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Evaluation of Nephroprotective Effects of Hydroalcoholic Extract of Cyperus scariosus Linn. in Gentamicin-induced Acute Kidney Injury in Wistar Albino Rats


1 Department of Pharmacology, Sir Takhtsinhji General Hospital, Government Medical College, Bhavnagar, India
2 Department of Pathology, Sir Takhtsinhji General Hospital, Government Medical College, Bhavnagar, India
*Corresponding author: C. B. Tripathi, Department of Pharmacology, Sir Takhtsinhji General Hospital, Government Medical College, Bhavnagar, India. Tel: +91-9825951678, Fax: +91-2782422011, E-mail: [email protected].
Jundishapur Journal of Natural Pharmaceutical Products. 11(3): e34452 , DOI: 10.17795/jjnpp-34452
Article Type: Research Article; Received: Nov 7, 2015; Revised: Mar 19, 2016; Accepted: May 14, 2016; epub: Aug 27, 2016; collection: Aug 2016

Abstract


Background: Gentamicin is a commonly used antibiotic for the treatment of Gram-negative infections, but nephrotoxicity limits its use. Cyperus scariosus Linn. (CS) has been found to have antioxidant properties in vitro.

Objectives: The aim of this study was to evaluate the nephroprotective effects of CS in gentamicin-induced acute kidney injury (AKI).

Methods: The animals were divided into nine groups. AKI was produced with a 100 mg/kg intraperitoneal (i.p.) injection of gentamicin for 7 days. Next, α-lipoic acid 100 mg/kg i.p. served as the active control, while the test drug (CS) was given in two doses (150 mg/kg and 250 mg/kg orally) for 10 days. Distilled water, 1 ml/day orally for 7 days, served as the vehicle control. The protective and curative effects, respectively, were assessed by the administration of CS before and after the induction of AKI. The effects of CS on AKI were assessed by serological and histopathological parameters.

Results: Serum creatinine was significantly increased (P < 0.05) while 24 hours urine output, urine creatinine, and serum protein were significantly decreased (P < 0.05) in rats treated with gentamicin as compared to the control group. As the active control, α-lipoic acid showed nephroprotective effects on the urinary, serological, and histopathological parameters. C. scariosus Linn., as a preventive and curative therapy, restored urine output and altered the serological parameters non-significantly compared to the disease-control group. It also preserved the normal kidney architecture, as evidenced by histopathological parameters.

Conclusions: In our study, hydroalcoholic extract of Cyperus scariosus Linn. offered nephroprotection in the form of AKI prevention and for the treatment of established AKI.

Keywords: Gentamicin; Acute Kidney Injury; Oxidative Stress; Nephroprotective; Cyperus scariosus Linn.

1. Background


Acute kidney injury (AKI) is a clinical syndrome characterized by a rapidly declining glomerular filtration rate (GFR), imbalances in electrolytes and in the acid-base level, derangement of extracellular fluid volume, and retention of nitrogenous waste products, often associated with reduced urine output (1). AKI develops rapidly, within a few hours to weeks, and is usually reversible. It can occur either in previously normal kidneys (classic AKI) or in patients with pre-existing chronic renal disease, such as glomerulonephropathies and other syndromes affecting the kidneys (‘acute-on-chronic’ renal failure).


AKI is a significant problem, affecting approximately 5% of all hospitalized patients (2) and 5% - 25% of all patients admitted to intensive care units (3, 4). A significant number of patients with AKI ultimately progress to end-stage renal disease (ESRD), resulting in dialysis and renal transplantation (5). Aminoglycoside antibiotics, including gentamicin, are commonly used in the treatment of various Gram-negative infections. A major complication with the use of this group of drugs is nephrotoxicity, accounting for 10% - 15% of all cases of acute renal failure (6). It has been proposed that aminoglycoside accumulation in renal proximal tubular epithelial cells causes membrane structural disturbances and cell death by involving reactive oxygen species (ROS) (7). ROS produce cellular injury and necrosis via several mechanisms, including peroxidation of membrane lipids, protein denaturation, and DNA damage (8). Renal accumulation of gentamicin and lysosomal phospholipidosis disrupts normal renal function and is implicated in the induction of nephrotoxicity (9, 10).


Dietary antioxidants are able to counteract reductions in the GFR and thus the severity of the tubular damage induced by gentamicin (11, 12). Cyperus scariosus R. Br. (Cyperaceae family; known locally as nagarmotha) is a delicate grass widely distributed in India, especially in Chhattisgarh, Bihar, Orissa, West Bengal, and Uttar Pradesh. Nagarmotha is also found in the eastern and southern areas of the Indo-Pak subcontinent, and in Bangladesh, South Africa, China, and the Pacific Islands; it is reported to be useful as a cordial, tonic, emmenagogue, vermifuge, diuretic, diaphoretic, and desiccant (13-15). Nagarmotha is prescribed by local herbal medicine practitioners to treat a variety of diseases, including diarrhea, epilepsy, gonorrhea, syphilis and liver damage (13-15). In one study, CS was found to contain phenols, flavonoids, and DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenger activity, all of which contribute to its antioxidant properties (16).

2. Objectives


The aim of this study was to evaluate the nephroprotective effects of a hydroalcoholic extract of CS on gentamicin-induced AKI in Wistar albino rats.

