Investigation of Protective Effect of Hydroalcoholic Extract of Walnut Tree Leaves ( Juglans regia ) on Gentamicin-Induced Nephrotoxicity in Male Wistar Rats

Abstract

Objective/background

The main side effect of gentamicin, an aminoglycoside antibiotic, is kidney toxicity. This side effect leads to electrolyte disorders and retention of nitrogen waste products such as urea and creatinine. This study aimed to investigate the protective effect of the hydroalcoholic extract of the walnut tree (Juglans regia) on gentamicin-induced nephrotoxicity in male Wistar rats.

Methods

The experiment involved five groups of rats: a control group, a gentamicin-treated group, and three groups treated with different doses of walnut leaf extract (100, 200, or 400 mg/kg) via intragastric gavage for 14 consecutive days (once per day). After 14 days, blood samples were collected and analyzed for various biochemical markers. Kidney tissue was also examined histopathologically.

Results

Findings showed that gentamicin significantly increased levels of uric acid (P < .001), urea (P < .001), creatinine (P < .01), albumin (P < .001), and lipid profile while decreasing the activity of antioxidant enzymes SOD (P < .001) and CAT (P < .001) compared to the control group. It also caused kidney tissue damage. Treatment with walnut leaf extract could significantly reduce urea in all treatment groups. However, the reduction in creatinine levels after treatment with walnut leaf extract wasn’t significant. Further, walnut leaf extract at doses of 200 and 400 mg/kg could significantly reduce albumin and uric acid levels (P < .001). Walnut leaf extract significantly improved lipid profile. Also, walnut leaf extract significantly increased SOD and CAT activity in all doses. Walnut leaf extract significantly reduced kidney tissue damage, including inflammation, necrosis, and tubular degeneration.

Conclusion

The results of this study conclude that walnut tree leaf extract positively affects kidney function and histology in gentamicin-treated rats. This extract or similar compounds could be an antioxidant treatment for people with renal impairment or chronic kidney disease.

Graphical abstract

This is a visual representation of the abstract.

Keywords

gentamicin nephrotoxicity oxidative stress rat in vivo

Introduction

The Juglans regia tree is a prominent member of the deciduous tree family, commonly found in temperate countries. It is cultivated on a large scale in southern Europe, the northern part of Africa, eastern Asia, the USA, and western Latin America.¹ The fruit of this tree is highly sought-after and widely consumed as an integral component of the Mediterranean diet. In addition to dried seeds (nuts), many parts of the walnut tree, such as husks, skins, green skins (epicarps), and leaves, are utilized in the cosmetic and medicinal sectors.²

Walnut leaves are regarded as a reservoir of bioactive substances such as phenolic acids and flavonoids.³ A study in 2013 determined the flavonoids, phenolic acids, and juglone in walnut tree leaves through HPLC analysis and declared that the concentrations of rutin, catechin hydrate, and myricetin are high and the concentrations of epicatechin aglycones and quercetin are low. Ellagic acid and then trans-cinnamic, chlorogenic, and caffeic acids were the dominant phenolic acid constituents of the walnut leaves. Besides, Juglone's content was between 44.55 mg and 205.12 mg/100 g in fresh weight.⁴ Due to their antidiarrheal, anthelmintic, antiseptic, and astringent characteristics, they are extensively utilized in traditional medicine to treat skin inflammations, hyperhidrosis, and wounds.⁵ Recently, studies have been conducted to describe the anti-radical and antibacterial activities of different varieties of J. regia.^6,7 They are considering the widespread use of gentamicin in infections. A study by Carvalho et al in 2010 showed that the methanolic extract of J. regia seeds has higher total phenolic content and 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than the green husk and leaves. However, the methanolic extract of leaves showed stronger suppression of 2,20 –azobis (2-amidinopropane) dihydrochloride (AAPH) induced oxidative hemolysis than the husk and seed extract.⁸ Another study showed that J. regia male flowers have a high free radical scavenging property and the is a correlation between this property and its total phenolic content.⁹

