DNase I-Like Endonuclease in Rat Kidney Cortex That Is Activated during Ischemia/Reperfusion Injury
Alexei G. Basnakian*,
Norishi Ueda*,
Gur P. Kaushal*,
Marina V. Mikhailova* and
Sudhir V. Shah*
*University of Arkansas for Medical Sciences, Little Rock, Arkansas, and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas.
Correspondence to Dr. Alexei G. Basnakian, Department of Internal Medicine, Division of Nephrology, University of Arkansas for Medical Sciences, 4301 W. Markham St., Slot 501, Little Rock, AR 72205. Phone: 501-257-1000, ext. 54861; Fax: 501-257-5827; E-mail: basnakianalexeig{at}uams.edu
ABSTRACT. Ischemia/reperfusion is known to result in DNA fragmentationand cell death in kidney tubular epithelium, but the endonucleasesresponsible for this DNA damage have not been identified. DNAsubstrate gel analysis of extracts from normal rat kidney cortexrevealed the presence of a DNase with an apparent molecularmass of 30 to 34 kD. This enzyme is not a dimer of the previouslydescribed nuclear 15-kD endonuclease in kidney cells. Partiallypurified DNase exhibited characteristics similar to those ofrat DNase I. The DNase was able to digest circular DNA (endonuclease),required both Ca2+ and Mg2+ ions, and was inhibited by Zn2+and by aurintricarboxylic acid; it was not inhibited by G-actin.Rat kidneys were subjected to 40 min of ischemia, followed by0, 1, 4, 16, or 48 h of reperfusion. The activity of the DNasein cytosolic and nuclear extracts, the 200-bp ladder-generatingactivity, and 3'OH strand breaks in nuclear DNA were simultaneouslyincreased after ischemia, during the first hours of reperfusion.Oxidative DNA damage, measured as 8-hydroxydeoxyguanosine content,did not coincide with endonuclease-generated DNA breaks. OxidativeDNA damage was increased during ischemia and gradually decreasedduring reperfusion. Phosphorothioated DNase I antisense oligodeoxynucleotideintroduced into cultured NRK-52E rat kidney epithelial cellsinhibited DNA fragmentation and attenuated cell death inducedby hypoxia/reoxygenation in vitro. The data indicate that theDNase I-like endonuclease may contribute to DNA fragmentationin reperfused rat kidneys.
Damage to renal tubular epithelial cells in response to ischemicor toxic insults occurs as a result of either apoptosis or acombination of apoptosis and necrosis (1). DNA fragmentationhas been considered a turning point that makes cell death irreversible(2,3). DNA strand breaks in tubular epithelial cells have beendemonstrated 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 kidneycortex has been detected within hours after reperfusion (6).Iwata et al. (4) demonstrated DNA ladder formation characteristicof endonuclease activation in postischemic rat kidneys by usingthe terminal deoxynucleotidyl kinase end-labeling assay. Itwas further demonstrated that isolated perfused rat kidneyssubjected to hypoxia develop DNA strand breaks in tubular epithelium,as detected with histochemical techniques based on the terminaldeoxynucleotidyl transferase reaction (5). These data indicatedthe involvement of a DNase in DNA fragmentation during ischemia/reperfusion;however, no particular DNase has been linked to the DNA damagein renal ischemia/reperfusion injury.
Recent studies in our laboratory provided evidence of a rolefor an endonuclease in hypoxia/reoxygenation injury to freshlyisolated tubules and chemical hypoxic or oxidant injury to LLC-PK1cells (7–9). Hypoxia/reoxygenation resulted in an increasein DNA-degrading activity with an apparent molecular mass of15 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 activityand provided marked protection against hydrogen peroxide-inducedcell death (9) and partial protection against antimycin A-inducedcell death (7). Taken together, these data provide evidenceof a role for one or several endonucleases in DNA damage andcell death in hypoxia/reoxygenation injury. Importantly, celldeath could be attenuated by endonuclease inhibition, suggestingthat the endonuclease is involved in the reversible stage ofcell death.
In this study, we examined DNase activities in kidney cortexduring ischemia/reperfusion, using DNA substrate-sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Todetect limited DNA fragmentation, we used the highly sensitiverandom oligonucleotide-primed synthesis (ROPS) assay. We identifieda 30- to 34-kD cytosolic endonuclease that was constitutivelypresent in rat kidney cortex and was upregulated during ischemia/reperfusion.The biochemical characteristics of this enzyme were identicalto those of DNase I. Anti-DNase I antisense treatment of normalepithelial rat kidney cells in vitro provided protection againstDNA fragmentation and cell death induced by hypoxia/reoxygenation.
