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Objective To explore the role and mechanism of pumilio homolog 1 （PUM1） in hypoxia/reoxygenation induced cell injury in human renal tubular epithelial cells （HK-2）. Methods A hypoxia/reoxygenation （H/R） model of HK-2 cells was established in vitro. PUM1 expression was knocked down through small interfering RNA （siRNA）. Cells were randomized into three groups of control， hypoxia/reoxygenation （H/R） and H/R+siRNA. Western blot （WB） method was used for detecting the expression level of PUM1 protein. Cell Count Kit 8 （CCK-8） was employed for detecting cell viability. Hydrogen peroxide （H2O2）， malondialdehyde （MDA） and superoxide dismutase （SOD） were used for evaluating the levels of oxidative stress. Flow cytometry was utilized for detecting the level of cell apoptosis. Results As compared with control group， protein expression level of PUM1 in H/R 3 h group （1.76 ± 0.11 vs 0.98 ± 0.05）， H/R 6 h group （2.89 ± 0.14 vs 0.98 ± 0.05） and H/R 12 h group （3.78 ± 0.08 vs 0.98 ± 0.05） gradually spiked with the prolongation of hypoxic time. As compared with H/R group， knocking down the expression of PUM1 significantly improved the cell viability （73.67 ± 3.42 vs 29.60 ± 2.94）， oxidative stress [H2O2:（13.53 ± 0.85）μmol/L vs （22.43 ± 1.12）μmol/L， MDA: （16.03 ± 0.70）μmol/L vs （31.20 ± 1.50）μmol/L， SOD: （34670 ± 1800）U/L vs （5730 ± 1220）U/L] and apoptotic level [（14.89 ± 1.65）% vs （39.71 ± 1.94）%] after H/R in H/R+si-PUM1 group. Conclusion PUM1 is up-regulated in H/R induced HK-2 cells and its inhibition may alleviate H/R injury through reducing oxidative stress and lowering cell apoptosis levels.

Angiotensinogen (AGT) expression in renal proximal tubular cells (RPTCs) and intrarenal tumor necrosis factor-α (TNF-α) levels are increased in hypertension and renal diseases However, the contribution of TNF-α to AGT expression in RPTCs has not been established. Therefore, the objective of the present study was to determine influence of TNF-α on AGT expression in RPTCs. Human kidney-2 (HK-2) cells, immortalized human RPTCs, were treated with several concentrations of TNF-α up to 24 h. AGT mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. Activation of nuclear factor-κB (NF-κB) by TNF-α was evaluated by Western blot analysis, immunocytochemistry, and electrophoretic mobility shift assay (EMSA). TNF-α suppressed AGT mRNA expression in a dose- and time-dependent manner. Maximum AGT mRNA reduction was caused by 40 ng/ml of TNF-α (0.52 ± 0.09, ratio to control, at 24 h) and at 24 h (0.66 ± 0.05, ratio to control, by 10 ng/ml TNF-α). TNF-α reduced AGT protein accumulation in the medium between 8 and 24 h (0.62 ± 0.13 by 40 ng/ml TNF-α, ratio to control). TNF-α activated and induced translocalization of p50 and p65, which are NF-κB subunits. Elevated formation of p50/p65 and p50/p50 dimers by TNF-α were observed by EMSA and supershift assay. Gene silencing of p50, but not p65, attenuated the effect of TNF-α on reduction of AGT expression in RPTCs. These results indicate that TNF-α suppresses AGT expression through p50/p50 homodimer formation in human RPTCs, suggesting a possible counteracting mechanism that limits excessive intrarenal AGT production. [ABSTRACT FROM AUTHOR]