BASIC RESEARCH
Protective effect of N-acetylcysteine on cyclosporine A-induced changes in lipid hydroperoxide levels and renal dysfunction in rats

Introduction
Cyclosporine A (CsA) is the most common immunosuppressive drug used for the prevention of allograft rejection [1]. Cyclosporine has improved patient and graft survival rate in solid organ transplantation and has been increasingly applied with considerable clinical benefits [2, 3]. Therapeutic benefits of cyclosporine are limited by the occur-rence of chronic nephrotoxicity. Acute renal dysfunction with cyclosporine therapy was recognized at the time of its first use in clinical renal transplantation [4, 5]. The exact mechanism of CsA-induced nephrotoxicity remains obscure. Clinical and experimental studies have revealed that several mechanisms may be involved [4, 6, 7]. Cyclosporine-induced nephrotoxicity seems to be caused by reduction in renal blood flow caused by arteriolar constriction [8]. Cyclosporine A nephro-toxicity is characterized by progressive renal dysfunction, afferent arteriolopathy and inflammatory cell influx [9]. Several lines of evidence suggest that cyclosporine increases hypoxia, decreases glomerular filtration rate and increases intra-renal vascular resistance [10].
Cumulative data suggest a role for reactive oxygen metabolites as one of the postulated mechanisms in the pathogenesis of CsA nephrotoxicity [11-13]. It is known that CsA increases renal nerve activity, resulting in vasoconstriction [14]. In addition, CsA brings about vasoconstriction in isolated renal arterioles by direct actions [15]. The vasoconstriction is reported to be due to CsA action in blocking mitochondrial calcium release, inducing increased intracellular calcium which causes vasoconstriction. These changes could lead to renal hypoxia reoxygenation injury and production of reactive oxygen free radicals [16, 17]. In vitro and in vivo studies indicate that CsA reduces renal microsomal NADPH cytochrome P450 and renal reduced/oxidized glutathione ratio in the kidneys. The extent to which the adverse effects of cyclosporine are related to the immuno-suppressive mechanisms of the drug is controversial [18]. Cyclosporine A is known to alter the production of many biologically active agents, such as endothelin, nitric oxide (NO) and transforming growth factor-b1 (TGF-b1), which have been implicated in adverse renal effects [6, 10, 18]. Endothelial dysfunction and reduced activity of endothelial derived nitric oxide might be one of the mechanisms underlining the functional effects of CsA on glomerular filtration [19].
N-acetylcysteine (NAC) is a thiol-containing antioxidant agent. N-acetylcysteine scavenges oxidants directly and replenishes intracellular glutathione [20]. Stimulation of glutathione following administration of NAC results in greater supply of glutathione for detoxification of oxygen free radicals and other foreign substances [21]. There are few reports showing the protective effect of N-acetylcysteine against oxygen free radical mediated injuries in the liver, heart and lungs [21, 22]. N-acetylcysteine has proven to be renopro-tective in toxic and ischaemic acute renal failure, although results have not been conclusive. It has been reported that antioxidant effects of NAC are able to prevent the increase in plasma peroxynitrite after ischaemia and NAC ameliorates the renal failure induced by inferior vena cava occlusion [23, 24]. N-acetylcysteine is reported to enhance the biological effects of nitric oxide and is known to have positive effects in reversing the haemodynamic disturbances in the renal circulation in acute renal failure [25]. A diet rich in natural substances reduces the risk of diseases associated with an increase in oxidative stress [26, 27].
The aim of this study was to observe the effect of NAC in reducing the cyclosporine-induced alterations in lipid hydroperoxides, total antioxidant levels and renal histopathology, and our research hypothesis was: NAC will increase the renal total antioxidant levels and ameliorate the cyclosporine- induced renal damage.

Material and methods
Animals
Adult male Sprague-Dawley rats weighing between 200 and 250 γ were housed two per cage with food and water ad libitum for two weeks before the beginning of the experiment. The animals were kept on husk bedding with a 12-h dark/light cycle. The animals had free access to standard rodent food and water. The study was conducted between 15/10/2006 and 20/05/2007 at the International Medical University, Kuala Lumpur, Malaysia. All experiments were performed in accordance with institutional guidelines for the ethical care of animals. The study protocol was designed in accordance with the 1996 revised form of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH), United States, and the study received approval from the Institutional Ethics Committee.
