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The nephroprotective potential of selected synthetic compound against gentamicin induced nephrotoxicity

BMC Pharmacology and Toxicology volume 25, Article number: 68 (2024) Cite this article

Abstract

Background

Nephrotoxicity, the rapid impairment of kidney function caused by harmful drugs and chemicals, affects about 20% of cases and is projected to become a leading cause of death by reactive oxygen species (ROS). Gentamicin (GM), an aminoglycoside antibiotic is one of the well know drugs/chemicals to cause nephrotoxicity both in humans and animals.

Methods

A study on the effects of a synthetic phenolic compound, called 5-a, on GM-induced nephrotoxicity in male Wistar albino rats was conducted. The rats were grouped into five groups: normal control (NC), GM control (GM), positive control (GM + Dexa), treatment I (GM + 5-a 5 mg/kg) and treatment II (GM + 5-a 10 mg/kg). Throughout the experiment, the rats’ weights were monitored, and at its conclusion, their serum and kidney tissues were analyzed for renal function indicators and inflammatory markers. The study also included histopathological evaluations, molecular docking studies, blood and urine analyses for electrolyte changes, and behavioural assessments for central nervous system impact.

Results

2-{5-[(2-hydroxyethyl)-sulfanyl]-1,3,4-oxadiazol-2-yl} phenol (5-a) significantly protected against renal damage by reducing inflammatory markers, improving antioxidant defences, and decreasing kidney injury, particularly at higher doses. The findings suggest that compound 5-a, due to its anti-inflammatory and antioxidant properties, could be a promising therapeutic option for reducing gentamicin-induced nephrotoxicity and potentially for other kidney disorders in the future.

Conclusion

These findings highlight the therapeutic effects of compound 5-a in alleviating gentamicin-induced nephrotoxicity.

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Introduction

The kidneys plays a vital role in various essential functions [42], including the regulation of extracellular fluid balance to maintain optimal hydration and acting as a primary site for the accumulation of xenobiotics, such as harmful environmental chemicals, thanks to its unique biochemical and physiological characteristics [26]. With a high renal blood flow rate [36] and a capacity for accumulating diverse solutes during urine formation, the kidneys are particularly vulnerable to exposure to various toxic substances. It is crucial in maintaining homeostasis and help eliminate toxic metabolites from the body [15, 16]. Kidney diseases are a significant health concern globally, with oxidative stress, inflammation, apoptosis, and fibrosis being key underlying pathological processes [20]. Nephrotoxicity can be defined as when the kidneys get damaged quickly because of harmful drugs and chemicals [12]. Some medicines can damage the kidneys in different ways, such as injuring the tiny filters in the kidneys, causing inflammation, or blocking the small blood vessels [33]. About 19–26% of kidney injuries in hospitals happen because of drugs or chemicals [28]. Some examples of the injuries include damage to the cells, changes in blood flow, inflammation, muscle breakdown, and blood clots in the tiny vessels of the kidneys [45]. Gentamicin (GM) is one of the most effective aminoglycosides against gram-negative bacterial infection in humans and animals [9]. One of the major complications associated with GM in nephrotoxicity/renal failure accounting for 10–30% of patients/cases [6]. Nephrotoxicity induced by GM is a complex phenomenon characterized by an increase in plasma creatinine and urea levels and severe proximal renal tubular necrosis, followed by deterioration of renal function [2]. The toxicity of aminoglycosides, including GM, is believed to be related to the generation of reactive oxygen species (ROS) in the kidneys [1]. Heterocyclic compounds have inspired scientists to synthesize innovative molecules and evaluate their biological properties. Among heterocyclic ring-containing molecules, oxadiazole is one of the most promising moieties [24]. Compounds containing 1,3,4-oxadiazole cores have a broad biological activity spectrum including antibacterial, antifungal, analgesic, anti-inflammatory, antiviral, anticancer, antihypertensive, anticonvulsant, and anti-diabetic properties [11].

