Jayesh B. Dhodi, Deepavali R. Thanekar, Snehal N. Mestry, Archana R. JuvekarInstitute of Chemical Technology (University Under Section-3 of UGC Act 1956), NP Marg, Matunga, Mumbai-400019, India

Carissa carandas Linn. fruit extract ameliorates gentamicin-induced nephrotoxicity in rats via attenuation of oxidative stress

Jayesh B. Dhodi, Deepavali R. Thanekar, Snehal N. Mestry, Archana R. Juvekar*
Institute of Chemical Technology (University Under Section-3 of UGC Act 1956), NP Marg, Matunga, Mumbai-400019, India

ARTICLE INFO ABSTRACT

Article history:

Received 6 February 2015

Received in revised form 8 February 2015 Accepted 9 February 2015

Available online 10 February 2015

Keywords:

Gentamicin

Carissa carandas

Nephrotoxicity

Oxidative stress

Kidney hypertrophy

Objective: To elucidate the mechanism of action of methanolic extract of Carissa carandas fruits (MCCF) in attenuation of diabetic nephropathy using gentamicin induced nephrotoxicity model. Methods: Extract was daily administered to Sprague Dawley rats at doses of 100, 200 and 400 mg/kg for 8 days along with intramuscular injection of gentamicin (80 mg/kg). After completion of the study, serum was analyzed for blood urea nitrogen, albumin and creatinine; urine (24 h) was analyzed for albumin and creatinine. Kidney was evaluated for its biochemical and morphological changes. Results: Extract at doses of 200 and 400 mg/kg significantly normalized the nephrotoxic biomarkers in serum and urine and increased the kidney antioxidant activities which were altered due to gentamicin toxicity. The histological findings reveal that MCCF was capable of protecting the kidney against gentamicin toxicity. Conclusions: Extract ameliorated oxidative stress generated by gentamicin administration, which is one of the mechanisms for its preventive action against diabetic nephropathy.

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1. Introduction

Gentamicin is a well known potent broad spectrum aminoglycoside antibiotic used against gram negative infection[1]. Nephrotoxicity is one of the serious side effects of gentamicin affecting 15-30% of patients after several days of treatment[2]. Evidences indicate that the renal toxicity of gentamicin is due to its selective accumulation in the renal proximal convoluted tubules and its long term stay subsequently leading to loss of brush border integrity[3,4]. Suppression of Na(+)K(+) ATPase and DNA synthesis in the proximal tubules and generation of reactive oxygen species associated with increase in lipid peroxidation induces nephrotoxicity[5-7]. Oxidative stress is mainly implicated in gentamicin induced acute renal injury in rodents, thus it can prove to be one of the best experimental models to assess the efficacy of natural antioxidants against nephrotoxicity[8].

Carissa carandas (C. carandas), commonly known as karanda, belongs to the family Apocynaceae[9,10]. It contains various chemical constituents such as carissol, carissic acid, ascorbic acid, lupeol, β‐sitosterolserine, glutamine, alinine, valine, phenylalanine and glycine[11]. A novel triterpenic alcohol, carissol has been reported to be present in the fruit of this plant[12]. In addition to this, antioxidant properties of the plant are also reported[13]. A previous study carried out in our research lab on methanolic extract of fruits of C. carandas (MCCF) demonstrated its ameliorative effects in diabetes induced nephropathy in rats by increasing the antioxidant parameters. In-order to understand the mechanism of action of MCCF, we further evaluated its effect on oxidative stress generated by gentamicin induced nephrotoxicity.

2. Materials and methods

2.1. Plant material and preparation of extract

C. carandas fruits were collected in the month of April,from Vangao, Maharashtra, India and were authenticated by Dr. Ganesh Iyer (Ruia College, Botany department, Mumbai). Voucher specimens (No. 2011/03) of the sample were deposited at the Department of Pharmacology, Institute of Chemical Technology, Mumbai.

Fruits collected were washed with distilled water and air dried under shade. The resulting dried fruits were ground into powder. The powder was initially subjected to defatting using petroleum ether and further extracted with methanol using Sohxlet apparatus (percent yield obtained 28.8%). The extract obtained was dried under vaccum with the help of rotary evaporator till a constant weight was obtained, and later stored in an airtight container for subsequent use.