3. Methods


All of the experiments were performed after approval from the Institutional Animal Ethics Committee (IAEC), Government Medical College, Bhavnagar, Gujarat, India (Approval No: IAEC 36/2014) and in accordance with the guidelines issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. Wistar albino rats (both sexes, weight 250 ± 50 g) were procured from the central animal house of the institute. They were housed in standard transparent polycarbonate cages and kept under a 12-hour light/dark cycle with controlled room temperature (25 ± 2°C). The animals were allowed to acclimatize to the laboratory conditions for at least one week before starting the experiments. They were provided a standard laboratory diet and were handled under the guidelines of Good Laboratory Practice (GLP).


3.1. Drugs and Chemicals

Dried hydroalcoholic extract of CS root was procured from Kuber Impex, Indore, India. To obtain fresh solution for dosing, the dried extract powder was dissolved in distilled water and 50 mg/mL of solution was prepared. Gentamicin solution vials were procured from Sir Takhatsinhji General Hospital, Bhavnagar (Nirma Limited, Sachana, Gujarat, India; batch no. 5A40015) and α-lipoic acid was procured from Sigma Chemical Company (St. Louis, MO, USA). It was dissolved in ground nut oil and 50 mg/ml of solution was prepared before the dosing.


3.2. Experimental Design and Procedure

Seventy-two rats were randomly assigned (using a trial version of Rando software version 1.2) to nine equal groups as follows:


Group I (Normal control): received distilled water in doses of 1 mL/day orally for 7 days.


Group II (Disease control): received gentamicin intraperitoneally (i.p.) in doses of 100 mg/kg for 7 days.


Group III (Test control): received hydroalcoholic extract of CS in doses of 250 mg/kg orally for 10 days.


Group IV (Active control - Preventive): received 100 mg/kg of α-lipoic acid i.p. for 10 days and gentamicin 100 mg/kg i.p. started on the 4th day (the first dose of α-lipoic acid was considered day 1) and given up to 10th day.


Group V (Active control - Curative): received 100 mg/kg of gentamicin i.p. for 7 days, followed by 100 mg/kg of α-lipoic acid i.p. for 10 days.


Groups VI and VII (Preventive): received CS orally in doses of 150 and 250 mg/kg, respectively, for 10 days; gentamicin 100 mg/kg i.p. was started on the 4th day and given up to 10th day.


Groups VIII and IX (Curative): received CS orally 150 and 250 mg/kg, respectively, for 10 days after i.p. injections of gentamicin 100 mg/kg for 7 days.


3.3. Outcome Measures
3.3.1. Urinary Parameters

On day 0, rats were kept in metabolic cages (B.I.K. Industries, Mumbai, India) with water and rations available ad libitum, and a 24-h urine collection was done to obtain baseline urinary parameters. At the end of the study period (7th day in groups I and II; 10th day in groups III, IV, VI, and VIII; and 17th day in groups V, VIII, and IX), a 24-hour urine collection was again performed to measure urine output and urinary creatinine.


3.3.2. Biochemical Parameters

The animals were anesthetized with ketamine (75 mg/kg) and xylazine (10 mg/kg) and blood was collected through the retro-orbital plexus from the inner canthus of the eye using a capillary tube, into plain and EDTA vacuum containers. The serum was separated by centrifugation and used for the estimation of blood urea, serum creatinine, electrolytes (sodium and potassium), liver function tests (serum glutamic oxaloacetic transaminase [SGOT] with the UV kinetic method, serum glutamic pyruvic transaminase [SGPT], and alkaline phosphatase [ALP] with the PMP-AMP kinetic method), serum proteins (Biuret method), serum bilirubin (modified Jendrassik and Groff method), and hemoglobin.


The animals were sacrificed according to the norms for the didactic scientific practice of animal vivisection. The kidneys were removed, and one kidney from each animal was kept in 10% (v/v) formaldehyde for further histopathological analysis while the other kidney was used for the antioxidant estimation.


3.3.3. Antioxidant Estimation in Kidney Tissue Homogenate Preparation
3.3.3.1. Kidney Homogenate Preparation

The kidneys were weighed and cross-chopped with a surgical scalpel into fine slices, then chilled in cold 0.25 M sucrose solution and quickly blotted with filter paper. The tissue was minced, then homogenized in ice-cold 10 mM Tris HCL (10% [w/v], 0.1 M, pH 7.4) with 25 strokes of a tight Teflon pestle of glass homogenizer at a speed of 2,500 rpm. The prolonged homogenization was designed to disrupt the cells in order to release the soluble proteins, leaving the nonsoluble particles as sediment.


The tissue homogenate was then centrifuged at 5,000 rpm at 4°C using a compact, high-speed, refrigerated centrifuge (Kubota 6500, Japan). The obtained supernatant was analyzed for levels of superoxide dismutase (SOD) by the method of Misra and Fridovich (17), glutathione (GSH) by the method of Morton et al. (18), and the marker of lipid peroxidation of malondialdehyde (MDA) by the method of Slater and Sawyer as described below(19).


3.3.3.2. Superoxide Dismutase (SOD)

Before starting the procedure, all reagents were kept refrigerated and added in the cold condition. For the procedure, 0.5 mL of tissue homogenate was diluted with 0.5 mL of distilled water. 0.25 ml ethanol and 0.15 mL chloroform were added. Blank was prepared similarly without tissue homogenate. The mixture was shaken for 1 minute and centrifuged at 2000 rpm for 20 minutes. The supernatant was separated. 0.5 mL of this supernatant was taken in another test tube and 1.5 ml of carbonate buffer and 0.5 mL of EDTA were added. Blank was similarly prepared. The reaction was initiated by the add