Antioxidant ameliorates cisplatin-induced renal tubular cell death through inhib - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:42 |只看该作者 |倒序浏览 |打印

作者：Kazuhiko Tsuruya, Masanori Tokumoto, Toshiharu Ninomiya, Makoto Hirakawa, Kohsuke Masutani, Masatomo Taniguchi, Kyoichi Fukuda, Hidetoshi Kanai, Hideki Hirakata, and Mitsuo Iida作者单位：Department of Medicine and Clinical Science, Graduate School of MedicalSciences, Kyushu University, Fukuoka 812-858 Japan ! L+ |2 ?; C! s, d
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      【摘要】
   We have recently demonstrated the direct involvement of the deathreceptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell(RTC) death. Reactive oxygen species are thought to be a major cause ofcellular damage in such injury. The aim of this study was to examine themechanism through which antioxidants ameliorate cisplatin-induced RTC death, with special emphasis on death receptor-mediated apoptotic pathways. Cisplatinwas added to cultures of normal rat kidney (NRK52E) cells or injected in rats.NRK52E cells and rats were also treated with dimethylthiourea (DMTU), ahydroxyl radical scavenger. We then examined the mRNA levels of death ligandsand receptors, caspase-8 activity, cell viability, cell death, renal function, and histological alterations. RT-PCR indicated cisplatin-induced upregulationof Fas, Fas ligand, and TNF- mRNAs and complete inhibition by DMTU invitro and in vivo. Cisplatin increased caspase-8 activity of NRK52E cells, andDMTU prevented such activation. Exposure to cisplatin reduced viability ofNRK52E cells, examined by WST-1 assay, and increased apoptosis and necrosis ofthe cells, examined by terminal deoxynucleotidyl transferase dUTP nick-endlabeling assay and fluorescence-activated cell sorter analysis. DMTU abrogatedcisplatin-induced changes in cell viability and apoptosis and/or necrosis.Cisplatin-induced renal dysfunction and histological damage were alsoprevented by DMTU. DMTU did not hinder cisplatin incorporation into RTCs. Ourresults suggest that antioxidants can ameliorate cisplatin-induced acute renalfailure through inactivation of the death receptor-mediated apoptoticpathways. " _2 i2 L7 s8 w) N4 Y* r4 M
      【关键词】 Fas tumor necrosis factor receptor reactive oxygen species apoptosis necrosis, z! m" S4 h6 q! y8 q
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CISPLATIN IS ONE OF THE MOST potent anticancer drugs used in thetreatment of solid tumors( 46 ). The cytotoxicity ofcisplatin is considered to be due to a combination of factors, including peroxidation of the cell membrane( 52 ), mitochondrialdysfunction ( 8 ), inhibition ofprotein synthesis ( 32 ), andDNA injury ( 27 ). However, themost common adverse effect limiting the use of cisplatin is nephrotoxicitythat develops primarily in the S3 segment of the proximal tubule( 4 ). Although reactive oxygenspecies (ROS) have been considered to play a central role in this injury( 30, 35, 36 ), the exact roles of free radicals and the mechanisms underlying the beneficial effects of free radicalscavengers have not been fully evaluated.: b, E' h- N2 E/ x$ H' d8 ?

On the other hand, cellular damage during acute renal failure (ARF) inducedby ischemia-reperfusion injury or nephrotoxic agents in the kidney isconsidered to consist mainly of proximal tubular necrosis( 56 ). Recent studies havedemonstrated that apoptosis, a form of cell death characterized by DNAfragmentation, as well as necrosis might play a crucial role in thepathogenesis of ARF ( 15, 43, 50 ). A number of proteinsystems regulate the apoptotic events. One of these systems operates through the TNF receptor family, including Fas antigen (Fas, CD95, APO-1) and TNFreceptor 1 (TNFR1). Both Fas and TNFR1 contain conserved death domains intheir cytoplasmic tails, which mediate defined protein-protein interactions( 24, 54 ), allowing recruitment ofother death domain-containing proteins such as Fas-associated death domainprotein (FADD). The association of FADD with Fas or TNFR1 results inrecruitment of caspase-8, activation of which consequently leads to cell death( 6, 41 ). Although a number ofreports have documented Fas expression on renal tubular cells (RTCs) andupregulation of Fas expression during acute and chronic renal failure( 7, 16, 19, 26, 43, 45, 47 ), whether RTC apoptosisunder those conditions depends on the FasFas ligand (FasL) pathway remainscontroversial. Information regarding the involvement of TNFR1 in apoptosis ofRTC is limited and still preliminary( 12, 18, 38 ). Recently, we investigated the involvement of the death receptor-mediated apoptotic pathways in thepathogenesis of cisplatin-induced ARF and demonstrated that ablation of Fas orTNFR1 protects RTCs against cisplatin toxicity( 57 ).

