N-acetylcysteine improves renal hemodynamics in rats with cisplatin-induced nephrotoxicity

J. Appl. Toxicol. 2010; 30: 15–21 Copyright © 2009 John Wiley & Sons, Ltd.

Research Article

Received 2 June 2009, Revised 29 June 2009, Accepted 29 June 2009, Published online in Wiley InterScience: 13 August 2009

(www.interscience.wiley.com) DOI 10.1002/jat.1465

N-acetylcysteine improves renal hemodynamics in rats with cisplatin-induced nephrotoxicityAly M. Abdelrahman,a* Suhail Al Salam,b Ahmed S AlMahruqi,a Ishaq S. Al husseni,c Mohamed A Mansourd and Badreldin H. Alia

ABSTRACT: This work investigated the e) ect of N-acetylcysteine (NAC), on renal hemodynamics in cisplatin (CP)-induced nephrotoxicity in Wistar–Kyoto (WKY) rats. The animals were divided into four groups (n = 5 or 6). The 5 rst and second groups received normal saline (control) and intraperitoneal (i.p.) N-acetylcysteine (500 mg kg−1 per day for 9 days), respectively. The third and fourth groups were given a single intraperitoneal (i.p.) injection of CP (5 mg kg−1) and an i.p. injection of CP (5 mg kg−1) together with i.p. NAC (500 mg kg−1 per day for 9 days), respectively. At the end of the experiment, rats were anesthetized and blood pressure and renal blood : ow were monitored, followed by intravenous (i.v.) injection of norepinephrine (NE) for mea-surement of renal vasoconstrictor responses. CP caused a signi5 cant reduction in renal blood : ow but did not a) ect NE-induced renal vasoconstriction. In addition, CP signi5 cantly increased plasma concentrations of urea and creatinine and urinary N-acetyl-β-D-glucosaminidase (NAG) activity and kidney relative weight. CP decreased body weight and creatinine clearance. Histopathologically, CP caused remarkable renal damage compared with control. NAC alone did not produce any signi5 cant change in any of the variables measured. However, NAC signi5 cantly ameliorated CP-induced hemodynamic, biochemical and histopathological changes. The concentration of platinum in the kidneys of CP + NAC treated rats was less than in CP-treated rats by 37%. The results show that administration of i.p. NAC (500 mg kg−1 per day for 9 days) reversed the renal hemodynamic changes as well as the biochemical and histopathological indices of CP-induced nephrotoxicity in WKY rats. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: cisplatin; N-acetylcysteine; nephrotoxicity; renal blood 5 ow; rats

*Correspondence to: A. M. Abdelrahman, Department of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, PO Box 35, Al-Khod, Muscat 123, Sultanate of Oman.Email: [email protected]

aDepartment of Pharmacology and Clinical Pharmacy, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman

bDepartment of Pathology, Faculty of Medicine, UAE University, Al Ain, UAE

cDepartment of Physiology, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman

dDepartment of Soils, Water and Agricultural Engineering, College of Agriculture and Marine Sciences, Sultan Qaboos University, Al-Khod, Sultanate of Oman

INTRODUCTION

Cisplatin [cis-diamminedichlorolatinum (II)]-based combination therapy regimens are used in the treatment of many types of cancer such as testicular, ovarian and head and neck cancer (Delord et al., 2009). The toxic e< ects of the drug in man and animals include nephrotoxicity, ototoxicity, neurotoxicity and bone marrow suppression but the chief factor limiting its use is nephrotoxicity (Hanigen and Devarajan, 2003; Ali and Moundhri, 2006; Pabla and Dong, 2008). The kidneys accumulate CP in the renal tissue more than in any other organ, resulting in necrosis of the proximal renal tubules and apoptosis in the distal nephron (Arany and Sa@ rstein, 2003). The molecular mechanisms underly-ing CP-induced apoptosis in renal tubular cells are not yet fully understood and the evidence suggests that reactive oxygen species are involved (Santos et al., 2007; Yao et al. 2007). CP depletes renal reduced glutathione (GSH) concentration and superoxide dismutase (SOD) activity, leaving the renal tissues vul-nerable to damage by oxygen radicals that are responsible for the induction of tubular epithelial cell damage (Ali et al., 2007). Prevention of CP-induced nephrotoxicity would reduce morbidity and complications, decrease hospitalization costs and may allow administration of higher dosage of this e< ective antitumor drug with added therapeutic potential (Sheikh-Hamad et al., 1997). Various approaches were used to reduce these side e< ects, includ-ing hydrating the patients during CP treatment (Pabla and Dong, 2008). In addition, many agents were tried in humans and animals

