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Selective ETA receptor blockade protects against cisplatin-induced acute renal failure in male rats
European Journal of Pharmacology 730 (2014) 133–139
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Selective ETA receptor blockade protects against cisplatin-induced acute renal failure in male rats Mai M. Helmy a,n, Maged W. Helmy b, Dina M. Abd Allah c, Ahmad M. Abo Zaid b, Mahmoud M. Mohy El-Din a a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Azarita, PO Box 21521, Alexandria, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Alexandria, Egypt c Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Azarita, Alexandria, Egypt b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 October 2013 Received in revised form 26 February 2014 Accepted 6 March 2014 Available online 14 March 2014
The present study aims to investigate the possibility that inhibiting the physiological function of endothelin-1 (ET-1) by blocking its receptors would significantly decrease the nephrotoxic effect of cisplatin. Therefore the study was designed to investigate the effect of treatment with BQ-123, the selective endothelin receptor-A (ETA) blocker, and bosentan, the non-selective endothelin receptor blocker, on the cisplatin-induced structural, functional, and biochemical alterations in the rat kidney. Rats were divided into four groups: control (given a single dose of normal saline, i.p.), cisplatin (given a single dose of cisplatin, 6 mg/kg, i.p.), cisplatinþ BQ-123 (1 mg/kg, i.p.), and cisplatin þbosentan (30 mg/kg, orally via gavage). Each of the two blockers was administered in two doses; 1 h before and one day after the cisplatin dose. Acute cisplatin administration resulted in significant increases in blood urea nitrogen and serum creatinine concentrations at 96 h following cisplatin injection. Increased concentrations of malondialdehyde, tumor necrosis factor-α (TNF-α) and caspase-3, decreased nitric oxide (NO) production and superoxide dismutase (SOD) activity in kidney homogenates were observed at 96 h following cisplatin injection, in addition to a typical ‘acute tubular necrosis’ pattern. BQ-123 ameliorated the structural and functional injuries caused by cisplatin mainly via restoring SOD activity, in addition to other antioxidant parameters, NO, TNF-α and caspase-3 concentrations. This study further proves that ETA but not ETB receptors are involved in cisplatin-induced nephrotoxicity. The selective ETA antagonist BQ-123 ameliorated the cisplatin-induced deleterious effects and showed reno-protective effect against cisplatin-induced acute renal damage. & 2014 Elsevier B.V. All rights reserved.
Keywords: Cisplatin Endothelin-1 ETA receptors BQ-123 SOD Nephrotoxicity
1. Introduction Although cisplatin is a highly effective anti-neoplastic agent, its nephrotoxicity is a major clinical problem (Saleh and ElDemerdash, 2005). Cisplatin causes tubular injury through multiple mechanisms, including hypoxia, the generation of free radicals, inflammation, and apoptosis. Additionally, significant interactions among these various pathways may occur in cisplatin injury (Yao et al., 2007). Endothelins have a key role in vascular homeostasis. Three isoforms of endothelin have been identified: the originally described endothelin-1 (ET-1) and two similar peptides, ET-2 and ET-3. Two endothelin receptor subtypes, termed ETA and ETB, have been cloned and sequenced. ETA receptors have a high affinity for ET-1 and a low affinity for ET-3 and are located on
n
Corresponding author. Fax: þ 203 487 3273. E-mail address: [email protected] (M.M. Helmy).
http://dx.doi.org/10.1016/j.ejphar.2014.03.002 0014-2999/& 2014 Elsevier B.V. All rights reserved.
smooth muscle cells, where they mediate vasoconstriction. ETB receptors have approximately equal affinities for ET-1 and ET-3 and are located on vascular endothelial cells, where they mediate release of prostaglandins-I2 (PGI2) and nitric oxide (NO) (Takaoka et al., 2000). Both receptor subtypes belong to the G proteincoupled seven-transmembrane domain family of receptors. Endothelins cause dose-dependent vasoconstriction in most vascular beds. There is increasing evidence that endothelins participate in a variety of cardiovascular diseases (Takaoka et al., 2000), including hypertension, cardiac hypertrophy, as well as in renal failure (Kone, 2004). They act on the kidneys to cause vasoconstriction and decrease glomerular filtration rate and sodium and water excretion (Takaoka et al., 2000). Interestingly, increased level of both circulating ET-1 and urinary excretion of ET-1 have been observed in patients treated with nephrotoxic immunosuppressive agents as cyclosporine A and tacrolimus (Slowinski et al., 2002). Other nephrotoxic agents, such as cisplatin, also increase urinary excretion of ET. Moreover, expressions of ET-1 have been found to be elevated in
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mice with cisplatin-induced acute renal failure (ARF), suggesting that ET-1 might play a role in the pathogenesis of such a disease (Lee and Ahn, 2008). ET receptor antagonism improved renal failure after ARF (Gellai et al., 1994). Treatment with an ETA receptor antagonist before or post-ischemia has been demonstrated to lessen glomerular dysfunction in rats (Wilhelm et al., 2001). Buyukgebiz et al. (1996) mentioned that inhibiting function of ET via an ETA receptor antagonist might prevent reperfusion injury in kidney transplantation. Bosentan is a non-selective endothelin receptors antagonist that blocks both ETA and ETB. In contrast, BQ-123 {2-[(3R,6R,9S, 12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,11,14pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid} is an ETA receptor selective antagonist (Buyukgebiz et al., 1996). In clinical trials, bosentan and other non-selective antagonists as well as ETA receptor selective antagonists have produced beneficial effects on hemodynamics and other symptoms in heart failure, pulmonary hypertension, and essential hypertension (Wilhelm et al., 2001). Therefore, the current study aimed at investigating the possibility that inhibiting the physiological function of ET-1 by blocking its receptors would significantly decrease the nephrotoxic effect of cisplatin. We, therefore, evaluated the reno-protective effects of both the non-selective ET receptors antagonist bosentan and the ETA receptor selective antagonist BQ-123 against cisplatin-induced nephrotoxicity.
