Zizyphus jujuba protects against ibuprofen-induced nephrotoxicity in rats

Abstract

Context. Zizyphus jujuba Mill. (Rhamnaceae) has long been used for the treatment of anxiety and insomnia in Chinese traditional medicine. The edible part is the fruit. Different parts of Z. jujuba possess medicinal properties such as anti-inflammatory, anticancer and antifertility.

Objectives: This study evaluated the therapeutic effect of Z. jujuba fruit aqueous extract (ZE) on nephrotoxicity induced by ibuprofen (IBP) in rats.

Materials and methods: Male Sprague-Dawley rats were grouped as normal saline (control), ZE (500 mg/kg), IBP (400 mg/kg) and ZE + IBP-treated groups. After five days of oral administration, rats were sacrificed. The protective effect of ZE was evaluated by measuring kidney biomarkers, and histopathological changes of kidney were observed. Kidney antioxidant enzymes such as superoxide dismutase, catalase (CAT), glutathione S-transferase (GST) and lipid peroxidase were investigated.

Results: Administration of IBP resulted in a significant increase in urea and creatinine (p < 0.05) and a significant decrease in albumin and total protein (p < 0.05). Damage in glomeruli and proximal convoluted tubules was observed. IBP also increased CAT (p < 0.05) and GST (p < 0.001) activities compared to the control group. Administration of ZE with IBP significantly decreased serum urea and creatinine (p < 0.05) and reduced the severity of kidney damage. There was also a significant increase in the activities of CAT (p < 0.05) and GST (p < 0.001).

Discussion and conclusion: These results indicated that Z. jujuba aqueous extract could have a therapeutic role in reducing nephrotoxicity induced by ibuprofen.

• Antioxidant capacity
• jujube
• kidney biomarkers
• renal impairment

Introduction

Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) used commonly to relieve pain, tenderness, swelling and stiffness caused by osteoarthritis and rheumatoid arthritis (Kantor, 1979). Several studies showed a correlation between ibuprofen and renal impairment. Regular use of NSAIDs may increase the risk of chronic kidney disease in some high-risk groups (Sandler et al., 1991). Short course of ibuprofen might result in acute renal failure in patients with asymptomatic, mild chronic renal failure (Whelton et al., 1990). Children with underlying illnesses or those who receive ibuprofen for a prolonged period while dehydrated are at high risk of renal dysfunction (Buck, 2000).

Ziziphus jujuba Mill. (Rhamnaceae), also known as the Chinese date, is a thorny plant that is widely distributed in Europe and Southeastern Asia. The edible part of this plant is the fruit. Different parts of Z. jujuba possess medicinal properties. The seed essential oil has anti-bacterial (Al-Reza et al., 2010), anxiolytic (Hsieh et al., 2000), antioxidant and antilisterial (Al-Reza et al., 2009) properties and a hair growth promoting effect (Yoon et al., 2010). Z. jujuba leaf extract showed significant weight reducing, hypophagic and hypolipidemic properties in sucrose-induced obese rats (Ganachari & Shiv, 2004a). It also has anti-ulcer (Ganachari & Shiv, 2004b) and hypoglycemic (Erenmemisoglu et al., 1995) activities. Z. jujuba fruit extract has antioxidant (Chang et al., 2010; Taati et al, 2011), anticancer (Huang et al., 2007) and anti-complementary (Lee et al., 2004) activities. The methanol fruit extract has a hepatoprotective effect in rats treated with paracetamol and thioacetamide (Kumar et al., 2009). The objective of this study was to evaluate the protective effect of the aqueous extract of Z. jujuba fruit on nephrotoxicity induced by ibuprofen.

Materials and methods

Chemicals

Thiobarbituric acid was from Acros Organics (Fair Lawn, NJ). All kits for biochemical assays were from Teco Diagnostics (Anaheim, CA). Superoxide dismutase Assay Kit-WST was from Sigma-Aldrich (Buchs, Switzerland).

Preparation of Z. jujuba aqueous extract

Fresh ripened fruits of Z. jujuba were collected from the city of Irbid, Jordan, in October 2011. The plant was identified by Prof. Jamil Lahham (plant taxonomist), Department of Biological Sciences, Yarmouk University, Jordan. The fruits were dried in shade. Three-hundred grams of the dried pulp were extracted with 800 ml of water by grinding using a blender. The resulting suspension was centrifuged at 4 °C for 20 min at 4000 g. The supernatant was lyophilized and stored at −20 °C.

