n-Acetylcysteine protects against diazinon-induced histopathological damage and apoptosis in renal tissue of rats

Abstract

Diazinon (DZN) is a member of organophosphorus insecticides that has cytotoxic effects on different organs. n-Acetyl cysteine (NAC) is a widely used antioxidant in clinical, in vivo and in vitro studies. We evaluated the protective role of NAC against DZN-induced toxicity in kidney tissue of Wistar rats. 30 male Wistar rats were divided into 5 groups of control, single dose of DZN, continuous dose of DZN, single doses of DZN + NAC and continuous doses of DZN + NAC. Kidney function test (blood urea nitrogen, creatinine and uric acid) was provided. Levels of malondialdehyde (MDA), total antioxidant capacity (TAC) and total sulfhydryl (T-SH) were determined in renal tissues. Renal cells apoptosis was detected using TUNEL assay. The mRNA expressions of apoptosis, oxidative stress and inflammatory mediators, including B-cell lymphoma-2 (Bcl2), Bcl-2-associated X protein (Bax), superoxide dismutase (SOD), catalase (CAT), Interleukin 10 (IL-10), Tumor necrosis factor-α (TNF-α), Caspase-3 and Caspase-8 were analyzed in kidney tissues using Real Time PCR method. Chronic exposure to DZN was associated with severe morphological changes in the kidney, as well as impairment of its function and decreased kidney weights. Continues treatment with DZN significantly decreased the percentage of renal apoptotic cells as compared to rats treated with continuous dose of DZN alone (17.69 ± 3.67% vs. 39.46% ± 2.44%; p < 0.001). Continuous exposure to DZN significantly decreased TAC and T-SH contents, as well as SOD and CAT expression, but increased MDA contents in the kidney tissues (p < 0.001). A significant increase was observed in mRNA expression of Bax, Caspase-3, Caspase-8, as well as TNF-α following exposure to DZN, but the expression of IL-10 and Bcl2 was significantly decreased. NAC can protect kidney tissue against DZN-induced toxicity by elevating antioxidants capacity, mitigating oxidative stress, inflammation and apoptosis.

Introduction

Diazinon (DZN), O,O diethyl O (6-methyl-2- (1-methyleneyl) 4-pyramidinyl), also known as an organophosphorus pesticide, is a compound widely used for pest control [1]. This insecticide causes irreversible inhibition of acetylcholinesterase enzyme and consequently results in acetylcholine accumulation in synaptic cleft [1, 2]. In addition, DZN can induce oxidative stress through the induction of lipid peroxidation, overproduction of reactive oxygen species (ROS), and depletion of antioxidant defense systems [3–5]. Previous studies have reported that DZN stimulates ROS generation by activating macrophages and increasing the level of inflammatory mediators such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) [6–8]. In oxidative stress, the redox balance is disturbed due to overproduction of free radicals, decrease in antioxidant defense systems, or both [9]. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) are associated with the antioxidant defense system, and their activity may be affected by DZN [3, 10, 11]. Diazinon may also induce cell apoptosis through multiple mechanisms such as oxidative stress, promoting mitochondrial deficiency, and reducing the mitochondrial membrane potential [12, 13].

