Protective Effect of Camel Milk on Gentamicin-inducedNephrotoxicity:From Renal Biomarkers to Histopathology Evidence

Document Type : Biochemistry and Pharmacology

Authors

1 DVM Student, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

2 Department of Basic Sciences, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran.

3 Department of Pathobiology, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

4 Cancer Biology Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran

5 Department of Clinical Sciences, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

Abstract

 
BACKGROUND: Gentamicin is an effective antibiotic with some important side effects, such as nephrotoxicity. There is evidence of renoprotective effects and antioxidant properties for camel milk.
OBJECTIVES:
In this study, the impact of camel milk on the nephrotoxicity induced by gentamicin was evaluated.
METHODS:
The present study was performed on four groups of six Wistar rats. Group 1(C), as the control group, received exclusively normal saline injections and the rats in group 2 (GM) received intraperitoneal gentamicin injections at the dose of 100 mg/kg for the last ten days. The animals in group 3 (CM) were fed by 5 mL/rat/day of camel milk through gavage for 15 days. Group 4 (MGM) was fed camel milk only for the first five days followed by gentamicin injections for 10 days. Serum urea, creatinine, and superoxide dismutase (SOD) were measured and kidneys were studied histopathologically.
RESULTS:
Increased concentrations of urea and creatinine along with the decreased level of SOD were found in the GM group. Histopathologic changes, such as eosinophilic casts in the tubular lumen, capillary congestion, glomerulonephritis, necrosis, interstitial nephritis, and edema were more common in the GM group, in comparison with the C, CM, and MGM groups (p <0.05). The elevations in serum urea and creatinine (p <0.05) were significantly prevented by the co-administra-tion of camel milk and gentamicin. Moreover, a significant increase in the serum activity of SOD was revealed in the GM group (p <0.05). Camel milk significantly prevented tissue injury, in comparison with the GM group (p <0.05).
CONCLUSIONS:
Our results demonstrated that gentamicin-induced histological and biochemical alterations in the kid-ney decreased significantly due to camel milk consumption

Keywords


Article Title [فارسی]

اثر محافظتی شیر شتر بر نفروتوکسیستی ناشی از جنتامیسین: ازبیومارکرهای کلیوی تا شواهد هیستوپاتولوژی

Authors [فارسی]

  • فرشته عرب 1
  • سعیده نعیمی 2
  • عباس جواهری وایقان 3
  • احد محمدنژاد 4
  • محمود احمدی همدانی 5
1 دانشجوی دکترای عمومی دامپزشکی، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران
2 گروه علوم پایه. دانشکده دامپزشکی. دانشگاه سمنان. سمنان. ایران
3 گروه پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه سمنان، سمنان، ایران
4 مرکز تحقیقات بیولوژی سرطان، موسسه سرطان ایران، دانشگاه علوم پزشکی تهران، تهران، ایران
5 گروه علوم بالینی، دانشکده دامپزشکی ، دانشگاه سمنان، سمنان، ایران
Abstract [فارسی]

زمینه مطالعه:  جنتامیسین یک آنتی‌بیوتیک موثر بوده اما اثرات جانبی مهمی از جمله مسمومیت کلیه دارد. شواهدی وجود دارد که نشان می‌دهد شیر شتر دارای اثرات محافظت کلیوی و اثرات آنتی‌اکسیدانتی است.
هدف: در این مطالعه، اثر شیر شتر بر مسمومیت کلیوی ناشی از جنتامیسین ارزیابی شده است.  
روش کار: 24 قطعه موش صحرایی نژاد ویستار به چهار گروه مساوی تقسیم شدند: گروه 1: گروه کنترل (دریافت‌کنندۀ نرمال سالین)، گروه 2: دریافت کننده جنتامیسین (mg/kg100)، گروه 3: دریافت‌کنندۀ شیر شتر (5 میلی‌لیتر در روز به مدت 15 روز، خوراکی) و گروه 4: تجویز جنتامیسین و شیر شتر (5 روز اول شیر شتر و سپس 10 روز جنتامایسینmg/kg 100). پس از اتمام دورۀ درمان، نمونۀ سرم اخذ و میزان اوره، کراتینین و سوپراکسید دسموتاز (SOD) اندازه‌گیری شد. سپس برای بررسی تغییرات پاتولوژی، از کلیه‌ها مقاطع هیستوپاتولوژی تهیه و بررسی شدند.
نتایج: جنتامیسین موجب افزایش اوره، کراتینین و کاهش SOD شد. تغییرات معنی‌داری نیز در هیستوپاتولوژی مانند کست‌های ائوزینوفیلی در لومن توبولی احتقان مویرگی، گلومرولونفریت، نکروز، نفریت بینابینی و ادم در کلیه‌ها مشاهده شد (05/0p < /em><). اوره و کراتینین سرم به‌طور معنی‌داری توسط تجویز هم‌زمان شیر شتر کاهش یافت (05/0p < /em><). یک افزایش معنی‌دار در فعالیت SOD سرم در مقایسه با گروه دوم مشاهده شد(05/0p < /em><). همچنین شیر شتر به‌طور معنی‌داری موجب جلوگیری از آسیب بافتی کلیه در مقایسه با گروه دوم شد.
نتیجه‌گیری نهایی: نتایج مطالعه حاضر نشان داد که شیر شتر می‌تواند موجب کاهش معنی‌دار تغییرات بیوشیمیایی و هیستولوژی ناشی از جنتامیسین در کلیه شود.

