THE PROTECTIVE EFFECT OF CARNOSINE ON KIDNEYS OF ALBINO RATS IN METHOTREXATE INDUCED OXIDATIVE INJURY

Objective: This study aimed to determine the anti-oxidant effect of carnosine in methotrexate (MTX) induced nephrotoxicity in albino rats. Materials & Methods: 40 male albino rats are used in this study and were equally divided into four groups. The negative control group received only saline orally, while the positive control group received carnosine (5 mg/kg) orally for 7 days. On the other hand the MTX group received a single dose (20 mg/kg) MTX intraperitoneally. The MTX+ carnosine group received the same doses of MTX and carnosine. On the seventh day, blood samples and kidney tissues were obtained for assessment of biochemical markers of the kidney, oxidative stress markers, and histopathological examination. Results: MTX group compared with the control groups (both negative and positive) and the MTX + carnosine group showed significant higher values of both BUN and serum creatinine. This significance was found between MTX group and all other studied groups for BUN levels; and between the MTX group and only the control groups for creatinine levels. The serum superoxide dismutase (SOD) levels were relatively higher in MTX than the other groups while the serum malonedialdehyde (MDA) was significantly higher in the MTX group compared to the other groups. Histopathological examination of the renal tissues showed glomerulosclerosis, marked damage of renal tubules, proteineous material in the renal tubules, and marked cellular infiltration in MTX group. Conclusion & Recommendations: MTX administration involves oxidative stress causing structural and functional damage in albino rats’ kidney tissue. Carnosine administration reduced the MTX-induced oxidative stress and nephrotoxicity through its antioxidant properties. Carnosine may be regarded as a promising agent to alleviate MTX-induced renal toxicity.


INTRODUCTION
Methotrexate (MTX) is used as an anti-cancer drug in higher doses such as acute lymphoblastic leukemia, lymphoma, carcinoma of the breast, osteogenic sarcoma, and cancer of the head and neck region (Choudhury et al., 2000; Miyazaki et al., 2003;Findlay et al., 2008;D'Adamo, 2011).However, lower doses of methotrexate have been used in the treatment of rheumatic diseases due to its immunosuppressant effect (Gisondi and Girolomoni, 2007;and Renna, 2014).
MTX depletes folic acid thus affecting the purine metabolism which is responsible for both therapeutic and toxic effects of MTX.The use of MTX is associated with deleterious effects on different organs such as the kidney, liver, testis and bone marrow.As MTX is mainly eliminated through the kidneys nephrotoxicity is more common to occur more than other side effects which limits its therapeutic uses in many condition (Lameire et al., 2011).
The mechanism of renal toxicity has been attributed to direct toxic effect of MTX as well as reactive oxygen species (ROS)  Carnosine (β-alanyl-l-histidine) was discovered in 1900 as an abundant non-protein nitrogen-containing compound of skeletal muscle and nervous tissue.The physiological roles of carnosine has been linked to its biochemical properties include pHbuffering, antioxidant capacity and its potential to protect against formation of lipid oxidation end-products (Boldyrev et al., 2013).
For these reasons, the therapeutic potential of carnosine supplementation has been tested in numerous diseases in which oxidative stress is involved.Furthermore, it has been used in physiological states accompanied by oxidative stress and showed promising preclinical and clinical results (Ahshin- Majd et al., 2016;Prokopieva et al., 2016;and Yamashita et al., 2018).
This study is designed to assess the protective properties of carnosine on methotrexate induced nephrotoxicity.This was carried through the evaluation of both biochemical and histopathological parameters with special focus on the antioxidant status and lipid peroxidation product (MDA) Forty male, albino rats, weighing of 200-220 gm aged 3 months were provided from animal laboratory and used in this experiment.All animals maintained at a constant temperature (30 +/-3 °C) in a controlled room where light/dark cycle was maintained.The rats had free access to food and water.Animals were left for 7 days for acclimatization the then distributed randomly into four groups each consisted of ten rats:

MATERIALS & METHODS
(I) At the end of the seventh day, all the experimental rats were anesthetized and euthanized by decapitation.Blood samples were collected for estimation of biochemical parameters and both kidneys were quickly removed and fixed in 10% neutral formalin solution for histopathological examinations.
Biochemical analysis Blood samples were collected and left at room temperature for 10 minutes before centrifugation to separate the serum and then aliquoted for analysis.Serum creatinine and blood urea nitrogen (BUN), in addition to serum superoxide dismutase (SOD) and serum malondialdehyde (MDA) were studied.
1-Serum creatinine and blood urea nitrogen (BUN) levels were measured using a colorimetric method, employing commercial kits by an auto analyzer.
2-Determination of serum superoxide dismutase (SOD) activity an indicator of antioxidant response, which was analyzed primarily based on reduction of nitroblue tetrazolium (NBT) compound.Activity of the enzyme was expressed as units per milliliter of plasma (Sun et al., 1988).
3-Determination of serum malondialdehyde (MDA) levels were measured with the thiobarbituric acid reaction according to Dahle's method using spectrophotometer and MDA levels were expressed in nanomoles per milliliter (Dahle et al., 1962).
Histopathological analysis Kidney samples were fixed in 10% buffered formalin, embedded in paraffin and then cut at 4-5 microns thickness.The cut sections were then stained with hematoxylin and eosin (HE) to be examined by light microscopy.Histopathological changes were diagnosed in a blinded manner by a specialized pathologist unaware of the study groups.Glomerulosclerosis, degeneration of tubular epithelium, presence of proteinous material in the lumen of renal tubules, and presence of inflammatory reaction were evaluated according to the severity of lesions using a 0-3 scoring system, where 0= normal; 1= normal glomeruli and slight degeneration in tubular epithelial cells, mild inflammatory reaction; 2= moderate glomerulosclerosis & tubular degeneration, deposition of proteneous material and moderate inflammatory reaction; 3= severe glomerulosclerosis & tubular degeneration, extensive proteinous material in tubular lumen, and severe inflammatory reaction.

Statistical analysis
The collected data were tabulated and analyzed using SPSS version 22 software (Spss Inc, Chicago, ILL Company).Quantitative data were expressed as mean ±standard deviation.ANOVA was used as tests of significance.Significant ANOVA and Krauskal Wallis were followed by post -hoc test to detect significant pairs.The accepted level of significance in this work was stated at 0.05 (P <0.05 was considered significant).

Biochemical results
The mean value ± SD of BUN levels was higher in the methotrexate group (group III) compared with all other groups in this study and the probability value was found highly significant (P<0.0001) as shown in table 1.This significance was further studied by post-hoc test and the significance was found between group III and all other studied groups as shown in table 2. Similarly, the mean value ± SD of the serum creatinine levels was higher in the group III compared with all other studied groups and the probability value was also found highly significant (P<0.0004) as shown in table 3.This significance was found only between group III and both the control groups (group I and group II) as shown in table 4.
As regard the serum value ± SD of SOD enzyme activity levels was relatively higher in group III than the other studied groups.However, the probability value was insignificant (P>0.05) as shown in table 5. On the other hand, the serum value ± SD of MDA level in group III was significantly higher than all other groups in this study (P<0.0001)as shown in table 6.This significance was further studied by post-hoc test which showed significance pairwise comparison between every group and each other (p<0.0001).

