High Glucose Induces Apoptosis Via Up-regulates Bim Expression In Proximal Tubule Epithelial Cells

IntroductionDiabetic kidney disease (DKD) is the most common cause of end-stage kidney disease worldwide. Increasing prevalence of diabetes has made the need for useful treatment of DKD critical. Many studies indicated that DKD was mainly associated with glomerulapathy, while our and others’preliminary investigation showed that the apoptosis of proximal tubular cells played an important role in DKD and tubulopathy occurs earlier than glomerulopathy. Intervention of tubulopathy may prevent the development of DKD much easily. However, how the tubulopathy happens or its mechanism of pathway is not clear presently. That is vital for investigating the target for its treatment.Autophagy is a major catabolic pathway involved in degrading and recycling macromolecules and damaged organelles to maintain intracellular homeostasis. Emerging body of evidence also implicates impaired autophagic activity in the pathogenesis of diabetic kidney disease, associated with an increase in p62/SQSTMl, in both proximal and distal tubular cells of both type 1 and type 2 diabetic animals.The functional role of autophagy in the kidneys is also currently under intense investigation. It has been revealed that autophagy has a renoprotective role in several animal models including aging or acute kidney injury. Emerging body of evidence suggests that targeting the autophagic pathway to activate and restore autophagy activity may be renoprotective. Impairment of autophagy activity lead to cellular injury responses including apoptosis, While the relationship between apotosis and autophagy in the pathogenesis of diabetic kidney disease is still limited.The BCL-2 protein family regulates apoptosis through a balanced activity of pro-and antiapoptotic family members. Among BCL-2 family of proteins, Bim is a proapoptotic protein with only one Bcl-2 homology (BH3) domain. In response to death stimuli, Bim is required for the activation of cell death pathways mediated by BAX and BAK. Recently, it was reported that Bim depletion increases autophagosome synthesis in cells and in vivo, and Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites. Bim expression is increased under high glucose, and promotes the apoptosis of retinal pericytes. However, the role of Bim in the development and progression of DKD has not been addressed previously.So the objective of this study is to investigate the switch control of apoptosis and autophagy in HK-2 cells.Steps and MethodsStep 1We cultured HK-2 cells in different concentration of sugar, and test the expression of Bim protein, and cell apoptosis and autophagy.HK-2 cells were divided into two groups:group NG, normal glucose group (D-glucose 5.5mmol/L+24.5mmol/L mannitol) and HG group (30.0mmol/L D- glucose). We detected Bim mRNA expression (using the method of RT-PCR), Bim protein(using Western Blot), Cell apoptosis (using TUNEL method); active-caspase 3(using Western Blot), the ratio of LC3-II/LC3-I(Using Western Blot).Step 2We decreased the expression of Bim protein by way of Transfection of Bim siRNA, up-regulated Bim protein by plasmid transfection method, then analysed HK-2 cell apoptosis and autophagy.NG group and HG group of HK-2 cells were divided into the following four groups:siNC group (transfected with no significance of siRNA), sibim group (transfected with Bim siRNA), sibim+plasmid group (transfected with Bim plasmid and siRNA-resistant plasmid), plasmid group (transfected with Bim over-expression plasmid). We detected Bim protein(using Western Blot), Cell apoptosis (using TUNEL method), active-caspase 3 (using Western Blot), the ratio of LC3-Ⅱ/LC3-Ⅰ(Using Western Blot).Step 3In the premise of the inhibition of the expression of Bim, we down regulate the autophagy activity and analysed the apoptosis of HK-2 cells.The sibim subgroup of HG group were divided into two groups:control group (only with DMSO) and the experimental group (3-MA group 3-MA). We detected Cell apoptosis (using TUNEL method) and active-caspase 3(using Western Blot).Quantitative Realtime-PCR(RT-PCR)Synthesis of Bim induced by HG was measured by quantitative RT-PCR. HK-2 cells were stimulated with 30mM glucose for the indicated times. Afterward, cells were washed with PBS followed by RNA extraction. Total