​Evaluation of Terminalia arjuna in Comparison to Taurine against Experimental Nephrotoxicity due to Cisplatin in Rats

Cisplatin is a highly effective and widely used anti-neoplastic drug against various solid tumors (Erhan et al., 2019). The anticancer behaviour of cisplatin originates from its ability to bind to N-7 of purine bases of cellular DNA, which leads to formation of mono-adducts that are transformed into inter-strand cross links and intra-strand cross links by reaction of second reactive site of the drug with the second nucleobase (Waseem et al., 2014). Despite being a potent anticancer drug, cisplatin elicits dose and time dependent nephrotoxicity limiting its clinical utility in 25–35% of hospitalized patients undergoing chemotherapy (Ramya et al., 2013).

The mechanisms by which cisplatin exerts its cytotoxic effects involve inflammation, oxidative stress and apoptosis (Abdel daim​ et al., 2019). Cisplatin becomes highly reactive within the cell and conjugates with molecules such as DNA. Intra- and inter-strand cross-linking of DNA by cisplatin blocks DNA replication and gene transcription. Thus, DNA damage is a critical component of toxicity. Due to its low molecular weight and uncharged character, unbound cisplatin in the plasma is freely filtered by the glomerulus. Most of the cisplatin is trapped within the renal cortex (Launayvacher et al., 2008). The concentration of cisplatin in the proximal tubular cells is 5 times higher than the serum concentration and thus such an accumulation of cisplatin in kidney contributes to its nephrotoxicity (Ozkok and Edelstein, 2014).

Medicinal plants play a very important role in primary health-care system. According to WHO, 60% of the world’s population relies on herbal medicine. TA is an ayurvedic plant with important medicinal value. It is commonly known as Arjuna, which belongs to family Combretaceae (Amalraj and Gopi, 2017). Medicinal properties of TA include antioxidant, anti-inflammatory, anti-carcinogenic and gastro-protective effects (Verma and singh 2013). The aqueous extract of TA possesses protective action against renal dysfunction by antioxidant mechanism with enhanced levels of GSH activity, decreased level of LPO (Manna et al., 2006). As a consequence of these properties, TA can have immense potential in preventing the oxidative damage produced by cisplatin (Sherif, 2015).

Taurine is the major intracellular sulphur containing ß-amino acid with diverse cytoprotective activity and is important for the regular functioning of organs (Brosnan and Brosnan, 2006). It plays a major role in bile acid conjugation, osmoregulation, cell proliferation and prevention of oxidant-induced injury in many cells (Bouckenooghe et al., 2006). Taurine treatment inhibits oxidative stress and apoptosis. Taurine also exerts nephroprotective effects, due to its antioxidant and membrane stabilization effects (Han and Chesney, 2012). Objectives of investigation are to study the nephrotoxicity due to cisplatin and to evaluate and compare nephroprotective potential of TA with taurine.

Chemicals
 
All chemicals were of analytical grade and they are obtained from Qualigens Pvt. Ltd., Mumbai and SRL Pvt. Ltd., Mumbai, India.
 
Plant material and preparation of leaf extract
 
The fresh leaves of TA plant were collected from Hyderabad, India. The plant species were authenticated by Scientist, Agricultural College, Hyderabad, India. Those leaves were washed and dried at room temperature for 40 days and then powdered using a mechanical blender. Then, 1gram powder was mixed with 100 ml of boiled distilled water and stirred on hot plate for 20 minutes. Thereafter, extract was filtered through Whatman No.1 filter paper. It was kept at 4°C for further use (Raj et al., 2020).
 
Animals and experimental design
 
This experiment was conducted in November 2020, at College of Veterinary Science, PVNR TVU, Hyderabad, Telangana, India. Total of 36 male Wistar rats aged about 90 days with an average body weight of 170 ± 10 g were obtained from Vyas labs, Hyderabad. They were divided into 6 equal groups (n=6) with different treatments (Table 1) and were housed in polypropylene cages, under controlled environmental conditions. The animals were maintained on standard balanced diet with drinking water ad libitum. All the experimental procedures and protocols used in this study were reviewed and approved by the IAEC (No.5/22/C.V.Sc., Hyd. IAEC- Rats/29.02.2020) and were in accordance with the guidelines of the CPCSEA.



