Damage to renal tubular epithelial cells in response to ischemic or toxic insults occurs as a result of either apoptosis or a combination of apoptosis and necrosis (1). DNA fragmentation has been considered a turning point that makes cell death irreversible (2,3). DNA strand breaks in tubular epithelial cells have been demonstrated in both renal ischemia/reperfusion injury in vivo (4–6) and hypoxia/reoxygenation models in vitro (7–9). In ischemia/reperfusion injury, DNA fragmentation in the kidney cortex has been detected within hours after reperfusion (6). Iwata et al. (4) demonstrated DNA ladder formation characteristic of endonuclease activation in postischemic rat kidneys by using the terminal deoxynucleotidyl kinase end-labeling assay. It was further demonstrated that isolated perfused rat kidneys subjected to hypoxia develop DNA strand breaks in tubular epithelium, as detected with histochemical techniques based on the terminal deoxynucleotidyl transferase reaction (5). These data indicated the involvement of a DNase in DNA fragmentation during ischemia/reperfusion; however, no particular DNase has been linked to the DNA damage in renal ischemia/reperfusion injury.
Recent studies in our laboratory provided evidence of a role for an endonuclease in hypoxia/reoxygenation injury to freshly isolated tubules and chemical hypoxic or oxidant injury to LLC-PK1 cells (7–9). Hypoxia/reoxygenation resulted in an increase in DNA-degrading activity with an apparent molecular mass of 15 kD, which preceded nuclear DNA fragmentation and cell death (8). In vitro, this DNA-degrading activity was Ca2+-dependent. In cultured cells, zinc sulfate inhibited the endonuclease activity and provided marked protection against hydrogen peroxide-induced cell death (9) and partial protection against antimycin A-induced cell death (7). Taken together, these data provide evidence of a role for one or several endonucleases in DNA damage and cell death in hypoxia/reoxygenation injury. Importantly, cell death could be attenuated by endonuclease inhibition, suggesting that the endonuclease is involved in the reversible stage of cell death.
In this study, we examined DNase activities in kidney cortex during ischemia/reperfusion, using DNA substrate-sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). To detect limited DNA fragmentation, we used the highly sensitive random oligonucleotide-primed synthesis (ROPS) assay. We identified a 30- to 34-kD cytosolic endonuclease that was constitutively present in rat kidney cortex and was upregulated during ischemia/reperfusion. The biochemical characteristics of this enzyme were identical to those of DNase I. Anti-DNase I antisense treatment of normal epithelial rat kidney cells in vitro provided protection against DNA fragmentation and cell death induced by hypoxia/reoxygenation.
Materials and Methods
Male Sprague-Dawley rats (220 to 280 g) were anesthetized with Nembutal (50 mg/kg; Midwest Medical Supply, Earth City, MO). Bilateral renal pedicle occlusion with smooth vascular clamps for 40 min was used to induce kidney ischemia, as previously described by Baliga et al. (10). The temperature of the ischemic kidneys was maintained at 38°C (normal rat body temperature). Control rats were subjected to an identical operation but without occlusion of the renal pedicles. After the period of ischemia, the incisions were sutured and the rats were returned to their cages. Reflow was maintained for 1, 4, 16, or 48 h. Immediately after removal, kidneys were frozen in liquid nitrogen for further study. Renal function was monitored with plasma urea nitrogen and creatinine level assessments.
Tissue Homogenates, Cell Nuclei, and Extracts
Kidney cortex tissue was homogenized in 50 mM Tris-HCl (pH 7.9), 0.25 M sucrose, 10 mM CaCl2, 0.7 μg/ml pepstatin A, 0.5 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.7 μg/ml leupeptin (buffer A). For DNA autohydrolysis, tissue homogenates were incubated for 30 min at 37°C, and then DNA was isolated as described below. To obtain cytosolic extracts, tissue homogenates were centrifuged at 15,000 × g for 10 min. Cell nuclei from rat kidney cortex or rat brain were isolated as described previously (11). Kidney cell nuclei were isolated via precipitation through a discontinuous gradient of 1.8 M sucrose, 50 mM Tris-HCl (pH 7.9), 10 mM CaCl2, 5 mM 2-mercaptoethanol (buffer B), at 70,000 × g for 90 min. Nuclei were then washed three times via low-speed centrifugation in buffer A and were resuspended in the same buffer. For protein extraction, the nuclear suspension was diluted in buffer A to a final concentration of 0.5 mg DNA/ml, lysed by slow addition of an equal volume of 2 M NaCl in buffer A, and then sonicated (5 × 30 s). The lysate was incubated for 30 min on ice. DNA was precipitated from the extract by centrifugation at 195,000 × g for 18 h. Protein, containing endonuclease activity, was precipitated in the presence of 80% saturated ammonium sulfate and was stored at −20°C. Protein concentrations were determined by using the Bradford method (12).
