Alleviation of doxorubicin-induced nephrotoxicity by Clerodendrum volubile leaf extract in Wistar rats: A preliminary study

Introduction Chemotherapeutic drugs which are toxic to naturally dividing cells have found a useful role in treating tumors (1). Majority of these cytotoxic agents lack the ability to precisely differentiate between normal and cancerous cells, resulting in accumulation of these agents in healthy tissues which gives birth to deleterious clinical consequences (2). As an important antitumor, the anthracycline antibiotic, doxorubicin (DOX) is commonly used to treat a variety of malignant neoplasms including breast cancer, leukemia and solid tumors (3). However, due to its severe side effects, such as cardiotoxicity, nephrotoxicity and hepatotoxicity (4-7), the use of DOX as a chemotherapeutic agent in medicine has been limited. It is now understood that the multi-organ injury of DOX is partially due to its oxidative damage (7-9). Based on this, the use of antioxidant compounds (natural or synthetic) has been deployed as a therapeutic approach to control renal injury induced by DOX (10-12). Several studies have been conducted and documented to show the protective effects of naturally occurring substances with potent antioxidant properties against DOXinduced nephrotoxicity (13-15). Clerodendrum volubile P. Beauv (Family: Lamiacea) is widely found growing in many deciduous forests across http://www.herbmedpharmacol.com doi: 10.15171/jhp.2020.xx


Introduction
Chemotherapeutic drugs which are toxic to naturally dividing cells have found a useful role in treating tumors (1). Majority of these cytotoxic agents lack the ability to precisely differentiate between normal and cancerous cells, resulting in accumulation of these agents in healthy tissues which gives birth to deleterious clinical consequences (2). As an important antitumor, the anthracycline antibiotic, doxorubicin (DOX) is commonly used to treat a variety of malignant neoplasms including breast cancer, leukemia and solid tumors (3). However, due to its severe side effects, such as cardiotoxicity, nephrotoxicity and hepatotoxicity (4)(5)(6)(7), the use of DOX as a chemotherapeutic agent in medicine has been limited. It is now understood that the multi-organ injury of DOX is partially due to its oxidative damage (7)(8)(9). Based on this, the use of antioxidant compounds (natural or synthetic) has been deployed as a therapeutic approach to control renal injury induced by DOX (10)(11)(12). Several studies have been conducted and documented to show the protective effects of naturally occurring substances with potent antioxidant properties against DOX-induced nephrotoxicity (13)(14)(15). Clerodendrum volubile P. Beauv (Family: Lamiacea) is widely found growing in many deciduous forests across West Africa (16). Its common names are 'Obenetete' among the Urhobo and Itsekiri tribes of the Niger-Delta of Nigeria, 'Marugbo' or 'Ewe ata' among the Yoruba tribes of Ondo State in South western areas of Nigeria (17)(18)(19). In the traditional system of medicine, the plant is used for the treatment of several diseases like diabetes, rheumatism, arthritis, edema and gout (16,(20)(21)(22). Some of the reported pharmacological activities of C. volubile leaf includes: antioxidant (21,23), hepatoprotective (24,25), antihypertensive (18), neuroprotective (26), and cancer chemopreventive (27) activities. However, to the best of our knowledge, no study has been carried out on the nephro-protective activity of the plant against DOXinduced toxicity in rats. Therefore, this preliminary study was conducted to investigate the ameliorative effects of C. volubile against oxidative stress toxicity induced by administration of DOX.

Plant collection and authentication
Fresh leaves of C. volubile were purchased from Oja Oba Market in Akure, Nigeria. The plant was identified and authenticated at the Department of Biology, Federal University of Technology (FUTA), Akure, Nigeria. The voucher number (FUTA/BIO/0121) was assigned.
Preparation of plant extract Powdered leaves (500 g) of C. volubile was extracted with 1 L of methanol at room temperature for 24 hours and evaporated to yield the crude extract. The combined methanol extract was filtered and with the aid of rotary evaporator concentrated to obtain the crude extract from which a stock solution was prepared and administered to rats at a concentration of 200 mg/mL. Acute toxicity study The mean lethal dose (LD 50 ) of the methanolic extract of C. volubile leaf was investigated in rats (weighing 160-180 g) following the method of Chinedu et al (28).

Experimental animals and treatment
Apparently healthy adult male Wistar rats about 160-180 g in weight were purchased from the College of Medicine, Ekiti State University, Ado-Ekiti Nigeria. Animals were kept under a natural conditions (12 h light/12 h dark) throughout the experimental period. They were fed standard pellets and water, ad libitum. In this study, animal care was upheld gently in agreement with established guidelines as provided in the Guide for the Care and the Use of Laboratory Animals and in line with the University institutional Ethics Committee and Standards on animal care and experiments.

