The Effect of Sildenafil on Cisplatin Nephrotoxicity in RatsBadreldin H. Ali1, Aly M. Abdelrahman1, Suhail Al-Salam2, Munjusha Sudhadevi2, Ahmed S. AlMahruqi1, Ishaq S. Al-Husseni3, Sumiya Beegam1,

Subramanian Dhanasekaran4, Abderrahim Nemmar4 and Mansour Al-Moundhri5

1Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanateof Oman, 2Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab

Emirates, 3Department of Physiology, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman,4Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates, and

5Department of Medicine, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman

(Received 9 January 2011; Accepted 26 April 2011)

Abstract: Sildenafil, the first drug for erectile dysfunction, has cardiopulmonary protective actions. A recent study hasreported that sildenafil given intraperitoneally (i.p.) attenuated cisplatin (CP)-induced nephrotoxicity. Here, we evaluatedwhether sildenafil, given by two different routes and at two different doses, can attenuate CP-induced nephrotoxicity andwould also affect renal haemodynamics in CP-treated rats. Six groups of rats were treated with saline (controls), CP [5 mg ⁄ kg,intraperitoneally (i.p.) once], sildenafil (0.4 mg ⁄ kg ⁄ day, i.p. for 5 days), sildenafil (0.4 mg ⁄ kg ⁄ day i.p. for 5 days) plus CP(5 mg ⁄ kg, i.p., once), sildenafil [10 mg ⁄ kg ⁄ day, subcutaneous (s.c.) for 5 days] or sildenafil (10 mg ⁄ kg ⁄ day, s.c. for 5 days)plus CP (5 mg ⁄ kg, i.p. once). Five days after the end of the treatments, urine was collected from all rats, which were thenanaesthetized for blood pressure and renal blood flow monitoring. This was followed by intravenous (i.v.) injection of norepi-nephrine for the measurement of renal vasoconstrictor responses. Thereafter, blood and kidneys were collected for measure-ment of several biochemical, functional and structural parameters. CP reduced body-weight and renal blood flow but did notaffect norepinephrine-induced renal vasoconstriction. It increased the plasma concentrations of urea and creatinine, andreduced creatinine clearance. CP caused extensive renal tubular necrosis, increased urine volume and N-acetyl-b-D-glucosaminidase activity. When sildenafil (0.4 mg ⁄ kg ⁄ day, i.p. for 5 days) was combined with cisplatin, there was a dramaticimprovement in renal histopathology, reduction in N-acetyl-b-D-glucosaminidase and increase in renal blood flow. However,sildenafil (10 mg ⁄ kg ⁄ day, s.c. for 5 days) did not affect CP nephrotoxicity, suggesting the importance of dose and route selec-tion of sildenafil as a nephroprotectant.

Cisplatin (CP) is widely used, often in combination withradiation and other drugs, against malignant, solid, epithelialtumours [1–3]. The major limitation of its use is the develop-ment of resistance by tumours [4] and the cumulative, dose-dependent severe nephrotoxicity that can culminate in acuterenal failure [5,6]. Despite intensive prophylactic measures,irreversible renal damage occurs within days in approxi-mately one-third of CP-treated patients [7–9].

The mechanism of action of CP-induced nephrotoxicity isnot fully understood [6,10]. Renal tubular injury and death(apoptosis and necrosis) are the main pathological events inCP nephrotoxicity [6]. Although the exact mechanism under-lying CP-induced tubular death is not fully known, the drugis known to damage cell mitochondria, arrest the cell cyclein the G2 phase, inhibit ATPase activity and alter the cellulartransport system. All of these actions eventually induceapoptosis, inflammation, necrosis and cell death [3,4,11]. Thenephrotoxicity of CP is mediated, at least partly, via c-glut-amyl transpeptidase (GGT), p53 and enhanced tumour

necrosis factor-a (TNF-a) production [8,12,13]. It has alsobeen proposed that CP binds to glutathione (GSH) and thesubsequent CP-GSH complex in tubules stimulates renallipid peroxidation [14,15]. CP interacts with thiol groups andmacromolecules, and causes significant oxidant loading onthe kidney through both xanthine oxidase activation andimpaired antioxidant defence system, which results in accel-erated oxidation reactions in the kidney tissues [16,17]. Ni-trosative stress is also involved [18]. However, the source ofthe O�2 has not been identified.