Blood collection
 
After completion of experiment, the blood samples were collected on last day of study from retro-orbital plexus of experimental rats for analysis. Then, the animals were euthanized and kidney tissues were collected immediately and kept in ice cold phosphate buffer. A small portion of the kidney was homogenized with tissue homogenizer to make 10% homogenate to assay the tissue antioxidants. Pieces of tissues from kidney were immediately kept in 10% of formalin fixative to study histological alterations.
 
Biochemical analysis
 
BUN and serum creatinine were estimated by kits from ERBA diagnostics Ltd, Surat, India. Malondialdehyde has been identified as the product of lipid peroxidation that reacts with thiobarbituric acid to give a red compound absorbing light maximally at 535 nm. GSH was estimated on the reaction of reduced glutathione with 5-5’ dithiobis-2-nitrobenzoic acid to give a compound that absorbs light at 412 nm. Protein carbonyls were estimated based on the reaction of amino carbonyls with 2, 4-dinitrophenyl hydrazine to form hydrazones, which can be detected spectrophotometrically at 372 nm.

Quantitative sandwich enzyme immunoassay technique is followed to analyze TNF –alpha and IL-10. The ELISA kits were procured from Krishgen Bio systems, Mumbai, India.
 
Histology
 
For light microscopy examination, the formalin fixed kidney tissues were dehydrated through ascending grades of alcohol, cleared in three changes of xylene and were embedded in paraffin. Kidney sections each of 4 micron thickness were cut and stained with H and E stain.
 
Statistical analysis
 
Data were subjected to statistical analysis by applying one-way ANOVA usingthe SPSS; version 21. Differences between means were tested using Duncan’s multiple comparison testand significance was set at P<0.05.

The results obtained in the present experiment are presented as below.
 
Sero-biochemistry
 
The concentration of BUN and serum creatinine (mg/dl) in group 2 (96.81 ± 0.11 and 1.49 ± 0.035, Respectively) was significantly (P < 0.05) higher when compared to non toxic groups 14th day, while treatment groups 5 (53.13 ± 0.12 and 0.88 ± 0.017, Respectively) and 6 (52.98 ± 0.14 and 0.83 ± 0.014, Respectively) showed significantly (P < 0.05) lower values in comparison to group 2 (Table 2).


 
Antioxidant profile
 
The concentration of GSH (nm/mg) revealed a significant (P < 0.05) decrease in toxic control group 2 (17.41±0.12) when compared to non toxic groups, whereas treatment groups 5 and 6 (28.54 ± 0.18 and 28.95 ± 0.17, respectively) revealed a significant (P<0.05) improvement as compared to toxic control group 2 (Table 2).

The concentration of TBARS and protein carbonyls (nm/mg) revealed a significant (P < 0.05) increase in toxic control group 2 (18.18 ± 0.12 and 9.71 ± 0.09) when compared to non toxic groups, whereas treatment groups 5 (12.54 ± 0.08 and 5.97 ± 0.10, Respectively) and 6 (12.23 ± 0.23 and 6.08 ± 0.16, Respectively) showed significantly (P < 0.05) lower values in comparison to group 2 (Table 2).
 
Cytokine profile
 
The concentration of TNF-α and IL-10 (pg/mg tissue) revealed a significant (P < 0.05) increase in toxic control group 2 (22.35 ± 0.24 and 14.95 ± 0.26, Respectively) when compared to non toxic groups, whereas treatment groups 5 (11.04 ± 0.21and 12.06 ± 0.21, Respectively) and 6 (11.11 ± 0.17 and 12.23 ± 0.19, Respectively) showed significantly (P < 0.05) lower values in comparison to group 2 (Table 3).


 
Histopathology
 
Normal histology of the rat kidney tissue was found in the non toxic groups (Fig 1). Renal tissue sections from rats treated with cisplatin showed moderate variations in glomerular structure (Fig 2), mild increase in bowman’s space with significant shrinkage of some glomerular tufts, degenerations in glomerular capsule and mononuclear cell infiltration with moderate peritubular inflammation (Fig 3). The kidney tissue sections of the cisplatin treated rats exhibited marked acute tubular necrosis and marked dilation and degenerations of tubules and vacuolation (Fig 4), whereas rats treated with TA justified the protective action by minimizing the inflammation of mononuclear cells, tubular necrosis (Fig 5) and by showing mild tubular degenerative changes (Fig 6). Renal histology in taurine treated group showed mild infiltration of inflammatory cells (Fig 7) and congestion (Fig 8).



