Manganese Superoxide Dismutase Attenuates Cisplatin-Induced Renal Injury: Importance of Superoxide
Christopher A. Davis*,
Harry S. Nick and
Anupam Agarwal*
*Department of Biochemistry and Molecular Biology, Department of Medicine, and Department of Neuroscience, University of Florida, Gainesville, Florida.
Correspondence to Dr. Anupam Agarwal, Assistant Professor of Medicine, Division of Nephrology, Hypertension, and Transplantation, Box 100224 JHMHC, 1600 SW Archer Road, University of Florida, Gainesville, FL 32610. Phone: 352-392-4008; Fax: 352-392-3581; E-mail: agarwal{at}nersp.nerdc.ufl.edu
ABSTRACT. Cisplatin is a potent chemotherapeutic agent thatis used to treat many human malignancies. Unfortunately, inaddition to side effects such as ototoxicity, anaphylaxis, andbone marrow suppression, a significant percentage of patientsreceiving cisplatin develop severe nephrotoxicity. Mitochondrialdysfunction that is mediated via the generation of reactiveoxygen species has been implicated in the pathogenesis of cisplatin-inducedrenal injury. To address the mechanism, it was hypothesizedthat overexpression of antioxidant enzymes, such as mitochondria-localizedmanganese superoxide dismutase (MnSOD) or mitochondria-targetedcatalase (mito-Cat), would be cytoprotective in cisplatin-inducedcell injury. To this end, human MnSOD or a mito-Cat vector werestably transfected into human embryonic kidney 293 cells. Cellsthat overexpressed MnSOD exhibited significantly less cell roundingand detachment compared with both mito-Cat and vector controlsafter exposure to 20 µM cisplatin. Cell injury as assessedby DNA fragmentation and annexin V binding assays was significantlydecreased in the cells that overexpressed MnSOD compared withvector alone and mito-Cat. In addition, elevated levels of MnSODwere strongly associated with increased clonogenic potentialafter cisplatin challenge. Thus, overexpression of MnSOD, andnot catalase, protects against cisplatin-induced renal epithelialcell injury. These results demonstrate the importance of reactiveoxygen species in the mechanism that underlies cisplatin-inducedrenal injury and specifically implicate the superoxide radical,and not hydrogen peroxide, as the mediator.
Cisplatin is a chemotherapeutic agent that is used in the treatmentof a variety of solid-organ cancers, including those of thehead, neck, testis, ovary, and breast (1). Unfortunately, inaddition to causing bone marrow suppression, ototoxicity, andanaphylaxis, 28 to 36% of patients receiving an initial dose(50 to 100 mg/m2) of cisplatin develop acute renal failure (1,2)due to its preferential accumulation within the proximal tubulecells in the outer medulla of the kidney (2,3). The cellularevents in cisplatin-mediated nephrotoxicity, including decreasedprotein synthesis, membrane peroxidation, mitochondrial dysfunction,and DNA injury, are a consequence of free radical generationand the inability to scavenge such molecules (4–6). Thesecontentions are supported by a variety of studies, includingthose that demonstrate a protective role for free radical scavengers,such as vitamin E, catalase, and glutathione, in cisplatin-mediatedcytotoxicity (7–12). Of particular interest is work byMcGinness et al. (11) that demonstrated a reduction in cisplatin-mediatednephrotoxicity in rats that were treated with the superoxidedismutase (SOD) mimetic, orgotein.
Studies from our laboratory and others have demonstrated a protectiverole for the mitochondrial-localized manganese superoxide dismutase(MnSOD) in several models of free radical-mediated cell injury(13–15). In light of the clear role that reactive oxygenspecies (ROS) and the mitochondria play in the apoptotic cascade(16) and, more importantly, in cisplatin-induced renal cellinjury (4,17,18), we hypothesized that overexpression of MnSODprovides protection against cisplatin-mediated cytotoxicityin cultured renal epithelial cells. Therefore, in an attemptto define the specific role of ROS in cisplatin-mediated toxicity,we challenged renal epithelial cells that stably expressed MnSODwith cisplatin. In addition, we generated cells that overexpresseda mitochondria-targeted catalase (mito-Cat) to evaluate thefree radical specificity of cisplatin-induced renal cell injury.Our results demonstrate that overexpression of MnSOD, and notcatalase, provides protection against cisplatin in renal epithelialcells, a result that strongly implicates a role for the mitochondria-derivedsuperoxide radical in cisplatin-mediated nephrotoxicity.