Kidney Ischemia/Reperfusion
Male Sprague-Dawley rats (220 to 280 g) were anesthetized withNembutal (50 mg/kg; Midwest Medical Supply, Earth City, MO).Bilateral renal pedicle occlusion with smooth vascular clampsfor 40 min was used to induce kidney ischemia, as previouslydescribed by Baliga et al. (10). The temperature of the ischemickidneys was maintained at 38°C (normal rat body temperature).Control rats were subjected to an identical operation but withoutocclusion of the renal pedicles. After the period of ischemia,the incisions were sutured and the rats were returned to theircages. Reflow was maintained for 1, 4, 16, or 48 h. Immediatelyafter removal, kidneys were frozen in liquid nitrogen for furtherstudy. Renal function was monitored with plasma urea nitrogenand 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, 0.5µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.7µg/ml leupeptin (buffer A). For DNA autohydrolysis, tissuehomogenates were incubated for 30 min at 37°C, and thenDNA was isolated as described below. To obtain cytosolic extracts,tissue homogenates were centrifuged at 15,000 x g for 10 min.Cell nuclei from rat kidney cortex or rat brain were isolatedas described previously (11). Kidney cell nuclei were isolatedvia precipitation through a discontinuous gradient of 1.8 Msucrose, 50 mM Tris-HCl (pH 7.9), 10 mM CaCl2, 5 mM 2-mercaptoethanol(buffer B), at 70,000 x g for 90 min. Nuclei were then washedthree times via low-speed centrifugation in buffer A and wereresuspended in the same buffer. For protein extraction, thenuclear suspension was diluted in buffer A to a final concentrationof 0.5 mg DNA/ml, lysed by slow addition of an equal volumeof 2 M NaCl in buffer A, and then sonicated (5 x 30 s). Thelysate was incubated for 30 min on ice. DNA was precipitatedfrom the extract by centrifugation at 195,000 x g for 18 h.Protein, containing endonuclease activity, was precipitatedin the presence of 80% saturated ammonium sulfate and was storedat -20°C. Protein concentrations were determined by usingthe Bradford method (12).
DNA-SDS-PAGE
DNase activity was detected by DNA substrate-SDS-PAGE using11.5% SDS-polyacrylamide gels containing heat-denatured nickedcalf thymus DNA (Clontech, Palo Alto, CA), as described previously(7,8). Gels were incubated in the presence of 5 mM MgCl2 and2 mM CaCl2 and were stained with ethidium bromide. DNase wasidentified as a black band on a bright red background of undigestedDNA. The molecular mass of the enzyme was assessed in the presenceof Kaleidoscope prestained standards (Bio-Rad, Hercules, CA).
Preparative Electrophoresis of Proteins
Preparative SDS-PAGE was performed with 11.5% SDS-polyacrylamidegels without DNA, as described by Laemmli (13). Protein bandsin samples and molecular mass standards were observed with copperstaining (14), cut out with a sharp blade, crushed in a Teflon-glasshomogenizer, and electroeluted into Centricon-10 filter units(Amicon, Beverly, MA), using Amicon concentrators. The proteinsolution was then concentrated to 1 mg/ml by centrifugationin the Centricon-10 units, dialyzed overnight against a storagebuffer containing 50% glycerol, 10 mM Tris-HCl (pH 7.7), and0.5 mM dithiothreitol, and stored at -20°C. At that stage,the DNase could be stored at -20°C for 2 mo without anydetectable loss of activity.
Plasmid Incision Assay
This assay was used for detection and quantification of endonucleaseactivity (circular DNA cannot be hydrolyzed by exonucleases),as described previously (15). Endonuclease activity was measuredin 20-µl samples containing 1 µg of plasmid pBR322DNA, 10 mM Tris-HCl (pH 7.7), 25 µg/ml bovine serum albuminfraction V, 0.5 mM dithiothreitol, 5 mM MgCl2, 2 mM CaCl2, and2 µl of DNase. After a 1-h incubation at 37°C, thereaction was stopped with the addition of 5 µl of 1% SDS,100 mM ethylenediaminetetraacetate (EDTA). Digested DNA wasthen subjected to 1% agarose gel electrophoresis at 7 V/cm for1 h at room temperature. The gel was stained with 0.5 µg/mlethidium bromide solution for 20 min and was photographed underfluorescent light. A scanning densitometer (Fotodyne Inc.) wasused to quantify the relative amounts of endonuclease-treatedplasmid DNA present as covalently closed circular DNA (formI), open circular DNA (form II), and linear DNA (form III).One DNase/endonuclease unit was defined as the amount of enzymerequired to convert 1 µg of DNA form I to DNA forms IIand III.