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The present study was conducted on 3 month old Wistar rats for a period of 14 days to evaluate the protective role of TA and taurine against cisplatin induced nephrotoxicity. The biochemical markers, oxidative stress and cytokines were assessed. Histopathology of kidney tissues were conducted to draw possible conclusions at the end of the experiment. The increase in kidney biomarkers is observed in toxic group due to exposure to cisplatin is a function of the overall balance between the degree of oxidative stress and the antioxidant capability (Hosseinian et al., 2016). Rashed et al., (2011) also reported the elevation of serum creatinine and BUN following cisplatin injection. This is a clear indication of renal toxicity caused by cisplatin as the markers are released by the damaged kidney into circulatory system. In the study of Sherif (2015), administration of TA had a protective effect leading to improvement in renal function as indexed by marked decline in serum creatinine and BUN in comparison to cisplatin group. Marked recovery was observed in the markers after treatment with TA and taurine The antioxidant system is responsible primarily for defense against ROS, and the formation and elimination of ROS in the cells is accomplished by the radical scavenging system, which includes GSH, TBARS and protein carbonyls (Ekinci akdemir et al., 2017). Karimi et al., (2018) reported increased MDA levels and decreased GSH levels in cases of cisplatin induced toxicity. Oxidative stress caused by cisplatin induced excessive production of ROS leads to peroxidation of membrane lipids and depletion of antioxidant enzymes. NADPH oxidase is an enzyme system, which enhances cisplatin mediated ROS generation and cytotoxicity (El Sawalhi and Ahmed, 2014). Lipid peroxidation causes glomerular injury, increases fluidity and permeability of tubular membranes and triggers hypertrophy of renal tubules (Al kahtani et al., 2014). The natural antioxidant arjunolic acid was shown to improve cisplatin induced nephrotoxicity via significant enhancement of antioxidant activity of renal GSH and significant reduction of renal MDA levels (Sherif, 2015; Elsherbiny et al., 2016). Our findings revealed that TA and taurine administration increased GSH levels, suppressed the increase in MDA and protein carbonyls concentration observed in cisplatin group.

Inflammation plays a crucial role in the pathogenesis of cisplatin induced nephrotoxicity (Zhang et al., 2007). TNF-α plays a key role in mediating the inflammatory injury in cisplatin-induced toxicity (Yousef and Hussien, 2015). Concurrent with the induction of a stress-activated inflammatory response, many agents with anti-inflammatory properties are generated that may inhibit tissue damage (Deng et al., 2001). IL-10 is a multifunctional anti-inflammatory cytokine that has been reported to attenuate different renal pathologies (Kitching et al., 2002). TA and taurine at the doses administered significantly normalized the elevated levels of inflammatory cytokines and thus protected the kidney from inflammatory insult.

These results are further substantiated from histopathology, which revealed normal histology of a rat kidney tissue found in control group. However, histopathology of renal tissue sections from rats treated with cisplatin alone showed shrunken glomeruli, mononuclear cell infiltration, including acute tubular necrosis and marked dilation of proximal convoluted tubules. The cisplatin-induced histological alterations are in agreement with previous studies (Athira et al., 2016 and Malik et al., 2016). TA and taurine administration for 14 days significantly improved the cisplatin induced histological changes and showed minimum tubular damage, less necrotic damage and less accumulation of inflammatory cells.

In conclusion, the study revealed that cisplatin induces nephrotoxicity by oxidative stress, thus impairing the sero-biochemical parameters and histoarchitecture of kidney. The treatment with TA and taurine showed significant improvement in the function of kidney tissue protection and improvements in cisplatin induced toxicity due to the presence of marked antioxidant potential.

The authors are thankful to College of Veterinary science, PVNR TVU, Rajendranagar, Hyderabad, for providing necessary facilities for research work.