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Published ahead of print on August 5, 2007
J Am Soc Nephrol 18: 2544-2553, 2007
© 2007 American Society of Nephrology
doi: 10.1681/ASN.2006080896
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Basic Research

Induction of Renal Endonuclease G by Cisplatin Is Reduced in DNase I-Deficient Mice

Xiaoyan Yin*, Eugene O. Apostolov*, Sudhir V. Shah*,{dagger}, Xiaoying Wang*, Konstantin V. Bogdanov*, Timea Buzder*, Anna G. Stewart* and Alexei G. Basnakian*,{dagger}

* Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, and {dagger} Central Arkansas Veterans Healthcare System, Little Rock, Arkansas

Correspondence: Dr. Alexei G. Basnakian, University of Arkansas for Medical Sciences, Department of Internal Medicine, Division of Nephrology, 4301 W. Markham Street, #501, Little Rock, AR 72205. Phone: 501-257-5052; Fax: 501-257-4822; E-mail: basnakianalexeig{at}uams.edu

Received for publication August 24, 2006. Accepted for publication May 24, 2007.


   Introduction
Top
 Introduction
RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Nephrotoxicity from the chemotherapeutic drug cisplatin is associated with DNA fragmentation and cell death. We have recently demonstrated that DNase I knockout mice are significantly protected against cisplatin nephrotoxicity, but it is unknown whether the DNA fragmentation that occurs is produced by DNase I or another endonuclease. In this study we assessed the expression of several endonucleases involved in cell death after injection of cisplatin and found that the expression of endonuclease G (EndoG) increased whereas the expression of DNase I decreased almost to zero. Immunostaining showed that some nuclei contained both fragmented DNA and EndoG, suggesting that EndoG may cause DNA fragmentation induced by cisplatin. The increase in expression of EndoG was greater in wild-type mice than in DNase I knockout mice, indicating a potential link between the two endonucleases. In support of such a link, overexpression of DNase I in cultured mouse tubular epithelial cells also induced EndoG. Furthermore, gene silencing of EndoG in vitro provided significant protection against cell death. Taken together, our data suggest that both DNase I and EndoG mediate cisplatin injury to tubular epithelial cells.

The nephrotoxicity of cisplatin (cis-diamminedichloroplatinum II) is the major factor limiting the use of this drug in the chemotherapy of cancer.1–3 It is known that cisplatin accumulates in the kidney more that in other organs, and its toxicity to the kidney is dosage dependent.4 The mechanism of cisplatin nephrotoxicity is not completely understood. It is known to be associated with DNA fragmentation determined either by a 200-bp DNA ladder in agarose5,6 or by using the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay.7–9 These types of DNA fragmentation are commonly attributed to apoptosis mediated by an unknown cell death endonuclease that is capable of internucleosomal DNA fragmentation and generation of 3'OH ends available for the TdT reaction.10 Cell death endonucleases are a recently recognized group of enzymes that includes deoxyribonuclease I (DNase I), DNase {gamma}, caspase-activated DNase (CAD), endonuclease G (EndoG), and DNase II.11–15 They act both premortem, leading to cell death, and postmortem, providing a "clean-up" after cell death.10 Because these two processes cannot be easily distinguished and there are no specific inhibitors for the endonucleases, the use of knockout (KO) models is the only available tool to study the role of premortem endonuclease-mediated events in vivo.

We previously showed that DNase I is the most active cell death endonuclease among cell death endonucleases in the kidney.16,17 Renal injury by ischemia-reperfusion was associated with the induction/activation of DNase I.18,19 The suppression of DNase I by antisense was protective against hypoxia/reoxygenation injury to renal tubular epithelial cells in vitro.18 Our recent study of cisplatin-induced renal injury demonstrated that DNase I KO mice are protected against cisplatin.17 It remains unclear whether DNase I is the sole enzyme that produces DNA fragmentation and induces cell death or these processes require the participation of another endonuclease.

In this study, we demonstrated that DNA fragmentation induced by cisplatin strongly depends on the contribution of EndoG. EndoG is a nuclear-encoded mitochondrial enzyme that is known to be released from its original location, translocate to nucleus, and degrade nuclear DNA during caspase-independent apoptosis.20,21 Importantly, we showed that the presence of DNase I before cisplatin injury was necessary for the induction of EndoG, which therefore serves as the executioner of DNase I–mediated cell death in the kidneys of mice that are treated by cisplatin. The expression of DNase I-cyan fluorescence protein (DNase I–CFP) fusion protein in immortalized mouse proximal tubule (TKPTS) cells caused the induction of EndoG, which provided additional evidence for the role of DNase I in EndoG regulation. In support of the role of EndoG as a downstream executioner of DNA fragmentation and kidney cell death, the EndoG activation was associated with increased DNA fragmentation in vivo, and the silencing of EndoG by anti-EndoG short interference RNA (siRNA) or peroxisome proliferator–activated receptor {alpha} (PPAR-{alpha}) agonist provided protection against the cisplatin injury of TKPTS cells in vitro.


