Effect of aqueous extract of Rheum ribes on cisplatin-induced nephrotoxicity in rat.

OBJECTIVE: The purpose of the present study was to examine whether Rheum ribes extract prevents cisplatin-induced nephrotoxicity.
MATERIALS AND METHODS: THE ANIMALS WERE DIVIDED INTO THREE GROUPS: Group A considered as control group, group B were treated with cisplatin (3 mg/kg B.W. for 3 alternative days), and group C further to cisplatin received the aqueous extract of Rheum ribes (150 mg/rat).
RESULTS: Blood urea nitrogen (BUN) level increased in group B on days 14 and 42 compared to day 0 (P < 0.001); it was also increased in group B vs. group A on day 14 (P < 0.001). Rheum ribes extract decreased the serum BUN level on day 14 compared to group B (P < 0.001). Serum creatinine level in group B had a similar profile as serum BUN level but Rheum ribes had no effect on blood creatinine level. Serum cholesterol level was increased in group B on days 14 and 42 compared to day 0 (P < 0.001). Also, cholesterol level was significantly increased in group B when compared to group A on day 14 (P < 0.001). Rheum ribes decreased the blood cholesterol level on day 42 in comparison to group B (P < 0.001). Serum glucose level was increased in group B on days 14 and 42 vs. day 0 (P < 0.001). Also, glucose level was significantly increased in group B when compared to group A on day 42 (P < 0.001). Rheum ribes increased the serum glucose level on days 14 and 42 compared to day 0 (P < 0.05). Histology of kidneys exposed to cisplatin showed renal injury, but Rheum ribes had no effect on the kidney architecture.
CONCLUSION: Cisplatin-induced nephrotoxicity was confirmed in our study. Although Rheum ribes had some effects on biochemical parameters; its effect on renal histology in injured kidney was insignificant.

Cisplatin (cis-diamminedichloroplatinum II; CDDP) is an important chemotherapeutic agent that has been widely used for its potent cytotoxic effects upon a variety of tumor types including testicular, ovarian, and cervical carcinoma.[12] However, dose-dependent and cumulative nephrotoxicity is the major side effect of this compound, sometimes requiring a reduction in dose or dis-continuation of treatment. Approximately 25-35% of patients develop evidence of nephrotoxicity following a single dose of cisplatin.[3]

Rheum ribes (Rhubarb) from Polygonaceae family, is a species of perennial and stout herbs that are distributed in the temperate and subtropical regions of the world, chiefly in Asian countries.[4]

Rheum ribes is a native plant of Iran which grows in several provinces including Khorasan and is locally known as “Rivas”.[5] The thick leaf-stalk of Rheum ribes is used as vegetable, jam or syrup by local people.[6] Leaf-stalk powder and roots are used in Iranian traditional medicine to relieve disorders such as: Gastric illnesses, constipation, headache, kidney and bladder pain, uterine pain and liver disorders; it also is used to increase appetite and bile secretion.[7] The roots of Rheum ribes are also used to treat diabetes, hemorrhoids, ulcer, diarrhea.[4] Hypocholesterolemic effects of both ethanolic and aqueous extracts of Rheum ribes in rabbit have been also reported by Hadjzadeh, et al.[8]

The roots of Rheum ribes, collected from Bingöl, contain tannins (8%) and anthracene derivatives (0.025%). According to the results of the chemical study on the roots of the plant collected from Erzincan, chrysophanol, physcion, rhein, aloe-emodin, physcion-8-O-glucoside, aloe-emodin-8-O-glucoside, sennoside A and rhaponticin have been isolated.[9] In a quantitative study from 400 g of rhizomes and roots of Rheum ribes, these compounds were found: chrysophanol (7 mg), physcion (14 mg), rhein (9 mg), aleo-emodine (29 mg), physcion-8-O-glucoside (12 mg), aleo-emodin-8-o-glycoside (35 mg), sennoside-A (4 mg) and rhaponticin (9 mg).[9] In another study, the aerial parts of Rheum ribes, collected from Hakkari, have also been studied, and chrysophanol, physcion, emodin, quercetin, 5-desoxyquercetin, quercetin 3-O-rhamnoside, quercetin, 3-O-galactoside and quercetin 3-O-rutinoside have been isolated.[10]

In view of the fact that there is no systematic study on the effect of aqueous extract of Rheum ribes on cisplatin-induced nephrotoxicity in rat, the aim of the present study was to examine the protective effect of Rheum ribes on cisplatin-induced toxicity in rats.

Materials and Methods

Plant material and preparation of extract

The aerial parts of Rheum ribes, i.e., thick leaf-stalk with newly growing leaves were obtained from heals of Zoshk (southwest of Mashhad, Iran) in June and identified by botanists in a herbarium of Ferdowsi University. The plant was cleaned, chapped into small pieces and dried while protected from light and wetness. The dried powder (120 g) was mixed with distilled water in a balloon; the balloon was shaken for two days. The preparation was then filtered off a Gauze mesh and the solvent was dried by evaporation under reduced pressure at 40°C. The final product yielded an 8% w/w dried extract.