Apoptosis is a genetically programmed cell death regulated by intrinsic or extrinsic pathways. The intrinsic pathway (mitochondrial) depends on B-cell lymphoma 2 (Bcl2) family proteins, while the extrinsic pathway is regulated by death receptors [14, 15]. Mitochondrial-dependent pathway is regulated by anti-apoptotic proteins such as Bcl2 and pro-apoptotic proteins such as Bcl-2-associated X protein (Bax) [16]. When the balance between pro- and anti-apoptotic proteins is disturbed, pro-apoptotic proteins are oligomerized in the outer membrane of the mitochondria. Then, cytochrome-c is released from the mitochondria and subsequently activates Caspases. In the extrinsic pathway, the death ligand binds to death receptors, thereby activating caspase-3 through caspase-8 as an initiator of extrinsic pathway [17, 18]. Regarding the importance of oxidative stress in apoptosis induction, antioxidants play pivotal roles in ameliorating oxidative stress. n-acetylcysteine (NAC) is one of the most important and powerful antioxidants that is found in plant species such as onions. This chemical also acts as a rich source of cysteine and glutathione [19, 20]. Recent studies have reported the protective effect of NAC on kidney tissues; however, the exact mechanism is not well understood [16]. For instance, Shah et al. [21] reported that NAC supplementation not only improves DZN-induced histopathological changes in the kidney tissue but also modulates the levels of serum blood urea nitrogen (BUN), creatinine (Cr), and uric acid. In addition, de Faria Guimaraes et al. [22] reported that NAC therapy for a period of 3 months significantly improved renal function in patients with nephropathic cystinosis. Some studies found that NAC supplementation preserved renal function in patients undergoing hemodialysis [23]. We predict that NAC exerts a protective role on renal failure by mitigating oxidative stress and renal cells apoptosis. Research has shown that NAC can ameliorate oxidative stress through free radicals scavenging [24, 25]. To the best of our knowledge, the effect of DZN on kidney tissue (especially its effects on oxidative stress and apoptosis) and the protective role of NAC on DZN-induced toxicity is not fully understood. Therefore, we explroe the effects of acute and chronic exposure to DZN on oxidative stress and apoptosis of renal cells, in addition to the protective role of NAC against DZN-induced nephrotoxicity in Wistar rats.

Materials and methods

Study setting, design, and population

In the present study, 30 Wistar rats aged 8–10 weeks old and 100–150 g body weight were assigned into 5 experimental groups. Then, they were kept under standard conditions (12 h dark/light cycle, temperature of 20 ± 2 °C, humidity of 55 ± 5%, and free access to standard diet and water). Rats in experimental groups were treated with DZN and NAC as follows: D1 (control group only fed with chow diet and water for 30 days), D2 (single dose of DZN; 70 mg/kg); D3 [fed with continuous dose of DZN; 70 mg/kg for 30 days, D4 (co-treated with single dose of DZN (70 mg/kg) + NAC (60 mg/kg)], and D5 [co-treated with continuous dose of DZN (70 mg/kg) + NAC (60 mg/kg)]. DZN and NAC were given to experimental groups by gavage at 10 a.m. DZN and NAC concentrations were selected based on previous studies [26, 27]. Figure 1 shows the experimental design of the study.

Fig. 1.

Flow chart showing the experimental design of the animal studies (Left side) and histopathological examination of kidney tissue in all experimental groups (Right side). Hypertrophy of epithelial cells of renal tubules along with degeneration of tubular epithelia and simultaneous infiltration of mononuclear cells were observed in rats that continuously exposed to DZN (D3). Also, dilation of renal glomeruli was observed following exposure to continuous dose of DZN (D3). Co-treatment with NAC significantly improved all of these adverse effects (D4 and D5). D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC. × 20 magnification

Collection of blood and tissue samples

After completing the treatment period, the animals were anesthetized using xylazine (10 mg/kg) and ketamine (30–50 mg/kg). Next, the biochemical parameters were examined by obtaining blood samples from the abdominal aorta, and serum samples were separated by centrifugation at 3000 rpm for 5 min. The studied genes in kidney tissues of experimental groups were compared, and the levels of oxidant/antioxidant parameters were examined by dissecting left kidneys and storing them at − 70 °C immediately. Finally, the right kidneys of the studied rats were weighed and fixed in 10% neutral-buffered formalin for histological examinations.

Histopathological analysis

For histopathological examinations, the right kidneys of the experimental groups were fixed in 10% formalin for 48 h. Afterward, the tissues were embedded in paraffin, dissected into 5 μm-thick samples, and stained with Hematoxylin and Eosin (H & E). For assessing morphometric characteristics, digital images by magnification of × 400 were taken from slides by a digital camera attached to the microscope. In this study, the captured images were processed using Adobe Photoshop software to enhance their contrast. Ultimately, glomerulus and renal tubules were assessed using ImageJ software.

Detection of apoptotic cells by TUNEL assay

A Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay Kit (In Situ Cell Death Detection Kit, POD; Roche, Germany) was used to detect and quantitate renal cells undergoing apoptosis in different groups.

