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

Document Type: Original Article


1 Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, JS 226001, P. R. China

2 Medical College of Nantong University, Nantong, JS, P. R. China


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-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.


Main Subjects

1. Xiong K, Liao H, Long L, Ding Y, Huang J, Yan J. Necroptosis contributes to methamphetamine-induced cytotoxicity in rat cortical neurons. Toxicol In Vitro 2016; 3:163-168.
2. Himmelfarb J, Ikizler TA. Acute kidney injury: changing lexicography, definitions, and epidemiology. Kidney Int 2007; 71:971-976.
3. Jie Zhou CX, Wenwen Wang, Xinglu Fu, Liang Jinqiang, Yuwen Qiu, Jin Jin, Jingfen Xu, Zhiying Huang. Triptolide-induced oxidative stress involved with Nrf2 contribute to cardiomyocyte apoptosis through mitochondrial dependent pathways. Toxicol Lett 2014; 230: 454-466.
4. Andreas Hartmann J-DT, Stéphane Hunot, Kristy Kikly, Baptiste A. Faucheux, Annick Mouatt-Prigent, Merle Ruberg, Yves Agid, Etienne C. Hirsch. Caspase-8 is an effector in apoptotic death of dopaminergic neurons in Parkinson’s disease, but pathway inhibition results in neuronal necrosis. J Neurosci 2001; 21:2247–2255.
5. Michael Fricker AV, Aviva M. Tolkovsky, Guy C. Brown. Caspase inhibitors protect neurons by enabling selective necroptosis of inflamed microglia. J Biol Chem 2013; 288:9145-9152.
6. Liu T,  Bao YH, Wang Y, Jiang JY. The role of necroptosis in neurosurgical diseases. Braz J Med Biol Res 2015; 48:292-298.
7. Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 2000; 1:489-495.
8. Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 2005; 1:112-119.
9. Degterev A, Hitomi J, Germscheid M, Ch’en IL, Korkina O, Teng X, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol 2008; 4:313-321.
10. Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009; 137:1112-1123.
11. Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC, et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009; 325:332-336.
12. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012; 148:213-227.
13. Mohan V, G SP, Meravanigi G, N R, Yerramshetty J. Adaptation of the Oswestry Disability Index to Kannada language and evaluation of its validity and reliability. Spine (Phila Pa 1976) 2016; 41:E674-680.
14. Herrera-Gutierrez ME, Seller-Perez G, Sanchez-Izquierdo-Riera JA, Maynar-Moliner J, group Ci. Prevalence of acute kidney injury in intensive care units: the “COrte de prevalencia de disFuncion RenAl y DEpuracion en criticos” point-prevalence multicenter study. J Crit Care 2013; 28:687-694.
15. Le Dorze M, Legrand M, Payen D, Ince C. The role of the microcirculation in acute kidney injury. Curr Opin Crit Care 2009; 15:503-508.
16. Mehta RL, Pascual MT, Soroko S, Savage BR, Himmelfarb J, Ikizler TA, et al. Spectrum of acute renal failure in the intensive care unit: the PICARD experience. Kidney Int 2004; 66:1613-1621.
17. Ye YC, Wang HJ, Yu L, Tashiro S, Onodera S, Ikejima T. RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy. Int Immunopharmacol 2012; 14:674-682.
18. Poetz O, Luckert K, Herget T, Joos TO. Microsphere-based co-immunoprecipitation in multiplex. Anal Biochem 2009; 395:244–248.
19. Jain K, Suryakumar G, Prasad R, Ganju L, Singh S. Enhanced hypoxic tolerance by Seabuckthorn is due to upregulation of HIF-1α and attenuation of ER stress. J Appl Biomed 2015;14,71-83
20. Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol 2008; 9:378-390.
21. Nagahara Y, Shiina I, Nakata K, Sasaki A, Miyamoto T, Ikekita M. Induction of mitochondria-involved apoptosis in estrogen receptor-negative cells by a novel tamoxifen derivative, ridaifen-B. Cancer Sci 2008; 99:608-614.
22. Festjens N, Vanden Berghe T, Cornelis S, Vandenabeele P. RIP1, a kinase on the crossroads of a cell’s decision to live or die. Cell Death Differ 2007; 14:400-410.
23. Vandenabeele P, Declercq W, Van Herreweghe F, Vanden Berghe T. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal 2010; 3:re4.
24. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell 2008; 132:344-362.
25. Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev 2004; 18:2195-2224.
26. Thapa RJ, Basagoudanavar SH, Nogusa S, Irrinki K, Mallilankaraman K, Slifker MJ, et al. NF-kappaB protects cells from gamma interferon-induced RIP1-dependent necroptosis. Mol Cell Biol 2011; 31:2934-2946.
27. Bertrand MJ, Vandenabeele P. RIP1’s function in NF-kappaB activation: from master actor to onlooker. Cell Death Differ 2010; 17:379-380.
28. Ting AT, Pimentel-Muinos FX, Seed B. RIP mediates tumor necrosis factor receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis. EMBO J 1996; 15:6189-6196.
29. Gomes LC, Di Benedetto G, Scorrano L. During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat Cell Biol 2011; 13:589-598.