Isolation of DNA and Quantification of DNA Strand Breaks
DNA extraction from tissues, homogenates, or nuclei was performedby using the method described by Ausubel et al. (16). To avoidadditional oxidative DNA damage, the DNA was not dried in theopen air after phenol extraction, as recommended by Finneganet al. (17). Instead, the DNA solution was dialyzed overnightagainst 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-generated3'-hydroxy DNA strand breaks in vitro (15). Briefly, 0.25 µgof 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, and0.5 U of Klenow polymerase (New England Biolabs, Beverly, MA),in a total volume of 25 µl, for 30 min at 16°C. Thereaction was stopped with an equal volume of 12.5 mM EDTA, and15-µl aliquots were applied to DE81 paper. Nonincorporatedprecursors were eluted from the paper with 0.5 M sodium phosphatebuffer (pH 6.8). Labeled DNA absorbed on the paper was measuredby scintillation counting.
8-Hydroxydeoxyguanosine Analysis
DNA was hydrolyzed to deoxynucleosides as described previously(18). The amounts of 8-hydroxydeoxyguanosine (8-OHdG) and deoxyguanosinein the deoxynucleoside mixture were analyzed by HPLC (model600; Waters, Milford, MA), with the simultaneous use of an electrochemicaldetector (Colouchem II; ESA) and an ultraviolet detector (PhotodiodeArray Detector model 969; Water, Chelmsford, MA). The separationconditions were as follows: µ-Bondapak C18 analyticalcolumn (5-µm particle size, 3.9 x 300 mm; Waters); flowrate, 1 ml/min; eluant, 10% aqueous methanol containing 6.25mM 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 DNAwas expressed as the number of 8-OHdG residues per 105 deoxyguanosineresidues.
Treatment of NRK-52E Cells with Antisense Oligodeoxynucleotide
NRK-52E normal rat kidney cells were routinely maintained inDulbecco’s modified Eagle’s medium (Life Technologies,Rockville, MD) supplemented with 5% fetal bovine serum. Aftertrypsinization, 6 x 106 cells were electroporated with 10 µMlevels of a 20-bp, phosphorothioated, rat DNase I antisenseoligodeoxynucleotide (ODN) targeted to the beginning of theDNase 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 weretransfected with sense ODN (5'-GATGAGGTACACAGGGCTGA-3'). Cellswere allowed to recover for 24 h in complete Dulbecco’smodified Eagle’s medium and were then subjected to glucose-free/serum-freemedium saturated with 5% CO2/95% N2 (by bubbling hypoxia for40 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 measuredin trypan blue exclusion assays, and DNA breaks were quantifiedby using the ROPS assay, as described above.
Identification of 30-kD DNase in Rat Kidney Cortex
Nuclear extracts prepared from rat kidney cortices containedtwo major DNases, with molecular masses of 30 to 34 kD and 15to 18 kD, as analyzed by DNA-SDS-PAGE (Figure 1). Diffusionof the proteins during extended incubation of the gels madeit difficult to more precisely identify the molecular masses.On the basis of its molecular mass, the smaller DNase band wassimilar to the 15-kD endonuclease observed in LLC-PK1 cellsby Ueda and colleagues (7–9). The total activity of the30-kD DNase in the isolated nuclei was much less than that ofthe 15-kD enzyme. However, the specific activity of the 30-kDDNase in cytosolic extracts was much higher, and the 15-kD endonucleasecould not be detected. The substrate gels revealed no otherDNases in kidney cortex. Because treatment of the 30-kD enzymeat 100°C in 1% SDS, 1% 2-mercaptoethanol, for 10 min beforeelectrophoresis did not result in 15-kD enzyme, it was consideredunlikely that the 30-kD DNase is a dimer of the 15-kD endonuclease.For determination of cation requirements and the effects ofinhibitors on enzyme activity, the 30-kD DNase was partiallypurified from cytosolic extracts by using preparative SDS-PAGE.Substrate gel electrophoresis demonstrated that the resultingpreparation contained a single DNase band of approximately 30kD (Figure 1).
