Ethanol Extract of Crataeva nurvala Stem Bark Reverses Cisplatin-induced Nephrotoxicity

Abstract

Generation of free radicals in the kidney cortex plays an important role in the pathogenesis of cisplatin-induced dysfunction of renal proximal tubule cells. Previous studies carried out showed that an alcohol extract of Crataeva nurvala stem bark possessed antioxidant activity in rats, hence the present work aimed at evaluating the possible effect of the alcohol extract of C. nurvala on cisplatin-induced dysfunction model of renal proximal tubule cells by oxidative stress. The alcohol extract was administered orally for ten days at two dose levels of 250 and 500 mg/kg body weight, five days after administration of a single i.p. dose of cisplatin (5 mg/kg). Renal dysfunction was evaluated histologically by light microscopy and biochemically by measuring the concentrations of blood urea nitrogen, serum creatinine, lipid peroxidation, glutathione and catalase activity in the kidney cortex. The results suggest that the plant extract (250 and 500 mg/kg) was effective in significantly altering the indices of cisplatin induced dysfunction of renal proximal tubule cells under oxidative stress by decreasing the concentration of blood urea nitrogen, creatinine and lipid peroxidation. The increased glutathione and catalase activity are indicative of the antioxidant properties of C. nurvala stem bark extract.

Keywords:

• Crataeva nurvala
• cisplatin
• nephrotoxicity
• nephroprotection
• oxidative stress
• antioxidants

Introduction

Crataeva nurvala Buch.-Ham (Capparidaceae), one of the medicinal plants recorded in the Indian system of medicine, is well recognised for its antilithic properties (Nadakarni, 2000). Clinically the plant has been evaluated for its role in the management of urolithiasis (Anand et al., 1993), neurogenic bladder, obstructive uropathies and chronic urinary infections (Baskar et al., 1996). In vivo studies have shown that C. nurvala stem bark has a protective role against free radical induced toxicity in experimental urolithiasis (Baskar et al., 1996) and hepatotoxicity (Sunitha et al., 2001).

Numerous investigations have reported that the renal proximal tubule cell dysfunction, a limiting adverse event associated with the antineoplastic cisplatin, is due to the involvement of oxidative stress via free radical formation (Goldstein et al., 1983; McGinness et al., 1978; Sugihara et al., 1987; Zhong et al., 1990). Review of the scientific literature showed the absence of any experimental data to justify the protective role of the stem bark of this plant in nephrotoxicity. Thus, the present investigation was undertaken to evaluate the efficacy of the alcohol extract of C. nurvala stem bark on free radical-induced renal tubular damage by cisplatin.

Materials and Methods

Plant material

The botanically identified plant material was collected in the month of September 2002 from Udupi district, Karnataka, India. A voucher specimen no. PP 516 has been preserved in the herbarium of the College of Pharmaceutical Sciences, Manipal, India.

Drugs and chemicals

Cisplatin was obtained from Dabur Pharmaceuticals, New Delhi, India. Biochemicals used were of analytical reagent grade and obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chemicals of high purity were obtained from E.Merck (India) Pvt. Ltd, Mumbai, India.

Preparation of extracts

The shade-dried stem bark powder (1 kg) was exhaustively extracted with 95% ethanol in a Soxhlet apparatus, concentrated in vacuo and the dried extract (yield 3%) used for experimental studies.

Phytochemical screening

Preliminary phytochemical screening (Harborne, 1984) revealed the presence of terpenoids, alkaloids, carbohydrates, phenolic compounds, tannins, fixed oils and fats.

Animals

Thirty male Wistar rats, weighing 170–210 g, bred in the Central Animal House, M.A.H.E, Manipal, were used for the study. The animals were fed a pellet diet (Hindustan Lever Ltd., India) and water ad libitum, and maintained in an environmental temperature of 21–23°C under a constant 12 h light and dark cycle. The study was conducted after obtaining institutional animal ethical committee clearance (No. IAEC/KMC/12/2002–2003).

Treatment schedule

Three days after acclimatisation to the laboratory conditions, the rats were randomly assigned to five equal experimental groups as follows.

• Group 1: Rats were administered a single i.p. dose of 0.9% NaCl (2 ml/kg) and after five days were treated orally with 2% gum acacia (2 ml/kg/day) for ten days. Control group-vehicle solvent.

• Group 2: Rats were administered a single i.p. dose of 0.9% NaCl (2 ml/kg) and after five days treated orally with an alcohol extract of C. nurvala (500 mg/kg/day) suspended in 2% gum acacia for ten days.

