Toxicology 230 (2007) 64–75

Parthenolide reduces cisplatin-induced renal damage

Heloısa D.C. Francescato a, Roberto S. Costa b,Cristoforo Scavone c, Terezila M. Coimbra a,∗

a Department of Physiology, Faculty of Medicine of Ribeirao Preto, Ribeirao Preto. Av. Bandeirantes,3900. CEP: 14049-900, Ribeirao Preto, SP, Brazil

b Department of Pathology, Faculty of Medicine of Ribeirao Preto, Ribeirao Preto. Av. Bandeirantes,3900. CEP: 14049-900, Ribeirao Preto, SP, Brazil

c Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo,Av. Prof Lineu Prestes, 1524, 05508-900, Sao Paulo, SP, Brazil

Received 22 June 2006; received in revised form 25 October 2006; accepted 30 October 2006Available online 6 December 2006

Abstract

Inflammatory events contribute to cisplatin-induced renal damage. Cisplatin promotes increased production of reactive oxygenspecies, which can activate nuclear factor-�B (NF-�B) that lead to increased expression of proinflammatory mediators whichcould intensify the cytotoxic effects of cisplatin. In this study, we evaluated the effect of parthenolide, a selective inhibitor ofNF-�B, on renal damage caused by cisplatin use. A total of 94 male Wistar rats were divided into six groups: Group A (18 rats)were treated with saline; Group B (12 rats) received dimethylsulfoxide plus saline (the solvent for parthenolide); Group C (12rats) received parthenolide (3 mg/kg) plus saline; Group D (20 rats) received cisplatin (5 mg/kg, i.p.); Group E (12 rats) receiveddimethylsulfoxide plus cisplatin (5 mg/kg, i.p.); and Group F (21 rats) received parthenolide (3 mg/kg) plus cisplatin (5 mg/kg, i.p.).Dimethylsulfoxide or parthenolide were administered at 24 h and 1 h prior to cisplatin injection, and again at 24 h and 48 h after. At2, 3 and 5 days after saline or cisplatin injection, blood and urine samples were collected for measurement of creatinine, sodiumand potassium and the kidneys removed for histological, morphometric, electrophoretic mobility shift assay (EMSA), apoptosis andimmunohistochemical studies. Cisplatin-treated rats presented higher plasma creatinine, as well as greater immunostaining for ED1

(macrophages/monocytes) and NF-�B in the renal cortices and outer medullae. The increase of NF-�B activation was confirmedby EMSA. Cisplatin-injected rats also presented higher urinary levels of lipid peroxidation and acute tubular necrosis. All of thesealterations were reduced by treatment with parthenolide. This effect seems to be related, at least in part, to the restriction of renalinflammatory process observed in parthenolide + cisplatin treated rats.© 2006 Elsevier Ireland Ltd. All rights reserved.

l failur

Keywords: Cisplatin; NF-�B; Inflammation; Parthenolide; Acute rena

∗ Corresponding author. Tel.: +55 16 3602 3021;fax: +55 16 3633 0017.

E-mail address: tmcoimbr@fmrp.usp.br (T.M. Coimbra).

0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.tox.2006.10.025

e; Nephrotoxicity

1. Indroduction

Cisplatin therapy is highly effective chemotherapeu-tic agent against a wide range of tumors (Meyer and

Madias, 1994). However, the primary side effect of cis-platin use, nephrotoxicity, has been reported to be adose-limiting factor (Blachley and Hill, 1981). Cisplatinis taken up by renal tubular cells, reaching its highest

ed.

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oncentrations in the proximal tubular cells of the innerortices and outer medullae, especially in the S3 seg-ent (Blachley and Hill, 1981; Chopra et al., 1982). Asresult, these are the major sites of cisplatin-induced

enal damage (Blachley and Hill, 1981; Leibbrandt et al.,995). Cisplatin provokes loss of tubular epithelial cellsy necrosis and apoptosis, followed by inflammatory cellnfiltration and fibroproliferative changes (Lierberthalt al., 1996; Taguchi et al., 2005). Higher number ofacrophages was observed in renal cortices and outeredullae from the cisplatin-treated rats (Francescato

t al., 2004; Taguchi et al., 2005). Macrophages canntensify the cytotoxic effects of cisplatin through theeneration of radical oxygen species, nitric oxide androinflammatory cytokines (Sean Eardley and Cockwell,005).

Cisplatin-induced kidney damage has been correlatedith the formation of free radicals and with oxidative

tress which can activate the nuclear factor-�B (NF-B) (Hannemann and Baumann, 1990; Martindale andolbrook, 2002; Xiao et al., 2003; Zhong et al., 1990).F-�B is present in the cytoplasm of every cell type in

n inactive form (Guijarro and Egido, 2001; Hill andreisman, 1995). Upon stimulation, NF-�B is releasedrom an inhibitory subunit (I�B) and translocates intohe nucleus, where it promotes the transcriptional acti-ation of target genes. A variety of extracellular stimulire able to induce the activation of NF-�B such asngiotensin II, lipopolysaccharide, free radicals releasedy damaged tissues, interleukin-1� and tumor necrosisactor-� (Guijarro and Egido, 2001; Hill and Treisman,995). NF-�B activation may contribute to renal dam-ge provoked by cisplatin by inducing the synthesis ofnflammatory substances that lead to kidney damage.arthenolide is a sesquiterpene lactone derived fromexican-Indian medicinal plants that can prevent the

ctivation of NF-�B by a variety of stimuli in cul-ured cells (Heinrich et al., 1998; Lopez-Franco et al.,002) and in some models of inflammatory diseasesEsteban et al., 2004; Sheehan et al., 2002). Althoughhere are evidences that parthenolide can interfere withome enzymes of the I�B phosphorylation complex, theolecular mechanisms of this drug is not well estab-

ished (Hehner et al., 1998; Herrera et al., 2005; Kwokt al., 2001).