DNase activity was detected by DNA substrate-SDS-PAGE using 11.5% SDS-polyacrylamide gels containing heat-denatured nicked calf thymus DNA (Clontech, Palo Alto, CA), as described previously (7,8). Gels were incubated in the presence of 5 mM MgCl2 and 2 mM CaCl2 and were stained with ethidium bromide. DNase was identified as a black band on a bright red background of undigested DNA. The molecular mass of the enzyme was assessed in the presence of Kaleidoscope prestained standards (Bio-Rad, Hercules, CA).
Preparative Electrophoresis of Proteins
Preparative SDS-PAGE was performed with 11.5% SDS-polyacrylamide gels without DNA, as described by Laemmli (13). Protein bands in samples and molecular mass standards were observed with copper staining (14), cut out with a sharp blade, crushed in a Teflon-glass homogenizer, and electroeluted into Centricon-10 filter units (Amicon, Beverly, MA), using Amicon concentrators. The protein solution was then concentrated to 1 mg/ml by centrifugation in the Centricon-10 units, dialyzed overnight against a storage buffer containing 50% glycerol, 10 mM Tris-HCl (pH 7.7), and 0.5 mM dithiothreitol, and stored at −20°C. At that stage, the DNase could be stored at −20°C for 2 mo without any detectable loss of activity.
Plasmid Incision Assay
This assay was used for detection and quantification of endonuclease activity (circular DNA cannot be hydrolyzed by exonucleases), as described previously (15). Endonuclease activity was measured in 20-μl samples containing 1 μg of plasmid pBR322 DNA, 10 mM Tris-HCl (pH 7.7), 25 μg/ml bovine serum albumin fraction V, 0.5 mM dithiothreitol, 5 mM MgCl2, 2 mM CaCl2, and 2 μl of DNase. After a 1-h incubation at 37°C, the reaction was stopped with the addition of 5 μl of 1% SDS, 100 mM ethylenediaminetetraacetate (EDTA). Digested DNA was then subjected to 1% agarose gel electrophoresis at 7 V/cm for 1 h at room temperature. The gel was stained with 0.5 μg/ml ethidium bromide solution for 20 min and was photographed under fluorescent light. A scanning densitometer (Fotodyne Inc.) was used to quantify the relative amounts of endonuclease-treated plasmid DNA present as covalently closed circular DNA (form I), open circular DNA (form II), and linear DNA (form III). One DNase/endonuclease unit was defined as the amount of enzyme required to convert 1 μg of DNA form I to DNA forms II and III.
Isolation of DNA and Quantification of DNA Strand Breaks
DNA extraction from tissues, homogenates, or nuclei was performed by using the method described by Ausubel et al. (16). To avoid additional oxidative DNA damage, the DNA was not dried in the open air after phenol extraction, as recommended by Finnegan et al. (17). Instead, the DNA solution was dialyzed overnight against an excess of deionized water or 10 mM Tris-HCl (pH 7.9), 0.1 mM EDTA. The ROPS assay was used for quantification of endonuclease-generated 3′-hydroxy DNA strand breaks in vitro (15). Briefly, 0.25 μg of heat-denatured DNA was incubated in 10 mM Tris-HCl (pH 7.5) with 5 mM MgCl2, 7.5 mM dithiothreitol, 0.5 μl of [32P]dCTP (3000 Ci/mmol), 0.05 mM levels of the three other dNTP, and 0.5 U of Klenow polymerase (New England Biolabs, Beverly, MA), in a total volume of 25 μl, for 30 min at 16°C. The reaction was stopped with an equal volume of 12.5 mM EDTA, and 15-μl aliquots were applied to DE81 paper. Nonincorporated precursors were eluted from the paper with 0.5 M sodium phosphate buffer (pH 6.8). Labeled DNA absorbed on the paper was measured by scintillation counting.
