Amelioration of cisplatin-induced nephrotoxicity by extracts of Hemidesmus indicus and Acorus calamus

Abstract

Administration of commonly used anticancer drug cisplatin [cis-diamminedichloroplatinum (II)] at pharmacologically relevant concentrations (12 mg/kg body weight) resulted in severe renal toxicity as evidenced from histopathological observations and biochemical alterations in the renal tissue. The extracts of medicinal plants Hemidesmus indicus L. (Apocynaceae) and Acorus calamus L. (Araceae) protected the renal tissue effectively from cisplatin-induced toxicity. Treatment of cisplatin-administered animals with the plant extracts could prevent the drug-induced oxidative damage in the renal tissue as evidenced from the decreased levels of lipid peroxidation and enhanced activities of the antioxidants in the renal tissue. Cisplatin treatment increased serum urea level to 41.3 ± 2.86 mg/dL and administration of the extracts of H. indicus and A. calamus brought down the level to 34.54 ± 0.37 and 30.12 ± 0.95 mg/dL, respectively. Serum creatinine levels were increased to 1.1 ± 0.02 mg/dL following cisplatin administration, and treatment with extracts of H. indicus and A. calamus brought this down to 0.76 ± 0.09 and 0.61 ± 0.06 mg/dL, respectively. The histopathological observations indicated that treatment with the H. indicus and A. calamus extracts restored the cisplatin-induced structural alterations in the renal tissue.

Keywords::

• Acorus calamus
• Hemidesmus indicus
• nephrotoxicity
• oxidative stress
• renal damage

Introduction

cis-Diamminedichloroplatinum (II) (cisplatin) a platinum co-ordinate complex, is a widely used antineoplastic agent for the treatment of metastatic tumors of the testis, metastatic ovarian tumors, lung cancer, advanced bladder cancer, and many other solid tumors (Sweetman, 2002). However, the clinical usefulness of this drug is limited by the development of nephrotoxicity, a side effect that may be produced in various animal models (Antunes & Darin, 2000; Chirino et al., 2004; Kim et al., 1997; Weijl et al., 2004). Cisplatin (CP) is retained particularly in proximal tubule epithelial cells at the corticomedullary junction, which are the main sites where the toxic effect of CP is manifested. Oxidative stress caused by increased generation of free radicals and caspase-mediated apoptosis play a major role in nephrotoxicity and renal dysfunction that progressively develop in response to CP treatment (Arany & Safirstein, 2003).

CP-induced nephrotoxicity is also closely associated with an increase in lipid peroxidation in the kidney tissues. This antitumoral drug also causes generation of reactive oxygen species (ROS), such as superoxide anion and hydroxyl radical that deplete the GSH (reduced glutathione) levels and inhibit the activity of antioxidant enzymes in renal tissue. This ROS may produce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage (Kim et al., 1997; Mora et al., 2003). Glutathione, an endogenous free thiol, has been reported to decrease cisplatin nephrotoxicity (Somani et al., 1995). The depletion of GSH seems to be a prime factor that permits lipid peroxidation (Younes & Siegers, 1981). Administration of superoxide dismutase (SOD) and antioxidants (GSH) ameliorates cisplatin nephrotoxicity in experimental animals (Anderson et al., 1990). Superoxide dismutase plays an important role in the dismutation of superoxide anions by catalyzing their conversion to hydrogen peroxide and singlet oxygen. Glutathione peroxidase activity was decreased in the rat renal mitochondria incubated with cisplatin and has been correlated to disturbances in GSH metabolism (Sugiyama et al., 1989).

Previous studies reported that there are several natural agents that may reduce the cisplatin-induced nephrotoxicity through the mechanism of free radical scavenging. Quercetin, a common antioxidant bioflavonoid in fruits and vegetables, has potent cytoprotective effects against cisplatin-induced nephrotoxicity in cultured renal proximal tubular epithelial cells (Kaushal et al., 2001). Sugiyama et al. (1989) reported that green tea tannin plays a major role as an antioxidant that scavenges the reactive radicals generated from cisplatin in experimental animals.