The kidneys are an essential organ whose functions include maintaining body homeostasis and detoxifying and excreting toxic metabolites and drugs.¹⁰ Kidney injury is a severe condition that is typically linked to high death rates mostly caused by cancer and cardiovascular disease.¹¹ It happens from chemotherapy and antibiotics. Kidney damage can lead to inefficiency, apoptosis, and necrosis of renal tubule epithelial cells. Kidney diseases are one of the leading public health issues that are increasingly spreading and are considered among the 12 most common causes of death in the world.¹² The kidneys’ ability to concentrate and metabolize chemicals makes them susceptible to toxic damage.¹³

Gentamicin (GN) is an aminoglycoside antibiotic frequently used to manage infections that arise from aerobic gram-negative bacteria. Despite its effectiveness in eradicating Gram-negative bacteria, nephrotoxicity and ototoxicity caused by GN are considered major side effects limiting its efficacy.^14,15 Given gentamicin's continued widespread usage and effectiveness in treating infections, it is crucial to identify strategies for mitigating its nephrotoxic effects. The exact and comprehensive mechanism of gentamicin-induced kidney damage remains a subject of discussion and an incomplete narrative.¹⁶ The medication has the potential to build up in the cells lining the tubules of the epithelium, resulting in many consequences. These include the initial deterioration of the brush boundary in the epithelial cells, followed by severe tubular necrosis, programmed cell death activation, and extensive protein breakdown.¹⁶ GN induces cell death by producing free radicals, phospholipidosis, activation of the extracellular calcium-sensing receptor, energy disruption, decreased blood flow to the kidneys, and inflammation.¹⁷

Gentamicin-induced nephrotoxicity results in oxidative stress due to heightened Reactive oxygen species (ROS) generation and reduced enzyme antioxidant defense.¹⁸ This leads to the buildup of free radicals and subsequent oxidative cascade events. Specifically, the kidney is more susceptible to reactive oxygen species (ROS) due to its elevated lipid content, resulting in lipid peroxidation inside the renal tissue.¹⁹

Multiple studies have examined the nephroprotective effects of substances with antioxidant properties in animals and plants. It has been demonstrated that certain plants with antioxidant and anti-inflammatory mechanisms possess nephroprotective benefits.^20–22 The objective of this study is to examine the potential protective properties of the hydroalcoholic extract derived from the walnut tree (Juglans regia) as a cost-effective and highly efficient chemical in countering the nephrotoxic effects generated by gentamicin in male Wistar rats. To our knowledge, there is no study available assessing this property of J. regia leaves. However, only a study in 2018 revealed the nephron-protective and antioxidant effects of J. regia vegetable oil against lead-induced nephrotoxicity.²³

Materials & Method

Collection, Identification, and Preparation of Plants

The leaves of walnut trees were gathered from trees in Damavand, a city inside the Tehran province. The Plant Herbarium Research Center identified the plant sample. The herbarium sample of Juglans regia L Iranian walnut plant from the Juglandaceae family with herbarium voucher number NO: 1650_AUPF is kept in Tehran Medical Sciences Islamic Azad University.

Preparation Method of Hydroalcoholic Extract

Walnut leaves are dried in the shade and then turned into powder. One hundred grams of the obtained powder was poured into a one-liter beaker, and 96% ethanol was added to cover the powder's surface. After 24 h, the solution was filtered. In the next step, 75% alcohol was added to the remaining residue and filtered after 24 h. The filtered solution of the first and second stages was mixed and concentrated by a vacuum distillation device at a temperature of 50 degrees Celsius and a rotation speed of 70 revolutions per minute to one-third of the initial volume.²⁴

The concentrated solution is decanted with 50 ml of chloroform three times to separate the protein, fat, and chlorophyll. The solution obtained from the last step was dried in an autoclave at a temperature below 50 degrees Celsius and under sterile conditions. In this way, after a few days, the dry powder of the extract is prepared. The dried powder was stored at 4 degrees Celsius.²⁵

Experimental Design

The experiment was performed on adult Wistar rats weighing 230 and 250 g. The animals were purchased from Institute Pasteur, providing unlimited water and food access. During the experiment, rats were housed in a polycarbonate cage with an ambient temperature of 22 °C, 12 h of daylight, and 12 h of darkness. According to Code of Ethics 3604, animal testing was supervised by the animal labor ethics committee of Tehran Medical Sciences, Islamic Azad University. The current study randomly divided twenty-five rats into five groups (five in each), including a control group, a group with single dose Intraperitoneal injection of gentamicin for 14 days, and three experimental groups with different doses of walnut leaf extract. The experimental treatment groups were administered a daily oral dose of walnut leaf extract mixed in .5 ml of distilled water for 14 consecutive days via intragastric gavage.

