Protective effect of N-acetylcysteine on kidney as a remote organ after skeletal muscle ischemia-reperfusion 1

PURPOSE: To investigate whether N-acetylcysteine has a protective effect against renal injury as a remote organ after skeletal muscle ischemia-reperfusion in rats. METHODS: Twenty Wistar male rats were divided randomly into two experimental groups: group ischemia-reperfusion (group I) and group ischemia-reperfusion + N-acetylcysteine (group II). After ketamine and xylazine anesthesia, femoral artery was exposed. All animals were undergone 2h of ischemia by occlusion femoral artery and 24h of reperfusion. Rats that were treated with N-acetylcysteine given IV at a dose of 150 mg/kg-1, immediately before reperfusion. After 24h of reperfusion, the blood samples were collected and submitted for evaluation of plasmatic urea, creatinine values and then rats were euthanized and left kidney harvested for histopathological analysis under light microscopy. RESULTS: The urea (35±7.84 mg.dL-1), creatinine (1.46±0.47 mg.dL-1) values were significantly lower in group II (P=0.000). Renal histopathologic study in group I showed extensive distal and proximal tubular cells necrosis and sloughing of epithelial cells into the tubular lumen, cast formation in tubule and glomerul, glomerul fibrosis and hemorrhage. Histopathologically, there was a significant difference (p=0.037) between two groups. CONCLUSION: The N-acetylcysteine was able to decrease renal injury induced by skeletal muscle ischemia reperfusion in rats.


Introduction
Ischemic and reperfusion injury of the extremities may result in a systemic, severe and complex metabolic syndrome, manifested by acute renal failure, myoglobinuria, metabolic acidosis, hypercalemia and free radicals release 1 .The risk of revascularization in ischemic extremities may result to renal failure and a clinical entity as myonephropathic-metabolic syndrome 2 .
The pathogenesis of acute renal failure following rhabdomyolysis has been attributed to several mechanisms: 1) Myoglobin nephrotoxicity impair renal function, mainly when dehydratation, acidemia, or both coxists; 2) Primary reduction of glomerular filtration rate due to cortical and glomerular hemodynamic changes due to hypotension after restoration of the blood flow to the extremity; 3) Myoglobin cast producing tubular obstruction and tubular acute necrosis; 4) Release of oxygenderived free radicals mediating back leakage of filtrate through damaged tubular renal epithelium, with loss of renal excretory function 1 .
Multiple pharmacological agents such as iloprost, vitamin C, pentoxifylline and L-alanyl-glutamin are proposed to be useful against renal injury as a remote organ after hindlimb ischemia-reperfusion [2][3][4] .
N-acetyl cystenie is a small molecule containing a thiol group, which has antioxidant properties 5 .It has been suggested that N-acetylcystenie replenishes glutathione stores, increases superoxide dismutase activity, scavenges hydroxyl free radical and interferes autocatalytic lipid peroxidation 6 .N-acetylcysteine has proven to be renoprotective in experimental models of both toxic, and ischemic acute renal failure [7][8][9] .Recent clinical trials suggest that N-acetylcysteine may be effective in preventing contrast nephropathy [10][11][12] .
However its role in reducing the damage in kidney after skeletal muscle ischemia-reperfusion has not been addressed completely yet.
In this experimental study, we aimed to examine the protective effect of N-acetylcysteine on kidney as a remote organ damage after the skeletal muscle ischemia-reperfusion by assessing histopathological and functional analysis in rat model.

