The Effect of Sildenafil on Cisplatin Nephrotoxicity in Rats

Authors

  • Badreldin H. Ali,

    1. Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
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  • Aly M. Abdelrahman,

    1. Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
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  • Suhail Al-Salam,

    1. Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates
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  • Munjusha Sudhadevi,

    1. Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates
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  • Ahmed S. AlMahruqi,

    1. Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
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  • Ishaq S. Al-Husseni,

    1. Department of Physiology, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
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  • Sumiya Beegam,

    1. Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman
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  • Subramanian Dhanasekaran,

    1. Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates
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  • Abderrahim Nemmar,

    1. Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates
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  • Mansour Al-Moundhri

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

Abstract

Abstract:  Sildenafil, the first drug for erectile dysfunction, has cardiopulmonary protective actions. A recent study has reported that sildenafil given intraperitoneally (i.p.) attenuated cisplatin (CP)-induced nephrotoxicity. Here, we evaluated whether sildenafil, given by two different routes and at two different doses, can attenuate CP-induced nephrotoxicity and would 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 then anaesthetized for blood pressure and renal blood flow monitoring. This was followed by intravenous (i.v.) injection of norepinephrine for the measurement of renal vasoconstrictor responses. Thereafter, blood and kidneys were collected for measurement of several biochemical, functional and structural parameters. CP reduced body-weight and renal blood flow but did not affect norepinephrine-induced renal vasoconstriction. It increased the plasma concentrations of urea and creatinine, and reduced creatinine clearance. CP caused extensive renal tubular necrosis, increased urine volume and N-acetyl-β-d-glucosaminidase activity. When sildenafil (0.4 mg/kg/day, i.p. for 5 days) was combined with cisplatin, there was a dramatic improvement in renal histopathology, reduction in N-acetyl-β-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 selection of sildenafil as a nephroprotectant.

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

The mechanism of action of CP-induced nephrotoxicity is not fully understood [6,10]. Renal tubular injury and death (apoptosis and necrosis) are the main pathological events in CP nephrotoxicity [6]. Although the exact mechanism underlying CP-induced tubular death is not fully known, the drug is known to damage cell mitochondria, arrest the cell cycle in the G2 phase, inhibit ATPase activity and alter the cellular transport system. All of these actions eventually induce apoptosis, inflammation, necrosis and cell death [3,4,11]. The nephrotoxicity of CP is mediated, at least partly, via γ-glutamyl transpeptidase (GGT), p53 and enhanced tumour necrosis factor-α (TNF-α) production [8,12,13]. It has also been proposed that CP binds to glutathione (GSH) and the subsequent CP-GSH complex in tubules stimulates renal lipid peroxidation [14,15]. CP interacts with thiol groups and macromolecules, and causes significant oxidant loading on the kidney through both xanthine oxidase activation and impaired antioxidant defence system, which results in accelerated oxidation reactions in the kidney tissues [16,17]. Nitrosative stress is also involved [18]. However, the source of the inline image has not been identified.

Many drugs with varying mechanisms of action have been tested to see whether they could ameliorate or prevent experimental CP nephrotoxicity [6,19]. Among these, sildenafil has recently been tested [20]. In this single report, the drug was given intraperitoneally (i.p.) at a dose of 0.4 mg/kg immediately 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 different routes on CP-induced nephrotoxicity, and its effect on renal haemodynamics in CP-treated rats has not been reported so far.

In the present work, we aimed to verify the possible nephroprotective action of sildenafil, and further, to see whether the drug, given at different doses and by different routes, would also protect against CP nephrotoxicity and would affect renal haemodynamics in CP-treated rats. In view of the kinetic peculiarities of sildenafil [21], we used in the present work a different route (subcutaneous, s.c.) and dose (10 mg/kg, i.p.) to those employed by Lee et al. [20], in addition to those used by them.