Animal Grouping

Control group: intact animals with free availability to water and pellets (n = 5)

Gentamicin group: animals receiving gentamicin at a dose of 100 mg/kg body weight + daily treatment with 0.5 ml of distilled water (drug solvent) (n = 5)

Treatment groups 100 (3a), 200 (3b), 400 (3c): rats poisoned with gentamicin at a dose of 100 mg/kg bw + Walnut tree leaf extract administered orally once daily at doses of 100, 200, or 400 mg/kg bw (n = 15)

The doses of the extract have been chosen based on the previous literature to ensure that the extract may not cause toxicity in rats and at the same time might have the therapeutic effect.^26–28

Biochemical Analysis

Following a 14-day treatment period, specimens of blood were gathered through intra-cardiac puncture after administering anesthesia via a 100 mg/kg dose of ketamine (Rotexmedica Co., Germany) and a 10 mg/kg infusion of xylazine (Alfasan Co., Holland).²⁹

Measuring the serum levels of Chol, TG, HDL, and LDL in the serum, based on the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) method and using the respective specific kits, with the help of the device Autoanalyzer Italy, BT4500 was performed (1999, Moss and Henderson). The levels of uric acid, creatinine, urea, and albumin in the blood of rats were determined via laboratory kits supplied by Pars Azmoun, an Iranian firm.

Histopathology

During 14 days of therapy, morphological changes in the kidney were assessed in the control group, gentamicin group, and three experimental groups treated with 100, 200, and 400 mg/kg of walnut leaf extract. The organs were gathered and preserved using a 10% formalin solution. They were then enclosed in paraffin and sliced into slices that were 5 micrometers thick. The immovable portions were stained with hematoxylin and eosin (H and E) for analysis. The slices were examined utilizing a binocular microscope (Olympus CH-2, Tokyo, Japan), and photographs of the preserved organs were obtained.

Statistical Analyses of Data

The results are displayed as the average value plus or minus the standard deviation. The data was analyzed using one-way analysis of variance (ANOVA), and then the multiple comparisons were conducted using the Tukey HSD test. The p-value level was determined at P < .05.

Results

This study aimed to investigate the defensive impact of hydroalcoholic extract derived from the walnut tree (Juglans regia) on nephrotoxicity produced by gentamicin in Wistar rats.

The Effect of Walnut Tree Leaf Extract Treatment on Serum Urea Level in Rats Poisoned with Gentamicin

Results showed that gentamicin poisoning resulted in a substantial elevation in urea levels (P < .001) compared to the Control group. According to Figure 1a, this study demonstrated that administering walnut leaf extract at 100, 200, and 400 mg/kg reduced urea levels compared to the group poisoned with gentamicin. The observed changes were statistically significant in all three treatment groups, with dosages of 100 mg/kg (P < .01), 200 mg/kg (P < .001), and 400 mg/kg (P < .001).

Figure 1.

(a) The effect of walnut tree leaf extract treatment with A variety of doses (100, 200, and 400 mg/kg) on the amount of serum urea in rats poisoned with gentamicin (b) the effect of walnut tree leaf extract treatment with A variety of doses (100, 200 and 400 mg/kg) on the amount of serum creatinine in rats poisoned with gentamicin (c) the effect of walnut tree leaf extract treatment with A variety of doses (100, 200 and 400 mg/kg) on the amount of serum uric acid in rats poisoned with gentamicin (d) the effect of walnut tree leaf extract treatment with A variety of doses (100, 200 and 400 mg/kg) on the amount of serum albumin in rats poisoned with gentamicin.

