Protective effect of thymol on glycerol-induced acute kidney injury

Abstract

Acute kidney injury (AKI) is a syndrome characterized by an accelerating decrease in renal function in a short time. Thymol is one of the main components of thyme species and has a variety of pharmacological effects. Here, we investigated whether thymol could ameliorate rhabdomyolysis (RM)-induced AKI and its related mechanism. Glycerol was used to induce RM-associated AKI in rats. Rats received thymol (20 mg/kg/day or 40 mg/kg/day) gavage 24 h before glycerol injection until 72 h after injection daily. Kidney injury was identified by measuring serum creatinine (Scr) and urea levels and by H&E and PAS staining and immunohistochemistry (the expression of proliferating cell nuclear antigen (PCNA)). Renal superoxide dismutase (SOD), malondialdehyde (MDA), and oxidative stress-related Nrf2/HO-1 signaling pathways were measured. The expression of the inflammatory markers TNF-α, IL-6, MCP-1, and NF-κB was assessed by ELISA and western blotting. Finally, the expression of the PI3K/Akt signaling pathway was detected by western blotting. Glycerol administration induced obvious renal histologic damage and increased Scr, urea, and PCNA expression. Notably, thymol treatment attenuated these structural and functional changes and prevented renal oxidative stress, inflammatory damage and PI3K/Akt pathway downregulation associated with glycerol-induced AKI. In conclusion, thymol might have potential applications in the amelioration of AKI via its antioxidant and anti-inflammatory effects and upregulation of the PI3K/Akt signaling pathway.

Keywords:

• Rhabdomyolysis
• AKI
• oxidative stress
• inflammation
• PI3K/Akt

1. Introduction

Acute kidney injury (AKI), previously known as acute renal failure (ARF), was first replaced by the emergency medical community and the international society of nephrology [1] and is a syndrome characterized by an accelerating decrease in renal function in a short time. It is a crucial clinical problem with a high mortality rate, prolonged hospital stays and accelerated chronic kidney disease. Generally, the onset of AKI is hidden until the body cannot tolerate natremia [2]. Once diagnosed, the mortality of AKI is as high as 50% in the intensive care unit [3], and effective therapy to reverse or prevent progression is rarely mere. Rhabdomyolysis (RM)-induced AKI is named RM-mediated myoglobinuric renal damage, with 15% of all RM patients accounting for 40% of AKI cases [4]. It often develops after crush syndrome, exhaustive exercise, medications, infections, and toxins [5–8].

One of the most common experimental models to clarify AKI is the rat receiving a single intramuscular injection of glycerol, which induces RM [9,10]. Glycerol-induced AKI is marked by myoglobinuria, which is the main cause of renal damage in RM. In addition to myoglobinuria, enhanced renal vasoconstriction, ischemic tubular injury, tubular obstruction, and the direct toxicity of heme protein are involved. Although the pathogenesis of glycerol-induced AKI is complex and remains unknown, inflammatory mediators, oxidative stress, apoptosis, endothelial dysfunction, and lipid peroxidation are involved [11–13]. The pathological changes in AKI induced by RM are mainly focused on tubular epithelial cells, which are responsible for the regulation of water, electrolyte, and metabolic waste balance. Studies have recently shown that AKI induced by RM results in renal tubular oxidative stress, inflammation, and activation of the Toll-like receptor-4 (TLR-4) and NOD-like receptor protein-3 (NLRP-3) inflammasomes. In addition, oxidative stress was also involved in the PI3K signaling pathway [14], which is a well-known pathway that is related to the inflammatory response, oxidative stress, and apoptosis [15]. In recent years, research on the prevention and recovery of AKI has attracted much attention, although, no established therapy has been determined.

