Nephroprotective Activity of Ethanolic Extract of Carissa carandas Leaves against Gentamicin- Induced Acute Kidney Injury in Wistar Albino Rats

The present study was aimed to evaluate the nephroprotective activity of ethanolic extract of Carissa carandas Linn. Leaves (EECC) in Gentamicin-induced nephrotoxicity in Wistar albino rats. The renal damage was induced by Gentamicin (80mg/kg body weight, i.p.). Nephroprotective activity was investigated by the administration of EECC at two different doses (100 and 200mg/kg body weight, p.o) for 28 days and by assessing serum parameters, renal oxidative stress markers and histopathological studies. Gentamicin-treated animals showed an increase in serum creatinine, uric acid, urea, and malondialdehyde (MDA) levels and decrease in total protein, reduced glutathione (GSH), and catalase(CAT) compared to normal control animals, which indicates severe nephrotoxicity. Histopathological studies of kidney Gentamicin-treated animals showed extensive acute tubular necrosis and peri-tubular inflammation. Administration of EECC showed a significant improvement (p<0.05) in biochemical and oxidative stress markers compared to the disease group. EECC treated groups showed better histological appearance when compared to the disease group. Ethanolic extract of Carissa carandas Linn. Leaves showed significant nephroprotective activity against gentamicin-induced acute kidney injury. Original Research Article Rajesham et al.; JPRI, 33(58A): 279-286, 2021; Article no.JPRI.78143 280


INTRODUCTION
Acute kidney injury (AKI) or acute renal failure (ARF) is a clinical condition where suddenly kidneys will be unable to filter wastes and toxic substances from the blood properly. It is mainly caused due to exposure of kidneys to nephrotoxic agents like drugs, diagnostic agents like radiocontrast agents, biological agents such as Recombinant leucocyte and interferon's, and some chemicals like heavy metals. It involves reduced GFR which leads to increased concentrations of creatinine, urea, uric acid, BUN, etc. and decreased concentrations of proteins and urine which is toxic [1,2]. ARF is also caused by altered blood circulation to the kidneys, low blood pressure, direct kidney damage, muscle breakdown, hemolytic uremic syndrome, and blockade of the urinary tract.
Need of the present investigation is Acute renal failure (ARF) is a life-threating disease with high mortality percentage. Two important mechanisms of ARF are inflammation and oxidative stress. Plants are rich source of antioxidant compounds and have a strong anti-inflammatory activity, so they may be useful for the treatment of ARF. Some herbal medicines are effective against different models of experimentally induced ARF such as cisplatin, gentamicin, glycerol, and ischemia-reperfusion injury.
In the present study, Nephroprotective activity of ethanolic extract of carissa carandas leaves against gentamicin-induced acute kidney injury in wistar albino rats was investigated.   [16] and kept for maceration [17] for 7 days with occasional stirring. On the 8 th day, the solvent was filtered by pressing with a muslin cloth and was evaporated in a rotary evaporator at 40 o C. The resultant extract was put in a desiccator for further drying. The dried ethanolic extract of Carissa carandas Linn. (EECC) was preserved for further studies.

Experimental Animals
Adult, healthy, male Albino-Wistar rats with an average weight of 150-200gms were acquired. Animals have been provided with 24-hour access with water and standard nutritional pellets, prior to and during the treatment. They were acclimatized under a period of one week under approved laboratory environment, i.e., 25 o C±1 o C temperature, 45-55% RH and also free access to food and water, after which they were employed in the experiment.

Evaluation of Nephroprotective Activity
The animals were divided into five groups with six animals in each group (n=6) and the treatment is as follows.

Histopathological Studies
The isolated kidneys preserved in 10% formalin were embedded in paraffin wax and longitudinally sliced by the use of a microtome. They were stained using hematoxylin and eosin (H&E) stain and observed under a trinocular microscope [20].

In vivo Antioxidant Studies
Lipid per oxidation (LPO) was determined by using TBARS method and malondialdehyde, which is the final product of lipid peroxidation pathway was measured at 532nm [21]. Reduced glutathione (GSH) was estimated by reaction with DTNB (5'5-dithiobis (2-nitrobenzoic acid)) according to Ellman's method [22] and Catalase (CAT) was estimated by reaction with hydrogen peroxide [23].

Statistical Analysis
All the results were analyzed by using one-way ANOVA followed by Dunnett's multiple comparison tests [24].

Preliminary Phytochemical Screening
The preliminary phytochemical screening showed the presence of various phytochemicals like flavonoids, phenolic compounds, triterpenoids, tannins, saponins, amino acids, proteins, and carbohydrates in EECC.

Kidney Weights
Kidney weights of animals on Day 29 were elucidated in Table1. There is a significant decrease in kidney weights of animals treated with EECC 100mg/Kg and 200mg/Kg when compared to disease and normal control.