RNA was extracted with TRNzol-A+RNA isolation reagent according to the manufacturer’s instructions. Reverse transcription was performed with 1μg of total RNA and RevertAid First Strand cDNA Synthesis Kit. Primer sequences used were as follows:Bim forward primer:5’-ATT ACC AAG CAG CCG AAG AC -3’and reverse primer:5’- TCC GCA AAG AAC CTG TCA AT-3’; Primers specific for β-actin were used as a control (forward:5’-TGA CGT GGA CAT CCG CAA AG -3’and reverse:5’- CTG GAA GGT GGA CAG CGA GGT-3’). Quantitative RT-PCR was performed in a cycler using SYBR green. The cycling condition was as follows:initial denaturation at 95℃ for 10 min, followed by 40 cycles consisting of denaturation at 95℃ for 15 s and amplification at 60℃ for 30 s. The average CT value was calculated from triplicates of each sample. The threshold cycle for each sample was chosen from the linear range and converted to a starting quantity by interpolation from a standard curve run on the same plate for each set of primers. The Bim mRNA levels were normalized for each well to the P-actin mRNA levels using the 2-ΔΔCT method. Each experiment was repeated three times.Plasmid constructs and siRNAKnock-down of Bim expression was performed by RNA interference using specific siRNA oligonucleotides. The target sequences for Bim was as follow:CGG AGA CGA GTT TAA CGC TTA. For siRNA reverse transfection, cell suspension was transfected with siRNA at a final concentration of 100 nM using Lipofectamine 2000 transfection reagents according to the manufacturer’s instruction. Cells were kept for 48 h before experiments, and the efficiency of siRNAs was evaluated by Western blottingusing specific antibodies against Bim. To generate the plasmid encoding Bim siRNA-resistant wild type of Bim-EL, pCAGIG-siRNAR-BimEL (Bim-Res) mutants were made by site-directed mutagenesis (without amino acids change) corresponding to the sequence of Bim siRNA, using the human Bim cDNA as a template, and the PCR product was subcloned into pCAGIG expression vector (addgene) using the Xbal and BamHI sites. The construct were confirmed by DNA sequencing to exclude potential PCR-introduced mutations.Assessment of apoptosisFor TUNEL/DAPI analysis, HK-2 cells were plated on poly-D-lysine-coated cover glasses in 6-well plates and maintained in culture with 1640 medium supplemented with 10% FBS,0.5 mM glutamine, and 100 units/ml penicillin/streptomycin. cells were transfected with indicated siRNA by Lipofectamine 2000 reagent and experiments were conducted 2 d after transfection. HK2 cells were treated with different drugs as indicated. After 48 h in culture, the cell death was assessed using the In Situ Cell Death Detection Kit (Roche) and DAPI staining (Sigma-Aldrich). Cells were visualized with a fluorescence microscope. For each condition, images of 12 preselected fields (totaling 1500-2000 cells) were captured in a nonbiased manner from each of three separate assays. The fraction of dead cells was determined by dividing the number of nuclei exhibiting TUNEL staining by the number of DAPI-positive nuclei. The experiment was repeated three times.Western BlotProtein level was measured by Western blot. Briefly, Cells were washed twice with ice-cold PBS and harvested in TNE lysis buffer (10 mM Tris, pH 8.0,150mM NaCl,1mM EDTA,1% NP-40,10% glycerol with protease inhibitors) as above supplemented with protease inhibitor cocktail Ⅱ (Sigma). The protein concentration was determined using BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA) with BSA as a standard, equal amounts of protein were separated by SDS-PAGE electrophoresis, and transferred to PVDF membrane (Bio-Rad Inc.). Then the membranes were incubated in blocking buffer (0.2 mM Tris,137 mM NaCl,5% no-fat milk, and 0.1% Tween-20) for one hour and then probed at 4℃ overnight with indicated antibody. The membranes were rinsed with TBST buffer (0.1% Tween 20, 0.2 mM Tris, and 137 mM NaCl) and incubated with HRP-conjugated secondary antibody (1:5000) for one hour at room temperature, followed by chemiluminescent detection. For estimation of relative amounts of proteins, the exposed films of the immunoblots were scanned and band intensities were measured with Image J software (NIH).Statistical analysisAll statistical analysis was performed using SPSS software. Student’s t test was used to assess significance