Introduction

Nickel (Ni) plays a significant role in urban life activities and Ni pollution is a server problem in Sichuan, China, where most Ni was produced for the country. Excessive Ni exposure has neurotoxicity, hepatotoxicity, nephrotoxicity with oxidative stress, and abnormal apoptosis usually shown at the cellular level [1]. Especially, the kidney is the major organ in excreting metabolites and targets Ni toxicity [2]. Ni exposure has been studied to induce renal cell oxidative stress, proptosis, apoptosis, and ferroptosis and lead to kidney tissue damage [3,4,5,6]. Oxidative stress is usually accompanied with such phenomenon toxicologically. 4-tert-Butylphenol treatment decreased SOD and CAT and caused oxidative stress in common carp head kidneys [7]. Excess ammonia exposure downregulated SOD and GSH-Px, upregulated ROS and MDA, and so resulted in oxidative stress in chicken splenic lymphocytes [8] that induced tissue damage. It has been reported that Ni could decrease the activities of antioxidative enzymes and increase the levels of oxidation products [9, 10] in chicken. Ni exposure reduced GSH, elevated MDA, and induced oxidative stress in chicken cerebellum and thalami [11]. However, sulfur-containing amino acids are an essential component of the antioxidant system, which has some ligands that could reduce metal toxicity by chelation and reactivate the activities of enzymes [12, 13]. Sulfur-containing methionine has been reported already to be capable of preventing and treating the toxic effects caused by heavy metals on organisms [14]. It exerted endogenous antioxidation through self-redox reaction under the catalysis of methionine sulfoxide reductase (MSR) [15].

NF-κB pathway is one of the main immune pathways in mouse kidneys and is considered a typical proinflammatory signal pathway. However, this pathway not only has a proinflammatory function but also an anti-inflammatory function [16]. Excess environmental pollutants lead to an increase in inflammation-related factors and inflammatory responses in animals. It was reported that 4-octylphenol caused inflammatory responses in common carp gills with increased NF-κB, TNF-α, and IL-6 [17]. Zhou et al. (2023) mentioned that exposure to excess manganese resulted in inflammatory responses in common carp kidneys with upregulated TNF-α and IL-6 [18]. Chen et al. (2024) found that excess lead elevated IFN-γ and IL-6, leading to brainstem inflammatory response in chickens [19]. Previous studies have shown that methionine deficiency could induce oxidative stress accompanied by server inflammation. Further research showed that the covalent modification of protein subunit could ease the expression of NF-κB, thus inhibiting the occurrence of oxidative stress and inflammation. It suggested that methionine may help inhibit the expression of NF-κB pathway by the covalent modification of sulfur-containing and achieve its antioxidant activity [20,21,22].

Given the serious Ni pollution in Sichuan and overdoes, Ni exposure could trigger a series of renal dysfunction (oxidative stress), and previous studies have shown that sulfur-containing amino acids have good antioxidant activity. We designed this study to detect renal function, histological changes, cell cycle, apoptosis, oxidative stress, and NF-κB regulated cytokines in mice treated with different diets with nickle under the condition of methionine addition and deficiency by tissue sections, immunohistochemistry, TUNEL, biochemical, and ELISA methods. We checked the toxicological effect of excessive Ni exposure on mice kidney injury with emphasis on cell and tissue damage, oxidative stress, and NF-κB regulated inflammation and determined if dietary methionine addition alleviated Ni toxicity in mice.

Materials and Methods

Animals

One hundred 6-week-old ICR male mice (25 ± 3 g) were randomly and equally divided into 4 groups, with each 5 replicates (5 mice in each replicate). ICR male mice were provided by the Experimental Animal Center of North Sichuan Medical College (Sichuan, China). The mice were raised under standard conditions, temperature (23 ± 2 °C) and humidity (50 ± 10%) with alternating 12 h light/dark cycles.

