Cisplatin Nephrotoxicity Is Mediated by Deoxyribonuclease I

Alexei G. Basnakian,* Eugene O. Apostolov,* Xiaoyan Yin,* Markus Napirei,† Hans Georg Mannherz,† and Sudhir V. Shah*‡ *Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas; †Department of Anatomy and Embryology, Medical Faculty, Ruhr University–Bochum, Bochum, Germany; and ‡Central Arkansas Veterans Healthcare System, Little Rock, Arkansas

Cisplatin is commonly used for chemotherapy in a wide variety of tumors; however, its use is limited by kidney toxicity. Although the exact mechanism of cisplatin-induced nephrotoxicity is not understood, several studies showed that it is associated with DNA fragmentation induced by an unknown endonuclease. It was demonstrated previously that deoxyribonuclease I (DNase I) is a highly active renal endonuclease, and its silencing by antisense is cytoprotective against the in vitro hypoxia injury of kidney tubular epithelial cells. This study used recently developed DNase1 knockout (KO) mice to determine the role of this endonuclease in cisplatin-induced nephrotoxicity. The data showed that DNase I represents approximately 80% of the total endonuclease activity in the kidney and cultured primary renal tubular epithelial cells. In vitro, primary renal tubular epithelial cells isolated from KO animals were resistant to cisplatin (8 ␮M) injury. DNase I KO mice were also markedly protected against the toxic injury induced by a single injection of cisplatin (20 mg/kg), by both functional (blood urea nitrogen and serum creatinine) and histologic criteria (tubular necrosis and in situ DNA fragmentation assessed by the terminal deoxynucleotidyl transferase nick end-labeling). These data provide direct evidence that DNase I is essential for kidney injury induced by cisplatin. J Am Soc Nephrol 16: 697–702, 2005. doi: 10.1681/ASN.2004060494

isplatin (cis-diamminedichloroplatinum II) is com- this hypothetical endonuclease had never been cloned. So far, monly used for chemotherapy in a wide variety of deoxyribonuclease I (DNase I) is the most well known Ca/Mg- C tumors; however, it has nephrotoxicity as a major side dependent endonuclease that produces 3ЈOH DNA ends and is effect (1–3). After a single injection of cisplatin, 28 to 36% of involved in DNA fragmentation during cell death (15,16). patients develop dose-dependent nephrotoxicity (4). Cisplatin We showed previously that DNase I is highly expressed in has been shown to accumulate in the kidney to a greater degree the kidney and is upregulated during kidney injury induced by than in other organs (4). ischemia-reperfusion in vivo (17,18). The suppression of DNase Although a number of cellular targets of cisplatin have been I by antisense was protective against the hypoxia/reoxygen- identified (5), the mechanism of cisplatin nephrotoxicity is un- ation injury of renal tubular epithelial cells in vitro (17). In the clear. Multiple studies showed that cisplatin nephrotoxicity is present study, we chose to investigate the effect of DNase I associated with DNA fragmentation. It is visualized either by a inactivation in vivo using recently produced DNase I–deficient 200-bp DNA ladder in agarose gel (6,7) or by using the terminal mice (19) to determine whether DNase I is important for kidney deoxynucleotidyl transferase-mediated deoxyuridine triphos- injury induced by cisplatin. Unlike previously described phate (TdT) nick-end labeling (TUNEL) assay (8–10). The ap- DNase1 knockouts (KO) in 129ϫC57B1/6 background, the Ϫ Ϫ pearance of a 200-bp ladder indicates that the enzyme is an DNase1 / CD-1 mice used in the current study do not develop Ϫ Ϫ endonuclease, capable of internucleosomal fragmentation of lupus. Our experience showed that DNase1 / mice are nuclear DNA. This type of DNA fragmentation is usually at- healthy, suggesting that DNase I is dispensable in normal tis- tributed to apoptosis (11). Positive TUNEL staining, which is sues. That distinguishes DNase I from other cell death endo- also characteristic for apoptotic DNA fragmentation, suggests nucleases, for example DNase II or endonuclease G (EndoG), that the endonuclease produces 3ЈOH DNA termini, a substrate inactivation of which was lethal (20–22). The role of DNase I for TdT. Earlier studies suggested that the apoptotic DNA during tissue injury has not been previously studied in any fragmentation is catalyzed by a Ca/Mg-dependent endonucle- organ, including the kidney. ase (12). Despite several attempts to isolate the enzyme (13,14), Materials and Methods Animals Received June 23, 2004. Accepted November 19, 2004. DNase1Ϫ/Ϫ KO mice (CD-1 background) were obtained from T. Published online ahead of print. Publication date available at www.jasn.org. Moroy (University of Essen, Essen, Germany). The mice were bred as heterozygotes and genotyped by PCR as suggested by Napirei et al. Address correspondence to: Dr. Alexei G. Basnakian, University of Arkansas for (19). Female 8- to 12-wk-old (20 to 30 g) mice were used in all of the Medical Sciences, Department of Internal Medicine, Division of Nephrology, 4301 W. Markham Street, #501, Little Rock, AR 72205. Phone: 501-257-1000 ext. 54861; experiments. We administered 20 mg/kg cisplatin (cis-diamminedi- Fax: 501-257-4822; E-mail: [email protected] chloroplatinum II; Bedford Laboratories, Bedford, OH) in a single