Figure 1. Analysis of rat kidney cortex DNases by substrate gel electrophoresis. Lane 1, 30-kD DNase in the cytosolic extract from rat kidney cortex; lane 2, 30-kD DNase and 15-kD endonuclease in the nuclear extract from kidney cortex; lane 3, partially purified 30-kD DNase from the kidney cortex cytosolic extract. All samples were heated for 10 min at 100°C with sodium dodecyl sulfate and 2-mercaptoethanol before electrophoresis, as described by Laemmli (13).
Characterization of the DNase
Nuclear Ca2+/Mg2+-dependent endonucleases, which are commonlyassociated with apoptotic endonucleases, are inhibited by zincand aurintricarboxylic acid (ATA) (2,3,19,20). The 29- to 36-kDDNase 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 inhibitedby G-actin. The 30-kD DNase extracted from control rat kidneyswas strongly activated by Ca2+ and Mg2+ added together. Densitometricquantification demonstrated that the activity in the presenceof Ca2+ and Mg2+ added separately was approximately 5 timesless (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 DNaseI, did not inhibit the activity of the 30-kD DNase (Table 1).The DNase was identified as an endonuclease because it degradedcovalently closed circular DNA, which has no DNA ends (no substratefor exonucleases). The DNase provided both single- and double-strandbreaks in covalently closed circular DNA (form I), resultingin open circular (form II) and linear (form III) DNA, respectively.As documented with the ROPS assay (see below), the 30-kD endonucleasegenerated 3'OH/5'P DNA ends. The enzyme therefore was similarto both the Ca2+/Mg2+-dependent endonuclease observed in differenttissues and DNase I identified in rat parotid gland (21,22).On the basis of these data, it was concluded that the enzymeis a DNase I-like, Ca2+/Mg2+-dependent endonuclease that isdifferent from the 15-kD endonuclease.
Figure 2. Cation dependence and inhibition profile of the purified DNase I from rat kidney cortex. DNase activity was measured with the plasmid incision assay, using pBR322 plasmid as the substrate. Lane 1, 2 mM ethylenediaminetetraacetate, no cations (pH 5); lane 2, 2 mM CaCl2 (pH 7.5); lane 3, 2 mM MgCl2 (pH 7.5); lane 4, 2 mM CaCl2 plus 2 mM MgCl2 (pH 7.5); lane 5, 2 mM CaCl2 plus 2 mM MgCl2 plus 2 mM ZnCl2 (pH 7.5); lane 6, 2 mM CaCl2 plus 2 mM MgCl2 plus 1 mM aurintricarboxylic acid (pH 7.5); lane 7, 2 mM CaCl2 plus 2 mM MgCl2 plus 100 µg/ml G-actin (pH 7.5).
Two Hundred-Base Pair Ladder Formation In Vitro
Internucleosomal DNA fragmentation into a 200-bp ladder is considereda biochemical hallmark of apoptosis and is commonly used asa marker of apoptotic endonucleases (19). The 30-kD endonucleasethat had been isolated by using preparative SDS-PAGE was examinedfor 200-bp ladder formation. In this reaction, rat brain nucleiwere used as the substrate because they exhibit very low endogenousendonuclease activity, which can interfere with the tested DNase(11). Nuclei were incubated with or without (control) endonucleasefor 60 min at 37°C. As indicated in Figure 3, the endonucleasewas able to attack chromatin DNA internucleosomally, generating200-bp ladder fragments in vitro. No reduction of the endonucleasesize was observed with substrate gel electrophoresis after incubationwith nuclei (data not shown).
Figure 3. Apoptosis-like, 200-bp ladder, DNA fragmentation in isolated rat brain cell nuclei by purified rat DNase I from kidney cortex. Brain nuclei were isolated as described in Materials and Methods and were incubated for 1 h at 37°C without DNase (lane 1) or with partially purified, kidney 30-kD DNase at 10 U/mg DNA (lane 2) or 100 U/mg DNA (lane 3).