Keywords [فارسی]

  • شیر شتر
  • مسمومیت کلیه
  • جنتامیسین
  • استرس اکسیداتیو
  • موش صحرایی

Introduction

Gentamicin is an aminoglycoside antibiotic, which is used for some infections all around the world. Several significant side effects have been reported for the long-term usage of this antibiotic, two important of which are nephrotoxicity and ototoxicity. However, this medication is still effective against many gram-negative and gram-positive bacteria (Avent et al., 2011). The prevalence of kidney failure has been estimated as about 30% among patients who received gentamicin (Adil et al., 2016; Poulikakos and Falagas, 2013). Gentamicin induced nephrotoxicity following accumulation in the epithelial cells of renal tubules (Edwards et al., 2020; Oztopuz et al., 2019; Fujiwaraet al., 2009). Gentamicin caused apoptosis and necrosis in different in vitro and in vivo studies (Lopez-Novoa et al., 2011). The nephrotoxicity induced by gentamicin is a complex situation involving diverse pathways, such as reduced renal blood flow, oxidative stress, inflammation, nitric oxide generation, lipid peroxidation, nuclear factor kappa B pathway, apoptosis, and the decreased efficiency of kidney antioxidant enzymes, including SOD, catalase, glutathione peroxidase, and reduced glutathione (GSH)(Ulu et al., 2018; Amaral et al., 2018).Therefore, it is necessary to find therapeutic substances for limiting this damage. There are some strategies, such as antibiotic time control, diet changing, and prescription of some other medicines (Balakumar et al., 2010). As mentioned above, oxidative stress is one of the main reasons for renal damage. Antioxidant agents play an important role in preventing oxidative damage by neutralizing the effect of reactive oxygen metabolites on cellular components. Little attention has been paid to natural substances with antioxidant properties for protecting against nephrotoxic damage induced by gentamicin (Quiroset al., 2016;Boroushaki et al., 2012)and eligible impacts of these substances have attenuated the damages caused by gentamicin (Tavafi, 2013; Ali et al., 2011; Ali, 2003).

Camel milk has some renoprotective (Althnaian et al., 2013), hepatoprotective (Darwishet al., 2012), and anticancer (Habib et al., 2013) influences and is utilized for the treatment of some special diseases, namely autism (Al-Ayadhi and Elamin, 2013), diabetes (Mirmiran etal., 2017; Khan et al., 2013), and cow milk allergy (Maryniak et al., 2018; Ehlayel et al., 2011; El-Agamy et al., 2009). Moreover, camel milk is traditionally applied for treating tuberculosis, hypertension, gastroenteritis, B hepatitis, and some autoimmune diseases (Kumar et al., 2016a; Al-Ayadhi and Elamin, 2013) in Africa and the Middle East. Camel milk contains various protective proteins, water, and fat-soluble vitamins (Ismaili et al., 2019; Khalesi et al., 2017; Al-Ayadhi and Elamin, 2013; El-Hatmi et al., 2007). The amount of vitamin C, as an essential antioxidant vitamin, is much higher in camel milk than cow milk (Legesse et al., 2017; Yadavet al., 2015). Therefore, it seems that camel milk can reduce the side effects of gentamicin.

In the present study, the objectives were to evaluate the effect of camel milk consumption on gentamicin-induced biochemical changes in laboratory rats by assessing serum urea, creatinine, and SOD, as well as renal tissue alterations.

Materials and Methods

Medication and Camel Milk

Gentamicin vial of 2 mL containing 40mg/mL (Alborz Darou Pharmaceutical Company, Iran) was used. Camel milk was obtained manually every day from Camelus dromedary, Semnan province, Iran. Milk samples were collected in a sterile tube and transferred to the laboratory in cold flasks.

Animals

A total of 24 Wistar rats (250-300 gr) were randomly selected from the Research Center of Veterinary Faculty, Semnan University. The subjects were kept in the humidity of 60%-65%, temperature of 25°C, and 12 hour light/dark cycle ten days prior to the experiment. The rats were fed with a laboratory diet and had free access to freshwater. Animals were divided into four groups with six rats in each. The experiment was performed within 15 days.

