Subscribe Now Subscribe Today
Research Article
 

Flavonoids-rich Extract of Beta vulgaris Subsp. cicla L. var. Flavescens Leaf, a Promising Protector Against Gentamicin- induced Nephrotoxicity and Hepatotoxicity in Rats



Sherien Kamal Hassan, Nermin Mohammed El-Sammad, Abeer Hamed Abdel-Halim, Amria Mamdouh Mousa, Wagdy Khalil Bassaly Khalil and Nayera Anwar
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

Background and Objective: The major complication of gentamicin antibiotic is nephrotoxicity which limited its clinical use. The current study was conducted to evaluate the possible protective effect of Beta vulgaris L. subsp. cicla var. flavescens (Swiss chard) against gentamicin-induced nephrotoxicity and hepatotoxicity in rats. Methodology: Twenty four rats were divided into 4 groups, group 1served as control. Group 2 to group 4 were intraperitoneally (i.p.) injected with gentamicin at a dose of 80 mg kg–1 body weight daily for 15 days. Swiss chard extract was orally administrated 1 week before and along with gentamicin treatment to groups 3 and 4 at doses of 300 and 600 mg kg–1 body weight daily, respectively. Results: The results revealed that, gentamicin significantly altered serum levels of kidney and liver markers as well as tumor necrosis factor alpha (TNF-α). These results were associated with significant changes in urinary urea, creatinine, micro-total protein (Micro TP) and vascular epithelial growth factor (VEGF) levels. A significant decrease in renal and hepatic reduced glutathione (GSH) levels, glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) enzymatic activities with a significant decrease in the expression levels of SOD1 and GSH-Px genes were also observed. In contrary, a significant increase in malondialdehyde (MDA), protein carbonyl levels and caspase-3 gene expression levels were also detected in gentamicin treated rats. Pretreatment with Swiss chard, dose dependently, ameliorated such altered changes. In harmony with this line, Swiss chard greatly decreased the severity of renal tubular and hepatic necrosis induced by gentamicin. Conclusion: Swiss chard leaf extract can attenuate gentamicin-induced nephrotoxicity and hepatotoxicity possibly by its antioxidant, anti-inflammatory and anti-apoptotic properties.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Sherien Kamal Hassan, Nermin Mohammed El-Sammad, Abeer Hamed Abdel-Halim, Amria Mamdouh Mousa, Wagdy Khalil Bassaly Khalil and Nayera Anwar, 2018. Flavonoids-rich Extract of Beta vulgaris Subsp. cicla L. var. Flavescens Leaf, a Promising Protector Against Gentamicin- induced Nephrotoxicity and Hepatotoxicity in Rats. International Journal of Pharmacology, 14: 652-666.

DOI: 10.3923/ijp.2018.652.666

URL: https://scialert.net/abstract/?doi=ijp.2018.652.666
 
Received: October 12, 2017; Accepted: January 15, 2018; Published: February 22, 2019


Copyright: © 2018. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Gentamicin is an aminoglycoside antibiotic obtained from Micromonospora purpurea fungus and other related species. It has a broad-spectrum action against life threatening Gram-negative bacterial infections and some strains of Gram-positive bacteria1,2. Inspite of its beneficial importance as a potent bactericidal agent, its use has been restricted owing to its adverse effects causing ototoxicity, nephrotoxicity, hepatotoxicity and neuromuscular problems3,4.

Nearly 10-20% of patients under gentamicin therapy showed the signs of nephrotoxicity, particularly with high and extended dose4,5. Renal toxicity occurred due to selective accumulation of gentamicin in renal tubular cells which induces necrosis, apoptosis and destruction of cells which finally results in renal failure with sudden and sustained decrease in glomerular filtration rate2,6. Renal damage induced by gentamicin can be accompanied by liver injury which leads to transient hepatomegaly and increases in serum bilirubin, lactate dehydrogenase and transaminases7,8. So there is an urgent need to search for agents that can protect kidney and liver against such harmful actions.

For decades, the use of natural and dietary products as potent chemoprotective agents increases day after day for the continuous discoveries of novel therapies. Swiss chard (Chenopodiaceae, Beta vulgaris subspecies cicla L.var. flavescens), a herbaceous plant cultivated around the world, is one of these magnificent plants as it is an excellent renewable source of various nutrients as well as phytochemicals9. Chemical screening for Swiss chard leaves showed that they have impressive amount of minerals as potassium, iron, phosphorus, calcium, magnesium and rich in vitamins as vitamin A, B3, B5, B9, E, also they are enriched with phenolic acids and flavonoids10,11. Syringic and caffeic acids are the most abundant phenolic acids10. Apigenin glycosides, namely vitexin, vitexin-2-O-α-rhamnosyl and vitexin-2-O-β-xylosyl are the most abundant flavonoids in Swiss chard leaves12.

Recently, in prior study six flavonoids from the leaves of Swiss chard, two of which are novel natural products namely herbacetin 3-O-β-xylopyranosyl- (1"’→2")-O-β-glucopyranoside and 2’’,2’’’-di-O-α-rhamnopyranosyl vicenin II have been identified13.

Modern pharmacology indicated the importance of such bioactive flavonoids contained in Swiss chard and demonstrated their hypoglycemic effect14,15, anti-inflammatory, antihypertension and anticancer effects12,16, hypolipidemic and hepatoprotective activities13 as well as high antioxidant activity11. Folk medicine also showed that Beta vulgaris L. species were used for treating liver and kidney diseases by stimulating the hematopoietic and immune systems17.

However, no study has been reported in the available current literature concerning the protective effect of Swiss chard extract on gentamicin induced nephrotoxicity and hepatotoxicity. Therefore, the present study aimed to investigate the nephroprotective and hepatoprotective effects of that extract against gentamicin-induced toxicity in Sprague Dawley rats.

MATERIALS AND METHODS

Plant material and preparation of extract: Leaves of Swiss chard were collected from plants cultivated in the Nile Delta near Cairo. The plant was identified and authenticated by Dr. Mohamed El Gebali, National Research Centre, Cairo, Egypt. A voucher specimen (B 201) was deposited at the Herbarium of the National Research Centre. Fresh leaves collected (3 kg) were exhaustively extracted with 75% MeOH in water (v/v). The extract obtained was dried under vacuum to afford a sticky dark brown aqueous methanolic extract (350 g) that was used in the present study.

Experimental animals: Male Sprague-Dawley rats, weighing 180-220 g, were obtained from the animal house of National Research Centre, Egypt. They were kept in polycarbonate cages under standard laboratory conditions at temperature 23±2°C with relative humidity of 50-60% and on a 12 h light/dark cycle, with free access to food and tap water ad libitum. All experimental procedures were approved by the Medical Ethical Committee of the National Research Centre and the animals were handled in accordance with "Principles of Laboratory Animal Care" (NIH publication No. 85-23, revised 1985).

