Page 1 of 30 Articles in PresS. Am J Physiol Renal Physiol (December 26, 2007). doi:10.1152/ajprenal.00263.2007
Amelioration of Oxidative Mitochondrial DNA Damage and Deletion after Renal Ischemic Injury by the K-ATP Channel Opener Diazoxide
Running title: Amelioration of renal ischemic injury by diazoxide
Subject of manuscript: Transplantation
Zhaoli Sun *, Xiuying Zhang *,†, Kazushige Ito *, Yulin Li †, Robert A. Montgomery *, Shingo Tachibana *, George Melville Williams * * †
Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA The Key Laboratory of Pathobiology, China’s Ministry of Education and Department of
Pathology, Norman Bethune School of Medicine, Jilin University, Changchun, China
Corresponding author: Zhaoli Sun, MD., Ph.D. Department of Surgery Johns Hopkins University School of Medicine, 720 Rutland Ave., Ross 749 Baltimore, MD 21205 Phone: 410 614-0491, Fax: 410 502-3812 Email:
[email protected]
Copyright © 2007 by the American Physiological Society.
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Abstract Renal ischemia was induced in the rat by clamping the renal artery for 45 minutes, and the ability of the K-ATP channel opener diazoxide (DZ) to ameliorate the renal ischemia reperfusion injury (IRI) was evaluated. In this model BUN and Cr were elevated at day two but returned closer to normal at day seven. Histological staining for reactive oxygen species (ROS) and for the presence of oxidized DNA, detected by the presence of the stable adduct, 8-hydroxy-2’deoxyguanosine (8-OHdG), was clearly positive in the cytoplasm of tubular cells, after just one hour of reperfusion and declined by day 7 after reperfusion. This finding was confirmed by ELISA methods which detected 8-OHdG in the mitochondrial fraction of kidney homogenates. Despite evidence of improved function measured by BUN and Cr on day 7, the early changes in tubules were remained alarming as we found mitochondrial DNA (mtDNA) had the common deletion, and the number of TUNEL positive tubular cells increased. There was continued activation of caspase-3, and abnormal levels of ROS were found in the mitochondrial fraction of cellular homogenates. The striking finding was that DZ provided prior to ischemia reduced or prevented both the acute and subacute deleterious effects associated with renal IRI. 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 IRI thereby reducing oxidative stress as measured by the presence of oxidized mtDNA and features of apoptosis.
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Introduction 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 (IR) injury is widely accepted (23, 29, 35). Free radicles (ROS) originate from several sources including NADPH, xanthine oxidasehypoxanthine, inflammatory cells, and from the 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 IR injury by intra-arterial injections of superoxide dismutase (SOD) were successful as were experiments providing allopurinal to the transplant to block the reaction between xanthine oxidase and hypoxanthine (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). Somewhat surprising, the role of agents opening the K-ATP channel found to be effective in ameliorating cardiac and neural IR injury (7, 9, 31, 34, 39) have been reported to be ineffective in kidney IR injury and even possibly injurious (8, 33, 36). Glibenclamide which closes K-ATP channels was reported to be effective in reducing renal IR injury (33). Struck by our findings that the K-ATP 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 IR injury. Our
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hypothesis was that the kidney was not unique, and that a significant IR injury would be ameliorated by the provision of DZ prior to ischemia. We subjected this idea to testing in rats subjected to a significant IR injury.
Material and Methods Animals Male SD rats were purchased from Harlan Sprague-Dawley (Indianapolis IN, USA) and used at 8-10 wk of age. Animals were maintained in pathogen-free facility of Johns Hopkins Medical Institutions. Animals were cared for according to NIH guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee.
Renal Ischemia/reperfusion The abdomen was opened in the mid-line and small clips place on the renal arteries for 45 minutes. After one hour of re-perfusion the left kidney was excised and portions instantly 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 days 2 and 7 and were tested for BUN and Cr. The rats were divided into six groups: l) Sham-operation without clamping of the renal artery, 2) Control operated animals receiving no drugs (Saline), 3). Animals given diazoxide (DZ, Sigma, 5mg/kg, im) dissolved in DMSO, 4) Same as 3) but also given 5-hydroxydecanoate (5-HD, Sigma, 5mg/kg, im) to block DZ, 5) DMSO alone to control for the anti- oxidative effects of DMSO, and 6) 5-HD alone to control the effects of 5-HD.
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Preparation of Mitochondria Mitochondrial fractions were isolated from kidneys using Mitochondria Isolation Kits (Sigma, St. Louis, MO) in accordance with the manufacturer's instructions. All procures were carried out at 0 to 2 degrees. For testing, the protein concentration was adjusted to 15-20 mg/ml.