for data within two groups. Multiple statistic comparisons were analyzed using one-way ANOVA followed by post hoc tests. Data are presented as mean ± SEM and significance was set at p<0.05.ResultsApoptosis is induced in response to high glucose (HG) in HK-2 cells.To examine the effect of high glucose on HK-2 cells, the cells were treated with glucose at different concentrations for varying time periods (Oh,24h,48h,72h). The effect of high glucose on apoptosis in HK-2 cells was detected by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) and western blot of active-Caspase3 (active-Casp3). In immunofluorescence staining for TUNEL (apoptotic cells), an increase in TUNEL-positive cells in HG-treated (30mM glucose) HK-2 cells was observed compared with control group (under normal glucose,5.5mM) in a time dependent manner. Also, High-glucose (30mM glucose) treatment induced significant time-dependent increase of active-Caspase 3.Autophagy has been thought to be a survival mechanism in response to various starvation and stress conditions. To investigate the role of HG on autophagy control of HK2 cells, we analyzed the protein expression of LC3 (also known as ATG8, its processing is believed to be a marker of autophagy) with a western blot assay. The presence of endogenous levels of LC3-II here most likely represents the basal level of autophagy that exists in all cells. In contrast, western blots of LC3 autophagy proteins indicated that the level of autophagy in HK-2 cells was not significantly affected.Bim expression is increased in response to high glucose (HG) in HK-2 cells.Bim is a proapoptotic member of the Bcl-2 family with significant role in the activation of cell death pathways. Three major isoforms [the extra long form of Bim (BimEL), the long isoforms of Bim (BimL) and the short isoform of Bim (BimS)] are produced by alternative splicing of 6 exons. These isoforms differ in size and have different apoptotic activity. BimEL is the main isoform that to be a key effecter molecular in apoptois regulation. Western blotting was performed to determine the involvement of BimEL in the regulation of HG-induced appotosis in renal proximal tubule cells. HG treatment caused an increase in BimEL protein expression. While the normal glucose treatment do not get detectable BimEL expression increase. In addition, another isoform of Bim protein—BimL, was also found to have increase expression upon HG treatment. Even though, BimEL is generally the prominent isoform when Bim level is up regulated by HG stimuli. To determine whether upregulation of Bim expression was a result of enhanced transcription, Bim mRNA was also analyzed by realtime RT-PCR. Increased levels of Bim mRNA were also detected.BIM knock-down cells by siRNA are protected from HG-mediated apoptosis, while promotes its autophagy.To investigate the function of Bim in HG-induced apopotosis, we knocked down endogenous Bim expression by siRNA. Bim siRNA transfection reduced about 21% endogenous Bim protein level to assessed by western blot analyses (n=3,*p< 0.05, Student’s t-test). Apoptosis of Bim-knocking down cells was not affected by HG compared with nonsense (NC) (nonsense) siRNA group, in which HG treatment could still increase the apoptosis rate.To prove that this phenotype is due to reduced Bim expression, but any unknown off-target effect, we created the siRNA-resistant Bim replacement vector that expresses BimEL protein (Bim-res) that contains silent mutations in the mRNA region that is targeted by the siRNA. Transient transfection experiments demonstrated that endogenous Bim was sensitive to the siRNA, but Bim-res encoded by the cDNA containing mismatches was abundantly expressed in the presence of Bim-siRNA. Importantly, the responding to HG upon apoptosis in Bim-siRNA transfection group was rescued by expression of the Bim-res. The result indicated that upregulated Bim expression was responsible for the apoptosis caused by HG. Recently, it has been reported that Bim works as a molecular link between autophagy and apoptosis, and Bim could negatively regulate autophagy. Interestingly, we found when Bim protein was knockdown by siRNA, the HK-2 cells showed a higher LC3-Ⅱ level even under normal glucose condition. What is more, when BimEL is overexpressed, the cell showed increased apoptosis level under normal