Experimental protocol

Four groups, each with six male Sprague-Dawley rats weighing 170–195 g, were used. Ibuprofen (IBP) doses used in two previous studies were 100 and 200 mg/kg for 14 d (Kumar et al., 2010) and 800 mg/kg for 5 d (Dodiya et al., 2011), and both doses caused renal damage. In this study, the dose of IBP used was 400 mg/kg. This concentration was used to induce renal damage within five days. The dose of ZE used was 500 mg/kg as reported by Kumar et al. (2009). The experimental design included one control and three experimental groups as follows:

1. Normal saline-treated control group. Rats received 0.9% NaCl.

2. ZE group. Rats received ZE (500 mg/kg).

3. IBP group. Rats received IBP (400 mg/kg).

4. ZE + IBP group. Rats received ZE (500 mg/kg) + IBP (400 mg/kg).

All treatments were applied by oral gavage. Animals were sacrificed on the sixth day, and blood samples were collected by the intracardiac puncture. Kidneys were immediately transferred into 0.1 M phosphate buffer solution pH 7.

Biochemical analysis

Serum samples were analyzed for urea, creatinine, albumin and total protein. Kidney homogenate was used to assay for catalase (CAT), glutathione S-transferase (GST), superoxide dismutase (SOD) and lipid peroxidation.

Catalase

CAT activity was measured as described by Aebi (1984). Diluted kidney homogenates (20 μl) were added to 2.98 ml of 30 mM H₂O₂ solution in 50 mM potassium phosphate buffer, pH 7.0. The change in the absorbance was monitored spectrophotometrically at 240 nm. One unit of CAT activity is defined as the amount of enzyme that catalyzes the decomposition of 1.0 µmol of H₂O₂ per minute, and the specific activity was expressed as unit per mg protein.

Glutathione S-transferase

GST activity was measured as described by Habig et al. (1974) by measuring the rate of conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione (GSH) as a function of time. Diluted kidney homogenates (20 μl) were added to 2.90 ml of 0.1 M phosphate buffer pH 6.5, 50 μl of 0.1 M GSH and 30 μl of 0.1 M CDNB. One unit of GST activity is defined as the amount of enzyme producing 1 nmol of 1-(S-glutathionyl)2,4-dinitrobenzene (GS-DNB) conjugate/min under the conditions of the assay. The specific activity of the enzyme was expressed as unit per mg protein.

Lipid peroxidase

Lipid peroxidase activity was measured as described by Ohkawa et al. (1979). The reaction mixture contained the following: 1.5 ml of 20% acetic acid, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 0.8% thiobarbituric acid, 0.77 ml of distilled water and 30 µl of the kidney homogenate. The reaction mixture was incubated at 95 °C for 1 h. Then it was cooled and centrifuged to get rid of the precipitate. The absorbance of the chromogen malondialdehyde (MDA) was read at 532 nm against a blank containing distilled water instead of the homogenate, and the concentration of MDA was calculated using the extinction coefficient 1.56 × 10⁵M⁻¹cm⁻¹.

Superoxide dismutase

SOD activity in the supernatant was determined by SOD Assay Kit-WST from Sigma. In this kit, a highly water-soluble tetrazolium salt produces a water-soluble formazan dye upon reduction with a superoxide anion. The rate of the reduction is linearly related to the xanthine oxidase activity and is inhibited by SOD. Activity of SOD, as an inhibition activity, was quantified by measuring the decrease in the color development at 440 nm. One unit of SOD activity is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.

Protein determination

Protein concentration of the kidney homogenate was measured according to Bradford method (Bradford, 1976), using bovine serum albumin as a standard.

Histological analysis

Kidney tissues were fixed in 10% formaldehyde immediately after killing. Sections were prepared and stained with hematoxylin and eosin for histological examination.

Statistical analysis

Data were expressed as means ± SEM. Statistical analysis of the data was done using one-way analysis of variance followed by Tukey’s test; p < 0.05 was considered significant.

Results

Body weight changes

The body weight gain in IBP group was significantly lower than that of the normal saline group (p < 0.05) (Table 1). Administration of ZE with IBP resulted in a significant increase in body weight gain compared with IBP group (p < 0.05).