DNA was hydrolyzed to deoxynucleosides as described previously (18). The amounts of 8-hydroxydeoxyguanosine (8-OHdG) and deoxyguanosine in the deoxynucleoside mixture were analyzed by HPLC (model 600; Waters, Milford, MA), with the simultaneous use of an electrochemical detector (Colouchem II; ESA) and an ultraviolet detector (Photodiode Array Detector model 969; Water, Chelmsford, MA). The separation conditions were as follows: μ-Bondapak C18 analytical column (5-μm particle size, 3.9 × 300 mm; Waters); flow rate, 1 ml/min; eluant, 10% aqueous methanol containing 6.25 mM citric acid, 12.5 mM sodium acetate, 15 mM sodium hydroxide, and 5 mM acetic acid (pH 5.3). The level of 8-OHdG in the DNA was expressed as the number of 8-OHdG residues per 105 deoxyguanosine residues.
Treatment of NRK-52E Cells with Antisense Oligodeoxynucleotide
NRK-52E normal rat kidney cells were routinely maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum. After trypsinization, 6 × 106 cells were electroporated with 10 μM levels of a 20-bp, phosphorothioated, rat DNase I antisense oligodeoxynucleotide (ODN) targeted to the beginning of the DNase I coding sequence, including the ATG site (5′-TCAGCCCTGTGTACCTCATC-3′), in 0.4-cm electrode-gap cuvettes in a Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA) at 960 μF and 300 V. Control cells were transfected with sense ODN (5′-GATGAGGTACACAGGGCTGA-3′). Cells were allowed to recover for 24 h in complete Dulbecco’s modified Eagle’s medium and were then subjected to glucose-free/serum-free medium saturated with 5% CO2/95% N2 (by bubbling hypoxia for 40 min at room temperature). After 1-h exposure in a 5% CO2/95% N2 atmosphere, cells were returned to normal oxygenation conditions. One hour later, cells were collected, cell death was measured in trypan blue exclusion assays, and DNA breaks were quantified by using the ROPS assay, as described above.
Identification of 30-kD DNase in Rat Kidney Cortex
Nuclear extracts prepared from rat kidney cortices contained two major DNases, with molecular masses of 30 to 34 kD and 15 to 18 kD, as analyzed by DNA-SDS-PAGE (Figure 1). Diffusion of the proteins during extended incubation of the gels made it difficult to more precisely identify the molecular masses. On the basis of its molecular mass, the smaller DNase band was similar to the 15-kD endonuclease observed in LLC-PK1 cells by Ueda and colleagues (7–9). The total activity of the 30-kD DNase in the isolated nuclei was much less than that of the 15-kD enzyme. However, the specific activity of the 30-kD DNase in cytosolic extracts was much higher, and the 15-kD endonuclease could not be detected. The substrate gels revealed no other DNases in kidney cortex. Because treatment of the 30-kD enzyme at 100°C in 1% SDS, 1% 2-mercaptoethanol, for 10 min before electrophoresis did not result in 15-kD enzyme, it was considered unlikely that the 30-kD DNase is a dimer of the 15-kD endonuclease. For determination of cation requirements and the effects of inhibitors on enzyme activity, the 30-kD DNase was partially purified from cytosolic extracts by using preparative SDS-PAGE. Substrate gel electrophoresis demonstrated that the resulting preparation contained a single DNase band of approximately 30 kD (Figure 1).