Analysis of kidney function parameter

The serum levels of BUN, Uric acid, and creatinine parameters were measured by colorimetric method using the Pars Azmun kit (Pars Azmun, Tehran-Iran).

Analysis of oxidative stress biomarkers

Kidney tissues were rinsed with normal saline and homogenized with liquid nitrogen to determine the levels of oxidative stress biomarkers. Next, the homogenates were incubated with RIPA buffer and protease inhibitor cocktail at pH 7.9 for 20 min on ice cold. Ultimately, the homogenates were centrifuged at 20,000 rpm for 20 min at 4 °C, and supernatants were aliquot in microtubes to assess the level of oxidative stress biomarkers. The Bradford method was used to measure the total proteins in tissue homogenates using Bovine Serum albumin as standard [28]. The levels of Total antioxidant Capacity (TAC) were measured by the ferric reducing of antioxidant power (FRAP) method [29]. Afterward, the colorimetric kit (ZB-0156-R9648, Germany) was used for evaluating the Malondialdehyde (MDA) level in tissue homogenates, and results were expressed as nmol/mg protein. Finally, we measured T-SH levels by a method described by Tietz [30].

Gene expression analysis

The expression of studied genes in experimental groups was assessed by extracting the total RNAs from the kidney tissues by RNX plus solution (Sinaclon, Iran) according to the manufacturer’s instruction. Also, cDNA was synthesized by applying the Revert Aid First Strand cDNA Synthesis Kit (Thermo Science, Germany) using 2 μg of total RNA. Subsequently, the expression of studied genes was measured using Ampliqon SYBR Green Master Mix on a Rotor-Gene 6000 (Corbett Research, Australia). The primers listed in Table 1 were used in this study, and GAPDH was used as the Housekeeping gene. Each sample was examined in triplicate for gene expression data. The Wilcoxon-Mann–Whitney test was applied to validate the homogeneity of gene expressions (p < 0.05). The groups were compared by calculating the quantification cycle threshold (Ct) of the treatment group in relation to the Ct of the control expressed on a logarithm basis. We considered genes with statistically significant change expression and a threshold with more than twofold as a differentially expressed genes.

Table 1.

Primer sequences of studied genes

Genes	Forward	Reverse
SOD	5′-TTCGTTTCCTGCGGCGGCTT-3′	5′-TTCAGCACGCACACGGCCTT-3′
CAT	5′-CTTCTGGAGTCTTTGTCCAG-3′	5′-CCTGGTCAGTCTTGTAATGG-3′
Caspase-3	5′-AAGCCGAAACTCTTCATCATTCA-3′	5′-GCCATATCATCGTCAGTTCCAC-3′
Caspase-8	5′-CTGACTGGCGTGAACTATGATG-3′	5′-CGTAGTGTGAAGATGGGCTGT-3′
Bax	5′-GAGGATGATTGCTGATGTGGATA-3′	5′-CAGTTGAAGTTGCCGTCTG-3′
Bcl2	5′-GAGGATTGTGGCCTTCTTTG-3′	5′-AGGTACTCAGTCATCCACA-3′
IL-10	5′-CAATAACTGCACCCACTTCC-3′	5′-ATTCTTCACCTGCTCCACTGC-3′
TNF-α	5′-GCCCAGACCCTCACACTC-3′	5′-CCACTCCAGCTGCTCCTCT-3′
GAPDH	5′-GCACCGTCAAGGCTGAGAAC-3′	5′-ATGGTGGTGAAGACGCCAGT-3′

Statistical analysis

All data were expressed as means ± SD. One-way analysis of variance (ANOVA) and the Post Hoc-Tukey test were used to compare the mean of all data between groups. Data were analyzed using SPSS software (version 19). A p < 0.05 was considered as statistically significant.

Results

Histopathological findings

Figure 1 presents the results of histopathological examination of kidney tissues in all experimental groups. Kidney sections of the control group had normal renal tubules and showed well-organized glomeruli without any pathological changes. Our observation showed that exposure to DZN caused hypertrophy of epithelial cells in renal tubules and degeneration of tubular epithelia with simultaneous infiltration of mononuclear cells. Additionally, our results revealed hyperemia of the medullary and cortical parts with mononuclear cell infiltration and dilation of renal glomeruli in rats receiving DZN. Surprisingly, co-treatment with NAC in rats exposed to DZN lowered the histopathological changes in kidney tissue, ameliorated hypertrophy of epithelial cells of renal tubules, dilation of renal glomeruli, and degeneration of tubular epithelia, and decreased infiltration of mononuclear cells.