to ameliorate this nephrotoxicity (Ali and Moundhri, 2006). Recently, some studies showed that CP toxicity alters renal hemo-dynamics in rats, probably due to altered renal adrenergic respon-siveness in CP-induced renal failure (Hye Khan et al., 2007, 2008).

N-acetylcysteine (NAC) is one of a large group of exogenous antioxidant drugs that protects against oxidative tissue injury. This e< ect may be directly related to the drug itself (e.g. inactiva-tion of hydroxyl radicals) or to the secondary induction of glu-tathione production (Fishbane et al., 2004). NAC was shown to ameliorate CP nephrotoxicity in humans (Skiekh-Hamad et al., 1997; Nisar and Feinfeld, 2002) and in rats (Appenroth et al., 1993; Dickey et al., 2005, 2008; Mishima et al., 2006). It has been shown that the level of protection of CP nephrotoxicity in rats is

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A. M. Abdelrahman et al.

www.interscience.wiley.com/journal/jat Copyright © 2009 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2010; 30: 15–21

dependent on the route of NAC administration and that only intravenous but not oral or i.p. administration was e< ective in protecting against CP-nephrotoxicity (Dickey et al., 2008). NAC was also shown to improve renal blood 5 ow in acute renal failure produced by inferior vena cava occlusion (Conesa et al., 2001).

Although a protective e< ect of NAC in CP-induced renal failure has been reported before (Nisar and Feinfeld, 2002; Mishima et al., 2006), there is no information on its e< ect on renal hemo-dynamics. The aim of the present study, therefore, was to inves-tigate the e< ect of i.p. NAC on renal hemodynamics in CP- induced renal failure in Wistar–Kyoto rats.

MATERIALS AND METHODSAnimals and Treatments

WKY rats (250–350 g) were obtained from the Animal House of the Sultan Qaboos University. They were maintained under a 12 : 12 h light : dark cycle and supplied with standard laboratory chow diet (contains normal sodium) and water ad libitum. The protocols were approved by the Animal Ethical Committee of Sultan Qaboos University, and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals, NIH publica-tion no. 85; 23, 1985.

The animals were randomly divided into four equal groups (n = 5 or 6). The @ rst group received normal saline (control). The second group received i.p. NAC (500 mg kg−1 per day for 9 days). The third group was given a single intraperitoneal (i.p.) injection of CP (5 mg kg−1). The fourth group received i.p. NAC (500 mg kg−1 per day for 9 days) and on the fourth day also received an i.p. injection of CP (5 mg kg−1). The doses of CP and NAC were selected to be in the range used in previous studies (Ali et al., 2007, 2009). Animals were sacri@ ced 5 days after CP treatment. The animals were weighed at the beginning and end of the experiments.

Hemodynamics Study

At the end of the treatment period, the rats were anesthetized with sodium pentobarbital (60 mg kg−1, i.p.). PE50 cannulae, @ lled with heparinized normal saline (25 IU ml−1 in 0.9% NaCl), were inserted into the right carotid artery for the measurement of blood pressure by a pressure transducer (TSD104A, Biopac Systems, Santa Barbara, CA, USA), and into the right jugular vein for the administration of drugs. Heart rate was derived electroni-cally from the upstroke of the arterial pulse pressure. An ultra-sonic probe (1RB, Hughes Sacks Electronik-Harvard Apparatus, March-Hugstetten, Germany) was placed around the left renal artery to measure renal blood 5 ow and was connected to a 5 ow meter (Hughes Sacks Electronik-Harvard Apparatus, March-Hugstetten, Germany).