Thirty-Kilodalton Endonuclease Activity during Ischemia/Reperfusion
Several studies reported DNA fragmentation in kidneys subjectedto ischemia/reperfusion (4–6). However, the DNA endonucleaseassociated with ischemia/reperfusion in kidney cells and responsiblefor DNA degradation was not identified. For determination ofwhether the 30-kD endonuclease could be associated with DNAfragmentation during reperfusion, the endonuclease activityin kidney extracts was measured with DNA-SDS-PAGE. Equal amountsof protein (10 µg) were loaded in each well, and the activityin the gel was developed in the presence of both Ca2+ and Mg2+ions. Cytosolic protein represented approximately 90% of totalprotein. Therefore, like cytosolic extracts, total kidney cortexprotein extracts contained a single band for 30-kD DNase inDNA-SDS-PAGE (Figure 4A). The mobility of the DNase band wasidentical in control, ischemic, and reperfused kidney extracts.In both total and nuclear extracts, the specific activity ofthe DNase was increased as early as 1 h after ischemia and reacheda maximum at 16 h of reperfusion (Figure 4, A and B). Cationrequirements for the 30-kD DNase from kidney cortices subjectedto ischemia/reperfusion were identical to those for the enzymefrom control rats (Table 1).
Figure 4. Thirty-kilodalton DNase activation and DNA damage during ischemia/reperfusion in rat kidneys. Kidneys were subjected in vivo to 40 min of ischemia (lane 0), followed by 1 h of reperfusion (lane 1), 4 h of reperfusion (lane 4), 16 h of reperfusion (lane 16), or 48 h of reperfusion (lane 48h). Lane St, standards; lane C, control kidneys. The DNase activities in total kidney cortex extracts (10 µg protein/well) (A) and in nuclear extracts (50 µg protein/well) (B) were measured by using substrate gel electrophoresis. Freshly isolated DNA was not visibly fragmented in an agarose gel (C) but was degraded to 200-bp ladder fragments after incubation of kidney cortex homogenates for 30 min at 37°C (D).
Freshly isolated DNA from rat kidneys subjected to ischemicinjury did not demonstrate any fragmentation when examined withagarose gel electrophoresis (Figure 4C). However, when tissuehomogenates were incubated at 37°C for 30 min, significantDNA degradation, resulting in a 200-bp ladder, was observed(Figure 4D). A peak of 200-bp ladder-generating activity wasobserved at 16 h of reperfusion. Therefore, although we observedthat the DNase capable of 200-bp ladder formation is locatedmainly in the cytosol, the nuclei contain enough DNase to degradenuclear DNA in tissue homogenates in vitro. As demonstratedin the next experiment, a portion of the DNase was probablyable 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 numberof endonuclease-generated 3'-hydroxy ends/breaks was observed(Figure 5). The number of endonuclease-generated DNA strandbreaks increased immediately after ischemia, with a maximumat 16 h of reperfusion, coinciding with the peaks of 30-kD endonucleaseactivity in cytosolic extracts and 200-bp ladder formation inkidney cortex homogenates.
Figure 5. Strand breaks and oxidative damage in DNA isolated from rat kidney cortices during ischemia/reperfusion. Sample numbering is as in Figure 4. Endonuclease-generated, 3'-hydroxy DNA strand breaks (upper) were measured by using the random oligonucleotide-primed synthesis (ROPS) assay, as described in Materials and Methods. Oxidative DNA damage was measured as 8-hydroxydeoxyguanosine (8-OHdG) content (lower). Each point is mean ± SEM (n = 4).
As few as 40 double-strand breaks/cell (approximately one completebreak/chromosome) is considered a lethal level for mammaliancells (23). The first DNA fragments observed during apoptosisare usually 300,000 and 50,000 bp in length (3). The ROPS assayis designed to quantify single- and double-strand breaks inDNA, and it allows determination of the average number of double-strandbreaks per cell. Data presented in Table 2 illustrate that thepotentially lethal level of 40 breaks/cell was reached withinthe first 1 h after ischemia. At the end of that period, theresultant DNA fragments were smaller than 50,000 bp. Therefore,despite the fact that DNase activity is maximized at 16 h, celldeath may occur much earlier, within 1 h after ischemia.
Table 2. Approximate number of double-strand 3‘-hydroxy breaks and average length of DNA fragments induced by ischemia/reperfusion in rat kidney DNAa
Oxidative DNA damage can be an alternative mechanism for theinduction of DNA fragmentation during ischemia/reperfusion.We measured oxidative DNA damage on the basis of the 8-OHdGcontent in DNA, which is the most commonly used marker of oxidativeDNA damage (18). Data presented in Figure 5 demonstrate thatoxidative DNA damage was increased during ischemia. During reperfusion,8-OHdG in DNA was repaired to a control level. This experimentdemonstrated that oxidative DNA damage did not coincide withDNA fragmentation and preceded the peak of endonuclease-generatedDNA breaks.