Thymol whose structural formula is shown in Figure 1, also named 2-isopropyl-5-methyl phenol, is one of the main components in thyme species. It has been used in basic research for many years since it has a variety of pharmacological effects, such as antioxidant, anti-inflammatory, antibacterial, antitumor, and antiallergenic properties, that are pharmacologically relevant. Recently, Zhang [16] revealed that thymol could exhibit an anti-virulence effect by targeting multiple Salmonella Typhimurium virulence factors for ion protease degradation. Furthermore, research has also clarified that thymol alleviates imidacloprid-induced testicular toxicity by regulating oxidative stress and the expression of steroidogenesis- and apoptosis-related genes in adult male rats [17]. In kidney injury models, thymol showed protective effects against cisplatin-induced renal tubular necrosis and in STZ-induced diabetic rats by suppressing renal oxidative stress [18,19]. In our previous research, we found that thymol alleviates the lipopolysaccharide-stimulated inflammatory response via downregulation of the RhoA-mediated NF-κB signaling pathway in human peritoneal mesothelial cells [20]. Given its important role in anti-inflammatory and antioxidant stress, in this study, we aimed to clarify the protective effect of thymol in RM-induced AKI and further elucidate the potential mechanisms through which thymol exerts its various effects.

Figure 1. Structure of thymol.

2. Materials and methods

2.1. Chemicals and reagents

Thymol was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China) and was dissolved in 0.1 mL olive oil to the desired concentration. All chemicals and reagents are underlined in this text unless otherwise provided by the central laboratory of Shandong Provincial Hospital affiliated with Shandong First Medical University.

2.2. Animals and experimental design

The experimental protocols for all animal studies were approved by the Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University. Eight-week-old male Sprague-Dawley rats (180–200 g body weight (BW), n = 32) were purchased from Shandong University Animal Center (Jinan, China). Rats were housed in a 12 h dark/light cycle animal facility with controlled temperature (20–25 °C) and humidity (40–70%). Food and water were given ad libitum throughout the study.

The rats were allowed to acclimatize for 1 week, after which they were deprived of water for 24 h before the experiment and randomized into four groups. Rats in the control group received saline (10 mL/kg) in each hind limb muscle; those in the AKI group received half the dose of glycerol (10 mL/kg, 50% v/v in sterile saline) in each hind limb muscle; rats in the AKI + Thy group received thymol (20 mg/kg/day or 40 mg/kg/day) gavage daily starting 24 h before glycerol injection; each regimen in AKI + Thy 20 and AKI + Thy 40 was administered thymol for four consecutive days using a gavage needle. Seventy-two hours after glycerol injection, all rats were sacrificed under general anesthesia using an intraperitoneal injection of pentobarbital sodium (150 mg/kg) and euthanized by decapitation. Body weight was measured and blood was collected through heart puncture before decapitation. The left side kidney was excised immediately, weighed, divided into four parts, and stored at −80 °C for biochemical analysis. The right kidney was cut into pieces and fixed in 10% neutral buffered formalin for histological studies.

2.3. Blood sample preparation and biochemical assays

We collected blood from eight rats in each group before decapitation. Blood samples were allowed to clot at room temperature for half an hour and centrifuged (3000 rpm for 10 min) to isolate the serum. Serum biochemical parameters of blood urea nitrogen (BUN), serum creatinine (Scr), calcium, magnesium, sodium, potassium, and phosphorus levels were measured by colorimetric methods adapted to an autoanalyzer.

2.4. Kidney sample homogenate and biochemical assays

The left kidney tissues were washed with normal saline, homogenized in normal saline at 1:9 (m/v) at 4 °C, and centrifuged at 1200 × g for 10 min. The supernatant with 10% tissue homogenate was used to determine the levels of biochemical markers such as malondialdehyde (MDA) and superoxide dismutase (SOD) as described in the manufacturer’s protocol (Jiancheng Biological Company, Nanjing, China).

2.5. Histopathological examination

After recording the kidney weight (KW) and morphological examination, the kidneys were fixed immediately in 10% formalin and embedded in paraffin. For general histology, specimens were cut into 2 μm sections and stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining as described in a previous study [20]. The quantitative results of renal interstitial damage were calculated as follows: 20 random high-power fields for each kidney slide of every rat in each group were examined to calculate the percentage of damaged renal tubules in the total renal tubules (n = 8 per group).

