to near sham-operated levels 1 day after reperfusion but rebounded to higher levels 7 days after reperfusion (Fig. 2B). Interestingly, treatment with DZ before occlusion reduced the generation of H2O2 by mitochondria to near sham-operated control levels at each time point (Fig. 2B). These results indicate that I/R injury increased mitochondrial ROS production, which persisted for ≥7 days, and that treatment with DZ before ischemia prevented the early and subacute effects.

Oxidative mtDNA damage and deletions. It is accepted that vulnerability to oxidative damage and subsequent mutations is 10–20 times greater for mtDNA than for nuclear DNA (11, 30, 38), and we reasoned that excessive ROS caused by I/R injury would cause mtDNA damage. We determine significant injury by 1) the presence of increased amounts of 8-OHdG, a well-accepted marker of oxidative DNA damage, and 2) the presence of mtDNA mutation/deletions by PCR. Using competitive ELISA, Nagakawa et al. (32) detected 8-OHdG at low levels in mtDNA of kidneys 1 h and 7 days after sham operation. In the kidney I/R model, 8-OHdG in mtDNA was dramatically increased 1 h after reperfusion and declined significantly 7 days after reperfusion (Fig. 3A). The levels of 8-OHdG in mtDNA were significantly lower in DZ-pretreated animals than in all other groups (P < 0.01). There was no significant difference between DMSO, saline, and DZ + 5-HD groups.


Figure 3
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  		Fig. 3. Oxidative mitochondrial DNA (mtDNA) damage and deletions in ischemic kidneys after reperfusion. A: measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in mtDNA. Extracted mtDNA from kidneys was enzymatically hydrolyzed into nucleosides, and levels of 8-OHdG were measured by ELISA. Values are means ± SE (n = 3 animals per group). *P < 0.05 vs. 1 h. **P < 0.01 vs. other control groups. B: immunohistochemical staining for 8-OHdG (brown). Original magnification x40. Photomicrograph is representative of 4 animals in each group. C: PCR analysis of mtDNA deletion. Template mtDNA from ischemic kidneys was amplified by 35 cycles using the primer pair between base pair 7835 and 13129. PCR amplification showed mtDNA deletion fragment in mtDNA recovered from ischemic kidneys pretreated with DMSO 1 h and 7 days after reperfusion. Each lane represents mtDNA extracted and pooled from 3 samples.

 
To localize expression of 8-OHdG in ischemic kidneys, kidney tissues were analyzed by immunohistochemical staining. Figure 3B shows a representative positive result in an ischemic kidney stained for 8-OHdG after 1 h of reperfusion. Levels of 8-OHdG declined by day 7 after reperfusion in animals treated with DMSO, DZ + 5-HD, and saline (see GoFig. 5). Only a few 8-OHdG-positive cells were recognized in ischemic kidneys pretreated with DZ. Although the method for 8-OHdG detection stains nuclear DNA as well as mtDNA, the staining was localized mainly in the cytoplasm, indicating that this oxidative adduct was present in mtDNA, but not in nuclear DNA (Fig. 3B). The glomeruli and blood vessels were spared.


Figure 4
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  		Fig. 4. Activation of apoptosis in ischemic kidneys after reperfusion. A: histological changes evaluated by hematoxylin-eosin (HE) staining and DNA fragmentation staining [TdT-mediated dUTP nick-end labeling (TUNEL)] with dUTP-FITC (green); nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (blue). Original magnification: x40 (HE) and x20 (fluorescent). Results are representative of 4 animals in each group. B: caspase-3 expression. Inactive caspase-3 precursor (32 kDa) and cleaved caspase-3, including p11 (11 kDa), p17 (17 kDa), and p20 (20 kDa), were quantified by Western blotting. Expression of cleaved caspase-3 proteins was significantly increased in kidneys 7 days after I/R in animals treated with DMSO, DZ + 5-HD, and saline. DZ pretreatment decreased cleaved caspase-3 expression (note absence of 11- and 17-kDa fragments and less 20-kDa fragment).