In this study, we evaluated the effects that partheno-ide has on cisplatin-induced renal damage in the rat.

e used for this study the same dose (3 mg/kg) used by

euter et al. (2002) in rat, similar dose (3.5 mg/kg) waslso used by Lopez-Franco et al. (2002) and Estebant al. (2004) in mice. It was observed that partheno-ide, at low doses, functions as an antioxidant that can

ology 230 (2007) 64–75 65

reduce the oxidative stress in culture cells. In contrast,at high doses, parthenolide by itself induces increase insuperoxide anion and causes oxidative-stress-mediatedapoptosis (Li-Weber et al., 2005).

2. Materials and methods

2.1. Animals and experimental protocols

A total of 94 male Wistar rats, each weighing 180–200 g,were divided into six groups: Group A (control, n = 17), receiv-ing a single injection of 0.9% saline (1 ml/100 g, i.p.); GroupB (DMSO, n = 12), receiving 50% dimethylsulfoxide (DMSO)i.p. (the vehicle for parthenolide) once a day for four daysand a single injection of 0.9% saline (1 ml/100 g, i.p.); GroupC (parthenolide, n = 12), receiving a single injection of 0.9%saline (1 ml/100 g, i.p.) and parthenolide (3 mg/kg/day, i.p.)(Sigma Chemical Company, St. Louis, MO., USA), diluted inDMS0 50% solution. Parthenolide at 3 mg/kg was given in a3 mg/ml solution, i.e. at a volume of 1 mg/kg, once a day for 4days; Group D (cisplatin, n = 20), receiving a single injectionof cisplatin in a volume equal to that of the saline injections(5 mg/kg, i.p.; Quiral Quımica do Brasil, Juiz de Fora, MG,Brazil); Group E (DMSO + cisplatin, n = 12), receiving 50%DMSO, i.p., once a day for four days and a single injection ofcisplatin (5 mg/kg, i.p.); and Group F (parthenolide + cisplatin,n = 21), receiving parthenolide (Sigma Chemical Company)diluted in DMS0 50% solution (3 mg/kg in a 3 mg/ml solu-tion), once a day for four days and a single injection of cisplatin(5 mg/kg, i.p.). The experimental groups are summarized inTable 1. The DMSO or parthenolide were administered at 24 hand 1 h prior to, and again at 24 h and 48 h after, the injec-tion of saline or cisplatin, the same volume was used for bothinjections (100 �l/100 g/injection). On post-injection days 2, 3and 5, the rats were killed. The kidneys were perfused throughthe aorta with phosphate-buffered saline (PBS; 0.15 M NaCland 0.01 M sodium phosphate buffer, pH 7.4) and removed forhistological, morphometric and immunohistochemical, elec-trophoretic mobility shift assay (EMSA) and apoptosis studies.Blood was collected to measure plasma creatinine. The samekidneys were used for histology, morphometric, apoptosis andimmunohistochemistry studies, while different animals wereused for nuclear protein extraction.

All experimental procedures were conducted in accordancewith the principles and procedures outlined in the NationalInstitutes of Health (NIH) Guide for the Care and Use of Lab-oratory Animals, and the Animal Experimentation Committeeof the University of Sao Paulo at Ribeirao Preto School ofMedicine approved the study protocol.

2.2. Lipid peroxidation in urine samples

Lipid peroxidation was estimated by measuring malondi-aldehyde (MDA) in urine samples collected from rats killed at48 h after i.p. injections of saline or cisplatin, using the LipidPeroxidation Assay Kit (Calbiochem, San Diego, CA, USA)

66 H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75

Table 1Experimental protocols

Groups Treatment Number of rats killed on day 2 Number of rats killed on day 3 Number of rats killed on day 5

A Saline 6 (LP, H/M, I, RF, A) 6 (EMSA) 6 (H/M, I, RF)B Saline + 50% DMSO* 6 (RF) – 6 (RF)C Saline + parthenolide 6 (RF) – 6 (RF)D Cisplatin 8 (LP, H/M, I, RF, A) 6 (EMSA) 6 (H/M, I, RF)E Cisplatin + 50% DMSO 6 (RF) – 6 (RF)F Cisplatin + parthenolide 8 (LP, H/M, I, RF, A) 6 (EMSA) 7 (H/M, I, RF)

gy/morp

* Solvent for parthenolide; LP: lipid peroxidation assay; H/M: histoloEMSA: eletrophoretic mobility shift assay.

(Liu et al., 1997). To correct the variation in urine concentra-tion, urinary MDA was defined as the urinary level of MDAdivided by milligrams of urinary creatinine.

