Introduction

Lead (Pb), an environmental pollutant and toxic agent, is among the heavy metals [1, 2]. Contaminated food, water, and air pollution are major sources of Pb toxicity [3, 4]. According to the World Health Organization, Pb is among the 10 most dangerous substances for public health [5]. Pb excretion is very difficult and can be stored in soft tissues, bones, and other important organs for a long time [6, 7]. In addition, it has toxic effects on many tissues, especially kidney [6, 8, 9]. Although the mechanism of Pb toxicity in the kidneys cannot be understood precisely, studies suggest that oxidative stress has an important contribution to Pb toxicity [10, 11]. Moreover, studies have reported that Pb can induce apoptosis by causing mitochondrial degradation and DNA damage [12, 13] (Fig. 1).

Fig. 1
figure 1

Schematic diagram of the experimental design

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In the treatment of toxicity caused by heavy metals, including Pb, chelators are used to excrete heavy metal from the body [7, 14]. It has been reported that chelators used in lead treatment, along with some undesirable side effects [15], did not have much effect on the lead accumulated in tissue [16, 17]. Therefore, research has focused on various alternative approaches to the treatment of Pb toxicity, particularly plant-based drugs [7].

Herbal products and their active components protect the tissues and organs from the attacks reactive oxygen species (ROS) and prevent the formation of oxidative stress [18, 19]. Thus, these substances are often among the research topics in metal detoxification [19]. Flavonoids, plant secondary metabolites, are the phenolic compounds which are abundant in foods and have antioxidant, antibacterial, anti-cancer, anti-mutagenic, and anti-inflammatory properties [20,21,22,23,24,25]. Chrysin (CR), whose chemical name is 5,7-dihydroxiflavone, is one of the flavonoids commonly used as a traditional medicine and found in many plant extracts, honey, and propolis [26, 27]. Much research demonstrates that CR has antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, anti-allergic, antiapoptotic properties [28,29,30]. CR has antioxidant properties since the hydroxyl groups in its structure have an elimination effect on free radicals [31, 32]. Furthermore, CR has been shown to have anti-inflammatory effect by lowering levels of certain cytokines, prostaglandin E (PGE), cyclooxygenase-2 (COX-2), and nitric oxide (NO) [33, 34]. The daily intake of CR is 0.5–3 g for people [35]. However, CR has been reported to cause toxicity even at low doses in the fish liver cell line [36].

There is no definite information in the literature on whether CR has a protective effect against kidney damage caused by lead, a toxic heavy metal. That’s why the present study was conducted to investigate the protective effects of CR on kidney damage caused by PbAc through using some biochemical and histopathological methods.

Materials and methods

Drugs and chemicals

All chemicals used in the experiment, including lead acetate (lead (II) acetate trihydrate [Pb(CH3CO2)2 3H2O], cas no: 6080-56-4, purity: 99.5–102.0%, mp: 75°C) and chrysin (5,7-dihydroxyflavone [C15H10O4], cas no: 480–40-0, purity 97%, mp: 284–286°C) were of the highest purity and obtained from Sigma-Aldrich Chemical Company (St Louis, MO, USA).

Animals

Thirty-five Sprague-Dawley male rats purchased from Atatürk University Medical Experimental Application and Research Center were used in the experiment. The animals were about 10–12 weeks old and weighed 250–270 g when the experiment began. The environment in which they were kept had 24 ± 1 ° C (room temperature) temperature, 45 ± 5% humidity, and 12-h light/dark cycles. They were fed ad libitum with standard laboratory feed and tap water. Rats were adapted to the environment for 1 week before drug administration. Approval was obtained from the Local Ethics Committee for Animal Experiments of Ataturk University in order to make the applications (Approval no: 2019-12/163).

Treatment protocol

PbAc and CR doses were determined with reference to previous studies [37, 38]. In this study, 5 different groups were formed with 7 male rats in each group. The groups were designed as follows:

  • Group 1 (control group): saline was given orally to rats for 7 days.

  • Group 2 (CR-50 group): 50 mg/kg/BW CR was given orally to rats for 7 days.

  • Group 3 (PbAc group): 30 mg/kg/BW PbAc was given orally to rats for 7 days.