The Effect of Walnut Tree Leaf Extract Treatment on Serum Creatinine Level in Rats Poisoned with Gentamicin

According to the results of this study, gentamicin poisoning caused a substantial rise (P < .01) in serum creatinine compared to the control group. Based on Figure 1b, treating animals with walnut tree leaf extract with concentrations of 100, 200, and 400 mg/kg caused a decrease in serum creatinine in comparison with the gentamicin group. However, these changes were not significant in any of the treatment groups.

The Effect of Walnut Tree Leaf Extract Treatment on Serum Uric Acid Level in Rats Poisoned with Gentamicin

Based on this research, gentamicin poisoning caused a significant increase (P < .001) in the amount of uric acid compared to the control group. According to Figure 1c, this study demonstrated that administering walnut leaf extract at 100, 200, and 400 mg/kg reduced blood uric acid levels compared to the group poisoned with gentamicin. These changes were significant only in the 200 and 400 mg/kg treatment groups (P < .001).

The Effect of Walnut Tree Leaf Extract Treatment on Serum Albumin Level in Rats Poisoned with Gentamicin

According to the results of this study, gentamicin poisoning caused a substantial rise (P < .001) in the amount of serum albumin compared to the control group. Based on Figure 1d, the results of this study also demonstrated that treating animals with 100, 200, and 400 mg/kg walnut tree leaf extract decreased the amount of albumin compared to the group poisoned with gentamicin. However, these changes were significant only in the 200 and 400 mg/kg treatment groups (P < .001).

The Effect of Walnut Tree Leaf Extract Treatment on Serum Triglyceride Level in Rats Poisoned with Gentamicin

The current investigation found that gentamicin poisoning led to a Considerable elevation (P < .001) in serum triglyceride levels compared to the control group. According to Figure 2a, the study found that administering walnut tree leaf extract at 100, 200, and 400 mg/kg to animals reduced triglyceride levels compared to the group poisoned with gentamicin. However, these changes were significant in the 200 and 400 mg/kg treatment groups (P < .001).

Figure 2.

(a) The effect of walnut tree leaf extract treatment with different doses (100, 200 and 400 mg/kg) on serum triglyceride levels in rats poisoned with gentamicin (b) the effect of walnut tree leaf extract treatment with different doses (100, 200 and 400 mg/kg) on serum cholesterol levels in rats poisoned with gentamicin (c) the effect of walnut tree leaf extract treatment with different doses (100, 200 and 400 mg/kg) on serum LDL levels in rats poisoned with gentamicin (d) the effect of walnut tree leaf extract treatment with different doses (100, 200 and 400 mg/kg) on serum HDL levels in rats poisoned with gentamicin.

The Effect of Walnut Tree Leaf Extract Treatment on Serum Cholesterol Level in Rats Poisoned with Gentamicin

The current investigation found that gentamicin poisoning led to a substantial elevation (P < .01) in blood cholesterol levels compared to the control group. According to Figure 2b, the findings of this study indicate that administering walnut leaf extract at doses of 100, 200, and 400 mg/kg to animals Reduced blood cholesterol levels compared to the group that was poisoned with gentamicin. These changes were significant in all three treatment groups in doses of 100 and 200 mg/kg (P < .01) and 400 mg/kg (P < .001).

The Effect of Walnut Tree Leaf Extract Treatment on Serum LDL Level in Rats Poisoned with Gentamicin

The current investigation found that gentamicin poisoning led to a Significant elevation (P < .001) in serum LDL levels compared to the control group. According to Figure 2c, the study found that treating animals with walnut leaf extract at 100, 200, and 400 mg/kg reduced LDL levels compared to the group poisoned with gentamicin. The changes in the treatment groups with 200 and 400 mg/kg were highly significant (P < .001).

The Effect of Walnut Tree Leaf Extract Treatment on Serum HDL Level in Rats Poisoned with Gentamicin

The current investigation found that gentamicin poisoning significantly reduced (P < .001) serum HDL levels compared to the control group. According to Figure 2d, this study found that treating rats with walnut tree leaf extract at 100, 200, and 400 mg/kg resulted in a dose-dependent rise in HDL blood serum levels compared to the poisoned group. These changes were observed specifically in the treatment groups. The results were statistically significant at 200 and 400 mg/kg (P < .001).