2.3. Nuclear protein extraction

Nuclear extracts were prepared as described by Nishio et al.,1998. The capsule was removed and the distinct regions wereidentified on the cut surface of a bisected kidney: a paler outerregion (the cortex) and a darker inner region (the medullae).The darker inner region (inner medullae) was then trimmed offwith scissors. Renal cortices and outer medullae from control,cisplatin, and cisplatin + parthenolide treated rats were homog-enized in ice-cold buffer containing 10 mM HEPES (pH 7.9),10 mM KCl, 0.1 mM ethylene diaminetetraacetic acid (EDTA),0.35 M sucrose, 0.5% nonidet P-40 (NP-40), 0.5 mM dithio-threitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)for 20 s. The mixture was centrifuged at 1500 × g for 25 minat 4 ◦C. The supernant was removed and the pellet was resus-pended in 10 ml of buffer containing 1 M HEPES (pH 7.9),2 M KCl, 0.5 M EDTA, 0.7 M sucrose and homogenized for20 s. This homogenate was centrifuged at 1500 × g for 30 minat 4 ◦C and the pellet resuspended in 10 ml of buffer contain-ing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA. After newcentrifugation, the pellet was resuspended in buffer contain-ing 20 mM HEPES, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mMEDTA, 25% glycerol, 0.5 mM DTT/PMSF) and incubated for20 min. This solution was centrifuged at 1500 × g for 30 minat 4 ◦C and the supernant was resuspended in buffer con-taining 20 mM HEPES, 50 mM KCl, 0.2 mM EDTA, 20%glycerol, 0.5 mM DTT/PMSF. The resulting supernants con-taining nuclear proteins were aliquoted and stored at −75 ◦C.Protein concentration was determined using the Biorad proteinreagent.

2.4. Electrophoretic mobility shift assay (EMSA)

Double stranded NF-�B consensus oligonucleotide (5′

– GAT CCA AGG GGA CTT TCC ATG GAT CCAAGG GGA CTT TCC AGT – 3′), were incubated with [�-32P]ATP at 35 ◦C for 45 min. The volume of the incubation was20 �l and the amount of [�32P] used was 17.95 × 10−9 Curie.The oligonucleotide end labeled with [�-32P] ATP was sepa-

hometry; I: Immuno-histochemistry; RF: renal function; A: apoptosis;

rated of unicorporated label with a G-50 sephadex spin column.The binding reaction was performed for 25 min at roomtemperature and contained 10 �g of nuclear protein extract.Samples were separated on a 5% SDS–PAGE gel (Berg et al.,2001). The autoradiographs were quantified by density analysisusing Image J NIH image software and expressed as arbitraryunits.

2.5. Antibodies

We used the following primary antibodies: a mouseanti-rat ED1 antibody to a cytoplasmic antigen present inmacrophages and monocytes (Serotec, Oxford, UK), a mon-oclonal anti-pJNK (c-jun-N-terminal kinase) antibody (SantaCruz Biotechnology, Santa Cruz, CA, USA) and a polyclonalNF-�B (p65) antibody (Santa Cruz Biotechnology).

2.6. Immunohistochemical studies

The kidneys from control, cisplatin-treated and patheno-lide + cisplatin-treated rats killed on postinjection days 2 and5 were submitted to immunohistochemical studies. The kid-neys were perfused through the aorta with PBS (pH 7.4) untilthey were blanched. The kidneys were then perfused with4% paraformaldehyde, fixed in the same for 2 h, postfixedin Bouin’s solution for an additional 4–6 h, rinsed with 70%ethanol to eliminate picric acid, dehydrated through a gradedalcohol series, embedded in paraffin, cut into 3-�m sections,deparaffinized and submitted to immunohistochemical stain-ing (Baroni et al., 2000; Kliem et al., 1996). The same fixationmethod was used for both immunohistochemical and histolog-ical studies.

The sections were incubated at 4 ◦C overnight with 1/200anti-NF-�B, polyclonal and 1/30 anti-pJNK, monoclonal, orfor 1 h with 1/1000 anti-ED1 monoclonal antibodies. The reac-tion product was detected with an avidin–biotin–peroxidasecomplex (Vector Laboratories, Burlingame, CA, USA). Thecolor reaction was developed with 3,3′-diaminobenzidine

(DAB; Sigma Chemical Company, St. Louis, MO, USA). TheED1 sections were counterstained with methyl green (SigmaChemical Company), dehydrated and mounted. The NF-�Band pJNK sections were counterstained with Harris hema-toxylin. We also performed new immunohistochemical studies

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or NF-�B using slices without counterstained in order to checkhe presence of staining in the nucleus. Nonspecific proteininding was blocked by incubation with 20% goat serum inBS for 20 min. Negative controls were created by replacing

he primary antibody with normal mouse immunoglobulin GIgG) or rabbit IgG, for monoclonal and polyclonal antibodies,t equivalent concentration. For evaluation of immunoperox-dase staining for NF-�B and pJNK, the renal cortices andhe outer medullae were graded semiquantitatively throughxamination of, respectively, 20 grid fields and 15 grid fieldseach field measuring 0.245 mm2), and the mean score per.245 mm2 per kidney was calculated (Kliem et al., 1996).he scores mainly reflected changes in the extent, rather than

he intensity, of the staining and depended on the percentagef grid fields showing positive staining: 0, less than 5%; I,–25%; II, 25–50%; III, 50–75%; and IV, >75%. It has beenemonstrated that the semiquantitative scoring system is notnly reproducible among different observers, but also that theata also are highly correlated with those obtained throughomputerized morphometry (Kliem et al., 1996). Infiltratingacrophages and monocytes were counted in the cortical and

uter medullary tubulointerstitium through examination of 35rid fields, measuring 0.245 mm2 each (20 from the renal cor-ices and 15 from the outer medullae), and the mean counts per.245 mm2 per kidney were calculated.