• Group 3: Rats were administered a single i.p. dose of cisplatin injection (5 mg/kg) and after five days treated orally with 2% gum acacia (2 ml/kg/day) for ten days.

• Group 4: Rats were administered a single i.p. dose of cisplatin (5 mg/kg) and after five days treated orally with C. nurvala extract (250 mg/kg/day) suspended in 2% gum acacia for ten days.

• Group 5: Rats were administered a single i.p. dose of cisplatin (5 mg/kg) and after five days treated orally with C. nurvala extract (500 mg/kg/day) suspended in 2% gum acacia for ten days.

On the 16th day, animals of all groups were anaesthetised with diethyl ether, and rapidly decapitated. Blood was collected and the serum was separated by centrifugation. The kidneys were excised, blotted on a filter paper and weighed. The cortex was dissected out and a part thereof placed in formalin saline for subsequent histological processing while the other part was used for biochemical estimations.

Assessment of renal function

The weight (g) of the animals was noted on the first and last day of treatment and the percentage change in body weight was calculated. Urea concentration in the blood was estimated by the modified Berthelot's method (Henry, 1963), using a kit obtained from Agappe Diagnostics, Maharastra, India. Creatinine level in serum was estimated by alkaline picrate method (Mayne, 1994; Tietz, 1986), using a kit obtained from Dr. Reddy's Laboratories, Hyderabad, India. Thiobarbituric acid reactive substances (TBARS) were estimated according to the method of Fraga et al. (1988), glutathione by the method of Ellman (1959), catalase activity according to the method of Sinha (1972) and protein in tissue homogenate by the method of Lowry et al. (1951).

Histological methods

Small pieces of the cortex of the left kidney of each animal were fixed in 10% neutral buffered formalin, dehydrated in graded alcohol and embedded in paraffin wax. Sections of 5 µm thickness were stained with haematoxyline and eosin (H & E) and subjected to microscopic examination for the presence of glomerular congestion, tubular casts, peritubular congestion, epithelial desquamation, blood vessel congestion, interstitial edema and inflammatory cells.

Statistical analysis

Results given as mean ± S.E.M. were analysed using one way ANOVA followed by post hoc Sheffe's test using SPSS computer software version 7.5. The statistical significance of difference was taken as P < 0.05.

Results

On the 16th day (Table 3), group 3 (cisplatin treated group) showed a significant reduction in normal body weight as compared to groups 1 and 2. Treatment of the animals in group 4 and 5 with the ethanol extract significantly reversed the cisplatin-induced decrease in body weight. (p < 0.05 vs Control -Group 1)

Rats of group 3 displayed a significant increase in blood urea nitrogen and serum creatinine levels on the 16th day as compared to group 1. Groups 4 and 5 showed a significant dose-dependent decrease in blood urea nitrogen and serum creatinine levels as compared to group 3 (Table 1).

Table 1 Effect of alcohol extract of Crataeva nurvala stem bark on cisplatin induced renal damage.

Groups no.	% change in body weight	Blood urea nitrogen (mg/dL)	Serum creatinine (mg/dL)
1	12.54 ± 2.46	38.79 ± 3.5	0.59 ± 0.04
2	13.8 ± 3.17	39.5 ± 3.86	0.56 ± 0.016
3	−17.5 ± 1.3^a	79.74 ± 3.15^a	1.57 ± 0.028^a
4	−3.18 ± 2.34^b	56.61 ± 4.37^b	0.95 ± 0.016^b
5	4.47 ± 1.85^b	43.85 ± 3.18^b	0.74 ± 0.024^b

Values are mean ± S.E.M.

^ap < 0.05 vs Control (Group 1).

^bp < 0.05 vs Cisplatin 16th day (Group 4).

One way ANOVA followed by post hoc Sheffe's test.

A significant elevation of lipid peroxides in group 3 animals was observed when compared to group 1, whereas groups 4 and 5 showed decreased lipid peroxide levels compared to group 3. As compared to group 3, animals treated with the higher dose of the ethanol extract (i.e., group 5) exhibited a significant (p < 0.05 vs cisplatin 16th day- Group 4) reversal of the decreased glutathione and catalase levels. In group 4, however, the increase was not found to be significant (Table 1).

Histological examination

On the 16th day, when compared to normal sections (Fig. 1) the cisplatin treated rat kidney sections of group 3 (Fig. 2) showed marked congestion of the glomeruli with numerous tubular casts associated with epithelial desquamation. Marked peritubular congestion and edema were also observed. The interstitium showed infiltration with inflammatory cells and congestion. These features suggest that cisplatin induces acute tubular necrosis. Group 5 showed complete normalization of kidney section (Fig. 4). However, mild glomerular, peritubular congestion and inflammatory cells were still apparent in group 4 (Fig. 3).

