Renoprotective effect of Corallocarpus epigaea in Nephropathy in Wistar Rats

 

Amruta V. Yadav, Mohd. Bashar Momin, Md. Awais Panjwani

Department of Pharmacology, MCE Society’s Allana College of Pharmacy Camp, Pune.

 

ABSTRACT:

The renoprotective effect of standardized hydroalcoholic extract of Corallocarpus epigaea was evaluated against cisplatin induced nephrotoxicity. The evaluation was performed on 36 Male Wistar rats. The acute toxicity study was performed in accordance with OECD 425 guidelines. Considering the safety of the extract the screening was done at three dose levels of 100, 200, 400mg/kg. The study was carried for period of 15 days. Vitamin E (100mg/kg) was used as a Standard nephroprotective agent. The kidney function test like serum uric acid, creatinine, albumin, (BUN) was performed. Oxidative stress studies like superoxide dismutase, malondialdehyde activity along with histopathological examination of kidneys was also conducted. Serum creatinine and Urea were elevated in Cisplatin treated group. Decrease in serum albumin was noted in cisplatin control as compared to normal control. Corallocarpus epigaea (100, 200, 400mg/kg) and Vitamin E (100mg/kg) decreased the serum creatinine and BUN and increased the serum albumin level. Decrease in activity of superoxide dismutase was observed in the Cisplatin group as compared to control. Corallocarpus epigaea (100, 200, 400mg/kg) and Vitamin E (100mg/kg) increased superoxide dismutase levels and decreased malondialdehyde levels as compared to Cisplatin group. Histopathological examination of the kidneys revealed the amelioration of Cisplatin induced renal tubular necrosis. Dose dependant increase in the activity was observed. Present investigation revealed that Corallocarpus epigaea resulted in attenuation of Cisplatin induced renal damage in rats.

 

 

 

1. INTRODUCTION:

Renal failure is caused by decrease in glomerular filtration rate, azotemia, changes in urine volume, biochemical and haemodynamic processes. Development of functional or structural kidney damage due to exposure to drugs toxins or other treatments may lead to nephrotoxicity. This results on functional consequences causing glomerular or tubular dysfunction attenuation of blood pressure and/or kidney, endocrine function.¹ Nephrotoxicity is attributed to wide spectrum of drugs like antibiotics, immunosuppressants, antineoplastic agents, nonsteroidal antiinflammatory drugs, heroin, cocaine, environmental pollutants.² Anticancer drug Cisplatin (CP) is used in cancer chemotherapy

 

Dose dependant nephrotoxicity is developed by CP that limits its clinical usage in cancer chemotherapy. CP is a strong cellular toxin, nephrotoxicity being its most important complication in experimental models. Necrosis of renal tubular cells by activation of extrinsic and intrinsic mitochondrial pathways is caused by CP. The P53 mediated proapoptic activation of proinflammatory pathways is involved in its pathogenesis. Infiltration of inflammatory cells along with proinflammatory pathways are major mechanisms in CP induced nephrotoxicity.³ CP is accumulated in the kidneys to a greater extent as compared to other organs. Moreover, it also serves as major route for its excretion. The CP concentration in proximal tubular epithelial cells is 5 times greater than serum concentration.⁴ Thus leading to the disproportionate accumulation of CP in kidney tissue and attributing to nephrotoxicity.⁵ Nephrotoxicity induced by CP is mediated by mitogen activated protein kinase (MAPK) intracellular signaling pathways. The MAPK pathways are activated by diverse extracellular physical, chemical stresses that are involved in regulation of cell growth, differentiation and survival. In the proximal and distal convoluted tubules CP promotes cellular damage by mechanisms including oxidative stress, DNA damage and apoptosis.⁶ CP is interlaced to glutathione and metabolized through a gamma glutamy transpeptidase and cysteine S conjugate β lyase dependent pathways to a reactive thiol. Accumulation in kidney takes place by peritubular uptake.⁷ Platinum compounds exert cytotoxic effects by interaction with DNA. In an aqueous environment, the chloride ligands of CP are replaced by water molecules where they generate a positively charged electrophile. On reaction with nucleophilic sites on intracellular macromolecules it forms DNA, RNA, and protein adducts. A large number of medicinal plants are claimed to be useful in renal failure in all traditional systems of medicine. Though the plant Corallocarpus epigae (CE) and its extracts have been extensively used traditionally, information from an organized search of published literature does not provide the evidence for its nephroprotective activities. In the present study, we have investigated the renoprotective activity of hydroalcoholic extract of Corallocarpus epigae (HAECE) that belongs to family Cucurbitaceae. CE is tendril-bearing climbing herbs and the root is yellowish white; it has a bitter taste. It possesses laxative properties, and also used in syphilitic rheumatism and dysentery.⁸ Ethnomedical information suggests that the roots and rhizomes are having many traditional claims especially in syphilitic, venereal complaints and chronic dysentery. It is also an effective remedy for diabetes, herpes, and Anthelmintic, rheumatism and snake bite. Decoction of powder root is beneficial in chronic mucous enteritis.⁹