Several medicinal plants can be employed to produce extracts exhibiting biological effects. Acorus calamus L. (Araceae) commonly known as sweet flag, and Hemidesmus indicus L. (Apocynaceae) (Indian sarsaparilla) are two important herbal drugs used in the ancient system of medicine. The fragrant oils obtained by alcohol extraction of the rhizome of A. calamus are mainly used in the pharmaceutical and oenological industries (Bertea et al., 2005). Prasad et al. (2006) reported the effect of A. calamus on nickel chloride (NiCl₂)-induced renal oxidative stress, toxicity, and cell proliferation response. Similar to that of A. calamus, the root extract of H. indicus also possesses strong antioxidant and radioprotective properties (Shetty et al., 2005). The root extract of this plant has been reported to be a potent chemopreventive agent in skin carcinogenesis (Sultana et al., 2003) and was also found to be effective against renal toxicity induced by ethanol and aminoglycoside antibiotics as well as ethanol-mediated hepatic toxicity in experimental animals (Kotnis et al., 2004, Saravanan & Nalini, 2007a, 2008). One of the major active principles in the root extract has been identified as 2-hydroxy 4-methoxy benzoic acid, which help to reduce the levels of liver collagen, hydroxyproline content, cross-linked fluorescence, shrinkage temperature and lipid peroxidation in ethanol-fed rats (Saravanan & Nalini, 2007b). The present study aimed to compare the modulating effect of A. calamus and H. indicus extract on lipid peroxidation and antioxidant defense mechanism in cisplatin-induced renal injury in rats.

Materials and methods

Animals

Male Swiss albino mice (6–8 weeks old, body weight 25–30 g) were purchased from Sri Venketeswara Enterprises, Bangalore, India and were kept under standard conditions of temperature and humidity in the center’s animal house facility. The animals were provided with standard mouse chow (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. All animal experiments in this study were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of the Government of India.

Chemicals

Nitroblue tetrazolium (NBT), reduced glutathione (GSH), 5′-5′dithiobis-(2-nitrobenzoic acid) (DTNB), EDTA and riboflavin were obtained from Sisco Research Laboratories, Mumbai, India. Hydrogen peroxide (H₂O₂) was obtained from Merck, Mumbai, India. Thiobarbituric acid (TBA), ascorbic acid, and bovine serum albumin were purchased from Sigma Chemical Company, St Louis, MO, USA. Cisplatin was provided from Samasth Pharma, Mumbai, India. All other chemicals were of analytical grade procured from reputed Indian manufacturers.

Preparation of the extracts

Authenticated dried rhizomes of Acorus calamus (Voucher specimen number Dan M.-5978(TBGT) D.Mathew, Scientist, Tropical Botanical Garden (TBGRI), Trivandrum, Kerala, India) and dried roots of Hemidesmus indicus (Voucher specimen number BSI 66441 (Amala Ayurvedic Hospital and Research Center) were obtained from Amala Ayurvedic Hospital and Research Center. The rhizomes and roots were powdered and 100 g of the powder was extracted with 50% ethanol at room temperature for 24 h. Extract was filtered through Whatman No: 1 filter paper and supernatant was evaporated in a rotary evaporator at 50°C under vacuum and then lyophilized. The yield of preparation of A. calamus was 19.3% and that of H. indicus was 12%.

Determination of nephroprotective activity

Animals were divided into five groups of six animals each. Group I treated with vehicle (distilled water) was kept as control. Group II was injected with a single dose of cisplatin (12 mg/kg body weight, intraperitoneally (i.p.), which is equivalent to the dose administered in human situations). Group III and Group IV were administered H. indicus extract (250 and 500 mg/kg body weight) along with cisplatin treatment. Group V was administered A. calamus extract (250 mg/kg body weight) along with cisplatin treatment. The extracts were administered by oral gavage, at intervals of 1, 24, and 48 h following cisplatin injection. Seventy-two hours after the cisplatin injection, animals were sacrificed using ether anesthesia; blood samples were collected by heart puncture for measuring serum urea and blood creatinine levels. Kidneys were quickly removed and washed with ice-cold normal saline and homogenates (10%, w/v) were prepared in PBS. A part of the homogenate was used for the estimation of GSH and lipid peroxidation. The remaining homogenate was centrifuged at 5000 ×g for 10 min at 4°C, after removal of the cell debris, the supernatant was used for the assay of super oxide dismutase (SOD), catalase, and giutathione peroxidase (GPx).

Blood creatinine was determined by the alkaline picric acid method (Toro & Ackermann, 1975) using a diagnostic kit (Agappe Diagnostic, Ernakulam, Kerala, India). Serum urea was determined by diacetyl monoxime (DAM) reagent (Henry, 1963) using a diagnostic kit (Agappe Diagnostic).

Reduced glutathione (GSH) level was measured colorimetrically using DTNB as the substrate (Moron et al., 1979). The concentrations of malondialdehyde (MDA) as indices of lipid peroxidation were assessed according to the method of Buege and Aust (1978). Superoxide dismutase activity was determined by the nitroblue tetrazolium reduction method of McCord and Fridovich (1969). GPx activity was determined by the method of Hafemann et al. (1974) based on the degradation of H₂O₂ in the presence of GSH. Catalase activity was determined from the rate of decomposition of H₂O₂, monitored by decrease of 240 nm following the addition of tissue homogenate (Aebi, 1983). Tissue protein was estimated according to the method of Lowry et al. (1951) using bovine serum albumin as standard.

