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Introduction

Cisplatin (Cisp) is one of the most widely used, potent chemotherapy drugs. Cisp and related platinum-based therapeutics are now being used for the treatment of tes-ticular, head and neck, ovarian, cervical, and many other types of cancer (Wang and Lippard, 2005; Cohen and Lippard, 2001; Siddik, 2003). The mechanism of the anti-cancer activity of Cisp is not completely understood, but a widely held view is that Cisp binds to DNA, leading to the formation of inter- and intrastrand cross-links (Cohen and Lippard, 2001; Ciccarelli et al., 1985; Heiger-Bernays et al., 1990; Jamieson and Lippard, 1999). Cross-linking results in defective DNA templates and arrest of DNA synthesis and replication. In rapidly dividing cells, such as cancer cells, cross-linking can further induce DNA damage. Mildly damaged DNA can be repaired, whereas

extensive DNA damage leads to irreversible injury and cell death.

The major limiting factor in the use of Cisp is its side effects in normal tissues, which include neurotoxicity, ototoxicity, nausea, vomiting, and nephrotoxicity. For years, various approaches have been attempted to cur-tail these side effects. One strategy is to synthesize and screen for novel Cisp analogs that have lower toxicity in normal tissues. In this direction, several Cisp analogs, such as carboplatin, have been identified with less severe side effects (Pasetto et al., 2006), but these are not as effective as Cisp. Another approach that has been used, with some success, is to hydrate patients during Cisp treatment (Cornelison and Reed, 1993; Bajorin et al., 1986). Despite these efforts, Cisp nephrotoxicity remains a major factor that limits the use and efficacity

ReseaRch aRtIcle

Protective effect of erythropoietin against cisplatin-induced nephrotoxicity in rats: antigenotoxic and antiapoptotic effect

Karima Rjiba-Touati1, Imen Ayed-Boussema1, Chayma Bouaziz1, Anis Belarbia2, Awatef Azzabi2, Abdelatif Achour2, Wafa Hassen1, and Hassen Bacha1

1Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Monastir, Tunisia, and 2Department of Nephrology, Dialysis, and Transplant, University Hospital of Sahloul, Sousse, Tunisia

abstractCisplatin (Cisp) is an active cytotoxic agent that was found efficient in the treatment of various types of solid tumors. Its nephrotoxic effect has been very well documented in clinical oncology. Erythropoietin (EPO), a renal cytokine-regulating hematopoiesis, has recently been shown to exert important cytoprotective effects in many experimental injuries. The aim of this study was to explore whether EPO would protect against Cisp-induced apoptosis in rat kidney. Adult Wistar rats were treated with saline solution as the control group, Cisp alone, EPO alone, or EPO with Cisp in different treatments: 1) EPO and Cisp simultaneously administrated to animals as a cotreatment; 2) EPO administered 24 hours before Cisp as a pretreatment; and 3) EPO administered 5 days after Cisp injection as a post-treatment. Our results have shown that Cisp induced renal failure, characterized with a significant increase in serum creatinine and blood urea nitrogen (BUN) concentrations. Cisp promoted kidney DNA fragmentation and apoptotic cell death. Apoptosis was revealed by an enhancement of proapoptotic protein (e.g., p53 and Bax) levels, decrease in antiapoptotic proteins (e.g., Bcl2 and Hsp27), and increase in caspase-3 activity. Treatments with EPO restored creatinine and BUN levels and inhibited Cisp-induced DNA damage in the kidney. Apoptosis was also reduced by the upregulation of antiapoptotic protein expressions, downregulation of proapoptotic protein levels, and reduction of caspase-3 activity.Keywords: Cisplatin, erythropoietin, nephroprotection, antigenotoxicity, antiapoptotic effect

Address for Correspondence: Hassen Bacha, Laboratoire de Recherche sur les Substances Biologiquement Compatibles (LRSBC), Faculté de Médecine Dentaire, Rue Avicenne, 5019 Monastir, Tunisia; Fax: +216 73 42 55 50; E-mail: hassen.bacha@fmdm.rnu.tn

(Received 05 January 2011; revised 02 February 2011; accepted 23 March 2011)

Drug and Chemical Toxicology, 2012; 35(1): 89–95© 2012 Informa Healthcare USA, Inc.ISSN 0148-0545 print/ISSN 1525-6014 onlineDOI: 10.3109/01480545.2011.589440

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of this drug in cancer therapy (Hanigan and Devarajan, 2003).

