Cystone is an Ayurvedic polyherbal preparation containing herbal extracts and minerals (Table 1). The plants used in the preparation are well known in Ayurvedic medicine for their beneficial actions on the kidney (Nadkarni, 1992). Cystone has been clinically used extensively for treating many urinary tract complications such as urolithiasis (Rai, 1960), burning micturition (Garg and Singh, 1985), neuro-ureterolithiasis (Misgar, 1982), urinary tract complications in pregnancy (Sengupta, 1987) and various other renal disorders (Sharma et al., 1983).

Cisplatin is a potent anticancer agent used in solid tumors of testes, ovary, breast, lungs, bladder etc (Rozeneweig et al., 1977). Its clinical use is limited by its renal toxicity (Madias and Harrington, 1978; Goldstein and Mayor, 1983). Although the mechanism of action of the renal toxicity of cisplatin is still not clear, it has been suggested that the oxygen free radicals play an important role. Cisplatin is known to cause increased lipid peroxidation in renal cortical slices (Inselmann et al., 1995). Many antioxidants such as sodium selenite (Baldew et al., 1989), hydroxy ethyl rutoside (Bull et al., 1988) etc., are known to reduce the renal toxicity caused by cisplatin. The crude extract of Ginkgo biloba L. (Family: Ginkgoaceae) protects kidney slices against cisplatin-induced lipid peroxidation and decreased uptake of p-aminohippuric acid (Inselmann et al., 1995). Our preliminary studies showed that Cystone inhibited the lipid peroxidation in renal cortical slices induced by cisplatin. Since Cystone is clinically used in various renal dysfunctions, we found it interesting to further investigate the effect of Cystone on the renal toxicity induced by cisplatin.

2.1 Animals

Male Wistar rats 8-10 weeks of age, weighing 200-230 g were used for the study. They were maintained on a standard diet (Lipton’s India, Calcutta) and water was given ad libitum.

2.2 Drug solutions

To 1000mg Cystone powder (The Himalaya Drug Co., Bangalore), 10ml distilled water was added and kept overnight at room temperature (25 ± 2°C) followed by boiling for 5 min. After cooling, the extract was filtered and the volume was made up to 10ml. A separate experiment showed that 1000mg of Cystone Powder yielded 210mg of water soluble extract. The filtrate (equivalent 100mg/ml to Cystone Powder) was used for the study.

Cisplatin (Sigma, St. Louis, MO) was dissolved in distilled water to give 1mg/ml solution and prepared freshly.

2.3 Lipid peroxidation in rat renal cortical slices

Lipid peroxidation in renal cortical slices was measured according to the method reported previously (Yamasaki et al., 1996). Immediately after the rats had been decapitated, the kidneys were removed and renal cortical slices were prepared using a razor blade in a kidney holder device to achieve a slice thickness of about 0.3-0.5 mm. Kidney slices of about 100mg/sample were incubated in buffer consisting of (in mM): NaCl (137), KCl (5.9), CaC1₂ (1.5),mgC1₂ (1.2), glucose (11.5), 2-[4-(2-hydroxyethyl)-1-piper-azinyl] ethanesulfonic acid (5.8), pH adjusted to 7.4 with NaOH.

Kidney slices were incubated in a sample volume of 4ml with cisplatin concentrations of 0.5, 1.0, 2.0 and 4.0 mM in the presence or absence of different concentrations of Cystone. Incubations were carried out for 120 min at 37°C. Controls were incubated under identical conditions without addition of cisplatin. At the end of incubations, renal cortical slices were taken from each sample to determine the amount of malondialdehyde (MDA) as indicator of lipid peroxidation. The kidney slices were quickly removed from the medium, blotted on filter paper, weighed and homogenized in 5ml sodium phosphate buffer (pH 7.4) at 2°C, centrifuged at 1000 g for 10 min at 2°C. To 1ml supernatant, 1ml thiobarbituric acid reagent (containing 0.375% thiobarbituric acid, 15% trichloroacetic acid and 0.25 N hydrochloric acid) was added and heated at 90°C for 45 min, cooled and absorbance was measured at 532 nm. The amount of lipid peroxidation was expressed as nmol of (MDA) using the molar extinction coefficient of 1.56 x 10⁵/M per cm as reported earlier by us (Rajakumar and Rao, 1993).

