Ameliorative Effect of Olea europaea Leaf Extract on Cisplatin-Induced Nephrotoxicity in the Rat Model

Background Olea europaea leaf extract (OELE) has potential health benefits and protects against cytotoxicity. This study investigated the possible ameliorative effect of OELE on cisplatin-induced nephrotoxicity in rats. Methods Rats were assigned into six groups; two groups received 150 mg/kg or 300 mg/kg of OELE, one group received a single dose of cisplatin (6 mg/kg) IP on the first day of the experiment, two groups received a single dose of cisplatin 150 mg/kg or 300 mg/kg of OELE on the first day then starting from the fifth day for 10 consecutive days, and one group acted as a control. Results and Conclusion. The findings showed that cisplatin-induced nephrotoxicity was evidenced by a significant increase in serum creatinine blood urea nitrogen (BUN) and a significant decrease in estimated creatinine clearance and potassium level, which corresponded with the alterations in the histopathology of the renal tissue. OELE significantly ameliorated the nephrotoxic effects of cisplatin as dose-dependent.


Introduction
Cisplatin is a platinum-derived chemotherapeutic agent frequently used as a standard antineoplastic drug for treating diferent types of malignancies in the lung, bladder, cervix, ovaries, and testes [1,2]. Cisplatin is eliminated through the renal system and accumulates in the renal tubules [3], leading to increased cisplatin concentrations in the proximal tubule cells that are fve times higher than the serum level [4]. Terefore, it is limited to patients with creatinine clearance greater than 60 ml/minute [5]. Although cisplatin induces various dose-limiting adverse efects, such as neurotoxicity, ototoxicity, gastrotoxicity, myelosuppression [2,3], cardiotoxicity [6], testicular toxicity [7], and hepatorenal toxicity [8], nephrotoxicity is the most prominent adverse efect encountered in clinical practice. Nephrotoxicity induced by cisplatin is often seen after 10 days of cisplatin administration and can present with various types of symptoms, including acute kidney injury (AKI), renal tubular acidosis, hypomagnesemia, hyperuricemia, and hypocalcemia [9]. Clinical evidence has revealed that onethird of patients experience AKI after cisplatin administration, along with increases in blood urea nitrogen (BUN), serum creatinine, reduced glomerular fltration rate (GFR), and disturbed electrolytes [10,11].
Olea europaea, also known as the olive tree, is an evergreen tree that is indigenous to the Mediterranean region, where it is widely utilized as traditional medicine for a variety of illnesses [12]. Among the diferent parts of the Olea europaea tree, the leaves have the highest antioxidant and scavenging capacity [13]. Te extracted leaves of Olea europaea have drawn attention since they have phenolic compounds that are linked to various pharmacological activities [14]. Several in vitro and in vivo studies have demonstrated wide range of health benefts of the extracted Olea europaea leaves, such as cytotoxic activity against human breast cancer cells [15], an antiproliferative efect on leukemia cells [16,17], an antiarrhythmic efect [18], a hypotensive efect [19], an anti-HIV efect [20], and an antimalaria efect [21]. Te wide range of these pharmacological activities is mostly linked to oleuropein and its bioactive byproduct, hydroxytyrosol, which comprises the major active compound in olive leaves [22]. Tis endogenous compound in OELE possesses empower antioxidant activities [14] that provide sufcient protection against the reactive oxygen species (ROS) resulted from cell damage or burst [22]. Several studies on the antioxidants of hydroxytyrosol and oleuropein have been performed. A recent study showed that both oleuropein and hydroxytyrosol have hypolipidemic and hepatoprotective efects against high-fat diet-induced metabolic disorders [23]. Te nephroprotective efect of Olea europaea leaf extract (OELE) has been reported on diclofenacinduced hepatotoxicity and kidney damage [24] and gentamicin-induced nephrotoxicity [25,26]. In addition, the ameliorative efect of the extracted Olea europaea leaves has been reported on nephrotoxicity induced by cyclosporin [27] and cyclophosphamide in an animal model [28]. In human embryonic renal epithelial "GP-293" cells, oleuropein has exhibited a protective efect against cisplatin-induced cellular toxicity [29]. However, the nephroprotective efect of diferent doses of OELE against cisplatin-induced nephrotoxicity in a rat model has not been evaluated yet. Tus, the aim of this study is to evaluate the ameliorative efect of diferent doses of the Olea europaea leaf extract against cisplatin-induced nephrotoxicity in rat models. It also aimed to study the effect of the extracted leaves and/or cisplatin on the body's and organs' weight and potassium level as a major complication of acute kidney injury.