Experimental design: Experimental animals were randomly divided into 4 equal groups of 6 animals each. The treatment schedule was as follows:

Samples preparation: After the last dose, animals were immediately kept in individual metabolic cages for 24 h urine collection. Urine samples were centrifuged at 1500 rpm for 10 min to remove debris and supernatant were stored at -20°C until analyzed. Blood samples were collected from Retro-orbital venous plexus of overnight fasted rats under ether anesthesia. Serum samples were separated by centrifugation at 3000 rpm for 15 min and stored at -20°C until assayed. The animals were then sacrificed by cervical dislocation, kidneys and livers were removed, rinsed with ice-cold normal saline, blotted with a piece of filter paper and weighed. A part of the kidney and the liver were immediately kept in 10% formalin solution for histopathological assay. Another part of the kidney and the liver were homogenized in 0.1 mol L–1 potassium phosphate buffer (pH 7.4) using tissue master TM125 (Omni International, USA) to obtain 1:10 (w/v) homogenate. After centrifugation at 3000 rpm for 10 min, the clear supernatant was stored at -80°C to be used for biochemical analysis. The remaining part of kidney and liver were stored at -80°C to be used for molecular analysis.

Biochemical analysis
Determination of kidney function tests: Serum and urinary levels of urea and creatinine were estimated with commercial kits developed by Spectrum Diagnostics Company (Egypt) based on the methods described previously19. Blood urea nitrogen was calculated using urea kit and creatinine clearance was calculated by the standard formula. Also, urinary micro TP and serum electrolytes, such as potassium, sodium and chloride were estimated using reagent kits purchased from Spectrum Diagnostics Company (Egypt) according to methods of Watanabe et al.20 and Mitchell et al.21, respectively.

Determination of liver function tests: Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP) and albumin (Alb) were assayed using reagent kits purchased from Spectrum Diagnostics Company, (Egypt) according to the methods of Reitman and Frankel22, Moss et al.23 Tietz24,25, respectively.

Determination of VEGF and TNF-α: Urinary VEGF and serum TNF-α were investigated by the enzyme-linked immunosorbent assay using kits obtained from Koma Biotec Inc, (Korea) according to the manufacturer’s instructions.

Estimation of oxidative stress biomarkers in renal and hepatic homogenates: The GSH was estimated according to the method of Beutler et al.26 after precipitating kidney and liver proteins with 10% metaphosphoric acid. GSH-Px, CAT and SOD activities were investigated according to the methods of Necheles et al.27, Aebi28 and Masayasu and Hiroshi29, respectively. Lipid peroxidation was assayed as MDA level in renal and hepatic tissues according to method of Lefevre et al.30. Protein carbonylation, a type of protein oxidation, was calculated by assay of Evans et al.31.

Estimation of total protein in renal and hepatic homogenates: The level of total protein in kidney and liver homogenates was estimated according to Lowry et al.32.

Gene expression analysis
Isolation of total RNA: TRIzolreg; Reagent (Invitrogen, Germany) was used to extract total RNA from liver tissues of rats according to the manufacturer’s instructions with minor modifications. Isolated total RNA was treated with one unit of RQ1 RNAse-free DNAse (Invitrogen, Germany) to digest DNA residues, re-suspended in DEPC-treated water and quantified photospectrometrically at 260 nm. Purity of total RNA was assessed by the 260/280 nm ratio which was between 1.8 and 2.1. Additionally, integrity was assured with ethidium bromide-stain analysis of 28S and 18S bands by formaldehyde-containing agarose gel electrophoresis (data not shown). Aliquots were used immediately for reverse transcription (RT), otherwise they were stored at -80°C.

Reverse transcription (RT) reaction:Complete Poly (A)+ RNA isolated from liver tissues was reverse transcribed into cDNA in a total volume of 20 μL using Revert AidTM First Strand cDNA Synthesis Kit (Fermentas, Germany). An amount of total RNA (5 μg) was used with a master mix. The master mix was consisted of 50 mM MgCl2,10×RT buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 10 mM of each dNTP, 50 μM oligo-dT primer, 20 IU ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 IU MuLV reverse transcriptase. The mixture of each sample was centrifuged for 30 sec at 1000 rpm and transferred to the thermocycler. The RT reaction was carried out at 25°C for 10 min, followed by 1 h at 42°C and finished with a denaturation step at 99°C for 5 min. Afterwards the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for cDNA amplification through quantitative real time-polymerase chain reaction (qRT-PCR).

Quantitative real time- PCR (QRT-PCR): QIAGEN’s real-time PCR cycler (Rotor-Gene Q, USA) was used to determine the cortex cDNA copy number.

Table 1:
Primer sequences used for qRT-PCR amplification

Table 2:
Effect of Swiss chard extract on body weight, relative kidney and liver weights in gentamicin administered rats
Data are expressed as Mean±SEM of 6 rats in each group. Values with superscripts are statistically significant at p<0.05. a: Indicates comparison versus control group, and b: Indicates comparison versus gentamicin treated group

Table 3:
Effect of Swiss chard extract on serum kidney markers in gentamicin administered rats
Data are expressed as Mean±SEM of 6 rats in each group. Values with superscripts are statistically significant at p<0.05. a: Indicates comparison versus control group, and b: Indicates comparison versus gentamicin treated group

The PCR reactions were set up in 25 mL reaction mixtures containing 12.5 mL 1×SYBRreg; Premix Ex Taq TM (TaKaRa, Biotech. Co. Ltd.), 0.5 mL 0.2 mM sense primer, 0.5 mL 0.2 mM antisense primer, 6.5 mL distilled water and 5 mL of cDNA template. The reaction program was allocated to 3 steps. First step was at 95°C for 3 min. Second step consisted of 40 cycles in which each cycle divided to 3 steps: (a) At 95°C for 15 sec, (b) At 55°C for 30 sec and (c) At 72°C for 30 sec. The third step consisted of 71 cycles which started at 60°C and then increased about 0.5°C every 10 sec up to 95°C. At the end of each qRT-PCR a melting curve analysis was performed at 95°C to check the quality of the used primers. Each experiment included a distilled water control. The sequences of specific primer used for SOD1, GSH-Px and caspase 3 genes are listed in Table 1. The RT-PCR quantitative values of genes were normalized on the bases of β-actin expression. At the end of each qRT-PCR a melting curve analysis was performed at 95°C to check the quality of the used primers. The relative quantification of the target to the reference was determined by using the 2-ΔΔCT method as follows:

•  Relative expression was calculated by 2-ΔΔCT

Histopathological investigation: The histopathologic examination was performed by light microscopy on kidney and liver tissue specimens that were fixed in 10% formalin. After fixation, the samples were processed to obtain 5 μm thick paraffin sections. Kidney and liver sections were stained with hematoxylin and eosin (H and E). Then the slides were examined by Olympus photomicroscope.