2. Materials and methods 2.1. Chemicals Bosentan monohydrate (free base) was generously gifted by Actelion Pharmaceuticals Ltd., Allscwil, Switzerland; cisplatin: Cisplatines Mylan 10 ml vial, Oncotec Pharma production, Germany; BQ-123: BQ-123 sodium salt 5 mg, purchased from Peptides international, Louisville, Kentucky, USA; other chemicals and reagents used in biochemical tests were of analytical grade. Details of quantitative analysis kits are under biochemical analysis section. 2.2. Experimental design The study was performed on adult male Sprague Dawley rats weighing 200–220 g, obtained from the Animal House of the Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Egypt. The animals were kept at a temperature of 2572 1C, on 12-h light/dark cycle (lights on at 7.00 am) and had free access to food and tap water before the experiment. The research experiments protocol was approved by the Animal Care & Use Committee (ACUC) at the faculty of Pharmacy, Alexandria University, Egypt. Animals were divided into four groups: (i) control (given a single dose of normal saline, i.p.), (ii) cisplatin (given a single dose of cisplatin, 6 mg/kg, i.p. (Dobyan et al., 1980), (iii) cisplatinþBQ-123 (1 mg/kg, i. p. (Garrido and Israel, 2002), and (iv) cisplatinþ bosentan (30 mg/kg, orally via gavage (Günal et al., 2006). Each of the two blockers was administered in two doses; 1 h before and one day after the cisplatin dose. The rationale of this schedule of dose administration was the maintenance of a steady sufficient plasma concentration of each of the drugs before, during and after the critical period of cisplatininduced toxicity, since the biochemical changes that occur in the kidney after cisplatin administration are of crucial importance in determining the extent of a nephrotoxic lesion (Borch and Pleasants, 1979). In fact, the dose of cisplatin used in the current study was lower than that used in previous experimental studies (El-Naga, 2014; Palipoch and Punsawad, 2013) and approximately double the minimal therapeutic dose that should be given to rats. Since the
therapeutic doses of cisplatin for an ideal man weighing about 70 kg ranged from 20 to 100 mg/m2 (Chabner et al., 2006) which is equivalent to 3.02–15.1 mg/kg rat according to Paget and Barnes table conversion factor. 2.3. Biochemical analysis 96 h after the cisplatin injection, rats were anaesthesized with thiopental sodium (50 mg/kg, intraperitoneally) El-Mas and AbdelRahman (1997) and blood samples were obtained from the orbital plexus (3 ml from each rat). Serum was recovered and stored at 70 1C until analyzed for determination of blood urea nitrogen and serum creatinine using the Randoxs assay kit (Randox Laboratories Ltd., United Kingdom). After collecting blood samples, rats were euthanized with an over dose of the anesthetic drug, the chest was opened, internal viscera were pulled aside. The kidneys were quickly removed, washed in ice cold saline, blotted dry, then weighed. The kidney homogenates were divided into portions and stored at 70 1C until analyzed for determination of renal malondialdehyde (Ottolenghi, 1959), superoxide dismutase (SOD) (Marklund and Marklund, 1974), and nitrite/nitrate (Guevara et al., 1998); tumor necrosis factor-alpha (TNF-α) was determined using RayBios Rat Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Ray Biotech Inc., Georgia, USA), and caspase-3 was determined using Bender MedSystems Caspase-3 instant ELISA kit (Bender MedSystems, Vienna, Austria, Europe) according to the manufacturer’s instructions. 2.4. Histological evaluation Following the animals’ sacrifice, tissues from the kidneys were carefully removed and preserved in 10% formalin for histological examination with haematoxylin and eosin (H&E) stain under a light microscope (Carleton, 1980; Tagboto and Griffiths, 2007). Histological evaluation of tubular necrosis was determined semiquantitatively using the methods modified from McWhinnie and coworkers (McWhinnie et al., 1986). Random fields were observed from each section using 100 magnification. Ten sections were examined for each kidney and a score from 0 to 3 was given to the tubular profile such that: 0 ¼normal histology, 1 ¼for interstitial nephritis and inflammation, 2 ¼for tubular atrophy and intratubular dilation with or without intralumenal hyaline casts, 3 ¼for renal tubular necrosis with “sloughing off” of the renal tubular lining cells. The total score for each kidney was calculated by addition of all 10 scores (Chatterjee et al., 2000; Hottendorf, 1984). 2.5. Statistics Results were expressed as mean 7SD of 7–8 rats. The One-Way Analysis of Variance (ANOVA or F test) followed by Student– Newman–Keuls post test was utilized. ANOVA was performed using a computer software program Graph PAD Instat (Version 1.13). The criterion for statistical significance was set at the 0.05 level.
3. Results 3.1. Effect of endothelin receptor blockers on cisplatin-induced deterioration in kidney functions Compared to control (saline-treated) group, there were significant increases in blood urea nitrogen and serum creatinine concentrations in rats treated with cisplatin at 96 h following cisplatin injection (20.69 71.45 vs. 62.6374.74 mg/dl and 0.257 0.012 vs. 1.23 70.037 mg/dl, respectively). Coadministration of BQ-123 with cisplatin resulted in a significant
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Fig. 1. Blood urea nitrogen (A), serum creatinine concentrations (mg/dl) (B), activity of superoxide dismutase (U/mg tissue protein) (C) and TNF-α concentration (pg/ml homogenate) (D) in kidney homogenates obtained from male rats at 96 h following the administration of saline (control), cisplatin (6 mg/kg, i.p.), cisplatin þ BQ-123 (1 mg/ kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose). Values are means of 7–8 observations expressed as mean 7 SD. n, # and þ denote significant difference (P o0.05) vs. control, cisplatin and cisplatin þ BQ-123 values, respectively.
reduction in blood urea nitrogen concentration compared to rats treated with cisplatin alone. However, the concentration of blood urea nitrogen in Cis þBQ-123 group still remained significantly higher than its corresponding value in the control group. On the other hand, co-administration of bosentan with cisplatin failed to produce any significant reduction in the concentration of blood urea nitrogen compared to cisplatin-treated rats (Fig. 1A). Concurrent treatment of rats with BQ-123 and cisplatin resulted in a significant reduction in serum creatinine concentration compared to rats treated with cisplatin alone; however this reduction is not complete since the serum creatinine concentration still remained significantly higher than its corresponding value in the
control group. On the other hand, co-administration of bosentan with cisplatin failed to produce any significant reduction in the concentration of serum creatinine compared to cisplatin-treated rats (Fig. 1B). 3.2. Effect of endothelin receptor blockers on cisplatin-induced intra-renal oxidative stress Malondialdehyde concentration was analyzed in different experimental groups as a marker of oxidative stress. Acute administration of cisplatin to male rats resulted in a significant increase in malondialdehyde concentration in kidney homogenate compared
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Fig. 2. Malondialdehyde (nmole/g tissue) (A), nitrite (nmole/g tissue) (B) and caspase-3 (ng/ml homogenate) (C) concentrations in kidney homogenates obtained from male rats at 96 h following the administration of saline (control), cisplatin (6 mg/kg, i.p.), cisplatin þ BQ-123 (1 mg/kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose). Values are means of 7–8 observations expressed as mean 7SD. n and # denote significant difference (P o0.05) vs. control and cisplatin values, respectively.