Animals and Treatment.
Adult healthy Wistar male rats weighing 160 ± 20 g were brought from the Biology Department, Sana'a University. Animals were housed in a group of fve in each metal cage under hygienic conditions at a temperature of 23 ± 2°C, relative humidity of 50-60%, and 12 h of light/dark cycles. All experiments were carried out in the light cycle according to the protocol of the Animal Care and Use Committee, National Research Council (Washington, 2011). Animals were identifed by tail labeling and provided with standard rodent food with free access to water ad libitum. All procedures were reviewed and approved by the ethical committee at the University of Science and Technology (reference number: EAC/UST231). Before starting the experiments, the animals were kept for two weeks to ensure acclimatization.

Induction of Acute Kidney Injury (AKI).
Acute kidney injury (AKI) was induced in rats by intraperitoneal administration of a single cisplatin dose (6 mg/kg; Zuviplat ® ) according to previous studies [30][31][32]. After the induction of AKI, the rat had free access to food and water.

Plant and Sample Preparation.
Te fresh leaves of Olea europaea were collected from the olive trees in the Sana'a region at the beginning of September 2016. Te leaves were thoroughly washed using tap water to remove dust and then dried at room temperature in a dark place for 14 days. Te dried leaves were ground using a blender to a fne powder and then packaged in a glass container and stored at 4°C for further analysis.

Preparation of Olea europaea Leaf Extraction.
Te airdried leaves (170 g) were extracted with 95% ethanol (1 : 25 w⁄ v) using a Soxhlet apparatus for 24 hours and concentrated using a rotary evaporator (Büchi ® , Rotavapor R-200) at 40°C. Te resulting extract, 120 g (a yield of 70.6%), was stored at 15-20°C in sealed desiccators, and the drying process was carried out in closed and controlled equipment using a Labconco freeze dryer to improve the quality of the fnal product [33]. Te required amount of the extracted Olea europaea leaves was dissolved in normal saline and administered to the animals by oral gavage according to their body weight [33,34].

Acute Oral Toxicity
Study. An acute oral toxicity study of the extracted leaves was carried out according to the OECD 425 guidelines and methods of Jaykaran et al. [37]. After a two-week acclimatization period, twelve rats weighing 180 ± 20 g were randomly assigned into three groups, with four rats in each group, as follows: Group I served as a control group, which has free access to rodent food and water; Group II was given 2 g/kg of the extract of Olea europaea leaves; and group III was given 5 g/kg of the same extract for 72 hours. Te extract was previously dissolved in water and given to the animals by oral gavage (once a day) for 72 hours. Rats from the two treatment groups were observed individually for any sign of toxicity after the treatment, as described in [37,38].

Study
Design. Tirty male Wistar rats with age 2-3 months, weighing 160 ± 20 g, were randomly assigned into six groups, with fve rats per group, as follows: the control group, which had free access to water for 15 days; the 2 International Journal of Nephrology cisplatin (CIS) group was given a single intraperitoneal dose (6 mg/kg) of cisplatin and utilized as a positive control; two groups (treated groups: CIS/OELE-150/kg and CIS/OELE-300/kg) were given a single dose of intraperitoneal cisplatin (6 mg/kg) on the frst day, and then 150 mg/kg and 300 mg/ kg of the extracted leaves of Olea europaea, starting from the 5th day to the 15th day; two groups were given 150 mg/kg and 300 mg/kg (OELE-150/kg, and OEL-300/kg) of OELE for 15 days according to the method [28] and utilized as a negative control. On the sixth day of the experiment, blood samples were withdrawn for nephrotoxicity evaluation, and at the end of the experiment (15th day), blood samples were collected using a heart puncture under halothane anesthesia, and both kidneys were dissected for histopathological study [28]. 2.11. Histopathology of the Kidney. Te animals were sacrifced by decapitation under light anesthesia using halothane in desiccator specialized for anesthesia on the same day of blood collection. Both kidneys were dissected immediately. Te organs were washed using normal saline water and weighed. Both kidneys were preserved in 10% phosphate bufer and formalin for histopathologic investigations [40]. Kidneys were embedded in parafn wax and chopped into small serial pieces, cut at 4 μm using a rotary microtome. Tey were stained with hematoxylin and eosin and then observed under a light microscope (Leica Microsystems, Germany) by a histopathologist in a blind manner. Diferent histopathological alterations, including tubular necrosis, proteinaceous casts, and medullary congestion, were examined. Te severity of these alterations was categorized as follows: normal renal tissue indicating no damage, mild damage characterized by the presence of unicellular or patchy isolated damage, moderate damage representing renal damage of less than 25% but greater than the mild, severe damage representing renal damage between 25% and 50%, and very severe damage indicating damage exceeding 50% of the renal tissue [40].