Experimental design
The rats were divided into five groups; (a) Control group: rats were given 0.9% NaCl as vehicle, (b) DOX group: A single dose of DOX (25 mg/kg; i. p.) was administered (29) and rats were sacrificed 4 days after DOX injection, (c-e) Methanolic extract of C. volubile (MECV)-treated DOX groups: rats were given MECV (at the dose of 125, 250 and 500 mg/kg body weight) orally, respectively viz: for 12 consecutive days; 8 days before, and 4 days after the DOX administration. At the end of treatment period, the animals were fasted overnight and then sacrificed. Blood samples were collected via cardiac puncture into dry tubes and thereafter centrifuged at 3000 × g for 10 minutes.
Tissue homogenate preparation The kidneys were dissected, excised and rinsed in 1.15% KCl, then, blotted with filter paper and the weighed. They were then placed in an iced-cold phosphate buffer (pH 7.4) and then homogenized. The resultant kidney homogenate was subjected to centrifugation at 12 000 × g for 15 minutes at 4°C to obtain the post-mitochondrial fractions which was kept at 4°C and used for further biochemical assays.
Determination of renal functions Blood samples were collected via cardiac puncture, centrifuged at 3000 × g for 10 minutes. Serum creatinine and blood urea nitrogen were measured as a marker of renal function, using colorimetric assay kits according to the manufacturer's instructions.
Biomarkers of oxidative damage Assessment of lipid peroxidation Lipid peroxidation was determined by estimating the thiobarbituric acid reactive substances formed (expressed as malondialdehyde [MDA] equivalents) following the method of Ohkawa et al (30). The level of MDA was deduced from the absorbance as described by Adám-Vizi and Seregi (31) and the unit given as nmol MDA/ mg protein. The reduced GSH estimation was carried out according to Jollow et al (32).

Antioxidant enzyme activities
The activity of superoxide dismutase (SOD) was evaluated using the method of Misra and Fridovich (33). The catalase (CAT) activities were investigated according to the procedure outlined by Sinha (34). The estimation of glutathione peroxidase (GPx) activity was carried out using the method of Lawrence and Burk (35).
Histological assessment Kidney tissues fixed in formalin were paraffin-embedded, cut at 5 μm thickness and stained with hematoxylin and eosin (H&E) stain. Histopathological examination of the stained tissue sections was carried out by a renal histologist, who was blinded to the sample groups (36).
Data analysis Data from this study are depicted as mean ± standard deviation. The analysis was performed by using oneway analysis of variance (ANOVA). Turkey's multiple comparison post hoc test was also carried out in all the groups using GraphPad prism 6.0 software package for Windows (37). The level of significance was placed at P < 0.05.

Acute toxicity
The results of the acute toxicity studies revealed the nontoxic nature of C. volubile methanolic leaf extract. Rats administered with C. volubile extract appeared normal and did not show any significant changes in behavior or neurological responses up to 3000 mg/kg body weight of the extract. There was no mortality or toxicity reaction at any of the doses used until the end of the experiment (data not shown).
Effect of treatment on kidney functions Serum urea and creatinine are indicators of kidney function. Our results showed that urea and creatinine levels were significantly increased in DOX group when compared to their corresponding control group. MECV + DOX showed decreased urea and creatinine levels in serum compared to DOX group (Figures 1 and 2).

Effect of treatment on biomarkers of oxidative damage
The antioxidant status of the kidney in normal and DOXinduced rats is shown in Table 1. The renal level of MDA increased significantly (P < 0.05) in the DOX-induced rats with concomitant decrease in GSH level. The pretreatment with MECV + DOX significantly reversed the effects of DOX.
Effect of treatment on antioxidant enzymes Administration of DOX led to significant reduction of SOD, CAT, and GPx contents compared to control group respectively in the kidney of rats. However, the concomitant administration of MECV + DOX restored enzyme activities near to the baseline valve recorded for the control group (Table 2).
Histological examination Histopathological examination with H&E staining revealed normal renal glomeruli and cortical tubules structures in the control group. However, DOX-treated group showed glomeruli distortion, filtration space obliterated disappear, tubules focal atrophy necrosis and exfoliation, and vascular congestion. MECV+DOX combination group showed little or no visible lesions in the observed group (Figure 3).

