Renal Failure, 29:257–263, 2007 Copyright © Informa HealthcareISSN: 0886-022X print / 1525-6049 onlineDOI: 10.1080/08860220601166487

257

LRNFLABORATORY STUDY

Effects of Different Doses of Hyperbaric Oxygen on Cisplatin-Induced Nephrotoxicity

Hyperbaric Oxygen and Cisplatin NephrotoxicitySecil AydinozDepartment of Pediatrics, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Gunalp UzunDepartment of Undersea and Hyperbaric Medicine, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Hakan CermikDepartment of Pathology, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Enes Murat AtasoyuDepartment of Nephrology, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Senol YildizDepartment of Undersea and Hyperbaric Medicine, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Bulent KaragozDepartment of Oncology, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Rifki EvrenkayaDepartment of Nephrology, Gulhane Military Medical Academy, Haydarpasa Teaching Hospital, Uskudar, Istanbul, Turkey

Cisplatin, an effective antineoplastic agent, frequentlyinduces acute renal failure in animals and humans. Hyperbaricoxygen (HBO) has been shown to prevent cisplatin-inducednephrotoxicity in rats. This study investigated the effect of twodifferent HBO regimes on renal functions, oxidative stress,and histopathological changes in rat kidneys after cisplatintreatment. Wistar rats were divided into five groups: control,HBO, cisplatin, cisplatin plus once daily HBO, and cisplatin

plus twice daily HBO. Cisplatin was given as a single intrap-eritoneal dose of 6 mg/kg, and HBO was applied for 60 min at2.5 atm for six days. HBO alone did not alter any biochemicalparameters or histopathological findings compared with thecontrol group. Cisplatin increased serum urea and creatininelevels and caused severe histopathological injury. In addition,cisplatin increased lipid peroxidation and impaired superoxidedismutase (SOD) and glutathione peroxidase (GSH-Px) activi-ties in kidney tissue. Once daily HBO after cisplatin treatmentslightly reduced serum urea and creatinine levels and attenu-ated histopathological injury. HBO also reduced lipid peroxi-dation and increased SOD and GSH-Px activities significantly.Although twice daily HBO was determined to be more effec-tive than once daily HBO on oxidative stress parameters, itincreased serum creatinine levels and histopathological injurycompared with the cisplatin group. It was concluded that HBOalone does not induce nephrotoxicity and oxidative stress in ratkidneys; once daily HBO may prevent cisplatin-induced neph-rotoxicity, an effect that is partially mediated by the modification

This work was partially supported by a research grant fromGulhane Military Medical Academy.

Address correspondence to Dr. Secil Aydinoz, GATAHaydarpasa Egitim Hastanesi, Cocuk Hastaliklari Servisi, TibbiyeCd. 34668, Uskudar, Istanbul, Turkey; Tel.: +90-216-542 2462;Fax: +90-216-348 7880; E-mail: saydinoz@yahoo.com

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of oxidant/antioxidant systems in the kidneys; and twice dailyHBO potentiates cisplatin nephrotoxicity by a ROS-independentmechanism.

Keywords acute renal failure, cisplatin, nephrotoxicity, hyperbaricoxygenation, reactive oxygen species

INTRODUCTION

Cisplatin is an antineoplastic agent that is widely pre-scribed for a variety of solid tumors, such as lung, testis,ovary, bladder, head, and neck cancer.[1] There are seriousside effects associated with cisplatin use, including neph-rotoxicity, neurotoxicity, and bone marrow suppression.Nephrotoxicity is seen in about 20% of patients and fre-quently limits the clinical use of cisplatin.[2] Although theunderlying mechanisms are not clear, recent evidencesuggests that reactive oxygen species (ROS) and the renalantioxidant defense system are involved in cisplatin-induced nephrotoxicity.[3,4] Cisplatin is able to induce theformation of ROS and reduce the activity of antioxidantenzymes in renal tissue.[5–9]. The formation of excessivelevels of ROS can lead to lethal or sub-lethal cellulardamage.[10]

Hyperbaric oxygen (HBO) therapy involves the inspi-ration of 100% oxygen at a pressure higher than normalatmospheric pressure (atm).[11] HBO increases the amountof oxygen dissolved in arterial blood and leads to hyper-oxia even in poorly perfused tissues. HBO therapy is usedas a primary therapy in arterial gas embolism, severe car-bon monoxide poisoning, gas gangrene, and decompres-sion sickness.[11] It is also employed as an adjunctivetreatment in problem wounds, necrotizing soft tissue infec-tions, refractory osteomyelitis, osteoradionecrosis, crushinjury, compromised skin grafts and flaps, and thermalburns.[11] It was recently shown that once daily administra-tion of HBO (60 min at 2.5 atm for seven days) protects ratkidneys against cisplatin-induced nephrotoxicity.[12] How-ever, the mechanism of the beneficial effect of HBO is notknown. Although it seems controversial, HBO has beenshown to attenuate oxidative stress in different experimen-tal models, including acute pancreatitis, acute distal colitis,and liver regeneration.[13–15] It was hypothesized that HBOprevents cisplatin-induced nephrotoxicity by inducing anti-oxidant enzyme activities and inhibiting lipid peroxidation.