2.4 Renal toxicity

Six groups of animals (n=8) were investigated. For Group 1 animals, cisplatin solution was given at a dose of 3mg/kg intraperitoneally (i.p.). Group 2, 3, 4 and 5 animals received Cystone 100, 250, 500 and 1000mg/kg, i.p. or orally 1 h before cisplatin 3mg/kg, i.p., respectively. Group 3 acted as vehicle control (distilled water). The body weight was recorded daily and on day 5, the blood was collected from retrorbital venous plexus to measure blood urea nitrogen (BUN), serum creatinine, total proteins and WBC count. BUN and serum creatinine were measured using commercial kits (Ranbaxy Diagnostics, New Delhi, India). WBC count was carried out using a hemocytometer. Serum protein was measured by Lowry’s method (Lowry et al., 1951). Urine was collected on day 5 for 6 h (initiated at 8 a.m.) using metabolic cages and analysed for creatinine and protein (Godkar, 1994).

2.5 Statistical analysis

Values are expressed as mean ± S.E.M. For comparison, ANOVA followed by Student’s Newman-Keuls test was used. Statistical significance was set at p<0.05.

3.1 Effect on lipid peroxidation

Incubation of renal cortical slices for 120 min with cisplatin resulted in increased MDA levels and this effect was found to be concentration dependent (Table 2). Addition of Cystone was found to protect cortical slices against cisplatin-induced lipid peroxidation (Table 2). Cystone at 100, 500 and 1000mg/ml inhibited the lipid peroxidation induced by cisplatin (2.0 mM) by 41.8, 62.7 and 71.6%, respectively.

3.2 Effect on the body weight of rats

When animals were given cisplatin 3mg/kg i.p., the weight of the animals decreased significantly. There was an average decrease of 12.5 ± 1.6 g on day 5 compared to control animals which gained 8.13 ± 1.3 g during the same period. When the animals were given Cystone 100, 250 and 500mg/kg i.p. 1 h before cisplatin 3mg/kg i.p., the decrease in the body weight on day 5 was not reversed significantly. Upon administration of Cystone at 1000mg/kg i.p., 1 h before cisplatin 3mg/kg i.p., the decrease in the body weight on day 5 was found to be only 5.63 ± 3.3 g. Thus, Cystone at 1000mg/kg i.p. was able to protect the animals against cisplatin-induced decrease in body weight. However, the protection was not complete.

3.3 Effect on BUN and serum creatinine

Administration of cisplatin 3mg/kg i.p., resulted in significant increase in BUN and serum creatinine compared to control animals (Table 3). When Cystone at 100, 250 and 500mg/kg i.p. was given 1 h before cisplatin, the BUN levels did not alter. Cystone at 1000mg/kg i.p. 1 h before cisplatin i.p., showed about 50% protection against the elevation of BUN. Similar results were obtained with serum creatinine. When cisplatin was given, the serum creatinine level was increased significantly compared to control animals. Cystone at 100, 250 and 500mg/kg i.p. did not protect increased SC levels induced by cisplatin 3mg/kg i.p. (Table 3). When Cystone 1000mg/kg i.p. was given 1 h before cisplatin, about 62% protection against the elevation of serum creatinine was shown.

Thus, the nephrotoxicity of cisplatin characterized by the elevation of BUN and serum creatinine was reversed to a significant extent by Cystone at 1000mg/kg i.p.

3.4 Effect on renal functions

The deterioration of the renal functions induced by cisplatin and the effect of Cystone is given in Table 4. There was a slight reduction in urine volume when cisplatin was given compared to control animals. Cystone did not show any significant effect. However, the urine to serum creatinine ratio (U_Cr/S_Cr) improved significantly when Cystone (1000mg/kg i.p.) was given 1 h before cisplatin. Similar results were obtained in the case of creatinine clearance (Cl_Cr). Cisplatin reduced Cl_Cr compared to control animals. When Cystone was given 1 h before cisplatin the value was found to be similar to control. In the case of urinary excretion of proteins Cystone was not able to inhibit the increased excretion of urinary proteins due to cisplatin.

3.5 Effect on WBC

Cisplatin caused significant reduction in the WBC count. However, Cystone did not show any protection against the reduction of WBC.