   RESULTS
Top
 Introduction
 RESULTS
DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Renal Endonucleases before Injury
This study began with the characterization of endonuclease activity in the kidneys. As determined by using the DNase I-specific single radial enzyme diffusion assay, inactivation of the DNase I gene (in DNase I KO mice) caused complete removal of DNase I activity from total kidney extracts (Figure 1A). Semiquantitative reverse transcriptase–PCR (RT-PCR) showed that DNase I is not expressed in KO mice, whereas other endonucleases are expressed at the same levels as in wild-type (WT) mice (Figure 1B). We found that in addition to DNase I, three other proapoptotic endonucleases—DNase II, CAD, and EndoG—are expressed in the kidney, whereas mRNA for DNase {gamma} could not be detected.


Figure 1
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  		Figure 1. Expression of DNase I and other endonucleases in wild-type (WT) and DNase I knockout (KO) mice. (A) Single radial enzyme diffusion assay showed that protein extracts from WT kidneys contain active DNase I, whereas it is absent in KO kidney extracts. Human recombinant DNase I (Dornase; Genentech, South San Francisco, CA) was used as positive control. (B) Semiquantitative reverse transcriptase–PCR (RT-PCR) of cell death endonucleases expressed in normal kidneys of WT and DNase I KO mice. Products of the RT-PCR reaction were produced and separated in agarose gel as described in the Concise Methods (top) and then quantified by densitometry (bottom; n = 4 to 6 per group). (C) Inactivation of CaMg-dependent endonuclease DNase I activity (CM) as measured using plasmid incision assay in total protein extracts of DNase I–/– mice. Mn-dependent endonuclease G (EndoG) activity (Mn) is the most prominent in these mice. DNase II activity measured in EDTA at pH 5 (E5) is very low. O, open circular DNA; L, linear DNA; c, covalently closed circular DNA; D, digested DNA.

 
In total protein extracts from nontreated WT mice, the strongest endonuclease activity was observed in the presence of Ca2+ and Mg2+ ions added together (Figure 1C). This is characteristic of DNase I, which is known to be the most active Ca/Mg-dependent endonuclease in mammalian tissues.17 Therefore, DNase I provides most of the endonuclease activity in the normal kidney. In kidney tissue extracts from KO mice, Mn-dependent endonuclease was the most prominent (Figure 1C, right), suggesting that EndoG, the only known Mn-dependent endonuclease, is the second major endonuclease in the absence of DNase I.

DNase I Is Suppressed during Cisplatin Injury In Vivo
For definition of the changes of renal endonucleases associated with cisplatin nephrotoxicity, renal failure was induced by a single cisplatin (20 mg/kg) injection as described in our previous study.17 Because cisplatin kidney injury has been reported in association with increased DNA fragmentation,5,7,17 it was expected that the endonuclease activity would be increased after cisplatin injection. Contrary to our expectations, the endonuclease activity of total kidney extracts measured using plasmid incision assay in the presence of Ca2+ and Mg2+ ions was decreased at day 1 and then had a tendency to recover on later days (Figure 2A). Because Ca/Mg-dependent activity was previously shown to be mainly represented by DNase I,17 these results suggested that DNase I activity is decreased after cisplatin injection. Mn-dependent endonuclease activity, which is known to belong to both DNase I22 and EndoG,15 remained unchanged after cisplatin treatment (Figure 2B). This suggested that while DNase I is downregulated, EndoG activity is increased during cisplatin injury. These data also showed that the increase of EndoG activity occurs only in DNase I–positive WT mice, whereas DNase I KO mice have very little induction of EndoG.


Figure 2
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  		Figure 2. Endonuclease activity in total kidney protein extracts from DNase I KO and WT mice that were treated with a single dose of cisplatin (20 mg/kg). Activity was measured using plasmid incision assay in the presence of Ca2+ and Mg2+ (A) or Mn2+ ions (B) as described in the Concise Methods (n = 6 in each point). (C) Activity in urine measured using plasmid incision assay in the presence of Ca2+ and Mg2+ (n = 6 in each point). (D) Zymogram gel of kidney protein extract isolated at days 0 and 4 after cisplatin injection.