The present study investigated the effect of HBO onrenal functions, oxidative stress, and histopathologicalchanges in rat kidneys after cisplatin treatment. In addi-tion, the efficacy of once daily versus twice daily adminis-tration of HBO was compared in the same experimentalmodel to determine the optimal treatment protocol.

MATERIALS AND METHODS

Experimental Design

A total of 40 female Wistar rats (160–210 g), pur-chased from the Istanbul University Medical FacultyExperimental Research and Diagnosis Center, werehoused at room temperature in a natural day/night cycleand were permitted to eat standard rat chow and drink tapwater ad libitum during the experimental period. Approvalfor the experiments was secured from the Gulhane Mili-tary Medical Academy Ethical Committee. The rats wererandomly divided into five groups, each consisting ofeight animals. The control group was given a placebo(saline 2 mL, intraperitoneally) on the first day of thestudy. Animals in the HBO group received only HBOtherapy for 60 min at 2.5 atm every day for six days. Thecisplatin group (CP) was given an intraperitoneal dose of 6mg/kg of cisplatin (Cisplatin DBL solution, 50 mg/50 mL,Orna, Istanbul, Turkey) on the first day of the study. Thecisplatin plus once daily HBO (CP+HBO) group wasgiven an intraperitoneal dose of 6 mg/kg of cisplatin andimmediately followed up with 60 min of HBO at 2.5 atmdaily for six days. The cisplatin plus twice daily HBO(CP+2HBO) group was given an intraperitoneal dose of6 mg/kg of cisplatin and immediately followed up with60 min of HBO at 2.5 atm twice a day for six days. Theinterval between the two HBO treatments in theCP+2HBO group was 8 hours. Body weight of all animalswas recorded daily during the experimental period. At theend of the study, the rats were sacrificed by cervical dislo-cation, and left nephrectomy for histopathological analysisand right nephrectomy for biochemical analysis were per-formed. Blood samples were collected using intracardiacpuncture.

Renal Function Assessment

The renal functions of rats were determined by meansof serum urea and creatinine levels. Blood samples werecentrifuged at 5000 rpm for 10 min, and sera were sepa-rated. Serum urea and creatinine levels were analyzedusing an autoanalyzer (Olympus® AU800).

Determination of Oxidative Stress

The right kidneys of rats were removed immediatelyafter cervical dislocation, put into tubes, frozen with liquidnitrogen, and stored at −70°C. The frozen kidney tissueswere homogenized in a phosphate buffer (pH 7.4) bymeans of a homogenizator (Heidolph Diax 900; Heidolph

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Hyperbaric Oxygen and Cisplatin Nephrotoxicity 259

Elektro GmbH, Kelhaim, Germany). The supernatant wasdivided into two to three parts, put in separate tubes, andstored at −70°C again. The protein content of kidneyhomogenates was measured using Lowry’s method, withbovine serum albumin as the standard.[16]

Lipid peroxidation was determined by measuring thethiobarbituric acid reactive substances (TBARS) level inkidneys, as described by Ohkawa.[17] This method wasused to obtain a spectrophotometric measurement of thecolor produced during the reaction to thiobarbituric acid(TBA) with malondialdehyde at 535 nm. For this purpose,2.5 ml of 100 g/L trichloroacetic acid solution was addedto 0.5 ml of homogenate in each centrifuge tube andplaced in a boiling water bath for 15 min. The mixture wascooled and centrifuged at 1000 g for 10 min. Next, 2 ml ofthe supernatant was added to 1 ml of 6.7 g/L TBA solutionin a test tube and placed in a boiling water bath for 15 min.The solution was then cooled and its absorbance measuredusing a spectrophotometer (Shimadzu UV-1601; Kyoto,Japan). TBARS levels were expressed as nmol/g-protein.

