Effect Of SREBPs On Lipid Accumulation In The Kidney Of Diabetic Rats

Objectives: Diabetic nephropathy is an important chronic complication of diabetes mellitus. Many factors are involved in the pathogenesis of this disease, for example, hyperglycemia, growth factors, inflammatory cytokines, oxidative stress and advanced glycation end products. Abnormal lipid accumulation has been testified to injury the kidney of diabetic patients and animal models.Sterol regulatory element-binding proteins (SREBPs) are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate triglyceride and cholesterol metabolism. Currently, there are three isoforms of SREBP that have been characterized, SREBP-1a, SREBP-1c and SREBP-2. Some renal injuries have been reported to involve in triglyceride accumulation and high expression of SREBP-1. The mechanism of lipid abnormal metabolism and the relationship between SREBP-1 expression and lipid deposit in diabetic kidney are still unclear.RNA interference (RNAi) is a specific and powerful tool that is used to silence gene expression in the study of gene functions and in the development of therapeutic strategies. Sustained RNAi can be achieved by constructing recombinant plasmid that synthesizes short-hairpin RNA (shRNA). A shRNA becomes siRNA, the final and functional product, through enzyme digestion of Dicer.In the present study, we investigated triglyceride accumulation and SREBP-1 expression using STZ-induced diabetic rats and HKC cells to clarify lipid deposit location and the correlation between triglyceride variation and SREBP-1 expression. Furthermore, we constructed two specific SREBP-1 shRNA plasmids and transfected them into HKC cells to silence SREBP-1 expression, which is the basis of further functional study.Methods1. The expression of SREBP-1 and SREBP-2 in the kidney of STZ-induced diabetic ratsSixty 5-week male Wistar rats were randomly divided into 3 groups: normal control group (n=25), diabetic group (n=25) and insulin treated diabetic group (n=10). The diabetic rats were injected intraperitoneally with 60 mg/kg body weight STZ in 50 mM sodium citrate solution (pH 4.5), and rats in the control group were injected with 50 mM sodium citrate solution. Normal control group and diabetic group rats were raised for 1 week, 2 weeks, 4 weeks and 8 weeks respectively until they were sacrificed. Insulin treated diabetic group were fed for 2 weeks. Blood samples were collected through abdominal aorta, and renal cortex was removed, cleaned, washed and immediately saved in buffered neutral formalin for histopathological sections, or saved in liquid nitrogen for frozen sections and extraction of protein.2. The expression of specific SREBP-1 recombinant plasmid in HKC cellsHKC Cells were maintained at 37℃in a humidified atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium containing penicillin/streptomycin (100 U/ml and 100μg/ml, respectively) and 10% fetal bovine serum (FBS), with a normal D-glucose concentration of 5mM. Prior to use, cells at 80% confluence were incubated in serum-free medium for 24 hours. In order to examine the direct effect of SREBP-1 on lipid droplets formation, HKC cells were randomly divided into 3 groups: untransfected control group, pcDNA3.1 vector control group and pcDNA3.1-SC1 group. Transient transfection of HKC cells was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Six hours after transfection, the medium was replaced by normal DMEM medium with 10% FBS. The HKC cells were collected and analyzed for lipid droplets and SREBP-1 and FAS expression 48 hours after transfection.3. Effect of high glucose on SREBP-1 expression and lipid droplet formation in HKC cellsTo investigate the effects of glucose on protein and mRNA expression of SREBP-1, serum-deprived HKC cells were cultured in DMEM containing 5mM glucose (normal glucose) or 30mM glucose (high glucose) for 12 h, 24 h, 48 h and 72 h. At the end of each time point lipid droplets and SREBP-1 expression were examined by Oil Red O staining, Western blot and RT-PCR. All experiments were repeated three times.4. Construction of eukaryotic vector expressing shRNA of human SREBP-1 gene and its inhibitory effect on HKC cellsTwo targeted sequences aimed at SREBP-1 gene were designed and the single-stranded DNA oligonucleotides for shRNA designated SREBP1-1 and SREBP1-2 were