2.6. Immunohistochemistry

For immunohistochemistry analysis, the paraffin specimens were cut into 4 μm sections. The sections were first dewaxed, after which heat-mediated antigen retrieval was performed by pressure boiling sections for 2.5 min in 10 mM sodium citrate (pH 6.0). The sections were allowed to cool for 60 min, followed by a brief wash in deionized water, and then rinsed twice in PBS. Then, the sections were incubated for 30 min in 5% goat serum in PBS containing 0.1% Tween and 0.5% BSA. The sections were incubated overnight at 4 °C with primary antibody-proliferating cell nuclear antigen (PCNA) (Proteintech, Rosemont, IL, 24036-1-AP, 1:100) at the appropriate dilution. The secondary antibody was used to detect primary antibodies, and DAB was used as the HRP substrate for signal detection. The specimens were then counterstained with hematoxylin for 1 min. All sections were incubated under the same conditions with the same concentration of antibodies and at the same time to have comparable immunostaining among the different experimental groups.

The quantitative calculation results of PCNA were as follows: 20 high-power fields were randomly selected for each animal to calculate the percentage of PCNA-positive expression in the total tubular epithelial cell nucleus.

2.7. Western blot analysis

Western blotting was performed according to methods previously described [20]. Briefly, 20 µg of total lysate from kidney tissue was subjected to 10–12% SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature and incubated at 4 °C overnight with the following primary antibodies: rabbit anti-PI3K and p-PI3K (1:1000; Abcam, Cambridge, UK), rabbit anti-AKT and p-AKT (1:1000; CST, Boston, MA), and anti-Nrf2 and anti-HO-1 (1:1000; Abcam, Cambridge, UK). Then, the membranes were washed with TBST buffer and incubated with HRP-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX) for 1 h at room temperature. Protein bands were detected using an ECL system and a Bio-Rad electrophoresis image analyzer (Bio-Rad, Hercules, CA).

2.8. Statistical analysis

Data are presented as the mean ± SD unless stated otherwise as described previously in our research [20]. One-way ANOVA was used to determine significant differences between groups. Dunnett’s test was used to perform multiple comparisons between the groups. A two-tailed p < .05 was considered statistically significant. Statistical analysis was performed using SPSS 22.0 software (SPSS Inc., Chicago, IL).

3. Results

3.1. Effect of thymol on the general condition

To evaluate the success of the AKI model and the preliminary effect of thymol, the following studies were conducted. First, we measured the BW and left KW of the rats separately and then calculated the KW/BW ratio. BW showed no significant difference between the four groups. Compared with the control group, the KW and KW/BW were both significantly higher in the other three groups. Compared with the AKI group, the KW and KW/BW were both significantly lower in the AKI + Thy20 and AKI + Thy40 groups. There was no difference between the two treatment groups (Table 1). Hence, the results showed that AKI induced by RM did not influence BW in 72 h, but caused kidney enlargement.

Table 1. Effect of thymol on BW, KW, and the KW/BW ratio in glycerol-induced AKI.

Index	CTR	AKI	AKI + Thy20	AKI + Thy40
Body weight (g)	212.95 ± 11.66	212.14 ± 15.86	209.09 ± 15.18	209.89 ± 15.15
Kidney weight (g)	0.6318 ± 0.0056	1.4829 ± 0.1623*	1.1691 ± 0.1088*^,^#	1.0977 ± 0.1849*^,#
KW/BW	0.2973 ± 0.0142	0.7017 ± 0.084*	0.5632 ± 0.0837*^,#	0.5245 ± 0.0919*^,#

BW was not changed in any of the treated groups, but the KW and KW/BW ratio significantly increased in the AKI groups. Thymol treatment showed significant and dose-dependent attenuation. Data are expressed as the mean ± SD.

* p < .05 compared with CTR.

^# p < .05 compared with the AKI group.

3.2. Effect of thymol on BUN and Scr

The two principal renal function biomarkers of BUN and Scr were measured in this study. The levels of serum BUN and Scr in the AKI group and the level of Scr in the two treatment groups were significantly higher than those in the control group. However, the levels of serum BUN and Scr in the AKI + Thy20 group and AKI + Thy40 group were significantly lower than those in the AKI group, and there was no difference between the two treatment groups (Table 2).

Table 2. Effect of thymol on renal function and serum electrolytes in glycerol-induced AKI.