Treatment and Sample Collection

The animals were randomly divided into 4 groups. As shown in Table 1, BC, MD, MN, and MDN groups were attributed to and equipped with freely accessed food and water by experimental requirements for 21 days. The diets in this study (normal or methionine deficiency) were made by Trophic Animal Feed High-tech Co., Ltd., China (Nantong, China), details of contents are referred to Miousse et al. (2018) [23]. After 3 weeks, all mice in each group were euthanized, urine samples were collected and aliquoted, blood samples were collected and were kept overnight to clot and then centrifuged at 3000 g for 10 min to separate the serum for the estimation of the parameters related to renal function. The kidney tissues were collected from the sacrificed animals and quickly fixed in 4% paraformaldehyde solution for histological observation (H.E), cell cycle (IHC), and apoptosis (TUNEL), and the other part was stored at − 80 °C for the detection of antioxidant enzymes activity, the oxidative product and inflammatory factors.

Table 1 Animal treatment
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Determination of Renal Function-Specific Markers

Blood serum was used to assess the contents of creatinine (Cr) (C011-2–1) and blood urea nitrogen (BUN) (C013-2–1). They were determined by the sarcosine oxidase method and urease method, respectively. β-N-acetyl-glucosaminidase (NAG) (A031-1–1) content was detected in the urine by colorimetric assay kits according to the manufacturer’s recommended protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The Observation of Histological Changes in the Kidneys

The fixed kidneys were dehydrated with 75%, 85%, 95%, and 100% alcohol, cleared with xylene, immersed, and embedded in melting paraffin. Then, sections with thicknesses of 5 µm were made and sealed after filming exhibition, baking sheet, dewaxing, and stained with hematoxylin/eosin (H.E.). Finally, photographs were taken under light microscopy to observe the histomorphological changes.

Detection of the Cellular Cycle by Proliferating Cell Nuclear Antigen (PCNA)—Immunohistochemistry (IHC)

The cellular distribution of PCNA was detected by the IHC method in the kidneys of the four groups. The number of PCNA-positive cells (brown) reflects the active level of cell proliferation. PCNA-positive cells were detected according to the instructions of the PCNA Cell Proliferation Detection Kit (E607250-0100, Sangon Biotech, Co., Ltd, Shanghai, China).

Detection of Apoptosis by Transferase-Mediated dUTP Nick-End Labeling (TUNEL)

Apoptotic cells in the kidney sections were detected with colorimetric apoptosis detection kits (G001-2, Nanjing Jiancheng Bioengineering Institute Nanjing, China). The pretreated samples were washed with PBS and dried. TdT enzyme reaction solution of 50 µl was added to each sample, reacted in humid and dark at 37 °C for 60 min, washed with PBS, and dried. Streptavidin HRP working solution of 50 µl was added to each sample, covered with glass slides reacting in humid and dark at 37 °C for 30 min, and washed with PBS. The DAB working solution of 60 ml was added to each sample, and the color reaction was taken at room temperature for 10 min and washed with PBS. Lastly, the sections were observed under a light microscope and photographed. The cells that ultimately express positive (turning brown) are apoptotic cells. When measured, dUTP connects to the end of 3′-OH with the broken DNA in apoptotic cells, producing a strong dark brown reaction that could be observed under a microscope.

Assay of Oxidative Biochemical Parameters in Renal Tissues

The homogenate of the kidneys was prepared and then centrifuged to obtain the supernatant. The biochemical indicators of SOD, CAT, GSH-ST, GSH, and GSH-Px were measured. Procedures were in strict accordance with the kit instructions, and the specific methods and formulas are as follows. The activity of superoxide dismutase (SOD) (A001-2) was detected with the hydroxylamine method; the activity of catalase (CAT) (A007-1–1) was detected with ammonium molybdate method; the activity of glutathione s-transferase (GSH-ST) (A004-1–1) was detected with colorimetric; the activity of glutathione (GSH) (A006-1–1) was detected with spectrophotometric method; the activity of glutathione peroxidase (GSH-Px) (A005-1) was detected with colorimetric. All the test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The Detection of MDA, LPO, and ROS

The oxidation products of malondialdehyde (MDA), lipid peroxide (LPO), and reactive oxygen species (ROS) were measured, procedures in strict accordance with the kit instructions, and the specific methods and formulas are as follows.

