Effects Of HIF-1α On Human Proximal Tubular Epithelial Cells Under Hypoxia And Involved Mechanisms

Background and objectiveAcute kidney injury is a common clinical syndrome. It is also an important cause of morbidity and mortality among critically ill patients. Ischemic injury is the major pathogensis in acute kidney injury, while hypoxia is the most critical ring-joint in the ischemic injury. Because of high oxygen consumption of tubular epithelial cells and the special vasculature system, the kidney is very susceptible to hypoxia, especially the proximal tubular epithelial cells. Therefore, hypoxic injury of renal tubular epithelial cells is paid close attention to, and it is the focus for investigation. Cobalt chloride (CoCl2) is one kind of iron chelating agents. CoCl2 is a common hypoxic mimics that has been widely used in experimental studies. To establish a stable and optimal celluar hypoxic model has important significance for the subsequent research. This part is aimed at the optimal hypoxic condition of proximal tubular epithelial cells for the subsequent research by comparing the growth inhibition rates and the expression of hypoxia-inducible factor-1α(HIF-1α) protein while the HK-2 cells are cultured at different concentrations of CoCl2 in cell culture medium and different times.Materials and methodsThe proximal tubular epithelial cell line (HK-2) was chosen as the experimental cell. Cellular hypoxia model was used as the experimental hypoxic approach, that is, CoCl2 was added into the cell culture medium. The HK-2 cells were incubated in cell culture mediums with different concentrations of CoCl2 (final concentrations of CoCl2 were 0,50,100, 150,300,600μM). The HK-2 cells were also incubated for different times, including 0,4,6,12,24 and 48h. MTT assay was used to detect the growth inhibition rates of cells. The nuclear proteins of HK-2 cells were collected, which was used in Western blot. So the expression of HIF-1αprotein could be measured.Results 1) The trend of the growth inhibition rates of HK-2 cells was rising, along with the increased concentrations of CoCl2 in the cell culture medium and the prolongation of the incubated time. Compared with cells incubated in cell culture medium with 600μM CoCl2, the growth inhibition rates of cells incubated in cell culture medium with 50-300μM CoCl2 were lower significantly(P<0.05). Although the growth inhibition rates of cells incubated in mediums with 50-300μM CoCl2 tend to rise, there was no significant difference among them(P>0.05). Meanwhile, the growth inhibition rates of cells incubated for 4-24h were lower than that of cells incubated for 48h.Although the growth inhibition rates of cells incubated for 4-24h tend to rise, there was no significant difference among them(P>0.05).2) When the incubated time was between 0 and 24h, the expression of HIF-1αprotein was rising along with the extension of time. And the peak of the expression of HIF-1αprotein was at 24h. The expression of HIF-1αprotein at 48h was less than that at 24h. When the concentration of CoCl2 in medium was between 0 and 150μM, the expression of HIF-1αprotein was rising along with the increasing concentration of CoCl2 in medium. And when the concentration of CoCl2 in the medium was 150μM, the expression of HIF-1αprotein could reach to the peak, which was more than that of 300μM CoCl2 and 600μM CoCl2.ConclusionsWhile the optimal cellular hypoxic model was that the concentration of CoCl2 in cell culture medium was 150μM and the incubation time was 24h, the viability of HK-2 cells was affected a little, and the expression of HIF-1αprotein could be the most. To establish an ideal model could provide solid basis for the subsequent research. Background and objectiveThe old phenomenon-gene silence widely exists in living nature. Gene silence is a protective mechanism for it can prevent virus or other external nucleic acid from invading and keep the stable heredity of the organism itself. Gene silence exists widely in eukaryotic cells. As a new method for gene silence, RNA interference (RNAi) shows enormous perspective in the study of genetic function. It is acceptable by scholars domestic and abroad for the application of RNAi to silence the target gene specially to study its function. RNAi has the characters of high specificity, high performance and safety. RNAi can study the function of the target gene by knockout it without destroying the genetic structure and gene mutation. However, the efficiency of transfection in different kinds of mammalian cells is different. Therefore, we need to chose optimal transfection reagent and optimize the transfecting condition for each cell line. Optimal dose of tranfection reagent and ideal concentration of siRNA play an important role in the efficiency of gene silence by RNAi. This part is aimed at evaluating the transfecting efficiency of transfection reagent and determining the ideal concentration of