Animals

Albino male rats (weighing 270-320 g) were purchased from animal house of Mashhad University of Medical Sciences, Iran. The animals were acclimatized under room temperature and humidity with a regular light/dark cycle. All experiments in this study were performed in accordance with the guidelines for the care and use of laboratory animals and the study was approved by Mashhad University of Medical Sciences.

Experimental groups

The animals were divided into three experimental groups and underwent following treatment:

Group A (n = 7): Control group, received distilled water (0.75ml/rat) for three alternate days.

Group B (n = 7): Received cisplatin (3 mg/kg, i.p.) for three alternate days.

Group C (n = 8): Received cisplatin (3 mg/kg, i.p.) for three alternate days; then two weeks after the first injection of cisplatin (day 0), the aqueous extract of Rheum ribes (150 mg/rat) was added daily in drinking water throughout the experiment.

Sample collection and biochemical assays

The serum samples were collected on the 0, 14 and 42nd day after the first injection of cysplatin. Blood samples were collected for determination of serum creatinine, blood urea nitrogen (BUN), cholesterol and glucose levels. The levels of these parameters were determined according to Olympus Kits in Olympus AU600 auto-analyzer (Olympus Corp., Tokyo, Japan).

Histological examination

At the end of the experiment (day 42), animals were decapitated and kidneys were excised, trimmed of connective tissues, rinsed with saline to eliminate blood contamination and preserved in 10% formalin solution for histological examination. After preparation and staining with hematoxylin and eosin (H and E), specimens were examined by a pathologist who was unaware of details of animal groups with light microscopy.

Statistical analysis

The values were expressed as mean ± SEM. The statistical analysis of data was realized by one-way analysis of variance (ANOVA), followed by Tukey multiple comparison tests using 5% level of significance. The statistical package used was statistical package for the social sciences (SPSS 15).

Results

Serum BUN level

As shown in Figure 1, serum BUN level was increased in group B on days 14 and 42 when compared to day 0 (P < 0.001). Also, BUN level was significantly increased in group B compared to group A on day 14 (P < 0.001). Treatment with Rheum ribes extract decreased the blood BUN level on day 14 compared to group B (P < 0.001).

Figure 1

Comparison of serum BUN levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+-treated group (150 mg/rat, n=8). aP <0.001 as compared to day 0. bP <0.001 as compared to control group. cP <0.001 as compared to day 14 in group B

Serum creatinine level

Serum creatinine level was increased in group B on days 14 and 42 when compared to day 0 (P < 0.001). Also, creatinine level was significantly increased in group B when compared to group A on day 14 (P < 0.001). Treatment of rats with Rheum ribes extract had no effect on the blood creatinine level [Figure 2].

Figure 2

Comparison of serum creatinine levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+-treated group (150 mg/rat, n=8). aP <0.001 as compared to day 0. bP <0.001 in comparison to control group

Serum cholesterol level

Serum cholesterol level was increased in group B on days 14 and 42 in comparison with day 0 (P < 0.001); it was also significantly increased in group B when compared to group A on day 14 (P < 0.001). Treatment with Rheum ribes extract decreased the blood cholesterol level on day 42 when compared to group B (P < 0.001) [Figure 3].

Figure 3

Comparison of serum cholesterol levels (mg/dl) in different groups of rats. Data are expressd as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+-treated group (150 mg/rat, n=8). aP <0.001 as compared to day 0. bP <0.001 as compared to day 14 in control group. cP <0.05 as compared to day 42 in cisplatin-treated group

Serum glucose level

As shown in Figure 4, serum glucose level was increased in group B on both days 14 and 42 in comparison to day 0 (P < 0.001). Also, glucose level was significantly increased in group B when compared to control group on day 42 (P < 0.001). Rheum ribes extract treatment increased the serum glucose level on days 14 and 42 compared to day 0 (P < 0.05).

Figure 4

Comparison of serum glucose levels (mg/dl) in different groups of rats. Data are expressed as mean±SEM. (Group A) Control group (n=7), (Group B) cisplatin-treated group (3 mg/kg, n=7), (Group C) cisplatin+-treated group (150 mg/rat, n=8). aP <0.001 as compared to day 0. bP <0.001 as compared to control group. cP <0.05 as compared to day 0 in the same group

Histopathologic parameters

The results of histopathologic examination of kidneys are shown in Figure 5. In control (normal) group, renal tissue sections had a normal morphology [Figure 5a]. Histological examination of kidneys exposed to cisplatin showed the distinctive pattern, which included widespread degeneration of tubular architecture, sloughing tubular epithelial cells from the basement membrane, tubular cell necrosis and intra-tubular cast formation especially in the outer medulla [Figure 5b]. Renal sections obtained from rats treated with Rheum ribes extract demonstrated features very similar to cisplatin-treated group; degeneration of tubular cells, basement membrane impairment and intra-tubular cast formation were found in this group.

