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

• Published in: Iranian journal of basic medical sciences Vol. 21; no. 8; pp. 863 - 868
• Main Authors: Zhu, Changlai, Liu, Yang, Guan, Zongyu, Zhou, Yi, Liu, Fang, Zhang, Tianyi
• Format: Journal Article
• Language: English
• Published: Iran Mashhad University of Medical Sciences 01.08.2018
• Subjects: Cell line; Hypoxia; Hypoxia-Reoxygenation; Morphology; Nec-1; Necrosome; Original; Receptor interacting protein; Necrosome; Cell line; Hypoxia-Reoxygenation Nec-1; Receptor interacting protein
• ISSN: 2008-3866; 2008-3874
• DOI: 10.22038/IJBMS.2018.26276.6444

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-52E, and further to investigate its possible mechanisms. Cells were cultured under different hypoxia-reoxygenation conditions . 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 1,3 (RIP1 and RIP3) and NF-κB were detected by Western-blot analysis. Co-immunoprecipitation (Co-IP) was conducted to investigate the formation of necrosome. Necrostatin-1 (Nec-1) 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). MTT assay indicated that hypoxia-reoxygenation treatment can cause a decrease in cell viability. Particularly, 6 hr of hypoxia and 24 hr of reoxygenation (H6R24 group) resulted in the lowest cell viability. Western-blot results indicated that the expression of RIP3 significantly increased in H6R24 group while the expression of NF-κB is decreased. Co-IP results demonstrated that the interaction between RIP1 and RIP3 was stronger in the hypoxia-reoxygenation induced group than the other groups, furthermore, treatment with Nec-1 reduced the formation of necrosome. TEM observation results showed that hypoxia-reoxygenation treated cells showed typical morphological characteristics of necroptosis and autophagy. Hypoxia-reoxygenation treatment can induce necroptosis in NRK-52E cells, and this effect can be inhibited by Nec-1. In addition, the mechanism of necroptosis induced by hypoxia-reoxygenation injury on cells may be related to the low expression of NF-κB.