DNase I Antisense ODN Inhibition of DNA Fragmentation and Cell Death Induced by Hypoxia
The 30-kD endonuclease was indistinguishable from DNase I. Becausethere are no specific inhibitors of DNase I or DNase I-likeendonucleases that could be used in vivo, we used antisenseassay to detect a cause-effect relationship between DNase Iand cell death. NRK-52E cells were transiently transfected invitro with phosphorothioated DNase I antisense ODN, as describedin Materials and Methods. The ODN inhibited DNA fragmentationand attenuated cell death induced by hypoxia/reoxygenation (Figure 6).This experiment suggests a cause-effect relationship betweenDNase I and cell death induced by hypoxia.
Figure 6. Antisense DNA inhibition of hypoxia/reoxygenation-induced DNA fragmentation and cell death in NRK-52E cells. Cells (6 x 106) were electroporated with 10 µM rat DNase I antisense oligodeoxynucleotide, as described in Materials and Methods. Cell death was measured by trypan blue exclusion, and DNA strand breaks were quantified with the ROPS assay. Each point is mean ± SEM (n = 4). C, control; S, sense; As, antisense. *P < 0.05, significantly different from control value.
Ischemia/reperfusion injury is known to cause DNA fragmentationand cell death in kidney tubular epithelium, but the exact mechanismof this DNA damage is not clear (4–6). In this study,we identified a 30-kD DNase/endonuclease associated with ischemia/reperfusioninjury 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 the30-kD band in DNA substrate (zymogram) gels for rat and mousetissues was DNase I (21,24,25). Since then, other DNase I-likeendonucleases have been described (20,26). DNase I is expressedin 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-kDendonuclease (29). Most closely related to DNase I is its homolog,DNase (26). However, the 30-kD endonuclease band in substrategels for kidney extracts does not belong to DNase , and it wasnot observed for recently generated DNase I-knockout mice (30).DNase cannot be identified in protein extracts by substrategel electrophoresis without prior HPLC purification (26).
In the absence of specific inhibitors for any of these endonucleasesor commercially available antibodies that could distinguishbetween DNase I, DNase , and other endonucleases, identificationis usually based on the biochemical characteristics of the enzymes(26). The 30-kD endonuclease that generated 3'-hydroxy breakswas activated by Ca2+ and Mg2+ added together, was inhibitedby Zn2+ and by ATA, and was not inhibited by G-actin. Its molecularmass was approximately 30 to 34 kD, as indicated by DNA-SDS-PAGE.The specific activity of the 30-kD endonuclease increased inischemia/reperfusion-injured kidneys shortly after ischemiaand reached maximal levels at 16 h of reperfusion. The endonucleaseis constitutively present in rat kidney cortex. The enzymesfrom control and reperfused kidneys were identical with respectto cation requirements and molecular mass. In kidneys subjectedto ischemia/reperfusion, oxidative DNA damage, as indicatedby 8-OHdG content, preceded endonuclease-mediated DNA fragmentation.We further demonstrated that the 30-kD DNase differs from the15-kD endonuclease in rat kidneys that we described previously(8). The 30-kD endonuclease is located mainly in cytosol, whereasthe 15-kD endonuclease is a nuclear enzyme. Unlike the 30-kDDNase, the 15-kD endonuclease is activated by Ca2+ alone. Theidentified DNase is unlikely to be a dimer of the 15-kD endonuclease,as demonstrated by substrate PAGE after heat treatment of theenzyme under reducing conditions. However, those findings donot exclude the possibility of other mechanisms for the generationof the 15-kD endonuclease from the 30-kD enzyme. For example,we previously observed partial proteolysis of rat endonucleasesfrom the 30-kD range to the 15- to 20-kD range (31), as wellas alternative splicing of rat DNase I pre-mRNA (32). It wasconcluded that the 30-kD endonuclease is a DNase I-like enzymesimilar to both the nuclear Ca2+/Mg2+-dependent endonucleaseobserved in different tissues (20,33) and DNase I from the ratparotid gland (21,22).