HIF-1αsiRNA to silence HIF-1α.Materials and methodsThe reagents for RNAi were SMARTpool HIF-1αsiRNA, siGLO siRNA negative control and DharmaFECT 1 transfection reagent, which were brought from Dharmacon Corporation in USA. The transfection process was done according to the manufacturer's protocol. The laser confocal fluorescence microscope was used to observe the transfecting efficiency. And fluorescent quantitation PCR was used to quantify the expression of HIF-1αmRNA in HK-2 cells.Results1) When 4μl transfection reagent and 100nM siGLO transfection indicator (the proportion of transfection reagent and siGLO was 4μl: 0.2nmol) transfected the HK-2 cells, the transfecting efficiency could reach to 95%-100%.2) When the final concentrations of HIF-1αsiRNA were 25,50 and 100nM, the efficiencies of HIF-la siRNA silencing HIF-la were 24.0±8.6%,43.0±4.2% and 70.0±4.2%, respectively.3) When HK-2 cells silenced HIF-1αgene suffered from hypoxia, the expression of HIF-1αmRNA in them account for 3.3% of that in HK-2 cells only suffering from hypoxia. That is to say, under hypoxia, the efficiency of HIF-1αsiRNA silencing HIF-1αcould reach to 96.7%.ConclusionsFor HK-2 cells, when the transfection reagent and siRNA mixed at the proportion of (4μl:0.2nmol) and the final concentration of siRNA was 100nM, the transfecting efficiency and silencing efficiency could be high. Furthermore, the silencing efficiency not only related with optimal transfection reagent and optimized transfecting conditions, but the degree of abundance of the target gene also contributed to it. Background and objectiveHypoxia is unfavorable for cell growth. But cells may maintain living, even proliferate, through a series of genetic and metabolic changes. Under hypoxia, organisms or cells appear a series of hypoxic response reactions which are mediated by hypoxia-inducible factor (HIF). As the key transcription factor regulating oxygen balance, HIF can regulate more than one hundred target genes so that organisms or cells can appear adaptive responses to the hypoxic or ischemic injury. At present, there are three members in HIF family, including HIF-1, HIF-2, HIF-3. Among the HIFs, HIF-1 widely distributes in mammalian and human cells. For renal hypoxic and ischemic injury, HIF-1 mainly expresses on tubular epithelial cells. HIF-1, which consists of a subunit andβsubunit, is a kind of heterodimer. a subunit is functional, andβsubunit is constitutive. Hypoxia and ischemia can induce the expression of HIF-1 to increase, and the downstream genes can be up-regulated. Up to now, the target genes of HIF-1 refer to many aspects, including glucose transportion, glycolysis, angiogenesis, cell fate, erythrocyte regeneration, extracellular matrix metabolism and fibrosis, etc. There are many studies about the effects of HIF-1 on tissue or cells under hypoxia or ischemia, but the conclusions are not identical. Some study showed that HIF-1 could protect tissue or cells from hypoxic or ischemic injury by inducing protective genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), erythropoietin (EPO) and heme oxygenase (HO-1). HIF-1 could ameliorate the dysfunction of energy metabolism, and it also could promote cells to survive and recover from hypoxic or ischemic injury. However, some studies showed the opposite conclusion. It showed that HIF-1 could promote apoptosis, aggravate renal injury, and develop kidney fibrosis. The effect of HIF-1 on the fate of human proximal tubular epithelial cells provokes much attention gradually. This part was aimed at exploring the effect of HIF-1αon the fate of HK-2 cells under hypoxia by silencing HIF-1αwith RNAi. Materials and methodsHK-2 cells were divided into five groups, including (1) Normoxia group, control normoxic cells were maintained in normal atmosphere; (2) Hypoxia group, medium with 150μM CoCl2 was added for a 24h-incubation after normoxic culture; (3) Transfection group, cells were treated only with DharmaFect 1 transfection reagent, then cells incubated in medium with 150μM CoCl2 for 24h; (4) Negative group, HK-2 cells were transfected with negative control siRNA, then cells incubated in medium with 150μM CoCl2 for 24h; (5) HIF-1αsiRNA group, HK-2 cells were transfected with HIF-1αsiRNA, then cells incubated in medium with 150μM CoCl2 for 24h. MTT assay was used to detect the growth inhibition rates of HK-2 cells. Apoptosis of HK-2 cells was detected by flow cytometry (Annexin/PI), TUNEL assay and Caspase-3 protein quantitation. Cellular necrosis was detected by lactate dehydrogenase (LDH) assay.Results1) The growth inhibition rates of HK-2 cells in hypoxia group, transfection group, negative group and HIF-1αgroup were 6.2±1.7%, 8.1±2.0%,4.6±3.5% and 22.1±1.2%. And the growth inhibition rate of HK-2 cells in HIF-1αgroup was significantly higher than those in hypoxia group, transfection group and negative group (P<0.05). But there is no significant difference among the