ROS release measurements ROS production in isolated mitochondria was measured by using Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188, Molecular Probes) according to the 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 temperaturecontrolled (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 sham-operation.
In situ detection of ROS production by histochemical staining Cytochemical procedures based on the 3,3’-diaminobenzidine (DAB) deposition technique were used on unfixed cryostat sections of kidney according to the method described (37). Briefly, renal tissue was immediately frozen in liquid N and cryostat sections (8µm) were cut immediately at a cabinet temperature of -250C. The sections are placed on Star-Frost adhesive slides and immediately are air dried for 3 minutes at room temperature. The incubation medium contained 10% weight/volume polyvinyl alcohol (PVA) dissolved in 100 mM Tris maleate buffer (pH 8.0). Sodium azide (5 mM) was added to inhibit endogenous myeloperoxidase activity. The
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following components were added shortly before incubation of the cryostat sections: 12.5 mM DAB and 2.5 mM MnCl2. All the compounds were added in strict order and thoroughly mixed from stock solutions into the PVA-containing medium. After incubation for 60 minutes 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 of 4µm were prepared from frozen tissue for 8-OHdG staining according to the method described before (32). Each representative section was stained with hematoxilin and eosin (HE). Anti-8-OHdG antibody (Institute for the Control of Aging, Japan) (1:100) were incubated at 4°C overnight. The slides were stained with diaminobenzamide tetrahydrochloride (DAB) and counterstained with haematoxilin. TUNEL staining was carried out using dUTPfluorescein isothiocyanate (FITC), according to the instructions of the manufacturer (In Situ Cell Death
Detection
Kit,
Roche).
For
Kir6.2
staining,
anti-Kir6.2
antibody
(Jackson
ImmunoResearch Lab, Inc.) (7.5µg/ml) were incubated at room temperature for 1.5 hours and followed by Texas red labeled donkey anti-rabbit IgG (Santa Cruz, 1:75) for 30 minutes.
ELISA assay for measurement of 8-OHdG levels in mtDNA The levels of 8-OHdG in 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, Japan Institute for the Control of Aging, Shizuoka, Japan). The determination range was 0.125-10ng/ml or 0.5-200ng/ml. The levels of 8-OHdG were expressed as amounts of 8-OHdG (ng) per mg mtDNA.
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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' (7,837 to 7,856-bp) and 5'-AAGCCTGCTAGGATGCTTC-3' (13,108 to 13,126-bp). The primer sets for control amplification of wild-type mtDNA were 5'-GGT TCTTACTTCAGGGGCCATC3' (15782 to 15892 bp) and 5'-GTGGAATTTTCTGAG GGTAGGC-3' (16279 to 16300 bp) (32). Sequence and numbering are based on the rat complete mitochondrial genomes (GenBank accession number AJ 428514). PCRs contained 0.2 mmol/l dNTP, 0.2 µmol/l of each primer, 1.25 unit Taq DNA polymerase (Qiagen), and 0.05µg total DNA as template in a 50µl reaction solution. The thermal cycling condition was started with one cycle at 940C for 3 min, and 6 cycles at 940C for 1min, 640C for 1min(-10C/cycle), 720C for 1min 30 seconds. This was followed by 29 cycles at 940C for 1min, 600C for 1min, 720C for 1 min 30 seconds, and 720C for final extension for 5 min. PCR products were electrophoresed on a 1.3% agarose gels and visualized with ethidium bromide staining.
Western Blot Analysis A 1:1000 dilution of caspase-3 antibody (sc-7148, Santa Cruze) used for western blotting to quantitate active caspase-3.
Monoclonal antibody against -actin (Ab-6, Oncogene Research
Products, MA) was used as controls for equal protein loading. Anti-Kir6.2 and anti-VDAC (Cell signaling, 1:1000) antibodies were used to quantitate the expression of Kir6.2 and VDAC (voltage-dependent anion channels) in mitochondria fractions. After reacting with the primary
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and secondary antibodies, the membrane was subjected to the colorimetric detection system by using a stabilized solution of BCIP and NBT (Promega, Madison, Wisconsin).
Statistics The results were expressed as mean values + SEM of n-independent experiments. Analysis was performed by ANOVA with p < 0.05 considered significant.
Results Renal function after ischemia/reperfusion In survival experiments, two of 40 rats died during the first week after ischemia/reperfusion (Fig. 1A). Both rats were in the untreated control group. Two days after the IR injury and left nephrectomy, the mean BUN and Cr levels were 150mg/dl and 3.6mg/dl respectively in the untreated animals while those receiving DZ prior to ischemia had values of 41mg/dl and 1.0mg/dl (P