Table 1. Effects of IBP and/or ZE treatments on body weight in rats.

Parameter	Saline	ZE	IBP	ZE + IBP
Initial body weight (g)	185.00 ± 5.77	178.33 ± 4.41	176.67 ± 6.67	178.33 ± 4.41
Final body weight (g)	200.00 ± 5.77	200.00 ± 2.89	166.67 ± 7.26	186.67 ± 4.41
Relative gain (%)	8.12 ± 0.25	12.31 ± 3.62^a	−5.7 1 ± 1.72^b	5.67 ± 0.65^a

Data shown as mean ± SEM. Different letters in the same row are significantly different (p < 0.05).

^aSignificantly different from the IBP group.

^bSignificantly different from the saline group.

Biochemical analysis

Ibuprofen at a concentration of 400 mg/kg significantly increased serum urea and creatinine when compared with saline (p < 0.05) (Table 2). IBP also reduced serum albumin and total protein (p < 0.05) (Table 2). When ZE (500 mg/kg) was administrated with IBP, it significantly reversed the elevations of urea and creatinine and the reductions in albumin and total protein (p < 0.05). The treatment with higher concentration of ZE (1000 mg/kg) did not show significant improvement in serum biochemical parameters (data not shown).

Table 2. Effects of IBP and/or ZE treatments on some serum biochemical parameters in rats.

Parameter	Saline	ZE	IBP	ZE + IBP
Urea (mg/dl)	40.70 ± 4.72	40.00 ± 3.53^a	94.24 ± 15.98^b	40.50 ± 1.33^a
Creatinine (µmol/l)	48.39 ± 1.89	42.48 ± 4.33^a	90.18 ± 14.03^b	54.03 ± 3.55^a
Albumin (g/dl)	3.87 ± 0.13	3.96 ± 0.12^a	2.69 ± 0.10^b	3.42 ± 0.13^a
Total protein (g/dl)	5.59 ± 0.18	5.56 ± 0.45^a	3.39 ± 0.25^b	5.27 ± 0.12^a

Data shown as mean ± SEM. Different letters in the same row are significantly different (p < 0.05).

^aSignificantly different from the IBP group.

^bSignificantly different from the saline group.

The activities of CAT and GST in rats treated with ZE were significantly higher than those of the control group (p < 0.05) (Table 3). Treatment with IBP also increased the activities of CAT (p < 0.05) and GST enzymes (p < 0.001). When ZE was coadministered with IBP, further increase in the activities of CAT (p < 0.05) and GST (p < 0.001) was observed. On the other hand, SOD and lipid peroxidase activities were not affected by IBP.

Table 3. Effects of IBP and/or ZE treatments on some antioxidant enzymes in rats.

Parameter	Saline	ZE	IBP	ZE + IBP
CAT (µmol/min/mg protein)	216 ± 16.5	367.33 ± 5.90^a	361.67 ± 19.01^a	397.67 ± 28.14^a
GST (nmol/min/mg protein)	167.33 ± 2.60	226.67 ± 17.03^a	284.00 ± 7.77^b	373.67 ± 11.09^b
SOD (U/mg protein)	48.67 ± 4.41	75.33 ± 4.67^a	34.00 ± 3.51	56.00 ± 4.16^a
MDA (µmol/min/mg protein)	1.93 ± 0.15	1.89 ± 0.12	1.41 ± 0.13	1.76 ± 0.12

Data shown as mean ± SEM.

^aSignificantly different from the saline group (p < 0.05).

^bSignificantly different from the saline group (p < 0.001).

Histological analysis

Histological examination of the kidney showed normal morphology of the glomeruli and tubules in both saline and ZE-treated groups (Figure 1A and B). In IBP-treated group, significant changes were observed including hypercellularity in some and shrinkage in other glomeruli lines, ischemia in proximal convoluted tubules and congestion (Figure 1C). Upon administration of ZE with IBP, normal architecture of the kidney was observed (Figure 1D).

Figure 1. Micrographs of rat kidney tissues: (A) Saline group, normal morphology; (B) ZE group, similar structure as saline group with normal tubules and glomeruli; (C) IBP-treated group, hypercellularity in some and shrinkage in other glomeruli lines, ischemia in proximal convoluted tubules and congestion and (D) ZE + IBP-treated group, normal morphology of tubules and glomeruli as saline group.

