Se Alleviated Oxidative Stress-Mediated Complex Poisoning Mechanism in Pb-Treated Chicken Kidneys: Inammation, Heat Shock Response, and Autophagy

Background: Lead (Pb) is a toxic environmental pollutant and can exerts toxicity in kidneys. It is known that selenium (Se) has an antagonistic effect on Pb poisoning. However, biological events during the process were not well understood in chicken kidneys. Methods: 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 Na 2 SeO 3 -added standard diet and potable water), the Pb group (offering standard diet and (CH 3 OO) 2 Pb-added potable water), and the Pb+Se group (offering Na 2 SeO 3 -added standard diet and (CH 3 OO) 2 Pb-added potable water). On 30 th , 60 th , and 90 th days, kidneys were removed to perform the studies of histological structure, oxidative stress indicators, cytokines, heat shock proteins, and autophagy in the chicken kidneys. Results: 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. Conclusion: Our research strengthens the evidence that Pb induced oxidative stress, inammation, heat shock response, and autophagy and Se administration alleviated Pb poisoning through mitigating oxidative stress in the chicken kidneys.


Background
Lead (Pb) is a well-known toxic environmental pollutant. Although measure has been taken to control Pb pollution, Pb poisoning is still an important health issue in many countries [1]. Continuous exposure of human beings and animals to this metal can induce health problems, and even lead to death [2]. Sancar (2019) found that the children exposure to Pb born between 1972 and 1973 in Dunedin, New Zealand may affect mental health in adulthood [3]. Exposure to Pb, even at low levels, was associated with chronic kidney disease in adults of UK [4]. Pb pollution led to millions of bird death every year around the world [5] and even drove California condors (an endangered species) to the edge of extinction from 1997 to 2010 [6].
Oxidative stress caused by prooxidant/antioxidant imbalance with reactive oxygen species overproduction plays a crucial role in renal injury, as it has been considered a central aggravating factor [7]. There are cross-talk between oxidative stress and in ammation in preeclampsia [8]. 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 [9]. Autophagy occurs at basal levels to preserve cellular homeostasis by recycling proteins and organelles which can also act in response to oxidative stress [10].
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 [11].
Pb induced oxidative stress which led to autophagy in the spleens of chickens [12] and mice [13]. Ge et al. (2018) reported that autophagy was intertwined with in ammation, and cytokines can help mediate this interaction [14]. Autophagy was decreased in nude mice with hepatocellular carcinoma and was inversely correlated with HSPs expression [15]. 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 [16]. As an antioxidant, it helps in maintaining intracellular redox balance. Our previous study found that Se could alleviate Pb-caused oxidative stress [17], heat shock response [18], and in ammatory damage [19] in chicken testes. In addition, recent studies reported that Se-yeast inhibited the initiation of autophagy and enhanced autophagic clearance in the brains of Alzheimer's disease mice [20]. 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.

Animals
Hyline chickens (1-day-old) were provided standard diet (containing 0.49 mg/kg Se) (D) and potable water (W) during 7 days acclimatization. Then, 180 healthy birds were randomly divided into four groups with 45 numbers: the control group (I group), the Se group (II group), the Pb group ( group), and the Pb + Se group ( group), respectively. I group was given D and W; II group received a diet enriched with Na 2 SeO 3 -added D (containing 1 mg/kg Se) (SeD) and W; III was group offered (CH 3 OO) 2 Pb 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 [21]; and grwas supplied with SeD and PbW. Na 2 SeO 3 and (CH 3 OO) 2 Pb 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 temperature 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 collection
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.

Histological analyses
Preparation of sections described in our present papers was processed as previously described [22]. 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.
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 ºC for 3 h (pH 7.2). The blocks were rinsed in 0.1 mol/L PBS, put in 1% osmium tetroxide at 4 ºC 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.

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 ºC 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-and LC3-), 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 ºC 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 alterations
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 Fig. 1 on 90th day. In I group ( Fig. 1(A1)) and II group ( Fig. 1(B1)), glomerular structure was clear and glomerular cavity was clearly visible. In III group ( Fig. 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 vacuolization occurred compared with I group. In IV group ( Fig. 1(D1), glomerulus was slightly swollen, the margin of renal cyst was little blurred, renal tubular epithelial cells were slightly swollen, and in ammatory in ltration decreased compared with group.
Ultrastructure results of chicken kidneys were as follows: Organelles were normal in group (Fig. (A2)) and group (Fig. (B2)). In group (Fig. (C2)), the nuclei were hyperchromatic and autophagosomes were visible after Pb treatment for 90 days. In group (Fig. (D2)), the number of autophagosome was less than that in group.