2.2. Experimental animals

2.2.1. Animals

After getting approval from Institutional Animal Ethics Committee (Protocol No. ICT/IAEC/2014/P-10 dated 05/07/2014), male Sprague Dawley rats were procured from Bharat Serum & Vaccines, Thane. Animals were placed in polypropylene cages in a controlled room temperature (22± 1) ℃ and relative humidity of 60-70% in registered animal house (87/1999/CPCSEA). Standard pellet diet (Amrut Brand, Sangali, India) and water ad libitum were provided to them. 2.2.2. Experimental protocol

Animals were randomLy divided into five groups (N=6 for each groups): Group I as control rats received water as a vehicle p.o.; Group II as negative control received gentamicin 80 mg/kg/day i.m.+vehicle; group III-V as treatment groups received MCCF (100, 200 and 400 mg/kg/day per oral respectively )+gentamicin 80 mg/kg/day i.m. simultaneously. The study was conducted for 8 days and thereafter the animals were kept in metabolic cages for 24 h for the collection of urine for biochemical analysis. Blood was collected from retro orbital plexus, serum was separated and analysed for blood urea nitrogen (BUN), albumin and creatinine levels. Rats were anesthetized, perfused with phosphate buffer pH 7.4 and both kidneys were isolated. Each kidney was cut into two halves and one half was fixed in 10% formalin solution for histological evaluation and other half was homogenized in phosphate buffer pH 7.4 to obtain 10% w/v homogenate for assessment of antioxidant parameters[8].

2.2.3. Renal function parameters

2.2.3.1. Serum and urine parameters

Serum was evaluated for creatinine, albumin and BUN levels. Tweenty four-hour urine was evaluated for creatinine and albumin value. The above mentioned parameters were evaluated using biochemical kits (ACCUREX, Biomedical Pvt. Ltd).

2.2.3.2. Kidney antioxidant parameters

Kidney homogenate was evaluated for lipid peroxidation levels, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) activities and kidney protein levels.

2.2.3.3. Estimation of lipid peroxidation

Formation of thiobarbituric acid reactive substances (TBARS) as a product of lipid peroxidation was estimated in the kidney. A volume of 0.2 mL of 10% tissue homogenate (0.1 mol/L phosphate buffer, pH 7.4) was added to 0.2 mL of sodium dodecyl sulphate (SDS), 1.5 mL acetic acid (20%, pH 3.5) and 1.5 mL of 0.8% TBA. The above reaction mixture was made up to 4 mL with distilled water and placed in boiling water bath for 60 min. Thereafter it was cooled under tap water. A mixture of Butanol: pyridine (15:1 v/v, 4 mL) was added to above mixture, mixed vigorously and centrifuged at room temperature for 10 min. 200μL of clear supernatant was collected and spectrophotometrically analyzed at 532 nm (epoch, synergy) against blank. The TBARS content was calculated and expressed as nmol malondialdehyde (MDA) formed per microgram protein[14,15].

2.2.3.4. Reduced glutathione

GSH levels in tissue homogenates were estimated using 5-5-dithiobis (2-nitrobenzoic acid) (DTNB) reagent. Tissue homogenate (10μL) was incubated with 200μL of DTNB and was read at absorbance of 412 nm. The amount of GSH in the sample was calculated inμg/mL from a standard curve obtained and represented in GSH per mg total tissue protein.

2.2.3.5. Superoxide dismutase

The assay mixture consisted of 180μL of phosphate buffer (pH 7.4), 10μL of pyrogallol (2.6 mmol/L in 10 mmol/L HCl) and 10μL of tissue homogenate in a total volume of 200μL. SOD activity was measured at 325 nm for 5 min (epoch, synergy) and expressed as units/mg protein. One unit of enzyme represents the enzyme activity that inhibits autooxidation of pyrogallol by 50%[15].

2.2.3.6. Catalase

The assay mixture consisted of 2.9 mL hydrogen peroxide (0.019 mol/L) and 0.1 mL tissue homogenate in a total volume of 3 mL. Changes in absorbance were recorded at 240 nm. Catalase activity was expressed as nmol H2O2consumed min-1mg-1protein[15].