.7. In situ detection of apoptosis using the TUNEL assay

The kidneys obtained from the rats killed on postinjectionay 2 were stained with terminal deoxynucleotidyl transferaseTdT)-mediated deoxyuridine 5 triphosphate-biotin nick endabeling (TUNEL) using a commercial kit (Oncogene Researchroducts, Boston, MA, USA) (34). Diaminobenzidine reactsith the labeled sample, generating an insoluble colored sub-

trate at the site of DNA fragmentation. The counterstainingas performed with methyl green (Sigma Chemical Company).issues treated with DNase I were used as positive controls, andections stained without terminal nucleotidyl transferase weresed as negative controls. The TUNEL-positive cells in theortical and outer medullary tubulointerstitium were countedn 35 grid fields, measuring 0.245 mm2 each (20 from the renalortices and 15 from the outer medullae), and the mean countser 0.245 mm2 per kidney were calculated.

.8. Light microscopy and morphometry

Histological sections (4-�m thick) were stained with Mas-on’s trichrome and examined under a light microscope.ubulointerstitial damage was defined as tubular necrosis,

nflammatory cell infiltrate, tubular lumen dilation or tubu-ar atrophy. Damage was graded according to Shih et al.

1988) on a scale of 0–4 (0 = normal; 0.5 = small focal areas;= involvement of less than 10% of the cortices and outeredullae; 2 = 10–25% involvement of the cortices and outeredullae; 3 = 25–75% involvement of the cortices and outeredullae; 4 = extensive damage involving more than 75% of

ology 230 (2007) 64–75 67

the cortices and outer medullae). The morphometric studieswere performed with a still camera connected to an imageanalyzer (KS 300; Kontron, Eching, Germany). In sectionsobtained from each kidney, the outer medullae were evaluatedin 20 grid fields measuring 0.245 mm2 each. The area surround-ing the tubular lumen was traced manually on a video screenand determined by computerized morphometry, thereby allow-ing the mean area per kidney to be determined (Baroni et al.,2000) and the mean area values were given in �m2. We alsoevaluated the number of tubules with cellular necrosis fromrenal cortices and outer medulla by grid fields (0.245 mm2).

2.9. Renal function studies

Beginning on post-injection day 1 or on post-injection day4, the rats were placed in metabolic cages, and 24-hour urineand blood samples were collected. The blood samples were col-lected in heparin-treated syringes. The 24-hour urine sampleswere used to measure creatinine, sodium and potassium, andplasma creatinine was measured by the Jaffe method (Haygen,1953). Sodium and potassium were measured in plasma andurine using flame photometry (model 262; Micronal, SaoPaulo, Brazil), urine osmolality was assessed by freezing pointdepression using a Osmometer (Fike OS, Norwood, Miss.,USA). The glomerular filtration rate (GFR) was evaluated byinulin clearance. On postinjection day 5, inulin clearance stud-ies were conducted in order to measure GFR in 6 controlrats, 6 cisplatin-treated rats and 6 cisplatin + parthenolide-treated rats. The rats were anesthetized with an i.p. injectionof sodium thionembutal (50 mg/kg). After tracheostomy, thefemoral artery and vein were cannulated to collect blood sam-ples and inject fluids. The ureters were cannulated to collecturine. Inulin was administered in a priming dose (12 mg/100 g),followed by a maintenance dose (30 mg/100 g/h). After anapproximately 60-minutes stabilization period, urine was col-lected for 1 h, and blood samples were collected at 30 min andat 1 h in heparin-treated syringes. These samples were used toassess levels of sodium, potassium and inulin. Inulin was mea-sured in plasma and urine using the method described by Fuehret al., 1955.

2.10. Statistical analysis

Data concerning urinary MDA content, plasma creatininelevels, urine volume, fractional excretion of sodium and potas-sium and tubular cell necrosis as well the data obtained throughhistology and in immunohistochemical studies for pJNK andNF-�B were analyzed statistically using the nonparametricKruskal–Wallis test followed by the Dunn post-test becausethey were not normally distributed. Those data are expressedas median and interquartile range (25–75%). Analysis of vari-

ance with the Newman–Keuls multiple comparisons test wasused for data regarding GFR, macrophage counts, EMSA andfor data obtained through morphometric studies. Those dataare expressed as mean ± S.E.M. The level of statistical signif-icance was set at P < 0.05.

68 H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75

Fig. 2. (A) NF-�B expression in nuclear extracts of cells of renal cor-tex and outer medullae from control rats (first three) and from ratstreated with cisplatin (CP, second three) or parthenolide + cisplatin

32

Fig. 1. Urine malondialdehyde (MDA) levels for saline-treated (con-trol), cisplatin (CP)-treated and parthenolide + CP-treated rats at 2 daysafter saline or CP injections. Horizontal line represent the median**P < 0.01 compared to controls; #P < 0.05 compared to CP, N = 7.

3. Results

3.1. Urinary MDA levels

Urinary MDA levels, which indicate the extent oflipid peroxidation, are shown in Fig. 1. Urinary MDAlevels, expressed as MDA urine level divided by mil-ligrams of urine creatinine, were significantly higher inthe kidneys of cisplatin-injected rats than in those of con-trol rats. This increase was reduced by treatment withparthenolide (Fig. 1).

3.2. Electrophoretic mobility shift assay (EMSA)

On post-injection day 3, the cisplatin-treated rats alsopresented higher NF-�B content in the nuclear extractsof renal cortices and outer medullae compared to thecontrols. This alteration was prevented by treatment withparthenolide (Fig. 2A and B).