Ϫ Ϫ ϩ ϩ intraperitoneal injection in DNase1 / and DNase1 / (wild-type [WT]) Results mice. The control mice received an injection of saline. All experiments Characterization of the Models were approved by the Animal Care and Use Committee of the Central To determine whether DNase I becomes important during Arkansas Veterans Healthcare System. kidney tissue injury, we started with an in vitro approach, considering that a significant effect of cisplatin can be observed Primary Tubular Epithelial Cells at the cellular level (1). In the kidney, the preferential damage Mouse tubular epithelial cells were freshly isolated from DNase I KO by cisplatin is located in tubular epithelial cells (2,3). To have an and WT mice as described by Nowak et al. (23) and cultured up to 10 d in vitro model for studying the role of DNase I in cisplatin renal (passages 1 and 2) before the experiment. The cells were treated in vitro injury, we used primary tubular epithelial (PTE) cells prepared with 8 ␮M cisplatin in serum-free media for 4 d. We assessed cell death as described by Nowak et al. (23). We did not observe any by lactate dehydrogenase (LDH) using the LDH release assay kit (Pro- mega, Madison, WI). Cytotoxicity was expressed as the ratio of the difference in morphology or viability between nontreated Ϫ/Ϫ ϩ/ϩ LDH release in the treated cell medium to that of the maximal LDH DNase1 and DNase1 PTE cells. release. Activation of caspase-3 was measured using the cell ELISA Zymogram gel electrophoresis of total protein extracts ob- procedure described by Frahm et al. (24). The cells were grown in tained from mouse kidneys or PTE cells showed that the 34-kD 96-well plates; permeabilized; and fixed with 4% paraformaldehyde, activity band corresponding to DNase I is absent in KO mice 0.012% saponin, and buffered phosphate saline for 10 min at room (Figure 1A). We measured the endonuclease activities of total temperature. Polyclonal rabbit anti-active caspase-3 (titer 1:1000; protein extracts from PTE cells and mouse kidneys using the Chemicon, Temecula, CA) was used as the primary antibody, which plasmid incision assay under conditions that, according to our was detected with secondary anti-rabbit antibody conjugated with HRP previous studies (17,18), are sufficient for all known cell death (titer 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). The HRP endonucleases. The activity in PTE cell extracts was 25 Ϯ 3 activity was measured with TMB substrate (Sigma, St. Louis, MO) at units/␮g protein in WT cells and 5 Ϯ 2 units/␮g protein in KO 450 to 540 nm using the Synergy HT-I microplate reader (Bio-Tek, Winooski, VT). After measurement and triple washing, the wells were cells (Figure 1B). The endonuclease activity of the total kidney Ϯ ␮ Ϯ ␮ reprobed with anti–actin-FITC antibody (titer 1:100; Santa Cruz), was 105 10 units/ g protein in WT mice and 15 2 units/ g washed again, and measured at 485/528 nm. All measurements were protein in KO mice (Figure 1B). We did not observe an activa- done in quadruplicate and were repeated at least three times in differ- tion of any known endonuclease in kidneys or PTE cells from ent plates. KO mice or an overexpression of any known endonuclease mRNA measured using reverse transcriptase–PCR (data not Histology and Histochemistry shown). Taken together, these data suggested that DNase I Kidney samples were fixed with 10% neutral formalin (Sigma) for represents the majority (approximately 80%) of the total 24 h, dehydrated, and embedded in paraffin. Sections of 3-␮m thickness Ca/Mg-dependent endonuclease activity both in the kidney were cut and stained with hematoxylin and eosin for routine histologic analysis. Tubular necrosis was assessed by measurement of the decreased number of tubular epithelial cells attached to the basement membrane as described by Solez et al. (25). The data were presented as the number of cell nuclei per mm2 as suggested by Ishikawa and Kitamura (26). We performed TUNEL staining using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s manual, followed by counterstaining with 10 ␮M 4Ј,6-diamidino-2-phenylindol (DAPI; Sigma) for 5 min. Slides were mounted under the ProLong Antifade Kit (Molecular Probes, Eugene, OR) and analyzed with a Carl Zeiss microscope with the green (fluo- rescein, TUNEL) or blue spectrum (DAPI).