The Effect of Walnut Tree Leaf Extract Treatment on the Level of Superoxidase Dismutase (SOD) Enzyme Activity in Kidney Homogenized Tissue in Rats Poisoned with Gentamicin

The study's findings indicate that gentamicin poisoning significantly reduced the superoxide dismutase enzyme activity compared to the control group (P < .001). According to Figure 3a, the study found that treating mice with 100, 200, and 400 mg/kg of walnut leaf extract resulted in higher enzyme activity than the gentamicin group. These changes were significant in all three treatment groups with a dose of 100 mg/kg (P < .05) and doses of 200 and 400 mg/kg (P < .001).

Figure 3.

(a) The effect of walnut tree leaf extract treatment with different doses (100, 200, and 400 mg/kg) on the level of super oxidase dismutase enzyme activity in kidney homogenized tissue in rats poisoned with gentamicin (b) the effect of walnut tree leaf extract treatment with different doses (100, 200 and 400 mg/kg) on the level of catalase (CAT) enzyme activity in kidney homogenized tissue in rats poisoned with gentamicin.

The Effect of Walnut Tree Leaf Extract Treatment on the Catalase (CAT) Enzyme Activity Level in Kidney Homogenized Tissue in Rats Poisoned with Gentamicin

The current study's findings indicate that gentamicin poisoning substantially reduced catalase enzyme activity compared to the control group (P < .001). According to Figure 3b, the study found that treating animals with 100, 200, and 400 mg/kg of walnut leaf extract increased catalase enzyme activity compared to the group poisoned with gentamicin. These changes were significant in all three treatment groups with 100, 200, and 400 mg/kg (P < .001).

Histopathology Results

As shown in Figure 4. (a) In the kidney cross-section in the healthy control group, the glomerulus (G) and healthy ureters (arrow sign) can be seen (H&E, 10X). (b) Kidney cross-section in the healthy control group, glomerulus (G), and healthy ureters (arrow sign) can be seen (H&E, 40X). As shown in (c), severe necrosis of the urinary tubes (arrow) can be seen in the cross-section of the kidney in the control group of the gentamicin patient (H&E, 40X). Also, the cross-section of the kidney in the control group of the gentamicin patient shows (d) infiltration of mononuclear inflammatory cells (arrowhead) and necrosis of the urinary tubes (arrow) (H&E, 40X). as shown in (e) In the kidney section of the experimental group of mice treated with a dose of 100 mg/kg of the extract, severe necrosis of urinary tubes (arrow) and inflammatory cells (arrowhead) can be seen (H&E, 40X). (f) In the kidney section of the experimental group of mice treated with a dose of 200 mg/kg of the extract, Significant infiltration of mononuclear inflammatory cells (arrowhead) and necrosis of urinary tubules (arrow) are seen (H&E, 40X). (g) In the kidney section of the experimental group of mice were treated with a dose of 400 mg/kg of the extract, Mild infiltration of mononuclear inflammatory cells (arrowhead) and mild necrosis of urinary tubules (arrow) are seen (H&E, 40X).

Figure 4.

Histopathology image of kidney tissue (a) An image of the kidney tissue from the control group at a magnification of 10x.b) The kidney tissue of a control patient group magnified 40x (c) The kidney tissue of gentamicin-treated patients in the control group (d) Control kidney tissue from a patient treated with gentamicin (e) Kidney tissue from patients treated with 100 mg/kg of the extract (f) The kidney tissue of a patient group treated with an extract dose of 200 mg/kg (g) The kidney tissue of patients treated with a dose of 400 mg/kg of the extract was examined.