2.2.4. Histological evaluation

Kidney was fixed with 10% neutral formalin phosphate buffer, dehydrated through graded alcohol series, embedded in paraffin, cut into 5μm sections and stained with hematoxylin and eosin and periodic acid schiff base (PAS) method. The slides were examined by light microscopy under 400× magnification for microscopic alteration of pathological significance.

3. Results

3.1. Body weight

No significant change in the body weight was observed in negative control group as well as the treatment groups as compared to the normal control (Table 1).

3.2. Kidney weight and kidney hypertrophy index

Kidney weight and kidney hypertrophy index of the negative control group was significantly increased as compared to normal control rats. MCCF at 100 mg/kg did not show any reduction in kidney weight and kidney hypertrophy. MCCF at 200 mg/kg and 400 mg/kg significantly decreased kidney weight and kidney hypertrophy in dose dependent manner as compared to negative control (Table 1).

Table 1Effect of MCCF on body weights, kidney weights and kidney hypertrophy in gentamicin nephrotoxicity (mean±SEM).

*: Treatment group was compared with negative control, P＜0.05;**: Treatment group was compared with negative control, P＜0.01;***: Treatment group was compared with negative control, P＜0.001;###: Negative group was compared with normal control, P＜0.001, using One-way ANOVA with Dunnett's test.

3.3. Blood and urine parameters

The negative control group demonstrated significant increase in urine albumin, BUN and serum creatinine levels and significant decrease in serum albumin and urine creatinine levels as compared to normal rats. Treatment groups normalized the altered urine and serum parameters due to gentamicin in a dose dependent manner. MCCF at 100 mg/kg showed significantly increased serum albumin and decreased serum creatinine levels. MCCF at 200 mg/kg significantly increased BUN and serum creatinine levels and significantly decreased serum albumin and urine creatinine levels. MCCF at 400 mg/kg significantly increased urine albumin, BUN and serum creatinine levels and significantly decreased serum albumin and urine creatinine levels (Table 2).

Table 2Effect of MCCF on serum and urine parameters of gentamicin nephrotoxicity

*: Treatment group was compared with negative control, P＜0.05;**: Treatment group was compared with negative control, P＜0.01;***: Treatment group was compared with negative control, P＜0.001;###: Negative group was compared with normal control, P＜0.001, using one-way ANOVA with Dunnett's test.

3.4. Kidney antioxidant levels and lipid peroxidation.

GSH levels, SOD and catalase activities were significantly decreased in the gentamicin induced nephrotoxic rats as compared to the normal rats. Similarly, increased lipid peroxidation levels were found in the gentamicin induced nephrotoxic rats in comparison with the normal rats. MCCF dose dependently increased GSH, Catalase and SOD activities in the treated rats compared to negative control but significant reduction was found at 400 mg/kg. MCCF significantly decreased lipid peroxidation level in kidney homogenates at all dose levels (Figure 1).

3.5. Histopathological evaluation

Normal rats did not show any abnormal morphological changes in H&E (Figure 2) and PAS (Figure 3) stained kidney specimens. According to H&E stained kidney specimens negative control group demonstrated severe degeneration of proximal convoluted tubules, necrosis, hyalinisation of arterioles and scarring of tubules and moderate infiltration of MNC. MCCF at 100 mg/kg was not able to significantly protect the kidney from gentamicin induced renal injury but at 200 and 400 mg/kg, MCCF was able to reduce the hyalinization of arterioles and infiltration of MNC.

Kidney stained with PAS illustrated moderate intensity PAS positivity in glomeruli, moderate degree basement membrane thickening and moderate increase in glomerular size in negative control group. The treatment groups failed to reduce the PAS positivity staining intensity in glomeruli but were capable of reducing the basement membrane thickening at all dose levels. Glomerular size was found to be normal in 200 and 400 mg/kg MCCF treatment groups.