Although ROS-mediated damage has been commonly considered as necrosis,recent observations suggest the potential role of apoptosis under theseconditions. Thus excessive formation of ROS as well as depletion of cellularantioxidants resulted in apoptosis of different cell types( 21, 49, 51 ). With regard to theassociation between ROS and death receptor-mediated apoptotic pathways, recentstudies have shown that ROS modulate the expression of Fas or FasL in variouscells, such as endothelial cells( 53 ), hepatoma cells( 22 ), microglial cells( 59 ), and astrocytoma cells( 31 ). Therefore, it is thoughtthat ROS may influence apoptotic cell death by regulating the expression ofreceptors and ligands involved in the cell death process.+ ]0 C* E$ [. r+ ?5 s: H1 {% b2 u

Thus the involvement of ROS and the death receptor-mediated apoptoticpathways in cisplatin-induced RTC death is evident; however, little is knownabout the association of ROS and these pathways in RTC death during ARF. Basedon these findings, we postulated that ROS might induce RTC death through theactivation of death receptor-mediated apoptotic pathways in cisplatin-induced RTC death. In this study, we examined gene expressions of the death receptorsand ligands and activity of caspase-8 after cisplatin administration with orwithout a ROS scavenger. Our results suggest that the mechanism through whichantioxidants ameliorate cisplatin-induced ARF may involve inactivation of thedeath receptor-mediated apoptotic pathways.
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MATERIALS AND METHODS7 ?) r9 p1 a6 ~: X- E' u2 m2 a1 q

In Vitro Studies
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Cell culture. NRK52E cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI-1640 medium(GIBCO BRL, Grand Island, NY) containing 1% fetal bovine serum (GIBCO BRL) andwere subcultured using a 0.05% trypsin-EDTA solution consisting of (in mM) 137NaCl, 5.4 KCl, 5.5 glucose, 4 NaHCO 3, 0.5 EDTA, and 5 HEPES, pH 7.2(GIBCO BRL). The cells were incubated with various concentrations of cisplatinor vehicle (distilled water) for 24, 48, or 72 h with vehicle (distilled water) or dimethylthiourea (DMTU), a hydroxyl radical scavenger, and used invarious analyses such as caspase-8 activity assay, cell viability assay,fluorescence-activated cell sorter (FACS) analysis, and terminaldeoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.  Y- |! d+ {. ~

RT-PCR. Total RNA was extracted from NRK52E cells using the TRIzolmethod according to the protocol recommended by the manufacturer (GIBCO BRL).Equal amounts (2.0 µg) of DNA-free total RNA from each sample wereconverted to cDNA using 200 U of SuperScript II RT (GIBCO) with 500 ng ofoligo(dT) 12-18 primer (GIBCO), 0.5 mM of each dNTP (Promega,Madison, WI), and 40 U of RNasin (Promega) in a 40-µl reaction volume. Reverse transcription was performed at 22°C for 10 min, at 42°C for 45min, and at 95°C for 5 min. The reaction products (2.0 µl) weresubjected to PCR amplification using 1.25 U of Taq DNA polymerase(Promega) in a 50-µl reaction volume with 0.4 µM of each dNTP, 0.4 µM of each specific primer, and 2 mM MgCl 2. PCR was performed using the PerkinElmer (Foster City, CA) thermal cycler according to the instructionsprovided by the manufacturer. The published sequences of the rat and mouseFas, FasL, TNF-, TNFR1, and GAPDH mRNA were retrieved from the GenBankdatabase, and primer pairs for each were designed with oligonucleotidesoftware (Oligo Primer Analysis Software, version 5.0; National Biosciences, Plymouth, MN). Primer sequences and PCR conditions are shown in Table 1. We determined thenumber of cycles for PCR of each gene by checking whether the amount of PCRproduct increased linearly in proportion to the increase in number of cycles and was directly proportional to the number of cycles in the exponential rangeof amplification. The resulting amplification products were sequenced, and theidentities of the fragments were confirmed. Equal volumes of the amplificationproducts were analyzed by agarose gel (1.5%) electrophoresis with ethidium bromide (0.5 mg/ml) staining.& C2 s- h( e( f  b
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Table 1. Sequences of upstream and downstream rat primers for RT-PCRanalysis% q0 A: U: d, ~6 }( d! b  x
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Activity of caspase-8. NRK52E cells were exposed for 24, 48, or 72h to 8 µM cisplatin or vehicle in the presence of vehicle or 10 mM DMTU.Caspase-8 activity was then measured in these cells using a ApoAlert Caspase-8Colorimetric Assay Kit according to the protocol recommended by themanufacturer (Clontech, La Jolla, CA).
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Cell viability assay. Cell viability was determined by the WST-1 assay, as described by Kawahara et al.( 25 ). In brief, NRK52E cells(5 x 10 4 cells in 100 µl) were treated with various concentrations of cisplatin or vehicle for 72 h with various concentrations(0.1, 0.5, 2.0, or 10 mM) of DMTU, which was administered simultaneously withor 4, 8, 12, or 24 h after the initiation of cisplatin treatment. WST-1reagents,2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (Dojin Laboratories, Kumamoto, Japan), and 1-methoxy-5-methylphenazinium methylsulfate (Dojin Laboratories), were addedto the cells at final concentrations of 5.0 and 0.2 mM, respectively, andincubated for 1 h at 37°C. Using an enzyme-linked immunoadsorbent assayautoreader, the cell viability was determined by measuring the differencebetween absorbance at 450 and 620 nm., W: D2 u$ a8 I3 m% x# }$ l# ^