Erythropoietin (EPO) is a cytokine originally used for its effects on erythropoiesis, as it supports the survival, proliferation, and differentiation of erythroid progenitor cells. Recently, EPO has been found to protect the brain and spinal cord from ischemic injury (Celik el al., 2002), the peripheral nerve from diabetic damage (Bianchi et al., 2004), and the heart from acute ischemic/reperfusion injury (Calvillo et al., 2003; Cai el al., 2003). Given that EPO receptors are expressed on renal tubular epithelial cells (Westenfelder et al., 1999), it is possible that the sys-temic administration of EPO may also provide protection against acute renal damage. Indeed, EPO has been found to ameliorate ischemic and toxic renal injury (Vesey et al., 2004; Bagnis et al., 2001). However, it is interesting to establish the mechanism adopted by EPO to exert the renoprotective effect in Cisp-induced renal failure.

In this study, we looked for the protective effect of EPO on Cisp-induced nephrotoxicity in rats. Using biochemi-cal analysis, we evaluated renal injury. DNA damage was quantified by the comet assay. The antiapoptotic action of EPO was evaluated by the analysis of pro- and antiapop-totic proteins (e.g., p53, Bax, Bcl2, and Hsp 27) levels and caspase-3 activity. We investigated whether EPO Cisp would induce nephrotoxicity, genotoxicity, and apop-tosis when was administered before, simultaneously, or after Cisp administration.

Methods

ChemicalsCisp (cis-diamminedichloroplatinum II) was purchased from Sigma-Aldrich (Fontenay-sous-Bois, France). Experiments were performed with a commercially available preparation of EPO (Hemax®; Bio SIDUS S.A., Buenos Aires, Argentina). Mouse monoclonal anti-p53, anti-Bax, and anti-Bcl2 were from Invitrogen. The secondary antibody (phosphatase-conjugated), anti-mouse immunoglobulin were from Invitrogen (Carlsbad, California, USA). Caspase-3 assay system colorimet-ric (G-7351) was purchased from Promega (Madison, Wisconsin, USA). Z-Val-Ala-DL-Asp-fluoromethylketone (ZVAD-fmk; 100 µM) is a Bachem product (Bachem Bioscience, Inc., King of Prussia, Pennsylvania, USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was supplied by Molecular Probes (Cergy Pontoise, France). All other chemicals used were of analytical grade.

Animal treatmentsExperiments were performed on male Wistar rats in the weight range of 120–140 g, kept at controlled environmen-tal conditions at room temperature 22 ± 2°C and 12-hour light-dark cycles, and allowed free access to food and water, but were fasted overnight before treatment. For the time-course experiment, rats were divided at random into six groups, with 6 animals in each group. The control group received a single injection of saline solution (0.9%).

The EPO group was given only EPO, and the Cisp group was given only a single injection of Cisp. To test the effects of EPO on Cisp-induced nephrotoxicity, three treatment conditions were employed. In the cotreatment group, a single dose of EPO was administered simultaneously with Cisp. In the pretreatment group, a single dose of EPO was given 1 day before Cisp treatment. In the post-treatment group, a single dose of EPO was given 5 days after Cisp treatment. EPO (3,000 UL/kg body weight; b.w.) and Cisp (6 mg/kg b.w.) injection were administered by the intrap-eritoneal (i.p.) route.

After animals were sacrificed, blood was collected for creatinine and blood urea nitrogen (BUN) analysis and kidneys were immediately removed for histological anal-ysis, comet assay, caspase-3, and Western blot assays.

Estimation of serum creatinineSerum creatinine concentration was measured by the picric acid colorimetric method (Allcock et al., 1999). A picric acid solution was made by a 10-fold dilution of saturated picric acid with 1% NaOH. Serum (50 µL) was added to picric acid/NaOH solution (200 µL) in a micro-titer plate. Samples were incubated 15 minutes before being read at 490 nm in a microplate reader. A standard curve was prepared on the same assay plate to derive the creatinine concentration.