Materials and Methods

Animals.  Male Wistar rats of 250–300 g body-weight were obtained from the Animal House of Sultan Qaboos University. The rats were housed in polypropylene cages and maintained under standard conditions [22 ± 2°C; 12 L:12 D cycle (lights on at 07:00 hr); and about 60% humidity]. Standard laboratory chow diet containing normal sodium (Oman Mills, Muscat, Oman) and tap water was provided ad libitum. The study was approved by the University Animal Ethical Committee and was conducted in conformity with international laws and 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 were given the following treatments:

  • 1 Group A (n = 7): A single injection (2 ml/kg, i.p.) of normal saline (0.09% NaCl).
  • 2 Group B (n = 7): A single dose of CP (5 mg/kg, i.p.) together with normal saline (2 ml/kg, i.p.).
  • 3 Group C (n = 4): Sildenafil (0.4 mg/kg, i.p. for 5 days) together with 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) together with a single injection of CP (5 mg/kg, i.p.).
  • 5 Group E (n = 4): Sildenafil (10 mg/kg, s.c., for 5 days) together with 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) together with a single injection of CP (5 mg/kg, i.p.).

All rats were killed with an overdose of sodium pentobarbital 5 days after either CP or saline treatment. The animals were weighed at the beginning and end of the experiments. The relative kidney weight was calculated as: (kidney weight/body-weight) × 100.

Haemodynamics study.  This was carried out as previously done in our laboratory [22]. Briefly, at the end of the treatment period, the rats were anaesthetized with sodium pentobarbital (65 mg/kg) and PE50 cannulae, filled with heparinized normal saline (25 IU/ml in 0.9% NaCl), were inserted into the right carotid artery for the measurement of blood pressure using a pressure transducer (TSD104A; Biopac Systems, Santa Barbara, CA, USA) and into the right jugular vein for the administration of drugs. An ultrasonic probe (1RB; Hughes Sacks Electronik-Harvard apparatus, March-Hugstetten, Germany) was placed around the left renal artery to measure renal blood flow and was connected to a flowmeter (Hughes Sacks Electronik-Harvard apparatus). After a 30-min. stabilization period, baseline blood pressure and renal blood flow were monitored on a data acquisition system (MP150, Biopac Systems). Norepinephrine (0.5, 1, 2 and 4 μg/kg) was injected at 3-min. intervals. The magnitudes of the vasoconstrictor responses were expressed as percentage change in renal blood flow.

Biochemical indices of renal function.  The animals were placed in metabolic cages 1 day before being killed, and the amount of urine voided for 24 hr was collected. At the end of the haemodynamic study and under sodium pentobarbital anaesthesia, approximately 5 ml of heparinized blood was collected from the inferior vena cava and centrifuged at 900 × g for 10 min. at 4°C to obtain plasma. The plasma samples were kept frozen (−80°C) until analysis. The animals were then killed by an overdose of pentobarbital and kidneys were removed, blotted on a filter paper and weighed, and a part of the kidney was placed in formalin until histological analysis. Plasma creatinine and urea and urinary creatinine were measured spectrophotometrically using commercial kits purchased from Human GmbH (Wiesbaden, Germany) and N-acetyl-β-d-glucosaminidase activity by kits purchased from Diazyme, General Atomics (San Diego, CA, USA). TNF-α in kidney homogenate and plasma was measured by an ELISA technique using kits from R & D systems (Minneapolis, MN, USA).

Plasma-reduced GSH concentration was measured spectrophotometrically using kits from Sigma-Aldrich (St. Louis, MO, USA). The activity of l-γ-glutamyltransferase (GGT) in plasma was measured spectrophotometrically using kits from Randox (Antrim, UK).

Histopathology.  The kidneys were fixed in 10% neutral-buffered formalin, dehydrated in increasing concentrations of ethanol, cleared with xylene and embedded in paraffin. Five-micrometre sections were prepared from kidney paraffin blocks and stained with haematoxylin and eosin. The microscopic scoring of the kidney sections was carried out in a blinded fashion by a pathologist who was unaware of the treatment groups and assigned a score as described by Mohan et al. [23] which represents the approximate extent of necrotic area in the cortical proximal tubules on a scale of 0–4 (0, no necrosis; 1, a few focal necrotic spots; 2, necrotic area was about one half; 3, necrotic spots formed about two thirds percentage; 4, nearly the entire area was necrotic). The size of the necrosis was also estimated, and values were presented as means ± S.E.M.