Percentage of kidney apoptotic cells

Figure 2 illustrates each group’s TUNEL assay of renal cells’ apoptosis. All groups had a statistically significant difference in the mean percentage of renal apoptotic cells (p < 0.001). Rats in group D3 (39.46% ± 2.44%) exhibited a higher percentage of renal apoptotic cells compared to the other groups (Fig. 2E; p < 0.001). Co-treated with DZN + NAC in rats caused a significant decrease in the percentage of renal apoptotic cells (17.69 ± 3.67%) in the kidney tissue compared to rats treated with a continuous dose of DZN alone (p < 0.001). No significant difference was found in the percentage of renal cell apoptosis between D1, D2, and D4 groups (Fig. 2E).

Fig. 2.

TUNEL assay (D1–D5) and the percentage of renal apoptotic cells (E) in in different groups. (D1–D5) Co-treatment with NAC significantly decreased the number of apoptotic cell in the kidney of tissue of DZN-exposed rats. Arrows show the positive apoptotic cells. (E) Co-treatment with NAC significantly decreased the percentage of apoptotic cell in the kidney of tissue of DZN-exposed rats. D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC. ^*p < 0.001 and ^**p < 0.01 compared to control. D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC. × 400 magnification

Results of kidney weight and its function parameters

Table 2 compares the mean of kidney weight and its parameters between different groups. Oral administration of DZN in rats for 30 days caused a significant decrease in kidney weight by 14.29% compared to the controls (p < 0.01). Combined therapy with a continuous dose of DZN + NAC in rats resulted in a significant improvement in kidney weight by 11.30% compared to rats treated with a continuous dose of DZN alone (p < 0.05). Significant increases in the serum Cr, BUN, and uric acid levels were observed in rats treated with a continuous dose of DZN (89.47, 66.17, and 182.47%, respectively) compared to the controls (p < 0.001). Combined therapy with DZN + NAC caused a significant decrease in Cr, BUN, and uric acid values (37.04, 31.22, and 50%, respectively) in the kidney tissue compared to rats treated with continuous DZN alone (p < 0.01).

Table 2.

Comparison of the serum biochemical parameters between different groups

	D1	D2	D3	D4	D5	p-value
Kidney weight (g)	1.19 ± 0.09	1.15 ± 0.11	1.02 ± 0.04††	1.17 ± 0.09	1.15 ± 0.09	< 0.001
Serum Cr (mg/dl)	0.57 ± 0.064	0.61 ± 0.86	1.08 ± 0.14†	0.62 ± 0.09	0.68 ± 0.14†††	< 0.001
BUN (mg/dl)	41.23 ± 1.14	45.04 ± 1.21††	68.51 ± 2.71†	43.19 ± 1.87	47.12 ± 2.08†††	< 0.001
Uric acid (mg/dl)	0.97 ± 0.12	1.02 ± 0.18	2.74 ± 0.07†	0.99 ± 0.15	1.37 ± 0.08†††	< 0.001

D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC

^†p < 0.001

^††p < 0.01

^†††p < 0.05 compared to control

Results of oxidative stress parameters

The effect of DZN and co-treatment with NAC on oxidative stress parameters of kidney tissues are presented in Table 3. A significant reduction was observed in FRAP and T-SH levels in the kidney tissue of rats treated with a continuous dose of DZN (45.6% and 55.47%, respectively) compared to the control (p < 0.001). Co-treatment with DZN + NAC in rats caused a significant increase in renal FRAP and T-SH values (55.18% and 70.53%, respectively) compared to rats treated with a continuous dose of DZN alone (p < 0.001). The MDA contents were significantly enhanced in the kidney tissue of rats treated with DZN (84.45%) compared to the controls. Co-treatment with DZN and NAC in rats caused a significant decrease in MDA level in the kidney tissue (31.20%) compared to rats treated with a continuous dose of DZN alone (p < 0.01).