Discussion

A kidney removes toxic substances from the bloodstream and produces urine, controlling extracellular fluid levels. Therefore, the kidneys are vulnerable to nephrotoxic and xenobiotic agents, including drugs or environmental toxins that can cause various harmful effects.²⁵

In some people, the kidneys lose their ability for several reasons, and they suffer from kidney failure, which causes the accumulation of toxins and water and various symptoms. The kidney failure leads to electrolyte disorders and retention of nitrogen waste products such as urea and creatinine. The two main factors in kidney damage are oxidative stress and inflammatory reactions.²⁹ Antibiotics are often used to treat infections.³⁰ GN is a commonly employed aminoglycoside antibiotic for treating Gram-negative bacteria. The main side effect caused by the antibiotic gentamicin is its nephrotoxicity. Symptoms of this disease occur in about 30% of patients after seven days of treatment with gentamicin. Gentamicin-induced nephrotoxicity is associated with renal inflammation, elevated renal oxidative stress factors, and increased pathological signaling mechanisms.³¹

The details of gentamicin-induced nephrotoxic mechanisms are still unknown. Several studies have demonstrated different causes, such as tubular necrosis, phospholipidosis, oxidative stress, apoptosis, and elevated endothelin I level.³² as well as have shown reducing the function of kidney antioxidant enzymes such as superoxide dismutase (SOD) and reducing glutathione (GSH).³³

Accumulation of gentamicin in these cells eventually leads to morphological changes, functional disorders, and a rise of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the kidney.¹⁶ Free radicals also suppress the kidney's antioxidant system through protein oxidation and lipid peroxidation (LPO).¹⁶ To solve this problem (Gentamicin-induced nephrotoxicity), various recent studies have shown that using natural antioxidants is a suitable method to protect the kidney against gentamicin nephrotoxicity. Aside from that, low levels of antioxidants have been reported to accelerate the process of nephrotoxicity.³² Therefore, studies on new natural antioxidants that benefit gentamicin nephropathy are still crucial.

Nowadays, phenolic compounds are the most important natural antioxidants that help reduce the occurrence of diseases related to oxidative stress.³⁴ The seeds, green skins, and leaves of Persian walnut, Juglans regia L (JRL), are Affluent sources of antioxidants. Flavonoids, phenolic acids, and naphthoquinones are the main phenolic compounds in walnut leaves. Evidence suggests that the beneficial impacts of walnut tree leaf extract include antioxidant and anti-inflammatory effects.³⁵ Based on a HPLC/DAD analysis of study in 2007 on the aqueous extract of walnut tree leaves, 3-caffeoylquinic acid, 5-caffeoylquinic acid, 3-p-coumaroylquinic acid, 4-p-coumaroylquinic acid, p-coumaric acid, quercetin 3-galactoside, quercetin 3-pentoside derivative, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside were the phenolic compounds identified in the extract.⁵ Another study in 2023 declared that based on their HPLC analysis, quercetin, hydroquinone, 4-hydroxybenzoic acid, and caffeic acid in three fractions are available in the walnut leaves.³⁶ A further study manifested that quercetin-3-O-rhamnoside, quercetin-3-O-xyloside, quercetin-3-O-arabinoside, quercetin-3-O-galactoside, kaempferol-O-pentoside derivative and kaempferol-O-pentoside are the major components of the J. regia leaves extract based on their HPLC-ESI-MS/MS analysis. Also, they elucidated that the extract can decrease reactive oxygen species (ROS) and this might be due to its flavonoid content.³⁷

This exogenous antioxidant source (walnut leaf extract here) plays a crucial function in the balance of oxidative stress. Since our endogenous antioxidant function is insufficient, it provides additional antioxidant potential to living organisms. According to research, the green leaf extract of the walnut tree shows the highest antioxidant potential.³⁸ In the current study, gentamicin was used to induce kidney damage. Previous research shows that injecting gentamicin at a dose of 100 mg/kg intraperitoneally in rats can cause kidney damage.³⁹ In this study, in agreement with the previous findings, the dose of gentamicin was selected, and rats were poisoned with a dosage of 100 mg per kilogram intraperitoneally for two weeks. The results showed that gentamicin increases kidney health factors, including urea, creatinine, uric acid, and kidney damage. The kidney collects these factors from the blood plasma and excretes them through the urine, which increases the blood serum due to damage to the kidney and its dysfunction.