After a 30 min stabilization period, baseline blood pressure, heart rate and renal blood 5 ow were monitored on a data acqui-sition system (MP 150, Biopac Systems, Santa Barbara, CA, USA). Norepinephrine (NE) (0.5, 1, 2 and 4 µg kg−1) was injected at 3 min intervals. The magnitudes of the vasoconstrictor responses were expressed as percentage change in renal blood 5 ow.

Biochemical Indices of Renal Function

The animals were placed in metabolic cages one day before sac-ri@ ce and the amount of urine voided in 24 h was collected. At

the end of the hemodynamic study and under sodium pentobar-bital anesthesia, approximately 5 ml of heparinized blood was collected from the inferior vena cava and centrifuged at 900g for 10 min at 4 ºC to obtain plasma. The plasma samples were kept frozen (−80 ºC) pending analysis. The animals were then sacri-@ ced by an overdose of pentobarbital and kidneys were removed, blotted on @ lter paper and weighed, and a part of the kidney was placed in formalin pending histological analysis. Plasma creati-nine and urea and urinary creatinine were measured spectropho-tometrically using commercial kits purchased from Human GmbH (Wiesbaden, Germany) and N-acetyl-β-D-glucosaminidase (NAG) activity by kits purchased from Diazyme, General Atomics (San Diago, CA, USA). The kidney relative weight was calculated as (kidney weight/body weight) × 100.

Histopathology

The kidneys were @ xed in 10% neutral bu< ered formalin, dehy-drated in increasing concentrations of ethanol, cleared with xylene and embedded in paraP n. Five-micrometer sections were prepared from kidney paraP n blocks and stained with haema-toxylin 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 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 were about two-thirds; 4, nearly the entire area was necrotic).

Staining for apoptosis was performed using signal stain cleaved caspase-3 Immunohistochemical detection Kit (Cell Signaling Technology, Boston, Mass, USA). This kit was used to detect the activation of caspase using avidin–biotin immunoper-oxidase method to detect intracellular caspase-3 protein. Staining was performed on 5 µm paraP n longitudinal sections from left kidney by standard technique using rabbit anti–cleaved caspase 3 (clone Asp175, 1 : 50). Known positive control sections for anti–cleaved caspase 3 from breast carcinoma 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 mea-sured by inductively coupled plasma atomic emission spectrm-etry (Perkin Elmer, Shelton, Connecticut, USA). The procedure involves mineralization of the kidney tissue with a mixture of concentrated HNO3 and H2O2, followed by determination of plati-num in the extract, using inductively coupled plasma optical emission spectrometry, at an emission wavelength of 265.945 nm. Platinum atomic absorption spectrophotometer standard solu-tion (Sigma, St Loius, MO, USA) was used to construct a standard curve.

Statistical Analysis

All values are presented as means ± SEM. The data were analyzed by one way analysis of variance (ANOVA), followed by Newman–Keul’s test. A value of P < 0.05 was selected as the criterion for statistical signi@ cance. All statistical analyses were performed using GraphPad Prism version 4.03 (GraphPad Software Inc, USA).16

N-acetylcysteine and renal hemodynamics in CP-induced nephrotoxicity

J. Appl. Toxicol. 2010; 30: 15–21 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/jat

RESULTSHemodynamics

In rats treated with CP, there was a signi@ cant reduction in blood pressure, renal blood 5 ow and an increase in renal vascular resis-tance, as compared with the control (Table 1). In the CP + NAC group, NAC ameliorated the decrease in blood pressure and renal blood 5 ow and the increase in renal vascular resistance. NAC alone did not induce signi@ cant changes in blood pressure or renal blood 5 ow (Table 1). Intravenous administration of NE induced dose-dependent decreases in renal blood 5 ow in all studied groups (Table 2). There were no signi@ cant di< erences in NE-induced decrease in renal blood 5 ow between the four groups.

