Hepatic injury can be induced by the administration of carbon tetrachloride (CCl4) via the production of free radicals. The present work was initiated to investigate the kidney response to hepatic injury induced by CCl4 and its treatment by L-arginine. Female Swiss albino mice were supplied with L-arginine for 6 days (orally, 200 mg/kg body weight) prior or post to hepatic injury induction through i.p. injection with a single dose of CCl4 (20 mg/kg body weight) for 24 h. After hepatic injury induction, renal MDA content was significantly elevated while renal GSH level and the activities of antioxidant enzymes (GR, GPx, GST, catalase, and SOD) were significantly decreased. These results suggest that CCl4 not only induces hepatic injury but also induces kidney dysfunction side by side. Following the treatment with L-arginine, all levels were almost back to normal. Therefore, Larginine administration is found to be an effective protector of both liver and kidney against CCl4-intoxication.
L-arginine plays important roles in the metabolism of an organism. It is the precursor for the synthesis of proteins and other molecules of great biological importance. For young organisms L-arginine is an essential amino acid for optimal growth and development. Therefore it must be provided in the diet. For adults, L-arginine is a semi-essential or conditionally essential amino acid, especially in such conditions as trauma, burn injury, smallbowel resection, and renal failure [
Several experimental studies have revealed that the administration of L-arginine can reduce renal ischemia reperfusion injury to skeletal muscle, kidney, liver, heart and brain. Moreover, L-arginine administration improves cardiovascular, pulmonary, immune, digestive functions, protects against the early stages of cancerogenesis and causes protective hemodynamic alterations. However, in situations with a stimulated inducible nitric oxide synthase (iNOS), the administration of large amounts of L-arginine is considered to be hazardous for the patients [1,4]. L-arginine is beneficial in the treatment of hepatic injury [
Carbon tetrachloride (CCl4) administration produces pathological changes not only in liver but also in different organs [
Liver is the main target for CCl4 toxicity while the kidney is the main site of CCl4 accumulation. The pathogenesis of CCl4-induced renal dysfunction is not completely understood [
CCl4 and L-arginine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used throughout this investigation were of the highest analytical grade commercially available.
Forty female Swiss albino mice (25 ± 2 gm) obtained from Theodor Bilharz Research Institute, Cairo (Egypt) were used in this experiment. The experiment was performed according to the approved ethical rules. The animals were housed for about one month prior to experimental use. All mice were housed under constant conditions of a 12-hour light/dark cycle in a temperature and humidity controlled room, and were maintained according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health [
The animals were randomly divided into 4 groups of 10 mice each. CCl4 group; received a 6-day repeated oral dose of Tween 80 (1 ml/kg) diluted in distilled water (1%, v/v) then mice were injected with a single dose of CCl4 (20 mg/kg body weight, intraperitoneally (i.p.)) dissolved in corn oil (2%, v/v) as described previously [20,21] and sacrificed after 24 h of CCl4 injection. For protection group, mice were orally supplemented with a dose of Larginine (200 mg) in 1 ml Tween/kg body weight/day for 6 days, then injected i.p. with a single dose of CCl4 (20 mg/kg body weight) and sacrificed 24 h later. For curative group; mice were injected i.p. with a single dose of CCl4 (20 mg/kg body weight), then after 24 h mice were orally treated with 200 mg L-arginine in 1 ml Tween/kg body weight/day for 6 days, then sacrificed. Finally, normal control group was fed with the control diet and received the respective vehicles only (received a 6-day repeated oral dose of 1% Tween 80 (1 ml/kg) then a single i.p. dose of corn oil was injected then mice were sacrificed 24 h later).
The animals of each group were sacrificed by decapitation at the end of the 8th day of the experiment. Blood samples were collected and serum was separated by centrifugation at 5000 rpm, for 10 min and stored at 4˚C. The kidneys were removed by dissection, rapidly rinsed with ice-cold saline and dried on filter paper, homogenized in 0.9% NaCl solution using a homogenizer and the resultant homogenate (10%, w/v) was then centrifuged at 5000 rpm for 15 min at 4˚C and the resultant supernatant was used for the determination of biochemical parameters.