Table 3.

Comparison of oxidative stress biomarkers between different groups

	D1	D2	D3	D4	D5	p-value
FRAP (μM/g tissue)	649.8 ± 51.38	612.83 ± 32.87	353.49 ± 27.31†	628.11 ± 35.0	548.57 ± 52.9†††	< 0.001
MDA (nmol/mg protein)	2.38 ± 0.43	2.47 ± 0.64	4.39 ± 0.28†	2.41 ± 0.73	3.02 ± 0.31†††	< 0.001
T-SH (μM/g tissue)	14.71 ± 1.06	12.59 ± 1.86	6.55 ± 1.16†	14.63 ± 1.04	11.17 ± 1.19†††	< 0.001

D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC

^†p < 0.001

^††p < 0.01

^†††p < 0.05 compared to control

Results of gene expression analysis

The gene expression analysis results are presented in Table 4 and Fig. 3. A significant difference between groups was observed in the mRNA expression of studied genes (p < 0.001). Continuous exposure to DZN caused a significant increase in mRNA expression of Bax (3.59-fold), Caspase-3 (4.22-fold), Caspase-8 (3.26-fold), and TNF-α (3.68-fold) genes in kidney tissues of animals when compared to the controls (p < 0.001). Co-treatment with continuous dose of NAC + DZN significantly reduced the expression of Bax (1.95-fold; p = 0.014), Caspase-3 (1.85-fold; p = 0.037), Caspase-8 (1.69-fold; p = 0.048), and TNF-α genes (2.1-fold; p = 0.011) compared to rats treated with a continuous dose of DZN alone (Fig. 3). A significant decrease was noticed in expression of Bcl2 and IL-10 genes in animals that received continues dose of DZN by 2.55-fold and 2.52-fold, respectively (p < 0.001). Combined therapy with NAC + DZN significantly improved the mRNA levels of Bcl2 (2.15-fold; p = 0.009) and IL-10 (1.95-fold; p = 0.018) genes in kidney tissue compared to a continuous dose of DZN alone. Continuous exposure to DZN significantly decreased SOD and CAT expression by 2.52-fold and 2.35-fold, respectively (p < 0.001). In contrast, NAC supplementation improved the expression of SOD (1.95-fold; p = 0.025) and CAT (1.64-fold; p = 0.042) genes compared to a continuous dose of DZN alone.

Table 4.

Fold change ratio of the genes expression in each group

	Bax	Bcl2	Casp3	Casp8	IL-10	TNF-α	SOD	CAT
Single vs. Control	+ 1.31	− 1.05	+ 1.17	+ 1.22	− 1.12	+ 1.23	− 1.08	− 1.05
Continuous vs. Control	+ 3.59	− 2.55	+ 4.22	+ 3.26	− 2.52	+ 3.68	− 2.52	− 2.35
NAC + single vs. Control	+ 1.15	− 0.99	+ 1.04	+ 1.05	− 1.05	+ 0.98	− 0.98	− 0.98
NAC + continuous vs. Control	+ 1.84	− 1.19	+ 2.28	+ 1.93	− 1.29	+ 1.75	− 1.29	− 1.44
Single vs. continuous	− 2.75	+ 2.42	− 3.59	− 2.66	+ 2.24	− 2.98	+ 2.33	+ 2.23
Single vs. NAC + continuous	− 1.41	+ 1.13	− 1.94	− 1.58	+ 1.15	− 1.42	+ 1.20	+ 1.37
Single + NAC vs. single	− 1.14	+ 1.06	− 1.13	− 1.16	+ 1.07	− 1.26	+ 1.10	+ 1.07
Single + NAC vs. continuous	− 3.13	+ 2.56	− 4.07	− 3.10	+ 2.40	− 3.77	+ 2.56	+ 2.39
Single + NAC vs. NAC + continuous	− 1.61	+ 1.19	− 2.19	− 1.84	+ 1.23	− 1.80	+ 1.31	+ 1.46
Continuous + NAC vs. continuous	− 1.95	+ 2.15	− 1.85	− 1.69	+ 1.95	− 2.10	+ 1.95	+ 1.64

⁽⁺⁾means up-regulation, ⁽⁻⁾ means down-regulation

Fig. 3.

