Selenium Alleviated Oxidative Stress-Mediated Complex Poisoning Mechanism in Lead-Treated Chicken Kidneys: In ammation, Heat Shock Response, and Autophagy


 Lead (Pb) is a toxic environmental contaminant, and exerts renal toxicity. It is known that selenium (Se) performs antagonistic effect on Pb poisoning. However, biological events during the process were not well understood in chicken kidneys. The purpose of this research was to investigate mitigative mechanism of Se on Pb poisoning from point of view of oxidative stress, inflammation, heat shock response, and autophagy in chicken kidneys. One hundred and eighty male Hyline chickens (7-day-old) were randomly divided into the control group (offering standard diet and potable water), the Se group (offering Na2SeO3-added standard diet and potable water), the Pb group (offering standard diet and (CH3OO)2Pb-added potable water), and the Pb+Se group (offering Na2SeO3-added standard diet and (CH3OO)2Pb-added potable water). On 30th, 60th, and 90th days, kidneys were removed to perform the studies of histological structure, oxidative stress indicators, cytokines, heat shock proteins, and autophagy in the chicken kidneys. The experimental results indicated that Pb poisoning changed renal histological structure; decreased catalase, glutathione-s-transferase, and total antioxidative capacity activities; increased hydrogen peroxide content; induced mRNA and protein expression of heat shock proteins; inhibited interleukin (IL)-2 mRNA expression, and induced IL-4 and IL-12β mRNA expression; inhibited mammalian target of rapamycin mRNA and protein expression, and induced autophagy-related gene mRNA and protein expression in the chicken kidneys. Supplement of Se mitigated the above changes caused by Pb. In conclusion, Pb induced oxidative stress, inflammation, heat shock response, and autophagy and Se administration alleviated Pb poisoning through mitigating oxidative stress in the chicken kidneys.


Introduction
Lead (Pb) is a ubiquitous toxic environmental contaminant. Although laws and regulations has been implement to control Pb pollution, it is still an important health issue in many countries (Mohammadi et al., 2014). Continuous Pb exposure to human beings and animals constitutes various health risk, and even lead to death (Patrick 2006). Previous study has reported that the children exposure to Pb born between 1972 and 1973 in Dunedin and New Zealand may affect mental health in adulthood (Sancar 2019). Exposure to low level Pb was associated with chronic kidney disease has been studied in adults of Oxidative stress caused by prooxidant/antioxidant imbalance with reactive oxygen species (ROS) overproduction plays a crucial role in renal injury, as it has been considered a central aggravating factor (Thomas et al., 2019). There are cross-talk between oxidative stress and in ammation in preeclampsia (Tenorio et al., 2019). Heat shock proteins (HSPs) are a family of proteins produced by cells in response to exposure to stressful conditions and are primary mitigators of cell stress (Hang et al., 2018).
Autophagy occurs at basal levels to preserve cellular homeostasis by recycling proteins and organelles which can also act in response to oxidative stress (Roca-Agujetas et al., 2019). Oxidative stress, in ammation, heat shock response, and autophagy have been described in many studies as prominent factors in mediating many pathological alterations in response to toxic agents (Ayoub et al., 2017). Pb induced oxidative stress which led to autophagy in the spleens of chickens (Han et al., 2017) and mice (Corsetti et al., 2017). Autophagy was intertwined with in ammation, and cytokines can help mediate this interaction (Ge et al., 2018). Autophagy was decreased in nude mice with hepatocellular carcinoma and was inversely correlated with HSPs expression (Chen et al., 2019). However, it is not well characterized whether there is an interplay between these factors or any combination of them in mediating harmful mechanisms of pathological alterations in Pb-treated kidneys. Selenium (Se) is a necessary trace element for organisms (Sun et al., 2018). As an antioxidant, Se plays critical roles in maintaining intracellular redox balance. Our previous study found that Se could alleviate Pb-caused oxidative stress , heat shock response , and in ammatory damage  in chicken testes. In addition, recent studies reported that Seyeast inhibited the initiation of autophagy and enhanced autophagic clearance in the brains of Alzheimer's disease mice (Song et al., 2018). Although antagonistic effect of Se on Pb was investigated, underlying molecular mechanism remained to be elucidated. Therefore, in current study, we designed interaction model of Pb and Se in chickens and detected histological alterations, oxidative stress indexes, mRNA and protein expression of interleukins, HSPs, and autophagy-related genes to reveal antagonistic mechanism of Se on Pb in the chicken kidneys.