chemically synthesized and annealed to form double-stranded DNA. After pGenesil-1 vector was digested with restriction enzyme of BamH1 and Hind3 at 37℃for 3 hours, the double-stranded DNA was cloned downstream of U6 promoter in the expression vector by T4 ligase at 16℃overnight. The recombinant plasmids were amplified and chosen by transfected into E. coil strain DH5 alpha and extracted using the kit from Promega Co. If the plasmid was constructed successfully, one 400bp segment will be produced by digestion with Sal1 because of an enzyme site of Sal1 designed into the single-stranded DNA oligonucleotides. Finally, two constructs that were designated pGenesil-1-SREBP1-1 and pGenesil-1-SREBP1-2 respectively were sequence-verified before use. Meanwhile, a vector containing the non-specific siRNA was designed as negative control (HK).For RNAi research, HKC cells were divided into 4 groups: normal control group, HK control group, pGenesil-1-SREBP1-1 group and pGenesil-1-SREBP1-2 group. Transient transfection of HKC cells was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Six hours after transfection, the medium was replaced by high glucose DMEM medium (30mM glucose) with 10% FBS, and cells were incubated for an additional 48 h. Fluorescent microscopy was used to examine GFP expression and then the cells were collected for the extraction of protein and total RNA.Results1. Expression of SREBP-1 and SREBP-2 in the kidney of type 1 diabetic ratsCompared with normal control group rats, TG content of renal cortex was elevated markedly in the diabetic rats at all time points. No altered cholesterol content was found in the diabetic kidney. Red lipid droplets appeared in the renal proximal tubule epithelium, not glomerulus. From the results of immunohistochemistry, SREBP-1 was located in the cytoplasm and increased in the renal proximal tubular cells of diabetic rats. By semi-quantitative image analysis, SREBP-1 in the kidney of diabetic rats revealed higher level than that of control rats at all time points. The renal expression of SREBP-1 in diabetic rats peaked at week 2 with a final mean IOD of immunohistochemitry images of 80019.17±5698.50 that is 2.43 times higher than that of normal control rats. The protein of SREBP-1 has two kinds of forms: precursor segment and mature segment. From Western blot analysis, the precursor and mature segments both showed higher expression in the kidney of diabetic rats at all time points. At week 2, the diabetic rats showed highest expression of precursor and mature segments, and bands IOD ratio (SREBP-1/β-actin) were 0.673±0.027 and 0.670±0.028 respectively. The mRNA of SREBP-1 was found increased in the renal cortex of diabetic rats and was located in the cytoplasm of renal proximal tubular cells by in situ hybridization. On the contrary, renal tubular cells of control normal group rats showed only mild staining. After the 2 week injection of insulin, the increased expression of SREBP-1 mRNA and protein was markedly inhibited.No difference was found in SREBP-2 expression in the kidney between normal rats and diabetic rats by immunohistochemistry and Western blot.2. Effect of SREBP-1 expression on lipid droplets formation in HKCcellsIn order to confirm the effect of SREBP-1 gene on lipid metabolism in renal proximal tubule epithelial cells, we carried out in vitro research by transfection of SREBP-1 plasmid into HKC cells. From the results of RT-PCR, IOD ratio of SREBP-1 mRNA bands normalized by internal control GAPDH was 0.20±0.09 for control group, 0.30±0.07 for pcDNA3.1 group, 1.24±0.10 for pcDNA3.1-SC1 group. Compared with normal control group, the SREBP-1 mRNA expression was increased by 6.158 times in pcDNA3.1-SC1 transfection group. From the results of Western blot normalized byβ-actin, IOD ratio of SREBP-1 protein was 0.93±0.21 for normal control group, 1.16±0.25 for pcDNA3.1 group, 3.09±0.25 for pcDNA3.1-SC1 group. Compared with normal control group, the protein expression was up-regulated by transfection of pcDNA3.1-SC1 plasmid. From the Oil Red O staining, over-expression of SREBP-1 in HKC cells resulted in lipid droplet formation which was not seen in normal control HKC cells. To investigate whether FAS (fatty acid synthase) was involved in the regulation of SREBP-1 on triglyceride accumulation, we detected the FAS mRNA expression in HKC cells