 
Urine DNase I is an important marker of DNase activity in the kidney because it is secreted by tubular epithelium.23 The measurement of total CaMg-dependent endonuclease activity in urine showed that DNase I KO mice have only approximately 1% of endonuclease activity left, suggesting that urine DNase is represented almost entirely by DNase I (Figure 2C). The activity of DNase I in urine was decreased almost to zero within 4 h after cisplatin injection and remained suppressed up to 8 d after the impact, which explains the decrease of the total endonuclease activity. Urine DNase I also disappeared as determined by zymogram gel (Figure 2D).

EndoG Is Induced during Cisplatin Injury in Mice Expressing DNase I
As determined by Western blotting, the expression of DNase II and CAD was not changed after cisplatin injection (Figure 3A). In contrast, EndoG was strongly induced on day 1 (data not shown) and reached a maximum 4 d later after cisplatin injection. As described next, EndoG induction correlated with DNA fragmentation and tubular necrosis as determined by histology. Importantly, EndoG was induced more in DNase I WT mice than in KO mice, suggesting that DNase I may be necessary for the induction of EndoG (Figure 3B).


Figure 3
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  		Figure 3. Induction of EndoG during cisplatin injury in vivo. (A) Induction of EndoG in kidney extracts measured by Western blotting during cisplatin injury in mice (4 d after a single injection with 20 mg/kg cisplatin). The significant increase of EndoG makes its presence in control kidneys almost invisible in Western. caspase-activated DNase (CAD) and DNase II are not induced. (B) Induction of renal EndoG 4 d after cisplatin injection is higher in WT than in DNase I KO mice as measured using Western and quantified by densitometry (n = 8; *P < 0.01 between the cisplatin-treated samples).

 
Overexpression of DNase I In Vitro Causes Induction of EndoG
For studying whether DNase I can cause EndoG induction, cultured TKPTS cells were transfected with pECRF-DNaseI construct or empty pECFP vector as described previously. An alternative approach using silencing of DNase I seemed impossible considering the large number of DNase I–like proteins in cells.11,24 The expression of the fusion protein of CFP was detected by fluorescence microscopy. The immunostaining of EndoG showed that cells expressing DNase I-CRF have more EndoG, whereas cells expressing CRF have EndoG expressed at the control level (Figure 4, A and B). The expression of EndoG directly correlated with DNase I–CFP expression but not with CFP expression (Figure 4, C and D). These data suggest that DNase I is capable of inducing EndoG expression in tubular epithelial cells.


Figure 4
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  		Figure 4. Induction of EndoG by DNase I in TKPTS cells. Cells were transfected with pECFP-DNaseI plasmid. The expression of CFP or DNase I-CFP fusion protein and EndoG is presented in the integrated OD units (IOD) per cell. (A) TKPTS cells expressing DNase I–CFP fusion protein but not cells expressing CFP alone show the induction of EndoG. Bars = 5 µm. (B) Quantification of EndoG expression in the transfected cells. *P < 0.001 versus either control or CFP vector. (C) Direct correlation between DNase I–CFP and EndoG expressions. (D) Absence of correlation between CFP and EndoG expressions.

 
Immunohistochemical Detection of EndoG and DNA Fragmentation in Kidneys during Cisplatin Injury
Immunostaining of EndoG in normal mouse kidney showed that this enzyme is mainly located in thick limbs of the outer medulla and medullary rays (Figure 5A). In cortex, proximal tubules had the most prominent EndoG expression, whereas almost no expression was observed in glomeruli (Figure 5A, segment 1). After cisplatin injection, the amount of both EndoG and DNA fragmentation determined by TUNEL were strongly increased, and the number of tubular epithelial cell nuclei determined by 4',6-diamidino-2-phenylindole (DAPI) staining was decreased (Figure 5B). Some nuclei in either live or dead cells were both EndoG and TUNEL positive, indicating that nuclear import of EndoG is associated with the fragmentation of DNA and nuclei (Figure 5C).