Endonuclease Activity We prepared total kidney extracts or cell extracts and measured the endonuclease activity using the plasmid incision assay with pBR322 plasmid (New England Biolabs, Beverly, MA) as substrate as described previously (17). The total endonuclease activity was measured in 2 mM CaCl2, 5 mM MgCl2, 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothre- Figure 1. Endonuclease deficiency in deoxyribonuclease I–defi- Ϫ Ϫ itol. We accepted 1 unit as the amount of endonuclease capable of cient (DNase I / ) primary tubular epithelial (PTE) cells and ␮ converting 1 g of covalently closed supercoiled plasmid DNA to open animals. (A) Zymogram gel electrophoresis of DNase I in total circular or linear isoforms in1hat37°C. The protein was measured mouse kidney or PTE cell protein extracts performed as de- using the BCA protein assay (Pierce, Rockford, IL). BSA was used as a scribed previously (17). (B) Although specific endonuclease standard. Zymogram gel electrophoresis of total protein extracts was activity measured by plasmid incision assay is lower in PTE performed as described by us previously (17). cells, the total endonuclease activity is decreased approximately 5 times in kidney extracts and PTE cells from knockout Statistical Analyses (KO) versus wild-type (WT) mice (the number of independent Statistical analysis was performed with ANOVA and t test. Results cell culture preparations, n ϭ 6 in each cohort). *P Ͻ 0.01; **P Ͻ were expressed as mean Ϯ SEM. P Ͻ 0.05 was considered significant. 0.001. J Am Soc Nephrol 16: 697–702, 2005 Cisplatin Nephrotoxicity Is Mediated by DNase I 699

and in the PTE cells, and, thus, PTE cells can be used as a and monitored mortality, kidney failure, and tubular necrosis cellular model to study the role of DNase I in vitro. for 4 d. In our experiments, the animal deaths started at day 3. Our data showed that the mortality of KO mice at days 3 to 4 Protection of DNase I–Deficient PTE Cells against Cisplatin after cisplatin injection was markedly reduced compared with Injury In Vitro WT mice. It was 11.1% in the KO mice (n ϭ 18) versus 38.1% in To determine the role of DNase I in cisplatin injury in vitro, the WT mice (n ϭ 21; Figure 2B). We did not observe animal ␮ we treated PTE cells with 8 M cisplatin for 96 h. The treatment deaths in the control groups of WT and KO mice that received with this concentration of cisplatin was shown previously to an injection of saline (n ϭ 10 in each group). There was no induce apoptosis in renal tubular epithelial cells in vitro (27). mortality observed after day 4. Our experiments demonstrated that cisplatin induces irrevers- ible PTE cell death measured by LDH release, whereas PTE Protection of Kidney Function in DNase I–Deficient Mice cells isolated from KO mice showed a significant resistance to during Cisplatin Injury cisplatin injury. Cisplatin treatment induced LDH release from We assessed kidney function by blood urea nitrogen (BUN) 9.5 Ϯ 1.1% in nontreated controls to 19.4 Ϯ 1.0% in WT cells and creatinine measured in blood serum at days 0, 2, 4, and 8 (P Ͻ 0.05) and from 10.1 Ϯ 0.3% in controls to 11.2 Ϯ 0.9% of after the cisplatin injection. Neither functional parameters was total LDH in KO cells (in each group, n ϭ 6; Figure 2A). These different between the two genotypes before the experiment or data suggest that DNase I is essential for the direct cisplatin significantly changed 2 d after cisplatin injection. However at cytotoxicity to the kidney tubular epithelium cells in vitro.We day 4, surviving WT mice showed marked kidney failure both measured caspase-3 activation in DNase I KO and WT cells by serum BUN and creatinine, which increased up to 250 Ϯ 28 after 8 ␮M cisplatin injury. These experiments showed that and 2.7 Ϯ 0.5 mg/dl, respectively (Figure 3). DNase I KO mice active caspase-3 amount normalized by ␤-actin was not signif- showed significant protection of kidney function: The BUN was icantly different between DNase I WT and KO cells before 125 Ϯ 29 mg/dl and the creatinine decreased to 0.9 Ϯ 0.2 mg/dl impact (WT, 0.82 Ϯ 0.04; KO, 0.78 Ϯ 0.02) or after cisplatin (n ϭ 10 in each group, P Ͻ 0.05 versus WT mice for both BUN treatment (WT, 1.11 Ϯ 0.05; KO 1.05 Ϯ 0.03; mean Ϯ SEM, n ϭ and creatinine). Kidney function normalized at day 8 in both 4 in each group). Thus, after cisplatin exposure, caspase-3 was genotypes. almost equally and significantly activated in both types of cells (P Ͻ 0.001 in each group). These data suggest that even after Protection of Kidney Structure in DNase I–Deficient Mice caspase-3 activation, DNase I is required for cell death in this during Cisplatin Injury model and the contribution of DNase I as an upstream event to To determine the morphologic changes of the kidney after caspase-3 is negligible. cisplatin treatment, we performed histologic analyses of kidneys that were isolated from animals that survived to deter- Decreased Mortality of DNase I–Deficient Mice after mine tubular necrosis using established histologic criteria (25). Cisplatin Injection This analysis showed the profound renal tubular necrosis in A dose-dependent nephrotoxicity can be observed after a WT mice at day 4 (Figure 4). The necrosis was associated with single injection of cisplatin (4). In mice, cisplatin injury is usually associated with tubular necrosis and kidney failure starting from day 2 and reaching maximum at days 3 to 4, depending on the dose of the cisplatin (28,29). To determine the role of DNase I in kidney injury by cisplatin in vivo, we injected a single dose of cisplatin (20 mg/kg) into KO and WT mice intraperitoneally