FACS analysis. NRK52E cells were cultured with 8 µM cisplatin or vehicle for 72 h with vehicle or 10 mM DMTU. Nonattached cells in themedium were collected, attached cells were trypsinized and washed, and bothcell populations were incubated with FITC-conjugated annexin V and propidiumiodide (PI) in binding buffer at concentrations suggested by the manufacturer(PharMingen, San Diego, CA) on ice in the dark for 15 min. Stained cells wereanalyzed on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data were analyzed using CELL-QUEST software (Becton Dickinson).: u7 O# u* t. F9 S7 E  ?( {3 V

TUNEL assay. The TUNEL technique (ApopTag Plus kit, Intergen, Gaithersburg, MD) was used to detect DNA fragmentation of the cells in situaccording to the instructions provided by the manufacturer. Briefly, adherentcultured cells were fixed in 1% paraformaldehyde for 10 min at roomtemperature and ethanol for 5 min at -20°C, and endogenousperoxidase was quenched with 3.0% H 2 O 2 for 5 min. Thesections were incubated with terminal deoxynucleotidyl transferase (TdT) and amixture of digoxigenin-labeled nucleotides for 60 min. This was followed byincubation with anti-digoxigenin-peroxidase for 30 min and color developmentwith H 2 O 2 -diaminobenzidine for 3-6 min. Then, theslides were counterstained with hematoxylin. For positive controls, specimensof thyroid tissue were provided by Intergen. Negative controls were preparedby omission of terminal deoxynucleotidyl transferase enzyme from theincubation buffers.

In Vivo Studies; b4 p! n5 l# z% @6 `

Rat model of cisplatin-induced ARF. Male Sprague-Dawley rats weighing 200-250 g were obtained from Kyudo (Kumamoto, Japan). Animalswere housed individually in standard laboratory cages and treated with theprinciples outlined in the Guide for the Care and Use of LaboratoryAnimals (National Academy Press, 1986). Cisplatin was administered by asingle intravenous injection to rats at 8 mg/kg body wt. In addition to thesingle cisplatin injection, DMTU was intraperitoneally injected at 500 mg (4.8mmol)/kg body wt 1 h before the injection of cisplatin, followed byintraperitoneal injection of 125 mg (1.2 mmol)/kg body wt twice a day (CP DMTUgroup). DMTU was substituted by saline in the other rats (CP group). Theserats were killed before (control group; n = 6 rats) or 6 or 12 h( n = 4 rats/group) or 1, 2, 3, or 5 days ( n = 6 rats/group)after cisplatin injection by exsanguination, blood samples were centrifuged(3,500 g for 5 min), and plasma samples were collected formeasurement of plasma blood urea nitrogen (BUN) and creatinine (Cr). The kidneys were perfused in situ via the aorta with PBS, pH 7.4, and then excisedfor further analysis., _2 }9 U' R8 T& M! Y3 b1 E
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Measurement of plasma BUN and Cr. Plasma BUN and Cr levels were measured using the urease-UV and the alkaline picrate method, respectively.Measurements were conducted by SRL Laboratories (Tokyo, Japan).. T, N, S$ ]' v% G  p+ @* P( l
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Morphological assessment. Excised kidneys were immersed in Bouin's solution and fixed for 12-24 h, embedded in paraffin, and 3-µmsections were mounted on silane-coated glass slides. After deparaffinizationand rehydration, the tissues were stained with periodic acid-Schiff, and thedegree of morphological involvement in renal failure was determined usinglight microscopy, as described by Megyesi et al.( 37 ) with some modifications. Briefly, the following parameters were chosen as indicative of morphologicaldamage to the kidney after cisplatin injection: brush border loss, tubuledilatation, tubule degeneration, tubule necrosis, and tubular cast formation.These parameters were evaluated on a scale of 0-4, which ranged fromabsent (0), mild (1), moderate (2), severe (3), to very severe (4). Eachparameter was determined in six different rats.( O2 V* C2 Q! [' `, g

