| ObjectiveIschemic injury is the major pathogenesis in acute kidney injury, and hypoxia is the most critical ring-joint in the setting of ischemia. Because of the low oxygen tension in kidney and high oxygen demand of tubular cells, kidney is more vulnerable to ischemic/hypoxic injury. Therefore the renal ischemia/hypoxia plays a very important role in the progression of acute renal injury and chronic kidney disease. Hypoxia-inducible factor (HIF) is a key transcription factor in the regulation of oxygen homeostasis, which can regulate the expression of hundreds of kinds of target genes, making the organs or cells to be adaptive to ischemic/hypoxic injury. It has so far found that HIF family contains three members, including HIF-1, HIF-2 and HIF-3. Previous studies have shown that ischemia/hypoxia induced renal tubular epithelial cells to up-regulate the expression of HIF-1 and downstream genes of HIF-1, which can produce a protective effect, but the roles of HIF-2 in ischemic/hypoxic renal injury remain unclear. This study is designed to explore the roles of HIF-2 in acute ischemic/hypoxic renal injury in vitro.Materials and methods(1) HK-2, a human kidney epithelial cell line, was obtained from and cultured as suggested by ATCC. Cellular hypoxia was induced by CoCl2. The final concentration of CoC12 in cell culture medium was 150μmol/L. Cells were harvested at 0,6,12, 24,48 and 52h after drug treatments. Protein expression of HIF-2αwere tested by western blot.(2) The design and synthesis of HIF-2αsiRNA and negative control agent were from Shanghai Jima company, the DharmaFECTltransfection reagent (Dharmacon, USA) was applied in this study. The transfection process was done according to the manufacturer's protocol. According to our previous experiences on siRNA interference experiments in vitro, we chose a final concentration of 1 OOnmol/L of HIF-2αsiRNA to silence the expression of the HIF-2αin HK-2. Cells were cultured with normal medium, without any treatment, ie, the normal group (Group N); or only treated with CoCl2, ie, the hypoxia group (Group H); or treated with RNAi transfection reagent and CoCl2, ie, the transfection reagent alone group (Group T); or treated with negative control siRNA which targets none gene and CoCl2, ie, the negative control group (Group NC); or treated with both HIF-2αsiRNA and the CoCl2, ie, the HIF-2αsiRNA group (Group HR).48h after treatments, cells were harvested and western blot was applied to quantitate the protein expression of HIF-2αin each group. In addition, Real-time PCR was used to quantitate the mRNA expression of HIF-2α.(3)Apoptosis of HK-2 cells was detected by flow cytometry (Annexin/PI). Cellular necrosis was detected by lactate dehydrogenase (LDH) assay.(4) Using Western blot to quantitate the protein expression of HIF-2a, VEGF and Glut-1 in each group to explore the roles of HIF-2αin acute ischemic/hypoxic renal injury in vitro. HK-2 were treated with CoCl2 at a concentration of 150μmol/L for 6, 12,24,48 and 52h respectively, then celles were harvested and total protein were extracted. Using Western blot to quantitate the protein expression of HIF-2α, VEGF and Glut-1 after different time treatment of CoCl2 induced hypoxia in HK-2.Results(1) The time course of HIF-2αexpression:protein expression of HIF-2αin HK-2 was detectable by western blot at h24, h48 and h52 after CoCl2 treatment, reaching peak at h48. But the protein expression of HIF-2αdidn't differ between h24 and h52.(2) HIF-2αsiRNA interference:The transfection efficiency of siRNA was 95%-100%. The protein expression of HIF-2αin HR group was significantly lower than those in the H,T and NC group (P<0.01), but not N group. Protein expression of HIF-2αdidn't differ within H, T and NC group. The mRNA expression of HIF-2αin HIF-2αsiRNA group was significantly lower than those in the other four groups (P <0.05). mRNA of HIF-2αwas silenced by 88.0% with the HIF-2αsiRNA interference.(3) The apoptotic rates of HK-2 cells in the five groups were 2.45±0.39%,4.81±0.72%,5.82±0.54%,5.72±0.74%,7.95±0.40% in order by flow cytometry; The apoptotic rate in HR group was higher than those in the other four groups (P<0.01), and the apoptotic rate in N group was lower than those in the other four groups (P<0.01). The concentrations of LDH in mediums were 492.6±49.0U/L,687.1±55.4U/L,662.6±16.8U/L, 638.7±46.3U/L and 1330.7±26.1U/L in order. The concentration of LDH in HR group was significantly higher than those in the other four groups (P<0.01). And The concentration of LDH in N group was significantly lower than those in the other four groups (P<0.01).(4) The relationship between HIF-2αand VEGF:The protein expression of VEGF was significantly lower in HR group than those in H, T and NC group (P<0.01), but had no significant difference with N group. The expression of VEGF in H, T and NC group were significantly higher than those in N group (P<0.01). Group H, T and NC had the similar protein expression of VEGF. Within the first 48h, protein expression of VEGF increased gradually in a time-dependent way, which mean, the longer HK-2 cells were treated with chemical hypoxia, the higher was the protein level of VEGF. The peak value of VEGF was detected at h 48 and after then, protein VEGF started to decrease. At h52, protein level of VEGF returned to the same level as that at h24. The change of protein pattern of VEGF in HK-2 was consistent with the HIF-2α, in response to CoCl2 treatment.(5) The relationship between HIF-2αand Glut-1:The protein expression of Glut-1 in NC and 48hH group were significantly higher than those in N, HR and 24hH group (P<0.05). Glut-1 protein levels in 24hH and HR group were similar to N group. The protein expression of Glut-1 and HIF-2αprotein expression were consistent.Conclusions(1) HIF-2αprotein expression was upregulated in HK-2 24h after the induction of chemical hypoxia with CoCl2. The expression of HIF-2αprotein reached peak at h 48,then decreased.(2) The optimal mixing ratio for siRNA to transfection reagents was 4μl:0.2nmol, and the final concentration of siRNA was 100nmol/L, which could achieve a satisfactory transfection efficiency and silencing efficiency of HIF-2α. (3) Hypoxia could induce HK-2 cells apoptosis and necrosis. Silencing HIF-2αgene could significantly aggravate apoptosis and necrosis of HK-2 cells under hypoxia. It was demonstrated that HIF-2αhad the protective role on HK-2 cells under hypoxia.(4) Protein expression of VEGF were consistent with the protein expression of HIF-2a protein expression, as both VEGF and HIF-2αreached peak at h 48 after CoCl2 treatment. Moreover, protein level of HIF-2αwas downregulated after interference of HIF-2α. Thus HIF-2a palyed a role by regulating VEGF in acute ischemic/hypoxic renal injury in vitro.(5) Protein expression of Glut-1 in HK-2 increased after being treated with CoCl2 for 48h. Glut-1 was downregulated in response to HIF-2αsiRNA interference, which suggesting Glut-1 as a target gene to HIF-2αin acute ischemic/hypoxic renal injury in vitro.
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