Biochemical and Other Kidney Functional Parameters

Table 3 shows that CP caused a signi@ cant decrease in body weight and an increase in kidney relative weight but did not change the 24 h urine output. NAC alone did not alter any of the observed measurements when compared with the control group. Treatment of rats with CP + NAC caused a decrease in body weight which was signi@ cantly less than the decrease observed in CP-treated group. There was also no increase in kidney relative weight and 24 h urine output in relation to the control group. Table 4 shows that CP increased the plasma concentrations of creatinine and urea and urinary levels of NAG (expressed as NAG–creatinine ratio; Skalova, 2005) and decreased creatinine clear-ance. NAC alone did not alter any of the observed measurements when compared with the control group. In the CP + NAC group,

Table 1. E< ect of treatment with saline, N-acetylcysteine (NAC, 500 mg kg−1, orally for 9 days), CP (5 mg kg−1) or CP (5 mg kg−1) and NAC (500 mg kg−1 for 9 days) on mean arterial blood pressure (MAP), renal blood 5 ow (RBF) and renal vascular resistance (RVR) in sodium pentobarbital (60 mg kg−1) anesthetized Wistar–Kyoto rats

Experimental groups Baseline MAP (mmHg) Baseline RBF (ml min−1 kg−1) Baseline RVR (mmHg ml−1 min−1)

Saline (control) 92 ± 5 14.5 ± 2.5 26.6 ± 3.9NAC 92 ± 5 17.1 ± 2.9 22.1 ± 3.3CP 67 ± 5a,b 6.5 ± 1.2a,b 52.6 ± 11.9a,b

CP + NAC 88 ± 2c 15.1 ± 1.6c 23.4 ± 3.2c

Values are mean ± SEM (n = 5 or 6 rats).Data were analyzed using one-way ANOVA followed by Newman–Keul’s post-hoc test.aStatistically signi@ cant compared with control group (P < 0.05).bStatistically signi@ cant compared with NAC group (P < 0.05).cStatistically signi@ cant compared with CP group (P < 0.05).

Table 2. E< ect of treatment with saline, N-acetylcysteine (NAC, 500 mg kg−1, orally for 9 days), CP (5 mg kg−1) or CP (5 mg kg−1) and NAC (500 mg kg−1 for 9 days) on mean percentage decrease in renal blood 5 ow following intravenous administration of nor-epinephrine (NE) in sodium pentobarbital (60 mg kg−1) anesthetized Wistar–Kyoto rats

Experimental groups NE (0.5 µg kg−1) NE (1 µg kg−1) NE (2 µg kg−1) NE (4 µg kg−1)

Saline (control) 32 ± 5 46 ± 7 71 ± 7 86 ± 5NAC 35 ± 6 52 ± 6 68 ± 4 83 ± 6CP 38 ± 10 46 ± 9 71 ± 4 87 ± 3CP + NAC 52 ± 7 63 ± 3 81 ± 3 94 ± 2

Values are mean ± SEM (n = 5 or 6 rats).Data were analyzed using one-way ANOVA.

Table 3. E< ect of treatment with saline, N-acetylcysteine (NAC, 500 mg kg−1, i.p. for 9 days), CP (5 mg kg−1) or CP (5 mg kg−1) and NAC (500 mg kg−1 for 9 days) on body (BW), kidney weight and 24 h urine output (UOP) of Wistar–Kyoto rats.