Animal model
Hyline chickens (1-day-old) were provided standard diet (containing 0.49 mg/kg Se) (D) and potable water (Wang et al., 2017) during 7 days acclimatization. Then, 180 healthy birds were randomly divided into four groups with 45 numbers: the control group, the Se group, the Pb group, and the Pb+Se group, respectively. the control group was given D and W; the Se group received a diet enriched with Na2SeO3added D (containing 1 mg/kg Se) (SeD) and W; the Se group was offered (CH3OO)2Pb through drinking water (containing 350 mg/L Pb) (PbW), following median lethal dose of Pb acetate for cocks and the need of chicken experiment in toxicology (Vengris & Mare 1974); and the Pb+Se group supplied with SeD and PbW. Na2SeO3 and (CH3OO)2Pb were analytical reagent grade and were purchased from Tianjinzhiyuan Chemical Reagen Co., Ltd. Tianjin, China. According to feeding standard, the chickens were provided food and water ad libitum at a tempreture of 22 ± 2°C under 12 h-light/12 h-dark cycles in Laboratory Animal Center, Animal Medical College, Northeast Agricultural University (Harbin, China) until the end of experiment.

Tissue samples
On 30th, 60th, and 90th days of the experiment, respectively, 15 birds with 12 h fasting from each group were euthanized. Then the kidneys were immediately separated and cleaned with ice-cold saline. The rst part of the sample was immediately frozen in liquid nitrogen and stored at -80°C to detect mRNA and protein expression. The second part of the sample was homogenized to determine oxidative stress indexes. The third part of the sample was xed in 4% paraformaldehyde solution and stayed at least 24 h to perform microstructure observation. The last part of the sample was xed in 2.5% glutaraldehyde phosphate buffer saline to observe ultrastructure.

Microstructure
Preparation of sections described in our present papers was processed as previously described (Huang et al., 2019b). Brie y, the kidney tissues xed with paraformaldehyde solution were dehydrated in gradient alcohol (30,50,70,90, 100, and 100%), were embedded in para n, and were sectioned to nominal thicknesses of 4 µm. The sections were stained with hematoxylin and eosin. Finally, the sections were subjected to microscopic examination (Eclipse 80i, Nikon, Tokyo, Japan) and photographs were taken.

Ultrastructure
The samples were cut into blocks with the size of 1.0 × 1.0 × 1.0 mm and were immediately xed in 2.5% glutaraldehyde phosphate buffer saline at 4°C for 3 h (pH 7.2). The blocks were rinsed in 0.1 mol/L PBS, put in 1% osmium tetroxide at 4°C for 1 h, and were rinsed in 0.1 mol/L PBS again. The tissues were impregnated and were embedded with epoxy resins. The obtained sections were counterstained with uranyl acetate and lead citrate after ultrathin section. The ultrastructure of chicken kidneys was observed and was photographed using transmission electron microscope (Model JEM-1200EX, Jeol Jem, Japan).
The homogenate solution was centrifuged at 16, 000 g and 4°C for 5 min. Obtained supernatant was used for determining T-AOC, GST, and CAT activities and H 2 O 2 content in chicken kidneys using kits produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer's instructions. All samples were detected in duplicate in a single assay to avoid interassay variation.

Relative mRNA expression analysis
Primer sequence and Genbank accession numbers of detected genes were listed in Table 1. GAPDH served as internal reference gene. The special primers were synthesized by Invitrogen Biotechnology Co.