Index	CTR	AKI	AKI + Thy20	AKI + Thy40
Scr (μmol/L)	28.74 ± 6.47	167.53 ± 40.06*	73.08 ± 13.06*^,#	68.71 ± 13.43*^,#
BUN (mmol/L)	5.78 ± 1.16	28.76 ± 4.10*	8.03 ± 1.09^#	7.08 ± 1.71^#
Calcium (mmol/L)	2.16 ± 0.109	2.37 ± 0.057*	2.23 ± 0.152	2.18 ± 0.137^#
Magnesium (mmol/L)	0.82 ± 0.069	1.19 ± 0.068*	1.06 ± 0.160*	1.10 ± 0.198*
Sodium (mmol/L)	135.4 ± 6.511	137.5 ± 4.79	133.6 ± 4.11	135.6 ± 4.55
Potassium (mmol/L)	5.60 ± 0.440	6.65 ± 0.47*	5.71 ± 0.381^#	5.94 ± 0.557^#
Phosphorus (mmol/L)	2.54 ± 0.373	3.75 ± 0.458*	3.2 ± 0.384*	3.09 ± 0.489^#

A single intramuscular glycerol injection significantly increased the levels of Scr, BUN, calcium, magnesium, potassium, and phosphorus, whereas thymol treatment attenuated this alteration in renal function and serum electrolytes. Data are expressed as the mean ± SD.

* p < .05 compared with the control group.

^# p < .05 compared with the AKI group.

3.3. Effect of thymol on ion levels

AKI is associated with electrolytes and acid–base disorders. Even mild electrolyte disturbances can significantly increase patient morbidity and mortality. Therefore, serum electrolytes were also detected. Serum calcium was significantly higher in the AKI group than in the control group. The level of serum calcium in the AKI + Thy40 group was significantly lower than that in the AKI group, while the AKI + Thy20 group was not affected (Table 2).

Treatment with glycerol produced a significantly higher level of serum magnesium in the AKI and thymol intervention groups than in the control group. The level of serum magnesium in the AKI + Thy20 and AKI + Thy40 groups showed no significant differences compared with the AKI group. Meanwhile, the serum sodium level in all four groups showed no significant difference (Table 2). Glycerol injection produced a significantly higher level of serum potassium in the AKI group than in the control group. The level of serum potassium in the AKI + Thy20 group and AKI + Thy40 group was significantly lower than that in the AKI group (Table 2).

Glycerol treatment also resulted in a significantly higher serum phosphorous level in the AKI group than in the control group. The level of serum phosphorous in the AKI + Thy20 group and AKI + Thy40 group was significantly lower than that in the AKI group (Table 2).

3.4. Effect of thymol on gross and histopathological changes

As shown in Figure 2(A), the kidneys in the AKI group were enlarged, and the renal parenchyma was congested/darkening, while the normal kidneys were thin and bright. Thymol treatment can effectively reverse this phenomenon. Especially in the high dosage group, the kidneys were nearly normal. Therefore, it is easy to conclude that the protective effect of thymol is effective. H&E and PAS staining of renal tissues was examined under light microscopy for the histopathological assay (Figure 2(B,C)). The rats in the control group were found to be under normal physiological conditions. These animals had normal glomeruli and tubules. The glycerol-treated rats showed widespread damage in both the cortex and the medullar region of the kidney. The microscopic changes include the loss of microvilli, tubular dilatation and necrosis and detachment, naked tubular basement membrane, cellular micro debris into the tubular lumen, many tubular casts, and interstitial inflammatory infiltration. Thymol treatment dose-dependently significantly relieved the severity of renal lesions and renal tubular injury, as shown. The percentages of damaged renal tubules in the total renal tubules in the thymol treatment groups were significantly decreased compared with those in the AKI group (Figure 2(E)).

Figure 2. Gross and histopathological changes in different groups. (A) Isolated rat kidneys from the different experimental groups. (B, C) H&E and PAS staining (×200) showed tubular injury involving the loss of microvilli, tubular dilatation, tubular necrosis, and detachment (blue arrow), naked tubular basement membrane (black arrow), cellular micro debris into the tubular lumen (green arrow), tubular casts (yellow arrow), and interstitial inflammation. Thymol treatment significantly ameliorated the above tubular lesions. (D) The expression of PCNA detected by immunohistochemistry in renal tissue. (E) Quantitative analysis of tubular injury. (F) Quantitative analysis of PCNA-positive tubular epithelial cells. CTR: kidney section from the normal rats; AKI: kidney section from the AKI rats. AKI + Thy20: AKI rats treated with 20 mg/kg thymol. AKI + Thy40: AKI rats treated with thymol 40 mg/kg. Statistical significance: *p < .05 versus the control group; ^#p < .05 versus the AKI group. ^&p < .05 versus the AKI + Thy20 group.