Characterization of the DNase
Nuclear Ca2+/Mg2+-dependent endonucleases, which are commonly associated with apoptotic endonucleases, are inhibited by zinc and aurintricarboxylic acid (ATA) (2,3,19,20). The 29- to 36-kD DNase I, which is considered a possible apoptotic endonuclease, also is Ca2+/Mg2+-dependent and inhibited by Zn2+ and ATA (21). In contrast to bovine DNase I, rat DNase I is not inhibited by G-actin. The 30-kD DNase extracted from control rat kidneys was strongly activated by Ca2+ and Mg2+ added together. Densitometric quantification demonstrated that the activity in the presence of Ca2+ and Mg2+ added separately was approximately 5 times less (fivefold Ca2+/Mg2+ synergism) (Table 1). Addition of Zn2+ or ATA to the reaction mixture completely inhibited the endonuclease (Figure 2). G-actin, which is capable of inhibiting bovine DNase I, did not inhibit the activity of the 30-kD DNase (Table 1). The DNase was identified as an endonuclease because it degraded covalently closed circular DNA, which has no DNA ends (no substrate for exonucleases). The DNase provided both single- and double-strand breaks in covalently closed circular DNA (form I), resulting in open circular (form II) and linear (form III) DNA, respectively. As documented with the ROPS assay (see below), the 30-kD endonuclease generated 3′OH/5′P DNA ends. The enzyme therefore was similar to both the Ca2+/Mg2+-dependent endonuclease observed in different tissues and DNase I identified in rat parotid gland (21,22). On the basis of these data, it was concluded that the enzyme is a DNase I-like, Ca2+/Mg2+-dependent endonuclease that is different from the 15-kD endonuclease.
Two Hundred-Base Pair Ladder Formation In Vitro
Internucleosomal DNA fragmentation into a 200-bp ladder is considered a biochemical hallmark of apoptosis and is commonly used as a marker of apoptotic endonucleases (19). The 30-kD endonuclease that had been isolated by using preparative SDS-PAGE was examined for 200-bp ladder formation. In this reaction, rat brain nuclei were used as the substrate because they exhibit very low endogenous endonuclease activity, which can interfere with the tested DNase (11). Nuclei were incubated with or without (control) endonuclease for 60 min at 37°C. As indicated in Figure 3, the endonuclease was able to attack chromatin DNA internucleosomally, generating 200-bp ladder fragments in vitro. No reduction of the endonuclease size was observed with substrate gel electrophoresis after incubation with nuclei (data not shown).
Thirty-Kilodalton Endonuclease Activity during Ischemia/Reperfusion
Several studies reported DNA fragmentation in kidneys subjected to ischemia/reperfusion (4–6). However, the DNA endonuclease associated with ischemia/reperfusion in kidney cells and responsible for DNA degradation was not identified. For determination of whether the 30-kD endonuclease could be associated with DNA fragmentation during reperfusion, the endonuclease activity in kidney extracts was measured with DNA-SDS-PAGE. Equal amounts of protein (10 μg) were loaded in each well, and the activity in the gel was developed in the presence of both Ca2+ and Mg2+ ions. Cytosolic protein represented approximately 90% of total protein. Therefore, like cytosolic extracts, total kidney cortex protein extracts contained a single band for 30-kD DNase in DNA-SDS-PAGE (Figure 4A). The mobility of the DNase band was identical in control, ischemic, and reperfused kidney extracts. In both total and nuclear extracts, the specific activity of the DNase was increased as early as 1 h after ischemia and reached a maximum at 16 h of reperfusion (Figure 4, A and B). Cation requirements for the 30-kD DNase from kidney cortices subjected to ischemia/reperfusion were identical to those for the enzyme from control rats (Table 1).
Freshly isolated DNA from rat kidneys subjected to ischemic injury did not demonstrate any fragmentation when examined with agarose gel electrophoresis (Figure 4C). However, when tissue homogenates were incubated at 37°C for 30 min, significant DNA degradation, resulting in a 200-bp ladder, was observed (Figure 4D). A peak of 200-bp ladder-generating activity was observed at 16 h of reperfusion. Therefore, although we observed that the DNase capable of 200-bp ladder formation is located mainly in the cytosol, the nuclei contain enough DNase to degrade nuclear DNA in tissue homogenates in vitro. As demonstrated in the next experiment, a portion of the DNase was probably able to reach the nuclear DNA and degrade it in vivo.