Reagents
Cisplatin and calf serum were obtained from Sigma Chemical (St.Louis, MO). The cytotoxicity kit for lactate dehydrogenase (LDH)was purchased from Roche (Mannheim, Germany). The apoptosisdetection kit was obtained from R & D Systems (MinneapolisMN). A rabbit polyclonal anti-MnSOD antibody was generated inour laboratory by using an internal peptide coupled to hemocyanin.The anti-catalase antibody was purchased from Calbiochem (LaJolla, CA). pPCR-Script Amp (SK)+ was purchased from Stratagene(La Jolla, CA), and the mammalian expression vector pcDNA3.1/Zeo+and Zeocin were obtained from Invitrogen (Carlsbad, CA).
Cell Culture
Human embryonic kidney 293 (HEK 293) cells (ATCC, Rockville,MD) were cultured in Dulbecco’s modified Eagle’smedia that was supplemented with 4 mM glutamine, 25 mM HEPES,and 10% calf serum at 37°C in 90% room air/10% CO2.
Plasmid Construction for MnSOD and Mito-Cat Overexpression
An 853-bp MnSOD cDNA was generated by reverse transcriptase-polymerasechain reaction (PCR) from mRNA that had been isolated from humanrenal proximal tubule cells (Clonetics, Walkersville, MD) byusing oligonucleotides that contained HindIII (5') and EcoRI(3') restriction sites. The resulting product was ligated intopPCR-Script Amp SK(+) and then directionally cloned into theHindIII/EcoRI sites of pcDNA3.1/Zeo+ (pcDNA3.1/SOD; Figure 1A).For construction of the mitochondrially targeted catalase vector,the MnSOD mitochondrial leader sequence fused to a human catalasecDNA was removed by complete digestion with BsaI and NotI ofpSVZEOmspCAT (kindly provided by A. Melendez and A. M. Rodriguez,Center for Immunology and Microbial Disease, Albany MedicalCollege, Albany, NY) (19). This fragment was then placed downstreamof the cytomegalovirus promoter/enhancer of the mammalian expressionvector pcDNA3.1/Zeo+ that had been completely digested withNotI and partially digested with BsaI (pcDNA3.1/mito-Cat; Figure 2A).The integrity of all constructs was verified by DNA sequencing.
Figure 1. Plasmid construction and overexpression of manganese superoxide dismutase (MnSOD). (A) Schematic representation of the MnSOD expression vector pcDNA3.1/SOD and Northern blot analysis of HEK 293 cell transiently transfected with either vector alone or pcDNA3.1/SOD. The endogenous (4 Kb and 1 Kb) and the transgene-derived (approximately 1.2 Kb) transcripts are shown. CMV, cytomegalovirus; polyA, bovine growth hormone polyadenylation signal. (B) Individual clonal cell populations transfected with either vector alone or pcDNA3.1/SOD were analyzed by immunoblot analysis as described in Materials and Methods. The 22-kD MnSOD monomer is shown.
Figure 2. Plasmid construction and overexpression of mitochondria-targeted catalase (mito-Cat). (A) Schematic representation of the mito-Cat expression vector containing an MnSOD mitochondrial leader sequence fused to the human catalase cDNA. (B) Immunoblot analysis of stable cell populations probed with anti-catalase antibody as described in Materials and Methods. The 52-kD band represents the overexpressed catalase protein. (C) Mitochondrial and cytosolic fractionation of vector and mito-Cat cell lines reveals mitochondrial enrichment of catalase as shown by immunoblot analysis.
Stable Transfection of MnSOD and Mito-Cat Plasmids
One µg of each expression plasmid was stably transfectedinto HEK 293 cells by using Lipofectamine Plus (Life Technologies/BRL,Rockville, MD) according to the manufacturers instructions.Cells were cultured for 48 h and transferred to Dulbecco’smodified Eagle’s medium that contained HEPES (25 mM),glutamine (4 mM), and zeocin (150 µg/ml). Media that contained150 µg/ml zeocin was replaced every 3 d for approximately4 wk. Multiple zeocin-resistant colonies were isolated by clonalselection and subcultured without antibiotic before screeningfor MnSOD or catalase overexpression by immunoblot analysis.