Superoxide dismutase (SOD) activity was assayedusing the nitroblue tetrazolium (NBT) method describedby Sun.[18] The stock solution contained 10 mg of Cu,Zn-SOD from bovine liver dissolved in 10 ml of isotonicsaline and was diluted to 600 μg/L with distilled waterbefore it was used in the assay. The SOD assay reagentconsisted of a combination of the following reagents:80 ml of 0.3 mmol/L xanthine solution, 40 ml of0.6 mmol/L ethylenediaminetetraacetic acid (EDTA) solu-tion, 40 ml of 150 μmol/L NBT solution, 24 ml of400 mmol/L Na2CO3 solution, and 12 ml of bovine serumalbumin. The samples were subjected to ethanol-chloroform(62.5%/37.5%) extraction prior to the assay of enzymeactivity. Briefly, 400 μl of ice-cold ethanol/chloroformmixture was mixed thoroughly with 250 μl of sample.After vortexing for 30 seconds and centrifugation at 3000gat 4°C for 5 min, the upper aqueous layer was collected.The collected supernatant was diluted by a factor of 100,and 0.5 ml of the diluted solution was used for the assayby adding it to 2.5 ml of SOD assay reagent. NBT wasreduced to blue formazan by O2

-., which has a strongabsorbance at 560 nm. One unit (U) of SOD is defined asthe amount of protein that inhibits the rate of NBT reduc-tion by 50%. The calculated SOD activity was expressedas U/mg-protein.

Glutathione peroxidase (GSH-Px) activity was mea-sured using the method described by Paglia and Valentine,in which GSH-Px activity was coupled with the oxidationof NADPH by glutathione reductase.[19] The oxidation ofNADPH was measured spectrophotometrically at 340 nmat 37°C. The reaction mixture consisted of 50 mmol potas-sium phosphate buffer (pH 7), 1 mmol EDTA, 1 mmolNaN3, 0.2 mmol NADPH, 1 mmol glutathione, and 1 U/mL

of glutathione reductase. The absorbance at 340 nm wasrecorded for 5 min. The activity was the slope of the linesas mmol of NADPH oxidized per minute. GSH-Px activitywas presented as U/g-protein.

Histopathological Evaluation

Left kidney tissues were fixed in buffered 10% forma-lin for 12 hours and then embedded in paraffin wax. Fourmicron-thick sections were stained with periodic acid-Shiff(PAS) and hematoxylin and eosin (H&E). Histopathologi-cal evaluation was performed by a pathologist in a blindmanner. Tubular injury was assessed in PAS stained sectionsusing a semi-quantitative scale in which the percentage ofdistal tubules as epithelial necrosis and apoptosis was assigneda score: 0 = normal; 1 = <10%; 2 = 10−25%; 3 = 26−75%;4 = >75%.[20] The percentage of hyaline cast formation indistal tubules was calculated in 30 medullary areas in eachcase under 400 high-power multiplication using a lightmicroscope and scored using a semi-quantitative scale: 0 =normal; 1 = <10%; 2 = 10−25%; 3 = 26−75%; 4 = >75%.

Statistical Analysis

Data were expressed as mean ± SD. The biochemicaland histopathological data and percentage of weight losswere analyzed using one-way analysis of variance (ANOVA)followed by the LSD test. All statistical analyses were per-formed using SPSS 11.0 statistical software. A value ofp < 0.05 was considered statistically significant.

RESULTS

Renal Functions

Serum urea and creatinine levels of all groups areshown in Table 1. HBO alone did not alter serum urea andcreatinine levels compared to the control group (p < 0.05).Cisplatin treatment increased serum urea and creatininelevels significantly, indicating renal failure development(p < 0.05, compared to the control group). Compared tothe CP group, serum urea and creatinine levels in theCP+HBO group decreased; however, the differencebetween the groups was not statistically significant (p >0.05). In addition, the difference between the CP+HBOgroup and control group in terms of creatinine levels wasnot significant (p > 0.05). The serum creatinine level washigher in the CP+2HBO group compared to the CP andCP+HBO groups (p < 0.05). Serum urea levels were simi-lar in the CP and CP+2HBO groups (p > 0.05).

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Body Weight Loss

The body weights of the animals at the beginning ofthe study were similar (see Table 1) and increased slightlyin the control and HBO groups. However, in the CP,CP+HBO, and CP+2HBO groups, body weight losseswere observed after six days. Compared to the CP group(25.05 ± 5.22%), both once daily (CP+HBO group; 10.40 ±6.27%) and twice daily (CP+2HBO group; 11.45 ± 4.79%)HBO treatments significantly reduced body weight loss(p < 0.05). The difference between the CP+HBO andCP+2HBO groups was not statistically significant (p > 0.05).