Statistical Analyses.
All data were expressed as means ± standard error of the means (SEM). Statistical analysis was carried out using GraphPad Prism version 7.0 (GraphPad Software, Inc., San Diego, USA). Comparisons of biomarkers among the diferent study groups were evaluated by one-way ANOVA followed by Tukey's posttest. Two-way ANOVA followed by the Bonferroni posttest was also used to compare groups at diferent time points (the 6th day and the last day of the experiment as repeated-measures factors). Te results of this study are considered signifcant at a P value less than 0.05.

Findings of the Acute Oral Toxicity Study.
Administration of 2 g/kg and 5 g/kg of the extracted leaves to diferent groups of rats for 72 hours did not show signs of poisoning such as lacrimation, hair erection, convulsion, coma, or death, a signifcant diference in the body weights, compared to the control group. Te fndings showed no signifcant diference in the hemoglobin level (Hb) or white blood cell (WBC) and other biochemical studies compared with the control group. Tese fndings indicated that this plant has LD50 greater than 5 g/kg.

Efect of OELE and/or Cisplatin on Serum Creatinine Levels in Rats.
A single intraperitoneal dose of cisplatin (6 mg/kg) administered on the frst day of the experiment resulted in persistent AKI on the sixth and ffteenth days of the experiment, as evidenced by a signifcant increase in serum creatinine levels (P < 0.0001; Figure 1) compared to the control group. However, the administration of 300 mg/ kg of ethanol-extracted Olea europaea leaves on day 5 of the experiment showed a signifcant improvement (P < 0.0001) in serum creatinine when compared with the cisplatintreated group. Similar fndings were observed when a second blood sample was collected on the 15th day of the experiment. Te fnding revealed persistent kidney damage on the 15th day of cisplatin administration, as evidenced by a persistent increase in the serum creatinine levels (P < 0.0001), compared with the control group, as shown in Figure 2. However, continuous administration of 300 mg/kg of ethanol-extracted Olea europaea leaves showed a signifcant (P < 0.0001) improvement in serum creatinine levels compared with the cisplatin-treated group. On the other hand, the lower dose (150 mg/kg) of the extracted Olea europaea leaves did not show signifcant improvement in the levels of serum creatinine at the two time points (the 6th and the 15th days) of the experiment when compared with the cisplatin-treated group. Normal rats receiving either 150 mg/kg or 300 mg/kg of the extracted leaves alone did not show any alteration in serum creatinine levels compared to the control group.
International Journal of Nephrology Te efect of the time factor and continuous administration of the extracted leaves on serum creatinine is shown in Table 1. On the 15th day, all groups treated with cisplatin (cisplatin, CIS/OELE-150/kg, and CIS/OELE-300/kg) exhibited a signifcant decrease (P < 0.0001) in the mean serum creatinine levels compared to the levels observed on the 6th day of the experiment.

Efect of OELE and/or Cisplatin on Urea Levels in Rats.
Te ameliorative efect of diferent doses of OELE against cisplatin-induced nephrotoxicity was also evaluated by measuring blood urea levels on the 6th and 15th days of the experiment. Administration of cisplatin on day one resulted in AKI, as evidenced by the signifcant (P < 0.0001) increase in urea levels on both 6th and 15th days of the experiment when compared with the control group. However, continuous administration of 300 mg/kg of the extracted leaves by oral gavage on the 6th day resulted in a signifcant improvement in kidney function on the 6th ( ++ P < 0.01) and 15th ( + P < 0.05) days of the experiment when compared with the cisplatin-treated group. However, the lower dose of the extract (150 mg/kg) did not show a signifcant improvement in the urea levels on the 6th (P > 0.05) and 15th (P > 0.05) days of the experiment when compared with the cisplatintreated group, as shown in Figures 3 and 4. Table 2 presents the impact of cisplatin and/or extracted leaves on urea levels at two diferent time points. On the 15th day, all the groups treated with cisplatin (cisplatin, CIS/ OELE-150, and CIS/OELE-300) exhibited a signifcant decrease (P < 0.0001) in the mean blood urea levels compared to the levels observed on the 6th day of the experiment. Table 3 and Figure 5, cisplatin-treated groups showed a signifcant reduction in the estimated creatinine clearance compared to the control group (P value <0.0001). Administration of high doses of the extracted Olea europaea leaves showed nonsignifcant improvement in the estimated creatinine clearance. However, the low dose of the plant did not show any improvement in the estimated creatinine clearance.