Statistical analysis: Results were expressed as Means±SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) for multiple comparisons (SPSS, Version 19.0) followed by LSD test to detect differences between groups. The differences were considered statistically significant at p<0.05.

RESULTS

Effect of Swiss chard extract on body weight, relative kidney weight (RKW) and relative liver weight (RLW): Gentamicin administration caused a significant weight loss of the whole body and significant increase in RKW and RLW as compared with control group. Treatment with Swiss chard extract improved these changes, particularly with 600 mg kg–1 dose as compared with gentamicin intoxicated rats (Table 2).

Effect of Swiss chard extract on renal function biomarkers: The serum levels of kidney markers in control and experimental groups are shown in Table 3.

Table 4:
Effect of Swiss chard extract on urinary kidney markers in gentamicin administered rats
Data are expressed as Mean±SEM of 6 rats in each group. Values with superscripts are statistically significant at p<0.05. a: Indicates comparison versus control group, and b: Indicates comparison versus gentamicin treated group

Table 5:
Effect of Swiss chard extract on serum liver markers in gentamicin administered rats
Data are expressed as Mean±SEM of 6 rats in each group. Values with superscripts are statistically significant at p<0.05. a: Indicates comparison versus control group, and b: Indicates comparison versus gentamicin treated group

The levels of serum urea, blood urea nitrogen (BUN), creatinine and potassium were significantly increased in gentamicin induced rats as compared to control animals, while sodium levels were significantly decreased. Oral administration of Swiss chard extract to the gentamicin treated rats significantly improved the levels of these markers in a dose related manner. Chloride level was almost similar among the whole groups.

On the other hand, urinary urea, creatinine and creatinine clearance showed significantly decreased levels in gentamicin group as compared to control rats, while urinary Micro TP level showed a significant increase. Administration of Swiss chard extract significantly improved excretion of urea, creatinine and Micro TP levels compared to gentamicin treated rats. While, creatinine clearance was significantly improved only at a dose of 600 mg kg–1 of the extract (Table 4). Furthermore, nephrotoxicity induced by gentamicin was associated with a significant increase in urinary VEGF level, whereas, treatment with Swiss chard extract significantly lowered its level as compared to gentamicin group (Fig. 1).

Effect of Swiss chard extract on liver function markers: Serum AST, ALT and ALP activities in gentamicin group showed a significant elevation as compared to the control group. In contrast, TP and Alb concentrations were significantly decreased as a result of gentamicin administration. Treatment with Swiss chard extract (300, 600 mg kg–1) significantly reversed the serum level of AST, ALT, ALP, TP and Alb when compared with the gentamicin treated group (Table 5).

Effect of Swiss chard extract on tumor necrosis factor-α: Gentamicin intoxicated rats showed significant elevation in the serum TNF-α level as compared to control rats.

Fig. 1:
Effect of Swiss chard extract on urinary vascular epithelial growth factor in gentamicin administered rats
 
Values are expressed as Mean±SEM of 6 rats in each group. aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05

However, administration of Swiss chard extract at a dose of 600 mg kg–1 significantly decreased the level of TNF-α when compared with the gentamicin group. A minor improvement was also observed in TNF-α level with Swiss chard treatment at 300 mg kg–1 dose but without statistical significance in comparing to gentamicin group (Fig. 2).

Effect of Swiss chard extract on renal and hepatic oxidative stress biomarkers: In the present study, there was a significant decline in GSH levels and GSH-Px, CAT and SOD activities in the kidney and liver tissues of gentamicin group as compared to normal rats. The treatment with Swiss chard extract dose dependently increased the renal and hepatic antioxidant parameters in the treated rats compared to the gentamicin group (Table 6).

Table 6:
Effect of Swiss chard extract on renal and hepatic oxidative stress biomarkers in gentamicin administered rats
Data are expressed as Mean±SEM of 6 rats in each group. Values with superscripts are statistically significant at p<0.05. a: Indicates comparison versus control group, and b: Indicates comparison versus gentamicin treated group. UA: μg of GSH consumed min–1 mg–1 tissue, UB: μmol of H2O2 decomposed min–1 mg–1 tissue, UC: 50% inhibition of nitroblue tetrazolium reduction g–1 tissue

On the other hand, MDA and protein carbonyl levels were significantly higher in the kidney and liver tissues of gentamicin group as compared to control rats. Swiss chard extract pretreatment caused a dose-dependent decrease in both renal and hepatic MDA and protein carbonyl levels when compared to the gentamicin treated rats. The increase in MDA was nearly prevented by administration of 600 mg kg–1 Swiss chard extract (Table 6).

Effect of Swiss chard extract on renal and hepatic total protein: Administration of gentamicin resulted in a significant decrease in TP contents in the kidney and liver tissues as compared to normal rats. Pretreatment with Swiss chard extract significantly increased renal TP in renal tissues in a dose dependent manner as compared to gentamicin group, while in hepatic tissues both doses of Swiss chard extract had almost the same improving effect on hepatic TP (Fig. 3).

Effect of Swiss chard extract on the expression alterations of renal and hepatic genes encoding antioxidant and apoptotic related enzymes: Expression of SOD1, GSH-Px and caspase-3 genes quantified by real time-polymerase chain reaction (RT-PCR) is summarized in Fig. 4-6. The results showed that gentamicin treatment significantly decreased the expression levels of SOD1 and GSH-Px genes in liver and kidney tissues compared with those in control rats. In contrary, gentamicin administration significantly increased the expression levels of caspase-3 gene in liver and kidney tissues compared with the control group.

Fig. 2:
Effect of Swiss chard extract on serum tumor necrosis factor-α in gentamicin administered rats
 
Values are expressed as Mean±SEM of 6 rats in each group. aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05

Pretreatment with Swiss chard extract significantly increased the expression levels of renal and hepatic SOD1 and GPx genes with significant decrease in the expression levels of renal and hepatic caspase-3 gene in a dose dependent manner compared with the gentamicin group.

Effect of Swiss chard extract on renal histopathology: Pathologically, there is a positive correlation between oxidative stress of gentamicin and nephrotoxicity. Drug induced nephrotoxicity is associated with their accumulation in renal cortex, depending upon their affinity to kidneys and on kinetics of gentamicin trapping process. In the present study, histopathological examination of sections from kidney treated with gentamicin showed tubular, glomerular and epithelial changes. Tubules showed evident variability in tubular size, generalized and extensive vacuolar degeneration in 60% of epithelial cell lining of proximal convoluted tubules (hydropic degeneration).