Determination of BUNBUN was measured using a colorimetric assay kit, according to the manufacturer’s instructions (Stanbio Laboratory, San Antonio, Texas, USA). Urea in the sample was hydrolyzed by the enzyme urease to yield ammonia and carbon dioxide. Then, ammonium ions reacted with a mixture of salicylate, sodium nitroprusside, and hypochlolrite to yield a blue-green chromophore. The absorbance of this chromophore at 600 nm was propor-tional to urea concentration in the sample.

Single-cell gel electrophoresis (the comet assay)Determination of DNA damage by the alkaline comet assay was conducted according to Tice et al. (2000), with minor modifications (Picada et al., 2003). Each piece of kidney was placed in 0.5 mL of cold phosphate-buffered saline (PBS) and finely minced to obtain a cellular sus-pension. Kidney cell suspensions (5 µL) were embedded in 60 µL of 1% low-melting-point agarose and spread on agarose-precoated microscope slides. To lyse cellular and nuclear membranes of the embedded cells and allow for DNA unwinding in alkaline conditions, slides were immersed in ice-cold, freshly prepared lysis solution and left at 4°C overnight to improve the efficiency of DNA damage detection (Banath et al., 2002). Slides were then placed in an electrophoresis alkaline buffer (pH >13), and embedded cells were exposed to this alkali solution for 20 minutes to allow for DNA unwinding. Electrophoresis was performed in the same alkaline buffer for 20 min-utes by applying a 25-V electric field and adjusting the current to 300 mA. After electrophoresis, slides were

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neutralized with 0.4 M of Tris (pH 7.5) and DNA was stained with 50 µL of ethidium bromide (20 µg/mL). All steps were conducted in darkness to prevent additional DNA damage. Images of 100 randomly selected cells (50 cells from each of two replicate slides) were analyzed from each animal. Cells were also visually scored accord-ing to tail size into five classes, ranging from undamaged (0) to maximally damaged (4), resulting in a single DNA damage score to each animal and, consequently, to each group. Therefore, the damage index could range from 0 (completely undamaged, 100 cells × 0) to 400 (with maxi-mum damage, 100 × 4).

Protein extraction and quantificationKidney was homogenized with a Potter (glass Teflon) in the presence of 10 mM of Tris-HCl (pH 7.4) at 4°C and centrifuged at 4,000 rpm for 30 minutes at 4°C. The super-natant was collected for analysis, and protein concentra-tions were determined in kidney extract using the protein Bio-Rad assay (Bradford, 1976).

Western blot analysisEqual amounts of proteins (20 µg) were separated by 12% sodium dodecyl sulfate/polyacrylamide gel elec-trophoresis. Separated proteins were electroblotted in the transfer buffer (10 mM of Tris-base, pH 8.3, 96 mM of glycine, and 20% methanol). The membrane was then blocked in TBS (20 mM of Tris-HCl, pH 7.5, and 500 mM of sodium chloride), containing 5% (w/v) nonfat milk. The membrane was washed in TTBS (TTBS containing 0.3% Tween-20) for 30 minutes (3 times × 10 minutes), incubated 2 hours at room temperature with primary antibodies at the following dilutions: p53 (1:500 dilution), Bcl2 (1:1,000 dilution), Bax (1:1,000 dilution), and Hsp 27 (1:1,000 dilution), then washed and incubated with goat antimouse alkaline phosphatase-conjugated antibody at a 1:3,000 dilution for 2 hours. Next, the membrane was washed and the chromogenic substrate, BCIP/NBT, was added to localize antibody binding. Protein levels were then determined by computer-assisted densitometric analysis (Densitometer, GS-800; Bio-Rad Quantity One®). The experiment was performed at least three times and

expression of each protein was described throughout as the proportional increase or decrease relative to the levels observed in control treatment.