to saline-treated rats (43.5172.17 vs. 32.6771.03 nmole/g tissue). Co-administration of BQ-123 or bosentan with cisplatin resulted in a significant reduction in malondialdehyde concentration in kidney homogenate back to control value (Fig. 2A). Compared with salinetreated rats, there were significant decreases in renal SOD activity (Fig. 1C) and nitrate/nitrite level (Fig. 2B) in the cisplatin-treated group. Co-administration of BQ-123 with cisplatin resulted in a significant increase in SOD activity compared to rats treated with cisplatin alone. However, co-administration of bosentan with cisplatin failed to produce any significant increase in SOD activity compared to cisplatin-treated rats (Fig. 1C). On the contrary,
concurrent adminstration of either BQ-123 or bosentan with cisplatin resulted in a significant increase in nitrite level in kidney homogenate back to control value (Fig. 2B). 3.3. Effect of endothelin receptor blockers on cisplatin-induced changes in TNF-α and caspase-3 levels Changes evoked by the administration of cisplatin to rats showed that there were significant increases in levels of TNF-α and caspase-3 in kidney homogenates compared to control group. Co-administration of BQ-123 or bosentan with cisplatin resulted in
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a significant decrease in TNF-α concentration compared to rats treated with cisplatin alone. However, the concentration of TNF-α in cisplatin þ bosentan group still remained significantly higher than its corresponding value in the control group (Fig. 1D). Similarly, concurrent administration of either BQ-123 or bosentan with cisplatin resulted in a significant decrease in caspase-3 concentration compared to rats treated with cisplatin alone. However, the concentration of caspase-3 in both groups still remained significantly higher than its corresponding value in the control group (Fig. 2C). 3.4. Effect of endothelin receptor blockers on cisplatin-induced histological renal damage Histological examinations of the kidneys obtained from rats treated with cisplatin showed typical ‘acute tubular necrosis’ pattern (Dobyan et al., 1980; Saleh and El-Demerdash, 2005); namely, widespread tubular‐cell necrosis, tubular dilatation, and “sloughing off” of the renal lining cells (Fig. 3B). The total severity score for tubular damage was significantly reduced in rats administered BQ-123 or bosentan compared to the cisplatin-treated group (Fig. 3E).
4. Discussion There are few reports on the expression of the ET system in nephrotoxic ARF. In cisplatin-induced nephrotoxic ARF, previous reports demonstrated that renal damages were confined to the cortex, and that ET-1 and ETA receptors were up-regulated without change of ETB receptors (Lee and Ahn, 2008). However, the current study is the first to report the protective effect of the selective ETA receptor blocker, BQ-123, against cisplatin-induced ARF. Results of the current study demonstrated that acute administration of cisplatin (6 mg/kg) to male rats led to histological alterations of the renal tubular cells. These changes were associated with decline in renal functions, leading to increased concentrations of serum creatinine and blood urea nitrogen, and are in line with previous studies (Evenepoel, 2004; Lameire et al., 2005; Sahu et al., 2013). Furthermore, the findings of the current study point to the presence of oxidative stress and are in accordance with data in previous reports (Antunes et al., 2001; Mora et al., 2003; Sahu et al., 2013; Weijl et al., 1997). Despite the fact that the underlying mechanism of cisplatin-induced nephrotoxicity is still unclear, in vitro and in vivo studies provide strong evidence that implicate oxidative stress as a mediator of cisplatin-induced nephrotoxicity (Baliga et al., 1998; Kuhlmann et al., 1997; Sahu et al., 2013). Cisplatin was found to generate superoxide and hydroxyl radicals, and to stimulate renal lipid peroxidation (Kuhlmann et al., 1997). As a result, an imbalance between generation of oxygen-derived radicals and endogenous enzymatic and non-enzymatic antioxidants will occur leading to oxidative damage of cell components (Weijl et al., 1997). The role of NO in cisplatin nephrotoxicity is unclear. Previous studies had shown a decreased NO level measured as total nitrate/nitrite level following cisplatin administration (Saad et al., 2002; Saleh and El-Demerdash, 2005), which is in line with results of the current study. The decrease in NO synthesis induced by cisplatin can be explained by the detected pathological damage of glomerular endothelial cells. Previous studies suggested that changes in renal haemodynamics play an important role in cisplatin-induced nephrotoxicity. Renal injury by cisplatin has been associated with oxidative stress, inflammation, and apoptosis (Francescato et al., 2007; Iseri et al., 2007; Kiymaz et al., 2008; Ueki et al., 2012). Specifically, apoptosis is an important mode of cell death in cisplatin nephrotoxicity, and many studies including ours have demonstrated renal
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tubular cell apoptosis after cisplatin treatment. The binding of extracellular TNF-α to a cell surface receptor activates caspase 8, upregulates the pro-apoptotic protein Bax, and downregulates the anti-apoptotic protein Bcl-2 (Sahu et al., 2013; Tsuruya et al., 2003; Ueki et al., 2012). Caspase activation is thought to be important in the genesis of apoptosis. In particular, caspase-3, the execution caspase, is instrumental in the apoptotic process; it cleaves and activates poly (ADP-ribose) polymerase which leads to DNA fragmentation (Lau, 1999; Lieberthal et al., 1996). ET-1 directly inhibits Na-K-ATPase of renal tubular cells Garvin and Sanders (1991) and modulates a variety of signal transduction pathways through its receptors as well. Therefore, while there is little doubt that ET-1 plays a certain role in the pathogenesis of renal diseases, it is not yet clear whether ET-1 acts as a causative factor in various renal diseases including ARF. Lee and Ahn (2008) demonstrated increased ET-1 peptide and ET-1 mRNA