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The accumulation of copper (Cu) in the organisms could lead to kidney damage by causing mitochondrial dysfunction. Given that mitochondria are one of the targets of Cu poisoning, this study aimed to investigate the role of mitophagy in Cu‐induced mitochondrial dysfunction in renal tubular epithelial cells to understand the mechanism of Cu nephrotoxicity. Hence, the cells were treated with different concentrations of Cu sulfate (CuSO4) (0, 100, and 200 μM), and mitophagy inhibitor (Cyclosporine A, 0.5 μM) and/or 200 μM CuSO4 in the combination for 12 h. Results showed that Cu caused mitochondrial swelling, vacuoles, and cristae fracture; increased the number of mitochondrial and lysosome fluorescent aggregation points; upregulated the mRNA levels of mitophagy‐associated genes (LC3A, LC3B, P62, BNIP3, NIX, OPTN, NDP52, Cyp D LAMP1, and LAMP2) and protein levels of LC3II/LC3I, BNIP3, and NIX, downregulated the mRNA and protein levels of P62; reduced the mitochondrial membrane potential (MMP), ATP content, mitochondrial respiratory control rate (RCR), mitochondrial respiratory control rate (OPR), and the mRNA and protein levels of PGC‐1α, TOMM20, and Mfn2, but increased the mRNA and protein levels of Drp1. Besides, cotreatment with Cu and CsA dramatically decreased the level of mitophagy, but increased mitochondrial division, further reduced MMP, ATP content, RCR, and OPR, mitochondrial fusion and thereby reduced mitochondrial biogenesis. Taken together, these data indicated that Cu exposure induced BNIP3/NIX‐dependent mitophagy in duck renal tubular epithelial cells, and inhibition of mitophagy aggravated Cu‐induced mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

High molybdenum (Mo) and cadmium (Cd) are harmful to the body, but pulmonary toxicity induced by Mo and Cd co‐exposure is unknown. To assess the combined impacts of Mo and Cd on fibrosis through M1 polarization in the lung of ducks, 80 healthy 8‐day‐old Shaoxing ducks (Anas platyrhyncha) were randomly assigned to 4 groups and fed with containing unequal doses of Mo or/and Cd diet. Lung tissues were collected on the 16th week. Results indicated that Mo or/and Cd significantly increased their contents in the lungs, and led to trace elements disorder and histological abnormality, and oxidative stress accompanied by promoting contents of H2O2 and MDA and decreasing activities of T‐SOD, GSH‐Px, and CAT, then activated the TLR4/NF‐κB/NLRP3 pathway accompanied by upregulating Caspase‐1, ASC, IL‐18, IL‐1β, TLR4, NF‐κB, and NLRP3 expression levels, and disrupted M1/M2 balance to divert toward M1, which evoked the TGF‐β/Smad2/3‐mediated fibrosis by elevating TGF‐β1, Smad2, Smad3, COL1A1, α‐SMA, and MMP2 expression levels, and decreasing Smad7 and TIMP2 expression levels. The changes of the combined group were most obvious. To sum up, the research demonstrated that Mo or/and Cd may cause macrophages to polarize toward M1 by oxidative stress‐mediated the TLR4/NF‐κB/NLRP3 pathway, then result in fibrosis through the TGF‐β1/Smad2/3 pathway in duck lungs. Mo and Cd may worsen lung damage. [ABSTRACT FROM AUTHOR]

Molybdenum (Mo) is an essential element for human beings and animals; however, high dietary intake of Mo can lead to adverse reactions. Cadmium (Cd) is one of the major transitional metals which has toxic effects in animals. To investigate the co-induced toxic effects of Mo and Cd on oxidative damage and kidney apoptosis in duck, 120 ducks were randomly divided into control group and 5 treatment groups which were treated with a commercial diet containing different dosages of Mo and Cd. Kidney samples were collected on the 60th and 120th days to determine the mRNA expression levels of ceruloplasmin (CP), metallothionein (MT), Bak-1, and Caspase-3 by quantitative RT-PCR. Additionally, we also determined the antioxidant activity indexes and contents of Mo, Cd, copper (Cu), iron (Fe), zinc (Zn), and selenium (Se) in serum. Meanwhile, ultrastructural changes of the kidney were observed. The results showed that glutathione reductase (GR) activity and CP level in serum were decreased in combination groups. In addition, the antioxidant indexes were decreased in co-treated groups compared with single treated groups. The mRNA expression levels of Bak-1 and Caspase-3 increased in co-treated groups. The mRNA expression level of CP in high-dose combination group was downregulated, while the mRNA expression of MT was upregulated except for low-dose Mo group. Additionally, in the later period the content of Cu in serum decreased in joint groups while the contents of Mo and Cd increased. In addition, ultrastructural changes showed mitochondrial crest fracture, swelling, deformed nuclei, and karyopyknosis in co-treated groups. Taken together, it was suggested that dietary Mo and Cd might lead to oxidative stress, kidney apoptosis and disturb homeostasis of trace elements in duck, and it showed a possible synergistic relationship between the two elements. [ABSTRACT FROM AUTHOR]

Excess molybdenum (Mo) and cadmium (Cd) are widespread environmental and industrial metal pollutants. To evaluate the combined effects of Mo and Cd on calcium homeostasis and autophagy in duck kidneys. 160 healthy 7-day-old ducks (Anas platyrhyncha) were randomized into 4 groups and given to a basic diet, adding various doses of Mo or/and Cd for 16 weeks. On the 4th, 8th, 12th and 16th weeks, kidney tissues were collected. The study exhibited that Mo or/and Cd caused histological abnormality, reduced the activities of Ca2+ ATPase, Mg2+ ATPase, Na+-K+ ATPase and Ca2+-Mg2+ ATPase, K and Mg contents, and increased Na and Ca contents, upregulated CaMKKβ, CaMKIIɑ, CaN, IP 3 R, GRP78, GRP94, CRT mRNA levels and CaMKIIɑ, CaN, IP 3 R protein levels. Moreover, exposure to Mo or/and Cd notably promoted the amount of autophagosomes and LC3II immunofluorescence, upregulated AMPKα1, ATG5, Beclin-1, LC3A, LC3B mRNA levels and Beclin-1, LC3II/LC3I protein levels, downregulated mTOR, Dynein, P62 mRNA levels and P62 protein level. The changes of above indicators in combined group were more obvious. Overall, the results suggest that Mo and Cd co-exposure may can synergistically induce nephrotoxicity via causing calcium homeostasis disorder and autophagy in ducks. [Display omitted] • The co-toxicity of Mo and Cd was assessed in duck kidneys. • Mo and Cd could synergistically induce Ca2+ homeostasis imbalance. • Mo and Cd could synergistically induce Ca2+ homeostasis imbalance and autophagy. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an occupational and environmental pollutant, which mainly causes nephrotoxicity by damaging renal proximal tubular cells. To evaluate the effects of Cd on pyroptosis and the relationship between pyroptosis and apoptosis in duck renal tubular epithelial cells, the cells were cultured with 3CdSO 4 ·8H 2 O (0, 2.5, 5.0, or 10.0 μM Cd), N-acetyl-L-cysteine (NAC) (100.0 μM), Z-YVAD-FMK (10.0 μM) or the combination of Cd and NAC or Z-YVAD-FMK for 12 h, and then cytotoxicity was assessed. The results evidenced that Cd significantly increased the releases of interleukin-18 (IL-18) and interleukin-1β (IL-1β), lactate dehydrogenase (LDH) and nitric oxide (NO), relative conductivity and cellular reactive oxygen species (ROS) level. Simultaneously, Cd also markedly upregulated NLRP3, Caspase-1, ASC, NEK7, IL-1β and IL-18 mRNA levels and NLRP3, Caspase-1 p20, GSDMD and ASC protein levels. Additionally, NAC notably improved the changes of above indicators induced by Cd. Combined treatment with Cd and Z-YVAD-FMK remarkably elevated Bcl-2 mRNA and protein levels, inhibited p53, Bax, Bak-1, Cyt C, Caspase-9 and Caspase-3 mRNA levels and p53, Bax, Bak-1, Caspase-9/cleaved Caspase-9 and Caspase-3/cleaved Caspase-3 protein levels, increased mitochondrial membrane potential (MMP), decreased apoptosis ratio and cell damage compared to treatment with Cd alone. Taken together, Cd exposure induces duck renal tubular epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway, and inhibiting Caspase-1 dependent pyroptosis attenuates Cd-induced apoptosis. Image 1 • Cadmium (Cd) toxicity was evaluated in duck renal tubular epithelial cells. • Cd could induce pyroptosis via ROS/NLRP3/Caspase-1 pathway. • N-acetyl-L-cysteine (NAC) could inhibit pyroptosis induced by Cd. • Inhibiting Caspase-1-dependent pyroptosis might weaken Cd induced-apoptosis. [ABSTRACT FROM AUTHOR]

Copper (Cu) is a vital micronutrient required for numerous fundamental biological processes, but excessive Cu poses potential detrimental effects on public and ecosystem health. However, the molecular details linking endoplasmic reticulum (ER) stress and apoptosis in duck renal tubular epithelial cells have not been fully elucidated. In this study, duck renal tubular epithelial cells exposed to Cu sulfate (CuSO 4) (0, 100 and 200 μM) and a PERK inhibitor (GSK2606414, GSK, 1 μM) for 12 h were used to investigate the crosstalk between ER stress and apoptosis under Cu exposure. Cell and ER morphological and functional characteristics, intracellular calcium (Ca2+) levels, apoptotic rates, ER stress and apoptosis-related mRNA and protein levels were examined. The results showed that excessive Cu could cause ER expansion and swelling, increase the expression levels of ER stress-associated genes (PERK, eIF2α, ATF4 and CHOP) and proteins (p-PERK and CHOP), induce intracellular Ca2+ overload, upregulate the expression levels of apoptosis-associated genes (Bax, Bak1, Caspase9 and Caspase3) and the cleaved-Caspase3 protein, downregulate Bcl-xl and Bcl2 mRNA levels and trigger apoptosis. PERK inhibitor treatment could ameliorate the above changed factors caused by Cu. In conclusion, these findings indicate that excessive Cu could trigger ER stress via activation of the PERK/ATF4/CHOP signaling pathway and that ER stress might aggravate Cu-induced apoptosis in duck renal tubular epithelial cells. Image 1 • Copper (Cu) has toxic effect on duck renal tubular epithelial cells. • Cu could trigger endoplasmic reticulum (ER) stress via PERK/ATF4/CHOP pathway. • Cu could induce apoptosis in duck renal tubular epithelial cells. • ER stress might aggravate Cu-induced apoptosis via PERK/ATF4/CHOP pathway. [ABSTRACT FROM AUTHOR]

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH 4) 6 Mo 7 O 24.4H 2 O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H 2 O 2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress. • Toxicity of excessive Mo was assessed in duck renal tubular epithelial cells. • Excessive Mo could induce autophagy. • Inhibition of autophagy exacerbated oxidative stress induced by excessive Mo. • Inhibition of autophagy aggravated excessive Mo-induced mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Excess molybdenum (Mo) is harmful to the body, and the kidney is the vital target organ for Mo exposure. This study focused on the impacts of excess Mo on pyroptosis and the relationship between pyroptosis and apoptosis in kidney. The duck renal tubular epithelial cells were treated with (NH 4) 6 Mo 7 O 24 ·4H 2 O (0, 480, 720 and 960 μM Mo), N-acetyl- L -cysteine (NAC) (100 μM), Z-YVAD-fluoromethylketone (YVAD) (10 μM) and the combination of Mo and NAC or YVAD for 12 h. The LDH release and IL-1β, IL-18 contents of cell supernatant were detected by LDH and ELISA kits. The MMP and ROS level were measured using MMP and ROS kits by flow cytometry. The apoptotic rate of cell was detected by AO/EB counterstaining. Pyroptosis and apoptosis-related factors mRNA and protein levels were assayed by real-time qPCR and western blot, respectively. Excessive Mo markedly increased LDH, IL-18, IL-1β releases and induced overproduction of ROS, pyroptosis-related factors mRNA and protein levels. NAC and YVAD dramatically decreased pyroptosis induced by Mo. Simultaneously, YVAD significantly changed apoptosis-related factors mRNA and protein levels, and reduced cell apoptotic rate. Excessive Mo exposure can induce pyroptosis by the ROS/NLRP3/Caspase-1 pathway in duck renal tubular epithelial cells, and restraining pyroptosis of Caspase-1 dependence might weaken excess Mo-induced apoptosis. The study provides theoretical basis for excess Mo exposure nephrotoxic researches on waterfowl and the interplay between pyroptosis and apoptosis highlights a new sight into the mechanism of Mo-induced nephrotoxicity. ga1 • Toxicity of excess molybdenum (Mo) was assessed in duck renal tubular epithelial cells. • Excess Mo could induce pyroptosis via ROS/NLRP3/Caspase-1 pathway. • N-acetyl- L -cysteine (NAC) could inhibit pyroptosis induced by excess Mo. • Inhibiting Caspase-1-dependent pyroptosis might weaken excess Mo induced-apoptosis. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates autophagy and apoptosis are involved in the toxicity mechanism of heavy metals. Our previous studies showed that cadmium (Cd) could induce autophagy and apoptosis in duck kidneys in vivo, nevertheless, the interaction between them has yet to be elucidated. Herein, the cells were either treated with 3CdSO 4 ·8H 2 O (0, 1.25, 2.5, 5.0 μM Cd) or/and 3-methyladenine (3-MA) (2.5 μM) for 12 h and the indictors related autophagy and apoptosis were detected to assess the correlation between autophagy and apoptosis induced by Cd in duck renal tubular epithelial cells. The results demonstrated that Cd exposure notably elevated intracellular and extracellular Cd contents, the number of autophagosomes and LC3 puncta, up-regulated LC3A, LC3B, Beclin-1, Atg5 mRNA levels, and Beclin-1 and LC3II/LC3I protein levels, down-regulated mTOR, p62 and Dynein mRNA levels and p62 protein level. Additionally, autophagy inhibitor 3-MA decreased Beclin-1, LC3II/LC3I protein levels and increased p62 protein level. Moreover, co-treatment with Cd and 3-MA could notably elevate Caspase-3, Cyt C, Bax, and Bak-1 mRNA levels, Caspase-3 and cleaved Caspase-3 protein levels, and cell apoptotic rate as well as cell damage, decreased mitochondrial membrane potential (MMP), Bcl-2 mRNA level and the ratio of Bcl-2 to Bax compared to treatment with Cd alone. Overall, these results indicate Cd exposure can induce autophagy in duck renal tubular epithelial cells, and inhibition of autophagy might aggravate Cd-induced apoptosis through mitochondria-mediated pathway. Image 1 • Cd exposure can induce autophagy in duck renal tubular epithelial cells. • Autophagy inhibitor 3-MA exacerbates cell damage induced by Cd in duck renal tubular epithelial cells. • 2. [ABSTRACT FROM AUTHOR]

To investigate Molybdenum (Mo) and Cadmium (Cd) co-induced the levels of autophagy-related genes via AMPK/mTOR signaling pathway in Shaoxing Duck (Anas platyrhyncha) kidney, 60 healthy 11-day-old ducks were randomly divided into 6 groups, which were treated with Mo or/and Cd at different doses on the basal diet for 120 d. Kidney samples were collected on day 120 to determine the mRNA expression levels of adenosine 5′-monophosphate (AMP)-activated protein kinase α1 (AMPKα1), mammalian target of rapamycin (mTOR), Beclin-1, autophagy-related gene-5 (Atg5), microtubule-associated protein light chain A (LC3A), microtubule-associated protein light chain B (LC3B), sequestosome-1, and Dynein by real-time quantitative polymerase chain reaction. Meanwhile, ultrastructural changes of the kidney were observed. The results indicated that the mTOR and P62 mRNA expression levels were significantly downregulated, but the Atg5 and Beclin-1 mRNA levels were remarkably upregulated in all treated groups compared to control group, and their changes were greater in joint groups. Additionally, compared to control group, the Dynein mRNA expression level was apparently downregulated in co-treated groups, the LC3B, LC3A, and AMPKα1 expression levels were dramatically upregulated in single treated groups and they were not obviously different in co-treated groups. Ultrastructural changes showed that Mo and Cd could markedly increase the number of autophagosomes. Taken together, it suggested that dietary Mo and Cd might induce autophagy via AMPK/mTOR signaling pathway in duck kidney, and it showed a possible synergistic relationship between the 2 elements. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) and high Molybdenum (Mo) can lead to adverse reactions on animals, but the co-induced toxicity of Mo and Cd to liver in ducks was not well understood. To investigate the co-induced toxic effects of Mo combined with Cd on mitochondrial oxidative stress and apoptosis in duck livers. 240 healthy 11-day-old ducks were randomly divided into 6 groups (control, LMo group, HMo group, Cd group, LMoCd group and HMoCd group). After being treated for 30, 60, 90 and 120 days, liver mitochondrial antioxidant indexes, ceruloplasmin (CP), metallothionein (MT), Bak-1 and Caspase-3 genes mRNA expression levels, and ultrastructural changes were evaluated. The results showed that total antioxidative capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD) and xanthine oxidase (XOD) activities in experimental groups were decreased, whereas malondialdehyde (MDA) content and nitric oxide synthase (NOS) activity were increased compared with control group, and these changes of co-treated groups were more obvious in the later period of the experiment. The mRNA expression levels of CP, Bak-1 and Caspase-3 were up-regulated in experimental groups compared with control group and showed significant difference between co-treated groups and single treated groups. The mRNA expression level of MT in Cd group was higher than that in co-treated groups. Additionally, ultrastructural changes showed karyopyknosis, mitochondrial swelling, vacuolation and disruption of mitochondrial cristae in co-treated groups. Taken together, it was suggested that dietary Mo and Cd might lead to mitochondrial oxidative stress and apoptosis in duck livers, and it showed a possible synergistic relationship between the two elements. [ABSTRACT FROM AUTHOR]