Staining for apoptosis was performed with signal stain-cleaved caspase-3 immunohistochemical detection kit (Cell Signaling Technology, Boston, MA, USA). This was used to detect the activation of caspase using the avidin-biotin immunoperoxidase method to detect intracellular caspase-3 protein. Staining was performed on 5m paraffin sections from the left kidney by a standard technique using rabbit anticleaved caspase 3 (clone Asp175, 1:50) [24]. Known positive control sections for apoptosis were used. For negative control, primary antibody was replaced with normal rabbit serum.

Measurement of renal platinum concentration.  The concentration of CP (as platinum) in cortical tissue was measured by inductively coupled plasma atomic emission spectrometry (Perkin Elmer, Shelton, CT, USA). The procedure involves mineralization of the kidney tissue with a mixture of concentrated HNO3 and H2O2, followed by determination of platinum in the extract, using inductively coupled plasma optical emission spectrometry at an emission wavelength of 265.945 nm. Platinum atomic absorption spectrophotometer standard solution (Sigma, St. Louis, MO, USA) was used to construct a standard curve.

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

Statistical analysis.  All values are presented as means ± S.E.M. The data were analysed by one-way analysis of variance (anova), followed by Tukey–Kramer multiple comparison test. A value of p < 0.05 was selected as the criterion for statistical significance. All statistical analyses were performed with GraphPad Prism version 4.03 (GraphPad Software Inc, San Diego, CA, USA).

Results

Haemodynamic effects.

To induce acute renal failure, rats were treated with a single CP injection (5 mg/kg), and this resulted in significant reduction in renal blood flow and a decrease in blood pressure (table 1). Intravenous (i.v.) administration of norepinephrine-induced dose-dependent decreases in renal blood flow and CP did not have any significant effect on the vasoconstrictor effect of norepinephrine (table 2). Concomitant i.p. treatment with sildenafil (0.4 mg/kg/day for 5 days) reversed the decrease in blood pressure and renal blood flow induced by cisplatin (table 1). Treatment with sildenafil (10 mg/kg/day s.c. for 5 days) alone and in the presence of cisplatin reduced renal blood flow (table 1).

Table 1. 
The effect of saline, cisplatin and sildenafil on mean arterial blood pressure (MAP) and renal blood flow (RBF) in sodium pentobarbital (65 mg/kg)–anaesthetized Wistar rats.
  1. i.p., intraperitoneal route; s.c., subcutaneous route.

  2. Values are means ± S.E.M. (n = 4–7).

  3. 1Significantly different from control group (p < 0.05).

  4. 2Significantly different from cisplatin group (p < 0.05).

  5. 3Significantly different from sildenafil (0.4 mg/kg, i.p.) group (p < 0.05).

  6. 4Significantly different from cisplatin + sildenafil (0.4 mg/kg/day, i.p.) group (p < 0.05).

  7. 5Significantly different from sildenafil (10 mg/kg/day, s.c.) group (p < 0.05).

GroupsNBaseline MAP (mmHg)Baseline RBF/g (ml/min)
Control7133 ± 73.6 ± 0.3
Cisplatin (Cis, 5 mg/kg)7107 ± 811.7 ± 0.21
Sildenafil (Sil, 0.4 mg/kg/day, i.p.)4111 ± 123.8 ± 0.42
Cis + Sil (0.4 mg/kg/day, i.p.)5139 ± 323.1 ± 0.42
Sil (10 mg/kg/day s.c.)4142 ± 621.9 ± 0.51,3,4
Cis + Sil (10 mg/kg/day s.c.)5113 ± 1351.5 ± 0.31,3,4
Table 2. 
The effect of saline, cisplatin and sildenafil on mean % decrease in renal blood flow after intravenous administration of norepinephrine (NE) in sodium pentobarbital (65 mg/kg)–anaesthetized Wistar rat.
GroupsNE
0.5 μg/kg1 μg/kg2 μg/kg4 μg/kg
  1. i.p., intraperitoneal route; s.c., subcutaneous route.