Experimental groups
The rats were randomly divided into four groups (n = 8 in each group) and treated for 21 days. Group I (control group) received vehicle of CsA, i.e. olive oil orally. Group II rats were treated with CsA dissolved in olive oil orally (20 mg/kg body weight). This group served as a positive control. Group III received NAC alone intraperitoneally (20 mg/kg body weight). Group IV rats were treated with cyclosporine (20 mg/kg body weight) and NAC (20 mg/kg body weight). In group IV NAC was administered intraperitoneally 30 min before CsA administration. Cyclosporine A (Sandimmune from Novartis, Malaysia) was dissolved to give a final concentration of 10 mg/ml. N-acetylcysteine (Sigma-Aldrich, USA) was dissolved to give a final concentration of 10 mg/ml. The drugs were freshly prepared for administration.
Daily body weight and food intake were recorded throughout the experiment. After 24 h after the last treatment, body weight of the animals in all the groups were recorded and rats were sacrificed 24 h after the last dose (on the 22nd day) using sodium pentobarbital anaesthesia (40 mg/kg body weight; Sigma Aldrich, USA) and blood samples were collected through cardiac puncture. Serum was separated and frozen at –20°C for biochemical analysis. A midline abdominal incision was done and both kidneys were removed and weighed; the left kidney was perfused with ice cold saline (0.9% NaCl) and homogenized in chilled phosphate buffer. The homogenate was centrifuged at 4000 rpm for 30 min at +4°C and the supernatant collected was stored at –20°C until analysis of lipid hydroperoxides and total antioxidants. The right kidney was stored in 10% neutral buffered formalin for histopathological studies. From the serum samples, creatinine, blood urea nitrogen (BUN), urea and uric acid assays were done by spectro-photometric methods using QuantiChrom Assay Kits (BioAssay Systems. USA). Lipid hydroperoxides and total antioxidants in the serum and kidneys were estimated using ELISA kits (Cayman Chemicals. USA).
Histopathological examination of kidneys: For light microscopic evaluation, portions of right kidney were sectioned and fixed in 10% neutral phosphate-buffered formaldehyde and embedded in paraffin. These specimens were cut into sections of 4 µm and these sections were stained with haematoxylin and eosin. A minimum of ten fields for each kidney section slide were examined blindly by a pathologist who was unaware of the treatment regimens used. The kidneys were examined for glomerular and tubular alterations, tubular casts, tubular necrosis, and blood vessel changes. All histopathological parameters were graded as follows: none (–), showing no meaningful histopathological changes; mild (+), with occasional glomerulus showing reduction in size, occasional blood vessel thickening, few foci of dilatation and casts; moderate (++), showing glomeruli beginning to undergo atrophy at different foci, thickened blood vessels, dilated blood vessels and tubular casts at different foci throughout the kidney; severe (+++), with marked histopathological changes showing extensive glomerular atrophy and tubular casts, tubular necrosis, thickened blood vessels, areas of congestion of interstitium and blood vessels, and very large areas of extravasation of red blood cells into the interstitium.
Statistical methods
Data are presented as means ± SD. The statistical analysis was done using SPSS statistical software version 12. The non-parametric Kruskal-Wallis and Mann-Whitney tests were used as not all the parameters were normally distributed. Global comparison among the groups (control, cyclosporine alone, NAC alone and NAC with CsA) was done using the Kruskal-Wallis test. To further explore the effect of CsA on kidney toxicity and the nullifying effect of NAC, pair-wise comparisons were carried out using the Mann-Whitney test. A value of p < 0.05 was considered statistically significant.