Figure 5
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  		Figure 5. Immunolocalization of EndoG and DNA fragmentation in kidneys during cisplatin injury. (A) EndoG (red) is unequally distributed in the intact renal parenchyma—more in medullary rays and outer medulla (segment 2) and less in cortex (segment 1)—whereas inner medulla has the lowest expression of EndoG (segment 3). Proximal and distal tubules are shown by open and filled arrowheads, respectively. White arrow indicates glomerulus; yellow arrowheads indicate straight thick limbs, and green arrowheads indicate thin limbs. Bar = 100 µm. (B) EndoG/terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)/4',6-diamidino-2-phenylindole (DAPI) staining of mouse kidneys 4 d after injection of saline (Control) or 20 mg/kg cisplatin (Cisplatin). Cisplatin induced the overexpression of EndoG and increased DNA fragmentation (TUNEL staining). Bar = 10 µm. (C) Nuclear translocation of EndoG is seen in mouse kidney 2 d after cisplatin injection. The nucleus shown by open arrow is stained positively for EndoG and TUNEL and has less DNA stained with DAPI. Two other nuclei above are TUNEL negative and do not contain EndoG. Bar = 5 µm.

 
Induction of EndoG and Suppression of DNase I Are Associated with Tubular Cell Injury by Cisplatin In Vitro
To study the cause–effect relationship between EndoG and tubular epithelial cell death induced by cisplatin, we first refined an in vitro model of cisplatin injury. For that, we used TKPTS cells that were treated in vitro with 0 to 400 µM cisplatin for 24 h. This approach revealed the dosage-dependent suppression of DNase I and induction of EndoG as determined by cell ELISA (Figure 6, A and B). These data confirmed our previous observations in vivo regarding the cisplatin-induced suppression of DNase I and induction of EndoG. Significant release of EndoG to cytoplasm and its import to nuclei were observed even at low concentration of cisplatin (25 µM) as determined by immunocytochemistry (Figure 6C).


Figure 6
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  		Figure 6. Dosage-dependent decrease of DNase I (A), induction of EndoG protein (B), and intracellular redistribution of EndoG (C) in TKPTS cells after cisplatin (24 h) treatment in vitro. (A and B) Protein expression was measured using cell ELISA as described in the Concise Methods. (C) EndoG pool redistribution measured by quantitative immunocytochemistry. Whereas the total amount of EndoG is only slightly increased after 25 µM cisplatin treatment, EndoG leaks from mitochondria and appears in cytoplasm and nuclei (n = 3 per group; 10 view fields per point). *P < 0.05, **P < 0.001 versus control samples.

 
Inactivation of EndoG Is Cytoprotective against Cisplatin In Vitro
The induction of EndoG was associated with cell death, which was measured using lactate dehydrogenase (LDH) release. To determine whether EndoG is essential for the cisplatin injury to tubular epithelial cells in vitro, we used two approaches. In the first, siRNA was used for EndoG silencing (Figure 7, A and B). The EndoG silencing was confirmed by the real-time RT-PCR and Western blotting. In the second, we applied the PPAR-{alpha} agonist WY-14643, which was previously shown by us to inhibit the expression of renal EndoG in vivo.25 Both approaches demonstrated that the inhibition of EndoG expression is protective against cell death induced by cisplatin (Figure 7, B and C). We also attempted to determine whether inactivation of EndoG was protective against apoptosis using Annexin V/propidium iodide flow cytometry. This experiment showed that the number of Annexin V–positive apoptosis cells did not increase above 5% of total, and the correlation with EndoG silencing was absent (data not shown), suggesting that, in the used model, the inactivation of EndoG protects against necrosis, not apoptosis.


Figure 7
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  		Figure 7. Protection from cisplatin injury by EndoG silencing in TKPTS cells. TKPTS cells treated with anti-EndoG small interfering (siRNA) or WY-14643 are protected from cisplatin injury in vitro. Inhibition of EndoG protein expression was confirmed by real-time RT-PCR (A) and Western dot-blotting (B). Cells were treated with EndoG siRNA (50 µM), control siRNA (50 µM), or transfection agent only ("nontreated") for 72 h before cisplatin (25 µM) treatment for 24 h. Total RNA was used for real-time RT-PCR, and protein extracts were used for Western blotting with anti-EndoG or anti-actin antibody. (C) Cell death after treatment with cisplatin was measured by lactate dehydrogenase (LDH) release. (D) In separate experiments, cells were grown to confluence, and medium was changed to a serum-free medium containing PBS, vehicle (DMSO), or WY-14643 (30 µM) for 2 h. Cells were then exposed to 25 µM cisplatin for 24 h, and cell death was measured by LDH release. For all graphs, n = 3 to 6 in each group; *P < 0.05, **P < 0.001 versus control cells without cisplatin treatment; #P < 0.05, ##P < 0.01, ###P < 0.001 versus nontreated cells.