  • Group 4 (PbAc + CR-25 group): 30 mg/kg/BW PbAc was given orally to rats 30 min after 25 mg/kg/BW CR the administration for 7 days.

  • Group 5 (PbAc + CR-50 group): 30 mg/kg/BW PbAc was given orally to rats 30 min after the administration of 50 mg/kg/BW CR for 7 days.

Collection of samples

Twenty-four hours after the last drug administration, the animals’ body weights were measured and then they decapitated under mild isoflurane (IsoFlo; Abbott, Queenborough, UK) anesthesia. As soon as they were decapitated, blood samples were collected from their Vena jugularis. Blood samples were collected in anticoagulant free tubes and centrifuged at 1200×g for 15 min. Blood serum was used to determine renal function. After the kidneys from rats were washed with ice-cold physiologic saline (0.85% NaCl), one of them was stored at − 80 ° C for biochemical analysis and the other in 10% buffered formalin solution for histological examination until used.

Determination of serum urea and creatinine levels

Serum urea [39] and creatinine [40] levels were analyzed with a commercial kit (Diasis Diagnostic Systems, Istanbul, Turkey) according to the manufacturer’s instructions.

Preparation of tissue homogenates

To obtain homogenate from the kidneys, the tissues were diluted 1:20 v/w with phosphate-buffered saline (PBS; pH 7.4). The resulting mixture was rapidly homogenized with a tissue lysate device (TissueLyser II, Qiagen). The homogenate was then centrifuged at + 4°C and 3000 rpm for 30 min. The supernatant was used for biochemical analysis.

Determination of lipid peroxidation and antioxidant enzyme activities in kidney tissue

The level of lipid peroxidation was determined by analyzing the amount of malondialdehyde (MDA) at 532 nm according to the method developed by Placer et al. [41]. The amount of MDA was expressed as nmol/g tissue. Superoxide dismutase activity was measured by the method designed by Sun et al. [42]. The results were expressed as U/g protein. The measurement of catalase activity was performed according to the method of Aebi [43]. The results were expressed as catal/g protein. Glutathione peroxidase (GPx) activity was measured according to the method developed by Lawrence, Burk [44]. Results were expressed as U/g protein. The method developed by Sedlak, Lindsay [45] was used to determine the level of glutathione. Results were expressed as nmol/g of tissue. Total protein analysis was performed according to the method developed by Lowry et al. [46] using bovine serum albumin (BSA) as standard.

Determination of AQP-1 levels in renal tissue

Aquaporin-1 (AQP-1) levels was performed by using rat enzyme-linked immunosorbent assay (ELISA) kit (cat. no: 201-11-0566; assay range: 0.15–40 ng/ml) obtained from Sunred Biological Technology Company.

Determination of p53 levels in renal tissue

p53 levels were analyzed with rat ELISA kit according to the manufacturer’s instructions (Sunred, Shanghai, China) (cat. no: 201-11-0072; assay range: 0.05–10 ng/ml). The color intensity at the end of the procedures was read at 450 nm with the ELISA microplate reader (Bio-Tek, Winooski, VT, USA).

Determination of inflammatory response levels in kidney tissue

Interleukin-33 (IL-33) (cat. no: 201-11-3102; assay range: 1.5 - 400 ng/L), COX-2 (cat. no: 201-11-0297; assay range: 0.5–150 ng/ml), PGE-2 (cat. no: 201-11-0505; assay range: 0.05–15 ng/ml), nuclear factor kappa-B (NF-κB) (cat. no: 201-11-0288; assay range: 0.08–20 ng/ml), and inducible nitric oxide synthase (iNOS) (cat. no: 201-11-0741; assay range: 0.8–200 ng/ml) in renal tissue were measured by ELISA to determine the degree of inflammatory response levels in accordance with the manufacturer’s instructions (Sunred, Shanghai, China).

Determination of DNA damage level in kidney tissue

The degree of DNA damage in kidney tissue was determined by measuring the level of 8-hydroxy-2′-deoxyguanosine (8-OHdG) using a commercial kit (Sunred, Shangai, China) (cat. no: 201-11-0032; assay range: 0.05–20 ng/ml).