Many drugs with varying mechanisms of action have beentested to see whether they could ameliorate or prevent exper-imental CP nephrotoxicity [6,19]. Among these, sildenafilhas recently been tested [20]. In this single report, the drugwas given intraperitoneally (i.p.) at a dose of 0.4 mg ⁄ kgimmediately after a single i.p. injection of CP (5 mg ⁄ kg, i.p.)and was reported to attenuate the nephrotoxicity. However,the effect of sildenafil, given at different doses and by differ-ent routes on CP-induced nephrotoxicity, and its effect onrenal haemodynamics in CP-treated rats has not beenreported so far.

In the present work, we aimed to verify the possible neph-roprotective action of sildenafil, and further, to see whetherthe drug, given at different doses and by different routes,would also protect against CP nephrotoxicity and would

Author for correspondence: Abderrahim Nemmar, Department ofPhysiology, Faculty of Medicine and Health Sciences, United ArabEmirates University, P.O. Box 17666, Al Ain, United Arab Emirates(fax +971 3 7671966, e-mail anemmar@uaeu.ac.ae; anemmar@hotmail.com).

Basic & Clinical Pharmacology & Toxicology, 109, 300–308 Doi: 10.1111/j.1742-7843.2011.00724.x

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affect renal haemodynamics in CP-treated rats. In view ofthe kinetic peculiarities of sildenafil [21], we used in the pres-ent work a different route (subcutaneous, s.c.) and dose(10 mg ⁄ kg, i.p.) to those employed by Lee et al. [20], in addi-tion to those used by them.

Materials and Methods

Animals. Male Wistar rats of 250–300 g body-weight were obtainedfrom the Animal House of Sultan Qaboos University. The rats werehoused in polypropylene cages and maintained under standard con-ditions [22 € 2�C; 12 L:12 D cycle (lights on at 07:00 hr); and about60% humidity]. Standard laboratory chow diet containing normalsodium (Oman Mills, Muscat, Oman) and tap water was providedad libitum. The study was approved by the University Animal EthicalCommittee and was conducted in conformity with international lawsand policies (EEC Council directives 86 ⁄ 609, OJL 358, 1 December,12, 1987; NIH Guide for the Care and Use of Laboratory Animals,NIH Publications No. 85-23, 1985).

Treatments. In this study, rats were divided into six groups and weregiven the following treatments:

1. Group A (n = 7): A single injection (2 ml ⁄ kg, i.p.) of normalsaline (0.09% NaCl).

2. Group B (n = 7): A single dose of CP (5 mg ⁄ kg, i.p.) togetherwith normal saline (2 ml ⁄ kg, i.p.).

3. Group C (n = 4): Sildenafil (0.4 mg ⁄ kg, i.p. for 5 days) togetherwith a single injection of normal saline (2 ml ⁄ kg, i.p.).

4. Group D (n = 5): Sildenafil (0.4 mg ⁄ kg, i.p. for 5 days) togetherwith a single injection of CP (5 mg ⁄ kg, i.p.).

5. Group E (n = 4): Sildenafil (10 mg ⁄ kg, s.c., for 5 days) togetherwith a single injection of normal saline (2 ml ⁄ kg, i.p.).

6. Group F (n = 5): Sildenafil (10 mg ⁄ kg, s.c., for 5 days) togetherwith a single injection of CP (5 mg ⁄ kg, i.p.).

All rats were killed with an overdose of sodium pentobarbital5 days after either CP or saline treatment. The animals were weighedat the beginning and end of the experiments. The relative kidneyweight was calculated as: (kidney weight ⁄ body-weight) · 100.

Haemodynamics study. This was carried out as previously done inour laboratory [22]. Briefly, at the end of the treatment period, therats were anaesthetized with sodium pentobarbital (65 mg ⁄ kg) andPE50 cannulae, filled with heparinized normal saline (25 IU ⁄ ml in0.9% NaCl), were inserted into the right carotid artery for themeasurement of blood pressure using a pressure transducer(TSD104A; Biopac Systems, Santa Barbara, CA, USA) and into theright jugular vein for the administration of drugs. An ultrasonicprobe (1RB; Hughes Sacks Electronik-Harvard apparatus, March-Hugstetten, Germany) was placed around the left renal artery tomeasure renal blood flow and was connected to a flowmeter (HughesSacks Electronik-Harvard apparatus). After a 30-min. stabilizationperiod, baseline blood pressure and renal blood flow were monitoredon a data acquisition system (MP150, Biopac Systems). Norepineph-rine (0.5, 1, 2 and 4 lg ⁄ kg) was injected at 3-min. intervals. Themagnitudes of the vasoconstrictor responses were expressed aspercentage change in renal blood flow.