The nephroprotective potential of 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol (compound 5-a) as shown in (Fig. 1), against GM-induced nephrotoxicity has not yet been explored. Therefore, the present study was designed to investigate the nephroprotective effect of compound 5-a against GM-induced nephrotoxicity in rats. We hypothesized that compound 5-a may ameliorate GM-mediated nephrotoxicity, oxidative damage, and inflammation through its antioxidant and anti-inflammatory properties.

Fig. 1
figure 1

Structure of compound 5-a, 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol

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Materials and methods

Chemicals and reagents

The chemicals and reagents used in the current study included gentamicin, dexamethasone (Dexa), normal saline (NS 0.9%), and synthetic phenolic compound 5-a. The primary antibodies used in this study are tumour necrosis factor-alpha (TNF-α) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB). Additional immunohistochemistry-related consumables, namely the avidin-biotin-peroxidase complex ELISA kit, 3,3-diaminobenzidine (DAB), biotinylated secondary antibody, DPX mounting media, proteinase K. All additional compounds utilized in the experiment were of analytical grade.

Computational analysis

Computational techniques are vital for identifying toxicity and designing novel potential compounds, notably using software like ProTox-ii, Swiss ADMET (absorption, distribution, metabolism, excretion, and toxicity), and molecular docking methods. The ADMET and toxicity assessments show promising interactions with selected protein targets, indicating the potential of these compounds as effective treatments for kidney diseases [25].

Drug likeliness and ADMET prediction

Toxicity evaluation and ADMET were screened to examine ligand molecules’ pharmacological potential and toxicity levels, including the drug candidate 5-a. These molecules were stored in smiles format and analyzed using Swiss ADMET & ProTox-ii platforms for predictions on various pharmacokinetics, physicochemical properties, and drug-likeliness based on parameters such as lipophilicity, water solubility, and resemblance to known drugs. The drug-likeness of synthetic phenol compound 5-a was assessed against Lipinski’s rules through Molinspiration, focusing on factors like hydrogen acceptors and donors, molecular weight, and partition coefficient log P. Toxicity categorization ranged from highly toxic compounds [LD50 (Lethal Dose 50) ≤ 50 mg/kg] to non-toxic ones (LD50 > 5000 mg/kg), with ProTox-ii server predicting oral toxicity in rodents and assigning appropriate toxicity classes to the evaluated molecules. This comprehensive approach facilitated the identification of compound 5-a’s drug-likeness and potential toxicity profile, crucial for further drug development processes [43].

Molecular docking

AutoDock Vina software matched specific ligands with a protein’s active site. This process aimed to find different ways the ligand can fit and attach to the protein. The software prepared the proteins by adjusting hydrogen atoms to make the docking more accurate. Molecular docking is crucial in research, mainly because it is efficient and does not cost much, making it essential for designing drugs with the help of computers to understand how molecules interact and predict how well they bind together. The PyRx 0.8 software and the BIOVIA Discovery Studio visualizer 3.0 were used for docking analyses for this study. The 3D structures of the active compounds were taken from the PubChem online database, and the 3D X-ray crystal structures of proteins, including TNF-α and NF-κB, were also examined with the compound 5-a. Any existing ligands and water molecules were removed from the protein and then prepared by adding the necessary hydrogen bonds. This prepared protein was saved in a PDB (Protein Data Bank) file format and then, along with the ligands, used in PyRx software to analyze how well they fit together through docking [17].

Experimental animals

Twenty-five male Wistar albino rats weighing 200 to 220 g were procured from the NIH (National Institute of Health) Islamabad-Pakistan. Animals were acclimatized for seven days before the commencement of the experiments. The rats were housed under a 12-hour light/dark cycle, maintaining a consistent temperature and providing unrestricted access to water and food. All the animals were utilized in diverse experiments to assess the efficacy of drugs. During the urine collection phase, the rats had restricted access to food while having ad libitum access to water. All the experiments were conducted as per the approved protocols of the BioEthical Committee (BEC) of Quaid-i-Azam University (QAU) (Approval No: BEC-FBS-QAU2023-473), Islamabad-Pakistan, and according to the Guide for the Care and Use of Laboratory Animals (8th edition, National Academies Press).