3.3. Immunohistochemical studies

In the immunohistochemical studies performed onpost-injection days 2 and 5, higher numbers of ED1-positive cells and greater NF-�B and pJNK expressionwere seen in the renal cortices and outer medullae ofcisplatin-treated rats than in those of controls. The stain-ing for NF-�B was present in the nucleus and in thecytoplasm of the cells of renal cortex and outer medul-lae from cisplatin-treated rats (Fig. 3B, inset). We did notobserve staining in the nucleus of these cells in rats fromcontrol groups and in parthenolide + cisplatin-treated

animals (Fig. 3A and C, inset). On post-injection day2, parthenolide + cisplatin-treated rats presented lowernumbers of ED1 positive cells in the renal cortices andouter medullae compared to cisplatin-only group rats,

(P + CP, third three). Nuclear extracts were incubated with P-NF-�Bprobe, followed by EMSA; (B) densitometric analysis of the p50/p65bands presented in panel A. Results are expressed as mean ± S.E.M.,**P < 0.01 compared to control; ##P < 0.01 compared to CP, N = 6.

and this reduction persisted on postinjection day 5. Treat-ment with parthenolide also prevented the increasedexpression of NF-�B induced by cisplatin in the renalcortices on postinjection day 5 and in the outer medullaeon post-injection days 2 and 5. However, parthenolidetreatment had no effect on the increase of pJNK expres-sion induced by cisplatin injection (Tables 2 and 3;Figs. 3–5).

3.4. In situ detection of apoptosis

On post-injection day 2, the cisplatin-treated rats pre-sented higher numbers of TUNEL-positive cells in therenal cortices and outer medullae compared to the con-trols. This alteration was attenuated by treatment withparthenolide (Figs. 5 and 6).

3.5. Light microscopy and morphometric studies

The light microscopy study of the kidneys from ratskilled on post-injection days 2 and 5 revealed renal dam-age, characterized by marked tubular lumen dilation dueto flattening of tubular cells with brush border loss,inflammatory cell infiltration into the interstitium, tubu-lar cell necrosis, and vacuolization. On post-injectiondays 2 and 5, these alterations were less intense inthe kidneys obtained from rats treated with partheno-

lide + cisplatin. There is also smaller number of tubulewith cell necrosis in parthenolide + cisplatin treated ratscompared to cisplatin-only group rats (Fig. 7). The mor-phometric analyses performed on post-injection days 2

H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75 69

F rats ati g for Ni nal mag

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ig. 3. Immunolocalization of NF-�B in the renal outer medullae ofnjections. Inset: slices without counterstaining. Note that the staininntense in CP-treated rats than in parthenolide + CP-treated rats. Origi

nd 5 showed an increase in the tubular lumen area ofhe renal outer medullae of rats treated with cisplatin. Onost-injection day 5, this alteration was less pronouncedn the kidneys of rats treated with parthenolide + cisplatinompared to cisplatin only group rats (Table 4).

.6. Renal function

On post-injection days 2 and 5, cisplatin-injectedats presented elevated plasma creatinine levels andncreased urine volume and greater fractional excretion

able 2umber of ED1-positive cells per 0.245-mm2 grid field and immunostainin

Group A, control), cisplatin (CP)-treated (Group D) and parthenolide + CP-tre

roups control

DaysED1 5.21 ± 0.76NF-�B 0.47 (0.31; 0.59)pJNK 0.75 (0.69; 1.13)

DaysED1 4.46 ± 0.77NF-�B 0.43 (0.26; 0.65)pJNK 0.77 (0.71; 1.00)

ata for ED1 are expressed as mean ± S.E.M., and data for NF-�B and pJNP < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. #P < 0.05,

5 days after saline (A), cisplatin (CP) (B) or parthenolide + CP (C)F-�B (cytoplasm and nucleus, inset) and tubular necrosis are morenification: ×280, inset ×840.

of sodium and potassium. On post-injection day 2,these changes were less pronounced in the partheno-lide + cisplatin-treated rats compared to cisplatin-onlygroup rats (Table 5). In addition, the increases inplasma creatinine levels and fractional excretion ofsodium, as well the reduction in GFR, observed on post-injection day 5 were also attenuated by the treatment

with parthenolide (Table 6 and Fig. 8). Parthenolideprevented the decrease in urine osmolality cisplatin-induced on day 2 after the injection. However, thisalteration was not modified by parthenolide-treatment

g scores for NF-�B and pJNK in the renal cortices of saline-treatedated rats (P + CP, Group F) at 2 and 5 days after saline or CP injections

CP P + CP

16.28 ± 1.86*** 6.33 ± 1.15###

1.46 (1.11; 1.56)** 1.14 (0.95; 1.22)1.06 (1.03; 1.08)* 0.86 (0.83; 1.14)

25.17 ± 5.16** 4.85 ± 1.14###

1.66 (1.30; 1.95)** 1.12 (0.89; 1.20)*#

1.05 (0.92; 1.13)* 1.03 (0.97; 1.18)*

K are expressed as median and interquartile range (25–75%); n = 6.###P < 0.001 compared to CP group.