T-AOC, GST ,and CAT activities and H2O2 content in chicken kidneys
To assess the effect of Pb treatment on oxidative stress and mitigative effect of Se, oxidative stress indexes including T-AOC, GST, CAT, and H 2 O 2 were measured on 30th, 60th, and 90th days. As shown in Fig. 3, there were signi cant differences (P < 0.05) in T-AOC (Fig. 3 (A)), GST (Fig. 3 (B)), and CAT ( Fig. 3 (C)) activities and H 2 O 2 ( Fig. 3 (D)) content among different groups except there were no signi cant difference (P > 0.05) in T-AOC, GST, and CAT activities and H 2 O 2 content between group and group at three time points. T-AOC, GST, and CAT activities was lowest in III group, followed by in IV group, in group and group at three time points. Change tendency of H 2 O 2 content was opposite to that of T-AOC, GST, and CAT activities at three time points. In addition, T-AOC, GST, and CAT activities decreased signi cantly (P < 0.05) with the increase of treatment duration, but H 2 O 2 content showed opposite trend (P < 0.05) in III 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 (Fig. 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 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 (Fig. 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 group (Fig. 3). However, there was no signi cant difference (P > 0.05) in mRNA and protein expression of HSP27, HSP40, HSP60, HSP70, and HSP90 between I group and II group. In addition, mRNA expression of all the above detected HSPs increased signi cantly (P < 0.05) with the increase of treatment duration in 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 Fig. 5, a notable increase in mRNA and protein expression of ATG5, Beclin 1, Dynein, LC3-I, and LC3-II and decrese in mRNA and protein expression of mTOR was observed in the Pb-treated chicken kidneys (P < 0.05, vs. I group or II group). However, Se intervetion signi cantly decreased the expression of above autophagy-related genes except that mTOR was increased (P < 0.05).

Discussion
Frequent environmental and occupational exposure to Pb has been well-established to induce organ toxicity and subsequent adverse pathological consequences. Many of these diverse toxic effects are manifested at both cellular and molecular levels and share common mechanisms of action across various tissues and organs. Excessive Pb exposure can cause histological alterations of chicken kidneys [23] and renal damage of rats [24]. Pb treatment caused tubular degeneration, cell swelling, and in ammatory in ltration in rat kidneys [25]. 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.
It is well known that oxidative stress is a core mechanism of Pb toxicity due to imbalance in oxidant/antioxidant homeostasis [26]. Excessive Pb was absorbed to tissues which overproduced H 2 O 2 [27]. Similar result has revealed that H 2 O 2 level elevated in the particulate matter component-induced oxidative stress in transformed human airway epithelial cells [28]. H 2 O 2 , a typical oxidant, is capable of diffusing throughout the mitochondria and across cell membranes and producing many types of cellular injury [29]. Moreover, Pb depletes cells antioxidants, particularly thiolcontaining compounds (GST) and antioxidant enzymes (CAT and T-AOC) during oxidative stress [27]. GST is one of the predominant antioxidant enzymes against oxidative stress in living organisms [30]. CAT is a primary defense against oxidative stress, and can catalyze the conversion of H 2 O 2 into oxygen and water [31]. At the same time, it is a potential target of Pb [32]. T-AOC is used to measure the amount of free radical purge and evaluate antioxidant status [33]. It has been reported that changes in GST, T-AOC, CAT, and H 2 O 2 were associated with Pb poisoning in rat kidneys [34,35]. Pb can decrease T-AOC, GST, and CAT activities; and increase H 2 O 2 content; and cause oxidative stress in the bursa of Fabricius of chickens [36]. In our ndings, the damage was clearly demonstrated by the production of H 2 O 2 , which was accompanied by depletion in the antioxidant enzymes' (T-AOC, GST, and CAT) activities in the chicken kidneys upon exposure to Pb compared to non-treated group, suggesting that Pb caused renal injury and oxidative stress in the chicken kidneys. These results supported the fact that Pb toxicity induced renal injury by increasing oxidative stress, and similar phenomena had been reported previously [37][38][39]. The consequence of the decrease in the level of T-AOC, CAT and GST was due to direct binding of Pb with their sulfhydryl groups [40], altering their function or suppressing their activities by Pb [41]. In addition, the decrease in CAT activity was also attributed to scavenging of H 2 O 2 in Pb-intoxicated chickens. Therefore, the alterations of oxidative status, either by the overproduction of oxidants or de cit in antioxidant activity, was one of direct consequences of Pb toxicity in the chicken kidneys. In addition, we also found that T-AOC, CAT, GST, and H 2 O 2 changed in a time-dependent effect in the Pb-induced chicken kidneys. It suggested that oxidative stress was gradually strengthened with Pb treatment duration. Our previous experiment also reported that Pb had a time-dependent effect on T-AOC, GST, and CAT activities and H 2 O 2 content in chicken bursa of Fabricius [36].
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 [42]. 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 [43]. 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. As reported by Khatlab et al. (2019), reducing IL-2 expression level as the consequent of in ammatory response induced by Eimeria spp. challenge in broiler chickens [39]. 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. [44] found that lipopolysaccharide decreased CAT activity and increased H 2 O 2 content with the increase of IL-4 in chicken myocardial [44]. Abd El-Ghffar et al. (2018) reported that 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 stomaches [45]. In addition, we also found that IL-2, IL-4, and IL-12β mRNA expressed in a timedependent 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 [46]. 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 [46]. Increased levels of HSPs were an indicative of initiation of a stress response for mediating cellular protection and indicated tissue damage [47]. Heat shock response can protect against toxicity caused by excess heavy metals [48]. Previous studies reported that Pb increased HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in peripheral blood neutrophils [49] and hearts [50] of chickens. 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 [46]. 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 [46]. Besides, in ammation is itself a stimulus for upregulation of HSPs production [51]. 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 [52]. Song et al. (2017) reported that autophagy contributed to Pb-induced nephrotoxicity in primary rat proximal tubular cells [53]. Pb promoted protein levels of Beclin1, LC3-I, and LC3-II; and induced autophagy in rat hippocampi [54]. Han et al. (2017) reported that 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 [12]. 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. As reported by Wang et al. (2019), the increased mTOR expression were detected in weaned pigs compare with dietary tributyrin supplemented weaned pigs [55]. 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.
Previous studies have con rmed potent antioxidative and anti-in ammatory activities of Se. Some researches demonstrated that Se mitigated Pb-induced oxidative stress by means of increasing T-AOC, GST, and CAT activities; and decreasing H 2 O 2 content in Cyprinus carpio livers [56], chicken bursa of Fabricius [36], and chicken splenic lymphocytes [57]. Jiao et al. (2017) found that Se can mitigate increase of IL-4 and IL-12β mRNA expression, and the decrease of IL-2 mRNA expression in Pb-treated chicken bursa of Fabricius [36]. Xing et al. (2018)reported that Se alleviated the increase of IL-4 and IL-12β mRNA expression and the decrease of IL-2 mRNA expression caused by Pb in chicken neutrophils [49]. In addition, Se can mitigate Pb-caused increase of HSPs and autophagy. Wang et al. (2017) found that Pb poisoning induced mRNA expression of HSP27, HSP40, HSP60, HSP70, and HSP90; and Se administration alleviated the above HSPs changes in chicken testes [19]. Se exhibited signi cant antagonistic roles against Pb-induced increases of HSP (27,40,60,70, and 90) mRNA expression in peripheral blood neutrophils [49] and hearts [50] of chickens. Se was reported by one of the articles to alleviate spleen toxicity in a chicken model induced by Pb via the modulation of oxidative stress, in ammation, and autophagy [12]. Se alleviated protein increase of ATG5, Beclin1, Dynein, LC3-I, and LC3-II and protein decrease of mTOR in Cd-induced chicken pancreas [58]. In our study, all alterations caused by Pb were ameliorated by treatment with Se. Such effect was attributed to kidney tissue antioxidant capacity because of better antioxidant supply, thus reducing the oxidative damage represented by the reduction of T-AOC, GST, and CAT and the rise of H 2 O 2 . The ability of Se to neutralize oxidative stress 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 [59]. So 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.