Study design

Rats were divided into five groups, each consisting of five animals. The normal control group was given 0.9%NS orally for eight days. The negative control group was given GM 100 mg/kg IP for eight days. In the positive control group, rats were treated with 10 mg/kg Dexa IP, followed by 100 mg/kg GM an hour later for eight days. Treatment group I received 10 mg/kg of compound 5-a IP, plus 100 mg/kg GM after one hour, for eight days. Treatment group II received 5 mg/kg of compound 5-a and then 100 mg/kg GM in the same manner [32].

Collection of blood and serum

All animals were anaesthetized using a cocktail of xylazine (9 mg/kg) and ketamine (90 mg/kg) intraperitoneally and were euthanized by cervical dislocation after blood samples were taken from the animals using the cardiac puncture method on the ninth day of the study following American Veterinary Medical Association (AVMA) guidelines [38]. The samples were placed in eppendorf tubes and spun at 3500 rpm for 15 min in a centrifuge to separate the serum. The serum was tested for various markers such as creatinine (Cr), total protein (TP), blood urea nitrogen (BUN), albumin, and cytokines. Blood tests were also carried out to assess the complete blood count (CBC) to study the effect of the compound 5-a on these parameters [14].

Collection of organs

The kidneys were immediately excised after euthanizing the animals, washed in ice-cold saline, and blot-dried. About 200 mg tissue pieces were homogenized in 5 volumes of ice-cold phosphate-buffered saline (PBS, 50 mM, pH 7.4) containing a protease inhibitor cocktail. The homogenates were prepared using a Teflon homogenizer on ice and then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatants were collected to estimate oxidative stress parameters and enzymatic antioxidant assays.

Assessment of body weight

On the ninth day of the experiment, body weights were recorded to assess any alterations. Following blood collection, the kidneys were removed and weighed, and the outcome was expressed as the wet weight of the kidneys per 100 g of body weight, providing an indicator for evaluating changes in kidney weight [17].

Evaluation of renal index

The renal index was calculated after initiating kidney injury using GM. One hour before sacrificing the mice, their body weights were measured. Subsequently, renal tissues were dissected from various groups, washed with 0.9%NS, and weighed. The renal index (RW/BW ratio) was then determined, following a methodology previously documented [15].

Assessment of antioxidant and oxidative stress markers

In the evaluation of oxidative stress markers, the determination of Malondialdehyde (MDA) and Nitric Oxide (NO) in the tissue was conducted using a specific method [27]. Additionally, the estimation of Glutathione (GSH) and the determination of (GST), Catalase were assessed [29].

Assessment of urine biochemical parameter

Urine was collected in polypropylene tubes, and the volume was quantified. Subsequently, urine samples underwent analysis for protein, sodium, potassium, and CrCl utilizing Accurex Biomedical kits, following the manufacturer’s guidelines.

Evaluation of depression in AKI

Rats were carried out into the behavioural testing room one hour before for acclimatization before commencing the study. The behavioural testing was done during the light phase of the circadian cycle. The behavioural study was captured and analyzed by using a camera for investigation of depressive-related behaviour using the Force swim test, Light dark box test, open field test [44].

Histopathological analysis

A segment of the kidney tissue was extracted, rinsed with NS and conserved in 10% formalin solution. Then, the tissue was sliced in 4 μm thickness and embedded in paraffin wax solution for examination employing H & E and stain [22].

Immunohistochemistry analysis

Immunohistochemistry staining was conducted to explore the impact of compound 5-a on renal injury induced by GM. The kidney tissue underwent treatment with xylene and alcohol after being embedded in paraffin. After alcohol washing, the tissue was subjected to proteinase-K, NGS (normal goat serum), and primary and secondary antibodies (TNF-α & NF-κB), as previously documented [37].