Figure 5

Light photomicrographs of the kidney sections (H and E, ×200). (a) Kidney sections from control group with normal renal morphology. (b) The group B shows the distinctive pattern of renal injury

Discussion

As shown in the present study, histopathologic and biochemical data demonstrated that cisplatin induced renal failure lasting until the end of the experiment (day 42). Much attention has been focused on the direct toxic effects of cisplatin on renal tubular cells in vitro.11 In this setting, cisplatin induces DNA damages,[1213] mitochondrial dysfunction,[13] formation of reactive oxygen species,[14] caspase activation,[15] and either necrotic or apoptotic cell death, depending on the level of cisplatin.[1617] Inflammatory mediators appear to play an important role in pathogenesis of acute renal failure (ARF).[181920] Faubel, et al., demonstrated that cisplatin-induced acute renal failure was associated with increased in IL-1, IL-18, and IL-6 levels and neutrophil infiltration in kidney. However, inhibition of IL-1, IL-18, and IL-6 or neutrophil infiltration in the kidney was not sufficient to prevent cisplatin-induced ARF.[21]

As shown in Figures 1 and 2, cisplatin increased the serum BUN and creatinine levels and Rheum ribes was not able to decrease the creatinine levels but decreased the BUN levels on day 14. Maliakel, et al., demonstrated that administration of cisplatin significantly increased the serum creatinine and urea levels compared to normal group.[22] In agreement with our study, Hanzlicek reported that administration of Rheum officinalis orally as 75 mg tablet every 12 hrs in cats with chronic kidney disease had no effect on body weight, serum creatinine, or systemic blood pressure.[23]

It has been reported that glomerulosclerosis was reduced after administration of aqueous extract of Chinese rhubarb to rats which underwent subtotal nephrectomy compared to those given only plain water.[24] Rhubarb has long held an esteemed place in traditional Chinese herbalism as part of treatment protocols for patients with chronic renal failure (CRF). Chinese rhubarb has been investigated in preliminary clinical trials in China and shown to have beneficial effects on clinical symptoms and on serum BUN and creatinine levels in CRF patients. In a similar trial, a combined herbal protocol with Chinese rhubarb was shown to reduce BUN and clinical symptoms in patients with CRF underwent hemodialysis compared to controls treated with hemodialysis alone.[25] Chinese rhubarb extracts have significant potential to improve treatment protocols for patients with CRF. In one perspective clinical and experimental study, the progression rate of renal failure was reduced markedly in both Rhubarb and Rhubarb plus captopril group.[26]

It has been demonstrated that oxidative stress is increased in ill patients with acute renal failure and CRF.[2728] Cisplatin therapy also induces oxidative stress, principally involving reactive oxygen species, in renal tubular cells.[29] Oxidative stress is caused by various free-oxygen radicals including superoxide anion, hydrogen peroxide and hydroxyl radical.[30] The interaction of reactive oxygen species with cellular components may result in damage to DNA, proteins, and lipids. Considering the phenolic constituent profile of Rheum ribes, particularly their flavonoids, stilbenes and anthraquinones, they appear to provide a potential source of antioxidants.[31] The protective effect of Rheum ribes may be partially mediated by preventing the disease or cisplatin-induced decline of renal antioxidant status.

In other part of our study, the results indicated that Rhuem ribes extract decreased the serum cholesterol levels in cisplatin-treated rats. Yang, et al., showed urine protein, serum triglycerides, cholesterol and low-density lipoprotein cholesterol (LDL-C) were decreased in diabetic rats treated with Rhuem ribes. They concluded that Rhuem ribes exerts a beneficial effect on the experimental diabetic nephropathy.[32] In a randomized trial, Chinese rhubarb extract was found to lower total cholesterol and LDL-C levels and increase serum high-density lipoprotein cholesterol (HDL-C) and albumin levels in patients with CRF compared to controls.[33] Another study in rats with diabetic nephropathy found that rhubarb extract speeded nitrogen excretion and alleviated hyperlipidemia compared to control rats.[25] Hypocholesterolemic effects of both ethanolic and aqueous extracts of Rheum ribes in hypercholesterolemic rabbit have been also reported by Hadjzadeh, et al.[8]

Rheum ribes has been used as a traditional Turkish therapeutic agent for diabetes mellitus in Eastern Anatolia. ÖzbekIn H, et al., reported that the decoction extract of Rheum ribes roots possesses significant blood-sugar lowering activity in alloxan-induced diabetic mice. However, this extract did not show hypoglycemic action in healthy mice. This study has shown that Rheum ribes roots decoction extract is more potent than glibenclamide during the first hour following the application. In other study, Rheum ribes considered as potential candidates for amelioration/management of type 2 diabetes.[34]

In conclusion, cisplatin-induced nephrotoxicity was confirmed by our study, but daily administration of 150 mg/kg aqueous extract of Rheum ribes had a little effect on biochemical factors in this dose. This may partially be due to its time-course action on biochemical parameters. Thus, further investigation is needed to determine the proper dose and mechanism(s) of action of Rheum ribes.