Comparison of the normalized expression of different genes between groups. D1 control, D2 Single dose of DZN, D3 continuous dose of DZN, D4 single dose of DZN + NAC, D5 continuous dose of DZN + NAC; ^*p < 0.001; ^**p < 0.01; ^***p < 0.05 compared to control group

Discussion

Although the toxicity of DZN on various organs has been widely studied in animal models, the exact mechanisms by which this organophosphorus pesticide triggers different pathologies remain unclear. Recent evidence has indicated that the propagation of oxidative stress and inflammatory reactions associated with cell apoptosis is a major mechanism of DZN toxicity in different organs [8, 31, 32]. Literature has shown that NAC has a protective effect against oxidative stress and attenuates apoptosis through compensating antioxidant pools in the body [24, 25]. Thus, the present study was designed to evaluate the protective effect of NAC supplementation against DZN-induced renal toxicity. Our data showed that chronic exposure to DZN not only declined the kidney weights of exposed rats but also was significantly correlated to morphological and structural changes in this tissue. In addition, higher levels of serum BUN, uric acid, and Cr in DZN-exposed animals were observed, indicating impairment of renal function. Some studies reported higher serum BUN, uric acid, and Cr contents in DZN-exposed animals. These data indicate that DZN toxicity may be hazardous for the kidney tissue, and agricultural workers who are chronically exposed to DZN in the spraying season may have side effects in this tissue.

Although some studies have reported the toxicity effect of DZN on different organs, the exact underlying mechanisms are not well understood. The present study showed that chronic exposure to DZN is strongly associated with depletion of TAC and T-SH contents, as well as lower expression of SOD and CAT at mRNA and protein levels in the kidney tissue. These data mean that DZN can decline the antioxidant capacity of cells, especially SH groups, and disturb cellular redox capacity. Shiri et al. [13] illustrated that DZN exposure significantly reduced SH molecules such as GSH in vitro. Glutathione is a main cofactor for many enzymatic antioxidants, such as glutathione peroxidase (GPX) and glutathione-S-transferase (GST). Beydilli et al. [10] reported reduced activity of GPX and GST in the liver of rats after DZN exposure. Down-regulation and decreased activity of other antioxidants such as GST-α3, CAT, SOD, peroxiredoxin (PRDX), 3-Mercaptopyruvate sulfurtransferase (MPST), vitamin C, vitamin E, and β-carotene were reported in previous studies [33–36]. Given the critical role of antioxidants in redox system regulation, lower expression of these antioxidants caused by DZN may promote cellular susceptibility to free radicals and, consequently, oxidative damage and apoptosis [37]. To support this hypothesis, we found increased levels of MDA and overexpression of apoptosis-related genes and proteins in the kidney tissue of rats chronically exposed to DZN. Besides, DZN toxicity was associated with overexpression of pro-inflammatory cytokine TNF-α but down-regulation of anti-inflammatory mediator IL-10 in the kidney tissue. These data highlight the importance of oxidative stress and inflammatory reactions as the main mechanism of DZN-induced nephrotoxicity. This issue is subsequently associated with antioxidant depletion and apoptosis of renal cells. Several studies reported that DZN toxicity is associated with oxidative damage to DNA, lipids, and proteins in different organs [38, 39]. For example, Ahmadi-Naji et al. [6] found increased levels of MDA and protein carbonyl (as protein oxidation biomarkers) in the liver tissue of rats following exposure to DZN. Moreover, Yaghubi Beklar et al. [40] showed that exposure to DZN increased the MDA level and caused GSH depletion in the liver tissue of exposed rats. Boussabbeh et al. [39] reported that DZN caused apoptosis of large intestine cells by inducing lipid peroxidation, overproduction of ROS, and decreasing mitochondrial membrane potential and DNA damage. In another study, Oksay et al. [3] demonstrated that DZN exposure promotes oxidative stress in rat testis by increasing lipid peroxidation levels and reducing GSH, vitamin C, and vitamin E contents. Some studies indicated that DZN may increase the number macrophages and their activity at the site of injury. Since these cells are the main source of ROS and pro-inflammatory mediators such as IL-6 and TNFα [41], they may be a reason for higher expression of TNFα in the kidney tissue of DZN-exposed rats. Ogasawara et al. [41] demonstrated that DZN not only increases macrophage recruitment and production of proinflammatory markers such as IL-6 and TNFα, but also up-regulates expression of cyclooxygenase-2 and inducible nitric oxide synthase enzymes as a main source of ROS. Some studies reported increased number of other leukocytes such as lymphocytes and neutrophils following exposure to DZN [42, 43]. Therefore, these data emphasize that DZN may recruit and activate inflammatory cells such as macrophages and neutrophils with a subsequent release of pro-inflammatory cytokines that can recruit and activate other leukocytes in renal tissue. Activated leukocytes are rich sources of ROS, which in turn may overcome the antioxidant defense systems, causing oxidative stress and renal cells apoptosis.