Serum creatinine was measured to assess kidney function using Jaffe alkaline picrate method (Randox, UK) [
The results are expressed as mean ± SD. Statistical analysis between two groups was performed using Student’s t-test; P < 0.05 was considered statistically significant. The data were analyzed with GraphPad InStat version 2.03 (GraphPad Software, Inc., CA, USA).
CCl4 injection resulted in a significant increase in serum creatinine level compared with those of the control group. On the other hand, preor post-administration of L-arginine resulted in a significant decrease in the serum creatinine level (
MDA level, as a marker of free radical mediated lipid peroxidation injury, was significantly elevated in the kidney homogenate of CCl4-intoxicated mice compared with that of the normal group and it was significantly decreased by preor post-treatment with L-arginine to back again around the normal range. On contrary, renal GSH level was significantly decreased in CCl4-intoxicated mice as compared with the normal group and it was significantly increased by both pre and post-administration of L-arginine (
GR, GPx, GST, catalase and SOD (antioxidant enzymes) were evaluated as an index of antioxidant status of tissues. As shown in Figures 1-3, renal activities of GR, GPx, GST, catalase and SOD enzymes were significantly inhibited by CCl4 intoxication compared with those of normal mice group. The activities of the antioxidant enzymes were significantly enhanced by pre-treatment or post-treatment of CCl4-intoxicated mice with L-arginine compared with those of untreated CCl4-intoxicated mice group.
Previous studies reported that CCl4 induces hepatic injury by producing free radicals [31-33]. Here and in accordance with others [
Each value represents the mean ± SD of ten mice. *Significant difference at P < 0.001 level compared with the normal group. !Significant difference at P < 0.001 level compared with the CCl4 group.
L-arginine against the acute intoxication of CCl4 (data not shown).
Acute and chronic renal damage are also very common pathophysiologic disturbances caused by CCl4 [31, 34]. Repeated administration of CCl4 to the experimental animals not only produces liver cirrhosis but also pathological changes in different organs such as lung, spleen and kidneys [
Furthermore, the alteration in the antioxidant enzyme activities may be relevant to the ability of the liver and kidney to cope with oxidative stress after CCl4 poisoning.
The preor post-treatment with L-arginine significantly decreased serum creatinine to be around normal levels as well as kidney MDA. On the other hand, the kidney levels of GSH and antioxidant enzyme activities (SOD, catalase, GPx, GST and GR) significantly increased as compared to CCl4-intoxicated mice. It was hypothesized that dietary L-arginine supplementation attenuates oxidative damage in kidneys [
Each value represents the mean ± SD of ten mice. *Significant difference at P < 0.001 level compared with the normal group. !Significant difference at P < 0.001 level compared with the CCl4 group.
observed that oral administration of L-arginine before cyclosporine (CsA) injection produced a significant protection against nephrotoxity induced by CsA, prevented the rise of MDA as well as reduction of GPx activity and reduced GSH content in kidney tissue. These suggested that, L-arginine can ameliorate kidney dysfunction. In another study of Saleh and El-Demerdash [
The present study revealed that L-arginine treatment significantly decreased renal MDA level while enhanced renal GSH levels and increased the activities of antioxidant enzymes (SOD, catalase, GPx, GST and GR). The toxicity of CCl4 can be explained on the basis of oxidative stress. These results indicated that besides the heaptoprotective and hepatocurative action of L-arginine [
Finally, it can be concluded that a single CCl4 dose (20
mg/kg body weight) is able to induce hepatic injury and kidney dysfunction. In addition, the effect of CCl4 on kidney function depends on the functional status of the liver. L-arginine has protective effects on both liver and kidney against CCl4 toxicity and these protective effects can be interpreted via the inhibition of CCl4 derived free radicals production and the enhancement of antioxidant enzymes activities as well as through the antioxidant effects of L-arginine itself. Thus, clinical application of Larginine should be considered in cases with hepatic and renal CCl4-induced injury.