Histopathological results
The gross examination of the kidneys between the different studied groups did not show any significant difference.Normal renal architecture i.e. glomeruli and renal tubules (histopathological score = 0) was observed in both negative and positive control groups.On the other hand, the MTX group showed glomerulosclerosis, renal tubular damage, proteineous material in the renal tubules, and infiltration by inflammatory cells that ranged between moderate to marked (histopathological score ranged between 2 -3).Lastly, the MTX + carnosine showed relatively normal kidneys i.e. mild glomeruli and renal tubular degeneration, no proteneous materials in the lumen of the tubules, and mild inflammatory reaction (histopathological score ranged between 0 -1).The histopathological results in MTX group were significant with both control groups and also the MTX + carnosine group.    .However, under uncontrolled increase in ROS, they interact with biomolecules, leading to their oxidative modifications.Products of such modification usually lose ability to carry out their functions.These products serve as -markers of oxidative stress,‖ and they include carbonylated, nitrosylated, and glycated proteins; aggregates due to cross linking of protein molecules; products of lipid peroxidation such as malondialdehyde.All these products of oxidative damage are resistant to destructions and accumulate in cells affecting their vital functions.Therefore, their neutralization plays an important role in correction of the oxidative stress (Lin and Beal, 2006;and Menshchikova et al., 2006).
Although the human endogenous antioxidant response system can regulate the amount of ROS tightly and minimize related cellular damage (Kensler et al., 2007), the role of exogenous antioxidants is also important.It was found that exogenous antioxidants have a priming effect on the antioxidant response system.The both endogenous antioxidant response system and exogenous antioxidants allow for a more enhanced and efficient defense against detrimental redox modulations ( Kensler et al., 2007;and Niture et al., 2014).
Carnosine meets almost all requirements for an ideal antioxidant.It is characterized by being synthesized and contained in human muscle and nervous tissues.It is absorbed from the alimentary tract and has high bioavailability.In addition, carnosine does not carry the danger of overdose nor it accumulates in the organism during administration as it is metabolized by the carnosinase enzyme.This low molecular weight hydrophilic antioxidant acts through its direct action and has also an impact on the antiradical protection system of the organism (Boldyrev, 2009).
Several studies described the positive biological effects by its pHbuffering properties (Skulachev, 1992); buffering reactive oxygen species (Severin et al., 1984); and its ability of to form complexes with bivalent metals such as ions of copper, cobalt, manganese, cadmium (Brown and Antholine, 1979), and iron ions (Vladimirov, 1996).Further, the antiglycating and the anticross-linking properties of carnosine have been shown and are attributed to its antioxidant effects (Hipkiss et al., 1995;and Hobart et al., 2004).
In this study, BUN and creatinine were measured in the four groups as indicators of renal function.The mean value of BUN levels was significantly higher in the methotrexate group (group III) compared with all other studied groups (both control groups and MTX + carnosine group) (P<0.0001).This was in agreement with Armagan et al., 2015 and Asci et al., 2017 that found similar biochemical findings indicating the impairment of renal function induced by MTX and the protective effect of carnosine.
As regard the serum creatinine the other studied parameter for renal function, it was found that the mean values levels was also significantly higher in the group III compared with all other studied groups (P<0.0004).However, this significance was found only between group III and both the control groups.Asci et al., 2017 described similar results one side, and on the other side Armagan et al., 2015 stated that serum creatinine levels were significant in MTX group as compared to all other studied groups.
SOD is an important antioxidant enzyme against the deleterious effects of ROS such as lipid peroxidation (Wang et al., 2007).The serum mean value of SOD activity levels in this study was relatively higher in the MTX group compared with the other studied groups (P>0.05) which can be attributed to the oxidative damage caused by MTX.In a similar study, Yuksel et al., 2017 showed that both serum and tissues SOD levels were higher in methotrexate group compared with the other studied groups and only the serum SOD was significant.In contrast, Savran et al., 2017 found a significant increase in the serum SOD in the MTX group compared to other groups in their study.
MDA is an indicator of free radical generation and lipid peroxidation

CONCLUSION
The present study demonstrates the renal protective and antioxidant effects of carnosine through acting as a potent scavenger for free radicals and limiting lipid peroxidation

REFERENCES
Photomicrograph of renal tissue in negative control group (stained with H&E X 200) showing normal renal tissue (score = 0) Photomicrograph of renal tissue in positive control group (stained with H&E X 200) showing normal renal tissue (score = 0)

Devrim et al., 2005; Asvadi et al., 2011).
Acute renal failure and nephrotoxicity have been reported with the usage of MTX apecially in high doses (

Table ( 7
): Statistical analysis of histopathological results of the kidneys