glucose condition. All of those indicated that the level of Bim played very important role in HK-2 cells fate-destination; it is a sufficient regulator of HK-2 cells to switch them into autophagy or apoptosis. To investigate whether Bim is involved in autophagy regulation of HK-2 cells under HG conditions, we analyzed the protein expression level of LC3 with a western blot assay. Bim knocking-down resulted in a remarkable increase in the level of LC3-Ⅱ(LC3-Ⅱ to LC3-Ⅰ ratio), and what is more, in Bim knocking down condition, HG could induce a higher levels of LC3-Ⅱ. And the increase could be blocked by expression of siRNA-resistant Bim. That is to say, firstly, Bim down-regulation could induce autophagy in HK-2 cells; secondly, with Bim-depletion, HG could trigger HK2 cell autophagy but not apoptosis.Autophagy inhibitor 3-MA worsens the glucotoxicity in Bim knockdown cells by re-trigger apoptosis.To further investigate whether there is a switch between autophagy and apoptosis in HK-2 cells responding to HG, we treated Bim-knocking down cells with 3-methyladenine (3-MA), an inhibitor of autophagy, which could inbibit autophagosome formation. To our surprise, we found that the autophagy inhibitor 3-MA worsened the glucotoxicity in Bim knockdown cells by re-trigger apoptosis. The results suggest that when autophagy is inhibited, there is still a Bim-independent apoptosis pathway which might be active in response to HG condition.Our results provide several new insights into the mechanism of HG induced glucotoxicity injury in HK-2 cells. First, we found that high glucose (HG) could induce apoptosis by upregulate the expression of Bim protein in HK-2 cells without changing the basal autophagy ratio. Bim is essential for apoptosis induced by certain stimuli. Further, Bim is likely to have cytotoxic effects by inhibiting autophagy, and autophagy and apoptosis can be coregulated by Bim. Our result suggests that upon HG stimuli, Bim expression is upregulated, and this may initiate Bax/Bak-mediated mitochondria-dependent apoptosis. While on the other hand, the increased-expression of Bim could inhibit autophagy by interacting with Beclin 1, an autophagy regulator. As indicated, autophagy activity is protective for DKD, so the up-regulation of Bim make cells could not protect themselves by autophagy from glucotoxicity.Second, we found knocking down Bim by siRNA, could protect HK-2 cells from HG-mediated apoptosis, and promote its autophagy. What is more, when BimEL is overexpressed, the cell showed increased apoptosis level under normal glucose condition. The results indicated that Bim is a critical regulator of HK-2 cells fate decision upon HG stimuli; it can switch cells between apoptosis and autophagy. Given the impairment of autophagy is implicated in the pathogenesis of DKD; the deregulation of Bim might be one important cause. And by down-regulating Bim, cells could restore autophagy activity and protect from themselves from glucotoxicity.Third, we found autophagy inhibitor 3-MA could re-trigger apoptosis in Bim knockdown cells. This result suggested that there is still Bim-independent apoptosis pathway in HK-2 cells, and normally, it could by shut-up by autophagy.ConclusionsIn all, we showed that high glucose had a direct effect on apoptosis in HK-2 cells. Our study is the first study to provide evidences that high glucose induces apoptosis via up-regulates Bim expression in proximal tubule epithelial cells HK-2, and the upregulation of Bim would further impair autophagy activity of cells. Downregulation of Bim could restore cells the autophagy activity and protect from themselves from apoptosis. Also we found autophagy inhibitor 3-MA could re-trigger apoptosis in Bim knockdown cells. Not only might these findings provide insights into the mechanistic link between apoptosis and autophagy in HG condition, but also they might have implications of the new target for the prevention or treatment of diabetic kidney disease.Innovations:First, the first study to provide evidences that high glucose induces apoptosis via up-regulates Bim expression in proximal tubule epithelial cells HK-2.Second, downregulation of Bim by siRNA could restore cells the autophagy activity and protect from themselves from apoptosis.Third, autophagy inhibitor 3-MA could re-trigger apoptosis in Bim knockdown cells.