Statistical analysis

Data were presented as mean ± standard error mean (S.E.M.). GraphPad Prism version 8.0.1 for Windows was used to generate the graphs. The statistical package for social sciences (SPSS) version 25 for Windows analysed means. One-way analysis of variance (One-way ANOVA) is followed by multiple comparisons using the Duncan test. The level of statistical significance was set at p < 0.05.

Results

Determination of ADMET

Compound 5-a plays a crucial role in the clinical setting, showing mostly drug-likeness and pharmacokinetic properties using the SwissADMET software, as shown in Fig. 2. Toxicity prediction was carried out by ProTox-ii; the compound 5-a demonstrated ADMET profiles within an acceptable range, indicating their potential as effective drug candidates. It exhibited favourable human intestinal solubility (HIA), and the acute rat toxicity (LD50) = 2000 mg/kg and fell within the same category (Class-IV) considered safe, as shown in Fig. 3; compound 5-a was found to be non-toxic. LD:50 = 2000 mg/kg through the analysis on ProTox ii, showing compound 5-a is safe for evaluating its protective effect against nephrotoxicity. Lipinski’s Rule of Five analysed the drug-likeness properties of compound 5-a, which indicates that the compound 5-a met Lipinski’s criteria.

Fig. 2
figure 2

Determination of drug likeliness and pharmacokinetic properties

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Fig. 3
figure 3

Evaluation of toxicity by ProTox-ii

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Evaluation of 3-D interaction

The docking outcomes for chosen phytocompounds against target proteins, specifically TNF-α and NF-κB, revealed that compound 5-a demonstrated a superior binding affinity and more effective binding modes for the targeted receptors. The analysis of hydrogen bond interactions, hydrogen bond lengths, and hydrophobic interactions between the receptors was also evaluated. The molecular docking process predicted the ligand’s conformation, placement, and orientation in specified sites (commonly referred to as poses). It assesses the binding affinity carried out by Pyrex and Discovery Studio for a comparison of TNF-α and NF-κB for 3-D interaction with the compound 5-a, as shown in Fig. 4; Table 1.

Fig. 4
figure 4

3-D interaction of compound 5-a against selected macromolecule

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Table 1 Best pose dock analysis showing E-values (Kcal/mole) and hydrogen bond formed by interaction of compound 5-a against selected targets
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Body weight variation

The body weights in the induced nephrotoxicity model were determined. On the ninth day, assessments were conducted by measuring body weights to gauge any changes. Subsequently, blood collection took place, followed by the removal and weighing of the kidneys. The outcome was presented as the wet kidney weight relative to 100 g of body weight, providing a metric for evaluating alterations in the kidney. The rats administered with GM experienced a notable reduction in body weight compared to the normal control rats.

Conversely, rats treated with compound 5-a (5 and 10 mg/kg) exhibited a notable (P < 0.05) improvement in body weight. Moreover, the kidney weight of treated rats was significantly increased compared to negative control rats. In contrast, the kidney weight of rats treated with compound 5-a (5 and 10 mg/kg) showed a significant improvement compared to rats treated with GM alone, as shown in Fig. 5.

Fig. 5
figure 5

Effect of the compound 5-a against gentamycin induced renal injury in A; initial body weight and B; final body weight. Each value represents ± S.E.M (n = 5), analysed by using two-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Assessment of relative kidney index

As previously documented, the renal index was evaluated for the entire treatment group. The negative control’s renal index exhibited a notable rise (P < 0.05); nevertheless, the group treated with compound 5-a demonstrated a substantial reduction compared to the negative control. The effect was dependent on the dosage, as illustrated in Fig. 6.

Fig. 6
figure 6

Effect of compound 5-a against gentamycin induced renal injury in kidney weight. Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparison. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Where, 5-a; 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Effect on renal biomarkers

GM (100 mg/kg) group showed alteration in the levels of SrCr, BUN, CrCl, serum protein, serum bilirubin, ALT, AST as compared to saline group. Whereas the levels of of SrCr, BUN, CrCl, serum protein, serum bilirubin, ALT, AST were significantly restored in the treatment groups as shown in Table 2.