Serum Parameters
The serum levels of creatinine, uric acid, urea, BUN, total protein, albumin, and globulin were elucidated in Table 2.

Histopathological Studies
The results of histopathological studies were shown in Fig. 1. In normal control group, kidney showed normal glomeruli, normal proximal and distal convoluted tubules. Severe distortion of glomeruli, and apical blebbing of proximal and distal convoluted tubules, interstitial oedema and interstitial haemorrhages were seen in disease control group. In 100 and 200 mg/kg of EECC treated group animals showed moderate reduction in the tubular damage and glomerular distortion, interstitial oedema, interstitial haemorrhages compared to disease control group. No significant changes were found in standard treated group.

Antioxidant Studies
The significant values of LPO, GSH, and CAT were elucidated in Table 3.

DISCUSSION
The present study indicates phytochemical and pharmacological evaluation of nephroprotective action of ethanolic extract of Carissa carandas Linn. in the doses of 100 and 200 mg/kg body weight. Preliminary phytochemical screening of EECC indicated the presence of alkaloids, flavonoids, saponins, carbohydrates, proteins, amino acids, tannins, and phenolic compounds.
Gentamicin (GM) which is widely used aminoglycosides antibiotic, is recognized as possessing significant nephrotoxic potential in man and experimental animals. GM-induced nephrotoxicity is characterized by elevated levels of urea, creatinine, uric acid, and BUN in plasma. The nephroprotective study was carried out and the serum and parameters like creatinine, uric acid, urea, BUN, total protein, albumin, and globulin were measured to assess the nephroprotective activity of EECC. There are two factors on which serum and urine parameters depend. One is the glomerular filtration rate (GFR) and other is the degree of tubular reabsorption. The observed effect may be attributed to a mechanism like increasing the renal blood flow and the attendant increase in GFR. The present study demonstrates that EECC significantly increased the GFR compared to disease control animals. EECC reduced the serum creatinine, uric acid, urea, and BUN and increased serum protein, albumin, and globulin. These observations indicate an improved renal function by EECC.
In the phytochemical evaluation EECC contains tannins and flavonoids. Flavonoids are known to be anti-inflammatory action and Tannins proved as vasodilatory activity [25]. Renal vasodilatation can improve the GFR and urine output. This could be for a reason using decreased accumulation of Creatinine, urea and uric acid. Hence terpinoids, tannins and flavonoids present in the EECC may have contributed to the protection from renal damage induced by gentamicin by their anti-inflammatory, antioxidant and vasodilatory actions.
Antioxidant parameters like LPO, GSH, and CAT were also evaluated. Reduced activity of CAT and levels of GSH after treatment with gentamicin suppresses endogenous enzymatic antioxidant machinery. Treatment with EECC increased the activity of CAT and levels of GSH significantly compared to the disease control animals. It was also observed that an increase in LPO in the disease group is due to altered antioxidant machinery and higher susceptibility towards oxidative damage. However, EECC lowered LPO levels in EECC treated groups. As per the findings, the secondary metabolite, flavonoids is present in the plant which is antioxidant in nature.
Oxidative stress activation and inflammatory process constitute a major part of gentamicin nephrotoxicity. In fact, nephrotoxicity induced by gentamicin is associated with the mitochondrial dysfunction in renal tubular cells. It causes a marked impairment in the activity of mitochondrial respiratory enzymes including NADH dehydrogenase resulting in the excessive ROS generation [26]. Furthermore, gentamicin has been reported to stimulate O 2 and NO • generation through activation of inducible isoform of NO • synthase (iNOS) in kidney tissue [27]. Reactive free radicals result in the lipid peroxidation, leading to the alteration of membrane lipid bilayer arrangement and reduction in antioxidant defense mechanisms, causing cellular damage and necrosis [28]. AECZ significantly reduced the oxidative/nitrosative stress markers such as MDA and NO • and further boosted the endogenous antioxidant parameters including SOD, catalase, and GSH, suggesting its in vivo antioxidant  effects. This antioxidant effect of AECZ might contribute to its nephroprotective activity. This may be responsible for kidney protective activity.
The histopathological results obtained correlated well with the biochemical results and standard and EECC treated groups showed significant improvement when compared to disease control.

CONCLUSION
The neprotective activity of EECC may be due to the single or combined effects of flavonoids, terpenoids, saponins, tannins, and phenolic compounds. But there is a high chance that flavonoids or tannins must have shown significant nephroprotective activity because as per literature flavonoids and tannins do possess good action on the urinary system. Thus we can conclude that Carissa carandas Linn. Possess nephroprotective activity.

CONSENT
It is not applicable.