Results
There was a significant increase in BUN, serum creatinine, urea and uric acid levels in rats treated with cyclosporine alone for three weeks compared to control or NAC alone groups (p < 0.01). The results show that the level of creatinine, BUN, urea and uric acid was highest in rats treated with cyclosporine for 21 days (p < 0.01) when compared to the control group (Table I). Treatment with NAC alone did produce a significant decrease in creatinine (p < 0.05), urea (p < 0.01) and uric acid (p < 0.05) levels compared to control groups. When NAC was treated with CsA it produced a significant decrease in these blood parameters, compared to the CsA alone group (p < 0.01 and p < 0.05). But the level of these parameters remained significantly higher than in the control group (p < 0.01) and NAC alone (p < 0.01) groups.
When the total antioxidant levels were compared between the groups, a statistically significant reduction in the antioxidant levels both in the kidney and serum was recorded in the cyclosporine alone group compared to the control (p < 0.01) and NAC alone group (p < 0.01) (Table II). N-acetylcysteine treatment alone significantly increased (p < 0.01) the total antioxidants in the kidneys and serum when compared to the control group. Treatment with N-acetylcysteine with CsA significantly increased the total antioxidant levels (p < 0.01) compared to the cyclosporine alone group and the increase was significantly more than the control total antioxidant levels (p < 0.01). No significant difference in total antioxidant levels was seen in the serum and kidneys when the NAC with CsA treated group was compared with the NAC alone group. There was a significantly greater decrease in the antioxidant levels with cyclosporine treatment in the serum (62%) than in the kidneys (49%). Compared to cyclosporine alone, there was a more than 600% increase in total antioxidant levels in the NAC with CsA group, indicating the significant antioxidant effect of NAC (Table II).
Lipid hydroperoxide levels in the serum and kidneys increased significantly with cyclosporine treatment (p < 0.01) when compared to control levels and NAC alone treatment (p < 0.01). Treatment with NAC alone was able to reduce the lipid hydroperoxides (p < 0.05) compared to control rats. Cyclosporine treatment for three weeks increased the lipid hydroperoxide levels 117% in the serum and above 300% in the kidneys compared to the control group. N-acetylcysteine treatment along with cyclosporine was able to decrease these changes significantly (p < 0.01). Lipid hydroperoxides in the serum and kidneys remained significantly higher than in the NAC alone group (p < 0.01) but there was no statistically significant difference in lipid hydroperoxide level in the serum and kidneys between the control and the NAC with CsA group, indicating the nullifying effect of NAC on cyclosporine toxicity (Table II).
Kidneys of the cyclosporine alone group showed severe glomerular atrophy. There was thickening of blood vessels and congestion of vessels and interstitium (Table III, Figure 1A). Many tubules showed casts and moderate areas of necrosis and hyalinization. N-acetylcysteine alone treatment showed normal kidney histology as in the control rats. In the NAC with CsA treated groups, most of the glomeruli appeared normal (Table III, Figure 1B). There was occasional reduced glomerular size compared to the control and NAC alone group. Minimal blood vessel thickening was present and areas of congestion were also reduced. Concomitant treatment with NAC showed only mild tubular atrophy, and occasional presence of tubular casts. Moderate glomerular atrophy and mild interstitial oedema were also seen in the NAC with CsA group (Table III).

Discussion
In the present study, treatment of rats with cyclosporine for a period of three weeks resulted in a significant increase in blood urea nitrogen, creatinine, urea and uric acid level, suggesting significant functional impairment in the kidneys. These observations are in agreement with earlier studies where significant alterations in the level of BUN and creatinine were reported following chronic CsA treatment [28, 29]. Treatment of rats with NAC alone had resulted in improvement in renal function which was reflected by a significant decrease in blood urea, creatinine and uric acid levels when compared to control rats. This effect on renal function with NAC alone in normal rats supports the hypothesis that NAC improves the renal haemodynamics [23, 24]. When NAC was administered to the cyclosporine treatment groups, there was a significant improvement in renal function in the rats and the level of urea, BUN, uric acid and creatinine decreased significantly compared to the CsA treatment group. But the level of these substances remained higher than the normal control and NAC alone groups, indicating that NAC could not totally nullify the effect of CsA on renal function.