Biochemical indices of renal function. The animals were placed inmetabolic cages 1 day before being killed, and the amount of urinevoided for 24 hr was collected. At the end of the haemodynamicstudy and under sodium pentobarbital anaesthesia, approximately5 ml of heparinized blood was collected from the inferior vena cavaand centrifuged at 900 · g for 10 min. at 4�C to obtain plasma. Theplasma samples were kept frozen ()80�C) until analysis. The animalswere then killed by an overdose of pentobarbital and kidneys wereremoved, blotted on a filter paper and weighed, and a part of the

kidney was placed in formalin until histological analysis. Plasma cre-atinine and urea and urinary creatinine were measured spectrophoto-metrically using commercial kits purchased from Human GmbH(Wiesbaden, Germany) and N-acetyl-b-D-glucosaminidase activity bykits purchased from Diazyme, General Atomics (San Diego, CA,USA). TNF-a in kidney homogenate and plasma was measured byan ELISA technique using kits from R & D systems (Minneapolis,MN, USA).

Plasma-reduced GSH concentration was measured spectrophoto-metrically using kits from Sigma-Aldrich (St. Louis, MO, USA). Theactivity of L-c-glutamyltransferase (GGT) in plasma was measuredspectrophotometrically using kits from Randox (Antrim, UK).

Histopathology. The kidneys were fixed in 10% neutral-bufferedformalin, dehydrated in increasing concentrations of ethanol,cleared with xylene and embedded in paraffin. Five-micrometre sec-tions were prepared from kidney paraffin blocks and stained withhaematoxylin and eosin. The microscopic scoring of the kidney sec-tions was carried out in a blinded fashion by a pathologist whowas unaware of the treatment groups and assigned a score asdescribed by Mohan et al. [23] which represents the approximateextent of necrotic area in the cortical proximal tubules on a scaleof 0–4 (0, no necrosis; 1, a few focal necrotic spots; 2, necrotic areawas about one half; 3, necrotic spots formed about two thirdspercentage; 4, nearly the entire area was necrotic). The size of thenecrosis was also estimated, and values were presented as means €S.E.M.

Staining for apoptosis was performed with signal stain-cleaved cas-pase-3 immunohistochemical detection kit (Cell Signaling Technol-ogy, Boston, MA, USA). This was used to detect the activation ofcaspase using the avidin-biotin immunoperoxidase method to detectintracellular caspase-3 protein. Staining was performed on 5-lm par-affin sections from the left kidney by a standard technique using rab-bit anticleaved caspase 3 (clone Asp175, 1:50) [24]. Known positivecontrol sections for apoptosis were used. For negative control,primary antibody was replaced with normal rabbit serum.

Measurement of renal platinum concentration. The concentration ofCP (as platinum) in cortical tissue was measured by inductively cou-pled plasma atomic emission spectrometry (Perkin Elmer, Shelton,CT, USA). The procedure involves mineralization of the kidney tis-sue with a mixture of concentrated HNO3 and H2O2, followed bydetermination of platinum in the extract, using inductively coupledplasma optical emission spectrometry at an emission wavelength of265.945 nm. Platinum atomic absorption spectrophotometer stan-dard solution (Sigma, St. Louis, MO, USA) was used to construct astandard curve.

Drugs and chemicals. CP used was from Pharma GES (Unterach,Austria) and platinum standard solution from Sigma. Sildenafil wasa gift from Pfizer (NY, USA). The rest of the chemicals were of thehighest purity grade available.

Statistical analysis. All values are presented as means € S.E.M. Thedata were analysed by one-way analysis of variance (ANOVA), fol-lowed by Tukey–Kramer multiple comparison test. A value ofp < 0.05 was selected as the criterion for statistical significance. Allstatistical analyses were performed with GraphPad Prism version4.03 (GraphPad Software Inc, San Diego, CA, USA).