Histopathological examination of kidney tissue

Necropsy was performed for histopathological evaluation of the renal tissues and tissue samples were fixed in 10% formalin solution for 48 h. Tissues were embedded in paraffin blocks with routine-tracking procedures. From each block were taken 4-μm-thick cross-sections. All preparations were stained with hematoxylin-eosin (HE) and examined with a light microscope (Leica DM 1000, Germany).

Immunohistochemical examination of kidney tissue

Cross-sections obtained from kidney tissues were transferred to adhesive (poly-L-Lysin) slides for immunperoxidase examination and passed through xylol and alcohol series. After washing with phosphate-buffered saline (PBS), endogenous peroxidase was inactivated for 10 min at 3% H2O2 (Merck, K50505100 830 1.08600.1000). In the microwave oven set at 500 watts, the antigen in the tissues was released by exposure to retrieval solution (abcam, ab93678) for 2 × 5 min. Tissues were incubated with nephrin and tumor necrosis factor-α (TNF-α) antibodies (catalog no. Ab-216692, Abcam, UK, sc-52B83, Santa Cruz, USA) for 30 min,in an incubator set at 37 °C. Immunohistochemistry procedures were followed according to the kit instructions (AbcamHRP/DAB Detection IHC kit). 3-3 ’Diaminobenzidine (DAB) was used as chromogen. Background staining was performed with hematoxylin. Immuno-positivity of the samples was expressed as none (-), mild (+), moderate (++), and severe (+++).

Determination of elemental content of kidney tissue

In this study, ICP-MS NexION® 2000 (PerkinElmer® Inc., USA) device with quartz nebulizer gasifier, cyclonic spray chember, and integrated auto-sampler was used for the element analysis of samples. The washing solution containing 1% hydrochloric acid ultra-pure water was prepared using 18.3 MΩ ultra-pure water and the ICP-MS method was performed. In the preparation of the sample, 0.2 g were weighed, and transferred to the microwave oven teflon cups and added 10 mL nitric acid. ICP-MS calibration solutions were prepared by dilution with commercially available multi-element standards of 1% (nitric acid, ultra-pure water). In addition, ICP-MS calibration was performed before each measurement. With a peristaltic pump, the samples were sent to the cyclonic spray chamber with argon gas flow. ICP-MS NexION instrument software was used to control the instrument, including calibration, interferences, data collection, and data analysis. In addition to argon gas, helium gas was used to prevent interference.

Statistical analysis

Statistical analysis of biochemical data was done with one-way ANOVA test in IBM SPSS program (version 20.0; IBM Co, North Castle, NY). Tukey’s multiple comparison test was used for comparisons between the groups. All values were expressed as mean ± standard error (SEM), and p < 0.05 was considered significant. Kruskal-Wallis test, which is one of the nonparametric tests, was applied to the data obtained from histopathological examinations in order to determine the differences between the groups. The comparison of binary groups was done using Mann-Whitney U test. SPSS 13.0 package program was used for these statistical analyzes.

Results

Analysis results of serum urea and creatinine levels

The results indicated that PbAc increases serum urea and creatinine levels by disrupting kidney function (P < 0.05). CR was found to reduce serum urea and creatinine levels by decreasing kidney toxicity caused by PbAc in a dose-dependent manner (25 and 50 mg/kg/BW). It was determined that there was no significant difference between the control and CR groups. Furthermore, it was observed that the rats who lost weight with PbAc administration reached the weight of the animals in the control group with CR treatment. Serum urea and creatinine levels and body weights of all groups are presented in Table 1.

Table 1 Effects of CR on kidney function parameters and body weights in PbAc-induced nephrotoxicity in rats
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Analysis of lipid peroxidation and antioxidant markers in kidney tissue

It was determined that PbAc administration increased MDA levels by causing lipid peroxidation in kidney tissue and decreased the activities of antioxidant enzymes (SOD, CAT and GPx) and GSH levels. On the other hand, it was found that CR administration decreased lipid peroxidation; thus, MDA levels reduced. It was also detected that CR treatment significantly increased SOD, CAT, and GPx activities and GSH levels compared to PbAc group (P < 0.05). Lipid peroxidation and the levels of antioxidant markers in kidney tissue are given in Table 2.