RT-PCR. Total RNA was extracted by the TRIzol method from rat kidneys according to the protocol provided by the manufacturer, followed bythe manipulations described above in the in vitro study. Equal volumes of theamplification products were analyzed by agarose gel (1.5%) electrophoresiswith ethidium bromide (0.5 mg/ml) staining. Gels were photographed andanalyzed using the public domain National Institutes of Health (NIH) Image program( http://rsb.info.nih.gov/nih-image/ ).The results were normalized to the intensity of GAPDH bands.0 g9 D# j6 j; J! A/ H

Platinum contents of NRK52E cells and rat kidneys. Renal tissue was weighed and digested with 5 ml of 16 M HNO 3 overnight at roomtemperature and then heated in a heating block for 4-5 h at 140°C.The digested sample was adjusted to 10 ml with 0.1 M HCl. The cultured cellswere removed by scraping with a plastic scraper in ice-cold PBS andcentrifuged at 1,000 g for 5 min. The pellet was resuspended with PBSand disrupted by sonication. After centrifugation at 13,400 g for 5min, the protein content of the supernatant was determined using the Lowrymethod ( 34 ). The platinumcontents in the samples were measured using a spectrometer (model 1100flameless atomic absorption spectrometer, PerkinElmer, Norwalk, CT) monitoring at 265.9 nm. The latter measurements were conducted by SBS (Sagamihara, Japan).0 t- J; s7 a0 T& a
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Statistical analysis. Data are expressed as means ± SE. One-way ANOVA followed by Bonferroni's t -test was used to compare thevalues in the CP group with control values, and an unpaired t -testwas used to compare the values in the CP group with those of the CP DMTUgroup. One-way ANOVA followed by Bonferroni's t -test was used forcomparison between the same cisplatin concentrations for cell viability assaysand comparison between the same times after cisplatin administration forcaspase-8 assays. Statistical significance was accepted at P
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RESULTS1 Q1 y( |* g/ E

Effect of DMTU on Cisplatin-Induced Upregulation of FasL, Fas, andTNF- mRNA Expression in NRK52E Cells and Rat Kidneys1 k' \& ]/ r0 p3 }# k8 B
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In the in vitro study, low expression levels of FasL, Fas, and TNF- mRNAs were observed in NRK52E cells in the absence of cisplatin stimulation.The mRNA levels of all these genes increased in the cells after incubationwith 8 µM cisplatin for over 24 h and this increase was inhibited bytreatment with 10 mM DMTU. In contrast, the level of TNFR1 mRNA wassubstantial and constant irrespective of stimulation of cisplatin andtreatment with DMTU ( Fig.1 A ).

Fig. 1. Effects of cisplatin and dimethylthiourea (DMTU) on mRNA expression ofdeath receptors and their ligands in NRK52E cells and rat kidneys determinedby RT-PCR. A : representative data of Fas-Fas ligand (FasL), Fas,TNF-, and TNF receptor 1 (TNFR1) mRNA expression in NRK52E cells afterincubation with vehicle (control), 8 µM cisplatin (CP), or 8 µM and 10mM DMTU (CP DMTU) for the indicated time intervals. B : representativedata of FasL, Fas, TNF-, and TNFR1 mRNA expressions in control ratkidneys and in kidneys 6 h, 12 h, and 1 (1 d), 2 (2 d), 3 (3 d), and 5 days (5d) after CP. C : representative data of FasL, Fas, TNF-, andTNFR1 mRNA expression in control rat kidneys and in kidneys 3 and 5 days afterCP or CP DMTU. D : mean mRNA levels of these genes, quantified usingNational Institutes of Health Image software and normalized to the intensityof GAPDH bands, in kidneys before (C; open bars, n = 6) and 3 or 5days after CP (closed bars, n = 6) or CP DMTU (hatched bars, n = 6) are shown as a ratio of mean mRNA levels in control kidneys.Values are means ± SE. * P