The content of MDA (A003-1) was detected according to the thiobarbituric acid (TBA) method; the content of LPO (A106-1) in the supernatant was detected according to the spectrophotometric method; the content of ROS was determined with chemical fluorimetry (E004-1–1). All the test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Expression Levels of Related Inflammatory Factors in NF-κB Signal in the Kidneys

NF-κB signal relating factors—NF-κB (SEB824Mu), TNF-α (PAA133Mu01), IFN-γ (SEA033 Mu), IL-1a (SEA071Mu), and IL-6 (SCA079Mu) protein contents were measured with commercial kits (Wuhan Cloud-Clone Technology Co., Ltd., China).

Statistical Analysis

Data were expressed as mean ± SD of at least three independent experiments. Data were analyzed using SPSS software version 17.0. The statistical significance of the data was assessed using one-way ANOVA followed by Tukey’s test. The different lowercase letters represented significant differences between groups (P < 0.05), and the different capital letters represented extremely significant differences between groups (P < 0.01).

Results

Changes in Renal Function

The comparisons of serum BUN, Cr, and NAG levels among the groups for the evaluation of renal function and renal injury are shown in Table 2. The levels of Cr and the NAG value in the MD group were significantly higher than the BC group (P < 0.05), and the level of BUN was slightly higher than the BC group. The levels of Cr, BUN, and the NAG value in the MN group were extremely significantly or significantly higher than in the BC group (P < 0.01 or P < 0.05); the levels of Cr and BUN as well as NAG value in MDN group were extremely significantly higher than BC group (P < 0.01); the levels of BUN, Cr and the NAG value in MDN group were significantly higher than MD group, which indicated that Ni could increase the levels of BUN, Cr, and NAG; Compared with MDN group, the parameter level in MN group was lower and NAG value was significantly lower, which showed that methionine could decrease the trend of parameter level increased caused by Ni. Results showed that methionine deficient plus Ni could extremely significantly increase the levels of BUN, Cr, and the NAG value, and methionine could decrease this trend.

Table 2 The parameters of renal function and renal injury
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Histological Changes in the Kidney

Figure 1 shows the histopathological changes of different kidneys of the control (BC) and treatment (MD, MN, and MDN) mice. There were no obvious pathological changes in the kidney tubules and glomerulus in the BC group. Glomerulus partial atrophy and kidney tubules partial swelling deformation were found in the MD group, and the atrophy degree of the glomerulus was high. The swelling degree of the kidney was high in the MN and MDN groups, especially in the MDN group. Compared with the MD group, the injury degree in the MDN group was higher, which showed that Ni could induce the injury of kidney tubules and glomerulus. Compared with the MDN group, the injury degree in the MN group was lower, which showed that methionine could alleviate renal tissue damage. Results showed that methionine deficient plus Ni could extremely significantly induce renal injury. Also, treatment with methionine could prevent toxin-induced alterations and keep the pathological lesions almost close to the normal range.

Fig. 1
figure 1

Changes in the histological structure of the kidneys (H&E, × 100). Kidney sections from control mice showed a normal appearance of the glomerulus and tubules. Nickel-exposed mice showed loss of integrity (glomerulus atrophy and kidney tubules swelling deformation) (arrow). Methionine-treated mice showed protection of the glomerulus and tubules in the kidney (glomerulus atrophy and kidney tubules swelling deformation) (arrow). BC group (a and b); MD group (c and d); MN group (e and f); MDN group (g and h)

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The kidney section from control mice showed a normally organized appearance of glomerulus and tubules. Ni-exposed mice are showing a loss of integrity. In contrast, methionine-treated mice showed protection of glomerulus and tubules in the kidney.

The Protective Effects of Methionine on Ni-Induced Cell Cycle Change of the Kidney

IHC was used to evaluate the expression of PCNA in kidney tissues. The results are shown in Fig. 2. PCNA proteins were mainly found in the nucleus of glomerulus and tubule cells. The PCNA protein level in the BC group was extremely significantly higher than the other groups (MD, MN, MDN) (P < 0.01). Compared with MD and MN groups, the expression level in the MDN group was extremely significantly lower (P < 0.01), which indicated that Ni could inhibit the expression of PCNA protein, and methionine could relieve the inhibitory effects on PCNA protein expression caused by Ni. The positive expression rates of PCNA protein in different groups were 92% (257/280), 31% (97/314), 36% (116/320), and 26% (31/120), respectively (Fig. 3).