Efect of OELE and/or Cisplatin on Electrolytes in Rats.
Te data illustrated a signifcant reduction in the serum potassium levels in the cisplatin-treated group on the 6th day (P < 0.05; Figure 6). Also, a further reduction was detected on the 15th day (P < 0.001; Figure 7) of the experiment compared with the control group. However, the administration of 150 mg/kg or 300 mg/kg of OELE for the cisplatintreated group resulted in a further reduction in potassium levels on the 6th and 15th days of the experiment, and the lower dose of OELE resulted in a signifcant reduction in potassium levels on the 15th day compared with the cisplatin group. No signifcant diferences in the potassium levels were observed when the rats received diferent doses of the extracted leaves of Olea europaea alone compared with the control group on both 6th and 15th days of the experiment.
Te efect of the time factor on the potassium levels was studied in the presence of other variables. As shown in Table 4, the fndings showed no signifcant (P > 0.05) differences in the potassium levels between the diferent groups on the 6th and 15th days of the experiment.

Efect of OELE and/or Cisplatin on Rats' Weight.
As shown in Table 5, regular increases in the body weight of rats were observed in the control group. Tere was a signifcant (P < 0.01) increase in the weight of rats between day 1 and day 15 of the experiment. However, all the cisplatin-treated groups (cisplatin, CIS/OELE-150, and CIS/OELE-300) showed a reduction in weight on day 15 compared with the weight on day 1. Tis efect was signifcant (P < 0.01 and P < 0.0001) when cisplatin was concurrently administered with either 300 mg/kg or 150 mg/kg of the extract, respectively.  Oral administration of 300 mg/kg of OELE alone prevented weight gain, while a lower dose (150 mg/kg) of the same extract did not prevent weight gain as appeared on day 15 when compared with the weight of the same rats on day 1.

Efect of Cisplatin and/or OELE on the Weight of Rats'
Kidneys on the Last Day of the Experiment. At the end of the treatment period, a signifcant increase (P < 0.05) in the weight of both kidneys was detected in the cisplatin-treated group when compared with the control group. In addition,   International Journal of Nephrology 5 normal rats that received diferent doses of the extract for 15 days showed a signifcant increase in the weight of the kidneys at a higher dose (300 mg/kg; P < 0.0001) and a lower dose (150 mg/kg; P < 0.05) of the extract compared with the control group. However, administration of a higher dose (300 mg/kg) of the extracted leaves to the cisplatin-treated group resulted in a signifcant (P < 0.01) reduction in the weight of the kidneys on the last day of the treatment period compared with the cisplatin-treated group. No signifcant (P > 0.05) diference in the weight of the kidneys was detected when the cisplatin-treated rats received a lower dose (150 mg/kg) of the same extract, as shown in Figure 7.

Te Efect of Cisplatin and/or OELE on the Histology of the Kidney.
Histopathological study showed several structural changes and alterations in the hematoxylin and eosin-  stained kidney section of cisplatin-treated rats. Tese alterations were shown as moderate interstitial infammatory cells mainly lymphocytes with multiple foci of tubulitis along with multiple foci of coagulative necrosis and calcifcation, as shown in Figures 8(b)-8(f). However, administration of 300 mg/kg of the extracted Olea europaea leaves ameliorated cisplatininduced interstitial infammation of the renal tissue to the mild stage, as shown in Figure 8(h). Moreover, the lower dose (150 mg/kg) of the extracted leaves in cisplatin-treated rats revealed amelioration of the interstitial infammation but induced hemorrhage, as shown in Figure 8(g). Control rats and normal rats received either 300 mg/kg or 150 mg/kg of OELE and showed normal morphology and histological structure of glomeruli and renal tubule, as shown in Figures 8(a), 8(i), and 8(j).