There is considerable evidence that DNase I and DNase I-likeendonucleases can participate in cell death (3,25). COS cellstransiently transfected with rat parotid gland cDNA expressedDNase I and exhibited increased degradation of nuclear DNA intooligonucleosomal fragments (21). The role of DNase I or otherDNase I-like enzymes does not exclude the importance of otherendonucleases, for example, DNase II (28), in cell death. Itis currently accepted that several endonucleases may be responsiblefor DNA fragmentation during cell death (3,25,27,29). Theremay be more than one apoptotic endonuclease in the same tissue,and different cells may exhibit different spectra of endonucleasesavailable for DNA fragmentation during cell death (3,28,34).In addition, there is a possibility that the same endonucleasemay participate both in the induction of cell death and in thepost mortem cleaning process. Our experiments with antisenseODN demonstrated a link between DNase I and cell death in NRK-52Ecells during hypoxia/reoxygenation. The fact that inhibitionwas not complete may indicate that DNase I is not the only enzymein NRK-52E cells responsible for hypoxia-induced cell death.Our previous studies demonstrated that inhibition of endonucleasesin cultured cells provides protection against hypoxia-inducedcell death (7–9) and the 15-kD endonuclease plays an importantrole in this process.
It is unclear what causes activation of the 30-kD DNase afterischemia/reperfusion. On the basis of our data, we propose thatoxidative DNA damage, which precedes the peaks of DNA strandbreaks and DNase activity, may induce the enzyme. It was demonstratedthat oxidative DNA damage could lead to endonuclease activationassociated with cell death. Activation of the endonuclease andmassive DNA fragmentation were observed during apoptosis inx-irradiated lymphoid tissues (35). Direct oxidative DNA damageinduced by the radiation itself produced only approximately15% of all DNA breaks (36). However, apoptosis within the subsequent12 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 delayedafter ischemic injury. Although a potentially lethal level ofDNA strand breaks is reached shortly after ischemia, at leastseveral hours are required for activation of the DNase to maximalactivity. At exactly which level of accumulating DNA strandbreaks tubular epithelial cell death becomes irreversible andwhether endonuclease inhibition at the reversible stage couldminimize tubular epithelial cell death during acute renal failurehave yet to be studied. Ongoing investigations in our laboratoryare focused on determining whether the 30-kD endonuclease mayaffect the course of acute renal failure and whether DNase activationis specific for kidney cell injury.
Acknowledgments
This research was supported in part by a Merit Review Grantfrom the Department of Veteran Affairs and Grants RO1-DK47990and 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.
Edelstein CL, Ling H, Schrier RW: The nature of renal cell injury. Kidney Int 51: 1341–1351, 1997[Medline]
Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature (Lond) 284: 555–556, 1980[CrossRef][Medline]
Nagata S: Apoptotic DNA fragmentation. Exp Cell Res 256: 12–18, 2000[CrossRef][Medline]
Iwata M, Myerson D, Torok-Storb B, Zager RA: An evaluation of renal tubular DNA laddering in response to oxygen deprivation and oxidant injury. J Am Soc Nephrol 5: 1307–1313, 1994[Abstract]
Beeri R, Symon Z, Brezis M, Bensasson SA, Baehr PH, Rosen S, Zager RA: Rapid DNA fragmentation from hypoxia along the thick ascending limb of rat kidneys. Kidney Int 47: 1806–1810, 1995[Medline]
Schumer M, Colombel MC, Sawczuk IS, Connor J, O’Toole KM, Olsson CA, Wise GJ, Buttyan R: Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 140: 831–838, 1992[Abstract]
Hagar H, Ueda N, Shah SV: Endonuclease induced DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells. Kidney Int 49: 355–361, 1996[Medline]
Ueda N, Walker PD, Hsu S-M, Shah S: Activation of a 15 kDa endonuclease in hypoxia/reoxygenation injury without morphologic features of apoptosis. Proc Natl Acad Sci USA 92: 7202–7206, 1995[Abstract/Free Full Text]
Ueda N, Shah SV: Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J Clin Invest 90: 2593–2597, 1992
Baliga R, Ueda N, Shah SV: Increase in bleomycin-detectable iron in ischemia/reperfusion injury in rat kidneys. Biochem J 291: 901–905, 1993
Pravdenkova SV, Basnakian AG, James SJ, Andersen BJ: DNA fragmentation and nuclear endonuclease activity in rat brain after severe closed head injury. Brain Res 729: 151–155, 1996[CrossRef][Medline]