  2. Values are means ± S.E.M. (n = 4–7).

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

Biochemical and other kidney functional parameters.

Treatment with CP (5 mg/kg) resulted in significant reduction in body-weight and increase in urinary output. Sildenafil did 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 and decreased creatinine clearance (fig. 1). The concentration of urinary N-acetyl-β-d-glucosaminidase activity was also significantly 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 concentration of N-acetyl-β-d-glucosaminidase activity that was increased by CP (fig. 2).

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)
  1. i.p., intraperitoneal route; s.c., subcutaneous route.

  2. Values are means ± S.E.M. (n = 4–7).

  3. 1Significantly different from control group (p < 0.05).

  4. 2Significantly different from cisplatin (Cis) group (p < 0.05).

  5. 3Significantly different from Sil 0.4 mg/kg i.p. group (p < 0.05).

  6. 4Significantly different from Cis + Sil 0.4 mg/kg i.p. group (p < 0.05).

  7. 5Significantly different from Sil 10 mg/kg s.c. group (p < 0.05).

Control4.2 ± 1.50.8 ± 0.038.0 ± 0.7
Cisplatin (Cis, 5 mg/kg)−16.3 ± 5.511.0 ± 0.1220.8 ± 5.81
Sildenafil (Sil, 0.4 mg/kg/day i.p.)6.1 ± 3.420.7 ± 0.069.3 ± 1.4
Cis + Sil (0.4 mg/kg/day i.p.)−12.1 ± 3.31,31.0 ± 0.03313.1 ± 3.01
Sil (10 mg/kg/day s.c.)−18.7 ± 1.31,30.9 ± 0.069.7 ± 0.8
Cis + Sil (10 mg/kg/day s.c.)−21.2 ± 5.01,31.1 ± 0.08327.3 ± 5.41,3,4
Figure 1.

 Creatinine (A) and urea (B) plasma concentration and the creatinine clearance (C) in rats treated with saline, cisplatin or sildenafil. Each column and vertical bar represent 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.

Figure 2.

 Urinary N-acetyl-β-d-glucosaminidase (NAG) activity in rats treated with saline, cisplatin or sildenafil. Each column and vertical bar represent 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.

Figure 3.

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

Table 4 shows the concentration of TNF-α and GSH in plasma of rats treated with CP and sildenafil (0.4 mg/kg/day i.p. for 5 days). CP treatment induced a significant increase in plasma TNF-α that was not significantly antagonized by sildenafil.

Table 4. 
The effect of saline, cisplatin and sildenafil on plasma tumour necrosis factor (TNF-α) and glutathione (GSH) concentrations.
GroupsPlasma TNFα (pg/ml)Kidney TNFα (pg/ml)Plasma GSH (mmol/mg)
  1. i.p., intraperitoneal route; s.c., subcutaneous route.

  2. Values are means ± S.E.M. (n = 4–7).

  3. 1Significantly different from control group (p < 0.05).

  4. 2Significantly different from cisplatin group (p < 0.05).

Control51.8 ± 121.62 ± 0.264.1 ± 1.3
Cisplatin (5 mg/kg)218.4 ± 6212.05 ± 0.22.1 ± 0.3
Sildenafil (Sil; 0.4 mg/kg/day i.p.)58.0 ± 9.321.77 ± 0.142.4 ± 0.3
Cis + Sil (0.4 mg/kg/day i.p.)127.3 ± 461.89 ± 0.190.9 ± 0.11

Histopathological effects.

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

Figure 4.

 Representative light microscopy sections of renal tissues of rats given: Saline-treated rats (control) showed normal kidney architecture and histology and were given a score 0 (A) (table 4). The Cisplatin (CP)-treated group (B) had diffuse acute tubular necrosis in nearly the entire examined 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 kidney architecture 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 few focal 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).