Animal group Initial BW (g) Final BW (g) Percentage change in BW Kidney relative weight UOP (ml per 24 h)

Saline (control) 280 ± 2 290 ± 2 3.7 ± 0.3 0.71 ± 0.01 5.1 ± 1.2NAC 278 ± 2 277 ± 2 −0.1 ± 1.0a 0.72 ± 0.01 7.6 ± 1.4CP 281 ± 3 226 ± 4a,b −19.4 ± 1.3a,b 0.85 ± 0.02a,b 7.2 ± 2.6CP + NAC 292 ± 8 269 ± 8c −7.8 ± 1.6a,b,c 0.71 ± 0.02c 11.6 ± 1.8

Values are mean ± SEM (n = 5 or 6). Data were analyzed using one-way ANOVA followed by Newman–Keul’s post-hoc test. BW, body weight.aSigni@ cantly di< erent from control group (P < 0.05).bSigni@ cantly di< erent from NAC group (P < 0.05).cSigni@ cantly di< erent from CP group (P < 0.05).

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there were no di< erences in plasma creatinine, urea, creatinine clearance and urinary NAG in comparison to the control and NAC-treated rats.

Histopathology

The saline- and NAC-treated rats had normal kidney architecture and histology (Figs 1A and 2A) and (Figs 1B and 2B) respectively. The CP-treated group showed di< use acute tubular necrosis involving all the examined tissues (score 4) (Fig. 1C, D), while the glomeruli were not a< ected. There were swelling, vacuolization and sloughing of tubular cells into the tubular lumen (Fig. 1C, D). Tubular distention with esosinophilic necrotic material was seen in the majority of renal tubules (Fig. 1C, D). Many apoptotic cells were seen within the lumen of many renal tubules (Fig. 1C, D). Anti-cleaved casapase-3 immunohistochemical stain showed brown, granular, cytoplasmic staining of casapase-3 within apop-totic tubular cells, which were seen either within damaged tubular lining epithelium or falling in tubular lumen (Fig. 2C, D). There was interstitial edema and accumulations of leukocytes within dilated vasa recta (Fig. 1C, D). Rats in the CP + NAC groups showed dramatic improvement with only few focal areas of swallen, vacuolated cells, constituting about 10% of total exam-ined tissue @ elds and rare apoptotic cells (score 1) (Figs 1E and 2E). There was complete recovery with absence of apoptosis and necrosis in approximately 90% of total examined tissue @ elds (Figs 1F and 2F). Table 5 shows a semiquantitative analysis of renal histology of rats in the di< erent groups.

Renal Platinum Concentration

The concentration of platinum in the kidney tissues of CP-treated rats (15.12 ± 1.18 ppm wet weight) was higher that in CP + NAC treated rats (9.46 ± 0.85 ppm wet weight) (P < 0.01).

DISCUSSION

CP causes renal tubular injury through multiple mechanisms including hypoxia, the generation of free radicals, in5 ammation and apoptosis (Lee et al., 2009). In this study, CP-induced acute renal failure in WKY rats was con@ rmed by a signi@ cant increase

in plasma creatinine, urea and kidney relative weight and a large decrease in creatinine clearance and histologically by severe renal tissue damage. CP accumulation (as platinum) was detected in the renal tissue which in accordance with previous reports (Arany and Sa@ rstein, 2003; Ali et al., 2008). In this study, urinary NAG excretion, measured as a marker of tubular damage (Skalova, 2005), was also signi@ cantly increased. In addition, CP reduced renal blood 5 ow and increased renal vascular resis-tance, which is in accordance with previous studies (Winston and Sa@ rstein, 1985; Matsushima et al., 1998; Bagnis et al., 2001; Hye Khan et al., 2008). It was reported that a decrease in nitric oxide (NO) level contributes, at least, in part to the mechanism underlying cisplatin nephrotoxicity (Ali and Moundhri, 2006). In this study, CP caused a signi@ cant reduction in blood pressure that was associated with a reduction in body weight. This may be due to excessive loss of 5 uids as CP causes diarrhea (Bearcroft et al., 1999). Hye Khan et al. (2008) explained that the CP-induced hypotensive e< ect might be due to the excessive loss of 5 uid via the urine that would deplete the extracellular volume. However, in our study, CP-induced renal failure, which was con-@ rmed by hemodynamic, biochemical and histopathological tests, was not associated with a signi@ cant increase in 24 h urinary output as expected and previously reported (Ali et al., 2007, 2008). The reason for this is not understood but it is pos-sible that in this study CP induced loss of 5 uid due to diarrhea and a very large reduction (95%) in creatinine clearance may have contributed to the absence of an increase in urine volume. NE caused a dose-dependent decrease in renal blood 5 ow in all groups; however, CP did not potentiate this vascoconstrictor e< ect. This is di< erent from previous reports that found that CP potentiated the vasoconstrictor e< ects of NE in Wistar–Kyoto rats (Hye Khan et al., 2007). The reason for this discrepancy is not clear. It should be mentioned that in the previous study, NE was injected in the renal artery while we injected NE intravenously.