in cisplatin-treated mice. Although ET-1 exerts its action through ETA and/or ETB, expression of ET receptors varies depending on ARF models used. Lee and Ahn (2008) showed that the expressions of ET-1 and ETA receptors, but not ETB, were increased, suggesting that the individual components of the renal ET system are differentially regulated in cisplatin-induced nephrotoxic ARF. Therefore, drug targeting to ETA receptor could help lessen nephrotoxic ARF induced by cisplatin. The reno-protective effect of the ETA selective antagonist BQ-123 has been documented against other nephrotoxic drugs as cyclosporine A (Kivlighn et al., 1994) as well as against ischemia-induced ARF (Erdogan et al., 2006). Concurrent administration of BQ-123 with cisplatin resulted in significant reduction in serum blood urea nitrogen and creatinine levels compared to rats treated with cisplatin alone. This was supported by the histological sections showing normal morphology. Up to our knowledge, this study is the first to reveal that selective ETA receptor blockade could significantly attenuate cisplatin-induced nephrotoxicity. It could be concluded that ET-1-induced vasoconstriction via ETA may contribute significantly but not completely to cisplatin-induced nephrotoxicity; BQ-123 targets only one of the pathophysiologic pathways for cisplatin which is the haemodynamic tone. In addition, increased expression of both ET-1 and ETA receptors in mice shown previously by Lee and Ahn (2008) was not an adaptive defensive reaction against cisplatin pathophysiological effects; but rather contributed to the cisplatin-induced nephrotoxicity. Furthermore, the current study is the first to give evidence that the ETA receptor selective antagonist BQ-123 reverses the cisplatin-induced ARF and that this effect requires the presence of an intact SOD enzymatic activity and functional ETB receptors. A notion that could be evidenced by the results obtained from the concurrent administration of the non-selective ET-1 blocker bosentan with cisplatin, which failed to reverse the cisplatin-induced ARF manifested as increases in blood urea nitrogen and serum creatinine. Despite the fact that its co-administration was able to reverse cisplatininduced changes in MDA, NO, TNF-α and caspase-3. Furthermore, the contradictory results obtained from the two blockers used highlighted the importance of presence of functional ETB receptors to elucidate the protective effect of BQ-123 against cisplatininduced ARF; since the blockade of ETB receptors mediated vasorelaxation (Mazzuca et al., 2013) seems to nullify the expected beneficial effects of the simultaneous blockade of ETA receptors mediated vasoconstriction during cisplatin-induced acute renal damage.
5. Conclusion To our knowledge, this study is the first to give evidence that the ETA selective antagonist BQ-123 reverses the cisplatin-induced
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Fig. 3. Hematoxylin and eosin stained photomicrographs ( 100) of kidneys obtained from male rats at 96 h following the administration of saline (control, (A)), cisplatin (6 mg/kg, i.p., (B)), cisplatin þ BQ-123 (1 mg/kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose, (C)), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose, (D)). H&E stained photomicrographs of normal renal tubules (A) is compared with those revealing tubular necrosis and dilatation in up to one third to two thirds of the tubules. Sloughing off of the renal lining cells is also presented in up to one third of the tubules (B). The total severity score for tubular damage was significantly reduced in rats administered BQ-123 or bosentan compared to the cisplatin-treated group (E). n and # denote significant difference (Po 0.05) vs. control and cisplatin values, respectively.
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ARF mainly via restoring SOD activity, in addition to other antioxidant parameters, NO, TNF-α and caspase-3 levels. And that this protective effect requires the presence of functional ETB receptors.
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Hottendorf, G.H., 1984. Renal morphologic–functional correlations and drug development. In: Solez, K., Wheton, A. (Eds.), Acute Renal Failure: Correlations between Morphology and Function. Marcel Dekker, New York, pp. 419–427. Iseri, S., Ercan, F., Gedik, N., Yuksel, M., Alican, I., 2007. Simvastatin attenuates cisplatin-induced kidney and liver damage in rats. Toxicology 230, 256–264. Kivlighn, S.D., Gabel, R.A., Siegl, P.K., 1994. Effects of BQ-123 on renal function and acute cyclosporine-induced renal dysfunction. Kidney Int. 45, 131–136. Kiymaz, N., Yilmaz, N., Mumcu, C., Anlar, O., Ozen, S., Kayaoglu, C.R., 2008. Protective effect of sildenafil (Viagra) in transient spinal cord ischemia. Pediatr. Neurosurg. 44, 22–28. Kone, B.C., 2004. Nitric oxide synthesis in the kidney: isoforms, biosynthesis, and functions in health. Semin. Nephrol. 24, 299–315. Kuhlmann, M.K., Burkhardt, G., Kohler, H., 1997. Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application. Nephrol. Dial. Transplant. 12, 2478–2480. Lameire, N., Van Biesen, W., Vanholder, R., 2005. Acute renal failure. Lancet 365, 417–430. Lau, A.H., 1999. Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int. 56, 1295–1298. Lee, S., Ahn, D., 2008. Expression of endothelin-1 and its receptors in cisplatininduced acute renal failure in mice. Korean J. Physiol. Pharmacol. 12 (4), 149–153. Lieberthal, W., Triaca, V., Levine, J., 1996. Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am. J. Physiol. 270, F700–708. Marklund, S., Marklund, G., 1974. Involvement of superoxide anion radical in autoxidation of pyrogallol and a convenient assay for SOD. Eur. J. Biochem. 47, 469–474. Mazzuca, M.Q., Dang, Y., Khalil, R.A., 2013. Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca2 þ ]i in mesenteric microvessels of pregnant rats. Br. J. Pharmacol 169 (6), 1335–1351. McWhinnie, D.L., Thompson, J.F., Taylor, H.M., Chapman, J.R., Bolton, E.M., Carter, N.P., Wood, R.F., Morris, P.J., 1986. Morphometric analysis of cellular infiltration assessed by monoclonal antibody labeling in sequential human renal allograft biopsies. Transplantation 42, 352–358. Mora, L., Antunes, L.M., Franceskocato, H.D.C., Bianchi, M., 2003. The effects of oral glutamine on cisplatin-induced nephrotoxicity in rats. Pharmacol. Res. 47, 511–522. Ottolenghi, A., 1959. 