Discussion
Doxorubicin is an antibiotic and a strong anticancer drug with wide spectrum of therapeutic actions. However, a prominent limiting factor to the use of DOX in anticancer therapy is its adverse effect of nephrotoxicity.  Although the exact mechanism of action behind DOXinduced nephrotoxicity remains not fully elucidated, the formation of free radical, oxidative damage, and lipid peroxidation of the membranes is believed to be a major factor contributing to DOX nephrotoxicity (6,7). C. volubile leaf extracts have been documented to perform an important role in attenuating oxidative stress, scavenging free radicals and boosting of antioxidant defense systems (12,20,21,24,38,39). DOX-induced nephrotoxicity was observed in our study following the significant increase in serum urea and creatinine levels (Figures 1 and 2) which was confirmed by toxic histopathological changes when compared with the corresponding control group.
In the diagnosis of renal injury, urea and creatinine are the commonly used diagnostic markers of nephrotoxicity (40,41). The damaging effect to the renal tissues by DOX is characterized by an increase in the level of urea and creatinine in the serum. The results from this study (Figures 1 and 2) are similar to previous studies (29,43). Literature reports on medicinal plants and their derived bioactive components have pointed to significant improvements on DOX-induced nephrotoxicity through Values are expressed as mean ± standard deviation of mean (SD) for five rats in each group. * P < 0.05 compared to control group; ** P < 0.05 compared DOX-treated rats. Values in parenthesis represent % change; a % change relative to control; b % change relative to DOX. Values are expressed as mean ± standard deviation of mean (SD) for five rats in each group. * P < 0.05 compared to control group; ** P < 0.05 compared DOX-treated rats. Values in parenthesis represent % change; a % change relative to control; b % change relative to DOX. their antioxidant and free radical scavenging activities (29,42,44). In the current study, the role of another medicinal plant (C. volubile) with reported antioxidant and free radical scavenging activities on DOX-induced nephrotoxicity was investigated. From our results, MECV could significantly reduce serum urea and creatinine levels compared to DOX-treated group (Figures 1 and 2). This might be due to the free radical scavenging abilities and antioxidant effects of MECV which suppressed DOXmediated oxidative stress and tissue damage. Results from the histopathology (Figure 3) showed that DOX-treated group presented with marked damage of renal tubules showing visible lesions. This is in agreement with Kumral et al (29) and Mohebbati et al (44), who showed similar histopathological findings. DOX has been reported to induce oxidative stress in the kidney with the evidence of an increase in lipid peroxidation and alteration in the antioxidant status indices (6). Pre-treatment with MECV obviously reduced the significant elevation in MDA content caused by DOX and is in agreement with Eleiwa et al (45) and Omobowale et al (46) who demonstrated that extracts from the plant Spirulina platensis decreased MDA level in the kidney of rats induced by DOX. In addition, previous report on C. volubile plant (21,20,23,38,39,47) have shown its potential to boost antioxidant activities and attenuate oxidative stress. Consequently, it is not surprising that MECV is able to reduce MDA levels possibly by the presence of bioactive compounds which has been well reported to have antioxidant properties that can scavenge free radicals generation in vivo. These phenolic compounds are found ubiquitously in plants and they have been documented to confer many health benefits which the plants exhibit such as antioxidant, antidiabetic, anticarcinogenic, and anti-carcinogenic properties (22,(48)(49)(50). Some reported bioactive compounds in C. volubile leaves are phenolic acids (rosmarinic acid, garlic acid, chlorogenic acid, caffeic acid) and flavonoids (quercetin, rutin, isoquercitrin) (18,19,23). Chlorogenic acid, rosmarinic and rutin are the predominant compounds present in C. volubile leaf. Previous studies on chlorogenic acid and rosmarinic acid have demonstrated the renoprotective abilities in xenobiotic-induced kidney damage in mice (51). The protective ability of this plant against DOX-induced kidney damage observed in this study might be as results of the presence of these bioactive components present in the leaves of C. volubile. Previous studies have also shown that treatment with DOX significantly reduced renal GSH and this could bring about a short fall in the redox status pool of the cell (52). The interaction of the protein thiols and sulfhydryl groups of GSH with the resultant metabolites from DOX has been associated to this occurrence (53). The significant elevation of lipid peroxidation as shown in this study might also be responsible for the reduced GSH level. However, administration of MECV prior to DOX treatment significantly raised this reduced GSH level at the doses used in this study. Furthermore, the activities of the renal SOD, catalase and GPx were significantly reduced in the DOX-intoxicated rats, respectively ( Table 2). The decrease in the SOD activity could be due to the increased lipid peroxidation (MDA content) observed earlier (as shown in table 1), or the inactivation of the antioxidant enzymes which would ultimately result in accumulation of superoxide radicals, further initiating the lipid peroxidation process (54). The results obtained from this study corroborates report by Erukainure et al (55) who showed the antioxidant activities of an isolated iridoid glycoside from the leaves of C. volubile in brain and hepatic tissue. Similar antioxidant properties of C. volubile leaves have also been demonstrated in vivo by Molehin et al (24) and in vitro by Adefegha and Oboh (18) and Ogunwa et al (21). From histology point of view, pretreatment with MECV offered remarkable protection against kidney damage as shown in the intact renal cyto-architecture with no visible lesions.

Conclusion
In conclusion, data from this preliminary study may support the hypothesis that oxidative stress plays an important role in the mechanism of DOX-induced nephrotoxicity and that MECV has therapeutic potential in ameliorating renal injury induced by DOX possibly via antioxidant mechanism.

Author contributions
The study design, experimental and data analyses, and preparation of the manuscript was done by ORM. The author read and approved the final manuscript.

Conflict of interest
The author declares that he has no conflict of interest.

Ethical considerations
All the animals received care according to the criteria outlined in the Guide for the Care and the Use of Laboratory Animals prepared by EU Directive 2010/63/EU for animal experiments. The ethic regulations were followed in accordance with national and institutional guidelines for the protection of animals' welfare during experiments. The protocol for this study was approved by The Research Ethics Committee, Ebelola Bioenergetic Solutions, Osogbo Osun State Nigeria (EBS/LSR/A/7/2019/001).

Funding/Support
This research was financially supported by the author.