NAC is a thiol that is rapidly metabolized to L-cysteine, a direct precursor of the synthesis of intracellular glutathione. In vitro, NAC blocked CP-induced apoptosis through the caspase signal-ing pathway (Wu et al., 2005). NAC was shown to have a protec-tive e< ect in CP induced nephrotoxicity in rats (Appenroth et al., 1993; Dickey et al., 2005, 2008; Mishima et al., 2006). However, these reports have only shown bene@ cial biochemical and histo-pathological e< ects. This is the @ rst study to examine the e< ect

Table 4. E< ect of treatment with saline, N-acetylcysteine (NAC, 500 mg kg−1, orally for 9 days), CP (5 mg kg−1) or CP (5 mg kg−1) and NAC (500 mg kg−1 for 9 days) on levels of plasma creatinine (Cr) and urea, creatinine clearance and urinary NAG–creatinine ratio in Wistar–Kyoto rats

Experimental groupsPlasma Cr (µmol l−1)

Plasma urea (mmol l−1)

Creatinine clearance (ml min−1)

Urinary NAG (IU l−1)–creatinine (mg dl−1)

Saline (control) 77 ± 6 13.7 ± 0.7 2.2 ± 0.4 0.028 ± 0.003NAC 103 ± 6 11.8 ± 1.1 1.9 ± 0.3 0.033 ± 0.009CP 534 ± 33a,b 141.4 ± 17.7a,b 0.11 ± 0.04a,b 0.570 ± 0.154a,b

CP+ NAC 107 ± 9c 16.9 ± 1.6c 2.7 ± 0.55c 0.089 ± 0.0185c

Values are mean ± SEM (n = 5 or 6).Data were analyzed using one-way ANOVA followed by Newman–Keul’s comparison test.aStatistically signi@ cant compared with control group (P < 0.05).bStatistically signi@ cant compared with NAC group (P < 0.05).cStatistically signi@ cant compared with CP group (P < 0.05).

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of i.p. injection of NAC for 9 days on renal hemodynamics in CP-induced renal failure. In our study, NAC had a protective e< ect in CP-induced renal failure as assessed by classical histo-logical and biochemical parameters. It was also able to amelio-rate the reduction in renal blood 5 ow and the increase in renal vascular resistance caused by CP. The preserved renal blood 5 ow in rats treated with NAC in this study is not due to a direct vaso-dilator e< ect because NAC alone did not signi@ cantly a< ect renal blood 5 ow. It is possible that NAC reduced vascular resistance by potentiation of NO that is decreased in CP-induced renal failure. NAC was reported to improve renal blood 5 ow in acute renal failure produced by inferior vena cava occlusion by scavenging of free radicals and potentiation of NO (Conesa et al., 2001). NAC also ameeliorated the hypotensive e< ect of CP and partially reduced the reduction in body weight. It is possible that NAC reduced the loss of 5 uids induced by CP. NAC also ameliorated the increase in plasma creatinine and urea concentrations and the marked reduction in creatinine clearance. In addition, NAC ameliorated the increase in urinary NAG, indicating that it is