Caspase-3 activity assayActivity of caspase-3 were assayed in whole kidney tissue using a commercial kit (caspase-3 assay kit, Fluorimetric®; Sigma-Aldrich). In brief, equal quantities of proteins extracted (50 µg) were incubated for 4 hours at 37°C with 10 nmol of fluorogenic substrates (Ac-DEVD-AMC for caspase-3 substrate). Controls (including buffer and substrate alone) were assayed in parallel and subtracted from experimental values.

Statistical analysisData are expressed as means ± standard deviation. Statistical comparison between different groups were done using one-way analysis of variance, followed by Fischer’s post-hoc test, to detect the difference between various groups.

Results

Changes in BUN and serum creatinine levels by co-, pre-, and post-treatment with EPOTo evaluate the nephroprotective effect of EPO against Cisp-induced acute renal injury in the rat, BUN and serum creatinine levels, common indicators of acute renal failure, were measured. Our results clearly demon-strated that, at 5 days after Cisp treatment, the rat showed a marked deterioration in renal function, as reflected in enhanced levels of BUN and serum creatinine, as com-pared to the control group. As shown in Figure 1, BUN level increased from 6.58 ± 1.01 to 154.1 ± 7.53 mmol/L in the Cisp group and serum creatinine increased from 25.5 ± 2.88 to 376 ± 35.13 µmol/L as compared to the con-trol group. EPO treatments (co-, pre-, and post-treatment) resulted in a significant reduction in BUN and serum creatinine levels, compared to the Cisp-alone–treated group. Our data showed that EPO administration 24 hours before Cisp provides the best protection against Cisp nephrotoxicity; in fact, BUN level was reduced from

Figure 1. EPO ameliorates renal functional impairment after Cisp injury. Wistar rats were administered i.p. EPO (3,000 U/kg) simultaneously, 24 hours before, and 5 days after Cisp treatment (6 mg/kg i.p.). Animals were scarified and serum samples were collected for determination of serum creatinine and BUN-level concentrations. *P #P versus Cisp, aP bP.

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154.1 ± 7.53 mmol/L in Cisp treatment to 43.28 ± 10.5 mmol/L in the pretreatment condition. Serum creatinine levels decreased from 376 ± 35.13 to 113.6 ± 8.2 µmol/L.

Effect of EPO on cisplatin-induced genotoxic damageThe antigenotoxic property of EPO was assessed through the alkaline comet assay. Results of the visual scoring of total basic DNA damage are illustrated in Figure 2. We observed a significant increase of the total DNA damage on rat kidney treated with Cisp (6 mg/kg intraperitoneal; i.p). EPO treatment (in co-, pre-, and postcondition) was able to reduce DNA fragmentation caused by Cisp. The amount of DNA damage decreased significantly in the presence of EPO treatment, DNA damage was reduced by approximately 2- and 3-fold when rats were, respectively,

treated with EPO 24 hours before and 5 days after Cisp intoxication. The degree of DNA damage seemed to be less noticeable when rats were pretreated with EPO. No specific DNA fragmentation was detected in control and EPO alone groups.

Effect of EPO on cisplatin-induced apoptotic cell deathP53, Bax, Bcl-2, and Hsp27 expressionsTo look for the preventive effect of EPO on cell death, we have monitored the expression of pro- and antiapoptotic proteins in response to exposure to Cisp alone, EPO alone, or Cisp with EPO in different treatment conditions (Figure 3). Our results showed that whereas Cisp (6 mg/kg b.w., i.p) increased expression levels of p53 and Bax, two proapoptotic proteins; it decreased the amount of Bcl-2

Figure 2. Total DNA damage was measured by the alkaline comet assay in isolated rat kidney cells. DNA fragmentation in kidney rat cells was estimated after a single exposure to Cisp and EPO in different treatment conditions: Cisp alone, EPO alone, and EPO with Cisp in different treatment conditions (EPO administration was performed simultaneously, 24 hours before, and 5 days after Cisp treatment). EPO (3,000 UL/kg body weight) and Cisp (6 mg/kg body weight) injection were administrated by the i.p. route. *P #P versus Cisp, aP bP.