Results

Haemodynamic effects.To induce acute renal failure, rats were treated with a singleCP injection (5 mg ⁄ kg), and this resulted in significantreduction in renal blood flow and a decrease in blood pres-sure (table 1). Intravenous (i.v.) administration of norepi-

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nephrine-induced dose-dependent decreases in renal bloodflow and CP did not have any significant effect on the vaso-constrictor effect of norepinephrine (table 2). Concomitanti.p. treatment with sildenafil (0.4 mg ⁄ kg ⁄ day for 5 days)reversed the decrease in blood pressure and renal blood flowinduced by cisplatin (table 1). Treatment with sildenafil(10 mg ⁄ kg ⁄ day s.c. for 5 days) alone and in the presence ofcisplatin reduced renal blood flow (table 1).

Biochemical and other kidney functional parameters.Treatment with CP (5 mg ⁄ kg) resulted in significant reduc-tion in body-weight and increase in urinary output. Sildenafildid not significantly affect the reduction in either body-weight or the increase in urinary output (table 3). In plasma,CP increased creatinine and urea concentrations anddecreased creatinine clearance (fig. 1). The concentration ofurinary N-acetyl-b-D-glucosaminidase activity was also sig-nificantly increased (fig. 2). The activity of GGT in plasma

was insignificantly increased by CP (fig. 3). Sildenafil(0.4 mg ⁄ kg ⁄ day i.p. for 5 days) significantly reduced the con-centration of N-acetyl-b-D-glucosaminidase activity that wasincreased by CP (fig. 2).

Table 4 shows the concentration of TNF-a and GSH inplasma of rats treated with CP and sildenafil (0.4 mg ⁄ kg ⁄ dayi.p. for 5 days). CP treatment induced a significant increasein plasma TNF-a that was not significantly antagonized bysildenafil.

Histopathological effects.Histological observations of renal sections of all rats (figs 4and 5) showed the following:

As shown in table 5, in the cisplatin group, the mean per-centage of necrosis was 85.8 € 7.4 (given a score of 4),whereas in the sildenafil s.c. + cisplatin group, this was84.6 € 4.0 (given a score of 4), and in the sildenafil i.p. +cisplatin group 20.3 € 3.6 (given a score of 2).

Table 1.The effect of saline, cisplatin and sildenafil on mean arterial bloodpressure (MAP) and renal blood flow (RBF) in sodium pentobar-bital (65 mg ⁄ kg)–anaesthetized Wistar rats.

Groups NBaseline MAP

(mmHg)Baseline RBF ⁄ g

(ml ⁄ min)

Control 7 133 € 7 3.6 € 0.3Cisplatin (Cis, 5 mg ⁄ kg) 7 107 € 81 1.7 € 0.21

Sildenafil (Sil, 0.4 mg ⁄ kg ⁄day, i.p.)

4 111 € 12 3.8 € 0.42

Cis + Sil (0.4 mg ⁄ kg ⁄day, i.p.)

5 139 € 32 3.1 € 0.42

Sil (10 mg ⁄ kg ⁄ day s.c.) 4 142 € 62 1.9 € 0.51,3,4

Cis + Sil (10 mg ⁄ kg ⁄day s.c.)

5 113 € 135 1.5 € 0.31,3,4

i.p., intraperitoneal route; s.c., subcutaneous route.Values are means € S.E.M. (n = 4–7).1Significantly different from control group (p < 0.05).2Significantly different from cisplatin group (p < 0.05).3Significantly different from sildenafil (0.4 mg ⁄ kg, i.p.) group(p < 0.05).4Significantly different from cisplatin + sildenafil (0.4 mg ⁄ kg ⁄ day,i.p.) group (p < 0.05).5Significantly different from sildenafil (10 mg ⁄ kg ⁄ day, s.c.) group(p < 0.05).

Table 2.The effect of saline, cisplatin and sildenafil on mean % decrease in renal blood flow after intravenous administration of norepinephrine (NE) insodium pentobarbital (65 mg ⁄ kg)–anaesthetized Wistar rat.