 

2. MATERIALS AND METHODS:

2.1. Chemicals:

Standardized (HAECA) was procured from Kuber Impex, Indore. Cisplatin cis-diamine platinum (II) dichloride was purchased from S.K. Enterprises, Pune. All other chemicals used were of the analytical grade.

 

2.2. Animals:

Male Wistar rats, weighing 200 to 250g were used for the study. They were maintained under standard laboratory conditions (Temperature 23±20⁰C, relative humidity 45 to 55±10%, 12:12hrs Light/dark cycles) throughout the experiments. The animals were acclimatized for 1 week before the study and had free access to standard laboratory food and water ad libitum. All experimental procedures were conducted in accordance with the principles for the care and use of laboratory animals in research and approved by the institutional animal ethics committee. (ACP/IAEC/2018/01).

 

3. EXPERIMENTAL PROTOCOL:

3.1 Acute oral toxicity:

Healthy adult female swiss albino mice (18-25g) were subjected to acute oral toxicity studies as per OECD (Organization for Economic Cooperation and Development) guideline 425. The animals were fasted overnight and divided into groups with 6 animals in each group. CE was administered orally by oral gavages at different doses of 175, 550, 1750 and 2000mg/kg. The mice were then observed continuously for behavior and autonomic profiles for 4 h and for any sign of toxicity or mortality up to 48 hours.¹⁰

 

3.2 Cisplatin induced nephrotoxicity:

The nephroprotective activity was tested on six groups of albino Male Wistar rats each group consisting of six animals.

Group I- Served as normal control and received 0.9% NaCl.

Group II- Disease control rats received 0.9% NaCl and CP (6 mg/kg; i.p)

Group III-Received Vitamin E 100mg/kg as Standard and CP (6 mg/kg; i.p)

Group IV- Received HAECE (100mg/kg; p.o) and CP (6 mg/kg; i.p)

Group V- Received HAECE (200mg/kg; p.o) and CP (6 mg/kg; i.p)

Group VI- Received HAECE (400mg/kg; p.o) and CP (6 mg/kg; i.p)

 

On the 15th day, 2 hours after the administration of extracts and 0.9% NaCl, groups II-VI received CP (6 mg/kg i.p). At the end of the experimental period, i.e on the 16^th day rats were sacrificed by cervical dislocation. The blood was allowed to stand for 30 min at 37°C and then centrifuged to separate the serum to evaluate the biochemical parameters.

 

Kidneys were removed and washed with ice-cold normal saline and homogenates of 10%, w/v was prepared using chilled tris hydrochloride buffer (10mM, pH 7.4). The homogenate was centrifuged at 7000rpm at 25 minutes. The clear supernatant was used for the determination of (MDA) Malondialdehyde, Superoxide dismutase (SOD).

 

3.2.1. Estimation of biochemical parameters:

Jaffe’s alkaline picrate method was used for creatinine estimation. The intensity of the orange color produced was directly proportional to the concentration of creatinine in the sample and was measured spectrophotometrically at 520nm. Albumin determination was done by Biuret method. The intensity of the violet color produced is directly proportional to the concentration of albumin in the sample and was measured spectrophotometrically at 540nm. The Blood Urea was estimated by Diacetylmonoxime method. The intensity of the color produced is directly proportional to the concentration of urea in the sample and was measured spectrophotometrically at 540nm.¹³

 