Table 2 Effect of the compound 5-a and GM/Dexa on the renal biomarkers
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Effect on hematological parameters

In saline (0.15 μl/kg) group the levels of neutrophils, lymphocytes, WBC, RBC, Hb, Platelets were 32.6 ± 0.50%, 63 ± 0.70%, 11.5 ± 0.18 x 103/μl, 9.15 ± 0.005  x 10 6/μl, 15.3 ± 0.03 g/dL, 715.8 ± 1.24 x 103/μl respectively. In GM (100 mg/kg) the levels if of neutrophils, lymphocytes, WBC, RBC, Hb, Platelets were 45.3 ± 0.64%, 75.2 ± 0.86%, 24.2 ± 0.25 x 103/μl, 3.11 ± 0.008 x 106/μl, 10.3 ± 0.07 g/dL, 434.2 ± 1.15 x 103/μl respectively. In Dexa (10 mg/kg) + GM the levels of neutrophils, lymphocytes, WBC, RBC, Hb, Platelets were 37.12 ± 0.62%, 65.9 ± 1.01%, 9.7 ± 0.12 x 103/μl, 4.04 ± 0.005 x 106/μl, 11.4 ± 0.04 g/dL, 461 ± 0.70 x 103/μl respectively. In compound 5-a (5 mg/kg) + GM (100 mg/kg) the levels of neutrophils, lymphocytes, WBC, RBC, Hb, Platelets were 34.9 ± 0.53%, 61.2 ± 0.58%, 11.7 ± 0.24  x 103/μl, 8.07 ± 0.005 x 106/μl, 14.5 ± 0.07 g/dL, 694.8 ± 0.58 x 103/μl respectively. In compound 5-a (10 mg/kg) + GM (100 mg/kg) the levels of neutrophils, lymphocytes, WBC, RBC, Hb, Platelets were 33.3 ± 0.50%, 54 ± 0.70%, 13.8 ± 0.11 x 103/μl, 7.15 ± 0.015 x 106/μl, 13.6 ± 0.05 g/dL, 701.4 ± 0.92 x 103/μl respectively as shown in Table 3.

Table 3 Effect of the compound 5-a treatment on the hematologic profile
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Effect on antioxidant and oxidative stress markers

This examination involved evaluating both antioxidants and the level of oxidative stress throughout the study. The group subjected to GM displayed a significant reduction in antioxidant enzymes like GST, GSH, and Catalase, accompanied by a notable increase in oxidative stress indicators like NO and MDA were observed. Conversely, the groups administered either compound 5-a or Dexa exhibited a significant improvement in antioxidant levels, and promising reduction in oxidative stress markers i.e. MDA and NO compared to the GM group, efficiently alleviating oxidative stress in renal tissue as illustrated in Fig. 7.

Fig. 7
figure 7

Effect of the compound 5-a treatment on the antioxidants and oxidative stress markers on GM induced renal injury model. GST (A), GSH (B), Catalase (C), MDA (D), and NO (E). Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Urinary assessment

The administration of GM significantly modified urine electrolytes, including sodium, potassium, and urine protein. In addition, there was a notable decrease in sodium ions and an elevated potassium in the urine. However, the treatment with compound 5-a effectively restored the urine electrolytes, demonstrating normalization in comparison to the negative control, as mentioned in Fig. 8.

Fig. 8
figure 8

Effect of the compound 5-a treatment on the GM induced renal injury on A; urine protein, B; urine sodium, C; urine potassium. Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Evaluation of depression

Light and dark box test

Time spent in the light compartment shows normal behaviour, while time spent in the dark compartment shows the anxiety-like behaviour of the rats. There was a significant (P˂0.05) decrease in time spent in light compartment in GM treated group as compared to the normal control group. However, the administration of compound 5-a (10 mg/kg & 5 mg/kg) significantly (P < 0.05) improves the time spent in the light compartment compared to the GM group. The findings of the Light/ Dark box test are depicted in Fig. 9.