Fig. 3:
Effect of Swiss chard extract on renal and hepatic total protein in gentamicin administered rats
 

Values are expressed as Mean±SEM of 6 rats in each group. aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05


Fig. 4:
Effect of Swiss chard extract on expression of renal and hepatic SOD1 gene in gentamicin administered rats
 
Data are presented as Mean±SEM of 6 rats in each group, aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05

Coagulative necrosis, focal sloughing in luminal aspects, detached apical brush borders and evident hyaline casts were observed in 40% of tubular population. Also, there was tubular dilation in 30% of tubules. Glomeruli showed atrophy in 30% of their population. One fourth of renal glomeruli showed deposition of eosinophilic proteinaceous material. Bowman`s capsule was intact all around. Intersitium was edematous and vacuolar showing mild inflammatory exudates formed mainly of lymphocytes and some plasma cells (Fig. 7b-e).

On the other hand, the histopathological sections of kidney in animals treated with 300 mg kg–1 Swiss chard extract and gentamicin showed mild renal tubular impairment expressed in decreased deposition of tubular eosinophilic proteinaceous hyaline casts in the tubular lumina.

Fig. 5:
Effect of Swiss chard extract on expression of renal and hepatic GSH-Px gene in gentamicin administered rats
 
Data are presented as Mean±SEM of 6 rats in each group, aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05

Fig. 6:
Effect of Swiss chard extract on expression of renal and hepatic caspase-3 gene in gentamicin administered rats
 
Data are presented as Mean±SEM of 6 rats in each group. aStatistically significant as compared to control group, p<0.05, bStatistically significant as compared to gentamicin group, p<0.05

However, tubular epithelial cell size, tubular vaculation and tubular sloughing showed the same pathological changes as that in the gentamicin group. Glomerular changes considering glomerular atrophy and hyaline deposits were the same as gentamicin group. Interstitium was cleared up with no edema, no vascularity and no inflammatory cell exudates (Fig. 7f). Moreover, treatment with 600 mg kg–1 Swiss chard extract plus gentamicin showed abrupt and very evident improvement of all tubular changes. There was great improvement of vacuolar degeneration of tubular cell lining, no coagulative necrosis, no variability in tubular size and no hyaline casts.

Fig. 7(a-g):
Photomicrographs (H and E stain) of kidney sections from control and experimental rats, (a) Control group shows normal kidney histomorphology, (X100), (b) Gentamicin group shows glomerular atrophy (black arrow), variable size proximal tubules with cloudy swelling (green arrows) and focal shedded epithelial lining in some convoluted tubules (red arrow), (X400), (c) Gentamicin group shows tubular casts (green arrows), (X400), (d) Gentamicin group shows deposition of proteinaceous material within glomeruli (black arrows), (X400), (e) Gentamicin group shows diffuse interstitial inflammatory exudate formed mainly of lymphocytes and few plasma cells (black arrow), (X100), (f) Group treated with 300 mg kg–1 Swiss chard extract and gentamicin shows small and large glomeruli (black arrows) as well as diffuse and cloudy swelling in proximal convoluted tubules (green arrow), (X100) and (g) Group treated with 600 mg kg–1 Swiss chard extract and gentamicin shows almost regular and uniform glomerular size. However, some proximal convoluted tubules have cloudy swelling (green arrows) (X100)

Glomeruli and interstitium appeared as normal (Fig. 7g). Control rats showed normal architecture of glomeruli, proximal convoluted tubules, distal convoluted tubules and interstitium, (Fig. 7a). These histopathological findings were summarized in Table 7.

Fig. 8(a-e):
Photomicrographs (H and E stain) of liver sections from control and experimental rats, (a) Control group shows normal histological architecture of hepatic tissues, (X100), (b) Gentamicin group shows disturbed lobular pattern with focal necrosis in hepatocytes (black arrow), intervening hepatocytes exhibiting cloudy swelling and dilated congested sinusoids in between (green arrow), (X200), (c) Gentamicin group shows very evident hydropic degeneration within hepatocytes (black arrows), (X400), (d) Group treated with 300 mg kg–1 Swiss chard extract plus gentamicin shows evident cloudy swelling within hepatocytes (black arrow), (X400) and (e) Group treated with 600 mg kg–1 Swiss chard extract plus gentamicin shows almost normal lobular pattern, (X200)

Effect of Swiss chard extract on hepatic histopathology: Liver sections of rats treated with gentamicin revealed periportal hydropic degeneration characterized by cytoplasmic vacuoles. These vacuoles were small and large in size, clear and random in shape with sharp boundaries. There was focal necrosis dispersed in some hepatocytes. In addition, there were dilated congested sinusoids as well as inflammatory exudates in portal areas, (Fig. 8b and c). On the other hand, gentamicin group pretreated with 300 mg kg–1 extract revealed the same pathologic changes as gentamicin group but to a lesser extent, (Fig. 8d). While rats pretreated with 600 mg kg–1 extract revealed totally normal architecture, (Fig. 8e). These histopathological findings were summarized in Table 8.

Table 7:
Histopathological features of gentamicin induced nephrotoxicity in different treatment groups
Scoring was done as follows: None (-), mild (+), moderate (++) and severe (+++)

Table 8:
Histopathological features of gentamicin induced hepatotoxicity in different treatment groups
Scoring was done as follows: None (-), mild (+), moderate (++) and severe (+++)

DISCUSSION

In the current study, intraperitoneal administration of gentamicin at a dose of 80 mg kg–1 for 14 days caused a reduction in body weight and an increase in relative kidney and liver weights. These results are in agreement with those of previous study1. Body weight loss could be attributed to the increased catabolism occurring in acute renal failure which is accompanied by anorexia35. Moreover, gentamicin is deleterious to renal epithelial cells and may decrease water reabsorption causing dehydration and decrease in body weight36,2. However, gentamicin induced toxicity causes edema and inflammation in liver and kidneys which may be responsible for the increase in relative liver and kidney weights37,38. On the other hand, pretreatment with Swiss chard extract at 300 and 600 mg kg–1 provided a protective effect through increasing the body weight and decreasing relative kidney and liver weights.

The nephrotoxicity of gentamicin was characterized by a marked elevation in serum creatinine, urea and BUN levels as well as marked reduction in creatinine clearance. This impairment in glomerular function was accompanied by a decreased excretion of creatinine and urea as compared with the normal control. These findings were further confirmed by renal histological examination, which revealed tubular necrosis. The findings of the present study are in accordance with those described previously38,39. The intracellular accumulation of gentamicin in the renal proximal convoluted tubules causes severe proximal renal tubular necrosis40, which leads to diminished creatinine clearance and renal dysfunction417. Also, the increased serum creatinine level in gentamicin group was found to be an indicative of decrease in glomerular filtration rate, whereas the increased serum urea and BUN levels were found to be an indicative of parenchyma tissue injury after tubular necrosis4. Serum electrolytes were also disturbed significantly in gentamicin treated rats. These results are in agreement with other investigators42,43. The decreased level of sodium indicates kidney inability to conserve sodium and chloride ions44, while the increased level of potassium may be due to renal tubular epithelium lesions45.