3.2.2. Estimation of MDA:

2ml of the kidney homogenate was added to 2ml of 10% w/v trichloroacetic acid (TCA) and the mixture was allowed to stand in an ice bath for 15 min. The mixture was centrifuged at 2500rpm at 4°C for 15 min. 2ml of clear supernatant solution was mixed with 2ml of freshly prepared thiobarbituric acid. The resulting solution was heated in a boiling water bath for 10 min. It was cooled in an ice bath for 5 min. The color developed was measured at 532nm against reagent blank.¹⁴

 

3.2.3. Estimation of SOD:

0.5ml of kidney homogenate was diluted with 0.5ml distilled water to which 0.25ml of ice cold ethanol and 0.15ml of ice cold chloroform were added. The mixture was centrifuged at 2500rpm at 4°C for 15 min. To 0.5ml of supernatant, 1.5ml of carbonate buffer and 0.5 ml of EDTA solution were added. The reaction was initiated by the addition of 0.4ml of epinephrine and the change in optical density/min was measured at 480 nm against reagent blank.¹⁵

 

3.2.4. Histopathology:

Fixation of the kidney was done by cutting and fixing in Bouin’s fluid immediately after removal from the animal body. The tissues were fixed in Bouin’s fluid for about 24 hours. The tissues were washed in glass distilled water to remove excess of picric acid. Dehydration was done. Xylene was clearing agent. The tissues were then kept in molten paraffin embedding bath maintained at about 50^oC. Glass plate was smeared with glycerine. The molten paraffin wax was poured. The tissue was placed in the paraffin and oriented for section. Then the tissue was pressed gently and allowed the wax to settle. The paraffin block was kept in cold water. Section cutting was performed by rotatory microtome. The excess of paraffin was removed by trimming, leaving half cm around the tissue. Microtome knife was adjusted to the proper angle. The tissue was cut in range of about 7µm. The sections were stained as deparaffinization with xylene two times for five minutes. Dehydration by ethyl alcohol. Staining with Ehrlich’s Haemaoxylin for 15-20 minutes. Then the tissues were washed in tap water for 10 minutes. Rinsed with distilled water and stained with Eosin. Dehydration with ascending grades of alcohol. Finally, the tissues were cleared with xylene two times for about 3 minutes. DPX mountant was applied uniformly and microglass cover slides were spread. The slides were observed in Nikon microscope.¹⁶

 

3.2.7. Statistical Analysis:

The results were expressed as mean±SEM. Comparison between the groups was made by one way analysis of variance (ANOVA) followed by "Dunnett's Test". P<0.05 was considered to be significant.

 

4. RESULT:

 

Fig. 1. Effect of HAECE on BUN

 

 

Fig. 2. Effect of HAECE on Creatinine

 

 

Fig. 3. Effect of HAECE on Albumin

 

 

Fig. 4. Effect of HAECE on SOD

 

 

Fig. 5. Effect of HAECE on MDA

 

4.1. Histopathology of kidneys:

 

Fig 5.1 Normal Control

Fig 5.2 CP (6mg/kg)

 

 

Fig 5.3 Vitamin E 100mg/kg

Fig 5.4 HAECE 100mg/kg

 

 

Fig 5.5 HAECE 200mg/kg

Fig 5.6 HAECE 400mg/kg

 

4.2. Biochemical analysis:

Serum creatinine, BUN and MDA was significantly (p<0.001) elevated in the CP group when compared to control group. CP group exhibited significant (p<0.001) decrease in albumin and SOD when compared to control. Significant (p<0.001) increase in the serum albumin level was found in CE (100, 200, 400mg/kg) and Vitamin E (250mg/kg) compared to CP group. Significant (p<0.001) decrease in activity of SOD was observed in the CP group as compared to control. CE (400mg/kg) and Vitamin E (250mg/kg) significantly (p<0.001) increased SOD levels. CE (100, 200mg/kg) significantly (p<0.05) increased SOD levels. CE (100, 200, 400mg/kg) significantly (p<0.01) decreased MDA levels as compared to CP group. Dose dependent increased in the activity was observed.