Figure 1 Kidney section of group 1 rat (normal).

Figure 2 Kidney section of group 3 rat administered cisplatin (5 mg/kg) showing glomerular congestion. H & E. × 200.

Figure 3 Kidney section of group 4 rat administered alcohol extract (250 mg/kg) showing mild glomerular and peritubular congestion. H & E. × 200.

Figure 4 Kidney section of group 5 rat administered alcohol extract (500 mg/kg) showing marked reduction in the extent of tubular damage. H & E. × 200.

Discussion

Results from this investigation demonstrate that cisplatin-induced renal injury was evidenced by elevated biochemical markers, such as blood urea and serum creatinine, as well as by the histopathological features of acute tubular necrosis. The alcohol extract of C. nurvala, when administered at two dose levels, i.e., 250 and 500 mg/kg body weight, significantly reduced the increase in blood urea and serum creatinine and brought about a marked recovery in kidneys as evidenced microscopically.

Numerous in vivo and in vitro studies have demonstrated that reactive oxygen metabolites, viz., free radical species, superoxide, hydroxyl radical anion and hydrogen peroxide are important mediators of tissue injury (Fantone et al., 1992; Weiss et al., 1982; Varani et al., 1985; Fox et al., 1984). Oxygen free radicals have been implicated in several biological processes, potentially important in glomerular diseases (Shah et al., 1984; Shah et al., 1987). Previous reports suggest that cisplatin induces nephrotoxicity by initiation of lipid peroxidation and depletion of cellular thiols (Zhang et al., 1994; Bramley et al., 1995). Cisplatin inhibits the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathoine peroxidase) in rat kidneys (Sdzuka et al., 1992), thereby suggesting that cisplatin cytotoxicity results from generation of reactive oxygen species. The results obtained in this study correlate with previous reports that lipid peroxidation contributes to cisplatin induced nephrotoxicity. Effects of several antioxidants like N,N′ diphenyl–p-diphenylenediamine and butylated hydroxy anisole and thiols like dithiothreitol and glutathione on cisplatin induced lipid peroxidation and glutathione depletion (Kim et al., 1997) have been reported.

In the present work, an alcohol extract of C. nurvala was found to increase the glutathione level and catalase activity and to decrease the concentration of TBARS in the kidney cortex. Hence, the possible mechanism of nephroprotection by this plant may be attributed to its antioxidant and free radical scavenging properties.

To conclude, the results of the present study show that the alcohol extract of the stem bark of Crataeva nurvala possesses marked nephroprotective activity and may have a promising role to play in the treatment of acute renal injury induced by nephrotoxins, like cisplatin. Further work envisages evaluating the nephroprotective activity of this plant in chronic renal failure models.

Table 2 Effect of alcohol extract of Crataeva nurvala stem bark on biochemical variables indicative of oxidative stress in cisplatin induced renal damage.

Groups no.	Glutathione (µg/mg protein)	TBARS (mM/100 g tissue)	Catalase (µg of H2O2/min/mg protein)
1	4.39 ± 0.25	1.40 ± 0.09	358.39 ± 18.37
2	4.42 ± 0.3	1.45 ± 0.13	366.63 ± 21.18
3	1.16 ± 0.2^a	2.89 ± 0.15^a	198.14 ± 16.69^a
4	2.29 ± 0.26	1.92 ± 0.09^b	246.42 ± 25.86
5	3.35 ± 0.2^b	1.5 ± 0.05^b	310.96 ± 21.63^b

Values are mean ± S.E.M.

^ap < 0.05 vs Control (Group 1).

^bp < 0.05 vs Cisplatin 16th day (Group 4).

One way ANOVA followed by post hoc Sheffe's test.

Table 3 Effect of alcohol extract of Crataeva nurvala stem bark on histological features of cisplatin induced renal damage.

Histological features	Group 1	Group 2	Group 3	Group 4	Group 5
Glomerular congestion	−	−	++	+	−
Tubular casts	−	−	++	−	−
Peritubular congestion	−	−	++	+	−
Epithelial desquamation	−	−	++	−	−
Blood vessel congestion	−	−	++	−	−
Interstitial edema	−	−	++	−	−
Inflammatory cells	−	−	++	+	−

- normal; + mild; + + severe.

Acknowledgements

This study was supported by College of Pharmaceutical Sciences, MAHE, Manipal, India.