Fig. 9
figure 9

Effect of compound 5-a on depressive-like and anxiety-like behaviour in GM induced nephrotoxicity. A; Light/Dark box test, B; Forced swim test. Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Forced swim test

The administration of GM led to a significant (P < 0.05) increase in the duration of immobility compared to the control group that received the NS. The administration of compound 5-a 10 mg/kg results in a significant (P < 0.05) decrease in immobility time compared to the GM group. The compound 5-a with the dose of 5 mg/kg significantly (P < 0.05) improved the immobility to a lesser extent than the compound 5-a 10 mg/kg, as shown in Fig. 9.

Open field test

The analysis of depressive-like behaviours like number of grooming, rearing, and box crossing, was analyzed. The control group significantly (P < 0.05) improved the stress compared to the GM group. On the other hand, depression decreased significantly (P < 0.5) in the compound 5-a compared to the GM group as shown in Fig. 10.

Fig. 10
figure 10

Effect of the compound 5-a on anxiety-like behaviour in GM induced nephrotoxicity. A; No of box crossings, B; No of rearing, C; No of grooming in open field test. Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column are significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Histopathological analysis

Kidney

The histopathological examination of kidney tissues treated with GM revealed significant and widespread damage, including dilation and necrosis of renal tubules, glomerular sclerosis, cellular swelling, and oedema. This damage could be attributed to the production of reactive free radicals resulting from GM-induced oxidative stress. Conversely, kidneys treated with GM combined with compound 5-a at 10 mg/kg and 5 mg/kg showed restoration of normal kidney structure in rats, as depicted in Fig. 11.

Fig. 11
figure 11

Histopathological results of kidney sections stained with H & E in GM induced renal damage in rats (×40). Group A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (5 mg/kg): Group E, 5-a (10 mg/kg) significantly different (p ≤ 0.05)

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Liver

The histopathological analysis of the liver indicates that the typical structure of the liver is expected to remain intact in the normal control group as compared to the Dexa and GM, where significant morphological alterations are present, such as those caused by cirrhosis or necrosis along with fibrosis, except in liver tissues treated with compound 5-a, as shown in Fig. 12.

Fig. 12
figure 12

Histopathological results of liver sections stained with H & E in GM induced renal damage in rats (×40). Whereas Group A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (5 mg/kg): Group E, 5-a (10 mg/kg)

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Heart

There were morphological changes observed in the heart tissue due to the cardiotoxic effects of GM and Dexa; whereas there was significant improvement in the architecture of the heart tissues treated with compound 5-a as mentioned below in Fig. 13.

Fig. 13
figure 13

Histopathological results of heart sections stained with H & E in GM induced renal damage in rats (×40). Whereas Group A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (5 mg/kg); Group E, 5-a (10 mg/kg)

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Immunohistochemistry

Kidney

Immunohistochemistry staining of kidney tissues was conducted to detect the impact of TNF-α and NF-κB, and the results revealed an increased release of TNF-α and NF-κB in the GM group, whereas compound 5-a treatment with the dose of 10 mg/kg and 5 mg/kg significantly decreased the protein hyperexpression which indicates the nephroprotective effect of compound 5-a on kidney tissues as illustrated in Fig. 14.

Fig. 14
figure 14

Effect of compound 5-a on the relative expression of TNF-α & NF-κB in kidney tissue treated with GM. Group: A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (10 mg/kg). Each value represents ± S.E.M (n = 5), analyzed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Liver

The results indicated that GM and Dexa cause hepatotoxicity and increased the expression of TNF-α and NF-ΚB, which limits its clinical use. While treatment with compound 5-a significantly reduced the expression of TNF-α and NF-κB in liver tissues supported its protective role as mentioned in Fig. 15.