70 H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75

Table 3Number of ED1-positive cells per 0.245-mm2 grid field and immunostaining scores for pJNK and NF-�B in the outer medullae of saline-treated(Group A, control), cisplatin (CP)-treated (Group D) and parthenolide + CP-treated rats (P + CP, Group F) at 2 and 5 days after saline or CP injections

Groups control CP P + CP

2 DaysED1 4.10 ± 0.53 25.78 ± 6.19** 6.83 ± 1.45##

NF-�B 0.53 (0.37; 0.70) 1.49 (1.00; 1.71)* 1.00 (0.81; 1.14)#

pJNK 0.90 (0.78; 1.05) 1.17 (1.06; 1.27)* 1.12 (0.80; 1.44)*

5 DaysED1 4.52 ± 0.62 56.55 ± 8.90*** 19.45 ± 2.15###

NF-�B 0.64 (0.50; 0.70) 1.81 (1.55; 1.96)** 1.08 (0.98; 1.15)#

pJNK 0.90 (0.79; 1.10) 2.00 (1.88; 2.12)* 1.99 (1.89; 2.20)*

Data for ED1 are expressed as mean ± S.E.M., and data for NF-�B and pJNK are expressed as median and interquartile range (25–75%); n = 6.*P < 0.05, **P < 0.01, and ***P < 0.001 compared to control group. #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to CP group.

Fig. 4. Immunolocalization of ED1 in the renal outer medullae of rats killed at 5 days after saline (A), cisplatin (CP) (B) or parthenolide + CP(C) injections. Note that the staining for ED1 is more intense in CP-treated rats than in saline-treated or parthenolide + CP-treated rats. Originalmagnification: ×280.

Table 4Score for tubulointerstitial lesions (TIL) and tubular lumen area (TLA) in the renal outer medullae of saline-treated (Group A, control), cisplatin(CP)-treated (Group D) and parthenolide + CP-treated rats (P + CP, Group F) at 2 and 5 days after saline or CP injections

Control (n = 6) CP-2d (n = 7) P + CP-2d (n = 7) CP-5d (n = 7) P + CP-5d (n = 7)

TIL, score 0 3.00*** (2.00; 3.50) 1.00**,## (0.75; 1.50) 4.00*** (3.50; 4.00) 3.00*** (3.00; 3.00)TLA (�m2) 277.45 ± 31.88 670.60* ± 102.58 494.60 ± 44.32 1678.00*** ± 84.49 837.50**,## ± 124.40

Data for TIL are expressed as median and interquartile range (25–75%), and data for TLA are expressed as mean ± S.E.M. *P < 0.05, **P < 0.01,and ***P < 0.001 compared to control group. #P < 0.05, and ###P < 0.001 compared to CP group.

H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75 71

Fig. 5. Immunolocalization of pJNK (A and B) and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5 triphosphate-biotin nick endlabeling (TUNEL)-positive cells (C and D) in the renal outer medullae of rats killed at 2 days after saline cisplatin (CP) (A and C) or parthenolide + CP(B and D) injections. Original magnification: ×280.

Table 5Parameters of renal function for saline (Group A, control)-, DMSO (Group B), parthenolide (P, Group C), cisplatin (CP, Group D)-, DMSO + CP-(Group E), and parthenolide + CP (P + CP, Group F)-treated rats at 2 days after saline or CP injections

Pcreat V Uosm FENa+ FEK+

control (n = 6) 0.53 (0.45; 0.66) 9.00 (7.50; 11.00) 1661 (1485; 1882) 0.62 (0.59; 0.72) 31.12 (27.77; 36.17)DMSO (n = 6) 0.54 (0.28; 0.66) 11.00 (8.55; 15.50) 1509 (1056; 1566) 0.52 (0.48; 0.61) 46.61 (33.13; 52.63)P (n = 6) 0.58 (0.38; 0.59) 10.00 (9; 16.03) 1444 (1099; 1835) 0.68 (0.50; 0.71) 49.87 (37.19; 59.76)CP (n = 8) 1.09** (0.88; 1.48) 16.00* (13.50; 21.00) 1099* (748; 1288) 2.52** (1.21; 3.76) 167.70** (145.50; 204.40)DMSO + CP

(n = 6)1.12** (0.90; 1.2) 13* (10.51; 14.62) 1156* (889; 1658) 2.36** (1.43; 2.66) 161.38** (99.75; 188.92)

P + CP (n = 8) 0.73# (0.63; 0.86) 8.00# (7.00; 9.5) 1699# (1125; 1768) 0.83# (0.38; 1.62) 97.38# (60.26; 142.20)

Pcreat, plasma creatinine (mg/dl); V, urinary volume (ml/24 h); FE, fractional excretion. Data are expressed as median and interquartile range(25–75%). *P < 0.05, **P < 0.01 compared to control group. #P < 0.05 compared to CP group.

Table 6Parameters of renal function for saline (Croup A, control)-, DMSO (Group B), parthenolide (P, Group C), cisplatin (CP, Group D)-, DMSO + CP-(Group E), and parthenolide + CP (P + CP, Group F)-treated rats at 5 days after saline or CP injections

Pcreat V Uosm FENa+ FEK+

Control (n = 6) 0.45 (0.43;0.53) 9.00 (7.00; 11.00) 1370 (1078; 1686) 0.45 (0.39; 0.53) 39.82 (39.46; 47.07)DMSO (n = 6) 0.55 (0.44; 0.82) 11.00 (8.22; 15.50) 1036 (1031; 1858) 0.42 (0.35; 0.56) 41.39 (35.11; 41.63)P (n = 6) 0.50 (0.41; 0.62) 9.00 (7.60; 13.83) 1254 (1122; 1366) 0.61 (0.54; 0.73) 39.96 (31.15; 49.32)CP (n = 6) 1.09** (0.91; 1.30) 37.60* (31.00; 39.50) 599.5** (499; 640) 2.34* (1.59; 4.46) 180.20* (155.30; 201.30)DMSO + CP

(n = 6)1.15** (0.89; 1.20) 30* (27.25; 31.60) 586** (478; 765) 1.89* (1.38; 1.99) 161.11* (89.15; 181.90)

P + CP (n = 7) 0.84*,# (0.77; 0.94) 42.00** (35.00; 43.00) 630* (613.00; 634.00) 1.38*,# (1.09; 2.08) 97.24**,# (43.98; 138.20)

Pcreat, plasma creatinine (mg/dl); V, urinary volume (ml/24 h); FE, fractional excretion. Data are expressed as median and interquartile range(25–75%). *P < 0.05, **P < 0.01 compared to control group. #P < 0.05 compared to CP group.