In the in vivo study, low expression levels of Fas, FasL, and TNF- mRNAs were also observed in the kidneys of control rats. The mRNA levels ofthese genes were upregulated 12 h after cisplatin injection and peaked andwere maintained at a high level of expression from 1-5 days afterinjection ( Fig. 1 B ).Such an increase was inhibited by treatment with DMTU ( Fig. 1 C ). Similar tothe findings of the in vitro study, the TNFR1 mRNA level was substantial anddid not change after cisplatin injection irrespective of DMTU treatment( Fig. 1, B and C ). The signal densities were also quantified using theNIH Image program, and results were expressed relative to the intensity ofGAPDH bands. Mean mRNA levels of these genes in control kidneys and ininsulted kidneys 3 and 5 days after injection of cisplatin with or withoutDMTU are shown in Fig.1 D relative to the mean mRNA levels in controlkidneys.% `9 c+ I- S' }: B  p! R" Z
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Effect of DMTU on Cisplatin-Induced Increase in Caspase-8 Activity inNRK52E Cells

Incubation of NRK52E cells with 8 µM cisplatin for over 24 h resulted inaugmentation of caspase-8 activity. Caspase-8 activity was significantlyhigher in cisplatin-stimulated cells than in cells incubated with vehicle ateach time interval after 24 h. However, in cells incubated with 10 mM DMTU,only a slight increase was observed despite stimulation with cisplatin ( Fig. 2 ).

Fig. 2. Effects of cisplatin and DMTU on caspase-8 activity in NRK52E cells. Cellswere incubated as described in Fig.1 A for 24, 48, or 72 h. Caspase-8 activity is shown aspercent change from control cells, which were incubated with vehicle for 24 h.The experiments were performed in triplicate on 3 different occasions, andrepresentative data are shown. Values are means ± SE. ** P

In Vitro Effect of DMTU on Cisplatin-Induced RTC Apoptosis andNecrosis

A significant dose-dependent loss of cell viability was noted after 72-hincubation with various concentrations (2, 4, or 8 µM) of cisplatin. Theloss of cell viability was significantly inhibited by treatment with variousconcentrations (0.1, 0.5, 2, or 10 mM) of DMTU in a dose-dependent manner( Fig. 3 A ). Delayedtreatment with DMTU also provided protection against cisplatin-induced celldeath, although the protective effect became weaker with longer delays in thetreatment ( Fig.3 B ).
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Fig. 3. Effects of cisplatin and DMTU on viability of NRK52E cells determined byWST-1 assay. The results are expressed as the percentage of the value obtainedwithout cisplatin. The experiments were performed in triplicate on 3 differentoccasions, and the average values ± SE were plotted. A :dose-dependent killing of NRK52E cells by 72-h exposure to cisplatin and adose-dependent inhibition of the killing by DMTU, which was addedsimultaneously with cisplatin, are shown. B : time indicates delayedtime (h) of DMTU treatment after the initiation of cisplatin exposure. Notethe time-dependent attenuation of the effect of DMTU on inhibition of thekilling of NRK52E cells by 72-h exposure to 8 µM cisplatin. Values aremeans ± SE. * P   F# \+ F  h( T; I+ V
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To confirm that the loss of viable cells was due to an increased populationof nonviable cells, NRK52E cells, incubated with vehicle (control), 8 µMcisplatin and vehicle (CP), or 8 µM cisplatin and 10 mM DMTU (CP DMTU) for72 h, were stained with both PI and FITC-labeled annexin V and then analyzed by flow cytometry. PI can penetrate into necrotic or late apoptotic cells butnot viable or early apoptotic cells. Annexin V, a protein with high affinityfor phosphatidylserine, can bind to exposed phospholipids in apoptotic cells.Phosphatidylserine externalization is a feature of apoptosis induced byvarious drugs ( 28 ). In Fig. 4, the bottomleft cell population in each plot represents viable cells, which did notcontain PI-stained cells and annexin V-binding cells. The top left population comprises necrotic cells, representing cells stained for PI but notFITC-labeled annexin V. The bottom and top right populations correspond to apoptotic and late apoptotic cells, respectively. Compared withcells unstimulated by cisplatin, both annexin V-positive cells and PI-positivecells were increased after 72-h exposure to 8 µM cisplatin( Fig. 4, B and C ). Treatment with 10 mM DMTU markedly suppressed theincrease in annexin V-positive cells and PI-positive cells( Fig. 4 D ).
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Fig. 4. Effects of cisplatin and DMTU on renal tubular cell death determined byfluorescence-activated cell sorter analysis using annexin V-FITC and propidiumiodide. The experiments were performed in triplicate on 3 different occasions,and representative plots are shown. A : cells without staining wereconsidered as the negative control. Cells incubated with vehicle (control; B ), 8 µM cisplatin (CP; C ), or 8 µM cistplatin and 10mM DMTU (CP DMTU; D ) for 72 h were stained with FITC-conjugatedannexin V and propidium iodide, followed by analysis using a FACSCalibur flowcytometer. Percent data in top left, top right, and bottom right quadrants represent the proportion of necrotic cells andcells at late and early apoptosis, respectively.