Fig. 15
figure 15

Effect of compound 5-a on the relative expression of TNF-α & NF-κB in liver tissue treated with GM. Group A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (10 mg/kg). Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Heart

The immunohistochemistry of heart tissues indicated that compound 5-a possesses a protective effect on the heart compared to the GM group. The expressions of TNF-α and NF-κB significantly reduced in the treated group of compound 5-a, as shown in Fig. 16.

Fig. 16
figure 16

Effect of compound 5-a on the relative expression of TNF-α & NF-κB in heart tissue treated with GM. Group A, Normal control; Group; B, GM; Group C, Dexa; Group D, 5-a (10 mg/kg). Each value represents ± S.E.M (n = 5), analysed by using One-way ANOVA (Analysis of variance) using the Duncan test for multiple comparisons. The values having different alphabetical superscriptions in the column significantly different (p ≤ 0.05). Whereas 5-a 2-{5-[(2-hydroxyethyl) sulfanyl]-1,3,4-oxadiazol-2-yl} phenol, GM Gentamicin, Dexa Dexamethasone

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Discussion

Aminoglycoside antibiotics are frequently employed for the treatment of gram-negative bacterial infections [21, 40]. Nephrotoxicity has been a well-known adverse effect associated with aminoglycosides. Nephrotoxicity induced by GM, a commonly used aminoglycoside is identified by direct tubular necrosis, predominantly concentrated in the proximal tubule [34]. However, their application is limited due to the pronounced adverse impact on renal function [5, 35].

At the start of the study, toxicity and pharmacokinetic properties were checked using computational tools, and compound 5-a lies in the category of drug-likeliness according to Lipinski’s rule of five. The molecular docking was done against TNF-α and NF-κB, 3-D interaction with best pose and binding affinity via dock analysis was carried out to check the interaction between selected molecule and ligand that shows strong binding affinity of TNF-α & NF-κB, and a strong interaction with synthetic compound 5-a. This study observed that GM administration in rats showed a notable decrease in body and kidney weight and an increase in relative kidney weight [15]. The decreased body weight was due to reduced food intake and appetite loss [4]. Creatinine is a crucial indicator for evaluating renal function and GFR in clinical settings. The 5-a treated groups had improvement in renal parameters including SrCr and BUN compared to GM group who presented with renal injury. The SrCr and BUN increase in the GM group as opposed to the normal control group [3]. Whereas in the 5-a group 10 mg/kg and 5-a 5 mg/kg these levels were decreased. Serum biochemistry evaluated ALT, AST and bilirubin [39]. Rats injected with GM exhibited noteworthy alterations in blood chemistry. There were significant increase in ALT, AST and bilirubin compared to the normal control group, however treatment with compound 5-a significantly reduced these markers [19]. Elevated AST blood levels typically coincide with renal damage [4]. This model demonstrated significant changes in blood composition, including elevated levels of WBCs, lymphocytes, and neutrophils. Conversely, RBCs, platelets, and haemoglobin levels were notably reduced following the administration of GM and Dexa. However, these effects were mitigated in groups treated with compound 5-a, 10 mg/kg, and 5 mg/kg. These produced ROS also contribute to renal tubular necrosis and GM-induced acute renal failure [30]. GM has been demonstrated to cause inflammation by elevating the production of ROS, which has been reported to contribute to cell death in diverse pathological conditions and result in oxidative stress and inflammation by increasing the expression of TNF-α and NF-κB [13] as compared to compound 5-a that result by decreasing in the relative expression of the inflammatory mediators [35]. The impact of compound 5-a treatment on the alterations in serum electrolytes induced by GM was assessed. The groups treated with GM displayed significant fluctuations in electrolytes such as sodium, potassium and urine protein [8]. However, the groups that received compound 5-a or Dexa exhibited promising improvements in serum electrolytes compared to the negative control and significant reduction with compound 5-a. The depression was assessed with the light/dark box test which demonstrated a noticeable increase in the time spent in the light box after the administration of compound 5-a compared to GM. Furthermore, the immobility was significantly (P < 0.05) higher in the GM group than compound 5-a in the force swim test. Based on the open field test, the anxiety behavior in rats improved significantly in the compound 5-a group. Morphological alterations in the kidneys occur because of deposition of GM in the renal cortex, reflecting pathological conditions observed in experimental animals. We observed the morphological changes in the kidneys of GM-treated rats, including damage to both glomerular and tubular structures. The relative expression of inflammatory markers i.e. TNF-α and NFκB observed through the immunohistochemistry resulted in a major decrease in inflammatory markers in the compound 5-a group compared to the GM group [18]. Thus, administering GM for eight days exacerbated nephrotoxicity by increasing oxidative stress, inflammation, and markers related to endoplasmic reticulum stress [31]. The anti-inflammatory properties of compound 5-a in a toxicity model induced by GM play a pivotal role in developing acute kidney injury pathogenesis. In rats with experimentally induced kidney injury caused by GM, there was a significant enhancement in the survival rate [18]. The experiment thoroughly explored the use of synthetic phenolic compound 5-a in various disease conditions due to its minimal adverse effects [10]. Accordingly, the objective or the goal of the present study was to evaluate the nephroprotective effects of compound 5-a against GM induced nephrotoxicity [41]. The experimental study demonstrates that compound 5-a exhibited the potential to ameliorate GM-caused inflammation in renal tubules, oxidative stress, histopathological changes [23], and instability in kidney function [7].