Discussion

IBP at a concentration of 400 mg/kg body weight resulted in a serious renal toxicity as shown by the increase in serum urea and creatinine. Moreover, decreased serum levels of albumin indicated proximal tubular toxicity. The histological study supports the biochemical examination as the changes in kidney morphology were apparent. In the kidneys of IBP-treated rats, the glomeruli were degenerated with hypercellularity, in addition to renal tubular injury. In one previous study, treatment of rats with IBP (100 and 200 mg/kg) for 14 d resulted in acute tubular necrosis and degeneration of tubules (Kumar et al., 2010). Another study using IBP (800 mg/kg) for five days showed a significant increase in rats serum urea with tubular damage (Dodiya et al., 2011). In 1995, Kim et al. reported a case of acute renal insufficiency in a healthy 2-year-old boy who had ingested approximately 40 ibuprofen 200 mg tablets (approximately 640 mg/kg) (Kim et al., 1995).

In this study, ZE at a concentration of 500 mg/kg was able to prevent the increase in creatinine and urea caused by IBP, and hence to partially protect the kidney from damage. The protective effect of ZE was also demonstrated by the histological study since it was relatively able – when introduced with IBP – to prevent morphological changes in the tubules and glomeruli. It has been reported that ZE (500mg/kg) is effective in reducing hepatic damage caused by paracetamol and thioacetamide in rats, while the effect is much less with higher dose (1000 mg/kg) (Kumar et al., 2009). In agreement with this study, our results demonstrated that high dose of ZE (1000 mg/kg) did not show significant protection against renal damage induced by IBP as did the lower dose (500 mg/kg).

In this study, IBP significantly elevated the activity of CAT and GST, while it did not affect SOD and lipid peroxidase activities. When ZE was administrated with IBP, the levels of the antioxidant enzymes (CAT and GST) were further increased. Several studies revealed the increasing effect of IBP on antioxidant enzymes. van Lieshout et al. (1997) found that NSAIDs are able to elevate the detoxification potential of gastrointestinal tract tissues by increasing the expression of GSTs. Dabhi et al. (2008) found that IBP significantly elevated CAT level while it reduced SOD and lipid peroxidation in atherosclerotic animals. Another study demonstrated that ibuprofen resulted in a significant increase in the total SOD and CAT activities in the stomach, which means that there is no temporal relation between the antioxidant status of the stomach and the tissue damage following oral administration of IBP (Balasubramanian et al., 2004). The most common way by which NSAIDs can adversely affect renal function is by reduction in renal prostaglandins resulting from inhibition of the cyclooxygenase pathway (Dabhi et al., 2008). Inhibition of cyclooxygenase enzymes inhibits generation of free radicals during prostaglandins synthesis, which may be responsible for high CAT level and for reduction in lipid peroxidase and SOD levels (Dabhi et al., 2008). The reduction in renal prostaglandins caused by NSAID use may result in electrolyte imbalance, renal hypoperfusion and acute tubular necrosis or cortical necrosis, and in most cases, these changes are reversible (Buck, 2000).

Our study suggests that IBP damaging effect on kidney has no correlation with oxidative stress because IBP increases the activity of GST and CAT. It was reported that IBP acts to suppress oxidative damage in the Alzheimer’s disease brain through its capacity to inhibit NADPH oxidase-derived free radical production (Wilkinson et al., 2012). Furthermore, IBP showed promising antioxidant activity in atherosclerotic animals (Dabhi et al., 2008). It also suppressed oxidative stress in smokers and non-smokers (Zapolska-Downar et al., 2000).

Z. jujuba extract protects the kidney against IBP-induced nephrotoxicity as can be seen from the histological study and from kidney biomarkers. However, the levels of antioxidant enzymes cannot be used as an indication of the protective effect since they were elevated by IBP. This means that the antioxidant properties of Z. jujuba are not involved in the protection. The exact constituents responsible for this protective effect cannot be explained by this study.

Conclusion

Z. jujuba aqueous extract bears therapeutic potential against nephrotoxicity caused by IBP. A long-term treatment with ibuprofen could be safe if Z. jujuba aqueous extract is taken during the treatment. Further studies are recommended to elucidate the exact components of Z. jujuba that are responsible for this therapeutic effect.

Declaration of interest

The authors declare that there was no conflict of interest.