3.5. Effect of thymol on the expression of PCNA

The expression of PCNA was always observed in proliferating cell nuclei. In our research, the expression of PCNA was significantly higher in the AKI and thymol intervention groups than in the control group (Figure 2(D,F)). Moreover, there was a lower percentage of PCNA in the AKI + Thy40 group compared with the AKI group and the AKI + Thy20 group (p < .05), but there was no significant difference between the AKI + Thy20 group and the AKI group (Figure 2(D,F)).

3.6. Effect of thymol on renal oxidative stress

Oxidative stress was determined using kidney tissue SOD and MDA levels. SOD, a superoxide radical scavenger enzyme, decreased significantly in the AKI group compared with the control group (Figure 3(A)). Thymol treatment resulted in a significant increase in SOD compared with the AKI group (20 or 40 mg/kg) (Figure 3(A)). MDA levels increased significantly in the AKI group compared with the control group, but these levels were significantly inhibited by thymol treatment (20 or 40 mg/kg) (Figure 3(B)).

Figure 3. Effect of thymol on renal oxidative stress. (A, B) The expression levels of SOD and MDA in the kidneys of each group. Thymol treatment significantly reduced kidney MDA levels and increased kidney SOD levels compared with the AKI Group 72 h after glycerol injection. (C) Western blot detection showed the downregulated expression of Nrf2 and HO-1 in the AKI group, but thymol treatment increased their expression. Each bar represents the mean ± SD (n = 3). Statistical significance: *p < .05 versus the control group; ^#p < .05 versus the AKI group.

Oxidative stress-related pathways were also detected. Western blot analysis showed that the expression of Nrf2 and HO-1 was significantly decreased in the AKI group compared with the control group (p < .05 for each), whereas treatment with thymol significantly increased the expression of Nrf2 and HO-1 compared with rats in the AKI group (p < .05 for each) (Figure 3(C)).

3.7. Effect of thymol on the renal inflammatory response

To detect the local regional inflammatory response in the kidney, ELISA and western blotting were used to detect the related indices. ELISA results showed that RM induced by glycerol injection generated an excessive inflammatory response with a higher content of IL-6 and TNF-α compared with the control group. In contrast, thymol treatment significantly decreased the expression of the above factors (Figure 4(A,B)). Furthermore, western blot results showed that the expression of MCP-1 and NF-κB p65 was significantly upregulated in the AKI group, while thymol treatment markedly downregulated their expression (Figure 4(C)).

Figure 4. Effect of thymol on the renal inflammatory reaction. (A, B) The levels of IL-6 and TNF-α detected by ELISA were markedly upregulated in the AKI group compared with the control group, while thymol treatment at 20/40 mg/kg reversed the inflammatory reaction. (C) Western blot results showed that the expression of MCP-1 and NF-κB p65 was significantly upregulated in the AKI group, while thymol treatment markedly downregulated their expression. Each bar represents the mean ± SD (n = 3). Statistical significance: *p < .05 versus the control group; ^#p < .05 versus the AKI group.

3.8. Effect of thymol on the PI3K/Akt signaling pathway

The potential mechanisms involved in the effects of thymol on glycerol-induced AKI were investigated. The protein expression of total PI3K and AKT in each group was equal, while p-PI3K and p-Akt were significantly lower in the AKI group than in the control group and were markedly upregulated in the thymol treatment groups (20 or 40 mg/kg) compared with the AKI group (Figure 5).

Figure 5. Effect of thymol on the PI3K/AKT signaling pathway in glycerol-induced AKI. (A) In the AKI group, the expression of p-PI3K and p-AKT detected by western blot was significantly decreased compared with that in the control group, while thymol treatment significantly upregulated them. Each bar represents the mean ± SD (n = 3). Statistical significance: *p < .05 versus the control group; ^#p < .05 versus the AKI group.

