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

Abstract

Objectives: 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.

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 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).

Results: 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.

Conclusion: 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.

Keywords: Cell line; Hypoxia-Reoxygenation Nec-1; Necrosome; Receptor interacting protein.

Figure 1

The anti-proliferative effect of the hypoxia-reoxygenation intervention in NRK-52E cells. Cells were exposed to the indicated hypoxia-reoxygenation time. Cell survival rate was detected using MTT assay. (** P<0.05)

Figure 2

The grey value of RIP1 expression level in different times of exposure to hypoxia-reoxygenation. Grey value of H6R24 group was significantly higher than in other groups. The quantitative analysis results were shown as mean grey value±SD (** P<0.05)

Figure 3

The Co-immunoprecipitation assay was analyzed by the Western-blot method. Whole cell lysates were immunoprecipitated with anti-RIP3, and the Co-immunoprecipitated complexes were immunoblotted for RIP1 and RIP3 (IP=immunoprecipitation, IB=immunoblot). Quantitative analysis of RIP1 expression level in different groups was also shown (** P<0.05)

Figure 4

Western-Blotting analysis of RIP1 expression level in H6R24 and H6R24+Nec-1 groups. The quantitative analysis results were expressed as mean grey value±SD (** P<0.05)

Figure 5

NRK-52E cells were exposed to various reoxygenation times after 6 hr of hypoxia, and then NF-κB, RIP1 expression was detected by the Western-blot method

Figure 6

Ultrastructure features of NRK-52E cells in the control group (A, a) and H6R24 group (B, b). Scale bars, as indicated in the images, white arrowhead shows characteristic autophagosome