Modulatory effect of Mangifera indica against carbon tetrachloride induced kidney damage in rats

There is little scientific evidence on the local use of Mangifera indica in kidney diseases. This study investigated the reno-modulatory roles of the aqueous stem bark extract of Mangifera indica (MIASE) against CCl4-induced renal damage. Rats were treated intragastrically with 125, 250 and 500 mg/kg/day MIASE for 7 days before and after the administration of CCl4 (3 ml/kg of 30% CCl4, i.p.). Serum levels of electrolytes (Na+, K+, Cl−, HCO3−), urea and creatinine were determined. Renal tissue reduced glutathione (GSH), malondialdehyde (MDA), catalase (CAT), superoxide (SOD) activities were also assessed. The histopathological changes in kidneys were determined using standard methods. In CCl4 treated rats the results showed significant (p<0.05) increases in serum Na+, K+, Cl−, urea and creatinine. CCl4 also caused significant (p<0.05) decreases in renal tissue SOD, CAT and GSH and significant (p<0.05) increases in MDA. The oral MIASE treatment (125-500 mg/kg) was found to significantly (p<0.05) attenuate the increase in serum electrolytes, urea and creatinine. Similarly, MIASE significantly (p<0.05) attenuated the decrease in SOD, CAT and GSH levels and correspondingly attenuated increases in MDA. Mangifera indica may present a great prospect for drug development in the management of kidney disease with lipid peroxidation as its etiology.


Introduction
Kidney diseases occur in all age groups with incidence between 1.5 per million and 3.0 per million in children (Fogo, 2007). Among the causes of kidney diseases are congenital abnormalities of the kidney and urinary tract, focal segmental glomerulosclerosis, hemolytic uremic syndrome, immune complex diseases (Foreman & Chan, 1988), exposure to drugs and chemicals. One of our previous studies (Awodele et al., 2010) and several other studies have however underscored the significant role of oxidative stress and lipid peroxidation in kidney diseases (Reeder et al., 2002;Reeder et al., 2008). In addition to its role in renal diseases, lipid peroxidation has also been The tree Mangifera indica (family: Anarcardiaceae) is among the most economically and culturally important tropical rainforest medicinal plants in Asia and Africa, especially due to its edible fruits. It is widely known as Mango. Studies have reported Mangifera indica fruit (mango) to possess anti-diabetic, antioxidant, anti-viral, cardiotonic, hypotensive, and antiinflammatory properties (Barreto et al., 2008). The stem bark of Mangifera indica has been reported to exert several pharmacological activities with antispasmodic, analgesic, antipyretic, anti-oxidant, anti-tumor, anti-viral, anti-diabetic, anti-bone resorption and immunomodulatory effects (Kumar et al., 2009). These findings are very encouraging and indicate that this herb should be studied more extensively to confirm the results and reveal other potential therapeutic effects. In African traditional medicine, in particular among Yoruba, Hausa and Igbo communities in Nigeria, various parts of Mangifera indica trees are used in the treatment of different human and veterinary diseases, including malaria (Ene et al., 2010), dysentery, cough, typhoid fever infection (Alo et al., 2012). It is also an anti-diuretic, anti-emetic and cardiac herb (Barreto et al., 2008). A preliminary ethno-botanical survey of its use conducted among traditional herbalists in Lagos metropolis (Southwest Nigeria) showed that hot and cold water infusion of Mangifera indica stem bark is highly valued in the local management of both liver and kidney diseases. Recently, the aqueous stem bark extract of Mangifera indica was reported to offer protection against CCl 4 -induced hepatotoxicity in Wistar rats (Adeneye et al., 2015). Nevertheless, there is a dearth of scientific investigation into the possible protective role of the aqueous stem bark extract of Mangifera indica against nephrotoxicity. Thus the presented explorative study was aimed at confirming or refuting the value of the folkloric use of water infusion of Mangifera indica stem bark in the local treatment of renal diseases. Thus the reno-modulatory roles of 125-500 mg/kg/day of the Mangifera indica stem bark aqueous extract were investigated in CCl 4 -induced nephrotoxicity in adult Wistar rats.