72 H.D.C. Francescato et al. / Toxicology 230 (2007) 64–75

Fig. 6. Number of TUNEL-positive cells in renal cortices and outermedullae from saline-treated (control), cisplatin (CP)-treated andparthenolide + CP-treated rats (P + CP), 2 days after saline or CP injec-tions by grid fields measuring 0.245 mm2 each. Data are expressedas mean ± S.E.M.; **P < 0.01, ***P < 0.001 compared to controls;#P < 0.05 compared to CP (N = 6).

Fig. 7. Number of tubules with cellular necrosis in renal cortices andouter medullae from saline-treated (control) (median = 0), cisplatin(CP)-treated and parthenolide + CP-treated rats (P + CP), 5 days aftersaline or CP injections by grid fields measuring 0.245 mm2 each. Box

Fig. 8. Glomerular filtration rate (GFR) for saline-treated (control),

plots represent median (horizontal line) with quartile range, 25–75%(box) and range (T-bars). *P < 0.05 vs. control, **P < 0.01, ***P < 0.001compared to controls; #P < 0.05, ###P < 0.001 compared to CP (N = 6).

on day 5 post-injection (Table 6). In control rats, theadministration of DMSO or parthenolide alone had noeffect on renal function or structure (Tables 5 and 6).

4. Discussion

Activation of NF-�B might play a role in the

cisplatin-induced renal damage by inducing the syn-thesis of inflammatory substances (cytokines, growthfactors, adhesion molecules, and enzymes, as well aschemotactic factors for macrophages and monocytes)

cisplatin (CP)-treated and parthenolide + CP-treated rats (P + CP),5 days after saline or CP injections. Data are expressed asmean ± S.E.M.; *P < 0.05, ***P < 0.001 compared to controls;##P < 0.01 compared to CP (N = 6).

that provokes kidney damage. In the present study theincreased NF-�B activation in cisplatin-injected rats wasconfirmed by EMSA performed with nuclear extractsof renal cortices and outer medullae from these rats.Our EMSA studies showed that the treatment withparthenolide prevents cisplatin-induced increase in NF-�B binding nuclear protein, on day 3 after cisplatininjection, while this treatment only provoked a partialreduction of the immunostaining for NF-�B in renalcortex and outer medullae from the animals cisplatin-injected on days 2 and 5 after the injection. Probably,because EMSA is more specific to evaluate the NF-�Bactivation. This assay was performed only with nuclearextracts of renal cortex and outer medulla. Therefore,the results only reflect NF-�B nuclear translocation andbinding to DNA, while the results of the immunohisto-chemical studies were expressed in scores which alsoreflect the immunostaining for the inactive NF-�B formpresent in the cytoplasm of the cells. Reuter et al. (2002)also provoked the inhibition of NF-�B in rats with a doseof 3 mg/kg of parthenolide, similar dose (3.5 mg/kg) wasused by Lopez-Franco et al. (2002) and Esteban et al.(2004) to block NF-�B in mice.

The immunohistochemical studies also revealedhigher numbers of tubulointerstitial ED1-positive cells(macrophages/monocytes) in the renal cortices and outermedullae of the cisplatin-only group rats, whether onpost-injection day 2 or on post-injection day 5. Thisalteration was prevented or attenuated by the treatmentwith parthenolide. Interstitial macrophage infiltrationinto the kidneys is seen in all types of progressive renaldisease (Eddy, 1996; Geleilete et al., 2002; Nikolic-Patterson et al., 1994). Since macrophages are capable

of releasing peptides such as tumor growth factor(TNF)-�, interleukin-1�, endothelin, and angiotensinII, they might be involved in the inflammatory pro-cess and fibrosis observed in cisplatin treated rats. In

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H.D.C. Francescato et al

ddition, macrophages can produce various types of col-agen and other extracellular matrix components such asbronectin (Eddy, 1996; Vaage and Lindblad, 1990). In aecent study involving double-label immunohistochem-stry, we showed that proliferating macrophages (ED1-nd PCNA- positive cells) accounted for 88.16 ± 0.59%nd 72.44 ± 4.61% of the total macrophage populationsn the renal cortices of rats killed at 5 or 30 days, respec-ively, after treatment with gentamicin (Geleilete et al.,002). These cells express higher levels of CD68 andajor histocompatibility complex class II antigens than

o nonproliferating macrophages, indicating that theyre functionally active.