Table 2 Effects of CR on oxidative stress parameters in PbAc-induced nephrotoxicity in rats
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Analysis results of AQP-1 levels in kidney tissue

AQP-1 levels are given in Fig. 2. The results demonstrated that PbAc decreased AQP-1 levels due to the damage to the kidneys. Furthermore, CR administration was observed to significantly increase AQP-1 levels compared to PbAc group by alleviating kidney damage P < 0.05.

Fig. 2
figure 2

Effects of CR on AQP-1 levels in PbAc-induced nephrotoxicity in rats. Data of rats in each group were expressed as mean ± SEM (n = 7). Different letters (a–d) indicate statistical difference among the groups (p < 0.05)

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Analysis results of p53 levels in kidney tissue

p53 levels analyzed by ELISA method are given in Fig. 3. The results demonstrated that PbAc made cells undergo apoptosis by increasing the p53 levels. CR was found to protect the cells against apoptosis by suppressing p53 expression.

Fig. 3
figure 3

Effects of CR on p53 levels in PbAc-induced nephrotoxicity in rats. Data of rats in each group were expressed as mean ± SEM (n = 7). Different letters (a–d) indicate statistical difference among the groups (p < 0.05)

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Analysis of inflammatory response levels in kidney tissue

Analysis results of inflammatory markers of kidney tissue are presented in Table 3. The results showed that PbAc led to inflammation by significantly increasing IL-33, PGE-2, COX-2, NF-κB, and iNOS levels compared to the control group. Nevertheless, CR was found to reduce PGE-2, COX-2, NF-κB, and iNOS levels in a dose-dependent manner, thereby alleviating inflammation in the kidney. It was found that IL-33 levels did not make a significant difference between PbAc + CR-25 and PbAc + CR-50 groups.

Table 3 Effects of CR on inflammation parameters in PbAc-induced nephrotoxicity in rats
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DNA damage level

As stated in Fig. 4, it was observed that there were increases in 8-OHdG levels since PbAc damaged DNA. CR was observed to protect DNA from damage caused by PbAc and to limit the rise of 8-OHdG levels.

Fig. 4
figure 4

Effects of CR on 8-OHdG levels in PbAc-induced nephrotoxicity in rats. Data of rats in each group were expressed as mean ± SEM (n = 7). Different letters (a–d) indicate statistical difference among the groups (p < 0.05)

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Histopathological findings

Histopathological examination showed that the kidney tissues of the rats in the control and CR-50 groups had normal appearance (Fig. 5a–b). Mononuclear cell infiltration in the interstitium of the PbAc group, severe hydropic degeneration and necrosis in the tubules, severe hyperemia in the vessels, and hyaline cylinders in some tubulus lumens were observed (Fig. 5c). Hydropic degeneration and mild coagulation necrosis, mild mononuclear cell (MNH) infiltration, and hyperemia in interstitial areas were detected in moderate tubulus epithelium in the PbAc + CR-25 group (Fig. 5d). In the PbAc + CR-50 group, the lesions were very mild and statistically significant P ˂ 0.05 differences were found when compared to the PbAc group (Fig. 5e). Histopathological findings of all groups are summarized in Table 4.

Fig. 5
figure 5

a–e Histopathological examination of rat kidney tissue. a–b Control and CR-50 group: kidney tissue with normal histological structure; c PbAc group: tubular epithelium with hydropic degeneration (arrowheads), necrosis (thin arrows), mononuclear cell infiltration in interstitial areas (stars) and severe hyperemia (arrows) in vessels, hyaline cylinders in tubulus lumens (thick arrows). d PbAc + CR-25: mild mononuclear cell infiltration in interstitial areas (star), tubular epithelium with moderate degeneration (arrowheads), mild necrosis (thin arrow), hyaline cylinders in tubulus lumens (thick arrows). e PbAc + CR-50: tubular epithelium with mild hydropic degeneration (arrowheads), H&E; bar, 20 μm