When DNA strand breaks in visibly nondegraded DNA (Figure 4C) were analyzed with the ROPS assay, an increase in the number of endonuclease-generated 3′-hydroxy ends/breaks was observed (Figure 5). The number of endonuclease-generated DNA strand breaks increased immediately after ischemia, with a maximum at 16 h of reperfusion, coinciding with the peaks of 30-kD endonuclease activity in cytosolic extracts and 200-bp ladder formation in kidney cortex homogenates.
As few as 40 double-strand breaks/cell (approximately one complete break/chromosome) is considered a lethal level for mammalian cells (23). The first DNA fragments observed during apoptosis are usually 300,000 and 50,000 bp in length (3). The ROPS assay is designed to quantify single- and double-strand breaks in DNA, and it allows determination of the average number of double-strand breaks per cell. Data presented in Table 2 illustrate that the potentially lethal level of 40 breaks/cell was reached within the first 1 h after ischemia. At the end of that period, the resultant DNA fragments were smaller than 50,000 bp. Therefore, despite the fact that DNase activity is maximized at 16 h, cell death may occur much earlier, within 1 h after ischemia.
Oxidative DNA damage can be an alternative mechanism for the induction of DNA fragmentation during ischemia/reperfusion. We measured oxidative DNA damage on the basis of the 8-OHdG content in DNA, which is the most commonly used marker of oxidative DNA damage (18). Data presented in Figure 5 demonstrate that oxidative DNA damage was increased during ischemia. During reperfusion, 8-OHdG in DNA was repaired to a control level. This experiment demonstrated that oxidative DNA damage did not coincide with DNA fragmentation and preceded the peak of endonuclease-generated DNA breaks.
DNase I Antisense ODN Inhibition of DNA Fragmentation and Cell Death Induced by Hypoxia
The 30-kD endonuclease was indistinguishable from DNase I. Because there are no specific inhibitors of DNase I or DNase I-like endonucleases that could be used in vivo, we used antisense assay to detect a cause-effect relationship between DNase I and cell death. NRK-52E cells were transiently transfected in vitro with phosphorothioated DNase I antisense ODN, as described in Materials and Methods. The ODN inhibited DNA fragmentation and attenuated cell death induced by hypoxia/reoxygenation (Figure 6). This experiment suggests a cause-effect relationship between DNase I and cell death induced by hypoxia.
Ischemia/reperfusion injury is known to cause DNA fragmentation and cell death in kidney tubular epithelium, but the exact mechanism of this DNA damage is not clear (4–6). In this study, we identified a 30-kD DNase/endonuclease associated with ischemia/reperfusion injury in the kidney. As detected with DNA substrate gel electrophoresis, this enzyme is the major endonuclease in rat kidney tissue.
Studies performed in the 1980s and 1990s concluded that the 30-kD band in DNA substrate (zymogram) gels for rat and mouse tissues was DNase I (21,24,25). Since then, other DNase I-like endonucleases have been described (20,26). DNase I is expressed in all tissues studied and is highly expressed in the kidney (27). It is a cytosolic, neutral, Ca2+/Mg2+-dependent, Zn2+-inhibitable, 200-bp ladder-generating, 3′-hydroxy/5′-phosphate-endonuclease, which makes it very different from DNase II, which is an acidic, lysosomal, cation-independent, 3′-phosphate/5′-hydroxy-endonuclease (28), and from the caspase-activated, Mg2+-dependent, 40-kD endonuclease (29). Most closely related to DNase I is its homolog, DNase γ (26). However, the 30-kD endonuclease band in substrate gels for kidney extracts does not belong to DNase γ, and it was not observed for recently generated DNase I-knockout mice (30). DNase γ cannot be identified in protein extracts by substrate gel electrophoresis without prior HPLC purification (26).