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976[CrossRef][Medline]
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227: 680–685, 1970[CrossRef][Medline]
Lee C, Levin A, Branton D: Copper staining: A five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 166: 308–312, 1987[CrossRef][Medline]
Basnakian AG, James SJ: Quantification of 3'-OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay. DNA Cell Biol 15: 255–262, 1996[Medline]
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. New York, Wiley-Interscience, 1989
Finnegan MT, Herbert KE, Evans MD, Griffiths HR, Lunec J: Evidence for sensitisation of DNA to oxidative damage during isolation. Free Radical Biol Med 20: 93–98, 1996[CrossRef][Medline]
Mikhailova MV, Littlefield NA, Hass BS, Poirier LA, Chou MW: Cadmium-induced 8-hydroxydeoxyguanosine formation, DNA strand breaks and antioxidant enzyme activities in lymphoblastoid cells. Cancer Lett 115: 141–148, 1997[CrossRef][Medline]
Arends MJ, Morris RG, Wyllie AH: Apoptosis: The role of the endonuclease. Am J Pathol 136: 593–608, 1990[Abstract]
Ribeiro J, Carson DA: Ca2+/Mg2+-dependent endonuclease from human spleen: Purification, properties, and role in apoptosis. Biochemistry 32: 9129–9136, 1993[CrossRef][Medline]
Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald HR, Mannherz HG, Tschopp J: Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J 12: 371–377, 1993[Medline]
Polzar B, Mannherz HG: Nucleotide sequence of a full length cDNA clone encoding the deoxyribonuclease I from the rat parotid gland. Nucleic Acids Res 18: 7151, 1990[Free Full Text]
Radford IR: The level of induced DNA double-strand breakage correlates with cell killing after x-irradiation. Int J Radiat Biol Relat Stud Phys Chem Med 48: 45–54, 1985[Medline]
Lacks SA: Deoxyribonuclease I in mammalian tissues: Specificity of inhibition by actin. J Biol Chem 256: 2644–2648, 1981[Abstract/Free Full Text]
Polzar B, Peitsch MC, Loos R, Tschopp J, Mannherz HG: Overexpression of deoxyribonuclease I (DNase I) transfected into COS-cells: Its distribution during apoptotic cell death. Eur J Cell Biol 62: 397–405, 1993[Medline]
Shiokawa D, Ohyama H, Yamada T, Tanuma S: Purification and properties of DNase from apoptotic rat thymocytes. Biochem J 326: 675–681, 1997
Takeshita H, Mogi K, Yasuda T, Nakajima T, Nakashima Y, Mori S, Hoshino T, Kishi K: Mammalian deoxyribonucleases I are classified into three types, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. Biochem Biophys Res Commun 269: 481–484, 2001
Wang CC, Lu SC, Chen HL, Liao TH: Porcine spleen deoxyribonuclease II: Covalent structure, cDNA sequence, molecular cloning, and gene expression. J Biol Chem 273: 17192–17198, 1998[Abstract/Free Full Text]
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S: A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature (Lond) 391: 43–50, 1998[CrossRef][Medline]
Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG, Moroy T: Features of systemic lupus erythematosus in DNase I-deficient mice. Nat Genet 25: 177–181, 2000[CrossRef][Medline]
Lebedeva LG, Alexandrova SS, Votrin II, Basnakian AG: In vitro. proteolysis of endonucleases in rat liver nuclei extracts. Biochem Mol Biol Int 35: 433–440, 1995[Medline]
Basnakian AG, Singh AB, Shah SV: Rat kidney DNase I pre-mRNA is alternatively spliced both in 5'-untranslated region and in coding region [Abstract]. J Am Soc Nephrol 9: 573A, 1998
Stratling WH, Grade C, Horz W: Ca2+/Mg2+-dependent endonuclease from porcine liver: Purification, properties, and sequence specificity. J Biol Chem 259: 5893–5898, 1984[Abstract/Free Full Text]
Gaido ML, Cidlowski JA: Identification, purification and characterization of a calcium-dependent endonuclease (NUC18) from apoptotic rat thymocytes. J Biol Chem 266: 18580–18585, 1991[Abstract/Free Full Text]
Skalka M, Cejkova M, Matyasova J: The sensitivity of chromatin from thymuses and spleens of irradiated mice to alkaline solutions. J Folia Biol (Praha) 22: 335–342, 1976
Jones DP: The role of oxygen concentration in oxidative stress: Hypoxic and hyperoxic models.In: Oxidative Stress.,edited by Sies H, New York, Academic Press, 1985,pp 152–195
Received for publication April 17, 2001.
Accepted for publication November 30, 2001.
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Abstract
Introduction
(26). However, the 30-kD endonuclease band in substrate gels for kidney extracts does not belong to DNase 