4. Discussion

AKI is characterized by a rapid decline in the glomerular filtration rate and retention of nitrogenous waste products. In the clinic, AKI is a significant complication of RM. Glycerol-induced RM-mediated AKI is the most commonly used model for evaluating new approaches to treat or alleviate AKI [21–25]. In this study, intramuscular injection of glycerol in rats led to significantly increased BUN and Scr levels, and histopathological staining revealed intense necrosis and protein cast formation in the tubules. These results efficiently supported the development of glycerol-induced AKI in rats. However, pretreatment with thymol (20 mg/kg and 40 mg/kg) significantly attenuated all previously mentioned changes dose-dependently, suggesting its reno-protective effect in this model.

Glycerol injection also produced a significant increase in serum calcium levels in rats, whereas thymol treatment attenuated glycerol-induced hypercalcemia. Our findings were in accordance with earlier investigators who also observed hypercalcemia in glycerol-treated rats [26,27]. An important role of calcium in cell viability and integrity has long been confirmed. Alteration of calcium homeostasis may cause cellular injury through the activation of phospholipases, impairment of ion permeability, and their subsequent effect on the cytoskeletal structure and function [28]. Moreover, renal dysfunction also led to a significant increase in serum phosphorous and potassium levels in the AKI group, but thymol treatment improved renal function and subsequently decreased phosphorous and potassium levels.

The pathogenesis of RM-induced AKI needs to be further studied. Myoglobin-induced renal toxicity plays a key role in increasing oxidative stress, inflammation, endothelial dysfunction, vasoconstriction, and apoptosis [29]. In this process, lipid peroxidation-mediated oxidant injury and the proinflammatory cytokine-mediated inflammatory response play critical roles [30]. Therefore, inflammatory reactions and oxidative stress are the treatment targets in most previous studies to intervene in AKI induced by glycerol [21–25,31]. Thymol is a colorless crystalline monoterpene phenol used in traditional medicine due to its multiple functions, including anti-inflammatory and antioxidative properties.

Glycerol-induced renal failure is generally associated with a remarkable increase in MDA and ROS levels and a decrease in SOD levels [21–23]. SOD is a superoxide radical scavenger enzyme in the body that plays an important role in the oxidation and antioxidation balance in organisms. SOD can remove excessive free radicals and reduce the negative effects of free radicals on tissues. Additionally, MDA is known as a lipid peroxidation index marker, whose tissue levels indirectly reflect the severity of the cells attacked by free radicals. The kidney is highly vulnerable to oxidative stress-induced tissue injury, likely due to the abundance of long-chain polyunsaturated fatty acids in the composition of renal lipids [32]. In the present study, renal MDA levels significantly increased, and SOD levels significantly decreased in the AKI group compared with the control group. Interestingly, these markers were reversed upon thymol treatment, but not in a dose-dependent manner, which also indicated its powerful antioxidant capacity.

These results also revealed that the antioxidant-related signaling pathway should be involved in the antioxidant effects of thymol. Nrf2 is an important transcription factor responsible for antioxidant genes such as HO-1 or SOD by binding to antioxidant response elements (AREs) in the promoter regions of Nrf2 target genes [33,34]. HO-1 is a stress protein known to abate apoptosis and necrosis, which is upregulated by various stressors and protects cells against stressors [33–35]. In our research, thymol upregulated HO-1 via activation of Nrf2, leading to decreased levels of MDA and increased expression of SOD compared with those in the AKI group.

The pro-inflammatory response and leukocyte infiltration are also implicated in the pathogenesis of glycerol-induced AKI [13,36]. Previous studies reported a significant increase in NF-κB levels in the kidney after glycerol administration in rats [21,37,38]. Free radicals released by damaged tissues and inflammatory cells induce NF-κB activation [39]. NF-κB is a potent inflammatory mediator and plays a major role in proinflammatory cytokine and chemokine syntheses, such as TNF-α, IL-1β, IL-6, IL-8, and MCP-1 [21,37–39]. These cytokines contribute to kidney injury and dysfunction. Accordingly, we found that the levels of IL-6, TNF-α, MCP-1, and NF-κB p65 were significantly elevated in the AKI group, but these changes were attenuated by thymol treatment. As shown in our previous study [20], thymol inhibited the level of inflammatory cytokines by suppressing NF-κB expression in LPS-stimulated peritoneal mesothelial cells.