Fig. 2
figure 2

Changes in kidney tissues for PCNA expression. High PCNA expression in BC group (a) at × 400, weaker PCNA expression in MD (b) and MN (c) groups, and significantly weak in MDN group (d) at × 400 magnification. The expression rates of PCNA protein in different groups (Fig. 3). PCNA = proliferating cell nuclear antigen

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Fig. 3
figure 3

Changes in the expression rate of PCNA protein

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The Protective Effects of Methionine on Ni-Induced Apoptosis

Apoptosis was detected by TUNEL to explore the protective effects of methionine on Ni-induced apoptosis. As shown in Figs. 4 and 5, the more browner tissue cells, the more apoptotic cells (positive cells). There were very few positive cells in the BC group, and the apoptotic cells in the MD group were extremely significantly higher than in the BC group (P < 0.01). Compared with the MD group, the positive cells in the MDN group were extremely significantly more (P < 0.01), and the positive cells in the MN group were significantly lower than those in the MDN group, showing that methionine could relieve apoptosis caused by Ni.

Fig. 4
figure 4

Changes in Ni-induced apoptosis in kidney tissue. TUNEL assay, Bar = 50 µm. a BC; b MD; c MN; d MDN

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Fig. 5
figure 5

Changes in the number of positive cells in the TUNEL

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Changes in Antioxidant Enzymatic Activity

To investigate the activities of antioxidant enzymes, hydroxylamine, ammonium molybdate, and colorimetry methods were used to determine the activities of SOD, CAT, GSH, GSH-Px, and GSH-ST. As shown in Table 3, the activities of SOD, CAT, GSH, and GSH-Px in the MD group were extremely significantly lower or significantly lower than BC group (P < 0.01 or P < 0.05), the activities of SOD, CAT, GSH, GSH-Px, and GSH-ST in MN and MDN group were extremely significantly lower or significantly lower than BC group (P < 0.01 or P < 0.05). Compared with the MDN group, the activities of antioxidant enzymes were higher in the MD group, which indicated that Ni could reduce the activities of antioxidant enzymes. Compared with the MDN group, the activities of GSH in the MN group were significantly higher, indicating that methionine could increase the antioxidant enzymatic activities.

Table 3 Changes in antioxidant enzymatic activity
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Changes in MDA, LPO, and ROS

To determine the changes in oxidation products in kidney cells, TBA, spectrophotometry, and chemical fluorescence methods were used to determine the changes in MDA, LPO, and ROS levels. As shown in Table 4, the levels of MDA and ROS in the MD group were slightly higher than in the BC group, the levels of LPO in the MD group were significantly higher than in the BC group (P < 0.05), the levels of MDA, LPO, and ROS in MN and MDN group were extremely significantly higher or significantly higher than BC group (P < 0.01 or P < 0.05). In terms of the MD group, MN group, and MDN group, the levels of various oxidation products in the MDN group were significantly higher than those in the MD group and MN group, indicating that Ni could significantly increase the levels of various oxidation products, while methionine could reduce the increased levels of MDA, ROS, and LPO induced by Ni.

Table 4 Changes in MDA, LPO, and ROS
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Changes in the NF-κB Signal Associated with Inflammatory Cytokines

To explore changes in inflammatory cytokines in the NF-κB signal pathway, the ELISA method was used to determine the levels of NF-κB, TNF-α, IFN-γ, IL-1a, and IL-6. As shown in Table 5, the levels of NF-κB, IFN-γ, IL-1a, and IL-6 in the MD, MN, MDN groups were higher than BC group, the levels of TNF-α in MD group were significantly higher than BC group (P < 0.05), the levels of NF-κB, TNF-α, IFN-γ, IL-1a, and IL-6 in MN and MDN group were extremely significantly higher or significantly higher than BC group (P < 0.01 or P < 0.05). According to the data of the MDN group and MD group, the levels of inflammatory cytokines in the MDN group were significantly higher than that in the MD group, indicating that Ni could induce an increase in the levels of inflammatory cytokines. Similarly, the levels of IL-1a and TNF-α in the MDN group were significantly higher than those in the MN group (P < 0.05), indicating that methionine could reduce the levels of some inflammatory factors. Results showed that Ni could increase the levels of inflammatory cytokines in the NF-κB signal pathway, and methionine could normalize the levels of inflammatory cytokines.