Material and methods
The plant collection and identification, preparation of the plant extract, qualitative phytochemical analyses of aqueous stem bark extract of Mangifera indica (MIASE), acute oral toxicity test of MIASE using preliminary dose test of up and down procedure were carried out as previously reported by Adeneye et al. (2015).

Experimental animals
The purchase of experimental animals, acclimatization, housing and feeding were done as documented in our previous study (Adeneye et al., 2015). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U. S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) for studies involving experimen tal animals.

Experimental design
Drug-induced renal toxicity models applied in conducting this study used 30% carbon tetrachloride dissolved in olive oil according to the modified method of Lu et al.( 2002). The study was performed in two phases (chemopreventive and curative) with each phase involving 36 male Wistar rats. The rats in each model were grouped into six groups of six rats each -three control and three treatment groups (Adeneye et al., 2015).

Induction of CCl 4 -induced nephrotoxicity and oral drug treatment in the chemopreventive model
In this model of chemically-induced nephrotoxicity, rats were randomly divided into 6 groups of 6 rats each so that the weight differences within and between groups did not exceed ±20% (Adeneye et al., 2015). The treatment protocols included: Group I (Control): 10 ml/kg of 0.9% normal saline; Group II: 10 ml/kg of 0.9% normal saline; Group III: 10 mg/kg of ascorbic acid; Group IV: 125 mg/kg of MIASE; Group V: 250 mg/kg of MIASE; Group VI: 500 mg/kg of MIASE.
The aforementioned oral treatments were applied for seven consecutive days and twenty-four hours after the last oral pretreatment with ascorbic acid and graded doses of MIASE, the rats in groups II-VI were treated with single intraperitoneal injection of 3 ml/kg of 30% CCl 4 dissolved in olive oil. Ascorbic acid, being a known potent antioxidant and nephroprotectant, was used as standard reference drug. The treated rats were then sacrificed humanely forty-eight hours post-CCl 4 treatment.

Induction of CCl 4 -induced nephrotoxicity and oral drug treatment in the curative model
In this model of chemically-induced nephrotoxicity, the rats were also randomly divided into 6 groups of 6 rats each so that the weight differences within and between groups did not exceed ±20% (Adeneye et al., 2015). The treatment protocols included: Group I (Control) 1 ml/kg of 0.9% normal saline intraperitoneally; Group II: 3 ml/kg of 30% CCl 4 i.p 1 hour before oral treatment with 1 ml/kg of 0.9% normal saline; Group III: 3 ml/kg of 30% CCl 4 i.p 1 hour before oral treatment with 10 mg/kg ascorbic acid; Group IV: 3 ml/kg of 30% CCl 4 i.p 1 hour before oral treatment with 125 mg/kg of MIASE; Group V: 3 ml/kg of 30% CCl 4 i.p 1 hour before oral treatment with 250 mg/kg of MIASE; Group VI: 3 ml/kg of 30% CCl 4 i.p 1 hour before oral treatment with 500 mg/kg of MIASE.
Each treatment lasted 7 days. Twenty-four hours after the last treatment on day 7, the rats were sacrificed humanely under diethyl ether anesthesia (Adeneye et al., 2015).

Collection of blood samples and kidneys for renal tissue oxidative stress markers
The blood samples and kidneys for renal tissue oxidative stress markers were collected using the methods as described by Adeneye et al. (2015).

Determination of serum renal function parameters Serum creatinine determination
An aliquot of 0.5 ml of serum sample was added to 3.5 ml of picric acid. The mixture was centrifuged for 5 minutes. 3 ml of the supernatant was taken and to this 0.2 ml of 4N NaOH was added. The mixture was incubated for 1 minute and the absorbance was read at 520 nm. The concentration of creatinine was determined.
Serum urea determination 0.1 ml of serum sample was added into a universal bottle containing 19.9 ml of distilled water and the suspension was well shaken. 1 ml of the suspension was transferred into a test tube and 1 ml of color reagent was added followed by 1 ml of acid reagent. The mixture was heated in boiling water for 20 minutes. It was then cooled and the absorbance was read at 520 nm against blank.