Figure 2. Correlation of animal mortality and PTE cell death induced by cisplatin with the inactivation of DNase I. (A) WT Figure 3. Protection of kidney function in DNase I KO mice after and KO PTE cell death after 96 h of exposure with 8 ␮M cisplatin injection. We measured blood urea nitrogen and cre- cisplatin measured by lactate dehydrogenase (LDH) release atinine in blood serum collected from mice that survived after into media. (B) Animal mortality at days 3 to 4 after 20 mg/kg a single-dose intraperitoneal injection of 20 mg/kg cisplatin. cisplatin injection. *P Ͻ 0.05. *P Ͻ 0.05 versus WT. 700 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 697–702, 2005

Figure 4. Protection of kidney morphology in DNase I KO mice against cisplatin injury. Hematoxylin-eosin–stained kidney tissue slices showed signs of tubular necrosis (ૺ, tubular dilation; 4, loss of tubular epithelium; block arrows, casts in tubular lumen) in WT mice but not in KO mice. Scale bar ϭ 50 ␮m. tubular dilation, accumulation of casts, loss of tubular epithelium, and interstitial inflammation visualized by leukocyte in- filtration. Often, tubular basement membranes were ruptured and necrotic epithelial cells were present in the tubular lumen. Histologic analysis of kidneys that were isolated from KO survivors showed that these mice had almost no tubular necrosis. To quantify the kidney injury, we measured the density of tubular cells by a manual count of the tubular cell nuclei. It was shown to be significantly decreased in KO mice compared with the WT mice (Figure 5).

Attenuation of DNA Fragmentation in DNase I–Deficient Mice during Cisplatin Injury We measured DNA fragmentation using the TUNEL assay, which allows detection of 3ЈOH DNA ends commonly associated with endonuclease-mediated DNA fragmentation. The degree of DNA destruction in WT mice 4 d after cisplatin injection was high and very heterogeneous throughout the tissue. Im- portantly, TUNEL staining was strongly suppressed in KO mice compared with WT mice (Figure 6). In WT mice, nuclei counterstaining with DAPI showed a decreased number of Figure 6. Decreased DNA fragmentation induced by cisplatin in nuclei after cisplatin injury. Some nuclei looked partially de- the kidney in DNase I KO mice compared with WT mice. We formed. It is interesting that we observed a small number of measured in situ DNA fragmentation using terminal deoxynu- apoptotic bodies in WT mice that were treated with cisplatin. cleotidyl transferase-mediated deoxyuridine triphosphate (TdT) No apoptotic bodies were found in the cisplatin-treated KO nick-end labeling (TUNEL) assay (a, d, and g). Nuclei were coun- terstained with 4Ј,6-diamidino-2-phenylindol (b, e, and h). DNase mice. I–treated tissue sections were used as positive controls for the TUNEL assay (Control). Scale bars ϭ 20 ␮m.