Figure 3. Effect of EPO co-, pre-, and post-treatment on p53, Bax inhibition, and Bcl2 and Hsp27 induction in Cisp-treated kidney. (A) Immunoblotting of p53, Bax, Bcl2, and Hsp27 proteins. (B) Scanning densitometry analysis of p53, Bax, Bcl2, and Hsp27 levels in Cisp-treated kidney expressed as percent of control at the different times. Antiactin antibody was used as the loading control. Results were normalized with respect to actin band intensities in respective controls. Similar results were obtained in three independent sets of experiments. *P < 0.05 versus control, #P versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

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and Hsp27, two antiapoptotic proteins. EPO administra-tion (3,000 UL/kg b.w., i.p.) simultaneously, before, or after Cisp induced p53 and Bax inhibition, even when Bcl2 and Hsp27 levels were enhanced significantly.

Caspase-3 activationTo confirm that the apoptotic process was induced by Cisp and inhibited by EPO, we investigated caspase-3 activity. As shown in Figure 4, caspase-3 proteolytic activity was increased in Cisp rat kidney, as compared to the control group (90.88 ± 3.06 vs. 34.55 ± 2.59 pmol PNA/h/μg protein; P < 0.05). The activity of the execu-tioner caspase-3 was significantly lower in groups treated with EPO (co-, pre-, and post-treatment) (52.47 ± 2.24, 42.55 ± 3.06, and 64.97 ± 3.18 pmol PNA/h/μg of protein, respectively), as compared to the Cisp-treated group (90.88 ± 3.06 pmol PNA/h/μg of protein; P < 0.05). The measure of caspase-3 activity showed that pretreatment with EPO promoted a best protection against Cisp-induced apoptosis.

Discussion

Cisp is one of the most potent chemotherapeutic antican-cer drugs for the treatment of various cancers (Fuertesa et al., 2003; Siddik, 2003). In spite of its significant anti-cancer activity, the clinical use of Cisp is often limited by its undesirable side effects, mainly nephrotoxicity. Many efforts have been made to improve the therapeutic index of Cisp using pharmacological strategies, such as the administration of chemoprotectors, intensive hydration, and hypertonic saline (Skinner, 1995; Anand and Bashey, 1993; Pinzani et al., 1994). EPO, apart from its crucial role in hematopoiesis, has been shown to prevent heart, central nervous system, and kidney injuries (Simon et al., 2008; Sharples et al., 2004). In the present study, we provide strong evidence that EPO has a preventive effect against Cisp-induced damage in rat kidneys with an antigenotoxic and an antiapoptotic process.

Our results clearly demonstrate that the administra-tion of Cisp to rats caused an increased BUN and creati-nine levels in serum. These results are in agreement with

several studies showing that Cisp induced severe renal failure, characterized by enhanced BUN and serum crea-tinine levels (Parlakpinar et al., 2002; Jiang el al., 2007; Jenderny et al., 2010). In the present study, EPO treatment 24 hours before Cisp was the most effective treatment in preventing the damages caused by Cisp. Moreover, either co- or post-treatment with EPO provided renal protection against Cisp, as shown by a lower level of BUN and serum creatinine. This is in accord with Bagnis et al. (2001), who demonstrated that EPO enhances recovery after Cisp-induced acute renal failure in the rat. Recently, Esposito et al. (2009) have demonstrated that EPO precondition-ing was effective to protect the kidney against ischemia/reperfusion injury by reducing both tubular cell injury and interstitial infiltration.

It has been reported that one of the molecular mecha-nisms of Cisp-induced nephrotoxicity is the induction of apoptosis, particularly in renal tubular cells (Eastman, 1999; Faubel et al., 2004; Pabla and Dong 2008; Jiang et al., 2007). The exact mechanism of Cisp-induced apoptosis is not completely understood, but it has been described that after entering the cell, because of the decrease of chloride concentration, Cisp is aquated and turned into highly reactive platinum species. These species act by damaging DNA resulting from platination to form covalent platinum DNA adduct (Jamieson and Lippard, 1999; Siddik, 2003). DNA damage elicits a series of signal-transduction cascades, involving chromatin remodeling, which eventually lead to DNA repair, cell-cycle arrest, or apoptosis (Green and Almouzni, 2002). For this reason, the comet assay was performed to assess the possible antigenotoxic action of EPO. Our results showed that EPO decreased DNA damage level caused by Cisp in all treatment conditions (co-, pre-, or post-treatment). This is in agreement with Sun et al. (2004), who demonstrated that EPO (300 U/kg i.p.), administrated 24 hours before insult and for 2 consecutive days after insult, reduced DNA fragmentation of the immature central nervous system.