Methods
All rats of the present research were cared according to the norms of the Islamic Azad University Faculty of Specialized Veterinary Sciences Tehran Iran laboratory of animal experimentations; this investigation was approved by the Committee of Ethics in Research with animals in Department of Veterinary Surgery too.
Twenty Wistar male rats weighing 250-300 g (12-15 weeks old) were used in this study.All rats were kept at a constant room temperature under standard conditions with food and water ad libitum in individual plastic cages with soft bedding.Animals were divided randomly into two experimental groups of ten rats each: group ischemia-reperfusion (group I) and group ischemiareperfusion + N-acetylcysteine (group II).
Anesthesia was induced using intramuscular ketamine (50 mg/kg -1 ) plus xylazine (10 mg/kg -1 ).After induction of anesthesia, the left hind limb was completely clipped with an electric shaver.After clipping, disinfecting and dropping (using a sterile technique), a skin incision was made on medial surface of the left hind limb.After isolated the femoral artery and vein from the surrounding structures, femoral artery was exposed and clamped with a mini bulldog forceps.Before clamped the femoral artery, 250 IU heparin was administered via the jugular vein to prevent clotting.All animals were undergone 2h of ischemia by occlusion femoral artery with a vascular clamp and 24h of reperfusion 13 .Rats were maintained in a dorsal recumbency and kept anesthetized throughout the duration of the ischemic period.
Additional doses were given as necessary to maintain anesthesia during the experiment.Body temperature was maintained with a heating pad under anesthesia.In group II N-acetylcysteine (150 mg/kg -l ) was injected intravenous immediately before reperfusion.
Following the ischemic period, the vascular forceps was removed and then surgical site was routinely closed.After surgery, fluid losses were replaced by administration of 5ml of warm (37˚C) isotonic saline i.p, and rats were returned to their cages with food and water ad libitum during the reperfusion period.The analgesic nalbuphine hydrochloride (2 mg/kg -1 ) was used via subcutaneous during observation time.
After 24h of reperfusion, the blood samples were collected from jugular vein and submitted for evaluation of plasmatic urea, creatinine values and all animals were submitted to laparotomy and the left nephrectomy was performed for histopathological analysis under light microscopy, then rats were euthanized by overdose of intraperitoneal pentobarbital injection (300 mg/kg -l ).
The blood samples were analyzed by Automatic DADE Analyzer TM to determine the biochemical plasmatic measures of urea, creatinine.Renal tissues were placed in 10% formalin solution and processed routinely by embedding in paraffin then tissues were sectioned in 4 µm pieces and stained with Hematoxylin-Eosin stain.

Grading of severity of renal injury was carried out by
Protective effect of N-acetylcysteine on kidney as a remote organ after skeletal muscle ischemia-reperfusion Acta Cirúrgica Brasileira -Vol.27 (9) 2012 -613 were considered as statistically significant.

Results
All of rats tolerated operation and survived until the final study period.Data belonging to plasma urea and creatinine measurements from blood samples after reperfusion are shown in Table 1.Plasma urea and creatinine concentrations were significantly increased in rats group I, compared with group II (p=0.000).

