Hypoxia-reoxygenation induced necroptosis in cultured rat renal tubular epithelial cell line

Objective(s): The aim of this study is to explore the potential role of hypoxia/reoxygenation in necroptosis in cultured rat renal tubular epithelial cell line NRK-۵۲E, and further to investigate its possible mechanisms.Materials and Methods: Cells were cultured under different hypoxia-reoxygenation conditions                        in vitro. MTT assay was used to measure the cell proliferation of cells that were exposed to hypoxia-reoxygenation conditions at different time points. Receptor-interacting protein ۱,۳ (RIP۱ and RIP۳) and NF-κB were detected by Western-blot analysis. Co-immunoprecipitation (Co-IP) was conducted to investigate the formation of necrosome. Necrostatin-۱ (Nec-۱) was adopted to inhibit the occurrence of necroptosis. In addition, morphological changes of cells after hypoxia-reoxygenation interference were observed under transmission electron microscope (TEM).  Results: MTT assay indicated that hypoxia-reoxygenation treatment can cause a decrease in cell viability. Particularly, ۶ hr of hypoxia and ۲۴ hr of reoxygenation (H۶R۲۴ group) resulted in the lowest cell viability. Western-blot results indicated that the expression of RIP۳ significantly increased in H۶R۲۴ group while the expression of NF-κB is decreased. Co-IP results demonstrated that the interaction between RIP۱ and RIP۳ was stronger in the hypoxia-reoxygenation induced group than the other groups, furthermore, treatment with Nec-۱ reduced the formation of necrosome. TEM observation results showed that hypoxia-reoxygenation treated cells showed typical morphological characteristics of necroptosis and autophagy.Conclusion: Hypoxia-reoxygenation treatment can induce necroptosis in NRK-۵۲E cells, and this effect can be inhibited by Nec-۱. In addition, the mechanism of necroptosis induced by hypoxia-reoxygenation injury on cells may be related to the low expression of NF-κB.