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Table 4 Histopathological and immunohistochemical results of the effects of CR on PbAc-induced nephrotoxicity
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Immunohistochemical findings

As a result of immunohistochemical examination of the renal tissues, severe nephrin expression was observed in the tubulus epithelium in the control and CR-50 groups, but TNF-α expression was not observed (Fig. 6, 7a–b). Negative nephrin expression in tubular epithelium in PbAc group and severe TNF-α expression in interstitial tissues, perivascular, and glomeruli were determined (Fig. 6, 7c). Nephrine was slightly expressed in renal tubule epithelium of the PbAc + CR-25 group (Fig. 6d) and TNF-α expression was moderate in the interstitial area (Fig. 7d). In the PbAc + CR-50 group, nephrine expression was severe in the tubulus epithelium (Fig. 6e). Mild TNF-α expression was detected in the interstitial area (Fig. 7e). In this group, expression levels of immunohistochemical markers were statistically significant (P ˂ 0.05) differences when compared with PbAc group. Immunohistochemical findings are summarized in Table 4.

Fig. 6
figure 6

a–e Nephrin expression in the rat kidney tissue. a–b Control and CR-50 group: severe nephrin expression (arrowheads) in tubular epithelium; c PbAc group: negative nephrin expression; d PbAc + CR-25: mild nephrin expression (arrowheads) in tubulus epithelia; e PbAc + CR-50, moderate nephrin expression in tubular epithelium (arrowheads), IHC-P; bar, 20 μm

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Fig. 7
figure 7

a–e TNF-α expression in the rat kidney tissue. a–b Control and CR-50 group, negative TNF-α expression; c PbAc group: severe TNF-α expression (arrowheads) at interstitial intervals; d PbAc + CR-25: moderate TNF-α expression (arrowheads); e PbAc + CR-50: mild TNF-α expression at interstitial intervals (arrowheads), IHC-P; bar, 20 μm

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Levels of elements in kidney tissue

According to the data obtained by ICP-MS method, the PbAc and PbAc + CR-25 groups were found to have the highest Pb accumulation in kidney tissue compared to the control group (P < 0.05). It was determined that 50 mg/kg/BW administration of CR reduced Pb accumulation caused by PbAc. The groups with the highest K level were found to have CR administration with PbAc. In the PbAc and CR groups, K levels were significantly lower than the control group (P < 0.05), but there was no significant difference between them (P > 0.05). The PbAc group had the highest Na levels in the kidney tissue compared to the control group. In the CR-50 group, Na levels decreased significantly (P < 0.05), but there was no significant difference between CR administration with PbAc and control groups (P > 0.05). PbAc + CR-25 and PbAc + CR-50 were the groups with the highest Ca levels compared to the control group. While there was no significant difference between CR-50 and PbAc groups (P > 0.05), it was found that Ca levels decreased compared to the control group (P < 0.05). It was observed that CR decreased significantly Fe levels in kidney tissue compared to control group (P < 0.05). Cu and Zn levels were significantly higher in the PbAc group than in the control group (P < 0.05); nevertheless, it was found that CR administration reduced this increase caused by PbAc from the control group to low levels. Levels of all elements in the kidney tissue in different groups are given in Table 5.

Table 5 Effects of CR on renal element contents in PbAc-induced nephrotoxicity in rats
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Discussion

The protective effects of naturally occurring antioxidant substances against heavy metal toxicity have been the subject of many studies, and researchers have achieved promising results [4, 47, 48]. In this study, the protective effects of CR against kidney damage of rats exposed to PbAc orally were investigated. Biochemical, histopathological, and immunohistochemical results showed that PbAc caused damage in the kidneys. However, it was determined that the administration of CR to rats before PbAc administration had a positive effect on these results and had a protective effect against kidney damage.