 

Figure 5
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  		Fig. 5. Expression of mitochondrial ATP-dependent K+ (KATP) channel subunit Kir6.2. A: immunofluorescent staining for Kir6.2. Kir6.2 was widely distributed in renal tubular epithelial cells in sham-operated and DZ-pretreated animals. Kir6.2 expression declined in DMSO-pretreated animals 7 days after reperfusion. Results are representative of 4 animals in each group. B: Western blot analysis of Kir6.2 and voltage-dependent anion channel (VDAC) protein expression in mitochondrial fractions. Each lane represents mitochondria extracted and pooled from 3 samples.

 
Deletions in mtDNA are the probable consequences of oxidative injury combined with inadequate repair mechanisms. Template mtDNA from ischemic kidneys was amplified by 35 cycles using the primer pair between base pair 7835 and 13129. PCR amplification showed mtDNA deletion in DMSO-pretreated kidneys 1 h and 7 days after I/R (Fig. 3C). In contrast, no mtDNA deletion was detected in kidneys pretreated with DZ or in nonischemic kidneys.

Activation of apoptosis. Few TUNEL-positive cells were present in kidneys 1 h after I/R (Fig. 4A). However, TUNEL-positive tubular cells were plentiful 7 days after reperfusion, except in DZ-pretreated kidneys (Fig. 4A). In contrast to the oxidative mtDNA damage (8-OHdG positive) that occurred 1 h after reperfusion and declined by 7 days after reperfusion (Fig. 3B), apoptosis demonstrated by TUNEL-positive cells was absent at 1 h but present at day 7 (Fig. 4A). This is not surprising, since apoptosis requires protein synthesis and lags behind other acute markers of injury.

Inactive caspase-3 precursor (32 kDa) and cleaved caspase-3, including p11 (11 kDa), p17 (17 kDa), and p20 (20 kDa), were further quantified by Western blotting. Expression of cleaved caspase-3 proteins was significantly increased in kidneys 7 days after I/R in animals treated with DMSO, DZ + 5-HD, and saline (Fig. 4B). DZ decreased cleaved caspase-3 expression: there were no 11- and 17-kDa fragments and fewer 20-kDa fragments.

Expression of the mitochondrial KATP channel subunit Kir6.2. Recent studies have identified molecular candidates constituting the mitochondrial KATP channels. Kir6.2, a subunit of the mitochondrial KATP channel, shows impressive localization in the mitochondria in cardiomyocytes (16) and kidney tubular cells (50). Immunofluorescent studies revealed wide distribution of Kir6.2 in the cytoplasm of renal tubular cells (Fig. 5A). Interestingly, Kir6.2 expression declined in non-DZ-pretreated animals 7 days after reperfusion. Western blot analysis showed that Kir6.2 was expressed in the mitochondrial fractions of rat kidneys (Fig. 5B). As positive control, VDAC was expressed in the mitochondrial fractions.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Considerable evidence supports the beneficial role of DZ in I/R injury of the heart and nervous system, and the mechanisms are becoming better understood. However, there is a persistent idea that renal tubular cells may be different. This belief is based on studies reporting that KATP channel antagonists are beneficial in renal ischemia models (33, 36) and that KATP channel openers lack efficacy (49). We find it possible to reconcile these reports with our data because of different model systems. One study of renal tubular cells in culture reported beneficial effects of the KATP channel closer glibenclamide (36) and increased K+ loss and leak of lactate dehydrogenase in DZ-exposed cells. In an investigation similar to the present study, Pompermayer et al. (33) reported the effects of 45 min of ischemia in rats. They administered DZ 40 min before reperfusion, which meant that DZ was provided after renal artery occlusion. If this is so, it is unlikely that DZ penetrated the mitochondria. Zager et al. (49) found that a KATP channel opener, levosimendan, and DZ provided protection from renal failure induced by endotoxemia, while neither agent played an impressive role in brief or extended I/R injury in mice. The amount of DZ administered by these investigators was 100-fold less than that used in the present study. Thus the present studies differed in both dosage and timing, which we propose to explain the differences noted. We used DZ dissolved in DMSO as the carrier at a dose of 5 mg/kg given into the thigh muscle. The drug was well tolerated. To be effective, we reasoned from other studies that the drug should be given before ischemia so that the mitochondrial KATP channels would be open when first exposed to molecular oxygen on reperfusion. We chose 5-HD as a compound to offset DZ, because it is accepted as a more specific mitochondrial KATP channel blocker than glibenclamide (27). We found that 5-HD antagonized the effects of DZ completely, as predicted by studies in other tissue and organ systems. Indeed, our data conform to the growing number of reports demonstrating that mitochondrial KATP channels are redox sensitive, and an open channel effectively blocks the generation and release of ROS (9, 10).

