The renoprotective effect of thymol (TML) was investigated in rats challenged with gentamicin (GN). Rats received TML (20 mg/kg/day, p.o.) for 15 days, and GN (80 mg/kg/day, i.p.) starting from the 8^th day. TML significantly lowered serum creatinine and neutrophil gelatinase-associated lipocalin, and renal malondialdehyde, nitric oxide, tumor necrosis factor-α, interleukin-18, Bax, caspase-3, and caspase-9 in GN-challenged rats. In addition, TML caused a significant increment of renal total antioxidant capacity in rats received GN. Moreover, TML significantly ameliorated GN-induced histopathological kidney tissue injury, and significantly decreased nuclear factor-κB p65 and kidney injury molecule-1 expressions in kidneys of GN-challenged rats. It was concluded that TML guarded against CN-induced nephrotoxicity in rats via inhibition of oxidative stress, inflammation, and apoptosis.

thymol, gentamicin, kidney, rats

Gentamicin (GN), an antibiotic related to aminoglycosides, is commonly used to treat serious infections caused by aerobic Gram-negative bacilli. Despite its efficacy in bacterial eradication, GN-induced nephrotoxicity is considered a major adverse effect which limits its usefulness (Hayward et al. 2018; Srisung et al. 2017). Acute kidney injury (AKI) and dysfunction is a well-known dose-dependent adverse impact of GN, and can even occur after a single dose administration (Hayward et al. 2018). GN accumulates in the cells of renal proximal tubules and causes direct toxic effect. The precise procedures contributing to GN nephrotoxicity are not fully uncovered. However, a well-built evidence proposes that increased manufacturing of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are implicated in GN-induced AKI. This leads to unbalance in the oxidant/antioxidant status, devours endogenous antioxidants with subsequent increment of lipid peroxidation of cell biomembranes and increased malondialdehyde (MDA) generation (Galal and Abd El-Rady 2019; Famurewa et al. 2020). Oxidative stress upregulates the inflammatory cascades, including nuclear factor-κB (NF-κB) pathway, with subsequent increment in the generation of inflammatory cytokines, as tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) (Abdelrahman and Abdelmageed 2020). Besides, the use of antioxidants and anti-inflammatory compounds proved effective as renoprotective strategy against GN-induced AKI and dysfunction in prior investigations (Cao et al. 2019; Abdelrahman and Abdelmageed 2020; Aziz et al. 2020; Mahi-Birjand et al. 2020).

Thymol (TML), 2-isopropyl-5-methylphenol, is a monoterpenoid phenolic compound found mainly in the volatile oil of thyme. Previous investigations demonstrated that TML possessed a wide range of biological activities, including antioxidant, anti-inflammatory, antiapoptotic, antimicrobial, antidiabetic, anticancer, and cardioprotective effects (Kumari et al. 2019). It was also reported in the literature that TML impeded kidney injury and dysfunction induced by cisplatin in rats and mice (El-Sayed et al. 2015; Hosseinimehr et al. 2015), prevented the deleterious effects of high fat diet-induced diabetes mellitus on the kidneys of mice (Saravanan and Pari 2016), and inhibited streptozotocin-induced diabetic nephropathy in rats (Oskouei et al. 2019). Therefore, the current investigation was performed to demonstrate the possible protective effect of TML in GN-challenged rats, and to explore the probable mechanisms underlying this effect.

Previous investigations, in consistence with the present one, showed that GN caused AKI in rats due to oxidative/nitrosative stress (Galal and Abd El-Rady 2019; Famurewa et al. 2020). This was evidenced by the significant increase of kidney tissue MDA, the indicator of lipid peroxidation of biomembranes, and significant depletion of renal TAC. In addition, increased production of NO, the nitrosative stress biomarker, in kidney tissue denoted further injury due to nitration of cellular macromolecules. Prior studies also revealed that GN upgraded inflammatory cascades with increased production of inflammatory cytokines. This is in agreement with the current investigation, which revealed that GN activated NF-κB pathway and resulted in increased level of the active subunit, NF-κB p65 in rat kidneys. Nuclear translocation of NF-κB p65 activates gene transcription of inflammatory cytokines, as TNF-α and IL-18, leading to augmentation of inflammatory cascades and further damage of kidney tissue (Ansari et al. 2016; Abdelrahman and Abdelmageed 2020).

Similar to the current study, prior investigations revealed that TML, the active ingredient of thyme, performed as ROS scavenger, provided significant antioxidant effect, prevented biomembrane lipid peroxidation, and maintained endogenous antioxidant capacity (El-Sayed et al. 2015; Hosseinimehr et al. 2015). This was attributed to the inhibitory effect of TML on NADPH oxidase, the major source of generation of ROS, as hydrogen peroxide and superoxide anion (Kavoosi et al. 2012). Additionally, TML enhanced the biosynthesis of reduced glutathione (GSH), the major endogenous ROS scavenger, by upregulating the gene expression of γ-glutamylcysteine synthetase 1 enzyme, essential for GSH production (Ye et al. 2016). It was also reported in the literature that TML exerted antinitrosative effect by decreasing the production of RNS, as NO, through inhibition of inducible nitric oxide synthase enzyme (Wu et al. 2017). Besides, TML inhibited the NF-κB-dependent inflammatory responses and decreased the generation of TNF-α and other inflammatory cytokines through down-regulation of NF-κB p65 protein production (Chamanara et al. 2019).

Moreover, previous studies, similar to the present one, showed that GN activated the mitochondrial apoptotic pathway in the kidney causing increased release of Bax, the pro-apoptotic protein. This resulted in increased mitochondrial membrane permeability and the release of cytochrome C in the cytosol. Subsequent activation of apoptotic signals lead to up-regulation of cleaved caspase-9 activity, and finally cleaved caspase-3 activity, which resulted in execution of cell apoptosis (Sahu et al. 2014; Cao et al. 2019). It was also mentioned in the literature that TML inhibited the mitochondrial apoptotic pathway, and downregulated the activity of Bax and caspase family of proteases (El-Sayed et al. 2015; Fizur et al. 2015). This is in accordance with the current investigation, which revealed that TML significantly reduced the level of apoptotic biomarkers, Bax, caspase-9, and caspase-3, in the kidneys of GN-challenged rats.

In addition, NGAL, IL-18, and KIM-1 are considered novel sensitive diagnostic and prognostic biomarkers of AKI (Koza 2016; Beker et al. 2018). NGAL is produced by renal tubular cells as self-defensive mechanism upon exposure to nephrotoxic agents. IL-18 is a chemokine derived from the endothelial cells, and it is important in neutrophil recruitment and chemotaxis to the injured kidneys. KIM-1 is a proximal tubular cell transmembrane glycoprotein expressed upon exposure to harmful kidney stimuli, which may be involved in cleaning debris from injured renal cells (Koza 2016; Beker et al. 2018). The present work showed that TML significantly inhibited the GN-induced increments of serum NGAL, and renal IL-18 and KIM-1 in rats. Besides, the current investigation showed that TML prevented the histopathological damage induced by GN in rat kidneys.

TML significantly preserved kidney structure and function against GN insult in rats. The antioxidant, anti-inflammatory, and antiapoptotic properties of TML are the most probable contributing factors for this nephroprotective effect.

All the authors declare that there are no conflicts of interest.

The authors did not receive financial support from any funding agency.

The authors want to thank Mr. Mohamed Hamami, the technician of Pharmacology Lab, Faculty of Medicine, Minia University, for his help in the conduction of the practical part of this research.