2-Thiobarbituric acid (TBA) method. Arch. Biochem. Biophys. 77, 355–360. Palipoch, S., Punsawad, C., 2013. Biochemical and histological study of rat liver and kidney injury induced by cisplatin. J. Toxicol. Pathol. 26, 293–299. Saad, S.Y., Najjar, T.A., Daba, M.H., Al-Rikabi, A.C., 2002. Inhibition of nitric oxide synthase aggravates cisplatin induced nephrotoxicity: effect of 2-amino4-methylpyridine. Chemotherapy 48, 309–315. Sahu, B.D., Kuncha, M., Sindhura, G.J., Sistla, R., 2013. Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation and DNA damage. Phytomedicine 20 (5), 453–460. Saleh, S., El-Demerdash, E., 2005. Protective effects of l-arginine against cisplatin induced renal oxidative stress and toxicity: role of nitric oxide. Basic Clin. Pharmacol. Toxicol. 97, 91–97. Slowinski, T., Subkowski, T., Diehr, P., Bachert, D., Fritsche, L., Neumayer, H.H., Hocher, B., 2002. Interaction of the endothelin system and calcineurin inhibitors after kidney transplantation. Clin. Sci. 103 (48), 396S–398S. Tagboto, S., Griffiths, A.P., 2007. The evaluation of renal ischaemic damage: the value of CD10 monoclonal antibody staining and of biochemical assessments of tissue viability. BMC Clin. Pathol. 7, 5. Takaoka, M., Kuro, T., Matsumura, Y., 2000. Role of endothelin in the pathogenesis of acute renal failure. Drug News Perspect. 13, 141–146. Tsuruya, K., Ninomiya, T., Tokumoto, M., Hirakawa, M., Masutani, K., Taniguchi, M., Fukuda, K., Kanai, H., Kishihara, K., Hirakata, H., Iida, M., 2003. Direct involvement of the receptor-mediated apoptotic pathways in cisplatin-induced renal tubular cell death. Kidney Int. 63, 72–82. Ueki, M., Ueno, M., Morishita, J., Maekawa, N., 2012. Curcumin ameliorates cisplatin-induced nephrotoxicity by inhibiting renal inflammation in mice. J. Biosci. Bioeng. 115 (5), 547–551. Weijl, N.I., Cleton, F.J., Osanto, S., 1997. Free radicals and antioxidants in chemotherapy-induced toxicity. Cancer Treat. Rev. 23, 209–240. Wilhelm, S.M., Stowe, N.T., Robinson, A.V., Schulak, J.A., 2001. The use of the endothelin receptor antagonist, tezosentan, before or after renal ischemia protects renal function. Transplantation 27, 211–216. Yao, X., Panichpisal, K., Kurtzman, N., Nugent, K., 2007. Cisplatin nephrotoxicity: a review. Am. J. Med. Sci. 334, 115–124.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Selective ETA receptor blockade protects against cisplatin-induced acute renal failure in male rats Mai M. Helmy a,n, Maged W. Helmy b, Dina M. Abd Allah c, Ahmad M. Abo Zaid b, Mahmoud M. Mohy El-Din a a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Azarita, PO Box 21521, Alexandria, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Alexandria, Egypt c Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Azarita, Alexandria, Egypt b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 October 2013 Received in revised form 26 February 2014 Accepted 6 March 2014 Available online 14 March 2014
The present study aims to investigate the possibility that inhibiting the physiological function of endothelin-1 (ET-1) by blocking its receptors would significantly decrease the nephrotoxic effect of cisplatin. Therefore the study was designed to investigate the effect of treatment with BQ-123, the selective endothelin receptor-A (ETA) blocker, and bosentan, the non-selective endothelin receptor blocker, on the cisplatin-induced structural, functional, and biochemical alterations in the rat kidney. Rats were divided into four groups: control (given a single dose of normal saline, i.p.), cisplatin (given a single dose of cisplatin, 6 mg/kg, i.p.), cisplatinþ BQ-123 (1 mg/kg, i.p.), and cisplatin þbosentan (30 mg/kg, orally via gavage). Each of the two blockers was administered in two doses; 1 h before and one day after the cisplatin dose. Acute cisplatin administration resulted in significant increases in blood urea nitrogen and serum creatinine concentrations at 96 h following cisplatin injection. Increased concentrations of malondialdehyde, tumor necrosis factor-α (TNF-α) and caspase-3, decreased nitric oxide (NO) production and superoxide dismutase (SOD) activity in kidney homogenates were observed at 96 h following cisplatin injection, in addition to a typical ‘acute tubular necrosis’ pattern. BQ-123 ameliorated the structural and functional injuries caused by cisplatin mainly via restoring SOD activity, in addition to other antioxidant parameters, NO, TNF-α and caspase-3 concentrations. This study further proves that ETA but not ETB receptors are involved in cisplatin-induced nephrotoxicity. The selective ETA antagonist BQ-123 ameliorated the cisplatin-induced deleterious effects and showed reno-protective effect against cisplatin-induced acute renal damage. & 2014 Elsevier B.V. All rights reserved.
Keywords: Cisplatin Endothelin-1 ETA receptors BQ-123 SOD Nephrotoxicity
1. Introduction Although cisplatin is a highly effective anti-neoplastic agent, its nephrotoxicity is a major clinical problem (Saleh and ElDemerdash, 2005). Cisplatin causes tubular injury through multiple mechanisms, including hypoxia, the generation of free radicals, inflammation, and apoptosis. Additionally, significant interactions among these various pathways may occur in cisplatin injury (Yao et al., 2007). Endothelins have a key role in vascular homeostasis. Three isoforms of endothelin have been identified: the originally described endothelin-1 (ET-1) and two similar peptides, ET-2 and ET-3. Two endothelin receptor subtypes, termed ETA and ETB, have been cloned and sequenced. ETA receptors have a high affinity for ET-1 and a low affinity for ET-3 and are located on
n
Corresponding author. Fax: þ 203 487 3273. E-mail address: [email protected] (M.M. Helmy).
http://dx.doi.org/10.1016/j.ejphar.2014.03.002 0014-2999/& 2014 Elsevier B.V. All rights reserved.