Urine analysis showed significant excretion of urinary protein along with significant elevation in VEGF level in gentamicin intoxicated rats as compared to control. These results are in accordance with those obtained previously39,46. Also, it has been reported that gentamicin administration impaired the capacity of renal tubule to reabsorb low-weight proteins47. The novel urinary protein biomarker, VEGF, together with the more classical urinary parameter, total protein, may be useful biomarkers that are more sensitive than BUN and serurm creatinine in detecting minimal to mild renal damage and dysfunction48. However, pretreatment with Swiss chard extract, dose dependently, ameliorated the gentamicin-induced nephrotoxicity by decreasing the elevated levels of serum kidney markers (urea, creatinine and BUN) and increasing the level of creatinine clearance as well as restoring the levels of the serum electrolytes to near normal. In addition, Swiss chard treatment elevated urinary urea and creatinine levels and reduced urinary Micro TP and VEGF levels. These results revealed that the methanolic extract of Swiss chard exerted a functional protection against gentamicin-induced nephrotoxicity, thereby improving renal function.

Gentamicin induced hepatotoxicity was evidenced by increased serum levels of hepatic marker enzymes ALT, AST and ALP as well as decreased levels of serum total protein and albumin, the major serum protein synthesized by the liver. These findings are in accordance with those described previously3,49. The elevation in levels of hepatic enzymes in gentamicin treated rats could be attributed to damage in hepatocytes plasma membrane that leads to loss of the functional integrity and increase in cell permeability, which results in release of enzymes from the damaged hepatocytes into serum50. Leakage of AST and ALT from hepatic cell can occur as a secondary change to cellular necrosis51. Whereas, decreased levels of serum protein and albumin are indicative of damage in hepatic protein synthesizing subcellular structures52. Present results revealed that Swiss chard pretreatment significantly lowered serum levels of ALT, AST and ALP which suggested that Swiss chard extract improved the functional status of the hepatic cells by preventing leakage of intracellular enzymes through restoring cell membrane integrity. These effects could be attributed to the presence of the flavonoid glycosides in the extract13. In addition, the normalization of serum protein and albumin levels due to Swiss chard administration may be due to the well-functioning capacity of hepatocytes in protein synthesis.

In this study, gentamicin administered rats showed a significant elevation in serum TNF-α, one of cytokines regulating inflammation, which is in parallel with that described previously37. Cellular damage and necrosis stimulate the generation of inflammatory mediators by the injured cells as well as by immune cells, which induce migration and infiltration of leukocytes into the injured organs53. Inflammatory response is one of characteristics of gentamicin-nephrotoxicity52.

On the other hand, pretreatment with Swiss chard reduced the elevated level of serum TNF-α. This reduction in TNF-α level in Swiss chard treated rats (600 mg) was prominent than (300 mg) treated group. The TNF-α reduction might be attributed to anti-inflammatory property of Swiss chard extract which could be due to its flavonoids. The ability of polyphenolic compounds to inhibit TNF-production was previously demonstrated54. It has been declared that blockade of TNF-α is a valuable approach for managing nephrotoxicity55.

In the current study, gentamicin administration induced oxidative stress in rat liver and kidneys as evidenced by a significant decrease in the activities of renal and hepatic antioxidant enzymes (GSH-Px, CAT and SOD) as well as GSH levels coupled with significant increase in MDA level (marker of lipid peroxidation) as compared to normal control. These results are consistent with those reported previously 56,57. The expression levels of antioxidant related genes (SOD1 and GSH-Px) were also significantly decreased in gentamicin treated rats. Gentamicin administration increases the generation of ROS that is followed by reducing the activities of antioxidant enzymes58,59 and by depleting intracellular concentrations of GSH during the process of combating oxidative stress, which enhances lipid peroxidation60. On the other hand, pretreatment with Swiss chard significantly restored the activities of these antioxidant enzymes and GSH content as well as decreased the level of MDA in renal and hepatic tissues. Moreover, a significant increase in the expression levels of antioxidant related genes (SOD1 and GSH-Px) were also observed. These results could be attributable to the antioxidant activity of Swiss chard, which markedly attenuated the oxidative stress induced by gentamicin and led to the inhibition of lipid peroxidation, the maintaining of GSH levels at near normal values and the enhancement of the gene expression and activities of antioxidant enzymes. In the same line with our findings, it has been reported that Swiss chard exhibited high antioxidant activity that could be due to its phenolic compounds10. Moreover, polyphenolic compounds promote the anti-free-radical activity of several herbal extracts61. The capacity of Swiss chard extracts for radical scavenging could be attributed to their concentration of hydroxyl group in the phenolic compounds which regulate the expression of antioxidant’s mRNA. Therefore, the anti-free-radical capacity of Swiss chard extracts depends on the concentration of the phenolic compounds and their molecular structure. Thus, that explain why the high concentration of the Swiss chard extract (600 mg) is more effective than its low concentration (300 mg) on the antioxidant enzymes activities and the expression levels of related genes (SOD1 and GSH-Px).

Gentamicin treatment induced a significant decrease in total protein contents and significant increase in protein carbonyl contents (marker of protein oxidation) in renal and hepatic tissues compared to the control group. Our results are in agreement with those reported that the formation of protein carbonyl as a consequence of oxidative stress, may be an early marker for protein oxidation62,63. Gentamicin administration causes abnormal production of ROS which induces cellular injury and necrosis through membrane phospholipids peroxidation and protein denaturation64. Among ROS, hydroxyl radical is thought to be the most damaging species and the one mainly responsible for lipid and protein oxidation65. Pretreatment with Swiss chard afforded significant protection against the oxidative modification of proteins induced by gentamicin treatment. A lot of investigations reported that natural phenolic compounds, particularly flavonoids, have strong antioxidant potency and protective activity as well as free radicals generation prevention10,66.

The expression levels of the apoptotic enzyme gene (caspase-3) were significantly increased in the kidney and hepatic tissues of gentamicin-treated group. Present results are consistent with earlier reports which mentioned that gentamicin treatment elevated the expression level of caspase-3 through increasing the production of ROS which finally lead to apoptosis67,68. Apoptosis mediated by ROS is an important mechanism in gentamicin-induced cytotoxicity67,69. Caspases are endoproteases that have important roles in controlling apoptosis pathways in mammalian cells70. Pretreatment with Swiss chard extract suppressed gentamicin-induced apoptosis by reducing the expression levels of caspase-3. It has been indicated that Swiss chard extract, rich in flavonol glycosyls are responsible for the anti-mitotic and anti-apoptotic activities71.

CONCLUSION

It is concluded that the results of the present study indicated that the methanolic extract of Swiss chard possesses significant protective effect in a dose dependent manner against gentamicin-induced hepato-renal toxicity. The high antioxidant capacity of Swiss chard together with the anti-inflammatory and anti-apoptotic properties are supposed to be related to its flavonoid contents. Hence, Swiss chard could be a promising therapeutic agent in alleviating gentamicin-induced nephrotoxicity and hepatotoxicity in clinical trials.

SIGNIFICANT STATEMENT

This study discovers the possible protective effect of Swiss chard extract that can be beneficial in treatment of gentamicin toxicity in rats. This study will help the researcher to uncover the role of Swiss chard extract in reducing the side effects of gentamicin nephrotoxicity on long-term therapy that many researchers were not able to explore. Thus, a new theory on combination of gentamicin and Swiss chard extract may be arrived at.

REFERENCES
Abd El-Rahman, H.S.M., 2016. The effect of olive leaf extract and α-tocopherol on nephroprotective activity in rats. J. Food. Sci. Nutr., Vol. 6. 10.4172/2155-9600.1000479

Aboubakr, M. and A.M. Abdelazem, 2016. Hepatoprotective effect of aqueous extract cardamom against gentamicin induced hepatic damage in rats. Int. J. Basic Applied Sci., 5: 1-4.
CrossRef  |  Direct Link  |  

Abuelezz, S.A., N. Hendawy and S.A. Gawad, 2016. Alleviation of renal mitochondrial dysfunction and apoptosis underlies the protective effect of sitagliptin in gentamicin-induced nephrotoxicity. J. Pharm. Pharmacol., 68: 523-532.
CrossRef  |  Direct Link  |  

Aebi, H., 1984. Catalase in vitro. Meth. Enzymol., 105: 121-126.
CrossRef  |  PubMed  |  Direct Link  |  

Al-Asmari, A.K., R. Abbasmanthiri, A.M. Al-Elewi, S.S. Al-Omani and S.A. Al-Asmari, 2014. Camel milk beneficial effects on treating gentamicin induced alterations in rats. J. Toxicol. 10.1155/2014/917608

Azab, A.E., M.O. Albasha and A.S.I. Elsayed, 2016. Prevention of hepatotoxicity with Curcuma longa and Rosmarinus officinalis in gentamicin treated guinea pigs. Indo Am. J. Pharm. Res., 6: 4791-4802.

Balakumar, P., A. Rohilla and A. Thangathirupathi, 2010. Gentamicin-induced nephrotoxicity: Do we have a promising therapeutic approach to blunt it? Pharmacol. Res., 62: 179-186.
PubMed  |  Direct Link  |  

Beutler, E., O. Duron and B.M. Kelly, 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med., 61: 882-888.
PubMed  |  Direct Link  |  

Bibu, K.J., A.D. Joy and K.A. Mercey, 2010. Therapeutic effect of ethanolic extract of Hygrophila spinosa T. Anders on gentamicin-induced nephrotoxicity in rats. Indian J. Exp. Biol., 48: 911-917.
PubMed  |  Direct Link  |  

Chaware, V.J., 2012. Protective effect of the aqueous extract of phaseolus radiates seeds on gentamicin induced nephrotoxicity in rats. Indian. J. Res. Pharm. Biotechnol., 3: 73-75.

Dontabhaktuni, A., D.R. Taft and M. Patel, 2016. Gentamicin renal excretion in rats: Probing strategies to mitigate drug-induced nephrotoxicity. Pharmacol. Pharm., 7: 43-55.
CrossRef  |  Direct Link  |  

Dursun, E., B. Dursun, G. Suleymanlar and T. Ozben, 2005. Carbonyl stress in chronic renal failure: The effect of haemodialysis. Ann. Clin. Biochem., 42: 64-66.
CrossRef  |  Direct Link  |  

Edmnnd, J.L. and P.P. Christopher, 2008. Creatinine, Urea and Uric Acid. In: Tietz Fundamentals of Clinical Chemistry, 6th Edn., Carl, A.B., R.A. Edward and E.B. David (Eds.)., Saunders Elsevier, USA., pp: 63-72.

El-Kashef, D.H., A.E. El-Kenawi, G.M. Suddek and H.A. Salem, 2015. Flavocoxid attenuates gentamicin-induced nephrotoxicity in rats. Naunyn-Schmiedeberg's Arch. Pharmacol., 388: 1305-1315.
CrossRef  |  Direct Link  |  

El-Tantawy, W.H., S.A.H. Mohamed and E.N. Abd Al Haleem, 2013. Evaluation of biochemical effects of Casuarina equisetifolia extract on gentamicin-induced nephrotoxicity and oxidative stress in rats. J. Clin. Biochem. Nutr., 53: 158-165.
CrossRef  |  Direct Link  |  

Evans, P., L. Lyras and B. Halliwell, 1999. Measurement of protein carbonyls in human brain tissue. Methods. Enzymol., 300: 145-156.
CrossRef  |  Direct Link  |  

Gao, Z.J., X.H. Han and X.G. Xiao, 2009. Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves. Food Chem., 117: 342-348.
CrossRef  |  Direct Link  |  

Gaskill, C.L., L.M. Miller, J.S. Mattoon, W.E. Hoffmann and S.A. Burton et al., 2005. Liver histopathology and liver and serum alanine aminotransferase and alkaline phosphatase activities in epileptic dogs receiving phenobarbital. Vet. Pathol., 42: 147-160.
CrossRef  |  Direct Link  |  

Gautier, J.C., T. Gury, M. Guffroy, R. Masson and R. Khan-Malek et al., 2014. Comparison between male and female sprague-dawley rats in the response of urinary biomarkers to injury induced by gentamicin. Toxicol. Pathol., 42: 1105-1116.
CrossRef  |  PubMed  |  Direct Link  |  

Gautier, J.C., X. Zhou, Y. Yang, T. Gury and Z. Qu et al., 2016. Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin. Toxicol. Applied Pharmacol., 303: 1-10.
CrossRef  |  Direct Link  |  

Gennari, L., M. Felletti, M. Blasa, D. Angelino, C. Celeghini, A. Corallini and P. Ninfali, 2011. Total extract of Beta vulgaris var. cicla seeds versus its purified phenolic components: Antioxidant activities and antiproliferative effects against colon cancer cells. Phytochem. Anal., 22: 272-279.
CrossRef  |  Direct Link  |  

Geziginci-Oktayoglu, S., O. Oacan, S. Bolkent, Y. Ipci, L. Kabasakal, G. Sener and R. Yanardag, 2014. Chard (Beta vulgaris L. var. cicla) extract ameliorates hyperglycemia by increasing GLUT2 through Akt2 and antioxidant defense in the liver of rats. Acta Histochem., 116: 32-39.
CrossRef  |  Direct Link  |  

Han, M.S., I.H. Han, D. Lee, J.M. An and S.N. Kim et al., 2015. Beneficial effects of fermented black ginseng and its ginsenoside 20(S)-Rg3 against cisplatin-induced nephrotoxicity in LLC-PK1 cells. J. Ginseng. Res., 40: 135-140.
CrossRef  |  Direct Link  |  

Hashem, A.N., M.S. Soliman, M.A. Hamed, N.F. Swilam, U. Lindequist and M.A. Nawwar, 2016. Beta vulgaris subspecies cicla var. flavescens (Swiss chard): Flavonoids, hepatoprotective and hypolipidemic activities. Pharmazie, 71: 227-232.
CrossRef  |  Direct Link  |  

Hsu, C.H., C.H. Chen, C.C. Hou, Y.M. Sue and C.Y. Cheng et al., 2008. Prostacyclin protects renal tubular cells from gentamicin-induced apoptosis via a PPARα-dependent pathway. Kidney Int., 73: 578-587.
CrossRef  |  Direct Link  |  

Hur, E., A. Garip, A. Camyar, S. Ilgun and M. Ozisik et al., 2013. The effects of vitamin D on gentamicin-induced acute kidney injury in experimental rat model. Int. J. Endocrinol. 10.1155/2013/313528

Jain, D. and R. Somani, 2015. Silibinin: A bioactive flavanone in milk thistle ameliorate gentamicin induced nephrotoxicity in rats. Pharmacologia, 6: 38-44.
Direct Link  |  

Jain, D.P. and R.S. Somani, 2015. Antioxidant potential of hesperidin protects gentamicin induced nephrotoxicity in experimental rats. Austin J. Pharmacol. Ther., Vol. 3, No. 2.

Kahkonen, M.P., A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T.S. Kujala and M. Heinonen, 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem., 47: 3954-3962.
CrossRef  |  PubMed  |  Direct Link  |  

Kamel, M.A., Abdel H.I. Fadil and M.A. Noha, 2015. Prevention of hepato-renal toxicity with vitamin E, vitamin C and their combination in gentamicin treated rats. Int. J. Pharm. Sci., 5: 1289-1296.
Direct Link  |  

Kang, C.Q., H.Y Lee, D.Y. Hah, J.H. Heo, C.H. Kim, E. Kim and J.S. Kim, 2013. Protective effects of Houttuynia cordata Thunb on gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicol. Res., 29: 61-67.
CrossRef  |  Direct Link  |  

Kanner, J., S. Harel and R. Granit, 2001. Betalains-A new class of dietary cationized antioxidants. J. Agric. Food Chem., 49: 5178-5185.
CrossRef  |  PubMed  |  Direct Link  |  

Kawada, N., S. Seki, M. Inoue and T. Kuroki, 1998. Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and kupffer cells. Hepatology, 27: 1265-1274.
CrossRef  |  Direct Link  |  

Khalil, W.K. and H.F. Booles, 2011. Protective role of selenium against over-expression of cancer-related apoptotic genes induced by O-cresol in rats. Arch. Ind. Hyg. Toxicol., 62: 121-129.
CrossRef  |  PubMed  |  

Khan, M.R., I. Badar and A. Siddiquah, 2011. Prevention of hepatorenal toxicity with Sonchus asper in gentamicin treated rats. BMC Complement. Altern. Med., Vol. 11. 10.1186/1472-6882-11-113

Kim, Y., M.S. Han, J.S. Lee, J. Kim and Y.C. Kim, 2003. Inhibitory phenolic amides on lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells from Beta vulgaris var. cicla seeds. Phytother. Res., 17: 983-985.
CrossRef  |  Direct Link  |  

Kobori, M., Y. Takahashi, Y. Akimoto, M. Sakurai and I. Matsunaga et al., 2015. Chronic high intake of quercetin reduces oxidative stress and induces expression of the antioxidant enzymes in the liver and visceral adipose tissues in mice. J. Funct. Foods, 15: 551-560.
CrossRef  |  Direct Link  |  

Lefevre, G., M. Beljean-Leymarie, F. Beyerle, D. Bonnefont-Rousselot, J.P. Cristol, P. Therond and J. Torreilles, 1998. [Evaluation of lipid peroxidation by measuring thiobarbituric acid reactive substances]. Annales Biologie Clinique, 56: 305-319, (In French).
PubMed  |  Direct Link  |  

Li, H., F. Song, J. Xing, R. Tsao, Z. Liu and S. Liu, 2009. Screening and structural characterization of α-Glucosidase inhibitors from hawthorn leaf flavonoids extract by ultrafiltration LC-DAD-MSn and SORI-CID FTICR MS. J. Am. Soc. Mass. Spectrom., 20: 496-503.
CrossRef  |  Direct Link  |  

Li, J., Q.X. Li, X.F. Xie, Y. Ao, C.R. Tie and R.J. Song, 2009. Differential roles of dihydropyridine calcium antagonist nifedipine, nitrendipine and amlodipine on gentamicin-induced renal tubular toxicity in rats. Eur. J. Pharmacol., 620: 97-104.
CrossRef  |  Direct Link  |  

Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275.
PubMed  |  Direct Link  |  

Martines, G., L. Butturini, I. Menozzi, G. Restori, L. Boiardi, S. Bemardi and P. Baldassarri, 1988. Amikacin-induced liver toxicity: Correlations between biochemical indexes and ultrastructural features in an experimental model. Rev. Med. Univ. Navarra, 32: 41-44.
Direct Link  |  

Masayasu, M. and Y. Hiroshi, 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clin. Chim. Acta, 92: 337-342.
CrossRef  |  PubMed  |  Direct Link  |  

Mitchell, G.C., A.L. Vicky and J.K. Stacey, 2008. Electrolytes and Blood Gasese. In: Tietz Fundamentals of Clinical Chemistry, 6th Edn., Carl, A.B., R.A. Edward and E.B. David (Eds.)., Saunders Elsevier, USA., pp: 431-449.

Moreira, M.A., M.A. Nascimento, T.A. Bozzo, A. Cintra and S.M. da Silva et al., 2013. Ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species. Clin. Nutr., 33: 296-301.
CrossRef  |  Direct Link  |  

Moss, D.W., A.R. Henderson and J.F. Kachmar, 1987. Enzymes. In: Fundamentals of Clinical Chemistry 3rd Edn., Tietz, N.W. (Ed.)., W.B. Saunders, Philadelphia, pp: 346-421.