Notably, we further demonstrated that thymol protects rats from glycerol-induced kidney injury by activating the PI3K/Akt signaling pathway. Previous data suggest that the PI3K/Akt pathway plays an important role in RM-induced AKI [40]. PI3K/AKT plays a central role in many cellular processes, such as apoptosis, cell survival, proliferation, cell cycle progression, and metabolism [41]. Moreover, many studies have shown that the PI3K/Akt pathway plays a key role in the treatment of AKI by enhancing the activity of Nrf2 and reducing apoptosis [14,42–44]. Inhibition of the PI3K/Akt pathway accelerates renal tubular cell death induced by cisplatin [44]. In our research, thymol might upregulate Nrf2 expression via PI3K/Akt. The pathway also plays a diverse role in both the innate immune system and the adaptive immune system [45,46]. Therefore, the anti-inflammatory and antioxidative properties of thymol may partly depend on the activation of the PI3K/Akt pathway, but the exact mechanism needs further definition.

Interestingly, thymol can also inhibit the PI3K/AKT signaling pathway, which is upregulated in some disease states, such as in several cancer cell lines [47,48] and bleomycin-induced pulmonary fibrosis in mice [49]. In these circumstances, PI3K/AKT pathway activation promotes tumor proliferation and tissue fibrosis, and thymol plays antitumor or antifibrotic roles by suppressing it. This is contrary to our result. We speculate that thymol might modulate the expression of PI3K/AKT signaling according to its different pharmacological effects. Significantly, regardless of how the PI3K/AKT pathway is regulated, thymol generally exerts anti-inflammatory and antioxidative stress effects and prevents apoptosis.

The number of PCNA-positive tubular cells in the AKI and thymol treatment groups increased significantly compared with that in the control group. PCNA is an auxiliary protein of DNA polymerase δ that plays a fundamental role in initiating cell proliferation. PCNA expression is an index of renal tubular cell regeneration and DNA synthesis after injury. In our study, there were markedly fewer PCNA-positive tubular cells in the AKI + Thy40 group than in the AKI group, but the opposite tendency was observed in the AKI + Thy20 group. We speculated that high-dose thymol (40 mg/kg) could promote cell proliferation and accelerate the recovery of injured tubular cells, and the recovered cells no longer expressed PCNA. However, the effects of low-dose thymol (20 mg/kg) were weaker than those of high-dose thymol, so there were relatively fewer recovered cells and more proliferating cells.

There are also limitations in our research. First, the number of animals involved is relatively small. Second, the pretreatment duration of thymol is short. In addition, we did not conduct thymol treatment after AKI. Finally, we detected the expression of the PI3K/AKT signaling pathway when an inhibitor of this pathway was not added. That is, the rescue experiment was not performed in our experiment. Additionally, urinary collection, detection, and the protective role of thymol in vitro experiments were not conducted. We hope that these limitations will be addressed soon.

In summary, RM-induced AKI will continue to be a major health problem as long as natural disasters and injuries persist. We demonstrated for the first time that thymol alleviated kidney injury in glycerol-induced AKI. The underlying mechanisms for this effect might be related to preventing of the lipid oxidative-related pathway and inflammation response, possibly via the PI3K/AKT pathway. Therefore, our research suggests that thymol is a potential therapeutic agent against RM-related AKI.

Ethical approval

The animal experiments were performed according to the Guidelines of the Animal Care and Use Committee of the Shandong Provincial Hospital (No. 2018-030).

Consent form

Not applicable.

Author contributions

Qinglian Wang and Guanghui Qi: data curation, formal analysis, and writing the original draft. Fajuan Cheng: conceptualization and writing review and editing. Hongwei Zhou: investigation and writing review. Xiaowei Yang: investigation. Xiang Liu and Rong Wang: conceptualization and writing, review, and editing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Additional information

Funding

The project was supported by the Natural Science Foundation of Shandong Province to Xiang Liu [ZR2020MH079] and the National Natural Science Foundation of China [82100791] to Qinglian Wang.