Protective Effects of Neutrophil Gelatinase–Associated Lipocalin on Hypoxia/Reoxygenation Injury of HK-2 Cells

Neutrophil gelatinase–associated lipocalin (NAGL) was first extracted from neutrophil granules. Our previous study showed that the expression of NGAL mRNA and protein can be induced by hypoxia/reoxygenation. This study was designed to investigate the relationship between NGAL and hypoxia/reoxygenation injury pathologies in HK-2 cells. The effect of NGAL on the proliferation of HK-2 cell lines was analyzed with a MTT colorimetric assay. Cell-cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry after stained with propidium iodide and annexin V–fluorescein isothiocyanate. The expression of genes for apoptotic proteins Bcl-2, Bax, and caspase-3 was analyzed with real-time reverse-transcription polymerase chain reaction (RT-PCR). The expression of NGAL mRNA and protein was analyzed with real-time RT-PCR or Western blot, respectively. HK-2 cells were treated with hypoxia/reoxygenation. HK-2 cells exhibited an increase in the number of cells in the G0/G1 phase and a decrease in the number of cells in the S and G2/M phases. The proliferation index is decreased. When HK-2 cells were treated with 200 ng/mL recombinant NGAL and hypoxia/reoxygenation, there were no effects on cell-cycle distribution. The ratio of early apoptotic cells in the control and hypoxia/reoxygenation groups were 1.1% and 26.5%, respectively. After the addition of 200 ng/mL recombinant NGAL, the ratio of early apoptotic cells in the hypoxia/reoxygenation group dropped to 19.6%. The expression of Bax/Bcl-2 ratio and caspase-3 were significantly higher in the hypoxia/reoxygenation compared with the control group. After the addition of 200 ng/mL recombinant NGAL, the levels of Bax/Bcl-2 ratio and caspase-3 decreased significantly compared with the control group. The action of NGAL against hypoxia/reoxygenation injury was due to inhibiting the apoptosis via inhibition of the expression of the genes of proapoptotic proteins Bax, Bcl-2, and caspase-3.