smooth muscle cells, where they mediate vasoconstriction. ETB receptors have approximately equal affinities for ET-1 and ET-3 and are located on vascular endothelial cells, where they mediate release of prostaglandins-I2 (PGI2) and nitric oxide (NO) (Takaoka et al., 2000). Both receptor subtypes belong to the G proteincoupled seven-transmembrane domain family of receptors. Endothelins cause dose-dependent vasoconstriction in most vascular beds. There is increasing evidence that endothelins participate in a variety of cardiovascular diseases (Takaoka et al., 2000), including hypertension, cardiac hypertrophy, as well as in renal failure (Kone, 2004). They act on the kidneys to cause vasoconstriction and decrease glomerular filtration rate and sodium and water excretion (Takaoka et al., 2000). Interestingly, increased level of both circulating ET-1 and urinary excretion of ET-1 have been observed in patients treated with nephrotoxic immunosuppressive agents as cyclosporine A and tacrolimus (Slowinski et al., 2002). Other nephrotoxic agents, such as cisplatin, also increase urinary excretion of ET. Moreover, expressions of ET-1 have been found to be elevated in
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mice with cisplatin-induced acute renal failure (ARF), suggesting that ET-1 might play a role in the pathogenesis of such a disease (Lee and Ahn, 2008). ET receptor antagonism improved renal failure after ARF (Gellai et al., 1994). Treatment with an ETA receptor antagonist before or post-ischemia has been demonstrated to lessen glomerular dysfunction in rats (Wilhelm et al., 2001). Buyukgebiz et al. (1996) mentioned that inhibiting function of ET via an ETA receptor antagonist might prevent reperfusion injury in kidney transplantation. Bosentan is a non-selective endothelin receptors antagonist that blocks both ETA and ETB. In contrast, BQ-123 {2-[(3R,6R,9S, 12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,11,14pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl]acetic acid} is an ETA receptor selective antagonist (Buyukgebiz et al., 1996). In clinical trials, bosentan and other non-selective antagonists as well as ETA receptor selective antagonists have produced beneficial effects on hemodynamics and other symptoms in heart failure, pulmonary hypertension, and essential hypertension (Wilhelm et al., 2001). Therefore, the current study aimed at investigating the possibility that inhibiting the physiological function of ET-1 by blocking its receptors would significantly decrease the nephrotoxic effect of cisplatin. We, therefore, evaluated the reno-protective effects of both the non-selective ET receptors antagonist bosentan and the ETA receptor selective antagonist BQ-123 against cisplatin-induced nephrotoxicity.
2. Materials and methods 2.1. Chemicals Bosentan monohydrate (free base) was generously gifted by Actelion Pharmaceuticals Ltd., Allscwil, Switzerland; cisplatin: Cisplatines Mylan 10 ml vial, Oncotec Pharma production, Germany; BQ-123: BQ-123 sodium salt 5 mg, purchased from Peptides international, Louisville, Kentucky, USA; other chemicals and reagents used in biochemical tests were of analytical grade. Details of quantitative analysis kits are under biochemical analysis section. 2.2. Experimental design The study was performed on adult male Sprague Dawley rats weighing 200–220 g, obtained from the Animal House of the Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Egypt. The animals were kept at a temperature of 2572 1C, on 12-h light/dark cycle (lights on at 7.00 am) and had free access to food and tap water before the experiment. The research experiments protocol was approved by the Animal Care & Use Committee (ACUC) at the faculty of Pharmacy, Alexandria University, Egypt. Animals were divided into four groups: (i) control (given a single dose of normal saline, i.p.), (ii) cisplatin (given a single dose of cisplatin, 6 mg/kg, i.p. (Dobyan et al., 1980), (iii) cisplatinþBQ-123 (1 mg/kg, i. p. (Garrido and Israel, 2002), and (iv) cisplatinþ bosentan (30 mg/kg, orally via gavage (Günal et al., 2006). Each of the two blockers was administered in two doses; 1 h before and one day after the cisplatin dose. The rationale of this schedule of dose administration was the maintenance of a steady sufficient plasma concentration of each of the drugs before, during and after the critical period of cisplatininduced toxicity, since the biochemical changes that occur in the kidney after cisplatin administration are of crucial importance in determining the extent of a nephrotoxic lesion (Borch and Pleasants, 1979). In fact, the dose of cisplatin used in the current study was lower than that used in previous experimental studies (El-Naga, 2014; Palipoch and Punsawad, 2013) and approximately double the minimal therapeutic dose that should be given to rats. Since the
therapeutic doses of cisplatin for an ideal man weighing about 70 kg ranged from 20 to 100 mg/m2 (Chabner et al., 2006) which is equivalent to 3.02–15.1 mg/kg rat according to Paget and Barnes table conversion factor. 2.3. Biochemical analysis 96 h after the cisplatin injection, rats were anaesthesized with thiopental sodium (50 mg/kg, intraperitoneally) El-Mas and AbdelRahman (1997) and blood samples were obtained from the orbital plexus (3 ml from each rat). Serum was recovered and stored at 70 1C until analyzed for determination of blood urea nitrogen and serum creatinine using the Randoxs assay kit (Randox Laboratories Ltd., United Kingdom). After collecting blood samples, rats were euthanized with an over dose of the anesthetic drug, the chest was opened, internal viscera were pulled aside. The kidneys were quickly removed, washed in ice cold saline, blotted dry, then weighed. The kidney homogenates were divided into portions and stored at 70 1C until analyzed for determination of renal malondialdehyde (Ottolenghi, 1959), superoxide dismutase (SOD) (Marklund and Marklund, 1974), and nitrite/nitrate (Guevara et al., 1998); tumor necrosis factor-alpha (TNF-α) was determined using RayBios Rat Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Ray Biotech Inc., Georgia, USA), and caspase-3 was determined using Bender MedSystems Caspase-3 instant ELISA kit (Bender MedSystems, Vienna, Austria, Europe) according to the manufacturer’s instructions. 2.4. Histological evaluation Following the animals’ sacrifice, tissues from the kidneys were carefully removed and preserved in 10% formalin for histological examination with haematoxylin and eosin (H&E) stain under a light microscope (Carleton, 1980; Tagboto and Griffiths, 2007). Histological evaluation of tubular necrosis was determined semiquantitatively using the methods modified from McWhinnie and coworkers (McWhinnie et al., 1986). Random fields were observed from each section using 100 magnification. Ten sections were examined for each kidney and a score from 0 to 3 was given to the tubular profile such that: 0 ¼normal histology, 1 ¼for interstitial nephritis and inflammation, 2 ¼for tubular atrophy and intratubular dilation with or without intralumenal hyaline casts, 3 ¼for renal tubular necrosis with “sloughing off” of the renal tubular lining cells. The total score for each kidney was calculated by addition of all 10 scores (Chatterjee et al., 2000; Hottendorf, 1984). 2.5. Statistics Results were expressed as mean 7SD of 7–8 rats. The One-Way Analysis of Variance (ANOVA or F test) followed by Student– Newman–Keuls post test was utilized. ANOVA was performed using a computer software program Graph PAD Instat (Version 1.13). The criterion for statistical significance was set at the 0.05 level.