Groups

NE

0.5 lg ⁄ kg 1 lg ⁄ kg 2 lg ⁄ kg 4 lg ⁄ kg

Control )11.9 € 2 )19.0 € 3 )45.9 € 7 )78.0 € 5Cisplatin (Cis, 5 mg ⁄ kg) )17.2 € 10 )25.0 € 8 )55.6 € 11 )85.5 € 11Sildenafil (Sil, 0.4 mg ⁄ kg ⁄ day, i.p.) )11.1 € 5 )24.2 € 6 )45.7 € 12 )74.3 € 17Cis + Sil (0.4 mg ⁄ kg ⁄ day, i.p.) )12.3 € 6 )26.4 € 9 )51.8 € 11 )76.6 € 6Sil (10 mg ⁄ kg ⁄ day s.c.) )15.1 € 3 )25.1 € 7 )51.8 € 10 )74.7 € 8Cis + Sil (10 mg ⁄ kg ⁄ day s.c.) )28.4 € 4 )43.4 € 1 )77.3 € 1 )96.1 € 2

i.p., intraperitoneal route; s.c., subcutaneous route.Values are means € S.E.M. (n = 4–7).

Table 3.The effect of saline, cisplatin and sildenafil on % change in body-weight, kidney relative weight and urinary output (UOP).

Groups% change

in BWKidney relative

weightUOP

(ml ⁄ 24 hr)

Control 4.2 € 1.5 0.8 € 0.03 8.0 € 0.7Cisplatin (Cis,5 mg ⁄ kg)

)16.3 € 5.51 1.0 € 0.12 20.8 € 5.81

Sildenafil (Sil, 0.4 mg ⁄kg ⁄ day i.p.)

6.1 € 3.42 0.7 € 0.06 9.3 € 1.4

Cis + Sil (0.4 mg ⁄ kg ⁄day i.p.)

)12.1 € 3.31,3 1.0 € 0.033 13.1 € 3.01

Sil (10 mg ⁄kg ⁄ day s.c.)

)18.7 € 1.31,3 0.9 € 0.06 9.7 € 0.8

Cis + Sil (10 mg ⁄kg ⁄ day s.c.)

)21.2 € 5.01,3 1.1 € 0.083 27.3 € 5.41,3,4

i.p., intraperitoneal route; s.c., subcutaneous route.Values are means € S.E.M. (n = 4–7).1Significantly different from control group (p < 0.05).2Significantly different from cisplatin (Cis) group (p < 0.05).3Significantly different from Sil 0.4 mg ⁄ kg i.p. group (p < 0.05).4Significantly different from Cis + Sil 0.4 mg ⁄ kg i.p. group(p < 0.05).5Significantly different from Sil 10 mg ⁄ kg s.c. group (p < 0.05).

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Control rats treated with saline showed normal kidneyarchitecture and histology, and was given a score of 0 (fig. 4A,table 5); there was an absence of apoptotic bodies (fig. 5A).

The CP-treated group (fig. 4B) had diffuse acute tubularnecrosis in nearly the entire examined tissue areas and wasgiven a score of 4 (table 5). In this group, there was tubulardistention with necrotic material and many apoptotic cellsinvolving all the examined tissue (fig. 5B).

The group treated with sildenafil (0.4 mg ⁄ kg, i.p. daily for5 days) had normal kidney architecture and normal histol-ogy, as shown in fig. 4C and was given a score of 0 (table 5).No apoptotic bodies were seen in this group (fig. 5C).

The group treated with CP together with sildenafil(0.4 mg ⁄ kg, i.p. daily for 5 days) showed dramatic improve-ment compared with the group treated with cisplatin alone(fig. 4D). There were only few focal areas of degenerated

vacuolated cells and acute tubular necrosis constitutingabout 20% of the total tissue fields examined (score 1,table 5) as well as few apoptotic cells (fig. 5D).

The group treated with sildenafil (10 mg ⁄ kg, s.c. daily for5 days) had normal kidney architecture and normal histol-ogy, as shown in fig. 4E and was given a score of 0 (table 5);there were only a few apoptotic cells (fig. 5E).

As shown in fig. 4F, the group treated with CP togetherwith sildenafil (10 mg ⁄ kg, s.c. daily for 5 days) had diffuseacute tubular necrosis in nearly the entire examined tissueareas and was given a score of 4 (table 5) and many apopto-tic cells involving all the examined tissue (fig. 5F).