Oxidative Stress

Oxidative stress parameters of the animals are pre-sented in Table 2. HBO therapy alone did not alter kidneyMDA levels, SOD, or GSH-Px activities compared to thecontrol group (p > 0.05). Cisplatin treatment significantlyincreased MDA levels and reduced SOD and GSH-Pxactivities compared to the control and HBO groups (p < 0.05).

Compared to the CP group, MDA levels increased andSOD and GSH-Px activities decreased significantly in theCP+HBO and CP+2HBO groups (p < 0.05). The differ-ences between the CP+HBO and CP+2HBO groupsregarding all oxidative stress parameters were statisticallysignificant (p < 0.05).

Histopathological Findings

Tubular injury and hyaline cast formation scores arepresented in Table 3. HBO alone did not induce histo-pathological injury. Cisplatin treatment increased bothtubular injury and hyalin cast formation compared to thecontrol and HBO groups (p < 0.05). Tubular injury andhyalin cast formation scores decreased in the CP+HBOgroup compared to the CP group, although this differencewas not significant (p > 0.05). Compared to the CP andCP+HBO groups, tubular injury and hyaline cast forma-tion scores were significantly higher in the CP+2HBOgroup (p < 0.05). Sample histopathological sections areshown in Figures 1–3.

Table 1 Serum urea and creatinine levels and body weights of all experimental groups

Groups (n=8)

Serum urea (mg/dL)

Serum creatinine (mg/dL)

Body weight (g)

Day 1 Day 6

Control 56.3 ± 5.8 0.34 ± 0.05 172.5 ± 8.8 175.0 ± 10.7HBO 63.6 ± 15.1 0.40 ± 0.07 168.7 ± 20.3 172.5 ± 19.8CP 292.1 ± 220.4*† 2.90 ± 3.15*† 170.0 ± 14.1 127.1 ± 11.5CP+HBO 188.1 ± 134.6*† 1.26 ± 0.91|| 183.7 ± 22.6 163.7 ± 15.1CP+2HBO 274.0 ± 59.9*† 5.48 ± 3.02*†‡§ 181.8 ± 21.3 160.6 ± 16.1

Data are expressed as mean ± SD.p < 0.05 compared with: the control group,* the hyperbaric oxygen (HBO) group,† the cisplatin (CP) group,‡ the cisplatin plus once

daily HBO (CP+HBO) group,§ and the cisplatin plus twice daily HBO (CP+2HBO) group.||

Table 2 Renal thiobarbituric acid reactive substances (TBARS) levels, superoxide dismutase (SOD), and

glutathione peroxidase (GSH-Px) activities of all experimental groups

Groups (n=8) TBARS (nmol/mg protein) SOD (U/mg protein) GSH-Px (U/g protein)

Control 0.61 ± 0.04 9.81 ± 1.07 37.00 ± 1.83HBO 0.61 ± 0.04 10.12 ± 1.05 35.53 ± 3.35CP 1.46 ± 0.17*†§|| 4.73 ± 1.09*†§|| 9.42 ± 2.01*†§||

CP+HBO 1.31 ± 0.11*†‡|| 6.26 ± 1.55*†‡|| 16.72 ± 1.62*†‡||

CP+2HBO 0.82 ± 0.07*†‡§ 9.58 ± 0.78‡§ 32.41 ± 4.59*†‡§

Data are expressed as mean ± SD.p < 0.05 compared with: the control group,* the hyperbaric oxygen (HBO) group,† the cisplatin (CP) group,‡ the cisplatin plus once

daily HBO (CP+HBO) group,§ and the cisplatin plus twice daily HBO (CP+2HBO) group.||

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Hyperbaric Oxygen and Cisplatin Nephrotoxicity 261

DISCUSSION

Using an experimental model, the effect of differentdoses of HBO on cisplatin-induced nephrotoxicity in ratswas evaluated. Single dose cisplatin injection impairedrenal functions and caused severe histopathological injury,indicating the development of acute renal failure. Whileonce daily HBO slightly attenuated cisplatin-inducedacute renal failure, twice daily HBO aggravated cisplatinnephrotoxicity.