3. Results 3.1. Effect of endothelin receptor blockers on cisplatin-induced deterioration in kidney functions Compared to control (saline-treated) group, there were significant increases in blood urea nitrogen and serum creatinine concentrations in rats treated with cisplatin at 96 h following cisplatin injection (20.69 71.45 vs. 62.6374.74 mg/dl and 0.257 0.012 vs. 1.23 70.037 mg/dl, respectively). Coadministration of BQ-123 with cisplatin resulted in a significant
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Fig. 1. Blood urea nitrogen (A), serum creatinine concentrations (mg/dl) (B), activity of superoxide dismutase (U/mg tissue protein) (C) and TNF-α concentration (pg/ml homogenate) (D) in kidney homogenates obtained from male rats at 96 h following the administration of saline (control), cisplatin (6 mg/kg, i.p.), cisplatin þ BQ-123 (1 mg/ kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose). Values are means of 7–8 observations expressed as mean 7 SD. n, # and þ denote significant difference (P o0.05) vs. control, cisplatin and cisplatin þ BQ-123 values, respectively.
reduction in blood urea nitrogen concentration compared to rats treated with cisplatin alone. However, the concentration of blood urea nitrogen in Cis þBQ-123 group still remained significantly higher than its corresponding value in the control group. On the other hand, co-administration of bosentan with cisplatin failed to produce any significant reduction in the concentration of blood urea nitrogen compared to cisplatin-treated rats (Fig. 1A). Concurrent treatment of rats with BQ-123 and cisplatin resulted in a significant reduction in serum creatinine concentration compared to rats treated with cisplatin alone; however this reduction is not complete since the serum creatinine concentration still remained significantly higher than its corresponding value in the
control group. On the other hand, co-administration of bosentan with cisplatin failed to produce any significant reduction in the concentration of serum creatinine compared to cisplatin-treated rats (Fig. 1B). 3.2. Effect of endothelin receptor blockers on cisplatin-induced intra-renal oxidative stress Malondialdehyde concentration was analyzed in different experimental groups as a marker of oxidative stress. Acute administration of cisplatin to male rats resulted in a significant increase in malondialdehyde concentration in kidney homogenate compared
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Fig. 2. Malondialdehyde (nmole/g tissue) (A), nitrite (nmole/g tissue) (B) and caspase-3 (ng/ml homogenate) (C) concentrations in kidney homogenates obtained from male rats at 96 h following the administration of saline (control), cisplatin (6 mg/kg, i.p.), cisplatin þ BQ-123 (1 mg/kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose). Values are means of 7–8 observations expressed as mean 7SD. n and # denote significant difference (P o0.05) vs. control and cisplatin values, respectively.
to saline-treated rats (43.5172.17 vs. 32.6771.03 nmole/g tissue). Co-administration of BQ-123 or bosentan with cisplatin resulted in a significant reduction in malondialdehyde concentration in kidney homogenate back to control value (Fig. 2A). Compared with salinetreated rats, there were significant decreases in renal SOD activity (Fig. 1C) and nitrate/nitrite level (Fig. 2B) in the cisplatin-treated group. Co-administration of BQ-123 with cisplatin resulted in a significant increase in SOD activity compared to rats treated with cisplatin alone. However, co-administration of bosentan with cisplatin failed to produce any significant increase in SOD activity compared to cisplatin-treated rats (Fig. 1C). On the contrary,
concurrent adminstration of either BQ-123 or bosentan with cisplatin resulted in a significant increase in nitrite level in kidney homogenate back to control value (Fig. 2B). 3.3. Effect of endothelin receptor blockers on cisplatin-induced changes in TNF-α and caspase-3 levels Changes evoked by the administration of cisplatin to rats showed that there were significant increases in levels of TNF-α and caspase-3 in kidney homogenates compared to control group. Co-administration of BQ-123 or bosentan with cisplatin resulted in
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a significant decrease in TNF-α concentration compared to rats treated with cisplatin alone. However, the concentration of TNF-α in cisplatin þ bosentan group still remained significantly higher than its corresponding value in the control group (Fig. 1D). Similarly, concurrent administration of either BQ-123 or bosentan with cisplatin resulted in a significant decrease in caspase-3 concentration compared to rats treated with cisplatin alone. However, the concentration of caspase-3 in both groups still remained significantly higher than its corresponding value in the control group (Fig. 2C). 3.4. Effect of endothelin receptor blockers on cisplatin-induced histological renal damage Histological examinations of the kidneys obtained from rats treated with cisplatin showed typical ‘acute tubular necrosis’ pattern (Dobyan et al., 1980; Saleh and El-Demerdash, 2005); namely, widespread tubular‐cell necrosis, tubular dilatation, and “sloughing off” of the renal lining cells (Fig. 3B). The total severity score for tubular damage was significantly reduced in rats administered BQ-123 or bosentan compared to the cisplatin-treated group (Fig. 3E).