Table 5 Changes in the NF-κB signal associated with inflammatory cytokines
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Discussion

Tissue sections, immunohistochemistry method, TUNEL, biochemical method, and ELISA were used to detect renal function, histological changes, cell cycle, apoptosis, oxidative damage, and NF-κB inflammatory cytokines. As shown in Fig. 6, excessive Ni intake would lead to renal dysfunction and structural injuries, and the change of the levels of biomarkers related to oxidative stress, such as increasing the content of ROS, LPO, and MDA in renal tissues. It increased the apoptosis, nephritis, and cell cycle changes. Methionine relieved the oxidative stress, renal dysfunction, and apoptosis caused by Ni with NF-κB signal pathway involved.

Fig. 6
figure 6

The mitigative effects of methionine on nickel-induced kidney injury of mice

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In this study, we found that Ni could induce glomerulus atrophy, kidney tubules swell, and kidney dysfunction, and methionine could alleviate renal tissue damage, which showed in histological change of the kidney. The changes of BUN, Cr, and NAG. The BUN, Cr, and NAG levels in the MDN group were significantly higher than in the MD group, indicating that Ni could increase the BUN, Cr, and NAG levels. Compared with the MDN group, the content level in the MN group was lower, and the NAG value was significantly lower. It suggested that methionine could decrease the trend of parameter level increase caused by Ni. Previous studies have shown that glomerulus atrophy, kidney tubule swelling, and renal dysfunction in rats after Ni treatment may be caused by the accumulation of Ni. Its metabolites in the tissue might impair the filtration of urea and creatinine, causing their elevation in the blood. Ni had a high affinity for metallothionein, and methionine, as a sulfur-containing amino acid, could bind to Ni. Possibly, methionine could reduce the accumulation of Ni in tissues by direct binding.

At the same time, the adverse effects of Ni on renal function may also be related to oxidative stress and excessive production of free radicals, which can lead to the oxidation of biomolecules (lipids, proteins, DNA) and ultimately lead to renal cell death. Meanwhile, methionine could reduce oxidation levels [24,25,26]. Then, we speculated that kidney damage may be related to cellular oxidative stress and apoptosis. The results showed that Ni could cause an increase in oxidation products (MDA, LPO, and ROS) and reduce the activities of antioxidant enzymes (SOD, CAT, GSH, GSH-Px, and GSH-ST). At the same time, the TUNEL assay showed that the number of positive cells in the MDN group was significantly higher than that in the MD group, indicating that Ni could cause apoptosis. The results also showed that methionine could alleviate the increase of oxidative products, such as MDA, LPO, and ROS, and the decrease of the activities of antioxidant enzymes, such as SOD, CAT, GSH, GSH-Px, and GSH-ST caused by Ni. Simultaneously, the TUNEL assay showed that methionine could alleviate Ni-induced apoptosis. Related research showed that antioxidants can significantly inhibit TNF-induced ROS accumulation and inhibit NF-κB-induced kinase (NIK) and IKK induced by TNF-α, and inhibiting the activation of NF-κB affects inflammation and apoptosis [27, 28]. Methionine is an antioxidant and can inhibit oxidative stress and apoptosis. Methionine was the precursor of S-adenosylmethionine (SAM), SAM (the precursor of GSH) has an antioxidant effect on NF-κB activation, and SAM can inhibit the increase of MDA (MDA was the main lipid peroxidation product, which was a mutagenic and carcinogenic compound). The possible mechanism of SAM’s antioxidant effect is Fe2+ chelation and the increase of GSH synthesis. GSH was the main non-enzymatic antioxidant in cells, which could reduce peroxide and free radicals (alkyl, hydrogen peroxide, etc.). The role of SAM in increasing GSH levels could be attributed to the sulfur-transfer pathway, which converted SAM into homocysteine and then converted to cysteine, which was one of the three main amino acids of GSH [29].