Muthuraman, A., S.K. Singla, A. Rana, A. Singh and S. Sood, 2011. Reno-protective role of flunarizine (Mitochondrial permeability transition pore inactivator) against gentamicin induced nephrotoxicity in rats. Yakugaku Zasshi, 131: 437-443.
Direct Link  |  

Nale, L.P., P.R. More, B.K. More, B.C. Ghumare, S.B. Shendre and C.S. More, 2012. Protective effect of Carica papaya L. seed extract in gentamicin induced hepatotoxicity and nephrotoxicity in rats. Int. J. Pharm. Bio. Sci., 3: 508-515.
Direct Link  |  

Necheles, T.F., T.A. Boles and D.M. Allen, 1968. Erythrocyte glutathione-peroxidase deficiencyand hemolytic disease of the newborn infant. J. Pediatr., 72: 319-324.
CrossRef  |  Direct Link  |  

Ninfali, P. and D. Angelino, 2013. Nutritional and functional potential of Beta vulgaris cicla and rubra. Fitoterapia, 89: 188-199.
CrossRef  |  Direct Link  |  

Noorani, A.A., K. Gupta, K. Bhadada and M.K. Kale, 2011. Protective effect of methanolic leaf extract of Caesalpinia bonduc (L.) on gentamicin-induced hepatotoxicity and nephrotoxicity in rats. Iran. J. Pharmacol. Therapeut., 10: 21-25.
Direct Link  |  

Oki, T., M. Masuda, S. Furuta, Y, Nishiba, N. Terahara and I. Suda, 2002. Involvement of anthocyanins and other phenolic compounds in radical-scavenging activity of purple-fleshed sweet potato cultivars. J. Food Sci., 67: 1752-1756.
CrossRef  |  Direct Link  |  

Padmini, M.P. and J.V. Kumar, 2012. A histopathological study on gentamycin induced nephrotoxicity in experimental albino rats. J. Dental Med. Sci., 1: 14-17.
Direct Link  |  

Palm, C.A., G. Segev, L.D. Cowgill, B.E. LeRoy, K.L. Kowalkowski, K. Kanakubo and J.L. Westropp, 2016. Urinary neutrophil gelatinase-associated lipocalin as a marker for identification of acute kidney injury and recovery in dogs with gentamicin-induced nephrotoxicity. J. Vet. Internal Med., 30: 200-205.
CrossRef  |  Direct Link  |  

Pyo, Y.H., T.C. Lee, L. Longedra and R.T. Rosen, 2004. Antioxidant activity and phenolic compounds of Swiss chard (Beta vulgaris subspecies cicla) extracts. Food Chem., 85: 19-26.
CrossRef  |  Direct Link  |  

Rajalingam, D., R. Varadharajan and S. Palani, 2016. Evaluation of hepatoprotective and antioxidant effect of Combretum albidum G.Don against CCl4 induced hepatotoxicity in rats. Int. J. Pharmacol. Pharm. Sci., 8: 218-223.
Direct Link  |  

Rajashekar, V., E.U. Rao and P. Srinivas, 2012. Biological activities and medicinal properties of gokhru (Pedalium murex L.). Asian Pac. J. Trop. Biomed., 2: 581-585.
CrossRef  |  Direct Link  |  

Randjelovic, P., S. Veljkovic, N. Stojiljkovic, L. Jankovic-Velickovic, D. Sokolovic, M. Stoiljkovic and I. Ilic, 2012. Salicylic acid attenuates gentamicin-induced nephrotoxicity in rats. Sci. World. J. 10.1100/2012/390613

Randjelovic, P., S. Veljkovic, N. Stojiljkovic, L. Velickovic, D. Sokolovic, M. Stoiljkovic and I. Ilic, 2012. Protective effect of selenium on gentamicin-induced oxidative stress and nephrotoxicity in rats. Drug Chem. Toxicol., 35: 141-148.
CrossRef  |  Direct Link  |  

Reitman, S. and S. Frankel, 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28: 56-63.
CrossRef  |  PubMed  |  Direct Link  |  

Rodrigues, F.A.P., M.M.G. Prata, I.C.M. Oliveira, N.T.Q. Alves and R.E.M. Freitas et al., 2014. Gingerol fraction from Zingiber officinale protects against gentamicin-induced nephrotoxicity. Antimicrob. Agents. Chemother., 58: 1872-1878.
CrossRef  |  Direct Link  |  

Sahu, B.D., M. Kuncha, G.J. Sindhura and R. Sistla, 2013. Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress, inflammation and DNA damage. Phytomedicine, 20: 453-460.
CrossRef  |  Direct Link  |  

Saleh, A.A.S., 2014. Synergistic effect of N-acetyl cysteine and folic acid against aspartame-induced nephrotoxicity in rats. Int. J. Adv. Res., 2: 363-373.
Direct Link  |  

Servais, H., Y. Jossin, F. Van Bambeke, P.M. Tulkens and M.P. Mingeot-Leclercq, 2006. Gentamicin causes apoptosis at low concentrations in renal LLC-PK1 cells subjected to electroporation. J. Antimicrob. Agents Chemother., 50: 1213-1221.
CrossRef  |  PubMed  |  Direct Link  |  

Sodimbaku, V., L. Pujari, R. Mullangi and S. Marri, 2016. Carrot (Daucus carota L.): Nephroprotective against gentamicin-induced nephrotoxicity in rats. Indian J. Pharmacol., 48: 122-127.
CrossRef  |  Direct Link  |  

Tietz, N.W., 1990. Clinical Guide to Laboratory Tests. 2nd Edn., WB Saunders, Philadelphia, pp: 26-29.

Tietz, N.W., 1994. Fundamentals of Clinical Chemistry. 2nd Edn., WB Saunders, Philadelphia, pp: 692.

Tugcu, V., E. Ozbek, A.I. Tasci, E. Kemahli and A. Somay et al., 2006. Selective nuclear factor kappa-B inhibitors, pyrolidium dithiocarbamate and sulfasalazine, prevent the nephrotoxicity induced by gentamicin. BJU Int., 98: 680-686.
CrossRef  |  PubMed  |  Direct Link  |  

Watanabe, N., S. Kamei, A. Ohkubo, M. Yamanaka, S. Ohsawa, K. Makino and K. Tokuda, 1986. Urinary protein as measured with a pyrogallol red-molybdate complex, manually and in a hitachi 726 automated analyzer. Clin. Chem., 32: 1551-1554.
PubMed  |  Direct Link  |  

Yarijani, Z.M., H. Najafi and S.H. Madani, 2016. Protective effect of crocin on gentamicin-induced nephrotoxicity in rats. Iran. J. Basic. Med. Sci., 19: 337-343.
Direct Link  |  

Zein, H., A.E.M.S. Hashish and G.H.H. Ismaiel, 2015. The antioxidant and anticancer activities of Swiss chard and red beetroot leaves. Curr. Sci. Int., 4: 491-498.
Direct Link  |  

©  2020 Science Alert. All Rights Reserved