Prophylactic effect of coconut water (Cocos nucifera L.) on ethylene glycol induced nephrocalcinosis in male wistar rat

ARTICLE INFO _________________________________________________________ ___________________ Vol. 39 (1): 108-117, January February, 2013 doi: 10.1590/S1677-5538.IBJU.2013.01.14 IBJU | PROPHYLACTIC EFFECT OF COCONUT WATER ON ETHYLENE GLYCOL INDUCED NEPHROCALCINOSIS 109 coconut producing countries. Coconut water, the liquid endosperm of coconut, contains sugars, vitamins, minerals, proteins, free amino acids and growth promoting factors. It is a natural isotonic beverage and is acclaimed in the tropics for its numerous medicinal properties. Coconut water is an essential dietary ingredient of South India where the incidence of urolithiasis is very low (11). However, so far no systematic study has been reported regarding the antiurolithiatic property of coconut water. So the present study was designed to evaluate the efficacy of coconut water on ethylene glycol induced nephrolithiasis. MATERIALS AND METHODS Collection of coconut water Fresh coconuts were purchased from the market, broken carefully, liquid endosperm were collected and used for each day experiment. While purchasing coconuts from the market, the nuts stored in cold conditions and from the same stock were procured. Animals Male Wistar rats weighing 150-170 g were housed in polypropylene cages in a room maintained at 25 ± 1o C with alternate exposure to light and dark for 12 hours. The animals were acclimatized for one month in polypropylene cages under hygienic conditions. All procedures were done in accordance with ethical guidelines for care & use of laboratory animals and were approved by the local experimental animal ethical committee. The animals were provided standard animal feed and water ad libitum. The standard rat diet was acquired from Aashirwad Company (Ludhiana, Punjab, India), and the composition of the diet is given in Table-1. To induce urolithiasis in animals, they were exposed to 0.75% ethylene glycol in their drinking water for 7 weeks. The protocol to induce urolithiasis was adapted from Huang et al. (12), where the authors showed evidence that the administration of “0.75 % ethylene glycol” in drinking water can induce “urolithiasis” in male wistar rats. Rats were divided into three groups of six rats each and fed the following diet: Group 1: Normal rat diet (control). Group 2: Normal rat diet + 0.75% ethylene glycol (EG) mixed with tap water for 7 weeks ad libitum (12). Group 3: Normal rat diet + 0.75% EG + 10% Coconut water for 7 weeks ad libitum. Methods A 10% of kidney homogenate was prepared in 0.1 mM tris buffer (pH 7.4) and was used for assaying lipid peroxidation (13), antioxidant enzymes superoxide dismutase (14) and catalase (15). All the animals were kept in individual metabolic cages and urine samples were collected throughout 24hours, one day before sacrificing the animals. Blood was collected from orbital sinus under mild anesthetic conditions, using diethyl ether as an anesthetic agent and animals were sacrificed by cervical decapitation. Serum was separated by centrifugation at 3,000 x g for 15 minutes, analyzed for creatinine (code no. FREBCERM0082) and urea (code no. FRCER0034) by Erba Manheim diagnostic kits. For the expression studies, total RNA was isolated from kidney of rats using Trizole reagent (Gibco BRL, UK). Single stranded cDNA was synthesized using oligo dT 12-18 as primers and AMV reverse transcriptase. The amplification of different antioxidant enzymes was carried out by using specific primers viz, SOD (forward primer 5’GCA GAA GGC AAG CGG TGA AC-3’, reverse primer 5’TAG CAG GAC AGC AGA TGA GT-3’, 446 bp), catalase (forward primer 5’GCG AAT GGA GAG GCA GTG TAC-3’, reverse primer 5’GAG TGA CGT TGT CTT CAT TAG CAC TG-3’, 652 bp). GAPDH was chosen as house keeping gene (forward primer 5’-TCT AAG AAA CAT GGC GGT CC-3’ reverse primer 5’-CAG TTA GCA GGC CAG CAG AT3’, 197bp) (16-20). PCR products were resolved on 1% agarose gels. The bands were identified based on the product size using 100 bp ladder. The PCR products were quantitated by densitometric scanning using scion image software. The values were expressed as percentages with respect to control. The RT-PCR conditions were as follows: (1) reverse transcription, (30 minutes, 55o C for CAT, SOD and 50 minutes 45o C for GAPDH) (2) initial activation step, 2 minutes at 94o C (3) three step cycling (34 cycles for GADPH , 26 cycles for SOD IBJU | PROPHYLACTIC EFFECT OF COCONUT WATER ON ETHYLENE GLYCOL INDUCED NEPHROCALCINOSIS 110 Table 1 Composition of standard animal diet. Elements Concentration (ppm)