Urinary MDA levels were significantly higher inisplatin-only group rats than in the saline-only groupats, indicating that the free radicals released by dam-ged tissues might have contributed, at least in part, tohe observed activation of the NF-�B pathways in theseats. It was observed in culture cells that parthenolide,t low doses, functions as an antioxidant that can reducehe oxidative stress. In contrast, at high doses, partheno-ide by itself induces increase in superoxide anion andauses oxidative-stress-mediated apoptosis (Li-Webert al., 2005). In addition, parthenolide may have somenfluence on the regulation of intracellular redox state,ncreasing glutathione content (Herrera et al., 2005). Weound that the cisplatin-induced increase in MDA levelsas reduced by the addition of parthenolide (3 mg/kg),

ndicating that treatment with this dose of parthenolideffsets cisplatin-induced lipid peroxidation. In cisplatin-njected rats, NF-�B might have been activated by freeadicals released by damaged tissues or by inflammatoryells present in the renal tissue. Although the mech-nisms involved in the effect that parthenolide has onhe NF-�B system have not been fully established, therere evidences that parthenolide can interfere with somenzymes of the I�B phosphorylation complex (Hehnert al., 1998; Kwok et al., 2001).

It was also found that parthenolide can activate theNK pathway independently of NF-�B inhibition inreast cancer cells (Nakshatri et al., 2004). pJNK activa-ion can result in apoptosis and inflammation (Kaminska,005; Xia et al., 1995), which may contribute to the lostf the renal function. However, the effect of partheno-ide in JNK pathway was observed in cancer cells andan be different in normal cells. Our studies showed thatarthenolide treatment had no effect on pJNK activa-ion, besides this, this treatment provoked a decrease in

he number of apoptotic cells in renal cortex and outer

edullae from the cisplatin-treated rats. In vitro studieslso show that parthenolide can inhibit a common stepn NF-�B activation by preventing the TNF-�-induced

ology 230 (2007) 64–75 73

induction of I�B kinase (IKK)� and IKK�, withoutaffecting the activation of p38 and pJNK (Hehner et al.,1999). Therefore, the parthenolide-induced decrease inthe number of apoptotic cells may occur by a mecha-nism independent of pJNK activation such as decreasingNF-kB activation and oxidative stress induced by cis-platin. Meldrum et al. (2002) found that NF-�B mediatesthe ischemia-induced renal tubular cells apoptosis. Onthe other hand, Ralstin et al. (2006) and Steele et al.(2006) observed that parthenolide can induce apoptosisin cancer cells.

In the present study, the rats treated with cisplatin onlyalso presented renal functional and structural alterationswhich were reduced by the addition of parthenolide tothe treatment. This effect can also be related to the factthat parthenolide reduces the activation of NF-�B. Inthe cisplatin + parthenolide group rats killed on post-injection days 2 and 5, the increases in plasma creatininelevels, as well as the increases in fractional excretionof sodium and potassium, were less pronounced thanin the cisplatin-only group rats killed on the same daysafter cisplatin injection. Glomerular filtration rate eval-uated by inulin clearance at day 5 was also higher inparthenolide + cisplatin group rats compared to ciplatin-only group rats. In addition, the presence of tubularnecrosis and dilation in renal cortices and outer medul-lae were more intense in the cisplatin-only group ratsthan in the parthenolide + cisplatin group rats. This sug-gests that parthenolide plays a role in the inflammatoryprocess occurring during cisplatin-induced acute tubu-lar necrosis, minimizing the alterations in renal functionand structure. However, treatment with parthenolide didnot modify cisplatin-induced alterations in urine volumeand osmolality observed on day 5 after the injection,probably because these disturbances were provoked bydifferent mechanisms. There is some evidence that cis-platin can decrease the expression of collecting ductwater channels (aquaporins 2 and 3) (Kishore et al.,2000).

There are several studies showing the sequence ofevents that results in renal toxicity induced by cisplatin(Taguchi et al., 2005). I was observed that cisplatininduces first the oxidative stress and then cytotoxicityand increases cytokines expression. Inflammatory andfibroproliferative events were observed latter. We veri-fied in previous studies an increase of NF-�B expressionat 24 h after the cisplatin injection and in pJNK expres-sion at 48 h (Francescato et al., 2004). However, these

alterations were more intense on day 5. Residual areas offibrosis are observed in renal cortex and outer medullaefrom the kidneys of cisplatin-injected rats (Francescatoet al., 2004; Taguchi et al., 2005) and can contribute

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74 H.D.C. Francescato et al

to permanent loss of nephrons and abnormalities ofrenal function with long-term implications. Therefore,the reduction of the inflammatory process can prevent orattenuate the fibrosis and the impaired recovery of renalfunction observed post-cisplatin treatment.

The protective effect of parthenolide on the renalfunction and structure of cisplatin-treated rats wasincomplete, despite the fact that the parthenolide treat-ment was started prior to the injection of cisplatin. Thismight be because parthenolide is more effective at reduc-ing inflammation than at preventing tubular lesions, asevidenced by the fact that the rats treated with the combi-nation of cisplatin and parthenolide also presented acutetubular necrosis, albeit of a lesser intensity than thatobserved in cisplatin-only group rats.

Taken together, these data show that treatment withparthenolide, an NF-�B inhibitor, reduces the renal dam-age induced by cisplatin. This effect might be related,at least in part, to the reduction in the inflammatoryresponse observed in the cisplatin + parthenolide-treatedrats. These data also suggest that the inhibition of the NF-�B pathway may represent a novel therapeutic approachfor reduce the cisplatin-induced renal damage.

Acknowledgments

The authors are grateful to Cleonice G. A. da Silva,Adriana L. G. de Almeida, Erica Delloiagono, andLarissa de Sa Lima for their technical assistance.

Grants: Financial support provided by the Fundacaode Amparo a Pesquisa do Estado de Sao Paulo (FAPESP,Foundation for the Support of Research in the state ofSao Paulo; grant nos: 03/07488-7; 04/00735-1).

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