 

   DISCUSSION
Top
 Introduction
 RESULTS
 DISCUSSION
CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Our data showed that DNase I is one of the most active endonucleases in the kidney. This observation confirmed previous reports from our and other groups.16,17,22,23,26 DNase I was found to be highly expressed in the normal kidney along with EndoG and DNase II, whereas the expression of CAD was low. The expression of DNase {gamma} was not observed. These data support previous observations from Shiokawa and Tanuma,27 who reported a very low level of DNase {gamma} expression in the rat kidney.

Inactivation of DNase I in KO mice did not affect the expression of other endonucleases. We found that despite that at the moment of cisplatin injection the activity of DNase I is higher than activity of any other endonuclease, the exposure to cisplatin strongly downregulated DNase I in vivo and in vitro. The suppression of urine DNase I (secreted by tubular epithelium) was especially profound and extended: It did not recover even at day 8. A mechanism of this phenomenon is unknown, and alternative pre-mRNA splicing can be suggested as one of the mechanisms.19 DNase I KO mice were demonstrated in our recent study to be markedly protected against toxic injury induced by a single injection of cisplatin (20 mg/kg), by both functional and histologic criteria. In vitro, primary tubular epithelial cells isolated from DNase I KO mice were resistant to low-dosage (8 µM) cisplatin injury. These data provided direct evidence that DNase I is essential for kidney injury induced by cisplatin. It is interesting that the total activity of DNase I does not seem to be important for mediating cell death. It may be hypothesized that DNase I activity is already in excess and therefore deviations of activity are not as important as the presence of DNase I itself. In support of this, our latest studies of acetaminophen-induced injury to the liver, an organ with minor amounts of DNase I, showed that the inactivation of the DNase I gene is protective.28

Despite that DNase I was shown to be important for the cellular response to cisplatin injury, it was not clear whether it is the enzyme that produces premortem DNA fragmentation during the injury. This study demonstrated that whereas DNase I was decreased and other endonucleases remained unchanged after cisplatin treatment, EndoG was induced in both in vitro and in vivo models. We also observed EndoG translocation to nuclei at day 2 after cisplatin administration, before kidney necrosis was developed. The nuclear import of EndoG increased further with the necrosis, often observed in nuclei with fragmented DNA determined by the TUNEL assay.

Our unexpected finding was that the induction of EndoG by cisplatin was higher in WT mice than in DNase I KO mice, suggesting a positive regulatory link between the two endonucleases. The in vitro experiment confirmed that the overexpression of DNase I causes induction of EndoG. This places EndoG downstream of DNase I. Such a link had not been observed before and may require further investigation. We can speculate that the activation of the DNA damage pathway by DNase I–mediated DNA breaks may be one of the potential mechanisms of EndoG induction.

EndoG is a nuclease that has a unique site selectivity, initially attacking poly(dG).poly(dC) sequences in double-stranded DNA, as a result of which the enzyme got its name. EndoG resides predominantly in the mitochondria, where it is localized in the intermembrane space.29 Mature active 27-kD EndoG can be released from mitochondria during cell death. It then translocates to nuclei and cleaves nuclear DNA without apparent sequence specificity.20 As opposed to DNase I, this enzyme has a greater activity on single-stranded nucleic acid substrates ssDNA and RNA. It preferentially cleaves the noncanonical structures of DNA, damaged DNA, triplex DNA, and R-loops that appear during transcription.30

The abundance of DNase I and EndoG varies in different tissues, and kidney is one of the primary organs of their expression.22,31 Neither DNase I nor EndoG is vitally necessary for normal tissue development. At least in some models, complete inactivation of the endonucleases did not lead to any abnormalities.17,32 The inactivation of these enzymes in normal animals may become important if they are associated with other unknown genetic changes. This is evident from the fact that homozygous EndoG–/– knockout mice (129xC57BJ/6 background) were not viable,33 whereas EndoG null mice, developed recently in a C57BJ/6 background, were viable.32 Similarly, the inactivation of DNase I caused lupus only in 129xC57BJ/6 mice but not in CD-1 mice.17,34 It was described that after cisplatin or DNase I pretreatment, DNA becomes more susceptible to EndoG digestion.15,35

On the basis of the finding that EndoG is downstream of DNase I and the previous demonstration of the key role of DNase I in cisplatin nephrotoxicity,17 we tested the hypothesis that the inhibition of EndoG is cytoprotective against cisplatin injury. The two used approaches, a nonspecific inhibition of EndoG expression by WY-14643 and a specific silencing of EndoG by siRNA, showed that the inhibition of EndoG is cytoprotective against cisplatin injury in vitro, thus confirming the hypothesis.

