Protective role of NGAL against hypoxia-reoxygenation injury in HK-2 cell
10.3760/cma.j.issn.1009-9158.2011.12.020
- VernacularTitle:NGAL对肾小管上皮HK-2细胞的缺氧与复氧损伤保护作用
- Author:
Shuo YANG
;
Liyan CUI
;
Jie ZHANG
- Publication Type:Journal Article
- Keywords:
Cell hypoxia;
Kidney tubules;
Epithelial cells;
Acute-phase proteins
- From:
Chinese Journal of Laboratory Medicine
2011;34(12):1129-1134
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo explore the role and mechanism of NGAL in the process of hypoxia/ reoxygenation (H/R) injury in human renal tubular epithelial cell (HK-2).MethodsThe H/R model of HK-2 cells was established in vitro (oxygen concentration ＜ 0.1％ ).The expressions of NGAL in the cells of H/R group and control group were determined by WB and real-time RT-PCR.The cell models were treated with 50,100,200,400 and 1 000 ng/ml of recombinant NGAL respectively.Cell proliferation and apoptosis of the treated groups and control group were detected by the MTT assay and Annexin V-FITC/PI staining to determine the appropriate NGAL concentration.The cell cycle distributions in the H/R group,NGAL treated H/R group and control group were detected by flow cytometry,and the expressions of bax/bcl-2 and caspase-3 were measured by real-time RT-PCR.The experiments were triplicated to obtain the mean values.Results The protein expression of NGAL/actin in H/R group (5.875 ±0.081 ) was higher than that of the control group ( 1.513 ±0.032),the differences between the two groups had statistic significance (t =96.89,P ＜0.05). While the gene expressions of NGAL/actin in HL/R groups (12.32 ± 1.27 ) and control group ( 1.00 ±0.00) had statistical significance of difference ( t =15.40,P ＜ 0.05 ).In MTT assay,the absorption values of the H/R group and control group were 0.97 ± 0.03 and 0.56 ± 0.04,the differences had statistic significance( t =18.680,P ＜ 0.05 ).And the absorption value of cells treated with different concentrations of NGAL (50,100,200,400,1 000 ng/ml) were 0.56±0.04,0.53 ±0.03,0.56 ±0.04,0.53 ± 0.03,0.54 ± 0.02,respectively.There were no significant differences between the H/R group and NGAL treated groups ( F =0.978,P ＞ 0.05 ),but the differences of absorption values in the control group and other groups had statistical significance ( F =105.20,P ＜ 0.05 ).The ratios of early apoptotic cells ( Annexin V positive,PI negative) in the control group and the H/R group were ( 1.0 ±0.2) ％ and ( 27.6 ± 1.4 ) ％ respectively with a statistical significance of difference ( t =33.590,P ＜0.05 ).After the treatment of NGAL ( 50,100 ng/ml),the ratios were ( 27.8 ± 1.1 ) ％ and ( 26.4 ±1.3 ) ％ and there were no significant differences compared to the H/R group ( t =0.250,P ＞ 0.05 ).When the H/R model was treated with 200 ng/ml of NGAL,the ratio of early apoptotic cells dropped to ( 19.4 ± 0.6) ％,leading to a statistical significance of difference ( t =10.350,P ＜ 0.05 ).However,in the H/R model treated with high concentration of NGAL (400,1 000 ng/ml),the ratios were ( 19.3 ± 1.1 )％ and ( 18.9 ±0.5 ) ％,which had no significant differences compared to the cells treated with 200 ng/ml of NGAL (t =0.130,0.630; P ＞0.05).Thus the study chose 200 ng/ml as the appropriate treating concentration of NGAL.In the control group,H/R group and 200 ng/ml,NGAL treated HR group,PI values were (30.2 ±0.4)％,(22.1 ± 2.7 )％ and (23.2 ± 3.7 )％ respectively.There were no significant differences between the H/R group and NGAL treated group (t =0.510,P ＞0.05),but there was still statistical significance in difference among the control group and the treated groups ( F =8.28,P ＜ 0.05 ).The ratio of bax/bcl-2 and the expression of caspase-3 detected by real-time RT-PCR were 1.00 ± 0.00,1.00 ± 0.00 in the control group,5.83 ±0.33,8.13 ±0.20 in the H/R group and 2.52 ±0.07,1.89 ±0.02 in the NGAL treated H/R group.The bax/bcl-2 ( F =485.30,P ＜ 0.05 ) and caspase-3 ( F =3456.78,P ＜ 0.05 ) did have statistical significances of difference among the three groups.ConclusionsNGAL acts as a protective factor against hypoxia-reoxygenation injury by regulating pro-apoptotic genes.It provides a new idea and evidences for the treatment of AKI caused by ischemia-reperfusion injury.