During a study, gentamicin was utilized to induce nephrotoxicity in rats. This study showed that the induction of gentamicin (with a dose of 80 mg/kg every fourteen days intraperitoneally) to rats, through the induction of oxidative stress, lipid peroxidation, and DNA damage, causes serious kidney damage. Gentamicin induction increased total bilirubin, urea, and creatinine in the serum of rats poisoned with gentamicin.⁴⁰ Multiple studies have mitigated Gentamicin's toxicity. Gentamicin decreased the activity of antioxidant enzymes, specifically catalase and superoxide dismutase, in kidney samples of poisoned rats. This was due to the induction of oxidative stress and cellular damage. These findings align with the outcomes of research conducted in 2011.⁴¹

Histological results indicate the effect of GN on kidney cells and the creation of inflammatory cells, degeneration of nephron tubes, and tissue necrosis. According to Babaeenezhad et al, a similar pattern was also observed.³⁹

In this study, hydroalcoholic extract of the walnut tree was administered at three different dosages (100, 200, and 400 mg/kg) to three groups to reduce uric acid, creatinine, and urea levels. This suggests that kidney function was enhanced in a dose-dependent manner. In the study of the effect of the hydroalcoholic extract on the reduction of blood serum urea, this reduction was significant in all three doses of the extract. Still, in the case of creatinine, this reduction was insignificant in any of the groups, although it caused a reduction in the level in all three groups. Creatinine. In the case of uric acid, it caused a dose-dependent decrease, although this decrease was significant in the 200 and 400 mg/kg dose groups. Majidi et al's research results confirm the reducing effect of walnut leaf extract on urea. However, studies still need to be done on creatinine and uric acid with the gavage of the hydroalcoholic extract of walnut tree leaves.

In the normal metabolic process of the body, free radicals are produced by the cells. These free radicals are also neutralized by antioxidants produced by cells. In general, the body can keep free radicals and antioxidants in balance. When this balance is disturbed in favor of free radicals, oxidative stress occurs.⁴²

Superoxide dismutase (SOD) and catalase are crucial intracellular antioxidant systems that combat oxidative stress induced by free radicals. These systems rely on glutathione to carry out their defensive function. Under normal conditions, excess free radicals are neutralized with these enzymes, and the cell maintains the oxidation-regeneration balance.⁴³

Studies have shown that SOD and catalase act as antioxidants and anti-inflammation in the body and neutralize free radicals.⁴⁴ In this research, we also saw that the antioxidant function increased with the consumption of the extract and improved the performance of SOD and CAT enzymes, which is probably due to the presence of strong antioxidant compounds in the active phytochemicals of walnut trees leaf extract, such as flavonoids, juglone, and Phenolic compounds.

According to the results of this research, with the consumption of the hydroalcoholic extract of the walnut tree, it was observed that the lipid profile of rats improved, including HDL, LDL cholesterol, and triglyceride. The results of the review studies by Mirzababai and his colleagues are in the same direction.⁴⁵ The primary characteristics of diabetes encompass hyperinsulinemia and insulin resistance accompanied by dyslipidemia (32). Additionally, there are deviations in the levels of high-density lipoprotein cholesterol (HDL-C) and cholesterol. A significant risk factor for cardiovascular disease is low-density lipoprotein (LDL-C) and total cholesterol (TC), as well as triglycerides (TG).⁴⁶ Many chronic conditions, including diabetes and cardiovascular diseases (CVD), are caused by oxidative stress, which flavonoids and polyphenols help prevent and treat.⁴⁷ An investigation was conducted on Juglans regia (walnut) leaves, which are abundant in polyphenolic chemicals and flavonoids.⁴⁸ The phytochemicals in walnut tree leaves have demonstrated antioxidant, anti-inflammatory, and anti-cancer properties, mostly via neutralizing free radicals.^49,50 The effects of reducing glucose and body fat following the administration of walnut tree leaf extract have been reported, and it has been proposed as a promising clinical treatment in diabetic rats.^51,52 Based on its favorable outcomes in individuals with type 2 diabetes, this extract can be utilized independently or in conjunction with other botanicals to enhance the therapeutic effects on glycemic and lipid profiles in Patients with type 2 diabetes.⁵³ Nevertheless, the existing information from clinical trials examining the efficacy of walnut tree leaf extract on glucose and blood lipid levels in diabetic individuals is inconclusive.^48,54–56 Rabiei et al⁴⁸ No notable effect was observed on blood glucose levels from consuming 200 mg of walnut tree leaf extract daily, According to the findings of Hosseini et al Nevertheless, their study did demonstrate improvements in blood and fat composition.