DZ (7-chloro-3-methyl-4H-1,2,4-benzothiadiazine 1,1-dioxide) was originally developed as an antihypertensive agent but was found to induce hyperglycemia by reducing insulin secretion (16). Thus, while suggesting that DZ prevents I/R injury by opening the mitochondrial KATP channels, we must acknowledge that DZ has many other areas of activity (6, 13), including some on cell membrane KATP channels. To provide more evidence that the protective effect of DZ is via opening of the mitochondrial KATP channel, we used 5-HD as a mitochondrial KATP channel blocker. Our results showed that 5-HD, when administered in conjunction with DZ, reversed the protective effect of DZ on kidney damage and function in I/R injury. However, 5-HD alone had no affect on kidney I/R injury. High, but not low, doses of DZ (49) have protective effects, suggesting relatively weak stimulatory effects of DZ on the mitochondrial KATP channel.

DZ has been found to limit I/R injury and apoptosis in a number of in vivo and in vitro models (1, 19, 25, 41, 44). The mechanism is thought to be similar in some ways to that produced by ischemic preconditioning (14). The precise steps leading to decreased ROS production by an open mitochondrial KATP channel remain unknown, because it is difficult to decipher primary and secondary effects. For example, DZ treatment before ischemia results in stabilization of the mitochondrial membrane potential, decreasing Ca2+ uptake (15, 17), preventing the opening of the mitochondrial permeability transition pore (5, 20) and release of cytochrome c (24, 26), and stabilizing mitochondrial morphology. Additionally, mitochondrial KATP channel activity effectively prevents the development and release of ROS (9, 10), which could be the initiator of all the deleterious effects of reperfusion via the local destructive actions of these reactive molecules. Mitochondrial KATP channel activity is regulated under normal conditions by the cellular thiol redox status. Treatment with H2O2 activates the KATP channel in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor (10). This may explain why a large dose of DZ is needed, because the efficacy of DZ requires widespread opening of the mitochondrial KATP channels.

Activation of mitochondrial apoptotic pathways and mitochondrial injury during renal I/R have been well documented (3, 42, 45, 46, 47). The present studies suggest that excessive mitochondrial ROS production is paramount in reperfusion injury. This concept is supported by the finding of oxidative mtDNA injury 1 h after reperfusion and the presence of ROS detected by DAB in tubules, but not in glomeruli or vascular structures. Blocking tubular ROS production by DZ treatment was associated with amelioration of all the parameters of renal injury. To explain these findings, we propose that I/R injury caused a major breakdown of the respiratory chain, resulting in a burst of ROS generated by the deranged respiratory chain, which overwhelmed cellular antioxidants, resulting in direct toxicity.

In the single-kidney rat model we used in the present study, others reported a second wave of injury 7–10 days after ischemia. We also found evidence of ongoing tubular injury at 7 days. This has been attributed to various mechanisms acting in concert, including activation of adhesion molecules such as ICAM-1 and selectins, reduction of nitric oxide production by endothelial cells, and secretion of cytokines including TNF-{alpha} and IL-6 by tubular cells (2, 4). These processes culminate in inflammation and, in some models, the activation of complement. Although interstitial inflammation was not prominent in our specimens, we found continued caspase-3 activation and a marked increase in TUNEL reactivity at 7 days. These findings were associated paradoxically with improved serum BUN and Cr values and diminished evidence of DNA oxidation as measured by 8-OHdG. The relative absence of 8-OHdG at 7 days suggests that either base excision and/or repair enzymes were effective or, more likely, on the basis of the finding of large numbers of TUNEL-positive cells, a significant proportion of cells with oxidized mtDNA were deleted by apoptosis. However, the deleterious effects of DNA oxidation were noted in mtDNA examined by PCR. The mtDNA deletions we found in the rat were similar to those reported in rats and humans (12, 30, 43, 48). They are flanked by two homologous repeats that span a region encoding five kinds of tRNA. Included in the omissions are respiratory enzyme subunits for complexes I, IV, and V. As a consequence, the I/R-induced injury to mtDNA is likely to be progressive. Tubule cells surviving with multiple mtDNA deletions at 7 days are likely to have an unstable mitochondrial genome and impaired mitochondrial respiratory function, resulting in ever-worsening oxidative damage. We speculate that mtDNA deletions may explain the progressive tubular atrophy seen in all types of chronic renal failure, including allograft rejection.