The rats in group 1(C), as the control group, received intraperitoneal (IP) injections of0.2 mL normal saline for 15 days. Group 2 (GM) was injected IP with 100mg/kg gentamicin on the last ten days of the experiment. The subjects in group 3(CM) received camel milk at the dose of 5 mL/rat/day orally for fifteen days. Group 4 (MGM) received 5 mL/rat/day of camel milk orally on the first 5 days of the experiment followed by IP injection of 100 mg/kg gentamicin for ten days.

Ethical Approval

The research was reviewed and approved by the Ethics in Research Committee of Semnan University, Iran with the ethics code ofE19-95-01-30. The research was conducted following the World Medical Association Declaration of Helsinki.

Collecting Blood Serum

At the end of the experiment, rats were euthanized by deep anesthesia. A volume of 4 mL of blood samples was collected by cardiac puncture and the sera were separated and stored at -20 ͦC until analysis.

Biochemical Analysis

Serum urea and creatinine were measured using commercial kits (Pars Azmun Co., Tehran, Iran), according to the instructions of the manufacturer. The SOD kit (ZellBio Co., CAT No. ZB-SOD-96A) converts superoxide anion to hydrogen peroxide and oxygen through enzymatic reactions. Finally, the product makes a color, which is measured colorimetrically at 420 nm. The SOD is known as one of the most effective antioxidant enzymes in the body.

Histopathologic Examination

The right kidney was removed and fixed in 10% formalin. The tissues were processed and stained by hematoxylin and eosin to observe the tubular, glomerular, vascular, and interstitial alterations. These changes were classified as zero (absence of alteration), 1 (mild alteration), 2 (moderate alteration), and 3 (severe alteration).

Statistical Analysis

Descriptive statistics were presented using the SPSS software version 15 (SPSS Inc., Chicago, Ill., USA). Two-way repeated measures analysis of variance (ANOVA) was used to evaluate significant changes in the groups. Bonferroni post-hoc test was applied as a correction for multiple comparisons. Histopathologic non-parametric data were analyzed by the Kruskal-Wallis test and the significant differences between the two groups were compared by the Mann-Whitney test. P-value

Results

Blood Biochemical Factors

Serum creatinine and urea in the GM group were significantly higher than the C and CM groups (P<0.05) (Figures 1 and 2). There was a significant decrease in the urea and creatinine of the MGM group, in comparison with the GM group (P<0.05). The activity of SOD, as an antioxidant enzyme, diminished in the GM group. However, camel milk could increase the activity of this enzyme in the MGM group, compared to the GM group (P<0.05) (Figure 3).

  

 

                       

Figure 1. Effect of camel’s milk on gentamicin -induced renal dysfunction as measured by serum creatiniin. Data are expressed as means ± SD for all groups. (n = 6). There are significant different between (a) and (b). C indicate control, GM indicate gentamicin alone, CM indicate camel’s milk alone and MGM indicate gentamicin and camel’s milk.

 

Figure 2. Effect of camel’s milk on gentamicin -induced renal dysfunction as measured by serum urea. Data are expressed as means ± SD for all groups. (n = 6). There are significant different between (a) and (b). C indicate control, GM indicate gentamicin alone, CM indicate camel’s milk alone and MGM indicate gentamicin and camel’s milk.

  

Figure 3. Effect of camel’s milk on gentamicin -induced oxidative steres as measured by SOD. Data are expressed as means ± SD for all groups. n = 6. There are significant different between (c) and (ad). C indicate control, GM indicate gentamicin alone, CM indicate camel’s milk alone and MGM indicate gentamicin and camel’s milk.

 


Histopathology

Histopathological study revealed that gentamicin administration caused a marked injury in the renal tissue of the GM group. Moreover, the co-administration of camel milk and gentamicin in the MGM group led to reduced alterations (Figure 4 and Table 1). The rate of cellular degenerative changes, such as vacuolization and swelling in the GM group were significantly higher than the C and CM groups (P<0.05). There were no significant differences in terms of degenerative alterations between the GM and MGM groups (P<0.05). Significantly elevated eosinophilic casts in the tubular lumen, capillary congestion, glomerulonephritis, and necrosis were observed in the GM and MGM groups, in comparison with the C and CM groups (P<0.05). On the other hand, the rate of these histopathologic signs significantly diminished in the MGM group, compared to the GM group (P<0.05).Furthermore, the occurrence of interstitial nephritis and edema significantly augmented in the GM group, in comparison with the C and CM groups (P<0.05). The latter injuries were significantly inhibited in the MGM group, compared to the GM group (P<0.05).