Although in normal kidney cells EndoG and DNase I may be dispensable, it is clear that during cisplatin injury the presence of both endonucleases aggravates the injury. At the moment of cisplatin injection, the activity of DNase I is higher than any other endonuclease in the kidney. DNase I may introduce first ssDNA breaks after being passively translocated to nuclei as suggested by Polzar et al.12 After the initial DNA damage produced by DNase I or cisplatin, DNA becomes more susceptible to EndoG digestion.15,35 After this moment, which immediately follows the cisplatin impact, the abundance of active DNase I is very low and the rest of the DNA damage is apparently produced by EndoG. Our in vitro data showed that EndoG is important for cell death induced by cisplatin. Similar to previous observations in other models,20,36 cisplatin induced the release of EndoG from mitochondria and its nuclear import. The translocation of EndoG occurred even at a low concentration of cisplatin (25 µM) that was not associated with significant induction of EndoG synthesis. Higher concentrations of cisplatin (200 to 400 µM) induced EndoG in a dosage-dependent manner. In vivo, the nuclear import of EndoG could be rarely visualized by histology, apparently because this maneuver caused prompt cell death. The presence of DNase I seems to be important only at the moment of injury, whereas variations of its activity after the initial impact by cisplatin do not matter. Further studies may determine whether this applies to other cell death endonucleases.

Taken together with the protection of DNase I KO mice against cisplatin, our data suggest that both endonucleases are essential for the tissue injury. DNase I acts immediately after cisplatin impact. Although the presence of DNase I is crucial for the kidney cisplatin injury, the activation of it does not seem to be necessary. During the inactivation of DNase I, EndoG is activated, possibly by DNase I, and it executes the DNA fragmentation, leading to the death of tubular epithelium.


   CONCISE METHODS
Top
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
DISCLOSURES
 REFERENCES
 
Animals
DNase1–/– KO mice (CD-1 background) were obtained from T. Moroy (University of Essen, Essen, Germany). The mice were bred as heterozygotes and genotyped by PCR as suggested by Napirei et al.34 Female 8- to 12-wk-old (20 to 30 g) mice were used in all experiments. Cisplatin (Bedford Laboratories, Bedford, OH) was administered in a single intraperitoneal injection 20 mg/kg in both DNase1 KO and WT DNase1 mice. Control mice were administered an injection of saline. The loss of kidney function and structural renal injury in WT mice and the absence of renal injury in DNase1 KO mice were confirmed as described by us previously.17 All experiments were approved by the Animal Care and Use Committee of the Central Arkansas Veterans Healthcare System.

RT-PCR
Semiquantitative RT-PCR was performed as described before.37,38 All primers were designed to have similar reassociation characteristics and located in the coding sequence approximately 300 to 450 bp apart to allow quantification by densitometry. Primers for endonucleases were as follows: 5'-AACTCAATCGGGACAAACCT-3' and 5'-GTTGATGTGACTGTGGTGT-3' for DNase I, 5'-TCTGTGTGTCCCTCCCGTTC-3' and 5'-GTCCTCTGTGGCACTGAAAG-3' for DNase II, 5'-GCGAGGATGACTCTGTGC-3' and 5'-CGGGGGCATTGGGCATCAC-3' for EndoG, 5'-GCTGGCGAGACCTGGCAT-3' and 5'-TTCTGGAGTACAGAGGCGGC-3' for CAD, and 5'-TCACGAAGAAGCACAACATA-3' and 5'-AAACAAACTTGGGGTCCGTC-3' for DNase {gamma}. All PCR products were sequenced to ensure their identity.

Real-Time RT-PCR
Our previously described protocol was followed.39 Briefly, 1 µg of total RNA was reverse-transcribed in a 50-µl reaction followed by real-time RT-PCR in a 25-µl reaction using SmartCycler (Cepheid, Sunnyvale, CA). Reaction mix was prepared using Platinum SYBR Green qPCR Supermix-UDG (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Two-temperature cycles with annealing/extension temperature at 62°C for EndoG and 64°C for 18s were used. The fluorescence was measured at the end of the annealing step. The melting curve analyses were performed at the end of the reaction (after 45th cycle) between 60 and 95°C to assess the quality of the final PCR products. The threshold cycle (Ct) values were calculated by fixing the basal fluorescence at 15 units. cDNA samples were diluted for real time 1:5 and 1:200 for EndoG and 18s respectively. Three replicate reactions were performed for each sample, and the average Ct was calculated. The standard curve of the reaction effectiveness was performed using the serially diluted (5 points) mixture of all experimental cDNA samples for EndoG and 18s separately. Calculation of the relative RNA concentration was performed using Cepheid SmartCycle software (version 2.0d, Sunnyvale, CA). Data are presented as ratio of EndoG/18s mRNA.

