[Protective effect and mechanism of neutrophil gelatinase-associated lipocalin against hypoxia/reoxygenation injury of HK-2 renal tubular epithelial cells].
Objective To study the role of neutrophil gelatinase-associated lipocalin (NGAL) in hypoxia/reoxygenation injury and its regulatory effect on autophagy in HK-2 renal tubular epithelial cells. Methods Three sets of designed NGAL short-hairpin RNA (shRNA) sequences were synthesized and transfected into HK-2 cells. The expression levels of NGAL mRNA and protein were analyzed by quantitative real-time PCR and Western blotting, respectively. Then, the HK-2 cells with the best NGAL mRNA interference were selected to establish the hypoxia/reoxygenation model. The levels of microtubule-associated protein 1 light chain 3II (LC-3II) and beclin-1 were detected by Western blotting. Besides, the viability of cells was tested by Cell Titer-Blue and CCK-8 assay. Results Three sets of shRNA plasmids carrying silenced NGAL gene were successfully constructed and transfected into HK-2 cells. The expressions of NGAL mRNA and protein in these cells were significantly lower than those in the controls with blank vector. After NGAL-silenced HK-2 cells were subjected to hypoxia/reoxygenation, the levels of LC-3 II and beclin-1 were lower than those in the controls with blank vector, whereas the levels of LC-3 II and beclin-1 in were higher than those in the ones in which 400 ng/mL recombinant NGAL was added. Cell Titer-Blue and CCK-8 assays showed that the viability of NGAL-knockdown HK-2 cells was significantly lower than the controls. Conclusion NGAL may plays protective role towards HK-2 cells in the process of hypoxia/reoxygenation by enhancing autophagy.
Department of Laboratory Medicine, Third Hospital of Peking University, Beijing 100191, China.
Yinmei
Y
Zhang
Department of Laboratory Medicine, Third Hospital of Peking University, Beijing 100191, China. *Corresponding author, E-mail: bjmuzhangjie@ hotmail.com.
Jie
J
Zhang
D001343:Autophagy; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D000071068:Lipocalin-2 / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
D000071068:Lipocalin-2; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D010100:Oxygen