Accession number
Primer sequence Size of the products (bp) Reverse5'-AGCCTTCACTACCCTCTTG-3' Total RNA was extracted from kidney tissues with TRIzol reagent following the method provided by the manufacturer (Invitrogen, China). Spectrophotometer (Healthcare Bio-Sciences AB, Sweden) was used to determine RNA purity. OD260/OD280 was between 1.8 and 2.1, and met the experimental requirements.
Complementary DNA (cDNA) was synthesized with PrimeScript™ RT reagent Kit (TaKaRa, Japan) in a volume of 60 µL (containing 5 µg of the total RNA) according to the manufacturer's instructions.
Obtained cDNA was diluted vefold with sterile water and was kept at -20°C until next step.

Western blot analysis
Kidney tissues (about 50 mg) were cut from each kidney and washed in saline, and then were sliced and homogenized in sodium dodecyl sulfate (SDS) lysate. Homogenate solution was centrifuged and were extracted supernatant. Protein quanti cation was detected with BCA protein assay kits (Thermo Scienti c, USA). Then, proteins were put into SDS-PAGE gel and were transferred to the membranes of nitrocellulose at 200 mA for 1 h. The membranes were put into 5% skim milk to block at 4°C for 12 h. The antibodies were diluted to 1:1000 (HSP27), 1:1000 (HSP40), 1:1000 (HSP60), 1:500 (HSP70), 1:500 (HSP90), 1:100 (LC3-I and LC3-II), 1:500 (Dynein and mTOR), and 1:1000 (ATG5 and Beclin 1), respectively. After being washed for four 5-min periods with PBST, the membranes reacted with secondary antibodies against rabbit IgG (1:1000, Santa Cruz, USA) at 37°C for 1 h. Then the membranes were washed for four 5-min periods. Western blotting detection kits (Thermo Scienti c, USA) were used for detecting protein expression. The membranes were exposed X-ray lms. Then, protein levels were analyzed using image VCD gel imaging system (Beijing Sage Creation Science and Technology Co. Ltd., Beijing, China). The GAPDH signal was used as an internal reference.

Statistical analysis
All experiment data were presented as the mean ± standard deviation (SD). One-way and two-way analyses of variance (ANOVA) were performed using SPSS (version 21.0, SPSS Inc., Chicago, IL, USA). Kruskal-Wallis ANOVA test and Mann-Whitney U test were used to compare difference among multiple groups. Statistical signi cance was assigned at P < 0.05.

Histology aerations
To explore the effect of Pb on chicken kidneys and mitigative effect of Se on Pb poisoning, chickens were treated with Pb and Se for 90 days. Histology alterations of chicken kidneys were shown in Figure 1 on 90th day. In the control group (Figure 1(A1)) and the Se group (Figure 1(B1)), glomerular structure was clear and glomerular cavity was clearly visible. In the Pb group (Figure 1(C1)), glomerulus was swollen, the boundaries of renal cyst were unclear, renal tubular epithelial cells were swollen, in ammatory cells in ltrated extensively, and vascularization occurred compared with the control group. In the Pb+Se group Relative mRNA expression of IL-2, IL-4, and IL-12β To investigate the effects of Pb and Se on the in ammation of chicken kidneys, the expression of IL-2, IL-4, and IL-12β was detected on 30th, 60th, and 90th days (Figure 3). Pb treatment caused a notable decrease in IL-2 mRNA expression and increase in IL-4 and IL-12β mRNA expression in chicken kidneys (P < 0.05). Se administration signi cantly induced IL-2 mRNA expression and reduced IL-4 and IL-12β mRNA expression (P < 0.05). In addition, IL-2 mRNA expression decreased signi cantly (P < 0.05) and IL-4 and IL-12β mRNA expression increased signi cantly (P < 0.05) with the increase of treatment duration in the Pb group.
Relative mRNA and protein expression of HSP27, HSP40, HSP60, HSP70, and HSP90 To detect the effect of Pb and Se on heat shock response in chicken kidneys, mRNA and protein expression of HSP27, HSP40, HSP60, HSP70, and HSP90 was measured on 30th, 60th, and 90th days ( Figure 4). Pb treatment led to notably increase (P < 0.05) in mRNA and protein expression of HSP27, HSP40, HSP60, HSP70, and HSP90 in the chicken kidneys, while chickens with Se administration showed signi cant recovery (P < 0.05) of mRNA and protein expression of HSPs as compared with the Pb group ( Figure 3). However, there was no signi cant difference (P > 0.05) in mRNA and protein expression of HSP27, HSP40, HSP60, HSP70, and HSP90 between the control group and the Se group. In addition, mRNA expression of all the above detected HSPs increased signi cantly (P < 0.05) with the increase of treatment duration in the Pb group.
Relative mRNA and protein levels of ATG5, Beclin 1, Dynein, LC3-I, LC3-II, and mTOR To determine mitigative effect of Se on autophagy in Pb-treated chicken kidneys, the expression of autophagy-related genes including ATG5, Beclin 1, Dynein, LC3-I, LC3-II, and mTOR was evaluated on 90th day. As shown in Figure 5, a notable increase in mRNA and protein expression of ATG5, Beclin 1, Dynein, LC3-I, and LC3-II and decrease in mRNA and protein expression of mTOR was observed in the Pb-treated chicken kidneys (P < 0.05, vs. the control group or the Se group). However, Se intervention signi cantly decreased the expression of above autophagy-related genes except that mTOR was increased (P < 0.05). In this study, we found that Pb exerted toxicity in chicken kidneys according to typical features of pathological alterations after Pb treatment, such as swollen glomeruli, in ammatory in ltration, and vascularization.