Histopathological examinations of kidney from all the treated groups were evaluated using light microscopy. The tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 5 μm and stained with hematoxylin-eosin. The histopathological examinations were carried out at Sudharma Metroplis Pathological Laboratory, Thrissur, Kerala, India.

Statistical analysis

The results are presented as Mean ± SD of the studied group. Statistical analyses of the results were performed using ANOVA with Tukey-Kramer multiple comparisons test.

Results

Administration of cisplatin to mice was found to induce a marked renal failure, characterized by a significant increase in serum urea and blood creatinine levels. As shown in Table 1, blood creatinine and serum urea concentrations were significantly increased in the cisplatin-treated group compared to the control group. The concentrations of blood creatinine and serum urea in the H. indicus extract (500 mg/kg body weight) treated group were reduced to 49.09% and 27.55%, respectively, with respect to the cisplatin-treated group. Similarly the concentrations of blood creatinine and serum urea in the A. calamus extract (250 mg/kg body weight) treated group were reduced to 44.54% and 27.07%, respectively.

Table 1.  Effect of Hemidesmus indicus and Acorus calamus extracts on serum urea and blood creatinine levels in mice treated with cisplatin (*p <0.001 when compared with cisplatin alone treated group).

Treatments	Urea (mg/dL)	Creatinine (mg/dL)
Control	28.084 ± 0.47*	0.46 ± 0.0.03*
Cisplatin	41.3 ± 2.86	1.1 ± 0.02
Cisplatin + H. indicus (250 mg/kg bw)	34.54 ± 0.37*	0.76 ± 0.09*
Cisplatin + H. indicus (500 mg/kg bw)	29.92 ± 2.5*	0.56 ± 0.06*
Cisplatin + A. calamus (250 mg/kg bw)	30.12 ± 0.95*	0.61 ± 0.06*

Activities of three major enzymes of the antioxidant defense system namely SOD, catalase and GPx and levels of GSH were significantly decreased while the concentration of malonaldehyde (MDA) was found to be elevated in the cisplatin-treated group. Administration of H. indicus and A. calamus extracts was found to significantly elevate the decreased activities of SOD, catalase, and GPx. The activities of renal SOD, CAT, and GPx in the cisplatin alone, cisplatin plus H. indicus extract and cisplatin plus A. calamus extract administered groups are given in Table 2. Administration of these two extracts also inhibited the cisplatin-induced increase in the MDA levels as shown in Figure 1. It can be seen from Figure 2 that the decrease in the GSH levels in renal tissues induced by cisplatin can be prevented by the administration of H. indicus and A. calamus extract.

Table 2.  Effect of p.o. administration Hemidesmus indicus and Acorus calamus on cisplatin-induced decrease in renal antioxidant enzymes (NS, not significant; *p <0.01; **p <0.001 when compared with cisplatin alone treated group).

Treatments	SOD(U/mg protein)	Catalase(U/mg protein)	GPx(U/mg protein)
Control	6.49 ± 0.4**	5.67 ± 0.4**	19.73 ± 0.97**
Cisplatin	2.91 ± 0.94	3.06 ± 0.2	14.13 ± 1.4
Cisplatin + H. indicus (250 mg/kg bw)	3.49 ± 0.86^NS	4.18 ± 0.56**	16.16 ± 0.7**
Cisplatin + H. indicus (500 mg/kg bw)	4.17 ± 1.02*	5.2 ± 0.8**	18.81 ± 0.49**
Cisplatin + A. calamus (250 mg/kg bw)	5.58 ± 0.13**	4.75 ± 0.54**	18.53 ± 0.94**

Figure 1.  Effect of administration of H. indicus and A. calamus extract on cisplatin-induced lipid peroxidation. The lipid peroxides formed are expressed as nanomoles of MDA per mg potein ± SD. *p <0.001 when compared with cisplatin alone treated group.

Figure 2.  Effect of administration of H. indicus and A. calamus extract on cisplatin-induced GSH. Reduced glutathione (GSH) level was measured colorimetrically using DTNB as the substrate and expressed as nanomoles of GSH per mg potein ± SD. *p <0.001 when compared with cisplatin alone treated group.