Total Endonuclease and DNase I Activities
Total kidney extracts were prepared, and endonuclease activity was measured by the plasmid incision assay using pBR322 plasmid (New England Biolabs, Beverly, MA) as substrate as described previously.18 The reaction was carried out in 2 mM CaCl2, 5 mM MgCl2, 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent endonuclease activity or in 5 mM MnCl2, 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent activity. One unit of endonuclease was capable of converting 1 µg of covalently closed supercoiled plasmid DNA to open circular or linear isoforms in 1 h at 37°C. Protein was measured using the BCA protein assay (Pierce, Rockford, IL). BSA was used as standard. DNase I activity was determined using the single radial enzyme diffusion assay40 or by the zymogram gel electrophoresis of total protein extracts as described by us previously.18

Western Blotting
Total kidney extracts were prepared as described previously.18 Proteins (10 to 20 µg) were separated in 11.5% gel according to the Laemmli procedure.41 Electrophoresis was performed at 100 V for 2 h. Proteins were transferred to the nitrocellulose membrane in Novex transferring buffer (Invitrogen) at 40 V for 3 h. For dot-blotting, the electrophoresis step was omitted and the samples were applied directly to membrane (0.25 µg protein/dot). After soaking in the blocking solution overnight at 4°C, the membrane was incubated with polyclonal anti-EndoG (Chemicon, Temecula, CA) or other antibodies diluted 1:1000 and washed in Tris-buffered saline, and primary antibodies were detected with anti-rabbit IgG horseradish peroxidase (HRP) using a SuperSignal chemiluminescent kit (Pierce). For allowing quantification, filters were stripped with 100 mM beta-mercaptoethanol, 2% SDS, and 62.5 mM Tris (pH 6.7) at 50°C and rehybridized with anti-actin 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA). OD of bands was determined by densitometry in quadruplicate format and normalized by actin.

Immunohistochemistry and TUNEL Assay
Samples of kidney tissue (1.5 mm thick) were fixed with 10% neutral formalin (Sigma, St. Louis, MO) for 24 h, dehydrated, and embedded in paraffin. For EndoG, TUNEL, and DAPI staining, sections of 3 µm thickness were cut, dewaxed, rehydrated in PBS (10 mM sodium phosphate buffer [pH 7.4] and 140 mM NaCl), pretreated with proteinase K (20 µg/ml) for 20 min at 37°C, and probed with diluted 1:400 anti-EndoG antibody (Chemicon) in blocking buffer (1% BSA [wt/vol], 0.012% saponin [wt/vol], and PBS) at 4°C overnight. The primary antibody was detected the next day after triple washing with 0.05% Tween-20 in PBS (PBST) by probing with 5 µg/ml solution in the same blocking buffer of anti-rabbit Ig (IgG; goat) conjugated with AlexaFluor 594 (Molecular Probes, Eugene, OR) for 30 min at 37°C, and after the washing, sections were then analyzed with the TUNEL assay using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN). TUNEL were performed according to the manufacturer's protocol. Each section was probed with a reaction mixture of terminal deoxynucleotidyl transferase and anti-actin–FITC–labeled precursor in cacodylate-based buffer for 1 h at 37°C, rinsed with PBST twice and water once, and counterstained with 10 µM DAPI (Sigma) for 5 min. The slides were washed with water three times, mounted under a ProLong Antifade Kit (Molecular Probes), and analyzed using a Carl Zeiss microscope with x10, x40, and x100 objectives. Controls of the specific reaction of anti-EndoG antibody and nick-labeling were performed with substitution of the reagents with the buffers.

Cell Culture
TKPTS cells were obtained from Dr. Elsa Bello-Reuss (University of Texas Medical Branch, Galveston, TX) and were cultured as described previously.42 The cells were maintained in DMEM/Ham F-12 medium (Sigma) supplemented with 7% FBS (Hyclone, Logan, UT). Cells were maintained in a humidified incubator gassed with 95% air/5% CO2 at 37°C, fed at intervals of 48 to 72 h, and used within 1 d after confluence (except for the siRNA experiments described in the EndoG siRNA Silencing section).

Construction of Expression Vector and Transfection
Previously cloned DNase I gene19 was inserted in pECFP-N1 vector (Clontech, Mountain View, CA) upstream of the gene encoding CFP. TKPTS cells were transfected using Lipofectamine 2000 (Invitrogen), and the expression of DNase I–CFP fusion protein or CFP alone (control) was detected by fluorescence microscopy using a cyan filter.