Discussion
Ischemic injury occurs in the lower extremities during surgery or trauma.During ischemia, muscle cells cannot keep their membrane integrity and this causes releasing of calcium, phospholipid A2, formation of polyunsaturated fatty acids and fatty acids radicals.If the oxygenation is re-established at that stage of ischemia, fatty acid radicals react with oxygen and undergo lipid peroxidation reaction.This reaction increases the membrane permeability and also stimulates chemotaxis of leukocytes, which release oxygen-derived free radicals and proteolytic enzymes when activated.Activated leukocytes release a variety of inflammatory mediators, including cytokines, neutrophil proteases, and reactive oxygen species.All of these products cause damage to adjacent endothelial cells, and they have been thought to play key roles in tissue injury 15 .Restoring blood flow to the extremities exposed to ischemia can cause the reperfusion injury by leading to formation of oxygen-derived free radicals that lead to more muscle necrosis than that caused by the ischemia itself 16,17 .Reperfusion injury develops on remote organs such as lungs, heart, liver, and kidneys that threatening life and makes progress acute renal and respiratory failure, cardiac dysfunction, and even death resulting from systemic toxic effects of reperfusion products known as myonephropathic-metabolic syndrome [18][19][20] .
Yassin et al. 21explained that the restoration of blood flow to an acutely ischemic lower limb in rats may, paradoxically, result in systemic complications and unexpected mortality.There was a significant increase in plasma concentrations of urea, creatinine, aspartate transaminase, alanine transaminase, and lactic dehydrogenase in reperfused animals compared with controls.In Teruya et al. 3 experimental model of unilateral hindlimb ischemia was associated to significant increase of plasmatic urea (p<0.0399) and creatinine (p<0.0382)shown the function of renal impairment caused by the skeletal muscle ischemia/reperfusion injury.
N-acetylcysteine is a simple and inexpensive watersoluble molecule that contains a sulfhydryl residue.It has long been approved for the prevention of hepatic and renal damage following acetaminophen overdose 22 .This well documented effect traditionally has been attributed to the restoration of intracellular glutathione levels 23,24 , required for detoxification of an acetaminophen-derived toxic metabolite.N-acetylcysteine also may attenuate the course of hepatorenal syndrome, a renal vasoconstrictive response of indeterminate nature that develops during advanced liver failure.This effect, shown in experimental settings 25 and in a preliminary clinical report 26 , could imply a better preservation of liver function.However, direct renal protective mechanisms may play a role also, considering the ubiquitous distribution of acylases that catalyze the deacetylation of N-acetylcysteine 27 .Indeed, recent studies suggest that N-acetylcysteine may increase intracellular glutathione and ameliorate renal ischemia-reflow injury 28,29 , after inferior vena cava-occlusion 30 or kidney damage from cis-platinum 31 , cyclosporine 32 , and other nephrotoxic insults 33,34 .N-acetylcysteine has been reported recently to prevent radiocontrast nephropathy in high-risk patients 35 .
In the Nitescu et al. 36 study, treatment with N-acetylcysteine ameliorated the decline in glomerular filtration rate on day one, and reduced plasma creatinine by~40% on days one and three, after renal ischemia/reperfusion.A similar reduction in plasma creatinine was demonstrated by DiMari et al. 37 using high intravenous doses of N-acetylcysteine (1g/kg -1 ) immediately before and after, bilateral renal ischemia/reperfusion in rats.Teruya et al. 3 reported that pentoxifylline has some protecting effect on remote kidney injury due to hindlimb ischemia/reperfusion injury only in the early phase of reperfusion.In our study plasma urea and creatinine concentrations were significantly decreases in group II that treated with N-acetylcysteine.
Teruya et al. 3 reported that the mesangial enlargement and the tubular cells necrosis were evidences of the remote ischemia/reperfusion renal injury.Nitescu et al. 36 have previously demonstrated that N-acetylcysteine treated animals with renal ischemia/reperfusion injury showed a significant reduction in renal interstitial inflammation seven days after the ischaemic insult.Our data demonstrate that N-acetylcysteine significantly decreases the severity of acute renal injury after skeletal muscle ischemia-reperfusion injury in rats.

Conclusions
The temporary occlusion of the femoral artery in rats leaded to severe histological tubule-interstitial changes in group I.However, administration of the N-acetylcysteine treatment significantly decreased renal injury induced by skeletal muscle ischemia-reperfusion according to our histological and biochemical findings.These results suggest the possibility of clinical application of N-acetylcysteine on renal injury induced by skeletal muscle ischemia-reperfusion. Different dosages, alternate time protocols and way of N-acetylcysteine administration should be investigated in future studies.

a
pathologist who was blinded to the experiment and data.renal injury was graded into four grades as follows: grade 0 represents no diagnostic change; grade 1 demonstrated tubular cell swelling, brush border loss, nuclear condensation with up to 1/3 of tubular profile showing nuclear loss; grade 2 is as grade 1, but greater than 1/3 and less than 2/3 of tubular profile showing nuclear loss; and grade 3 displayed greater than 2/3 of tubular profile showing nuclear loss 14 .A total of four slides from each renal sample were randomly screened and the mean was accepted as the representative value of the sample.Statistical analyses were carried out using SPSS statistical software (version 11.2).Results were expressed as the mean +/standard deviation.The Mann-Whitney U-test and T-Test were employed to analyze two groups consecutively.Values of P<0.05

Figure 1 TABLE 2 -FIGURE 1 -
Figure 1 illustrates representative photomicrograph of the renal tissues from group I and Figure 2 illustrates representative photomicrograph of the renal tissues from group II that obtained 24h after reperfusion.Renal histopathologic study in group I showed extensive distal and proximal tubular cells necrosis and sloughing of epithelial cells into the tubular lumen, cast formation in tubule and glomerul, glomerul fibrosis and hemorrhage.Histopathologic examination confirmed the extent of renal change in the group II (1.6±0.84) was significantly lower than group I (2.4±0.70).

FIGURE 2 -
FIGURE 2 -Light microscopic view from renal tissues of the group II.Less tubular cells changes and more preservation of nearly normal structure (magnification of 4×10, H&E staining).

TABLE 1 -
Results of biochemical analysis.