The apoptosis and oxidative stress might be caused by inflammation. The experimental results also showed that inflammatory cytokines (such as NF-κB, TNF-α) in the MDN group were significantly higher than in the MD group, which showed that Ni could cause the increase of pro-inflammatory factors (such as NF-κB) in cells, thus causing inflammation. The pro-inflammatory factor tumor necrosis factor (TNF) can stimulate the accumulation of ROS and eventually lead to cell damage and even cell death. Studies have shown that Ni could increase the levels of mRNA expression in pro-inflammatory factors (NF-κB, TNF-α, and IL-6) and reduce the levels of mRNA expression in anti-inflammatory mediators (IL-2, IL-4, and IL-13) through NF-κB signal pathway induced tissue inflammatory response [5, 30,31,32], which relied on hypoxia-inducible factor (HIF)-1α to activate the expression of main signal transduction genes [33], and induced the expression of COX-2 (inflammatory reaction will stimulate the expression of COX-2), and could replace the iron in proline hydrooxygenase. At the same time, Ni could simulate hypoxia conditions and increase the levels of hypoxia-inducible factor (HIF)-1, activate JNK1, NFJB, and P38MAP kinases, and induce the expression of COX-2 [34]. NF-κB, one of the inflammatory cytokines, plays a key pathological role in inflammatory diseases. It can regulate the expression of various genes involved in inflammation, anti-apoptosis, and immune response [35,36,37]. Interestingly, NF-κB not only participates in the induction of inflammation, NF-κB activity was related to the increase of iNOS expression during inflammation, and in the later stage of this process, NF-κB activation was related to the expression of COX-2, which guided the synthesis of anti-inflammatory cyPGs (cyclopentenone prostaglandins) [38]. Our results showed that methionine can alleviate the inflammatory reaction of tissues, and relevant studies showed that the anti-inflammatory properties of methionine may be due to the inhibition of NF-κB activation [39]. Methionine could reduce the generation of TNF-α, IL-6, and IFN-β [40].

There was a close relationship between apoptosis and the cell cycle, and apoptosis might be due to changes in the cell cycle. The cell cycle was the core of maintaining multicellular homeostasis. The expression of PCNA was detected using the IHC method to investigate the change in the cell cycle induced by Ni. The cell cycle included the S phase (DNA replication), the M phase (mitosis and cell division), the G1 phase (cell cycle gap between M and S phases), and the G2 phase (cell cycle gap between S and M phases). Ni and its compounds could cause DNA damage and apoptosis. After DNA damage, cell cycle checkpoints were activated, resulting in cell growth arrest [41]. According to cell type, the effect of Ni on the cell cycle was different, and the effect of Ni on the renal cell cycle was at the G2/M stage [42]. Ni induced an increase in the proportion of G2/M phase cells in kidney cells [43] and prevented the G2/M phase cell cycle by increasing the mRNA expression of p53 and p21 and reducing the mRNA expression of cdc2 and cyclin B. The cell cycle transition from the G2 phase to the M phase was strictly regulated by the cdc2-cyclin B complex. Nickel might inhibit the cdc2-cyclin B complex by directly stimulating the expression of p53 and p21, thus inducing the accumulation of the G2/M phase (p53 can induce the upregulation of p21, thus inhibiting the activation of cdc2), p53 protein not only caused the cell cycle to stop at the G2/M phase of the cell cycle by directly stimulating the expression of p21 but also promoted cell apoptosis by upregulating the expression of Bax and downregulating the expression of Bcl-2 protein. However, Lee et al. (2001) [43] showed that the level of p53 protein did not change in normal rat kidney cells treated with nickel, and p53 did not participate in nickelNi-induced apoptosis and G2/M phase arrest. The mechanism of the effect of nickel on the renal cell cycle is still controversial. Our results showed that methionine could relieve the inhibitory effects of Ni on PCNA protein expression.

In conclusion, the NF-κB signal pathway is one of the main pathways of the renal inflammatory response, and methionine can, passing the NF-κB pathway, alleviate the inflammatory reaction, oxidative stress, cell cycle change, and apoptosis caused by Ni (Fig. 6).