Conclusion

The findings of this study suggest that compound 5-a demonstrated potential nephroprotective activity against GM-induced nephrotoxicity, due to its’ antioxidant and anti-inflammatory properties. Special attention should be paid to an in-depth study of this compound’s molecular mechanisms of action and acute and chronic in-vivo toxicological studies.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

5-a:

2-{5-[(2-hydroxyethyl)-sulfanyl]-1,3,4-oxadiazol-2-yl} phenol

AKI:

Acute Kidney Injury

AVMA:

American Veterinary Medical Association

CAT:

Catalase

DAB:

3,3’ Diaminobenzidine

Dexa:

Dexamethasone

GM:

Gentamycin

GSH:

Glutathione

GST:

Glutathione-S-transferase

H and E:

Haematoxylin and Eosin

IHC:

Immunohistochemistry

I.P.:

Intraperitoneal

LD:

Lethal Dose

MDA:

Malonaldehyde

NFκB:

Nuclear Factor Kappa-Lightchain-Enhancer of Activated B Cells

NO:

Nitric Oxide

ROS:

Reactive Oxygen Species

SrCr:

Serum Creatinine

TNF-α:

Tumor Necrosis Factor Alpha

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Acknowledgements

The authors are thankful to the Department of Pharmacy, Quaid-i-Azam University Islamabad, Pakistan, for helping in this research work.

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Authors and Affiliations

  1. Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan

    Sony Amir, Muhammad Abid, Muhammad Khalid Tipu & Nadeem Irshad

  2. Riphah Institute of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan

    Humaira Nadeem

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Contributions

S.A, M.A, and N.I designed and performed research including behavioural and biochemical assays. S.A, N.I and M.K.T analysed the data and drafted the manuscript. H.N provided the lab facilities and resources. N.I supervised the project. All authors read and approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

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Correspondence to Nadeem Irshad.

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The animals were housed in animal house of Quaid-i-Azam University. The experiments were carried out according to ARRIVE guidelines and the US National Institutes of Health, by the approval of the Quaid-i-Azam University (QAU) (Approval No: BEC-FBS-QAU2023-473), Islamabad, Pakistan. All animals were anesthetized using a cocktail of xylazine (9 mg/kg) and ketamine (90 mg/kg) intraperitoneally and were euthanized by cervical dislocation following AVMA guidelines. All efforts were made to minimize their suffering.

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Amir, S., Abid, M., Nadeem, H. et al. The nephroprotective potential of selected synthetic compound against gentamicin induced nephrotoxicity. BMC Pharmacol Toxicol 25, 68 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40360-024-00765-3

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BMC Pharmacology and Toxicology

ISSN: 2050-6511

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