Increased production of lipid hydroperoxides and a significant decrease in total antioxidants with CsA confirms the role of oxidative stress in CsA-induced nephrotoxicity. Parra et al. reported that treatment of CsA increased oxygen free radical production [30]. These oxygen free radicals modulate the filtration in the glomerulus and also influence the renal blood flow [31]. The endogenous antioxidant system operates to combat the oxygen free radicals and these antioxidants scavenge the oxygen free radicals. Decreased total antioxidants after CsA treatment proves that there is increased production of oxygen free radicals and endogenous antioxidants are being used up for scavenging the wide variety of these free radicals including O2–, H₂O2 and OH– [32].
Treatment with N-acetylcysteine along with CsA increased the total antioxidant level both in the serum and kidneys. N-acetylcysteine with CsA also decreased the lipid hydroperoxide levels. Treatment of rats with NAC alone showed a significant improvement in the total antioxidant levels and decreased the lipid hydroperoxide levels compared to control rats. The mechanism of action of NAC in reducing renal damage caused by CsA is not clear [33]. Protective effects of NAC against oxidative stress induced tubular damage might involve various chemical mechanisms. N-acetylcysteine directly scavenges superoxide radicals.
As a precursor of glutathione synthesis NAC significantly increases intracellular redox potential and improves the reductive states of critical regulatory protein thiol groups [23, 34, 35]. Marked protective effects of NAC administration against CsA-induced renal damage may be strongly associated with amelioration of the effects of oxidative stress [25]. N-acetylcysteine is also reported to stimulate vasodilatation in the kidneys, thereby reducing the renal vascular resistance and hypoxia/reoxygenation-induced formation of oxy-gen free radicals [36]. CsA-induced nephrotoxicity is associated with accumulation of cellular calcium, and calcium channel blockers have been shown to reduce CsA-induced kidney damage. N-acetyl-cysteine have also been shown to block calcium channels, maintain calcium homeostasis and improve renal function [37, 38].
There were characteristic morphological findings with CsA treatment, such as glomerular atrophy, blood vessel thickening and hyaline casts in the tubules. Concomitant treatment with NAC attenuated the CsA-induced structural and functional changes in the kidneys. Reactive oxygen species mediate peroxidation of lipid structures of the tissues, resulting in subcellular damage, as observed by histopathological examination in this study with CsA treatment for 21 days [4, 39]. Vasoconstriction produced by CsA produces local ischaemia which may lead to a number of cellular changes such as deterioration of membrane integrity and distinct histological changes in the renal structures [29]. Treatment with NAC along with CsA significantly reduced the pathological changes in the kidneys, suggesting the protective role of NAC in CsA-induced renal morphological damage. Decrease in structural damage with NAC treatment could be attributed to the NAC-induced decreased vascular resistance and enhanced tissue perfusion [36, 40]. Improved tissue perfusion with NAC decreased the formation of oxygen free radicals and minimized the CsA-induced cellular damage in renal tissues [41].
Thus, cyclosporine decreased the total antioxidants and increased the lipid hydro-peroxides, causing severe renal damage. There was also a significant histopathological change in the kidneys with three weeks of cyclosporine treatment. Concomitant treatment with NAC (20 mg/kg body weight) could prevent the toxic renal damage of CsA by increasing total antioxidants and reducing the lipid hydro-peroxides in the kidneys. In conclusion, our study confirms the hypothesis that oxidative stress is the main cause of renal damage induced by cyclosporine, and N-acetylcysteine, through its marked antioxidant activity and positive haemodynamic effects, significantly reduces the renal damage due to cyclosporine treatment. N-acetylcysteine may be considered for phar-macological therapy for cyclosporine-induced nephrotoxicity in humans.

Acknowledgments
The project received a Research Grant from International Medical University (IMV) in 2006.

References
1. Rezzani R. Cyclosporine A and adverse affects on organs: histochemical studies. Prog Histochem Cytochem 2004; 39: 85-128.