We conclude that renal tubular cells are similar to neurons, hepatocytes, and cardiomyocytes in their response to I/R injury and that a major upstream mechanism resulting in acute renal I/R injury is the failure of ischemic mitochondria to utilize and fully reduce molecular oxygen at reperfusion. This results in a burst of ROS documented to occur 1 h after reperfusion by the precipitation of DAB in the tubular cell cytoplasm in unfixed sections and the generation of H2O2 in mitochondria determined by the Amplex Red technique. Thus we are confident that ROS are produced rapidly in the mitochondria of tubular cells after reperfusion. We also found that the mitochondrial KATP channel opener DZ prevented the mitochondrial free radical generation during I/R. Because DZ is likely to act on the hypoxia-reperfusion component alone, it is unlikely to have an effect on established renal failure, regardless of cause. These studies provide a rationale for lessening the effects of planned renal ischemia, as in the situation of renal transplants. Under these circumstances, DZ or other mitochondrial KATP channel openers may have a major role by preventing acute and chronic oxidative injury.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Sun, Dept. of Surgery, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross 749, Baltimore, MD 21205 (e-mail: zlsun{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Akao M, Ohler A, O'Rourke B, Marban E. Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells. Circ Res 88: 1267–1275, 2001.[Abstract/Free Full Text]
  2. Boros P, Bromberg JS. New cellular and molecular immune pathways in ischemia/reperfusion injury. Am J Transplant 6: 652–658, 2006.[CrossRef][Medline]
  3. Castaneda MP, Swiatecka-Urban A, Mitsnefes MM, Feuerstein D, Kaskel FJ, Tellis V, Devarajan P. Activation of mitochondrial apoptotic pathways in human renal allografts after ischemia-reperfusion injury. Transplantation 76: 50–54, 2003.[CrossRef][Web of Science][Medline]
  4. Chatterjee PK, Thiemermann C. An in vivo model of ischemia/reperfusion and inflammation of the kidneys of the rat. Methods Mol Biol 225: 223–237, 2003.[Medline]
  5. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 341: 233–249, 1999.[CrossRef][Web of Science][Medline]
  6. D'hahan N, Moreau C, Prost AL, Jacquet H, Alekseev AE, Terzic A, Vivaudou M. Pharmacological plasticity of cardiac ATP-sensitive potassium channels toward diazoxide revealed by ADP. Proc Natl Acad Sci USA 96: 12162–12167, 1999.[Abstract/Free Full Text]
  7. Domoki F, Bari F, Nagy K, Busija DW, Siklos L. Diazoxide prevents mitochondrial swelling and Ca2+ accumulation in CA1 pyramidal cells after cerebral ischemia in newborn pigs. Brain Res 1019: 97–104, 2004.[CrossRef][Web of Science][Medline]
  8. Engbersen R, Moons MM, Wouterse AC, Dijkman HB, Kramers C, Smits P, Russel FGM. Sulphonylurea drugs reduce hypoxic damage in the isolated perfused rat kidney. Br J Pharmacol 130: 1678–1684, 2000.[CrossRef][Web of Science][Medline]
  9. Facundo HT, de Paula JG, Kowaltowski AJ. Mitochondrial ATP-sensitive K+ channels prevent oxidative stress, permeability transition and cell death. J Bioenerg Biomembr 37: 75–82, 2005.[CrossRef][Medline]
  10. Facundo HT, de Paula JG, Kowaltowski AJ. Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production. Free Radic Biol Med 42: 1039–1048, 2007.[CrossRef][Medline]
  11. Fraga CG, Shigenaga MK, Park JW, Degan P, Ames BN. Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine. Proc Natl Acad Sci USA 87: 4533–4537, 1990.[Abstract/Free Full Text]
  12. Fukagawa NK, Li M, Liang P, Russell JC, Sobel BE, Absher PM. Aging and high concentrations of glucose potentiate injury to mitochondrial DNA. Free Radic Biol Med 27: 1437–1443, 1999.[CrossRef][Web of Science][Medline]