Northern Blot Analysis
Total cellular RNA was isolated from transfected cells by usinga modification of the Chomczynski and Sacchi method (20). RNAwas quantified and size fractionated on a 1% agarose-formaldehydegel, blotted onto a nylon membrane, and hybridized with a 32P-labeledhuman MnSOD cDNA probe. The membrane was washed and subjectedto autoradiography.
Immunoblot Analysis
For immunoblot analysis, transfected cells were lysed with bufferthat contained Triton X-100 (1%) (Sigma). Protein concentrationwas determined by the bicinchoninic assay (Pierce, Rockford,IL), and 20 µg of total protein was electrophoresed on10% sodium dodecyl sulfate-polyacrylamide gels and transferredonto polyvinylidene difluoride membranes. Membranes were blockedand incubated for 1 h with anti-MnSOD (polyclonal, 1:600) oranti-catalase (1:500, Calbiochem) and then by washing and incubationwith peroxidase-conjugated goat anti-rabbit IgG antibody (1:10,000).Labeled protein bands were visualized by chemiluminescence (Pierce).
Subcellular Fractionation
To confirm mitochondrial targeting of overexpressed catalase,cytosolic and mitochondrial fractionation was performed as describedpreviously (21). Briefly, cells were washed with ice-cold phosphate-bufferedsaline (PBS), harvested with a cell scraper, and centrifugedat 200 x g for 5 min at 4°C. The pellet was resuspendedin 300 µl of ice-cold buffer A (20 mM HEPES [pH 7.5],10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid,1 mM ethyleneglycotetraacetic acid, 1 mM dithiothreitol, 250mM sucrose, and protease inhibitors). After incubation on icefor 20 min, cells were disrupted by dounce homogenization (Bpestle/40 strokes). The sample was centrifuged at 1000 x g for10 min at 4°C, and the supernatant was subjected to furthercentrifugation at 10,000 x g for 15 min. The resulting supernatant(cytosolic fraction) and the mitochondria-enriched pellet (resuspendedin buffer B, consisting of Tris [pH 8.0], 0.5% NP-40, and 5mM CaCl2) were subjected to immunoblot analysis as describedabove.
Cell Viability Assays
Cell viability was assayed by phase contrast microscopy, specificLDH release, and trypan blue exclusion. For LDH release, celllines were split into 24-well tissue culture dishes and incubatedat 37°C 24 h before challenge with 20 µM cisplatinor 500 µM hydrogen peroxide (H2O2) for the indicated times.LDH release was quantified per the manufacturer’s protocol.
Agarose Gel Electrophoresis for DNA Fragmentation
DNA fragmentation was performed as described previously (22).Briefly, control and treated cells from 100-mm plates were collectedby scraping and were centrifuged at 1000 x g for 5 min at roomtemperature. Cell pellets were resuspended in 50 µl ofTris-ethylenediamine- tetraacetic acid (TE) (10 mM Tris, 1 mMethylenediaminetetraacetic acid, pH 8.0), followed by the additionof 900 µl of lysis buffer (TE plus 0.5% Triton X-100)and incubation on ice for 15 min. The samples were centrifugedat 10,000 x g for 10 min, and supernatants were treated with50 µg/ml ribonuclease A for 1 h at 37°C. Next, 100µg/ml proteinase K and 0.5% sodium dodecyl sulfate wereadded and allowed to incubate for 2 h at 50°C. FragmentedDNA was extracted by using equal volumes of phenol/chloroformand ethanol precipitation. The nucleic acid pellets were resuspendedin 30 µl of TE and subjected to 2% agarose gel electrophoresisat 100 V for 1 h.
Measurement of Cell Size
Measurements of cell size after cisplatin treatment were assessedby changes in forward-angle light scatter by using a fluorescence-activatedcell sorter (FACScan; Becton Dickinson, San Jose, CA) and CellQuestsoftware version 3.3 (Becton Dickinson, San Jose, CA). Forward-anglelight scatter is directly proportional to cellular diameter,and a decrease in light scatter is indicative of a reductionin cell size (23). Therefore, normal versus apoptotic cellscan be distinguished by this method. Data are presented as themean differences in cells exhibiting less forward-angle lightscatter (smaller size) between the control and treated cellsin each of the cell lines.