2. Pourfarziani V, Einollahi B, Assari S, Kardavani B, Moghani Lankarani M, Kalantar E. A link between the outcome of living unrelated kidney transplantation and HLA compatibility: a preliminary report. Arch Med Sci 2007; 3: 108-11.
3. Andoh TF, Burdmann EA, Bennett WM. Nephrotoxicity of immunosuppressive drugs – experimental and clinical observations. Semin Nephrol 1997; 17: 34-5.
4. Can Li, Lim SW, Sun BK, Yang CW. Chronic cyclosporine nephrotoxicity: new insights and preventive strategies. Yonsi Med J 2004; 45: 1004-16.
5. Mason J. Pharmacology of cyclosporine (sandimmune). Pathophysiology and toxicology of cyclosporine in humans and animals. Pharmacol Rev 1990; 41: 423-34.
6. De Nicola L, Thomson SC, Wead LM, Brown MR, Gabbai FB. Arginine feeding modifies cyclosporine nephrotoxicity in rats. J Clin Invest 1993; 92: 1859-65.
7. Rezzani R, Rodella L, Buffoli B, et al. Change in renal heme oxygenase expression in cyclosporine A-induced injury. J Histochem Cytochem 2005; 53: 105-12.
8. Murray BM, Paller MS, Ferris TF. Effect of cyclosporine administration on renal hemodynamics in conscious rats. Kidney Int 1985; 28: 767-74.
9. Kopp JB, Klotman PE. Cellular and molecular mechanisms of cyclosporine nephrotoxicity. J Am Soc Nephrol 1990; 1: 162-79.
10. Takeshi FA, Bennett WM. Chronic cyclosporine nephrotoxicity. Curr Opinion Nephrol Hypertension 1998; 7: 265-70.
11. Durak I, Ozbek H, Elgün S. Cyclosporine reduces hepatic antioxidant capacity: protective roles of antioxidants. Int Immunopharmacol 2004; 4: 469-73.
12. Wolf A, Clemann N, Frieauff W, Ryffel B, Cordier A. Role of reactive oxygen formation in the cyclosporine A mediated impairment of renal functions. Transplants Proc 1994; 26: 2902-07.
13. Chander V, Singh D, Tirkey N, Chander H, Chopra K. Amelioration of cyclosporine nephrotoxicity by irbesartan, a selective AT1 receptor antagonist. Ren Fail 2004; 26: 467-77.
14. Hansen JM, Foghandersen N, Christensen NJ, Strandgaard S. Cyclosporine-induced hypertension and decline in renal function in healthy volunteers. J Hypertens 1997; 15: 319-26.
15. Lanese DM, Falk SA, Conger JD. Sequential agonist activation and site specific mediation of acute cyclosporine constriction in rat renal arterioles. Transplantation 1994; 58: 1371-8.
16. Lo Russo A, Passaquin AC, Andre P, Skutella M, Rüegg UT. Effect of cyclosporine A and analogues on cytosolic calcium and vasoconstriction: possible lack of relationship to immunosuppressive activity. Br J Pharmacol 1996; 118: 885-92.
17. Padi SS, Chopra K. Salvage of cyclosporine A-induced oxidative stress and renal dysfunction by carvedilol. Nephron 2002; 92: 685-92.
18. Inselman G, Hanneman J, Baumann K. Cyclosporine A induced lipid peroxidation and influence on glucose-6 phosphatase in rat hepatic and renal microsomes. Res Commun Chem Pathol Pharmacol 1990; 68: 189-203.
19. Shihab FS, Bennett WM, Isaac J, Yi H, Andoh TF. Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity. Kidney Int 2003; 63: 522-33.
20. Eylar E, Rivera-Quinones C, Molina C, Báez I, Molina F, Mercado CM. N-acetylcysteine enhances T cell functions and T cell growth in culture. Int Immunol 1993; 5: 97-101.
21. Thielemann LE, Oberhauser EW, Rosenblut G, Videla LA, Valenzuela A. Sulfur containing amino acids that increase renal glutathione protect the kidney against papillary necrosis induced by 2-bromothylamine. Cell Biochem Funct 1990; 8: 19-24.