The kidneys are highly vulnerable to toxic damage because they are exposed directly to blood plasma through their open fenestrae [49]. That’s why, it is thought that one of the organs primarily affected by toxic substances is kidneys and kidney function disorders occur. The measurements of serum urea and creatinine levels are frequently used to evaluate kidney function [50]. It is known that an increase in serum urea and creatinine levels is associated with kidney failure [51]. Abdel-Moneim et al. [49] reported that PbAc caused an increase in serum urea and creatinine levels possibly because it led to kidney dysfunction and kidney failure. In this study, it was detected that there was a significant increase in serum urea and creatinine levels in animals treated with PbAc. In addition, as a result of histopathological examinations, it was observed that PbAc caused severe hydropic degeneration and necrosis in the tubules. It was determined that CR administration alleviates PbAc-induced lesions in tissues and reduces urea and creatinine levels to normal levels.

The disruption of the balance between the antioxidant system in the body and the production of reactive oxygen species causes oxidative stress [52, 53]. The possible mechanism of Pb toxicity is thought to be oxidative stress [15]. Lipid peroxidation provides important contributions in determining renal cell damage. MDA is the degradation product of lipid peroxidation. On the other hand, SOD, CAT, and GPx are antioxidant enzymes that provide antioxidant defense in the body [54]. Studies reported that Pb, by linking to SH- groups of antioxidant enzymes, decreased the activities of these enzymes and caused the depletion of GSH, a non-enzymatic antioxidant and lipid peroxidation. Therefore, in the treatment of Pb toxicity, it is aimed both to remove Pb from the body and to prevent the occurrence of oxidative stress by cleaning the reactive oxygen species [15]. In the current study, it was seen that PbAc increased MDA levels by causing lipid peroxidation. It was observed that CH reduced lipid peroxidation, increased enzymatic and non-enzymatic markers and protected membrane integrity in kidney tissue due to the antioxidant properties of hydroxyl groups in its structure.

Aquaporins, which are water channel proteins, are transmembrane glycoproteins that allow the entry or release of water across the permeable epithelium, such as renal tubular epithelium [55, 56]. To date, many types of AQP have been cloned and characterized. The expression and/or physiological regulation of AQP-1, AQP-2, AQP-3, AQP-4, AQP-6, AQP-7, AQP-8, and AQP-11 are well documented in the kidney [57] AQP1 is abundantly expressed in the apical and basolateral areas of the proximal tubular cells and in the descending limb cells of the Henle loop. AQP1 helps quickly reabsorption of large quantities of filtered water [58]. It has been reported that toxicity and dysfunction in kidneys considerably affect the levels of AQPs and that AQP-1 levels in the kidney tissues of rats with different toxic agents dramatically decrease [19, 55, 59]. Nephrin is an important structural protein of the glomerular filtration barrier and is responsible for ultrafiltration [60, 61]. Decrease in nephrine levels causes glomerular dysfunction and proteinuria [60]. Preservation of nephrine expression is thought to be a potential therapeutic approach to alleviate podocyte loss and glomerular damage in glomerular disease [62]. In the present study, it was determined that PbAc had toxic effects in the kidneys, which led to kidney dysfunction and significantly reduced AQP-1 and nephrin levels. CR was found to reduce the toxic effects of PbAc and alleviate kidney dysfunction and increase AQP-1 and nephrin levels.

The mechanism of lead toxicity is quite complicated. However, according to the studies done, it has been reported that one of Pb’s toxicity mechanisms is the apoptosis pathway [63]. Apoptosis induced by various chemicals or environmental stimuli occurs under the control of many genes, including p53 [64]. Under normal conditions, p53 expression is kept in a low levels due to the extremely short half-life of the polypeptide. Nonetheless, in the cases where ROS levels increase and following the damage to DNA, p53 protein levels increase significantly in a short time [63]. PbAc can directly damage DNA, or by causing oxidative stress, it can indirectly damage cells and DNA [65]. This explains the situation in the current study that PbAc causes severe damage to the kidneys by inducing an increase in p53 levels. However, it has been determined that CR reduces p53 levels by alleviating oxidative stress and DNA damage due to its antioxidant effect, thus protecting kidneys from PbAc toxicity.