Figure 5.

 Representative immunohistochemical analysis (anticaspase-3, streptavidin-biotin immunohistochemical method) of renal tissues of rats given: Saline-treated rats (control) showed normal kidney architecture and absence of apoptotic bodies (A). The Cisplatin (CP)-treated group showed tubular distention with necrotic material and many apoptotic cells involving all the examined tissue (B). The group treated with sildenafil (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. daily for 5 days) showed dramatic improvement compared with the group treated with cisplatin alone. There were only few focal areas of degenerated vacuolated 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).

As shown in table 5, in the cisplatin group, the mean percentage of necrosis was 85.8 ± 7.4 (given a score of 4), whereas in the sildenafil s.c. + cisplatin group, this was 84.6 ± 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 5. 
Semiquantitative analysis of histology of kidneys from rats treated with saline, cisplatin or sildenafil.
Group% necrosis (mean ± S.E.M.)Score of necrosis
  1. i.p., intraperitoneal route; s.c., subcutaneous route.

  2. Values are means ± S.E.M. (n = 4–7).

Saline (i.p.) group, control00
Cisplatin (i.p.) group85.8 ± 7.44
Sildenafil s.c.00
Sildenafil s.c. + cisplatin (i.p.)84.6 ± 4.04
Sildenafil (i.p.) group00
Cisplatin (i.p.) + sildenafil (i.p.)20.3 ± 3.62

Control rats treated with saline showed normal kidney architecture 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 tubular necrosis in nearly the entire examined tissue areas and was given a score of 4 (table 5). In this group, there was tubular distention with necrotic material and many apoptotic cells involving all the examined tissue (fig. 5B).

The group treated with sildenafil (0.4 mg/kg, i.p. daily for 5 days) had normal kidney architecture and normal histology, 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 improvement compared with the group treated with cisplatin alone (fig. 4D). There were only few focal areas of degenerated vacuolated cells and acute tubular necrosis constituting about 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 for 5 days) had normal kidney architecture and normal histology, 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 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 (table 5) and many apoptotic cells involving all the examined tissue (fig. 5F).

Renal platinum concentration.

Measurement of CP (as platinum) in renal tissues of treated rats showed values similar to those in our previous study [22]. Concomitant treatment with sildenafil, at both doses, did not significantly (p > 0.1) affect the concentration (data not shown).

Discussion

As far as we are aware, this is the first study to show the action of sidenafil, a phosphodiesterase inhibitor, on renal haemodynamics in CP-induced renal failure in normotensive rats. The acute renal failure induced by CP was demonstrated by the decrease in renal blood flow, increase in plasma urea and creatinine, and decrease in creatinine clearance. In addition, the kidneys showed diffuse acute tubular necrosis and many apoptotic cells. Moreover, urinary N-acetyl-β-d-glucosaminidase activity excretion, measured as a marker of tubular damage [25], was also significantly increased. CP accumulation was also detected in the renal tissue. The decrease in renal blood flow induced by CP is in accordance with previous studies [22,26–28]. In our study, the renal vasoconstrictor responses of norepinephrine were not potentiated by CP. This is in accordance with our previous studies in which we did not observe a potentiation effect of the vasoconstrictor response to norepinephrine in normotensive rats [22,26]. This is different from previous reports that found that CP potentiated the vasoconstrictor effects of norepinephrine in Wister-Kyoto rats [29]. The reason for this difference is not clear, but it is to be noted that in the previous study, norepinephrine was injected in the renal artery while it was injected intravenously in our study. Similarly to previous reports [22,26,27], the haemodynamic measurements in this work have been conducted under anaesthesia. We are aware of the disadvantages of this, but conducting these measurements in rats in the awake state was not technically feasible for us. Nevertheless, we believe that our observations are valid as all the groups were anaesthetized and treated in the same manner.

