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25 January 2018 : Laboratory Research  

Neutrophil Gelatinase-Associated Lipocalin Attenuates Ischemia/Reperfusion Injury in an In Vitro Model via Autophagy Activation

Cai Yan1ABCDEFG*, Tang Yuanjie2G, Xu Zhengqun1B, Chen Jiayan1C, Li Kongdan1BC

DOI: 10.12659/MSM.908158

Med Sci Monit 2018; 24: LBR479-485

Abstract

BACKGROUND: The aim of this study was to investigate the protective effects of neutrophil gelatinase-associated lipocalin (NGAL) on hypoxia/reoxygenation (H/R) induced acute kidney injury (AKI) in vitro.

MATERIAL AND METHODS: We used NRK-52E cells and H/R treatments to mimic ischemia/reperfusion injury (IRI) in vitro. Experimental groups were: the control group, the H/R group, the 3-methyladenine (3-MA)+H/R group, the NGAL (0.25, 0.5, and 1 ug/mL)+H/R group, and the NGAL (0.25, 0.5, 1 ug/mL)+3-MA+H/R group. After 24 hours of culture, cell proliferation was analyzed by CCK-8 assay. Expression of LC3-II was detected by immunoblot assay. Autophagy was detected by electron microscopy.

RESULTS: The expression of LC3-II was increased in the H/R group compared with normoxic condition (p<0.05) and proliferation also improved. Autophagy was significantly inhibited by 3-MA, with downregulated of LC3-II, followed by decreased cell viability (p<0.05). We further detected the effect of different doses of NGAL in H/R induced injury, and found that low doses of NGAL alone slightly increased LC3-II protein accumulation, and autophagy was further induced with higher dose of NGAL treatment. Meanwhile, cell viability assays showed induced cell survival. We found that in the NGAL+3-MA group, cell viability assays revealed reduced cell damage, followed concomitantly with depressed autophagy. The formulation of autophagosomes were correlated with LC3-II protein expression in each group.

CONCLUSIONS: Autophagy plays a renoprotective role in H/R injury, as well in AKI. NGAL might be related to attenuated tubular epithelial cell damage via adjusting autophagy.

Keywords: Cell Hypoxia, Lipocalins

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750