Discussion
It is well known that oxidative stress is a core mechanism of Pb toxicity due to imbalance in oxidant/antioxidant homeostasis (Chander et  In ammatory response is the rst line of defense in response to all forms of cellular injuries and clears cellular damage and initiates cellular repair (Sochocka et al., 2017). But when in ammatory response is inappropriate it can lead to damage of surrounding normal cells. One of the events that occurred following oxidative stress is in ammatory response. It has been reported that increased oxidative stress might stimulate the expression of cytokines leading to increased in ammation (Reuter et al., 2010). IL-4 and IL-12β were proin ammatory mediators and IL-2 was anti-in ammatory one. Thus, in present study, IL-2, IL-4, and IL-12β were selected for mRNA expression analysis. We found that Pb treatment increased IL-4 and IL-12β and decreased IL-2 in the chicken kidneys, suggesting that Pb enhanced in ammatory process after oxidative stress in the chicken kidneys. The process of abnormal Pb invasion-caused oxidative stress triggered in ammatory response, through the cytokine production, such as IL-4 and IL-12β, which led to a reduction in the anti-in ammatory cytokine production, such as IL-2, and consequently, cells were damaged. In fact, in ammatory damage has been known to occur during the process of in ammation after Pb treatment, which was clearly seen from our histological results. Moreover, the increase of H 2 O 2 level could cause structural damage to membranes. Our ndings suggested a crosstalk between Pb-induced oxidative stress and in ammation. Other researchers also concluded that there was a relationship between oxidative stress and in ammation. Previous study has found that lipopolysaccharide decreased CAT activity and increased H 2 O 2 content with the increase of IL-4 in chicken myocardials (Liu et al., 2020). Also, H 2 O 2 content increased, GST and CAT activities decreased, and oxidative stress occurred which prompt expression of IL-4 in aspirin-treated mouse stomachs (Abd El-Ghffar et al., 2018). In addition, we also found that IL-2, IL-4, and IL-12β mRNA expressed in a time-dependent effect in the Pb-induced chicken kidneys, which suggested that in ammatory response was gradually strengthened with Pb treatment duration.
Oxidative stress is also responsible for activation of heat shock response (Kalmar & Greensmith 2009). HSPs also play a role in sensing oxidative stress, are involved in restoring physiological protein conformation during and after oxidative stress, and which are characteristic features of a number of pathological conditions. In response to oxidative stress, the expression of HSPs elevates dramatically which is notable as a pervasive adaptation mechanism in organisms that enables them to survive and adapt to different environmental stressors ( In the present study, we observed high expression of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) mRAN and protein caused by Pb exposure in the chicken kidneys, re ected the activation of this intracellular buffer system, which responds to oxidative stress when the antioxidant enzyme (T-AOC, GST, and CAT) exhaustion occurs (Kalmar & Greensmith 2009). The ndings of this study indicated that Pb exposure resulted in the activation of HSPs under burden of oxidative stress. Interestingly, increasing evidence suggests that there is a complementary regulation between HSPs and in ammation (Kalmar & Greensmith 2009). Besides, in ammation is itself a stimulus for upregulation of HSPs production (Humphries et al., 2005). Therefore, in our study, elevated HSPs, on the one hand, antagonized the mentioned Pb-induced oxidative stress, on the other hand, inhibited in ammation. In addition, we also found that HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression increased in a time-dependent effect in the Pb-induced chicken kidneys. It suggested that HSP response was gradually strengthened with Pb treatment duration.
Autophagy is an intracellular lysosomal degradation process, which plays an important role in regulating normal cell homeostasis, and is considered as one of cellular defense against increased oxidative stress (Zhang et al., 2017). Autophagy contributed to Pb-induced nephrotoxicity has been elucidated in primary rat proximal tubular cells (Song et al., 2017). Pb promoted protein levels of Beclin1, c and LC3-II; and induced autophagy in rat hippocampi (Zhang et al., 2012a). Pb increased mRNA and protein levels of ATG5, Beclin-1, Dynein, LC3-I, and LC3-II; decreased mRNA and protein levels of mTOR; and induced autophagy in chicken spleens (Han et al., 2017). Our present research is consistent with above studies. We found that Pb treatment promoted mRNA and protein expression of Beclin 1, Dynein, ATG 5, LC3-I, and LC3-II; and inhibited mRNA and protein expression of mTOR. Furthermore, we found typical features of autophagy, formation of autophagosome, through the ultrastructure of chicken kidneys. Molecular and histology evidence of our study demonstrated that Pb induced autophagy in the chicken kidneys.
Therefore, we concluded that elevated HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) were also a trigger for autophagy in Pb treatment group. could be due to facilitating chelation with Pb in the chicken kidney tissues, resulting in reduced Pb accumulation in the body through its potential antioxidant e cacy (Li et al., 2005). Se alleviated oxidative stress, which naturally alleviated these downstream events. Therefore, Se alleviates heat shock response and autophagy.