  13. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection. Circ Res 81: 1072–1082, 1997.[Abstract/Free Full Text]
  14. Gross GJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res 70: 223–233, 1992.[Abstract/Free Full Text]
  15. Gunter KK, Gunter TE. Transport of calcium by mitochondria. J Bioenerg Biomembr 26: 471–485, 1994.[CrossRef][Web of Science][Medline]
  16. Hansen JB. Towards selective Kir6.2/SUR1 potassium channel openers, medicinal chemistry and therapeutic perspectives. Curr Med Chem 13: 361–376, 2006.[CrossRef][Medline]
  17. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol 519: 347–360, 1999.[Abstract/Free Full Text]
  18. Hoshino T, Maley WR, Bulkley GB, Williams GM. Ablation of free radical-mediated reperfusion injury for the salvage of kidneys taken from non-heartbeating donors. A quantitative evaluation of the proportion of injury caused by reperfusion following periods of warm, cold, and combined warm and cold ischemia. Transplantation 45: 284–289, 1988.[Medline]
  19. Ichinose M, Yonemochi H, Sato T, Saikawa T. Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress. Am J Physiol Heart Circ Physiol 284: H2235–H2241, 2003.[Abstract/Free Full Text]
  20. Katoh H, Nishigaki N, Hayashi H. Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes. Circulation 105: 2666–2671, 2002.[Abstract/Free Full Text]
  21. Land W. Impact of the reperfusion injury on acute and chronic rejection events following clinical cadaveric renal transplantation. Clin Invest 72: 719, 1994.[CrossRef][Medline]
  22. Land W, Schneeberger H, Schleibner S, Illner WD, Abendroth D, Rutili G, Arfors KE, Messmer K. The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation 57: 211–217, 1994.[Web of Science][Medline]
  23. Lehr HA, Messmer K. Rationale for the use of antioxidant vitamins in clinical organ transplantation. Transplantation 62: 1197–1199, 1996.[CrossRef][Medline]
  24. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang XD. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479–489, 1997.[CrossRef][Web of Science][Medline]
  25. Liu D, Lu C, Wan R, Auyeung WW, Mattson MP. Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J Cereb Blood Flow Metab 22: 431–443, 2002.[Web of Science][Medline]
  26. Liu XS, Kim CN, Yang J, Jemmerson R, Wang XD. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86: 147–157, 1996.[CrossRef][Web of Science][Medline]
  27. Liu Y, Sato T, Seharaseyon J, Szewczyk A, O'Rourke B, Marbán E. Mitochondrial ATP-dependent potassium channels. Viable candidate effectors of ischemic preconditioning. Ann NY Acad Sci 874: 27–37, 1999.[CrossRef][Web of Science][Medline]
  28. Lum H, Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280: C719–C741, 2001.[Abstract/Free Full Text]
  29. Meneshian A, Bulkley GB. The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction. Microcirculation 9: 161–175, 2002.[CrossRef][Web of Science][Medline]
  30. Merriwether DA, Clark AG, Ballinger SW, Schurr TG, Soodyall H, Jenkins T. The structure of human mitochondrial DNA variation. J Mol Evol 33: 543–555, 1991.[CrossRef][Web of Science][Medline]
  31. Miura T, Miki T. ATP-sensitive K+ channel openers: old drugs with new clinical benefits for the heart. Curr Vasc Pharmacol 1: 251–258, 2003.[CrossRef][Medline]
  32. Nagakawa Y, Williams GM, Zheng Q, Tsuchida A, Aoki T, Montgomery RA, Klein AS, Sun Z. Oxidative mitochondrial DNA damage and deletion in hepatocytes of rejecting liver allografts in rats: role of TNF-{alpha}. Hepatology 42: 208–215, 2005.[CrossRef][Web of Science][Medline]