DNA damage induced by a variety of toxic agents usu-ally activates the tumor suppressor, p53 (Park et al., 2006; Ueno et al., 1999). Once activated, p53 regulates a number

Figure 4. Effect of rHuEPO treatment on caspase-3 activity in different rat groups. In rat kidneys with different treatments: vehicle, EPO, Cisp, and Cisp+EPO. Caspase-3 activity decreased significantly in rat kidney treated with EPO (co-, pre-, and post-treatment), compared with Cisp-only group. *P < 0.05 versus control, #P < 0.05 versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

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of downstream cellular processes, such as cell-cycle arrest, senescence, apoptosis, and DNA repair (Oren, 1999; Ljungman, 2000; Fuster et al., 2007). In apoptotic cells, p53 mediated transactivation of apoptosis-related genes, including proapoptotic Bcl-2 family members. Bcl2 family proteins are known to modulate apoptosis through the regulation of the mitochondrial apoptosis pathway (Cory et al., 2003). Bax is one of the promoters of apoptosis (Story and Kodym, 1998), whereas Bcl-2 func-tions as a major inhibitor of cell death, therefore guarding against apoptosis (Dixon et al., 1997; Story and Kodym, 1998; Cory and Adams, 2002). Further, several studies showed Hsp27 to be an antiapoptotic protein (Rashmi et al., 2003; Schepers et al., 2005; Casado et al., 2007). The correct balance of pro- and antiapoptotic proteins can determine whether or not apoptosis occurs (Wang et al., 1999).

In our study, we demonstrated that Cisp induced apoptosis in rat kidney. In fact, Cisp enhanced proapop-totic protein levels (e.g., p53 and Bax) and decreased antiapoptotic proteins level (e.g., Bcl2 and Hsp27). Further, our results showed that Cisp increased caspase-3 activity. Our findings are in accord with those of Tsuruya et al. (2003), who demonstrated that Cisp-induced neph-rotoxicity was shown to be associated with an increase in the proapoptotic protein, Bax, and a decrease in the antiapoptotic protein, Bcl-2. Rat treatment with EPO markedly decreased the ratio of pro- and antiapoptotic proteins and reduced caspase-3 activity. The decrease in proapoptotic factors with different EPO treatments might be the result of the capacity of EPO to upregulate of anti-apoptotic factors (Sharples and Yaqoob, 2006). Further, this protective effect of EPO can be observed in cotreat-ment as well as in pretreatment and in post-treatment conditions, but the pretreatment is the most effective. These findings are in accord with Yang et al. (2003), showing that EPO treatment before ischemia/reperfu-sion injury in rat decreased apoptotic cell death and was accompanied by decreased activities of caspase-3 and increased Bcl-2 protein expression.

Thus, our in vivo study provides strong evidence that EPO reduces Cisp-induced renal failure. These results indicate the central role of EPO in the protec-tion from the nephrotoxic effect of Cisp, especially in pretreatment condition, by an antigenotoxic and an antiapoptotic mechanism and point toward its clinical implications.

conclusion

In this study, we provide evidence that in addition to its well-known erythyropoieitic effect, EPO inhibits Cisp-induced tubular injury and apoptotic death in rat kidney. Further, we demonstrated that any type of treatment with EPO (co-, pre-, and post-treatment) decreased Cisp-induced apoptosis. However, the antiapoptotic action of EPO was more pronounced when administrated to rat 24 hours before Cisp treatment.

Declaration of interest

This research was supported by the Ministère Tunisien de l’Enseignement Supérieur et de la Recherche Scientifique et de la Technologie (Laboratoire de Recherche sur les Substances Biologiquement Compatibles: LRSBC).

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