Ameliorative Effect of Rutin on Gentamicin-Induced Nephrotoxicity in Murine Model

Abstract

Gentamicin is one of the widely used antibiotics. However, it suffers from drawback of nephrotoxicity, which is predisposed due to oxidative stress. Rutin is one of the major classes of flavonoids and phyto-antioxidant. It has demonstrated cardioprotective, hepatoprotective and neuroprotective effects. However, no studies pertaining to its nephroprotective activity have still performed. Present work was aimed to determine nephroprotective activity of Rutin. Dose of 100 mg/ kg and 200 mg/ kg was taken in present study. Gentamicin (80 mg/ kg) was used to induce nephrotoxicity. At the end of the study body weight, kidney weight, serum creatinine, serum urea, blood urea nitrogen and urine creatinine were determined along with histopathological studies. Administration of Rutin restored the levels of biomarkers. Histopathological studies also demonstrated protective effect on kidney. Thus it could be concluded that rutin demonstrates nephroprotective potential.

Keywords: Rutin; Nephroprotective; Histopathology; Markers

Introduction

Since last 50 years, aminoglycosides are being effectively used as an integral part of antimicrobial chemotherapy [1] and these drugs are still useful to treat various life threatening infections [2]. Low resistance rate, dose dependent effects, along with low cost are some of its merits [3]. However, Gentamicin causes dose dependent nephrotoxicity [4,5].

Gentamicin induced nephrotoxicity is a multifaceted process that is observed by augmentation of plasma urea and creatinine levels along with necrosis of renal tubules that ultimately leads to renal failure [6,7]. One of the reasons behind this manifestation includes increased oxidative stress [9]. Thus anticipation and diminution of oxidative stress seems to be a novel strategy in preventing such situation.

Flavonoids are polyphenol obtained from plants [9]. They are found abundantly in grains, fruits, vegetables, barks, roots, stem, flower, tea and wine [10]. These compounds have benzo-pyran nucleus. Flavonoids are produced by plants as a result of response to preclusion of microbial infection [11]. However, due to high antioxidant capacity, they are believed to have health promoting properties [12,13].

Rutin (3, 3’, 4’, 5, 7-pentahydroxyflavone-3-rhamnoglucoside; Vitamin P) is an important bioflavonoid has demonstrated a number of beneficial pharmacological effects [14]. Rutin is known for various therapeutic effects. It has effectively prevented cognitive impairments by ameliorating oxidative stress and neuro-inflammation in rat model of sporadic dementia of Alzheimer type [15] and established protective effect against cognitive deficits and brain damage in rats with chronic cerebral hypoperfusion [16]. Rutin demonstrated protective effect against reflux oesophagitis by the inhibition of gastric acid secretion, oxidative stress and inflammatory cytokine production [17].

It has reported to be neuroprotective [18], cardioprotective [19], nephroprotective, and protective effect against reproductive toxicity [20] along with anti-inflammatory [21] and antihyperglycemic [22] effects in experimental animals.

In the present context, in vivo protective activity of Rutin against Gentamicin induced nephrotoxicity was evaluated in albino Wistar rats.

Experimental

Drugs and chemicals

Rutin and Gentamicin were purchased from Central Drug House, Mumbai, India. All the other chemicals used were of analytical grade.

Animals

Healthy adult male wistar albino rats (5-6 month; 250-300 g) were used. The animals were housed in polypropylene cages and maintained under standard conditions. They were fed with standard rat pellet diet (Hindustan Lever Ltd, Mumbai India) and water ad libitum. All the animal experimental protocols were approved by Institutional Animal Ethics Committee.

Selection of dose

In accordance to the dose as determined by Motamedshariaty et al., [23], rutin in the dose of 100 and 200 mg/kg orally was used in the present study.

Experimental protocols [24]

Animals were divided into four groups containing five animals each and grouped as

Group I: Control

Group II: Gentamicin (80mg/kg)

Group III: Gentamicin (80mg/kg) + Rutin (100mg/kg)

Group IV: Gentamicin (80mg/kg) + Rutin (200mg/kg)

Except group I animals, other three groups received 100 mg/kg/ day gentamicin by the intraperitoneal (i.p.) route.

After daily dosing, on the day 10, individual rats were placed in separate metabolic cages for 24 h for urine collection to determine urine creatinine content.

Body weight

Body weights of these rats were monitored on 10^th day

Kidney function test

Blood urea nitrogen

Blood urea nitrogen was estimated as per manufacturer’s instructions (Span Diagnostic Ltd).

Serum creatinine

Creatinine was estimated using the method described previously by Broad and Sirota, using Jaffe’s reaction. To 1.0mL of serum and kidney homogenates, 8.0 ml of double-distilled water, 0.5mL of 2/3N sulfuric acid and 0.5ml of 10% sodium tungstate were added. This mixture was centrifuged at 4000 rpm for 15 min at 4oC, 5.0ml of the clear supernatant was taken to which 1.5ml of saturated picric acid and 1.5ml of 0.75N sodium hydroxide were added. The absorbance was read at 460 nm after 15 min in a Shimadzu UV- 1700spectrophotometer. Standard and blank were also processed similarly and the creatinine levels were expressed in milligram per deciliter [25,26].

Blood urea nitrogen [25] and serum creatinine [26] were estimated by method reported elsewhere.

Urea estimation

Urea was estimated by method reported by Natelson et al. [25]. Urea was estimated by the reported method. To 0.1ml of serum and kidney homogenates, 3.3ml of double-distilled water, 0.3ml of 10% sodium tungstate and 0.3mL of 0.67N sulfuric acids were added. The samples were centrifuged (4000 rpm, 15 min, 4^°C) and to 1ml of the supernatant, 1ml of double-distilled water, 0.4ml of diacetyl monoxime reagent and 0.6ml of sulfuric acid– phosphoric acid mixture were added. The samples were incubated in a boiling water bath for 30 min, cooled to room temperature and the absorbance was read at 480 nm in a Shimadzu UV-1700spectrophotometer (Tokyo, Japan) [27].

Histopathological inspection of kidney

Rats were sacrificed and both kidneys were isolated from each rat. The kidneys were processed for histopathological examination. The kidney were excised quickly and fixed in 10 % formalin and stained with haemotoxylin and eosin and then observed under microscope for degeneration, fatty changes, necrotic changes and evidence of nephrotoxicity if any.

Statistical Analysis

The results were expressed as mean ± SEM. Statistical analysis was carried out by using One way ANOVA followed by Dunnett’s’ test and p<;0.01, p<;0.001 was considered significant.

Results

Body weight

Administration of Gentamicin (80 mg/kg) caused decrement in weight of animals. However, administration of Rutin (200 mg/kg) prevented a significant reduction (P < 0.01) in weight loss as evident from Figure 1. Weight gain in group II animals were observed, which was prevented up to an extent significantly (P < 0.01) by administration of Rutin (200 mg/kg) (Figure 1).