Renal platinum concentration.Measurement of CP (as platinum) in renal tissues of treatedrats showed values similar to those in our previous study

0

100

200P < 0.001

P < 0.001

P < 0.001

Plas

ma

urea

(µM

)

0.0

0.1

0.2

0.3

0.4P < 0.05

P < 0.01P < 0.001

Cre

atin

ine

clea

ranc

e (m

g/m

in)

0

150

300

450

600P < 0.01

P < 0.01

ControlCisplatinSil 0.4 mg/kg i.p.Cis+Sil 0.4 mg/kg i.p.Sil 10 mg/kg s.c.Cis+Sil 10 mg/kg s.c.

Plas

ma

crea

tinin

e (µ

M)

B

A

C

Fig. 1. Creatinine (A) and urea (B) plasma concentration and the creatinine clearance (C) in rats treated with saline, cisplatin or sildenafil. Eachcolumn and vertical bar represent mean € S.E.M. (n = 4–7). Differences between the groups were assessed by analysis of variance followed bymultiple comparison test. p < 0.05 was considered significant.

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[22]. Concomitant treatment with sildenafil, at both doses,did not significantly (p > 0.1) affect the concentration (datanot shown).

Discussion

As far as we are aware, this is the first study to show theaction of sidenafil, a phosphodiesterase inhibitor, on renalhaemodynamics in CP-induced renal failure in normotensiverats. The acute renal failure induced by CP was demonstratedby the decrease in renal blood flow, increase in plasma ureaand creatinine, and decrease in creatinine clearance. In addi-tion, the kidneys showed diffuse acute tubular necrosis andmany apoptotic cells. Moreover, urinary N-acetyl-b-D-glu-cosaminidase activity excretion, measured as a marker oftubular damage [25], was also significantly increased. CPaccumulation was also detected in the renal tissue. Thedecrease in renal blood flow induced by CP is in accordancewith previous studies [22,26–28]. In our study, the renal vaso-constrictor responses of norepinephrine were not potentiatedby CP. This is in accordance with our previous studies inwhich we did not observe a potentiation effect of the vaso-constrictor response to norepinephrine in normotensive rats

0

1

2

3

4

5ControlCisplatinSil 0.4 mg/kg i.p.Cis+Sil 0.4 mg/kg i.p.

Plas

ma

GG

T (U

/L)

Fig. 3. Plasma l-c-glutamyltransferase (GGT) activity in rats treated with saline, cisplatin or sildenafil. Each column and vertical bar representmean € S.E.M. (n = 4–7). Differences between the groups were assessed by analysis of variance, followed by multiple comparison test. Therewas no significant difference between the four groups.

Table 4.The effect of saline, cisplatin and sildenafil on plasma tumournecrosis factor (TNF-a) and glutathione (GSH) concentrations.

GroupsPlasma TNF

a (pg ⁄ ml)Kidney TNF

a (pg ⁄ ml)Plasma GSH(mmol ⁄ mg)

Control 51.8 € 12 1.62 € 0.26 4.1 € 1.3Cisplatin (5 mg ⁄ kg) 218.4 € 621 2.05 € 0.2 2.1 € 0.3Sildenafil (Sil; 0.4 mg ⁄kg ⁄ day i.p.)

58.0 € 9.32 1.77 € 0.14 2.4 € 0.3

Cis + Sil (0.4 mg ⁄kg ⁄ day i.p.)

127.3 € 46 1.89 € 0.19 0.9 € 0.11

i.p., intraperitoneal route; s.c., subcutaneous route.Values are means € S.E.M. (n = 4–7).1Significantly different from control group (p < 0.05).2Significantly different from cisplatin group (p < 0.05).

0

10

20

30

40

50

60

70 P < 0.001 P < 0.001

P < 0.01

P < 0.001

ControlCisplatinSil 0.4mg/kg i.p.Cis+Sil 0.4mg/kg i.p.Sil 10 mg/kg s.c.Cis+Sil 10 mg/kg s.c.

Uri

ne N

AG

(IU

/L)

Fig. 2. Urinary N-acetyl-b-d-glucosaminidase (NAG) activity in rats treated with saline, cisplatin or sildenafil. Each column and vertical barrepresent mean € S.E.M. (n = 4–7). Differences between the groups were assessed by analysis of variance, followed by multiple comparison test.p < 0.05 was considered significant.