reducing proximal tubular necrosis. The results of this study are similar to those of a previous study on gentamicin nephrotoxic-ity in which it was shown that i.p. injections of NAC 500 mg kg−1 per day can have a nephroprotective e< ect (Ali et al., 2009). However, it is at variance with a study which showed that NAC 400 mg kg−1 given i.p. produced no renal protection in CP-induced renal failure (Dickey et al., 2008). Probably, the reason for this discrepancy is that in our study NAC was given for 9 consecutive days while in their study it was given once 30 min prior to CP injection (Dickey et al., 2008). NAC treatment reduced renal platinum concentration, which is in accordance with a pre-vious study (Appenroth et al., 1993). It is not known whether NAC increased renal excretion of platinium or somehow prevented its accumulation in the renal tissue. Appenroth et al. (1993) sug-gested that the protective e< ect of NAC on CP nephrotoxicity might be based on the formation of a complex unsuitable for tubular reabsorption.

In conclusion, NAC given i.p. for 9 days ameliorated CP-induced renal failure as demonstrated by reversing the reduction in

Figure 1. (A) Saline (IP) group, showing normal kidney architecture and histology, H&E. (B) NAC(IP) group, showing normal kidney architecture and normal histology, H&E. (C, D) CP (IP) group, showing acute tubular necrosis (thick arrow), tubular distention with necrotic material, and many apoptotic cells (arrow head), H&E. (E, F) CP(IP) + NAC(IP), showing dramatic improvement with few focal areas of degenerated vacuolated cells (thin arrow) in (E) constituting about 10% total tissue @ elds, and complete recovery with absence of apoptosis and necrosis in (F) which constitutes about 90% of total tissue @ elds, H&E.

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Table 5. Semiquantitative analysis of histology of kidneys of rats treated with cisplatin (CP) with or without N-acetylcysteine (NAC)

Group Score

Saline (control) 0NAC (500 mg kg−1 per day for 9 days) 0CP (5 mg kg−1) 4CP (5 mg kg−1) + NAC (500 mg kg−1 per day

for 9 days)1

Values in the table are scores given by a histopathologist unaware of the treatments. (0, no necrosis; 1, a few focal necrotic spots; 2, necrotic area was about one half; 3, necrotic spots were about two-thirds; 4, nearly the entire area was necrotic).

Figure 2. (A) Saline (IP) group, showing normal kidney architecture and histology, anti-caspase-3, strepavidin–biotin immunohistochemical method. (B) NAC (IP) group, showing normal kidney architecture and normal histology, anti-caspase-3, strepavidin–biotin immunohistochemical method. (C, D) CP (IP) group, showing acute tubular necrosis, tubular distention with necrotic material and many apoptotic cells (arrow head) with dark bown granular cytoplasmic staining for caspase-3, anti-caspase-3, strepavidin–biotin immunohistochemical method. (E, F) CP (IP) + NAC (IP), showing dramatic improvement with only one apoptotic cell (arrow head) in (E) and absence of apoptosis and necrosis in F, anti-caspase-3, strepavidin–biotin immuno-histochemical method.

renal blood 5 ow and the increase in renal vascular resistance. In addition, i.p. NAC ameliorated all the studied biochemical and histopathological changes induced by CP. It is not known whether the reduction of renal CP concentration by NAC inter-feres with its anticancer properties. More experiments on that aspect is needed before recommending NAC as an e< ective nephroprotectent drug in patients on platinum anticancer drugs.

Acknowledgments

This work was supported by a grant from Sultan Qaboos University (IG/MED/PHAR/09/02). The authors wish to thank the sta< of the Small Animal House at Sultan Qaboos University for their help and Professor Gerald Blunden for obtaining the PE50 tubing used in the work.20



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N-acetylcysteine improves renal hemodynamics in rats with cisplatin-induced nephrotoxicity

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