Figure 3 presents the histopathology of the renal tissues of mice following various treatments. Histopathological investigation showed that, the normal renal tissue architecture of the untreated mice (Figure 3A) was unaffected with normal glomeruli. In cisplatin-treated mouse kidney there is a decreased cellularity of the glomeruli and edema of the lining of epithelial cells in the renal tubules. Moreover, the nuclei of the lining cells show vaculation. The interstitial tissue also showed edema as can be evident from Figure 3B. The renal tissues of cisplatin-treated mice, when administered with extracts of H. indicus or A. calamus after the cisplatin treatment, showed normal glomerular, renal tubule and interstitial tissue appearance (Figures 3C, 3D).

Figure 3.  Micrograph of kidney of mice (A) control, (B) 72 h of cisplatin injection, (C) cisplatin and Hemidesmus indicus, (D) cisplatin and Acorus calamus. The tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 5 μm and stained with hematoxylin-eosin. The histopathological examinations were carried out using light microscopy (40×).

Discussion

Cisplatin is one of the most effective cancer therapeutic agents but the major side effect of this chemotherapeutic agent is nephrotoxicity. Boogaard et al. (1991) reported that a minimum dose of cisplatin (5 mg/kg body weight, i.p.) was sufficient to induce nephrotoxicity in rats. A higher dose of cisplatin (10 mg/kg body weight, i.p.) corresponds to the equivalent human dose currently being used in clinical practice. Administration of cisplatin shows significant increase in blood creatinine and serum urea concentrations compared to normal, which clearly indicates the intrinsic acute renal failure. The present study has shown that A. calamus extract (250 mg/kg bw) and H. indicus extract (500 mg/kg bw) significantly prevent the increase of cisplatin-induced serum creatinine and urea concentrations. However, treatment with H. indicus extract at 250 mg/kg body weight was not as effective when compared to the A. calamus extract of the same concentration.

The present study shows that cisplatin revealed a significant reduction in the renal antioxidant status, such as SOD, CAT, GPx activities, and reduced GSH concentration. These observations support the evidence that the inhibition of enzymes is reversible and part of the mechanism of nephrotoxicity in cisplatin-treated rats is related to depletion of the antioxidant system. The inhibition of the antioxidant system and this inhibitory effect was attenuated by the administration of these extracts.

GSH, a tripeptide synthesized by glutathione synthase, is thought to be one of the most effective primary antioxidants against oxidizing agents (Shang et al., 2003). Reduced glutathione maintains cellular sulfhydryl groups and other structural proteins in stable form. The superoxide radical is the most well-known oxygen-derived free radical (Yu, 1994) and can lead to the formation of additional reactive species H₂O₂, because of its non-ionized state, is able to diffuse through hydrophobic membranes and can form hydroxyl radicals that react with organic lipids to act like highly reactive free radicals. These can cause cellular damage and cell death. Reduced renal GSH can markedly increase the toxicity of cisplatin. The depletion of GSH also seems to be a prime factor that permits lipid peroxidation in the cisplatin-treated group. But treatment with these two extracts in cisplatin-treated mice rendered protection due to the increase in GSH concentration and could protect the renal cells from oxidants attack. Animal bodies possess intracellular defense against both hydrogen peroxides and superoxide anions. The first line of defense against these products is SOD. The function of this enzyme is to convert two superoxide radicals into oxygen and hydrogen peroxide (Gaetani et al., 1989).

Our results in renal tissues showed that cisplatin induced decline in SOD activities. The decreased SOD activity could cause the initiation and propagation of lipid peroxidation. Moreover, reduction in the activity of GPx during cisplatin administration also increases in the levels of peroxides. The decreased activity of CAT and GPx could enhance the lipid peroxidation (Amudha et al., 2006). Thus the levels of MDA, as a result of lipid peroxidation, increased in the cisplatin-treated animals. Treatment with A. calamus or H. indicus prevented the lipid peroxidation by enhancing the renal CAT, SOD and GPx activities.

Gentamicin and ethanol-induced oxidative stress and thereby caused renal toxicity in rats, and administration of H. indicus root extract has been shown to reduce the toxicity by preventing the free radical-mediated oxidative stress in the tissues (Kotnis et al., 2004; Saravanan & Nalini 2007a, 2007b).

In conclusion, the present study showed that administration of the extracts of either A. calamus or H. indicus resulted in amelioration of cisplatin-induced nephrotoxicity. The protective effect of these extracts is associated with their antioxidant properties, as they showed significant biological activities. Even though the potential usefulness of this herbal extracts is evident in cisplatin therapy from the present work, further studies are needed for the therapeutic application.

Declaration of interest

The authors express their gratitude to KSCSTE, Government of Kerala, India, for the financial support through a research grant award.