Serum electrolyte determination
Serum levels of sodium, potassium, chloride, calcium, bicarbonate and phosphate were determined using the ISE 6000 BYY SFRI spectrophotometer. When powered on, the machine carries out self-calibration for all parameters. When calibration is complete, the sample is placed into the probe and the tun button on the machine is pressed on the screen of the machine. The machine aspirates the sample and beeps with a screen display "remove sample". The machine then processes the sample and displays the result of the test. The results of the test are printed out, showing all the required electrolyte levels, namely: sodium, potassium, chloride, bicarbonate, calcium and phosphate.

Histopathology of the kidneys from treated rats
The remaining of the pair of kidneys harvested was gently but briskly rinsed in 0.9% normal saline and fixed in 10% formo-saline. The kidney histology was processed according to the methods described by Adeneye et al. (2015).

Statistical analysis
Statistical analysis was performed using Graph Pad Prism (Graph Pad Software -Version 5.0. Graph Pad Software Inc., La Jolla, California, U.S.A.). Data were expressed as mean ± S.D. for body weights and relative kidney weights and mean ± S.E.M. for biochemical and hematological assays. The data were analyzed using the one-way ANOVA for comparison between the control and treated groups and post hoc test conducted using Newman-Keuls'-test. The level of statistical significance was considered at p<0.05, p<0.001 and p<0.0001.

Plant extraction and phytochemical analysis of Mangifera indica aqueous stem bark extract
A yield of 15% was obtained. Alkaloids, tannins, cardiac glycosides, flavonoids, phlobatinnins, reducing sugars and saponins were contained in the extract as reported in our previous study (Adeneye et al., 2015). Table 1 shows that doses of up to 5 000 mg/kg of MIASE resulted in no mortality. However, behavioral toxicities such as body scratching, feed refusal, reduced locomotor activity, and watery stools were observed, as earlier reported in our study (Adeneye et al., 2015). Table 2 shows the effect of MIASE oral pretreatments on average body weight and relative organ weight of the kidney of CCl 4 -treated animals on days 1 and 7 of the experiment. Intraperitoneal treatment with CCl 4 caused significant (p<0.0001) weight loss and non-significant weight reduction in the relative organ weight of the kidneys of CCl 4 -treated rats compared to control (Table 2). However, oral pretreatments with 125-500 mg/kg/day of MIASE and subsequent intraperitoneal treatment with CCl 4 caused significant dose-dependent (p<0.05, p<0.001 and p<0.0001) further weight loss and non-significant alterations in the relative organ weights (kidneys) when compared with CCl 4 -treated (Group II) rats ( Table 2).

Effect of 125-500 mg/kg of MIASE on renal function parameters in rats with CCl 4 -chemoprevention
CCl 4 treatment caused significant (p<0.05, p<0.0001) increases in serum Na + , K + , Cl -, urea and creatinine while causing significant (p<0.05, p<0.001) decreases in serum HCO 3 levels compared to control rats (Table 3). However, oral pretreatments with 125-500 mg/kg of MIASE significantly (p<0.05, p<0.001) attenuated the increase in serum levels of Na + , K + , Cl -, urea and creatinine, while significantly (p<0.05, p<0.001) increasing the serum HCO 3 levels compared to CCl 4 -treated rats ( Table 3).  (Table 4). These results were comparable to the effect recorded for the 10 mg/kg of the standard antioxidant (ascorbic acid) used. However, 125 mg/kg/day of MIASE did cause significant alterations in the renal tissue levels of SOD, CAT, GSH and MDA when compared to the effect in the CCl 4 -treated rats of Group II (Table 4).

Histopathological results of oral pretreatment with 125-500 mg/kg MIASE on kidneys of CCl 4 -treated rats
Figures 1-6 show the histopathological findings of oral pretreatments with 125-500 mg/kg/day of MIASE on the   renal tissue of CCl 4 -treated rats. Single intraperitoneal treatment with CCl 4 caused glomerular atrophy with tubular swelling and necrosis (Figure 2) compared to normal renal architecture (Figure 1). With repeated daily oral pretreatments with 10 mg/kg of ascorbic acid and 125-500 mg/kg/day of MIASE, these histological changes were ameliorated and improved in a dose-related manner (Figure 3-6). Table 5 shows the effect of MIASE oral pretreatments on the average body weight and relative organ weight of the kidney of CCl 4 -treated, days 1 and 7 of the experiment. Intraperitoneal treatment with CCl 4 caused significant (p<0.0001) weight loss changes and non-significant increase in the kidney relative weight of CCl 4 -treated rats      compared to control rats (Table 5). However subsequent oral treatments with 125-500 mg/kg/day of MIASE after intraperitoneal treatment with CCl 4 caused further significant dose-dependent weight loss (p<0.05, p<0.001 and p<0.0001) and non-significant alterations in the kidneys compared with CCl 4 -treated rats (Group II) ( Table 5).