Annexin V Binding Assay
Annexin V binding assays were performed by using the apoptosisdetection kit (R & D Systems) per the manufacturer’sprotocol for analysis of adherent cells. Briefly, cell lineswere split into 6-well trays 24 h before incubation with 20µM cisplatin for 5 d. Both floating and adherent cells(removed with a 0.025% trypsin treatment) were collected andpelleted by centrifugation at 500 x g for 5 min. Cell pelletswere resuspended in complete media to allow for recovery fromthe trypsinization. The concentration of trypsin used does notinterfere with the annexin V assay, as shown by negative stainingof control cells. Cells were pelleted, resuspended in ice-coldPBS, and recentrifuged at 500 x g for 5 min. Control and treatedcells were then suspended in binding buffer that contained annexinV-FITC for 15 min. Unbound annexin V-FITC was removed by centrifugation,and the cells were resuspended in excess binding buffer. Ineach sample, a minimum of 10,000 cells were subjected to fluorescence-activatedcell sorting with a Becton Dickinson Biosciences FACScan andCellQuest software version 3.3.
Clonogenic Assay
To evaluate the ability of the vector, mito-Cat, and MnSOD celllines to survive and proliferate after a cisplatin challenge,equal numbers of cells were plated into 35-mm dishes 24 h beforeincubation with PBS (control) or 20 µM cisplatin. Twenty-fourhours later, cells were washed with PBS, trypsinized, and seededat a density of 5000 cells per 150-mm dish. Cells were maintainedin culture for 7 d in room air and 10% CO2 at 37°C. Thedishes were then washed with PBS, fixed with cold methanol,and stained with a 0.2% crystal violet solution. Colonies greaterthan 0.5 mm in size were counted by using a dissecting microscope.The clonogenic capability of each cell line is presented asthe percentage of colonies formed in treated versus controlcells.
Statistical Analyses
Data are presented as mean ± SEM. The t test was usedfor comparisons between two groups. For comparisons that involvedmore than two groups, we used ANOVA and the Newman-Keuls test.Significance is defined as P < 0.05.
Overexpression of MnSOD and Mito-Cat
In an attempt to explore the mechanisms of cisplatin-mediatedcytotoxicity in renal epithelial cells, we generated cell linesthat overexpressed human MnSOD by using the MnSOD expressionvector that is depicted in Figure 1A. To differentiate the exogenouslyderived transcript from the endogenous mRNA, we designed thepcDNA3.1/SOD construct to include only 853 nucleotides of thehuman MnSOD cDNA. Northern blot analysis of cells transientlytransfected with vector alone or pcDNA3.1/SOD demonstrates overexpressionof an approximately 1.2-Kb transcript (Figure 1A). Clonal cellpopulations of vector alone and pcDNA3.1/SOD were selected,and overexpression of MnSOD protein was confirmed by immunoblotanalysis (Figure 1B). Vector clone #2 and pcDNA3.1/SOD#5 werechosen for subsequent experiments.
Previous work has suggested that mitochondrial dysfunction isan early event in cisplatin-induced renal tubular toxicity andthat it is potentially mediated by the generation of ROS (4,17).We therefore chose to overexpress another antioxidant enzyme,catalase, with specific targeting to the mitochondrion. Therationale for developing the mito-Cat cell line was providedby the studies of Bai et al. (19), in which cells expressinga mito-Cat displayed increased resistance to oxidant-mediatedapoptosis as compared with cytosolic expression of the enzyme.In addition, the mito-Cat cells provided an additional toolto address the ROS specificity and consequently the cellularmechanism of cisplatin-mediated cytotoxicity. Immunoblot analysisafter clonal selection demonstrates overexpression of catalase(Figure 2B). Mito-Cat clone #3 was chosen for subsequent experiments.The subcellular localization of catalase in the mito-Cat cellline was confirmed by immunoblot analysis, which showed a significantenrichment of catalase in the mitochondrial compartment (Figure 2C).