  33. Pompermayer K, Souza DG, Lara GG, Silveira KD, Cassali GD, Andrade AA, Bonjardim CA, Passaglio KT, Assreuy J, Cunha FQ, Vieira MA, Teixeira MM. The ATP-sensitive potassium channel blocker glibenclamide prevents renal ischemia/reperfusion injury in rats. Kidney Int 67: 1785–1796, 2005.[CrossRef][Web of Science][Medline]
  34. Rajapakse N, Shimizu K, Kis B, Snipes J, Lacza Z, Busija D. Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats. Neurosci Lett 327: 208–212, 2002.[CrossRef][Web of Science][Medline]
  35. Rauen U, de Groot H. New insights into the cellular and molecular mechanisms of cold storage injury. J Investig Med 52: 299–309, 2004.[Medline]
  36. Reeves WB, Shah SV. Activation of potassium channels contributes to hypoxic injury in proximal tubules. J Clin Invest 94: 2289–2294, 1994.[Web of Science][Medline]
  37. Remans PH, van Oosterhout M, Smeets TJ, Sanders M, Frederiks WM, Reedquist KA, Tak PP, Breedveld FC, van Laar JM. Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis. Arthritis Rheum 52: 2003–2009, 2005.[CrossRef][Web of Science][Medline]
  38. Richter C, Park JW, Ames BN. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc Natl Acad Sci USA 85: 6465–6467, 1988.[Abstract/Free Full Text]
  39. Roseborough G, Gao D, Chen L, Trush MA, Zhou S, Williams GM, Wei C. The mitochondrial K-ATP channel opener, diazoxide, prevents ischemia-reperfusion injury in the rabbit spinal cord. Am J Pathol 168: 1443–1451, 2006.[Abstract/Free Full Text]
  40. Stadtman ER, Levine RL. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25: 207–218, 2003.[CrossRef][Web of Science][Medline]
  41. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial KATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res 85: 1146–1153, 1999.[Abstract/Free Full Text]
  42. Tanaka T, Nangaku M, Miyata T, Inagi R, Ohse T, Ingelfinger JR, Fujita T. Blockade of calcium influx through L-type calcium channels attenuates mitochondrial injury and apoptosis in hypoxic renal tubular cells. J Am Soc Nephrol 15: 2320–2333, 2004.[Abstract/Free Full Text]
  43. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet 6: 389–402, 2005.[CrossRef][Web of Science][Medline]
  44. Teshima Y, Akao M, Li RA, Chong TH, Baumgartner WA, Johnston MV, Marban E. Mitochondrial ATP-sensitive potassium channel activation protects cerebellar granule neurons from apoptosis induced by oxidative stress. Stroke 34: 1796–1802, 2003.[Abstract/Free Full Text]
  45. Viñas JL, Sola A, Hotter G. Mitochondrial NOS upregulation during renal I/R causes apoptosis in a peroxynitrite-dependent manner. Kidney Int 69: 1403–1409, 2006.[CrossRef][Web of Science][Medline]
  46. Wei Q, Alam MM, Wang MH, Yu F, Dong Z. Bid activation in kidney cells following ATP depletion in vitro and ischemia in vivo. Am J Physiol Renal Physiol 286: F803–F809, 2004.[Abstract/Free Full Text]
  47. Xie J, Guo Q. Par-4 is a novel mediator of renal tubule cell death in models of ischemia-reperfusion injury. Am J Physiol Renal Physiol 292: F107–F115, 2007.[Abstract/Free Full Text]
  48. Yowe DL, Ames BN. Quantitation of age-related mitochondrial DNA deletions in rat tissues shows that their pattern of accumulation differs from that of humans. Gene 209: 23–30, 1998.[CrossRef][Web of Science][Medline]
  49. Zager RA, Johnson AC, Lund S, Hanson SY, Abrass CK. Levosimendan protects against experimental endotoxemic acute renal failure. Am J Physiol Renal Physiol 290: F1453–F1462, 2006.[Abstract/Free Full Text]
  50. Zhou M, He HJ, Suzuki R, Tanaka O, Sekiguchi M, Yasuoka Y, Kawahara K, Itoh H, Abe H. Expression of ATP sensitive K+ channel subunit Kir6.1 in rat kidney. Eur J Histochem 51: 43–51, 2007.[Medline]



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