Discussion
Te renal system plays a crucial role in the regulation of blood volume and the excretion of toxic materials from the body. Kidneys have a high capacity level for drug uptake, making them vulnerable to drug toxicity and injury. An efcient therapeutic agent to attenuate cisplatin-induced nephrotoxicity is not yet available. Terefore, manipulation with an endogenous antioxidant by chemoprotective agents, such as natural products rich in antioxidants or supplementation with traditional antioxidants, has become an alternative modality. A single dose (6 mg/kg) of cisplatin was intraperitoneally injected into rats on the frst day of the experiment. Two doses (150 mg/kg and 300 mg/kg) of extracted Olea europaea leaves (OEL) were administered to diferent groups of rats by oral gavage, starting on the 5th day of cisplatin administration until the end of the experiment. Serum biomarkers about kidney function, as well as electrolyte changes, were measured on days 6 and 15 of cisplatin administration. Te outcomes of this work showed a signifcant increase in the serum creatinine, and blood urea nitrogen, and a signifcant decrease in the potassium level in cisplatin-treated rats was detected. However, administration of a higher dose of the extracted OEL ameliorated cisplatininduced AKI as evidenced by a signifcant reduction in the     International Journal of Nephrology serum creatinine and blood urea nitrogen. In other words, the reduction of toxic parameters means improvement in kidney function. Moreover, the weights of rats were studied in this experiment. It revealed that cisplatin and a higher dose of the extracted OEL prevented weight gain when administered separately and resulted in further reduction in body weight when administered concurrently. Furthermore, cisplatin and OELE caused a signifcant increase in rats' kidneys when administered separately compared to the control group. However, the weight of the kidney was signifcantly reduced when cisplatin and a higher dose of OELE were administered concurrently. Te fndings of histopathologic examination also confrmed the presence of renal tissue damage in the cisplatintreated rats. Tese damages were observed as interstitial infammation, lymphocyte infltration, multiple foci of tubulitis, coagulase necrosis, and calcifcation of the renal tissue. Tese consequences of cisplatin administration were ameliorated by the administration of diferent doses of OELE.
Te results of the acute toxicity study showed that the crude extract of OEL did not cause physical and behavioral changes as well as mortality during 72 hours of the follow-up toxicity period after administration of 2 g/kg and 5 g/kg of the extracted leaves to diferent groups. Te higher dose during the toxicity study represents more than 15 times the higher tested dose (300 mg/kg) of the extracted leaves. It has been revealed that a good candidate substance for further studies is one with a three times lethal dose (LD50) higher than the minimum efective dose 38, making the doses of the crude extract in this study suitable for further studies.
In the present study, a signifcant reduction in the body weight of all cisplatin-treated rats was found on the last day of the experiment. Te fndings of this study are in agreement with other studies that have shown a total body weight reduction after cisplatin administration [41][42][43][44]. It seems that weight reduction that occurs after cisplatin administration is attributed to cisplatin-induced anorexia and gastrointestinal disturbances [45,46]. Connected with weight reduction in cisplatin-treated rats, this study revealed a signifcant increase in kidneys' weight in this group compared with the control group. Tis study is in agreement with another fnding which shows a correlation between cisplatin treatment and an increase in kidney weight [43,47,48].
In this study, the administration of cisplatin signifcantly caused clinical and histopathological manifestations of kidney dysfunction, including weight loss, infltration of leukocytes, and necrosis. Moreover, cisplatin administration induced damage in the renal vasculature, leading to declining creatinine clearance. In this study, AKI was manifested as an increase in the serum creatinine level, blood urea nitrogen, and a decrease in the potassium level. Tis efect is in agreement with several experimental and clinical studies that showed that a single shot of cisplatin generated an overproduction of free radicals, which caused oxidative stress damage and polyunsaturated fatty acid peroxidation in the kidney tissue [43,49,50]. Diferent mechanisms of cisplatin-induced nephrosis have been illustrated in the literature.
Te frst underline mechanism of cisplatin-induced nephrosis is mostly attributed to the disturbances in the oxidative stress balance, which is manifested by the increase in the reactive oxygen species (ROS) production, especially superoxide anions and hydroxyl radicals and depleted in the enzymatic and nonenzymatic renal antioxidant defense system [51]. Excessive ROS results in destruction of cellular proteins and lipids as well as DNA [52]. However, administration of a higher dose of OELE caused improvement in the kidney function test which might be attributed to restore antioxidant defense capacity in the kidney. Te strong antioxidant activity of OEL has been linked to the presence of polyphenolic contents, including oleuropein, hydroxytyrosol, luteolin, and orthodiphenols; as a result, these compounds could prevent the generation of reactive oxygen species [53][54][55]. Te second underline mechanism involved in cisplatin-induced nephrotoxicity is the infammation of the renal tissue [56]. It has been revealed that cisplatin causes the promotion of a series of infammatory cytokines, such as IL-1β and TNF [57]. However, treatment using OEL prevented the production of infammatory cytokines, including TNF-α and IL-1β in many organs, including testicular tissue and gastric tissue [38,58,59]. Similarly, Al-Quraishy et al. reported that the gastroprotective efect of OELE was mediated by maintaining the antioxidant activity and restraining infammation of gastric mucosa by preventing the mobilization of the activated leukocytes and inhibiting nuclear factor-KB (NF-kB) [38]. In line with previous studies, the fndings of this study showed that OELE decreased cisplatininduced tubulointerstitial lesions, lymphocyte infltration, and coagulative necrosis of the renal tissue. Tis anti-infammation efect of OELE has a dosedependent efect.
Te third underline mechanism of cisplatin-induced acute renal damage is renal epithelial apoptosis [60]. Te potential molecular mechanisms of cisplatin-induced apoptosis are related to increase the expressions of Bax and p53 genes and suppressing the expression of Bcl-2. However, Olea europaea compounds have a benefcial efect against cisplatin-induced nephrotoxicity in vitro. For example, in the "GP-293" cell line, incubation of cisplatin-treated cells with oleuropein at a dose of 20 μg/ml could prevent cellular apoptosis by preventing the activation of caspase-3 and reducing Bax: Bcl2 elevation ratio [29]. Another study has shown that treatment with the extracted OEL prevents apoptosis by downregulating Bax and upregulating Bcl-2 in the testicular tissue of cisplatin-treated rats [58].
In this study, serum electrolyte imbalances were detected following cisplatin administration. For example, serum potassium was signifcantly decreased in cisplatin-treated groups as compared with the control group, and it showed that cisplatin had a cumulative efect on potassium reduction, meaning that the hypokalemic efect of cisplatin on the last day is more prominent compared with the potassium level on the 6th day of the experiment. Te higher dose of OELE (300 mg/kg) preserved potassium at a higher level compared with the lower dose of the extracted leaves. Tis might be related to the restoration efect of the kidney function by the higher dose of OELE, and thus maintained potassium level better than that of the lower dose. Te fndings of this study are in agreement with other fndings that showed a reduction in the potassium concentration in the cisplatin-treated group [42,47]. On the other hand, a study has shown that hyperkalemia is detected in rats following a single intraperitoneal cisplatin treatment [61].
In this study, a signifcant improvement in kidney function was observed in the second time frame when compared to the frst-time frame. Tis improvement might result from the ameliorative efect of this extract. Another explanation for this improvement was the washout that happened to the cisplatin concentration from the body after a period of 15 days of administration.