In the absence of specific inhibitors for any of these endonucleases or commercially available antibodies that could distinguish between DNase I, DNase γ, and other endonucleases, identification is usually based on the biochemical characteristics of the enzymes (26). The 30-kD endonuclease that generated 3′-hydroxy breaks was activated by Ca2+ and Mg2+ added together, was inhibited by Zn2+ and by ATA, and was not inhibited by G-actin. Its molecular mass was approximately 30 to 34 kD, as indicated by DNA-SDS-PAGE. The specific activity of the 30-kD endonuclease increased in ischemia/reperfusion-injured kidneys shortly after ischemia and reached maximal levels at 16 h of reperfusion. The endonuclease is constitutively present in rat kidney cortex. The enzymes from control and reperfused kidneys were identical with respect to cation requirements and molecular mass. In kidneys subjected to ischemia/reperfusion, oxidative DNA damage, as indicated by 8-OHdG content, preceded endonuclease-mediated DNA fragmentation. We further demonstrated that the 30-kD DNase differs from the 15-kD endonuclease in rat kidneys that we described previously (8). The 30-kD endonuclease is located mainly in cytosol, whereas the 15-kD endonuclease is a nuclear enzyme. Unlike the 30-kD DNase, the 15-kD endonuclease is activated by Ca2+ alone. The identified DNase is unlikely to be a dimer of the 15-kD endonuclease, as demonstrated by substrate PAGE after heat treatment of the enzyme under reducing conditions. However, those findings do not exclude the possibility of other mechanisms for the generation of the 15-kD endonuclease from the 30-kD enzyme. For example, we previously observed partial proteolysis of rat endonucleases from the 30-kD range to the 15- to 20-kD range (31), as well as alternative splicing of rat DNase I pre-mRNA (32). It was concluded that the 30-kD endonuclease is a DNase I-like enzyme similar to both the nuclear Ca2+/Mg2+-dependent endonuclease observed in different tissues (20,33) and DNase I from the rat parotid gland (21,22).
There is considerable evidence that DNase I and DNase I-like endonucleases can participate in cell death (3,25). COS cells transiently transfected with rat parotid gland cDNA expressed DNase I and exhibited increased degradation of nuclear DNA into oligonucleosomal fragments (21). The role of DNase I or other DNase I-like enzymes does not exclude the importance of other endonucleases, for example, DNase II (28), in cell death. It is currently accepted that several endonucleases may be responsible for DNA fragmentation during cell death (3,25,27,29). There may be more than one apoptotic endonuclease in the same tissue, and different cells may exhibit different spectra of endonucleases available for DNA fragmentation during cell death (3,28,34). In addition, there is a possibility that the same endonuclease may participate both in the induction of cell death and in the post mortem cleaning process. Our experiments with antisense ODN demonstrated a link between DNase I and cell death in NRK-52E cells during hypoxia/reoxygenation. The fact that inhibition was not complete may indicate that DNase I is not the only enzyme in NRK-52E cells responsible for hypoxia-induced cell death. Our previous studies demonstrated that inhibition of endonucleases in cultured cells provides protection against hypoxia-induced cell death (7–9) and the 15-kD endonuclease plays an important role in this process.
It is unclear what causes activation of the 30-kD DNase after ischemia/reperfusion. On the basis of our data, we propose that oxidative DNA damage, which precedes the peaks of DNA strand breaks and DNase activity, may induce the enzyme. It was demonstrated that oxidative DNA damage could lead to endonuclease activation associated with cell death. Activation of the endonuclease and massive DNA fragmentation were observed during apoptosis in x-irradiated lymphoid tissues (35). Direct oxidative DNA damage induced by the radiation itself produced only approximately 15% of all DNA breaks (36). However, apoptosis within the subsequent 12 to 72 h, which was associated with endonuclease activation, provided 85% of the DNA breaks (35).
In the kidney, cell death of the tubular epithelium is delayed after ischemic injury. Although a potentially lethal level of DNA strand breaks is reached shortly after ischemia, at least several hours are required for activation of the DNase to maximal activity. At exactly which level of accumulating DNA strand breaks tubular epithelial cell death becomes irreversible and whether endonuclease inhibition at the reversible stage could minimize tubular epithelial cell death during acute renal failure have yet to be studied. Ongoing investigations in our laboratory are focused on determining whether the 30-kD endonuclease may affect the course of acute renal failure and whether DNase activation is specific for kidney cell injury.
This research was supported in part by a Merit Review Grant from the Department of Veteran Affairs and Grants RO1-DK47990 and PO1-DK58324-01A1 from the National Institutes of Health. We thank Amar Singh, Ph.D., for comments and Ray Biondo, M.D., M.S., for editorial assistance.
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