Cytoprotective Effects of Mito-Cat in H2O2-Induced Cell Injury
To assess the functionality of the cells that overexpress mito-Cat,we challenged both vector and mito-Cat cells with 500 µMH2O2. As shown in Figure 3, cells that overexpressed catalasedemonstrated significant cytoprotection as assessed by phasecontrast microscopy (A) and LDH release (B) when compared withvector. In contrast, overexpression of MnSOD did not confercytoprotection to H2O2-induced cell injury (data not shown),as reported previously (24).
Figure 3. Functional assessment of mito-Cat cells. (A) Phase contrast microscopy of vector alone and mito-Cat after exposure to 500 µM H2O2 for 48 h. Magnification, x25. (B) Lactate dehydrogenase (LDH) release of vector alone and mito-Cat exposed to 500 µM H2O2 for the indicated times.
Overexpression of MnSOD but Not Catalase Is Cytoprotective in Cisplatin-Induced Injury
To explore the effects of MnSOD and catalase overexpressionin cisplatin-induced cell injury, we exposed vector alone, MnSOD,and mito-Cat cell lines to 20 µM cisplatin for 72 h, anexposure that primarily caused an apoptotic form of cell injury.As shown in Figure 4, significant cytotoxicity, as evidencedby cell rounding, detachment, and decrease in cell size, wasobserved in the vector and mito-Cat clones, whereas no visibletoxicity was evident in the cells that overexpressed MnSOD (Figure 4, D through F).Both floating and adherent cells excluded trypanblue after the 72-h exposure to cisplatin. In addition, no significantincrease in LDH release was observed in vector, MnSOD, and mito-Catcell lines that were exposed to cisplatin.
Figure 4. Cell morphology after exposure to cisplatin. Phase contrast microscopy of vector alone (A and D), mito-Cat (B and E), and MnSOD (C and F) challenged with either phospate-buffered saline (control) (A through C) or 20 µM cisplatin for 72 h (D through F). The insets in D through F represent higher magnification (x60) images of each treated cell line. Magnification, x25 for A through F.
To further evaluate the cytoprotective potential of MnSOD overexpression,we compared the extent of DNA fragmentation after exposure to20 µM cisplatin for up to 24 h. We demonstrate significantoligonucleosomal fragmentation of DNA in the vector and mito-Catoverexpression cell lines at 24 h after cisplatin treatmentconsistent with a predominantly apoptotic form of cell deathin our studies (Figure 5). Significantly less DNA ladderingwas observed in the cell line that overexpressed MnSOD, substantiatingthe cytoprotective effects of MnSOD expression.
Figure 5. DNA electrophoresis of cells exposed to cisplatin. Confluent monolayers of vector, mito-Cat, and MnSOD cells were exposed to 20 µM cisplatin for the indicated times, and gel electrophoresis for DNA fragmentation was carried out as described in Materials and Methods. A 100-bp DNA ladder was used as a marker.
In an effort to quantify cell injury, we employed two methodsof flow cytometric analysis, forward-angle light scatter measurementsand the annexin V binding assay. As further evidence of theapoptotic nature of cell injury, a significantly higher percentageof cells with decreased forward-angle light scatter were observedin vector cells and cells that expressed mito-Cat as comparedwith cells that overexpressed MnSOD (vector, 15.94 ±2.17; mito-Cat, 15.89 ± 7.34; MnSOD; 5.40 ± 3.88;P < 0.05; n = 4).
In early stages of cell injury, phosphatidylserine residues,normally located on the inner leaflet of the membrane bilayer,flip to the outer leaflet. The appearance of these flipped lipidmoieties can be quantified by fluorescence-activated cell sorteranalysis after incubation with annexin V-FITC in the presenceof calcium. In these experiments, cells were exposed to 20 µMcisplatin for 5 d followed by assessment of annexin V binding.Figure 6 is a representative histogram that demonstrates theappearance of a higher number of annexin V-positive cells inboth vector and mito-Cat cell lines that are indicative of increasedcell injury. Interestingly, cells that overexpressed MnSOD didnot demonstrate an increase in annexin V binding.
Figure 6. Annexin V binding in control and cisplatin-treated cells. A representative result of fluorescence-activated cell sorter (FACS; Becton Dickinson, San Jose, CA) analyses after annexin V binding of vector alone, mito-Cat, and MnSOD challenged with 20 µM cisplatin for 5 d. The top row of panels represents untreated cells (control), and the bottom row represents cells that were exposed to cisplatin. The x-axis shows increasing annexin V-FITC labeling, and the y-axis shows the percent cell count. Each panel results from a total of 10,000 input cells.
A quantitative representation that summarizes the results ofthe annexin V binding is shown in Figure 7. Cell injury as assessedby annexin V binding was significantly decreased in the cellsthat expressed MnSOD compared with vector alone (vector, 15.84± 5.12; MnSOD, 0.84 ± 1.42; P < 0.05) (Figure 7A).Similar cell injury was observed in vector alone and mito-Catcells, suggesting the inability of catalase to protect renalepithelial cells against cisplatin (vector, 20.79 ± 3.53;mito-Cat, 19.42 ± 5.89; P = NS) (Figure 7B).
Figure 7. Quantification of annexin V binding. Percentage cell death is shown as increasing annexin V binding in treated and control samples. Results represent four independent experiments performed each time in triplicate. The results are depicted as mean ± SEM, and the statistical significance is indicated.
To assess the growth potential of the vector, mito-Cat, andMnSOD cell lines after a 24-h, 20 µM cisplatin challenge,a clonogenic assay was performed. No differences in growth potentialwere observed in each of the control (untreated) cell lines.However, significant differences were observed in the abilityof cisplatin-treated cells to form colonies. Cells that overexpressedMnSOD showed significant clonogenic capability after cisplatincompared with vector and mito-Cat cell lines (vector, 0.47 ±0.25; mito-Cat, 2.86 ± 0.64; MnSOD, 51.84 ± 2.38;n = 4; P < 0.001) (Figure 8), demonstrating the ability ofMnSOD overexpression to abrogate cisplatin-mediated cytotoxicity.
Figure 8. Clonogenic potential of cells after exposure to cisplatin. Vector, mito-Cat, and MnSOD overexpressing cell lines were treated with 20 µM cisplatin for 24 h and assessed for their growth potential by using a clonogenic assay as described in Materials and Methods. Data are represented as the percent of colony formation from treated versus control cells from each respective group. The results represent three independent experiments performed each time in quadruplicate and are depicted as mean ± SEM. *Statistically different from vector and mito-Cat cell lines (P < 0.001, ANOVA).
The results presented in this work demonstrate the specificityof ROS generation in response to cisplatin and directly implicatethe superoxide radical, and not H2O2, as the mediator of cisplatin-inducedrenal cell injury.
A significant risk of dose-dependent nephrotoxicity exists forpatients who receive cisplatin for cancer chemotherapy. Severallines of evidence support a role for ROS in the pathogenesisof cisplatin-induced renal injury (8,9,18). In vitro assayshave documented the generation of superoxide radicals duringinteraction of cisplatin with DNA (25), a phenomenon that hasbeen implicated as the mechanism of cisplatin-mediated toxicityin cancer cells (12). In addition, a variety of oxygen radicalscavengers have been shown to protect against cisplatin-mediatedcytotoxicity in vitro and in vivo, further substantiating therole of ROS (7–12). The majority of these studies haveinvolved the addition of antioxidants in the form of reconstitutedprotein (SOD and catalase) to cells in culture (9). In thiswork, we report the selective and targeted overexpression oftwo antioxidant enzymes, MnSOD and mito-Cat, in renal epithelialcells. Phenotypic characterization of vector alone, MnSOD, andmito-Cat cell lines demonstrated the ability of overexpressedMnSOD to protect HEK 293 cells against an apoptotic challengeof cisplatin, and catalase, targeted to the mitochondrion (theorganelle where cisplatin-mediated injury is prominent), didnot afford cytoprotection. These data substantiate the specificityof MnSOD and indicate a significant role for the superoxideradical in cisplatin-induced nephrotoxicity.
The mechanism of cisplatin-mediated cell death, although notyet completely elucidated, is probably pleiotropic. Recently,Lieberthal et al. (9) demonstrated differences in proximal tubulecell phenotype in response to varying cisplatin concentrations.Low doses (<50 µM) of cisplatin resulted in apoptosis,but cells challenged with higher concentrations (50 to 800 µM)displayed a more necrotic phenotype with cytosolic swellingand an early loss of plasma membrane integrity. These resultsare not surprising, because the concentration-dependent mechanismof cisplatin-mediated cell death is similar to that observedwith other cytotoxic compounds (6,9). Our data support thesestudies in that a low dose of cisplatin (20 µM) resultedprimarily in apoptosis as evidenced by DNA laddering, decreasedcell size, the exclusion of trypan blue, lack of LDH release,and annexin V-positive labeling. In addition, the ability ofcells to proliferate after exposure to cisplatin was demonstratedby significantly higher colony formation in the cells that overexpressedMnSOD compared with the vector cell line or the cells that expressedmito-Cat.
The functionality of the mito-Cat cells was demonstrated bythe fact that these cells were resistant to H2O2 but showedno cytoprotection with cisplatin in our studies. Previous studiesby Bai et al. (19) have demonstrated the beneficial effectsof catalase overexpression in either the mitochondria or cytoplasmafter exposure to H2O2 and menadione. In contrast, overexpressionof catalase within the cytosol or mitochondrial compartmentsof Hep G2 cells was shown to exacerbate tumor necrosis factor(TNF)-mediated cell injury (26). We have not documented a significantincrease in cisplatin-mediated toxicity in the mito-Cat celllines as compared with vector alone; therefore, the mechanismof cisplatin-mediated cell death may involve a different pathwaythan that of TNF. Nonetheless, MnSOD provides protection againstboth cisplatin (this study) and TNF-mediated cytotoxicity (15).
Similar to these studies, St. Clair et al. (27) have shown thatoverexpression of MnSOD attenuates the toxic effects of paraquatwith no affect on endogenous copper/zinc SOD, catalase, or glutathioneperoxidase expression. These results are consistent with ourobservations with cisplatin in that overexpression of MnSODalso demonstrated the role of superoxide radicals in paraquatcytotoxicity. Furthermore, studies in mouse proximal tubularcells have demonstrated that antioxidants, including SOD andcatalase, protect these cells from ROS in apoptosis that isinduced by either growth factor deprivation (28) or cisplatin(9). Regarding a cellular mechanism that is associated withMnSOD cytoprotection, Manna et al. (29) have reported that MnSODblocks TNF-mediated activation of the transcription factors,NF-B and AP-1, as well as the induction of c-Jun protein kinaseand mitogen-activated protein kinase kinase. These investigatorsalso showed that MnSOD overexpression suppresses apoptosis thatis induced by okadaic acid, H2O2, and Taxol (Bristol-Myers Squibb)but not by the chemotherapeutic agents, vincristine, vinblastine,and daunomycin. This further demonstrates the unique specificityof MnSOD overepxression in cisplatin-mediated cytotoxicity inrenal epithelial cell injury.
In summary, we have demonstrated the ability of MnSOD to protectrenal epithelial cells against cisplatin, whereas cells thatoverexpress mito-Cat do not afford cytoprotection. Our resultsstrongly implicate the superoxide radical in cisplatin-mediatednephrotoxicity and provide the impetus for potential targetedgene therapy with MnSOD in high-risk settings of acute renalfailure.
Acknowledgments
We are grateful to Drs. J. Andres Melendez and Ana M. Rodriguez(Center for Immunology and Microbial Disease, Albany MedicalCollege, Albany, NY) for providing the pSVZEOmspCAT expressionplasmid. This work was supported by grants from the NationalInstitutes of Health (NIH), K08 DK02446 (to AA) and HL39593(to HSN). CD was supported by an NIH Tumor Biology traininggrant. We thank Vijayalakshmi Thamilselvan and Jorge E. Garciafor excellent technical assistance in these studies. This workwas presented in part at the meeting of the American Societyof Nephrology, Toronto, Canada, October, 2000 and appears inabstract form in J Am Soc Nephrol 11: 599A, 2000.
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Received for publication March 14, 2001.
Accepted for publication June 13, 2001.
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Abstract
Introduction
B and AP-1, as well as the induction of c-Jun protein kinase and mitogen-activated protein kinase kinase. These investigators also showed that MnSOD overexpression suppresses apoptosis that is induced by okadaic acid, H2O2, and Taxol (Bristol-Myers Squibb) but not by the chemotherapeutic agents, vincristine, vinblastine, and daunomycin. This further demonstrates the unique specificity of MnSOD overepxression in cisplatin-mediated cytotoxicity in renal epithelial cell injury. 
