A model of necroptosis in human renal tubular epithelial cells |
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Authors: | JIANG Fen CHEN Yuan-han LIANG Xin-ling LI Dong-feng XU Li-xia XIE Hong-ping HU Peng-hua LIU Shuang-xin SHI Wei |
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Affiliation: | 1.Division of Nephrology, the First Affiliated Hospital of Nanhua University, Hengyang 421001, China;2.Division of Nephrology, 3Medical Research Center, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China. |
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Abstract: | AIM:To make a model of necroptosis in human renal tubular epithelial HK-2 cells. METHODS:To induce necroptosis, HK-2 cells were treated with tumor necrosis factor α (TNF-α) followed by ATP depletion, and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was added to block the activity of caspase-8. The morphological changes of the cells were observed under light microscope and electronic microscope.The cell viability was detected by CCK-8 assay, and the marker of necroptosis was analyzed by Western blotting. RESULTS:In the cells treated with TNF-α followed by zVAD-fmk and antimycin A for 1 h, the morphological changes including the cell and organelle inflation, and membrane fragmentation, with a large amount of autophagysome, were observed.However, these abnormalities were markedly attenuated after treatment with Nec-1. Meanwhile, the cell viability was also significantly improved after using Nec-1. No similar variation was observed in other groups. In addition, the expression of LC3-II was significantly decreased in Nec-1+TNF-α+zVAD-fmk+ antimycin A (1 h) group compared with control group. CONCLUSION: TNF-α stimulation and energy depletion induce necroptosis in renal tubular epithelial cells.Nec-1 inhibits necroptosis in a caspase-independent pathway, and may have therapeutic potential to prevent and treat renal ischemia injury. |
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Keywords: | Necroptosis Renal tubular epithelial cells Microtubule-associated protein 1 light chain 3-II Necrostatin-1 |
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