There is growing evidence of the link between oxidative stress and inflammatory response. Oxidative stress provides important contributions to the inflammation process. It has been reported that oxidant molecules affect all phases of the inflammatory process, such as the release of endogenous danger signal molecules, their perception by natural immune cells from the Toll-like receptors (TLRs) and NOD-like receptor (NLRs) families, and the activation of signal pathways that initiate an adaptive cellular reaction to these signals [66]. The responses initiated by TLRs are transmitted by activation of NF-κB [67]. Therefore, oxidative stress activates NF-κB and initiates inflammation mechanism. This is one of the strongest evidence supporting the link between oxidative stress and inflammation in disease progression [68]. NF-κB stimulates the release of pro-inflammatory cytokines, particularly TNF-α. In addition, expression of iNOS and COX-2 proteins is regulated by NF-κB. Therefore, suppression of NF-κB is of great therapeutic importance [19, 69]. Liu et al. [70] reported that Pb affects kidney tissue and causes NF-κB activation and inflammation. Flavonoids play an important role in the regulation of cellular functions such as cell cycle signals and modulation of inflammatory pathways [71]. Rehman et al. [72] showed that CR effectively inhibited the increase in ferric nitrilotriacetate-mediated TNF-α, COX-2, iNOS, and PGE2 expressions. Kandemir et al. [73] reported that paracetamol-induced inflammation in kidney tissue improves CR and decreases IL-33 levels. Similar to the literature, in the present study, PbAc increased NF-κB, IL-33, PGE-2, COX-2, and iNOS levels in renal tissue due to oxidative stress. As a result, inflammation in the kidney tissue occurred. CR reduced the inflammation caused by PbAc and decreased NF-κB, IL-33, PGE-2, COX-2, and iNOS levels significantly compared to PbAc group. Immunohistochemical examination revealed that TNF-α was strongly expressed in the PbAc-treated group, while CR decreased TNF-α expression.

DNA is a highly sensitive macromolecule to oxidative damage [74]. 8-OHdG is a widely used biomarker for determining oxidative damage in DNA [75]. ROS is thought to play an active role in the formation of 8-OHdG [19, 75]. According to the data obtained from this study, PbAc increased the formation of 8-OHdG by causing oxidative damage in DNA. Also, CR improved PbAc-induced oxidative DNA damage with antioxidant properties, approximating the formation of 8-OHdG to that of the control group. Similarly, Rani et al. [76] reported that CR significantly reduced the 8-OHdG level dose-dependently.

In the studies evaluating the effectiveness of antioxidants as chelating agents, although antioxidants are reported to be not as effective as traditional chelators [77, 78], there are the studies showing that flavonoids have chelating properties in addition to their antioxidant properties [79]. It has been reported that CR is also capable of metal chelation [80]. In our study, we found that administration of 25 mg/kg/BW of CR did not make a significant difference in the amount of Pb in renal tissue compared to the PbAc group, but that of 50 mg/kg/BW reduced chelation of Pb significantly.

There is sufficient information that heavy metals, including Pb, may have adverse effects on the concentrations of essential metals. However, the information about the effect of electrolytes in the body is insufficient [81]. Xia et al. [82] reported that Pb has no effect on the amount of Cu and Zn in kidney tissue. Aksu et al. [83] stated that PbAc administration increases zinc accumulation in the kidney and has no effect on Cu and Fe levels and also increases Zn level with the use of phenolic compounds. In our study, it was found that PbAc significantly increased Cu, Zn, and Na amounts in kidney tissue and decreased K, Ca, and Fe amounts in comparison to the control group. However, it was seen that K and Ca increased; Fe, Cu, and Zn decreased in the kidneys of the rats given CR with PbAc, and Na did not make a significant difference compared to the control group. Given that Zn homeostasis is provided by the kidneys, the information that PbAc accumulates in the kidneys along with damage to the kidneys confirms our data [83]. Also, Pb causes Fe absorption to reduce by linking to similar areas with Fe [84]. This explains why PbAc reduces the amount of Fe in the kidney in the current study.

Conclusion

Our findings confirmed that PbAc led to toxicity in the kidneys because of inflammation and apoptosis associated with oxidative stress. It was also detected that toxicity decreased AQP-1 levels. However, it was concluded that the antioxidant, anti-inflammatory and antiapoptotic properties of CR also apply to PbAc-induced nephrotoxicity, and that CR is a promising compound in the treatment of renal toxicity. Still, the mechanism of this effect of CR needs to be supported by further studies.