EndoG siRNA Silencing
TKPTS cells were seeded in six- or 24-well plates and grown to 60 to 70% confluence. To knockdown EndoG mRNA, cells were transfected with siRNA duplexes (sense siRNA 5'-AUGCCUGGAACAACCUUGAdTdT-3' and antisense siRNA 5'-UCAAGGUUGUUCCAGGCAUdTdT-3') or control siRNA #1 (Dharmacon, Lafayette, CO). The cells were treated with 50 nM siRNA mixed with TransIT-TKO transfection reagent (Mirus, Houston, TX) according to the manufacturer's recommendations, in serum-free medium for at least 48h. After that, the transfection medium was removed and the cells were treated with cisplatin for 24 h. EndoG mRNA expression was measured using real-time RT-PCR.

Cell ELISA
Cell ELISA was performed as described by Frahm et al.43 The cells were seeded in a 96-well plate (10,000 cells/well) and grown in a complete medium for 24 h. The cells were washed with serum-free medium, permeabilized, and fixed with fixative (4% wt/vol paraformaldehyde, 0.012% saponin, and PBS) for 10 min at room temperature. After fixation, the cells were washed and rehydrated in PBS and the endogenous peroxidase activity was inhibited by exposure to 0.5% hydrogen peroxide for 30 min at room temperature. Then the cells were probed with the primary antibody in blocking buffer (2% BSA and PBS) for 2 h. After triple washing with PBST (0.05% Tween-20 and PBS), primary antibody (titers 1:500 to 1:1000) was detected with anti-rabbit antibody (Santa Cruz) conjugated with HRP. The HRP activity was measured with 3,3',5,5'-tetramethylbenzidine substrate (Sigma) at 450 to 540 nm using the Synergy HT-I microplate reader (Bio-Tek Instruments, Winooski, VT). After measurement and triple washing, wells were reprobed with anti–actin-FITC antibody (titer 1:10; Santa Cruz Biotechnology) and washed again, and the fluorescence was measured at 485/528 nm (excitation/emission). All measurements were done in quadruplicate per one marker per one cell line and were repeated at least three times in different plates. Negative controls of primary antibody were done by their substitution by blocking buffer.

Measurement of EndoG in Cellular Compartments
TKPTS cells that were treated with cisplatin or vehicle were washed twice with PBS and fixed with 4% paraformaldehyde and 0.12% saponin in PBS for 10 min. Cells were probed with rabbit anti-EndoG (1:400) and mouse anti-cyclooxygenase IV (1:200) antibodies, which were detected with goat anti-rabbit AlexaFluor 594 and goat anti-mouse AlexaFluor 488 conjugates, respectively. Nuclei were counterstained with DAPI. Slides were analyzed under magnification of 600 using an Olympus IX-81 microscope (Olympus America, Center Valley, PA). Images and acquisitions were made with a digital camera HAMAMATSU ORCA-ER (Hamamatsu Photonics K.K., Hamamatsu City, Japan) and software Slidebook 4.1 (SciTech Pty Ltd., Preston, Victoria, Australia). OD of cyclooxygenase IV signal was used as the marker of mitochondrion localization and subtracted from the EndoG optical signal. The result was considered "nonmitochondrial EndoG," and the rest of the signal was considered "mitochondrial EndoG." For measurement of EndoG presence in nuclei, images were traced by DAPI, and part of nonmitochondrial EndoG, which was co-localized with DAPI, was considered nuclear EndoG.

Cytotoxicity Assays
For measurement of cytotoxicity of cisplatin, the LDH release assay kit (Promega, Madison, WI) was used. The results were expressed as the ratio of LDH released by treated cells into medium to the total LDH. In the Annexin V/propidium iodide assay, Annexin V-FITC (Invitrogen) and propidium iodide (Sigma) were used as described by us previously.44

Statistical Analyses
Results were expressed as means ± SEM. The significance of difference in mean values within and between multiple groups was examined with an ANOVA for repeated measures followed by a Duncan's post hoc test. The t test was used to evaluate the significance of differences between two groups of experiments (SigmaStat; SPSS, Chicago, IL). P < 0.05 was considered statistically significant.


   DISCLOSURES
Top
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
REFERENCES
 
None.


   Acknowledgments
 
This research was supported in part by the PO1 DK58324-01A1 grant from the National Institutes of Health and VA Merit Review grants to S.V.S. and A.G.B.

A portion of this study was published as an abstract (J Am Soc Nephrol 15: 711A, 2004).

We thank Ray Biondo, MD, for editorial assistance.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   REFERENCES
Top
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 

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