 

5. DISCUSSION:

Kidneys are vital organs of the body. It maintains the homeostatic balance and excretes the nitrogenous waste in the form of urine. Nephrotoxicity is an adverse effect of certain antibiotics, anticancer drugs and other synthetic molecules.¹⁷ CP is highly effective and alkylating agent, used for the treatment of solid tumors. But the clinical utilization of this heavy metal complex is restricted due to its major toxicities.¹⁸ Tubular damage by CP is characterized by reduced glomerular filtration rate, increased serum creatinine and blood urea concentrations. The pathophysiological mechanism of CP involves accumulation of CP in the kidney and its conversion to nephrotoxins. It is also characterized by DNA damage, mitochondrial dysfunction oxidative stress, inflammatory response, activation of signal transducers and intracellular messengers.¹⁹ The kidney being the principal excretory organ for injected CP is drastically affected by its accumulation and retention of platinum. In the rat, the kidney excretes the drug rapidly within the first hour of its administration by glomerular filtration. The uptake of CP by the kidney is dependent on temperature and the normal consumption of oxygen. It can be inhibited by drugs that participate in the organic base transport system. CP lowers glomerular filtration rate in a dose-dependent manner, even after single drug exposure. The nephrotoxic effect of CP is proportional to the amount of drug accumulated.²⁰ Renal failure occurs gradually, usually 3 to 5 days after its administration. In the present study, the protective effect of HAECE was evaluated against CP induced nephrotoxicity and degree of functional alterations in the kidneys by histopathological and biochemical estimations. CP (6mg/kg) was used to induce nephrotoxicity. Vitamin E was one of the antioxidant standard used to ameliorate CP induced nephrotoxicity in rats. Creatinine is the by product of muscle metabolism. Most of the creatinine is being filtered by kidney and maintained in the normal range. Impairment in the kidney function rises creatinine level in the blood causing poor clearance of creatinine. Abnormally high levels of creatinine thus are predictor of possible malfunction of the kidneys. Urea and creatinine are the end product of protein and muscle metabolism. If the kidney fails to remove these markers, their concentration increases in the blood and causes excess of protein excretion in the urine.²¹ Microalbumuria is the Urinary albumin to creatinine ratio. A ratio greater than 30-300mg/gm of creatinine is called as microalbumuria.²² The elevation in these renal biomarkers may be caused due to the impaired renal functions, tubular obstruction, or the back leakage of the renal tubules^.23 Hypoalbuminaemia is the strongest predictor of death in patients with renal failure. Patients with lower serum albumin level have consistently higher morbidity rates.²⁴. Treatment with the HAECE at the dose level of 100, 200 and 400mg/kg significantly lowered the level of creatinine, Urea when compared with the CP treated group. It also augmented the reduced levels of albumin. Lipid peroxidation is the propagation of oxidative insult to polyunsaturated fatty acids consisting of free radicals.²⁵ Increase in TBARS damages DNA induced by H₂O₂.²⁶ (MDA) is the final product of the oxidation of polyunsaturated fatty acids that serves as an indicator for oxidative stress estimation.²⁷ SOD is the primary line of defense against free radical induced oxidative stress. It causes catalytic dismutation of highly reactive and potentially toxic superoxide radical to hydrogen peroxide.²⁸ These antioxidant enzymes defend against free radicals that mediate tissue or cellular damage.²⁹ Increased ROS production in renal tissue attributed to changes in levels of MDA and SOD. MDA levels were significantly increased in rats treated with CP when compared to control group. HAECE significantly attenuated the MDA levels in renal tissue probably due to its capacity to scavenge oxygen free radicals in the kidneys. Furthermore, it also increased the levels of SOD resulting in improvement of kidney function. Impairment in the histological features of the kidneys was substantiated by CP treatment. Our study demonstrated histological changes in proximal and distal convoluted tubules, which were signs of tubular necrosis and atrophy of the vascular component in glomerulus in CP group. Vitamin E treated rat kidney sections showed similar features to the normal control. Recovery of renal function was observed by treatment of HAECE. The experimental results revealed that the renoprotective activity of the extract.

 

6. CONCLUSION:

CP caused an elevation in biochemical renal parameters and histopathological features of necrosis. The administration of HAECE ameliorated CP induced renal damage. Further studies are required to characterize the phytoconstituents from HAECE & to study the exact mechanism of action.

 

7. CONFLICT OF INTEREST:

All authors have none to declare.

 

8. AUTHORS CONTRIBUTION:

All the authors contributed equally in preparing, editing, and reviewing the article.

 

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Received on 05.10.2019         Modified on 03.12.2019

Accepted on 11.02.2020         © RJPT All right reserved