Figure 2. Photomicrographs of kidney (Hematoxylin and Eosin staining under light microscope at 400× magnification).A: Control rats showing glomeruli and normal architecture; B: Negative control showing Degeneration of PCT, hyalinization of arterioles and Infiltration of MNC; C: MCCF treated group at dose 100 mg/kg showing scarring of tubules, degeneration of PCT and necrosis; D: MCCF treated group at dose 200 mg/kg showing scarring of tubules, degeneration of PCT and hyalinization of arterioles; E: MCCF treated group at dose 400 mg/kg showing Infiltration of MNC, degeneration of PCT and hyalinization of arterioles. D-Degeneration of PCT; MI-Infiltration of MNC; SC-Scarring of tubule; G-Glomeruli; H-hyalinization of arterioles; N-Necrosis.

Figure 3. Photomicrographs of kidney (Periodic Acid Schiff staining under light microscope at 400× magnification).A: Control rats; B: Negative control; C: MCCF treated group at dose 100 mg/kg; D: MCCF treated group at dose 200 mg/kg; E: MCCF treated group at dose 400 mg/kg.

4. Discussion

Oxidative stress is one of the factors responsible for acute renal injury[16]. Generation of reactive oxygen species (ROS) is implicated in the pathophysiology of gentamicin induced kidney damage. ROS causes cellular injury and necrosis via several mechanisms such as peroxidation of membrane lipids, protein denaturation and DNA damage[17]. As mentioned earlier, since Carissa carandas fruit extract was capable of ameliorating diabetes induced renal injury, we carried out this present study to elucidate whether MCCF was able to protect the kidney by attenuating oxidative stress.

Earlier studies have reported that rats treated with gentamicin have no effect on their body weight as compared with the normal control group. Our results were found to be in consistence with these reports[18].

Any abnormal stimulus to the kidney triggers an inflammatory response resulting in its increased weight which is estimated as kidney hypertrophy index. This value was found to be increased significantly in the negative control group. Treatment with MCCF at 400 mg/kg was capable of reducing the hypertrophy index thus indicating that the treatment was capable of controlling the inflammation.

Previous studies have reported that consequences of gentamicin induced renal injury are elevated levels of serum creatinine accompanied by its decreased excretion in urine and increased blood urea nitrogen. These are also considered as biomarkers of kidney damage. The negative control group illustrated the presence of these biomarkers thus confirming that the model was well established. Presence of albumin in the urine is also another important biomarker portraying the level of kidney damage[3]. MCCF treated groups was capable of normalizing the levels of the above mentioned biomarkers significantly in a dose dependent manner thus suggesting the protective nature of the extract. It is also evidenced by histological analysis of the kidney wherein it was observed that the extract at 400 mg/kg reduced the damage as compared to the negative control.

Gentamicin reduced the activities of important endogenous antioxidants such as superoxide dismutase, catalase and reduced glutathione. MCCF prevented the depletion of these antioxidant activities due to gentamicin. This apparent protective effect might be due to the ability of MCCF to neutralize the increase in free radicals generated by gentamicin[19].

ROS stimulates oxidation of macromolecules like lipids, DNA, proteins and carbohydrates. It also induces peroxidation of membrane lipids which may alter its membrane structure and function[20]. Lipid peroxidation is associated with decreased membrane fluidity and function, impaired mitochondrial and Golgi apparatus functions and inhibition of intracellular enzymes[21]. Lipid peroxidation measured as the level of MDA formed thus gives an indication of the level of oxidative stress. Gentamicin induced acute renal damage increased the levelof lipid peroxidation in kidney homogenate as seen in the negative control group. This increase in lipid peroxidation was significantly reduced by MCCF significantly in a dose dependent manner thus confirming that the fruit extract is capable of attenuating oxidative stress[8].

Taking into account the results obtained in the present study, MCCF portrays nephroprotective activity by ameliorating oxidative stress generated by gentamicin administration, thus establishing its mechanism of action.

Conflict of interest statement

The authors report no conflict of interest.

Acknowledgements

The authors are grateful to University Grant Commission, New Delhi, for providing the financial grant for this project, Dr. Ganesh Iyer for carrying out authentication of the plant and Dr. Sanjay Pawar for assisting in the histopathological analysis.

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*Corresponding author:Prof. Archana R. Juvekar, Professor, Pharmacology and Physiology, Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, (University Under Section-3 of UGC Act 1956), NP Marg, Matunga, Mumbai-400019, Maharashtra, India.