Our findings and the concepts of DZN pathogenesis on the kidney tissue might make a wise basis for using antioxidants that could protect kidney cells against DZN-induced nephrotoxicity. In the current study, we considered the protective effect of NAC supplementation to mitigate morphological changes, oxidative stress, depletion of antioxidants, apoptosis of renal cells, and dysregulation of apoptosis-related genes caused by DZN toxicity. We found that NAC supplementation significantly reversed the adverse effects of DZN toxicity on kidney tissues. These improvements were associated with a significant increase in TAC and T-SH values and overexpression of CAT and SOD enzymes. Meanwhile, they lead to a significant decrease in MDA contents in the kidney tissue. Interestingly, it was found that co-treatment with NAC not only attenuated the expression of pro-inflammatory cytokine TNFα but also increased the expression of anti-inflammatory mediator IL-10 in the kidney tissue. Furthermore, NAC modulated the expression of apoptosis-related genes in the renal tissue of DZN-exposed animals. The results showed that the levels of oxidative stress biomarkers and expression of apoptotic genes in the kidney tissue of rats receiving co-treatments DZN + NAC were still somewhat high. However, NAC supplementation significantly improved these abnormalities in this group compared to rats treated with continuous DZN alone. These data indicate that NAC supplementation can help mitigate oxidative stress and apoptosis of renal cells in subjects chronically exposed to DZN. As support to these findings, many studies found that NAC supplementation protects different organs by mitigating ROS production, oxidative stress, inflammation, and cell apoptosis. For example, Finamor et al. [44] showed that NAC can exert a protective effect against oxidative damage in the brain tissue of rats. A recent study has demonstrated that NAC supplementation inhibited apoptosis of irradiation-induced neural cells by inhibiting caspase-3 and ROS production [45]. Therefore, according to previous data and current results, antioxidant depletion, oxidative stress, inflammation, and changes in the expression of apoptosis-related genes are the main underlying mechanisms by which DZN causes damage to the kidney tissues. NAC supplementation reversed all of these adverse effects of DZN-induced kidney toxicity. Our findings support the idea that the toxicological effects of DZN on kidney tissue are mediated through the depletion of antioxidant levels, oxidative stress, and apoptosis of renal cells. Thus, antioxidant supplementation with NAC can be used to alleviate the detrimental effects of DZN on the kidney tissue.

In summary, our findings showed that chronic exposure to DZN is strongly associated with kidney function impairment, morphological changes in the kidney tissue, oxidative stress, inflammation, depletion of antioxidants, and apoptosis of renal cells. Interestingly, NAC supplementation protected kidney tissue against DZN-induced nephrotoxicity by elevating antioxidant capacity, mitigating oxidative stress and inflammation, and down-regulating apoptosis-related genes. One of the limitations of this study is the lack of Western Blot or Immunohistochemistry staining (IHC staining) to validate the upregulated genes at protein levels.

Funding

This research was supported by a grant provided by the Department of Nephrology and Immunology at Children’s Hospital of Soochow University, and Department of Pediatrics at Affiliated Hospital of Yangzhou University, Yangzhou Jiangsu, China.

Data availability

The datasets used to support the outcomes of this research are available from the corresponding author upon request.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest.