Will Nickel Affects Kidney ?

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Part Ⅱ：Nickel induces autophagy via PI3K/AKT/mTOR and AMPK pathways in mouse kidney

Heng Yin, Zhicai Zuo & et al.

ABSTRACT

Nickel (Ni), a widely distributed metal, is an important pollutant in the environment. Although the kidney is a crucial target of Ni (Nickel) toxicity, information on autophagy and the potential mechanisms of Ni-induced renal toxicity are still poorly described. As we discovered, NiCl2 could induce renal damage including a decrease in renal weight, renal histological alterations, and renal function injury. According to the obtained results, NiCl2 could obviously increase autophagy, which was characterized by the increase in LC3 expression and decrease of p62 expression. Meanwhile, the result of ultrastructure observation showed increased autolysosomes numbers in the kidney of NiCl2 (nickel chloride 2)-treated mice. In addition, NiCl2 increased mRNA and protein levels of autophagy flux proteins including Beclin1, Atg5, Atg12, Atg16L1, Atg7, and Atg3. Furthermore, NiCl2 induced autophagy through AMPK and PI3K/ AKT/mTOR pathways which featured down-regulated expression levels of p-PI3K, p-AKT, and p-mTOR and upregulated expression levels of p-AMPK and p-ULK1. In summary, the above results indicate involvement of autophagy in renal injury induced by NiCl2, and NiCl2 induced autophagy via PI3K/AKT/mTOR and AMPK pathways in mouse kidneys.

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Discussion

As the widespread environmental pollution, the toxicology of Ni (Nickel) has been confirmed, however, the underlying mechanisms are still largely unclear. As we all know, the kidney is a major target organ where Ni (Nickel) accumulates and intoxication occurs. In our previous studies, oxidative stress, cell cycle arrest, inflammation, and apoptosis were involved in NiCl2 (nickel chloride 2) toxicology in the broiler’s kidney (Guo et al., 2014, 2015, 2016a, 2016b, 2019). Autophagy plays a critical part in cellular homeostasis, and abnormal autophagy may cause apoptosis (Denton and Kumar, 2019). However, it is not clear whether autophagy was induced in the kidney by Ni (Nickel). In the current research, research results indicated enhancement of renal autophagy by excess NiCl2. In this study, the increase of LC3-II/LC3-I while the decrease of p62 indicates that NiCl2 (nickel chloride 2) treatment could induce autophagy. Meanwhile, ultrastructure observation results demonstrated that autolysosomes were increased in Ni-exposed in the kidney of mice. Similarly, it has been demonstrated that heavy metals (Cd, Cu, As, Pb) can induce autophagy in the kidney (Gu et al., 2018; Shi et al., 2019; Q. Guo et al., 2020; H. Guo et al., 2020; Wan et al., 2020). The process of autophagy is regulated by several evolutionarily-conserved genes called autophagy-related (ATG) genes. To further explore whether NiCl2 (nickel chloride 2) affects the proteins involved in the autophagy flux, we checked the levels of proteins associated with autophagy, such as Beclin1, Atg5, Atg12, Atg16L1, Atg7, and Atg3. Beclin1, an important gene facilitating initiate autophagy, has involvement during autophagy initiation (Wargasetia et al., 2015). Atg12-Atg5-Atg16L1 complex as an essential component for forming autophagosomes is in favor of the expansion of phagophores (Lystad et al., 2019). LC3-I is activated by ATG7 and conjugated to phosphatidylethanolamine by ATG3 for forming LC3-II, which is referred to as LC3 lipidation (Tan et al., 2020). Besides, the ATG12-ATG3 complex is found to play a crucial part in basic autophagy fluxes, endolysosomal transport, and endosome functions in the late nuclear period (Murrow et al., 2015). In this research, the results showed increased mRNA and protein levels of Beclin1, Atg5, Atg12, Atg7, Atg16L1, and Atg3 after NiCl2 treatment.

These results indicated that NiCl2 (nickel chloride 2) could induce renal autophagy through up-regulation of autophagy-related proteins. Similar results were also observed in recent studies of other heavy metals. Zou et al. (2020) have reported that Pb and Cd could increase expressions of proteins related to autophagy (Beclin-1, Atg5, and Atg7) in the liver of rats. Wan et al. (2020) have demonstrated that being exposed to Cu for the long term could induce autophagy and up-regulate the expression of Beclin1 and Atg5 in the kidney of rats. mTOR, a major negative regulator for autophagy, is responsible for nutrient depletion, low energy, or oxidative stress (Parzych and Klionsky, 2014). By inhibiting the ULK1-ATG13-FIP200 complex, particularly through inhibitory phosphorylation of ULK1, active mTOR van suppresses autophagy (Park et al., 2016). Furthermore, ULK1 could also phosphorylate Beclin 1, resulting in a canonical autophagy pathway induction (Russell et al., 2013). In the current research, reduction of p-mTOR and increase of p-ULK1 implies the capability of NiCl2 (nickel chloride 2) in inhibiting mTOR activities. This in turn activates ULK1 to initiate autophagy. Conforming to results obtained here, Huang et al. (2016) have also proposed Ni exposure inducing autophagy in Beas-2B cells by inhibiting mTOR. It has been considered that the classic PI3K/AKT-mTOR pathway serves as an important negative regulator in forming autophagosomes. The PI3K controls diverse cell processes such as growth, survival, metabolism, apoptosis, autophagy, and so on (Xu et al., 2020). AKT is a main PI3K effector. Phosphorylation of tuberous sclerosis complex 2 (TSC2) by activated AKT hinders the formation of inhibitory TSC1/TSC2 heterodimers, then activating TOR complex 1 (TORC1), a complex of mTOR with distinct functions (Wang et al., 2017). In this study, down-regulation of p-PI3K and p-AKT indicates that PI3K/AKT-mTOR pathway has involvement in autophagy induced by NiCl2. Furthermore, recent research suggests that negatively regulating mTOR, and activation of AMPK probably induces autophagy (Yang et al., 2018). Besides, AMPK can directly phosphorylate ULK1 to induce autophagy (Gwinn et al., 2008). Research findings demonstrated that protein levels of p-AMPK are increased by NiCl2, which reveals that NiCl2 activates AMPK/mTOR pathway in the kidney.

In summary, research findings indicated the involvement of autophagy in NiCl2 (nickel chloride 2)-induced renal injury and NiCl2 (nickel chloride 2) induced autophagy via AMPK and PI3K/AKT/mTOR pathways in mouse kidneys. However, the role of autophagy in Ni-induced kidney toxicity should be further explored.

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