22. Salom MG, Ramirez P, Carbonell LF, et al. Protective effect of N-acetyl-L-cysteine on the renal failure induced by inferior vena cava occlusion. Transplantation 1998; 65: 1315-21.
23. Conesa EL, Valero F, Nadal JC, et al. N-acetyl-L-cysteine improves renal medullary hypoperfusion in acute renal failure. Am J Physiol Regul Integr Comp Physiol 2001; 281: R730-7.
24. Cabassi A, Dumont EC, Girouard H, et al. Effects of chronic N-acetylcysteine treatment on the actions of peroxynitrite on aortic vascular reactivity in hypertensive rats. J Hypertens 2001; 19: 1233-44.
25. Efrati S, Dishy V, Averbukh M, et al. The effect of N-acetylcysteine on renal function, nitric oxide and oxidative stress after angiography. Kidney Int 2003; 64: 2182-87.
26. Middleton E Jr, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol Rev 2000; 52: 673-751.
27. Hamid Nasri. A positive correlation of serum homocysteine with leptin in maintenance hemodialysis patients. Arch Med Sci 2006; 2: 185-9.
28. Mason J. The pathophysiology of sandimmune (cyclosporine) in man and animals. Pediatr Nephrol 1990; 4: 554-704.
29. Tirkey N, Kaur G, Vij G, Chopra K. Curcumin, a dife-ruloylmethane, attenuates cyclosporine – induced renal dysfunction and oxidative stress in rat kidneys. BMC Pharmacol 2005; 5: 15.
30. Parra T, De Arriba G, Arribas I, et al. Cyclosporine A nephrotoxicity: role of thrombaxane and reactive oxygen species. J Lab Clin Med 1998; 131: 63-70.
31. Hughes AK, Stricklett PK, Padilla E, Kohan DE. Effect of oxygen species on endothelin-1 production by human mesangial cells. Kidney Int 1996; 49: 181-9.
32. Nath KA, Norby SM. Reactive oxygen species and acute renal failure. Am J Med 2000; 109: 665-78.
33. Dobashi K, Singh I, Orak JK, Asayama K, Singh AK. Combination therapy of N-acetylcysteine, sodium nitroprusside and phosphoramidon attenuates ischemia reperfusion injury in rat kidney. Mol Cell Biochem 2002; 240: 9-17.
34. Arouma OI, Halliwell B, Hoey BM, Bulter J. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, super oxide and hypochlorus acid. Free Radic Biol Med 1989; 6: 593-97.
35. DiMari J, Megyesi J, Udvarhelyi N, Price P, Davis R, Safirstein R. N-acetyl cysteine ameliorates ischemic renal failure. Am J Physiol 1997; 272: F292-8.
36. Boesgaard S, Aldershville J, Poulsen HE, Christensen S, Petersen D, Giese J. N-acetylcysteine inhibits angiotensin converting enzyme in vivo. J Pharmacol Exp Ther 1993; 265: 1239-44.
37. Ruggenenti P, Perico N, Mosconi L. Calcium channel blockers protect transplant patients from cyclosporine- induced daily renal hypoperfusion. Kidney Int 1993; 43: 706-11.
38. Tee LBG, Boobis AR, Davies DS. N-acetylcysteine for paracetamol overdose. Lancet 1986; 476: 331-2.
39. Nitescu N, Ricksten SE, Marcussen N, et al. N-acetyl-cysteine attenuates kidney injury in rats subjected to renal ischaemia-reperfusion. Nephrol Dial Transplant 2006; 21: 1240-7.
40. Alencar JL, Lobysheva I, Geffard M. Role of S-nitrosation of cysteine residues in long lasting inhibitory effect of nitric oxide on arterial tone. Mol Pharmacol 2003; 63: 1148-58.
41. Dragger LF, Andrade L, Barros de Toledo JF, Laurindo FR, Cesar LA, Seguro AC. Renal effects of N-acetylcysteine in patients at risk for contrast nephropathy: decrease in oxidant stress mediated renal tubular injury. Nephrol Dial Transplant 2004; 19: 1803-7.