Conclusion
In conclusion, this study reveals that a single intraperitoneal dose of cisplatin to Wistar rats induces histopathological alterations of the renal tissue, appearing as moderate interstitial infammation and coagulative necrosis and calcifcation along with AKI and electrolyte disturbances as evidenced by elevated serum creatinine and blood urea nitrogen and decrease in the serum potassium level. However, the administration of the extracted Olea europaea leaves markedly ameliorates cisplatin-induced acute renal injury as explored by improving the renal tissue and reducing creatinine and blood urea nitrogen levels in cisplatintreated rats, and this efect is obvious with a higher dose of the extract and at the second time point, which proved that the benefcial efect of this extract on renal function is both dose-dependent and time-dependent.

Limitation.
Tis study had several limitations. One limitation was that this study did not assess renal oxidative stress biomarkers, infammatory markers, and genetic testing. Tis limitation had consequences on the interpretation of the underline mechanism of the ameliorative efect of the crude extract. In addition, this study evaluated the efect of the crude extract, making the identifcation of the potential active compound that has the ameliorative efect another limitation. Terefore, further studies are recommended to address this limitation to understand the exact underline mechanism of this efect and to identify the potential active compound that exhibits the ameliorated efect of extracting Olea europaea leaves.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te study was carried out after getting permission from the Ethics Committee (reference no.: EAC/UST231), Faculty of Medicine and Health Science, University of Science and Technology, Sana'a, Yemen. Te Committee approved all study protocols according to the International Guide of Laboratory Animal Care and Use.

Conflicts of Interest
Te authors declare that they have no conficts of interest.