transfected with SREBP-1 specific plasmid by RT-PCR, and found that FAS mRNA expression level in HKC cells transfected with pcDNA3.1-SC1 was significantly higher than that in untransfected HKC cells.3. High glucose modulates SREBP-1 proteins expression in HKC cellsAccording to the results of in vivo experiment, we presumed that over-expression of SREBP-1 in renal proximal tubular cells was induced by high glucose condition. In order to confirm this hypothesis, HKC cells were stimulated with high glucose and detected the expression of SREBP-1 gene. HKC cells were cultured in media containing 5mM glucose (normal glucose levels) or 30mM glucose (high glucose levels) respectively for 12, 24, 48 and 72 hours to study the time-course effects of glucose on lipid metabolism. Accumulation of neutral lipids in cytoplasm was seen by Oil-Red O staining. We observed that lipid droplets started to appear 48 hours after high glucose stimulation. High glucose incubation resulted in significant up-regulation of SREBP-1 protein in cytoplasm and nuclei. . In comparison with normal glucose group, high glucose group showed much larger expression of SREBP-1 (p<0.01) at the corresponding time point. From the immunoblotting studies, precursor and mature forms of SREBP-1 protein were significantly higher in HKC cells of high glucose group than that of controls at all time points. The intensity of SREBP-1 precursor bands in high glucose group was 4.9 times higher than that of normal glucose group. Furthermore, the mature form expression in high glucose group was 6.5 times higher than that of normal control group at 48 h. We also performed experiments to test the existence of a time-dependent manner of glucose-induced changes in the precursor and nuclear forms of SREBP-1. The precursor content of SREBP-1 increased gradually after high glucose addition. This increase reached the maximum after 48 h and the density ratio of SREBP-1 precursor / ?-actin was 2.334. The changes in the mature form were parallel to the changes in precursor form of SREBP-1 in the HKC cells treated with high glucose. The density ratio of SREBP-1 mature form / ?-actin was 1.101 at 48 h that was also maximal. Increased expression of SREBP-1 mRNA was further confirmed by RT-PCR. We first assessed the effects of high glucose on SREBP-1 mRNA expression. Being consistent with the results observed at the protein level, a significant increase in SREBP-1 mRNA expression under high glucose condition at all time points was found by semi-quantitative RT-PCR analysis.4. Successful construction of SREBP-1 shRNA plasmidWe performed SREBP-1gene silencing research with the method of vector-mediated RNAi in HKC cells. Restriction endonuclease digestion of two recombinant shRNA plasmids by SalⅠgot a 400bp DNA fragment respectively. Besides, the sequencing-identification also confirmed that the target sequence had inserted into the predicted site precisely and the recombining plasmids were successfully constructed. 48 hours after transfection, GFP expression was observed though the comparative analysis of bright field image and corresponding fluorescent image and about 40% HKC cells were transfected successfully. Meanwhile, the SREBP-1 mRNA and protein were examined using the methods of RT-PCR and Western blot. Compared with normal control group, the SREBP-1 mRNA was significantly inhibited by24.11%, 36.15%, respectively, in recombinant plasmid groups. IOD ratio of precursor and mature segment of SREBP-1 protein bands in recombinant plasmid groups (SREBP-1/β-actin) were significantly lower than that of normal control group and HK negative control group.Conclusions①In summary, our results indicate high glucose may upregulate SREBP-1 expression at both mRNA and protein levels in renal proximal tubular epithelium, and cause renal triglyceride accumulation in diabetic kidney.②Increasing SREBP-1 by transfection of specific pcDNA3.1-SC1 plays an important role in lipid droplet formation in HKC cells by up-regulating FAS mRNA expression.③The up-regulative effect of high glucose on SREBP-1 mRNA and protein in HKC cells were confirmed by in vitro experiment.④The application of vector-mediated RNAi could significantly inhibit the expression of SREBP-1 in HKC cells, which is a promising tool for future research into the mechanisms of renal lipid accumulation in vivo.