4. Discussion There are few reports on the expression of the ET system in nephrotoxic ARF. In cisplatin-induced nephrotoxic ARF, previous reports demonstrated that renal damages were confined to the cortex, and that ET-1 and ETA receptors were up-regulated without change of ETB receptors (Lee and Ahn, 2008). However, the current study is the first to report the protective effect of the selective ETA receptor blocker, BQ-123, against cisplatin-induced ARF. Results of the current study demonstrated that acute administration of cisplatin (6 mg/kg) to male rats led to histological alterations of the renal tubular cells. These changes were associated with decline in renal functions, leading to increased concentrations of serum creatinine and blood urea nitrogen, and are in line with previous studies (Evenepoel, 2004; Lameire et al., 2005; Sahu et al., 2013). Furthermore, the findings of the current study point to the presence of oxidative stress and are in accordance with data in previous reports (Antunes et al., 2001; Mora et al., 2003; Sahu et al., 2013; Weijl et al., 1997). Despite the fact that the underlying mechanism of cisplatin-induced nephrotoxicity is still unclear, in vitro and in vivo studies provide strong evidence that implicate oxidative stress as a mediator of cisplatin-induced nephrotoxicity (Baliga et al., 1998; Kuhlmann et al., 1997; Sahu et al., 2013). Cisplatin was found to generate superoxide and hydroxyl radicals, and to stimulate renal lipid peroxidation (Kuhlmann et al., 1997). As a result, an imbalance between generation of oxygen-derived radicals and endogenous enzymatic and non-enzymatic antioxidants will occur leading to oxidative damage of cell components (Weijl et al., 1997). The role of NO in cisplatin nephrotoxicity is unclear. Previous studies had shown a decreased NO level measured as total nitrate/nitrite level following cisplatin administration (Saad et al., 2002; Saleh and El-Demerdash, 2005), which is in line with results of the current study. The decrease in NO synthesis induced by cisplatin can be explained by the detected pathological damage of glomerular endothelial cells. Previous studies suggested that changes in renal haemodynamics play an important role in cisplatin-induced nephrotoxicity. Renal injury by cisplatin has been associated with oxidative stress, inflammation, and apoptosis (Francescato et al., 2007; Iseri et al., 2007; Kiymaz et al., 2008; Ueki et al., 2012). Specifically, apoptosis is an important mode of cell death in cisplatin nephrotoxicity, and many studies including ours have demonstrated renal
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tubular cell apoptosis after cisplatin treatment. The binding of extracellular TNF-α to a cell surface receptor activates caspase 8, upregulates the pro-apoptotic protein Bax, and downregulates the anti-apoptotic protein Bcl-2 (Sahu et al., 2013; Tsuruya et al., 2003; Ueki et al., 2012). Caspase activation is thought to be important in the genesis of apoptosis. In particular, caspase-3, the execution caspase, is instrumental in the apoptotic process; it cleaves and activates poly (ADP-ribose) polymerase which leads to DNA fragmentation (Lau, 1999; Lieberthal et al., 1996). ET-1 directly inhibits Na-K-ATPase of renal tubular cells Garvin and Sanders (1991) and modulates a variety of signal transduction pathways through its receptors as well. Therefore, while there is little doubt that ET-1 plays a certain role in the pathogenesis of renal diseases, it is not yet clear whether ET-1 acts as a causative factor in various renal diseases including ARF. Lee and Ahn (2008) demonstrated increased ET-1 peptide and ET-1 mRNA in cisplatin-treated mice. Although ET-1 exerts its action through ETA and/or ETB, expression of ET receptors varies depending on ARF models used. Lee and Ahn (2008) showed that the expressions of ET-1 and ETA receptors, but not ETB, were increased, suggesting that the individual components of the renal ET system are differentially regulated in cisplatin-induced nephrotoxic ARF. Therefore, drug targeting to ETA receptor could help lessen nephrotoxic ARF induced by cisplatin. The reno-protective effect of the ETA selective antagonist BQ-123 has been documented against other nephrotoxic drugs as cyclosporine A (Kivlighn et al., 1994) as well as against ischemia-induced ARF (Erdogan et al., 2006). Concurrent administration of BQ-123 with cisplatin resulted in significant reduction in serum blood urea nitrogen and creatinine levels compared to rats treated with cisplatin alone. This was supported by the histological sections showing normal morphology. Up to our knowledge, this study is the first to reveal that selective ETA receptor blockade could significantly attenuate cisplatin-induced nephrotoxicity. It could be concluded that ET-1-induced vasoconstriction via ETA may contribute significantly but not completely to cisplatin-induced nephrotoxicity; BQ-123 targets only one of the pathophysiologic pathways for cisplatin which is the haemodynamic tone. In addition, increased expression of both ET-1 and ETA receptors in mice shown previously by Lee and Ahn (2008) was not an adaptive defensive reaction against cisplatin pathophysiological effects; but rather contributed to the cisplatin-induced nephrotoxicity. Furthermore, the current study is the first to give evidence that the ETA receptor selective antagonist BQ-123 reverses the cisplatin-induced ARF and that this effect requires the presence of an intact SOD enzymatic activity and functional ETB receptors. A notion that could be evidenced by the results obtained from the concurrent administration of the non-selective ET-1 blocker bosentan with cisplatin, which failed to reverse the cisplatin-induced ARF manifested as increases in blood urea nitrogen and serum creatinine. Despite the fact that its co-administration was able to reverse cisplatininduced changes in MDA, NO, TNF-α and caspase-3. Furthermore, the contradictory results obtained from the two blockers used highlighted the importance of presence of functional ETB receptors to elucidate the protective effect of BQ-123 against cisplatininduced ARF; since the blockade of ETB receptors mediated vasorelaxation (Mazzuca et al., 2013) seems to nullify the expected beneficial effects of the simultaneous blockade of ETA receptors mediated vasoconstriction during cisplatin-induced acute renal damage.
5. Conclusion To our knowledge, this study is the first to give evidence that the ETA selective antagonist BQ-123 reverses the cisplatin-induced
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40
Total severity score
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os
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Fig. 3. Hematoxylin and eosin stained photomicrographs ( 100) of kidneys obtained from male rats at 96 h following the administration of saline (control, (A)), cisplatin (6 mg/kg, i.p., (B)), cisplatin þ BQ-123 (1 mg/kg, i.p. at two time intervals; 1 h before and one day after the cisplatin dose, (C)), and cisplatin þ bosentan (30 mg/kg, orally at two time intervals; 1 h before and one day after the cisplatin dose, (D)). H&E stained photomicrographs of normal renal tubules (A) is compared with those revealing tubular necrosis and dilatation in up to one third to two thirds of the tubules. Sloughing off of the renal lining cells is also presented in up to one third of the tubules (B). The total severity score for tubular damage was significantly reduced in rats administered BQ-123 or bosentan compared to the cisplatin-treated group (E). n and # denote significant difference (Po 0.05) vs. control and cisplatin values, respectively.
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ARF mainly via restoring SOD activity, in addition to other antioxidant parameters, NO, TNF-α and caspase-3 levels. And that this protective effect requires the presence of functional ETB receptors.
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