Inflammation is the process of fighting the body against harmful factors, Such as infections, injuries, and poisons. When a harmful agent enters the body, the body releases factors that trigger the immune system response. Five primary symptoms are inflammation-related: redness, swelling, heat, pain, and loss of tissue function. Inflammatory reactions of the body include changes in vascular permeability, absorption and accumulation of leukocytes, and release of inflammatory mediators.

An inflammatory mediator is a messenger that enhances the inflammatory response by acting on blood vessels or cells at the injury site. Therefore, the presence of inflammatory and anti-inflammatory mediators creates a powerful immune system in the physiology of living organisms.⁵⁷ The present study showed that the administration of walnut tree leaf extract reduced the penetration of inflammatory cells into the kidney tissue compared to the gentamicin group.

Necrosis refers to the accumulation of diseased cells and tissues within a specific region of the body. Tissue necrosis is typically a result of the cessation of blood flow. Tissue necrosis can be caused by diseases or exposure to chemicals. An external factor such as inflammation, bacterial toxin, or trauma can lead to necrosis. Necrosis is a type of cell death that is always pathological (that is, it always occurs due to damage to the cell and not due to normal processes of the body, such as maintaining the shape of the tissue, etc).⁵⁸

The functional unit of the kidney is the nephron. When necrosis occurs, urine formation and elimination of body waste become problematic. As a result, toxic substances, urea, and creatinine increase, and kidney failure occurs.

In this research, the extract effectively decreased tissue necrosis in all treatment groups, demonstrating a considerable improvement compared to the group that received gentamicin. The histological results agreed with the biochemical results and indicated an improvement in the grade of tissue damage. In this research, kidney tissue damage was investigated in terms of tubular degeneration, infiltration of inflammatory cells, and tissue necrosis, and based on tissue results, it caused a dose-dependent decrease in tissue damage, especially at a dose of 400 mg/kg.

Conclusion

Based on the findings of this study, the histopathological results agreed with the serum results. Treatment with hydroalcoholic extract of walnut tree leaves in rats poisoned with gentamicin significantly improved renal serum factors, improved the endogenous antioxidant status of the body, and reduced the tissue damage level in the kidneys. According to this research, this extract or its similar phenolic compounds can be antioxidants for people with kidney disorders or chronic kidney disease. However, this study had some limitations including lacked long-term follow-up data to evaluate the chronic effects of walnut leaf extract. In addition, the exact mechanisms by which the extract exerts its protective effects are unclear, which requires more detailed studies to investigate the mechanism. Finally, this study did not control for possible confounding factors, such as changes in mouse metabolism and environmental factors that could affect the results.

Based on the findings of the current investigation and other studies that show the positive effect of walnut leaf extract on the improvement of laboratory indicators of rats poisoned with gentamicin, the following are suggested:

Measuring the effect of walnut tree leaf extract on the activity of SOD, COT enzymes, and microscopic examination of the liver tissue of rats.

Repetition of this study with doses higher than 400 mg per body weight of walnut tree leaf extract.

Measurement of other serum biochemical factors of rats, such as total and direct bilirubin.

Finally, after the final approval and implementation of these suggestions, the walnut tree leaf extract, the dose used in this study, can be used on human volunteers suffering from kidney failure.

The lack of sufficient information about the details of the nephrotoxic mechanisms caused by gentamicin, on the one hand, and the important role of kidneys in maintaining body health, on the other hand, shows the necessity of conducting studies such as the present research.

Footnotes

Acknowledgment

NA.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

All procedures followed the NIH Guide for the Care and Use of Laboratory Animals (NIH publication No. 85–23, revised in 1996) and were approved by the Tehran Medical Sciences Islamic Azad University Animal Care and Ethics Committee (NO: 1650_AUPF).

Informed Consent Statement

Not applicable.

ORCID iD

Saeed Aghebat_Bekheir

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