Discussion The degradation of cellular DNA by endonucleases is an important component of renal tubular epithelial cell death induced by ischemia or nephrotoxins. Several studies showed activation of endonuclease(s) after cisplatin injury in kidney cells, which was visualized by an increased 200-bp ladder DNA Figure 5. Quantification of tubular necrosis in DNase I KO and fragmentation (6,7,30,31) or TUNEL (8–10). Our previous stud- WT mice after cisplatin injection. The decreased number of ies indicated that endonucleases are important executioners of tubular epithelial cell nuclei was observed in WT mice (**P Ͻ renal cell death mechanisms induced by a variety of stimuli, 0.01) but not in KO mice. including hypoxia/reoxygenation in vitro (32), toxic injury in J Am Soc Nephrol 16: 697–702, 2005 Cisplatin Nephrotoxicity Is Mediated by DNase I 701 vitro (33), and ischemia/reperfusion of the kidney in vivo (17). promoted by nuclear membrane damage, which was shown to The inhibition of renal endonucleases by aurintricarboxylic be induced by cisplatin (41). acid, a pan-endonuclease inhibitor (34), or suppression of The use of DNase I KO mice allowed the determination of the DNase I using antisense was protective against DNA fragmen- endonuclease activity produced by DNase I in the kidney and tation and cell death in vitro (17). Takeda et al. (7) also showed the identification of its role in cisplatin injury using the cause– that the cisplatin-induced fragmentation of DNA in renal S3 effect approach. The DNase I–mediated kidney cell death was cells can be inhibited by aurintricarboxylic acid. These data evident by both functional and morphologic criteria. Although demonstrated a cause–effect relationship among endonuclease our data did not determine whether all DNA fragmentation activation, DNA fragmentation, and renal cell death. However, was produced by DNase I alone or was only guided by DNase the endonuclease responsible for DNA fragmentation in cispla- I, it demonstrated that inactivation of DNase I is protective tin-induced renal injury in vitro or in vivo has not been identi- against cisplatin-induced DNA fragmentation, whereas other fied. endonucleases participated in DNA destruction. DNase I was The mechanism of the cisplatin-induced cytotoxicity is not previously described as an endonuclease that participates in fully understood. It was shown that cisplatin is capable of apoptosis and is capable of cooperating with EndoG (16,37). binding to several cellular components, including membrane Further studies will be necessary to determine whether DNase phospholipids, thiols, cytoskeletal microfilaments, proteins, I induces renal tubular necrosis directly or through apoptosis RNA and DNA (5). Apparently, this may involve more than and whether it functions in cooperation with other endonucle- one mechanism of cell death. Indeed, both apoptosis and ne- ases. crosis were observed in cells treated with cisplatin (6,27). These Cell death endonucleases act both premortem, leading to cell death, and postmortem, providing a “clean-up” after cell death studies revealed that low doses of cisplatin induce apoptosis, (11). Importantly, our study showed that DNase I acts at the while high doses lead to necrosis (6,27). It was demonstrated premortem level because its inactivation caused protection of recently that apoptosis induced by cisplatin in cultured renal the kidney. This is an encouraging result for potential applica- tubular epithelial cells and epithelial cells of other origins is to tions of DNase I inactivation in future therapeutic strategies to a significant degree independent from p53 and caspases 3, 8, protect the kidney against toxic acute renal failure. and 9 (10,35). Because DNA fragmentation is observed in all of these models, it can be suggested that apoptosis and necrosis pathways, as well as caspase-dependent and caspase-indepen- Acknowledgments dent pathways, are likely to be shared at the level of cell death This research was supported in part by grant PO1 DK58324-01A1 endonucleases (2). Our measurements of caspase-3 activation from the National Institutes of Health and a grant from the Deutsche indicated that during low-dose cisplatin injury in vitro, DNase Forschungsgemeinschaft (Ma 807/12-2). Portions of this study were published as an abstract (J Am Soc Nephrol I does not use the caspase-3–mediated apoptosis pathway. 14: 567A, 2003). However, it is still possible that DNase I may be regulated by We thank G. Nowak of the University of Arkansas for Medical caspase-3. Sciences for assistance with the development of the primary cell culture Cell death endonucleases are a recently recognized group of technique. enzymes that include cytoplasmic DNase I and caspase-activated DNase (16,36), mitochondrial EndoG (37), lysosomal DNase II (38), and perinuclear DNase ␥ (39). In the kidney, References DNase I is highly expressed (18,40). We showed recently that 1. Reese DM: Anticancer drugs. Nature 378: 532, 1995 among other endonucleases, EndoG and DNase II are also 2. 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