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[22,26]. This is different from previous reports that foundthat CP potentiated the vasoconstrictor effects of norepi-nephrine in Wister-Kyoto rats [29]. The reason for this differ-ence is not clear, but it is to be noted that in the previousstudy, norepinephrine was injected in the renal artery while itwas injected intravenously in our study. Similarly to previousreports [22,26,27], the haemodynamic measurements in thiswork have been conducted under anaesthesia. We are awareof the disadvantages of this, but conducting these measure-ments in rats in the awake state was not technically feasiblefor us. Nevertheless, we believe that our observations arevalid as all the groups were anaesthetized and treated in thesame manner.

In this study, CP reduced body-weight, increased 24-hrurinary output and decreased blood pressure. In addition,we have shown that CP treatment increases significantly theconcentration of TNF-a in plasma. CP nephrotoxicityinvolves both inflammatory and oxidative stress processes[17,18]. It has been reported that CP induces (both in vitroand in vivo) necrosis in the tubular cells of the kidney by

generating large quantities of hydroxyl radicals, followed byincreased synthesis of TNF-a. A role for apoptosis afterinjection of cisplatin has also been suggested [30].

In this study, sildenafil (0.4 mg ⁄ kg ⁄ day, i.p. for 5 days),but not sildenafil (10 mg ⁄ kg ⁄ day, s.c. for 5 days) reversedthe histopathological changes induced by CP. In the groupof rats treated with CP and sildenafil (0.4 mg ⁄ kg ⁄ day i.p. for5 days), there were only few focal areas of degenerated vacu-olated cells and acute tubular necrosis constituting about20% of the total tissue fields examined and few apoptoticcells. Furthermore, the increase in urinary N-acetyl-b-D-glu-cosaminidase activity (a marker of renal tubular damage)excretion observed after CP treatment was completelyreversed by co-administration of sildenafil (0.4 mg ⁄ kg ⁄ day,i.p. for 5 days) with CP. In addition, sildenafil(0.4 mg ⁄ kg ⁄ day, i.p. for 5 days) attenuated the decrease inrenal blood flow and blood pressure induced by CP. Con-comitant treatment with sildenafil, at both doses, did not sig-nificantly affect the concentration of platinum, indicatingthat it does not increase renal excretion of CP. This demon-

A

C D

E F

B

Fig. 4. Representative light microscopy sections of renal tissues of rats given: Saline-treated rats (control) showed normal kidney architectureand histology and were given a score 0 (A) (table 4). The Cisplatin (CP)-treated group (B) had diffuse acute tubular necrosis in nearly the entireexamined tissue areas and was given a score of 4 (table 4). The group treated with sildenafil (0.4 mg ⁄ kg, i.p. daily for 5 days) had normal kidneyarchitecture and normal histology, as shown in (C), and was given a score of 0 (table 4). The group treated with CP together with sildenafil(0.4 mg ⁄ kg, i.p. daily for 5 days) showed dramatic improvement, compared with the group treated with cisplatin alone (D). There were only fewfocal areas of degenerated vacuolated cells and acute tubular necrosis constituting about 20% of the total tissue fields examined (score 1,table 4). The group treated with sildenafil (10 mg ⁄ kg, s.c. daily for 5 days) had normal kidney architecture and normal histology, as shown in(E), and was given a score of 0 (table 4). As shown in (F), the group treated with CP together with sildenafil (10 mg ⁄ kg, s.c. daily for 5 days)had diffuse acute tubular necrosis in nearly the entire examined tissue areas and was given a score of 4 (score 5, table 4).

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strates that sildenafil can protect against CP-induced nephro-toxicity. At the same time, it is suggested that sildenafil willnot affect the therapeutic action of CP through increasingurinary excretion of CP. The mechanism by which sildenafil