ROS are continuously formed in vivo in healthy sub-jects under physiological conditions. Various enzymes andnon-enzymatic compounds in the cell prevent ROS produc-tion and scavenge formed radicals to maintain a balance

between ROS and the antioxidant defense system. When theamount of ROS exceeds the capacity of the cell or organantioxidant defense system, the normal function is disruptedand tissue damage develops.[21] In this study, cisplatin treat-ment increased lipid peroxidation and significantlyimpaired antioxidant enzymes’ SOD and GSH-Px activitiesin rat kidneys. These results support the hypothesis thatexcessive formation of ROS and lipid peroxidation play animportant role in cisplatin-induced nephrotoxicity.[3,4]

A number of antioxidants have been reported to reduceoxidative stress and prevent cisplatin nephrotoxicity.[5–9]

Table 3 Tubular injury and hyalin cast formation scores of all

experimental groups

Groups (n=8)Tubular injury in

distal tubulesHyaline cast

formation

Control 0.25 ± 0.46 0.25 ± 0.46HBO 0.25 ± 0.46 0.25 ± 0.46CP 2.57 ± 1.40*†|| 2.29 ± 1.50*†||

CP+HBO 1.88 ± 0.99*†|| 1.88 ± 0.99*†||

CP+2HBO 3.50 ± 0.76*†‡§ 3.25 ± 0.89*†‡§

Data are expressed as mean ± SD.p < 0.05 compared with: the control group,* the hyperbaric

oxygen (HBO) group,† the cisplatin (CP) group,‡ the cisplatinplus once daily HBO (CP+HBO) group,§ and the cisplatin plustwice daily HBO (CP+2HBO) group.||

Figure 1. Epithelial necrosis (short black arrow), castformation, (long white arrow), and apoptotic cells (arrowhead) inthe distal tubulus due to cisplatin nephrotoxicity (×200 HE;cisplatin group).

Figure 2. Minimal epithelial necrosis (short black arrow) andapoptotic cells (arrowhead) in the distal tubulus. Some of thedistal tubulus shows dilatation (×200 HE; cisplatin plus oncedaily hyperbaric oxygen group).

Figure 3. Epithelial necrosis (short black arrow), castformation (long white arrow), and apoptotic cells (arrowhead) inthe distal tubulus (×200 HE; cisplatin plus twice daily hyperbaricoxygen group).R

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262 S. Aydinoz et al.

Similarly, the improved renal functions, reduced kidneyinjury, and reduced body weight loss with once daily HBOare accompanied by a significant reduction in lipid peroxi-dation and by an increase in SOD and GSH-Px activitiesin the kidneys. The beneficial effect of HBO on cisplatin-induced nephrotoxicity seems to be partially mediated bymodification of the oxidant/antioxidant system in the kidney.

It has been postulated that exposure to HBO can resultin increased ROS formation, which may induce oxidativestress in tissues.[22] However, whether HBO is an oxidantor an antioxidant agent is a controversial issue. In somestudies, HBO has been shown to induce oxidativeinjury,[23,24] while others have reported the opposite.[13–15]

Hink and Jansen suggested that some beneficial effects ofHBO, such as vasoconstriction, impairment of leukocyteadhesion, inhibition of lipid peroxidation, neovasculariza-tion, and antibacterial effects, are mediated by ROS.[25]

HBO reduces lipid peroxidation by increasing antioxidantenzyme activities[13–15] and by the oxygen mediated termi-nation mechanism.[26] In this study, although HBO alonedid not alter any oxidative stress parameters, both oncedaily and twice daily HBO regimes significantly reducedcisplatin-induced oxidative stress in kidneys. The effect ofHBO on oxidant/antioxidant systems may depend on thespecific environment and cellular state.

Twice daily HBO sessions increased SOD and GSH-Px activities and reduced lipid peroxidation significantlycompared to a single HBO session. Surprisingly, however,twice daily sessions of HBO led to the impairment of renalfunctions and an increase in tuberal damage. It may bespeculated that the pressure, duration, and frequency ofHBO determine the cellular and subcellular effects of thetreatment. Barth et al. showed that while once daily HBOaccelerates bone repair and vessel ingrowth, twice dailyHBO retards these processes.[27] Conconi et al. investi-gated the effects of different HBO regimes on the growthof the 3T3/J2 fibroblast cell line and determined that expo-sure to HBO at 2.5 atm for 15 min was ineffective, 30- and60-min exposures raised the proliferation rate, and 120-min exposure significantly reduced these parameters andraised the apoptotic rate of cultured fibroblasts.[28] Thereare mechanisms other than ROS in the pathogenesis of cis-platin-induced nephrotoxicity; Kruidering et al. showedthat although ROS formation occurs during cisplatin-induced toxicity, it is not the direct cause of cell death.[29]

Twice daily HBO may have induced a ROS-independentpathway and aggravated the toxic effects of cisplatin. Thiswarrants further investigation.

In conclusion, these findings suggest that HBO alonedoes not cause nephrotoxicity and oxidative stress in rat kid-neys; once daily HBO may attenuate cisplatin nephrotoxic-ity, and this effect is partially mediated by modification ofoxidant/antioxidant systems in kidneys; and twice daily

HBO potentiates cisplatin nephrotoxicity by a ROS-independent mechanism.

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