Effect of 125-500 mg/kg of MIASE on renal function parameters in CCl 4 -chemocurative rats
CCl 4 treatment caused significant (p<0.05, p<0.0001) increases in serum Na + , K + , Cl -, urea and creatinine, while causing significant (p<0.05, p<0.001) decreases in serum HCO 3 levels compared to control rats (Table 6). However, post-CCl 4 oral treatments with 125-500 mg/kg of MIASE significantly (p<0.001, p<0.0001) attenuated the increase in the serum levels of Na + , K + , Cl -, urea and creatinine dose-dependently, while significantly (p<0.0001) increasing the serum HCO 3levels at 500 mg/kg of MIASE compared to the CCl 4 -treated rats ( Table 6). Table 7 shows the effects of 125-500 mg/kg of MIASE on renal tissue antioxidant markers (SOD, CAT, GSH and MDA) in the post-CT treated rats. CCl 4 treatment caused significant (p<0.05 and p<0.0001) decreases of renal SOD, CAT and GSH while causing significant (p<0.001 and p<0.0001) increases in renal MDA values (Table 7). However, post-CCl 4 oral treatments with 125-500 mg/kg/day of MIASE significantly (p<0.05, p<0.001 and p<0.0001) reversed and improved the values of these markers when compared to values for the CCl 4 -treated rats, returning them to near normal values (Table 7).

Histopathological results of post-CCl 4 oral treatment with 125-500 mg/kg of MIASE on the renal tissue of CCl 4 -treated rats
Intraperitoneal CCl 4 treatment was associated with severe tubular swellings, tubular lumen obliterations and tubular necrosis ( Figure 8) when compared to normal renal architecture ( Figure 7). With repeated post-CCl 4 oral treatment with 10 mg/kg/day of vitamin C and 125-500 mg/kg/day of MIASE, there was a dose related amelioration in the CCl 4 -induced renal lesions (Figures 9-12).

Discussion
Exposure to carbon tetrachloride is on the increase due to environmental pollution. The exposure can come from the air, drinking water, foodstuffs and soil (ATSDR, 2005;IPCS, 1999). It could also be from certain industrial sites where carbon tetrachloride is still used or where previously industrial contamination had occurred (ATSDR, 2005). The liver and kidney are the major target organs for toxicity following acute inhalation or ingestion exposure to carbon tetrachloride (IPCS, 1998;1999). Liver damage can occur after 24 hours and in serious cases this can result in painful swollen liver, ascites, hemorrhages, hepatic coma and death (ATSDR, 2005;IPCS, 1999). Kidney damage with impairment in function normally occurs 2-3 weeks after exposure (IPCS, 1999), but in severe cases this can develop within 1-6 days in association with liver failure (ATSDR, 2005). Due to the fatality of kidney damage in affecting optimal human functions, attention should concentrate on strategies preventing the occurrence of kidney disease more than on palliative management. In the recent past, several research studies on preventive strategies of renal damage have been conducted. Mesery et al. (2009) demonstrated the chemopreventive and renal protective effects of ocosahexaenoic acid (DHA); Pracheta et al, (2012) showed the chemopreventive effect of hydroethanolic extract of Euphorbia neriifolia leaves against DENA-induced renal carcinogenesis in mice and Sharma & Janmeda (2012) documented the chemopreventive role of Euphorbia neriifolia (Linn) and its isolated flavonoid against N-nitrosodiethylamine-induced renal histopathological damage in male mice.
A preliminary ethno-botanical use survey conducted among traditional herbalists in Lagos metropolis (Southwest Nigeria) showed that hot and cold water infusion of Mangifera indica stem bark is highly valued in the local management of both liver and kidney diseases. However, this assertion has not been scientifically