Conclusion
Excessive Pb led to oxidative stress, which further triggered a defensive response including heat shock response, in ammatory response, and autophagy in the chicken kidneys. Se alleviated heat shock response, in ammatory response, and autophagy in the Pb-treated chicken kidneys. In addition, the effects of Pb poisoning had time-dependent manners in the chicken kidneys.  Effects of Pb, Se, and their co-treatment on oxidative stress indicators in chicken kidneys on 30th, 60th, and 90th days. T-AOC (A), GST (B), and CAT (C) activities and H2O2 (D) content were determined with commercial assay kits. Bars represent mean ± SD (n = 5/group). In the same time point, the bars sharing different lowercase letters represent statistically signi cant differences between the groups (P < 0.05); the bars with a common letter are not signi cant different (P > 0.05). In the same group, the bars sharing different uppercase letters represent statistically signi cant differences in the different time points (P < 0.05); the bars with a common letter are not signi cant different (P > 0.05).

Figure 4
Effects of Pb, Se, and their co-treatment on mRNA and protein expressions of HSPs in chicken kidneys. HSP27 (A), HSP40 (B), HSP60 (C), HSP70 (D), and HSP90 (E) mRNA expressions were determined by realtime PCR and their protein expressions (F) were determined using Western-blot. Bars represent mean ± SD (n = 5/group). In the same time point, the bars sharing different lowercase letters represent statistically signi cant differences between the groups (P < 0.05); the bars with a common letter are not signi cantly different (P > 0.05). In the same group, the bars sharing different uppercase letters represent statistically signi cant differences in the different time points (P < 0.05); the bars with a common letter are not signi cant different (P > 0.05).

Figure 5
Effects of Pb, Se, and their co-treatment on mRNA and protein expression of autophagy-related genes in chicken kidneys on the 90th day. ATG5, Beclin 1, Dynein, LC3-I, LC3-II, and mTOR mRNA expression (A) were determined by real-time PCR and their protein expression (B) were determined using Western-blot.
Bars represent mean ± SD (n = 5/group). Bars with different lowercase letters were signi cant different in different groups (P < 0.05).