DNA fragmentation determined by TUNEL assay was not seen in cells that werenot treated with cisplatin ( Fig.5 A ). After 72-h exposure to 8 µM cisplatin,TUNEL-positive nuclei were seen in the cells( Fig. 5 B ). Combinationtreatment of 10 mM DMTU and cisplatin prevented the cells from undergoing apoptosis ( Fig.5 C ).
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Fig. 5. Effects of cisplatin and DMTU on DNA fragmentation of NRK52E cells examinedby terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay. Brown staining indicates TUNEL-positive nuclei and bluestaining counterstained with hematoxylin indicates TUNEL-negative nuclei. Theexperiments were performed in triplicate on 3 different occasions, andrepresentative figures are shown. A : cells incubated with the vehicleonly. Note the lack of cell staining. B : cells incubated with 8 µMcisplatin for 72 h. Note the positively stained nuclei of apoptotic cells(arrows). C : cells incubated with 8 µM cisplatin and 10 mM DMTUfor 72 h. Note the lack of cell staining. Magnification, x 600.
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In Vivo Effect of DMTU on Cisplatin-Induced RTC Damage and RenalDysfunction- [# O, `  B$ w& a* _! q) ^+ [
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As shown in Fig. 6, plasmalevels of BUN and Cr were significantly increased after 3 and 5 days ofcisplatin injection, whereas treatment with DMTU significantly inhibited suchincreases in these parameters. Histological examination of the kidneys 3 daysafter cisplatin injection showed severe RTC damage such as brush border loss,degeneration, necrosis, and cast formation in the outer medulla (data notshown). The damage was more severe 5 days after the injection and wasassociated with tubular dilatation ( Fig.7 A ). These RTC injuries were also seen in the kidneys ofrats treated with DMTU but were less severe compared with rats that were nottreated with DMTU ( Fig.7 B ). Semiquantitative analyses of the histopathologicalchanges confirmed the aforementioned injury( Fig. 7 C ).Furthermore, tubular necrosis was observed not only in the S3 segment but alsoin S1 and S2 segments in the CP group, whereas such damage was restricted tothe S3 segment in the CP DMTU group.$ h$ @. v8 x' I  S

Fig. 6. Effects of cisplatin and DMTU on renal function of rats. Plasma levels ofblood urea nitrogen (BUN) and creatinine before (Con) and at 1, 2, 3, and 5days after injection of cisplatin with vehicle (CP) or CP DMTU. Values aremeans ± SE; n = 6 rats. * P

Fig. 7. Histopathological effects of cisplatin and DMTU on the rat kidney. A and B : representative sections from rat kidneys at 5 daysafter injection of cisplatin with vehicle ( A ) or with DMTU( B ). Magnification, x 200. C : quantitative evaluationof kidney damage at 5 days after injection expressed as relative severity on ascale from 0 to 4. Values are means ± SE values of sections from ratkidneys at 5 days after injection of cisplatin with vehicle (CP; n =6) or CP DMTU ( n = 6). Morphology was scored according to proximaltubule brush border loss (BB Loss), tubule dilation (Dilatation), tubuledegeneration (Degeneration), tubule necrosis (Necrosis), and cast formationwithin tubules (Casts). ## P - O0 v7 q3 T6 L# }$ @- E; T# l) @8 c

DMTU Does Not Alter Cisplatin Incorporation into RTCs

To exclude the possibility that DMTU might hinder cisplatin incorporationinto RTCs, we measured platinum levels in cultured RTCs treated with 8 µMcisplatin or 8 µM cisplatin plus 10 mM DMTU and kidneys of rats treatedwith 8 mg/kg cisplatin or cisplatin plus DMTU. After exposure to cisplatin,the platinum contents of both cultured RTCs and rat kidneys were similar irrespective of DMTU treatment, indicating that DMTU does not hinder cisplatinincorporation into RTCs ( Fig.8 ).
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Fig. 8. DMTU does not alter CP incorporation into renal tubular cells (RTCs).Platinum contents of NRK52E cells ( A ) treated with 8 µM cisplatin(CP; filled bars, n = 6) or 8 µM CP and 10 mM DMTU (CP DMTU;hatched bars, n = 6) and kidneys of rats ( B ) treated with 8mg/kg cisplatin (CP; filled bars, n = 6) or CP DMTU, (hatched bars, n = 6). Values are means ± SE. n.s., Not significant.+ G! l. B( P* U( j2 s* F5 e0 \  K

DISCUSSION) _0 y% `2 ]/ _  W

The major findings of the present study were 1) Fas, FasL, and TNF- mRNA levels and caspase-8 activity increased after cisplatin administration; 2) these cisplatin-induced changes were inhibited bytreatment with an antioxidant, DMTU; and 3) treatment with DMTUsuppressed cisplatin-induced apoptosis and necrosis of RTCs and preventedrenal dysfunction.
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DMTU is known as a powerful scavenger of hydroxyl radicals ( 61 ). Matsushima et al.( 36 ) demonstrated that DMTU isbeneficial in preventing the accumulation of malondialdehyde, tubular damage, and renal dysfunction through scavenging the hydroxyl radicals incisplatin-induced ARF. Later, the same group reported that the attenuation ofsuch injuries by treatment with DMTU was associated with less apoptotic celldeath ( 62 ). Similarly, ourdata revealed the beneficial effects of DMTU in the prevention of tubulardamage and renal dysfunction. We showed not only the protective effect butalso the therapeutic effect of DMTU; i.e., DMTU was also effective even whensuch treatment was delayed, although the protective effect became weaker with longer delays in treatment. These findings indicate that if there isinadvertent exposure to cisplatin, DMTU treatment should be consideredwhenever the treatment is delayed, although the effect is weak. We alsodemonstrated that such effects were associated with reductions in Fas, FasL,and TNF- mRNA levels and caspase-8 activity, which were increased bycisplatin. The involvement of ROS in death receptor-mediated apoptotic pathways has been reported in various cells recently( 22, 31, 53, 59 ); however, there is nodocumentation of such role in RTCs. Therefore, this is the first report thatshows the association between ROS and death receptor-mediated apoptotic pathways in RTCs.
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Although it has been well documented that apoptosis plays a crucial role inthe pathogenesis of cisplatin-induced ARF, intracellular pathways ofcisplatin-induced RTC apoptosis remain unknown. In this study, it is not clearwhether the effect of DMTU on the inhibition of RTC death is directly due toreduced levels of Fas, FasL, and TNF-. Therefore, one can argue that decreased expression levels of these genes by DMTU were merely secondaryphenomena related to amelioration of RTC injury. However, it has been reportedthat upregulation of Fas/FasL system is involved in ischemia- orcisplatin-induced apoptosis in rodent kidneys and cultured human proximaltubular cells ( 43, 45 ). We also recentlydemonstrated the direct involvement of the death receptor-mediated apoptoticpathways in the pathogenesis of cisplatin-induced RTC death, in which Fas - or TNFR1 -knockout mice were resistant to cisplatin( 57 ). In addition, we also showed in this study that cisplatin activated caspase-8 and DMTU suppressedsuch activation. On the basis of these findings, we consider thatdownregulation of Fas, FasL, and TNF- mRNAs by the antioxidant is not abyproduct but rather the underlying mechanism of action of DMTU in preventingcisplatin nephrotoxicity.
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If DMTU has a suppressive effect on cisplatin incorporation into RTCs, ourargument in this study becomes questionable. To exclude such a possibility, wemeasured platinum levels in cultured RTCs and rat kidneys treated withcisplatin with or without DMTU. After exposure to cisplatin, the platinum contents of both cultured RTCs and rat kidneys were similar irrespective ofDMTU treatment, indicating that DMTU does not hinder cisplatin incorporationinto RTCs. On the basis of these data, we can exclude the role of otheralternative pathways in mediating the effect of DMTU, ones through which DMTUinteracts with cisplatin in the incubation medium and the blood or peritoneum to form an inactive compound that limited cisplatin access to the cell.3 g. U/ Z4 {3 X' X, p4 u
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Recently, Choi et al. ( 13 )and Cummings and Schnellmann( 14 ) emphasized the role ofthe tumor-suppressor protein p53 in RTC apoptosis in chronic obstructiveuropathy and cisplatin-induced RTC apoptosis, respectively. Both reportssuggested that RTC apoptosis is mediated by p53-dependent and -independentpathways. p53 Can be a possible candidate as a transcriptional regulator ofFas. The involvement of p53 in initiating apoptosis after exposure to apleiotropic array of stimuli results in upregulation of proapoptotic membersof the bcl-2 family of proteins such as Bax or cell cycle inhibitor p21( 9 ). Recent data suggest thatp53-induced apoptosis occurs also through mechanisms other than Bax. Forexample, several laboratories have suggested the involvement of p53 inFas/FasL-induced apoptosis ( 3, 39, 40 ). On the other hand, thereare several lines of evidence indicating that p53 expression is increased byROS ( 42, 60 ). Thus it is possible thatcisplatin-induced upregulation of Fas in RTCs observed in this study might bemediated, in part, by p53, which could be activated by cisplatin-induced DNAdamage and ROS.
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On the other hand, Park et al.( 44 ) reported recently that cisplatin can induce RTC apoptosis via translocation of cytochrome c from the mitochondria to the cytosol and via activation and translocation ofthe proapoptotic molecule Bax. They also demonstrated a sixfold upregulationof caspase-9 activity by cisplatin, but not of caspase-8. Their resultsindicate the involvement of a mitochondrial pathway in cisplatin-induced RTCapoptosis in view of the perspective that activation of Bax can trigger asequence of events leading to alterations in mitochondrial permeabilitytransition, release of mitochondrial cytochrome c into the cytosol,and activation of caspase-9. It seems that our results contradict those of theabove study; however, we consider this not to be the case. That is, we suggestthat the death receptor-mediated pathway and mitochondrial pathway are bothinvolved in cisplatin-induced RTC apoptosis. However, with regard tocaspase-8, different results were observed between these two studies. Wesuspect that these different data are due to the use of differentmethodologies, i.e., different cells and different concentrations of cisplatinused. In particular, we believe that the concentration of cisplatin is ofcritical importance. In support of this argument, Lieberthal et al. ( 33 ) demonstrated that at ahigh concentration (800 µM), cisplatin caused necrotic cell death thatoccurred over a few hours, whereas at low concentration (8 µM) it resulted in apoptosis occurring over several days, after incubation of mouse proximaltubular cells with cisplatin. We used cisplatin at a low concentration,whereas Park et al. ( 44 ) usedmoderate concentrations of the drug (25-100 µM). On the basis ofthese findings, we suggest that low concentrations of cisplatin mainlyactivate the death receptor-mediated pathway, whereas moderate concentrationsmainly activate the mitochondrial pathway, although no direct evidence wasobtained in support of this conclusion. Alternatively, it is possible that themitochondrial pathway might be activated in part via the deathreceptor-mediated pathway. This is based on previous findings demonstratingthat activated caspase-8 propagates the apoptotic signal, as well as directlycleaving and activating downstream caspases, by cleaving the BH3Bcl2-interacting protein, which leads to the release of cytochrome c from mitochondria, triggering activation of caspase-9 in a complex with dATPand Apaf-1 ( 29 ). It is considered that activated caspase-9 then activates further "downstream caspases," including caspase-8, indicating that the death receptor pathway and mitochondrial pathway might in part be correlated with eachother.( g2 Q) L( I/ {/ D
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Hughes and Johnson ( 23 )demonstrated, using TUNEL assay, that the absence of functional Fas protectedRTC from undergoing apoptosis in the murine kidneys injured by unilateralureteric ligation and that such an effect was localized to the distal tubularcell compartment. In contrast, we recently demonstrated that the absence ofFas protected proximal tubular cells from undergoing apoptosis in the murinekidneys injured by cisplatin administration( 57 ). In this respect, Hughesand Johnson ( 23 ) argued thattheir results correlated with those of a previous study( 55 ), in which Fas was foundto be most strongly expressed in distal tubular epithelium in obstructedmurine kidneys. We also demonstrated using RT-PCR and FACS analysis thatcisplatin markedly induced Fas expression in cultured proximal tubular cells,and absence of Fas protected proximal tubular cells from undergoing apoptosisin the murine kidneys injured by cisplatin administration( 57 ). Thus Fas/FasL seems toplay a critical role in apoptosis of proximal tubular cells rather than distaltubular cells in cisplatin-induced RTC death. Therefore, it is considered thatour data are reasonable and do not contradict the results of Hughes andJohnson ( 23 ).

Whereas death receptor-mediated pathways can be activated by ROS, severalstudies have documented that such cascades can also generate ROS( 1, 2, 10, 11, 17, 20, 48, 58 ). That is, ROS can act assecond messengers during death receptor-mediated apoptosis( 5 ). Thus it is considered thatROS and death receptor-mediated apoptotic cascades can interactsynergistically with each other. We propose that such interaction provides thebest mechanism for the remarkable effect of antioxidants on cisplatin-induced RTC death.* U( T4 H9 ]1 M" B  h  Q/ s" X

In conclusion, we have demonstrated in the present study that treatmentwith DMTU, an antioxidant, inhibited upregulation of Fas, FasL, andTNF- mRNA expression and ameliorated cisplatin-induced RTC death andrenal dysfunction. Our findings suggest that ROS play a crucial role in thepathogenesis of RTC death through the activation of death receptor-mediatedapoptotic cascades during cisplatin-induced ARF.

ACKNOWLEDGMENTS0 c5 e* \- [/ ~! n
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We are grateful to H. Noguchi for technical assistance and Dr. F. G. Issafor editing the manuscript.+ m% X/ R. V" [9 ^: o2 I1 ~

Part of this study was conducted at the Morphology Core, Graduate School ofMedical Sciences, Kyushu University.9 u) z1 k. |( H8 Y
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