title: Protective effects of neutrophil gelatinase-associated lipocalin on hypoxia/reoxygenation injury of HK-2 cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Laboratory Medicine, Peking University Third Hospital, Beijing, China.
authors.forename: L Y
authors.initials: LY
authors.lastname: Cui
authors.forename: S
authors.initials: S
authors.lastname: Yang
authors.forename: J
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000209:Acute-Phase Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019610:Cytoprotection; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D000071068:Lipocalin-2; D054834:Lipocalins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020011:Protective Agents / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D051028:bcl-2-Associated X Protein / Q000235:genetics
chemical_list: D000209:Acute-Phase Proteins; C494826:BAX protein, human; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D020011:Protective Agents; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; D053148:Caspase 3