latter three groups (P>0.05).2) The apoptotic rates of HK-2 cells in the five groups were 0.5±0.1%, 2.8±0.8%,2.3±0.5%,3.0±0.1% and 3.3±1.0% in order by flow cytometry; The apoptotic indices were 2.8±0.6%,6.3±0.6%,5.8±0.6%,6.3±0.6% and 7.1±0.4% in order by TUNEL assay. The apoptotic rate and index in normoxia group were lower than those in the other four groups (P<0.05), but there was no significant difference among the other four groups (P> 0.05). Also, the expression of Caspase-3 protein in normoxia group was significantly lower than those in the other four groups (P<0.05), but there was no significant difference among the other four groups (P>0.05).3) The concentrations of LDH in mediums were 516.7±23.6U/L, 618.5±26.2U/L,618.8±46.8U/L,629.8±52.2U/L and 822.6±62.3U/L in order. The concentration of LDH in HIF-1αsiRNA group was significantly higher than those in the other four groups (P< 0.05). And The concentration of LDH in normoxia group was significantly lower than those in the other four groups (P<0.05).ConclusionsHypoxia could induce HK-2 cells apoptosis and necrosis. Silencing HIF-1αgene could significantly aggravate growth inhibition and necrosis of HK-2 cells under hypoxia. But silencing HIF-1αgene had no effect on apoptosis of HK-2 cells under hypoxia. It was demonstrated that HIF-1αhad the protective role on human proximal tubular epithelial cells under hypoxia. Background and objectiveHypoxia can mediate the expression of several kinds of genes and gene families in human proximal tubular epithelial cells. As the key transcription factor in hypoxic adaptative responses, HIF-1αcan protect kidney from the ischemic injury by inducing downstream protective genes. At present, about one hundred target genes were discovered to the downstream genes of HIF-1α. Among them, some genes can promote cell survival and recover from injury, such as genes with respect to energy metabolism and angiogenesis. Some studies indicated that VEGF was an important target gene of HIF-1αand could protect cells. VEGF could inhibit apoptosis of peri-tubular capillary endothelium, promote renal tubular epithelial in vitro survival, and promote proliferation of endothelium. Glut is one transmembrane glycoprotein distributing on the cell membranes. Glut is the glucose carrier for transmembrane transportation. Among Glut family, Glut-1 distributes widespread, which is the mainly carrier to transport glucose for mammalian cells. Glut-1 can promote glucose to diffuse into cells and promote energy metabolism. Some studies showed that overexpression of Glut-1 facilitate cells to be tolerant to hypoxia or ischemia and facilitate cells to recover from injury. Also it could make the cells under hypoxia survive. This part was aimed at exploring the possible molecular mechanism of effects of HIF-1αon proximal tubular epithelial cells under hypoxia by detecting the expression of Glut-1 and VEGF mRNA and protein.Materials and methodsHK-2 cells were divided into five groups, including (1) Normoxia group, control normoxic cells were maintained in normal atmosphere; (2) Hypoxia group, medium with 150μM CoCl2 was added for a 24h-incubation after normoxic culture; (3) Transfection group, cells were treated only with DharmaFect 1 transfection reagent, then cells incubated in medium with 150μM CoCl2 for 24h; (4) Negative group, HK-2 cells were transfected with negative control siRNA, then cells incubated in medium with 150μM CoCl2 for 24h; (5) HIF-1αsiRNA group, HK-2 cells were transfected with HIF-1αsiRNA, then cells incubated in medium with 150μM CoCl2 for 24h. Cells were collected from each group, then protein and RNA were extracted. The expressions of HIF-1α, HIF-2α, Glut-1, VEGF andβ-actin mRNA were detected by fluorescent quantitative PCR. The expressions of HIF-1α, HIF-2α, Glut-1, VEGF and tubulin protein were detected by Western blot.Results1) The expressions of HIF-1α, Glut-1 and VEGF mRNA of HK-2 cells in normoxia group and HIF-1αsiRNA group were significantly lower than those in the other three groups (P<0.05). The expression of HIF-2a mRNA of HK-2 cells in normoxia group was significantly lower than those in the other four groups (P<0.05), but there was no significant difference among the other four groups (P>0.05).2) The expressions of HIF-1α, Glut-1 and VEGF protein of HK-2 cells in normoxia group and HIF-1αsiRNA group were significantly lower than those in the other three groups (P<0.05). The expression of HIF-2a protein of HK-2 cells in normoxia group was significantly lower than those in the other four groups (P<0.05), but there was no significant difference among the other four groups (P>0.05).ConclusionsThe expressions of Glut-1 and VEGF in HK-2 cells synchronized with the expression of HIF-1α. The protective role of HIF-la to HK-2 cells under hypoxia might be mediated by the downstream genes Glut-1 and VEGF. However, there might be other mechanisms, so it needs further study in-depth.