In this study, CP reduced body-weight, increased 24-hr urinary output and decreased blood pressure. In addition, we have shown that CP treatment increases significantly the concentration of TNF-α in plasma. CP nephrotoxicity involves both inflammatory and oxidative stress processes [17,18]. It has been reported that CP induces (both in vitro and in vivo) necrosis in the tubular cells of the kidney by generating large quantities of hydroxyl radicals, followed by increased synthesis of TNF-α. A role for apoptosis after injection 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) reversed the histopathological changes induced by CP. In the group of rats treated with CP and sildenafil (0.4 mg/kg/day i.p. for 5 days), there were only few focal areas of degenerated vacuolated cells and acute tubular necrosis constituting about 20% of the total tissue fields examined and few apoptotic cells. Furthermore, the increase in urinary N-acetyl-β-d-glucosaminidase activity (a marker of renal tubular damage) excretion observed after CP treatment was completely reversed 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 in renal blood flow and blood pressure induced by CP. Concomitant treatment with sildenafil, at both doses, did not significantly affect the concentration of platinum, indicating that it does not increase renal excretion of CP. This demonstrates that sildenafil can protect against CP-induced nephrotoxicity. At the same time, it is suggested that sildenafil will not affect the therapeutic action of CP through increasing urinary excretion of CP. The mechanism by which sildenafil (0.4 mg/kg/day for 5 days, i.p.) attenuated the nephrotoxic effect of CP is not clear. Lee et al. [20] suggested that sildenafil attenuates experimental cisplatin-induced nephrotoxicity by preventing apoptosis. Indeed, in our study, sildenafil (0.4 mg/kg/day for 5 days, i.p) was shown to reduce the number of apoptotic cells induced by cisplatin. In addition, sildenafil was shown to have renoprotective effects against renal ischaemic/reperfusion injury, attenuating the renal tubular damage, decreasing apoptosis and suppressing the increases in the blood urea nitrogen and serum creatinine. The authors suggested that this protection was primarily because of the inhibition of apoptosis and necrosis, as revealed by increased iNOS/eNOS, and decreased activation of caspase-3, TUNEL-positive cells and the Bax/Bcl-2 ratio [31,32]. It has been reported that intravenous (i.v.) sildenafil in patients with heart failure was associated with a small decrease in systemic arterial blood pressure and a more substantial reduction in pulmonary arterial pressure [33]. In this work (table 1), sildenafil given i.p. (but not s.c.) caused a small and statistically insignificant decrease in blood pressure.

The administration of sildenafil (10 mg/kg/day s.c.) for 5 days resulted in a decrease in renal blood flow and creatinine clearance and did not reverse the nephrotoxic effect of cisplatin. The reason for this is not clear. In our study, sildenafil (10 mg/kg) reduced renal blood flow but did not have a significant effect on blood pressure. This might be due to the fact that sildenafil can cause dilation in some organs and tissues, and that this action was probably opposed by reflex vasoconstriction of renal blood vessels and reduced renal blood flow, leading to reduction in renal blood flow with a minimal effect on blood pressure. In this work, sildenafil (10 mg/kg) reduced the body-weight of treated rats. The reason for this action is not certain, especially in the absence of data on feed intake.

In conclusion, sildenafil given i.p. but not s.c. ameliorated CP-induced renal failure as demonstrated by reversing the reduction in renal blood flow and histopathological changes induced by CP. After thorough studies to select appropriate doses of sildenafil in patients with cancer, this drug may potentially be useful in mitigating the nephrotoxicity of cisplatin and also in potentiating its anticancer activity, as has been recently shown with the anticancer agent adriamycin [34].

Acknowledgements

This work was supported by a grant from Sultan Qaboos University (SQU) (IG/MED/PHAR/09/02). Thanks to Mr Tabisam Khan for measuring the platinum concentration, the staff of the Animal House at SQU for their help and Professor G. Blunden for reading the manuscript. The work that has been performed at the United Arab Emirates University was supported by funds of the Faculty of Medicine and Health Sciences. Thanks to Pfizer (NY, USA) for gifting us with sildenafil.

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