(0.4 mg ⁄ kg ⁄ day for 5 days, i.p.) attenuated the nephrotoxiceffect of CP is not clear. Lee et al. [20] suggested that silde-nafil attenuates experimental cisplatin-induced nephrotoxi-city by preventing apoptosis. Indeed, in our study, sildenafil(0.4 mg ⁄ kg ⁄ day for 5 days, i.p) was shown to reduce thenumber of apoptotic cells induced by cisplatin. In addition,sildenafil was shown to have renoprotective effects againstrenal ischaemic ⁄ reperfusion injury, attenuating the renaltubular damage, decreasing apoptosis and suppressing theincreases in the blood urea nitrogen and serum creatinine.The authors suggested that this protection was primarilybecause of the inhibition of apoptosis and necrosis, asrevealed by increased iNOS ⁄ eNOS, and decreased activationof caspase-3, TUNEL-positive cells and the Bax ⁄ Bcl-2 ratio[31,32]. It has been reported that intravenous (i.v.) sildenafilin patients with heart failure was associated with a smalldecrease in systemic arterial blood pressure and a more sub-stantial reduction in pulmonary arterial pressure [33]. In thiswork (table 1), sildenafil given i.p. (but not s.c.) caused a

A

C

B

D

E F

Fig. 5. Representative immunohistochemical analysis (anticaspase-3, streptavidin-biotin immunohistochemical method) of renal tissues of ratsgiven: Saline-treated rats (control) showed normal kidney architecture and absence of apoptotic bodies (A). The Cisplatin (CP)-treated groupshowed tubular distention with necrotic material and many apoptotic cells involving all the examined tissue (B). The group treated with sildena-fil (0.4 mg ⁄ kg, i.p. daily for 5 days) showed no apoptotic bodies (C). The group treated with CP together with sildenafil (0.4 mg ⁄ kg, i.p. dailyfor 5 days) showed dramatic improvement compared with the group treated with cisplatin alone. There were only few focal areas of degeneratedvacuolated cells and acute tubular necrosis constituting about 20% of the total tissue fields examined (score 1, table 4) and few apoptotic cells(D). The group treated with sildenafil (10 mg ⁄ kg, s.c. daily for 5 days) had normal kidney architecture and showed only few apoptotic cells (E).CP together with sildenafil (10 mg ⁄ kg, s.c. daily for 5 days) showed the presence of many apoptotic cells involving all the examined tissue (F).

Table 5.Semiquantitative analysis of histology of kidneys from rats treatedwith saline, cisplatin or sildenafil.

Group% necrosis

(mean € S.E.M.)Score ofnecrosis

Saline (i.p.) group, control 0 0Cisplatin (i.p.) group 85.8 € 7.4 4Sildenafil s.c. 0 0Sildenafil s.c. + cisplatin (i.p.) 84.6 € 4.0 4Sildenafil (i.p.) group 0 0Cisplatin (i.p.) + sildenafil (i.p.) 20.3 € 3.6 2

i.p., intraperitoneal route; s.c., subcutaneous route.Values are means € S.E.M. (n = 4–7).

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small and statistically insignificant decrease in bloodpressure.

The administration of sildenafil (10 mg ⁄ kg ⁄ day s.c.) for5 days resulted in a decrease in renal blood flow and creati-nine clearance and did not reverse the nephrotoxic effect ofcisplatin. The reason for this is not clear. In our study, silde-nafil (10 mg ⁄ kg) reduced renal blood flow but did not havea significant effect on blood pressure. This might be due tothe fact that sildenafil can cause dilation in some organs andtissues, and that this action was probably opposed by reflexvasoconstriction of renal blood vessels and reduced renalblood flow, leading to reduction in renal blood flow with aminimal effect on blood pressure. In this work, sildenafil(10 mg ⁄ kg) reduced the body-weight of treated rats. The rea-son for this action is not certain, especially in the absence ofdata on feed intake.

In conclusion, sildenafil given i.p. but not s.c. amelioratedCP-induced renal failure as demonstrated by reversing thereduction in renal blood flow and histopathological changesinduced by CP. After thorough studies to select appropriatedoses of sildenafil in patients with cancer, this drug may poten-tially be useful in mitigating the nephrotoxicity of cisplatinand also in potentiating its anticancer activity, as has beenrecently shown with the anticancer agent adriamycin [34].

AcknowledgementsThis work was supported by a grant from Sultan Qaboos

University (SQU) (IG ⁄ MED ⁄ PHAR ⁄ 09 ⁄ 02). Thanks toMr Tabisam Khan for measuring the platinum concentra-tion, the staff of the Animal House at SQU for their helpand Professor G. Blunden for reading the manuscript. Thework that has been performed at the United Arab EmiratesUniversity was supported by funds of the Faculty of Medi-cine and Health Sciences. Thanks to Pfizer (NY, USA) forgifting us with sildenafil.

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