| BackgroundProtein molecules on podocytes and slit diaphragm are key components of the glomerular filtration barrier in kidney. Studies have confirmed that podocyte proteins, including nephrin, podocin, synaptopodin and etc, are closely related to proteinuria. The injury of podocytes has been the highlight of the study on interpreting pathogenesis of the primary nephrotic syndrome and cell-specific antiproteinuric therapeutics. In our previous study, renal gene expression profile using gene chip technology was perfomed in children with primary nephrotic syndrome, whose pathologic feature was minimal change disease, MCD, and expression of angiopoietin-like protein 3 (ANGPTL3) in kidney was found obviously increased. And, similar results were achieved in adriamycin-induced nephrotic rats. Moreover, we found that ANGPTL3 can not only influence the adhesion and stability between the podocytes and glomerular basement membrane (GBM), but also induce cytoskeleton rearrangement of the podocytes in vitro. Our results indicated that ANGPTL3 may be an important factor for injury of structure and function of podocyte and slit diaphragm.ANGPTL3 is a member of the angiopoietin family. Its molecular weight is 70KDa (human), and it is mainly synthesized by liver cells and only micro-expressed in normal kidney tissue. Its biological function is mainly to promote angiogenesis and regulate lipid metabolism. The current studies for the ANGPTL3 are mainly focused on this field. ANGPTL3 can lead to elevated blood lipids by inhibiting lipase activity, particularly triglycerides and LDL-based. Intergrin-(33 is the major receptor of ANGPTL3. ANGPTL3 can play biological effects by binding to intergrin-?3. Our previous study also found that ANGPTL3 colocalized with intergrin-?3 in podocytes and can lead to rearrangements of podocytes cytoskeleton through intergrin-(33, which indicated intergrin-(33 is an important downstream receptor of ANGPTL3 in kidney. Whether ANGPTL3 can influence podocytes and lead to proteinuria in vivo has been unclear.The studies in vitro suggested that low expression of ANGPTL3 can protect the integrity of cytoskeleton structure of the podocytes. However, whether it can be validate in vivo still need further study. We intend to observe effects of ANGPTL3 expression on the kidneys through transgenetic animal model. The transgenetic technology via permanent integration of genes in repopulating spermatogonial cells in vivo has shorter period and simple operation process for generating transgenic mice. It was reported that the first generation of transgenic offspring by this way can be obtained after 60 days, and positive rate was more than 60%. Sperm chromosomes of F0 generation can be stably integrated in target genes by more than one year. In this study, the ANGPTL3-knockdown and ANGPTL3-upregulated mice model were establised using transgenetic technology via permanent integration of genes in repopulating spermatogonial cells in vivo, to elucidate the influence of expression of ANGPTL3 on renal structure and function in mice. Meanwhile, the feasibility and character of this transgenetic technology in order to establish a simple and effective platform for transgenetic animal model was evaluated. Furthermore, Adriamycin-induced nephrotic mice model was establised to observe influence of expression of ANGPTL3 on renal structure and function in the pathological condition in mice and initially explored the possible mechanisms. We validated our previous results from in vitro studies and provided a basic research evidence for the pathogenesis of nephrotic syndrome and the development of therapeutic targets. Part I Preparation and identification of transgenic mice with regulated expression of ANGPTL3 and observation of changes in their kidneyObjectives We intended to make ANGPTL3-knockdown and ANGPTL3-upregulated mice model via permanent integration of genes in repopulating spermatogonial cells in vivo. Then, we investigated the influence of expression of ANGPTL3 on renal structure and function in these mice models.Methods (1) Recombinant vectors of pcDNA3.1-ANGPTL3 and pcDNA6.2-GW/EmGFP-ANGPTL3-miRNA were constructed and linearized. Then, both kinds of linearized recombinant vectors 20ul (0.5ug/ul) were injected into testes of 20 mature male Balb/C mice and 10 KM mice, respectively. After electroporation, reset testes and remove the other testes (F0); (2) 2 F0 Balb/C mice (injected by pcDNA6.2-GW/EmGFP-ANGPTL3-miRNA) were sacrificed after 1,3,6 and 12 month respectively to observe expression of green fluorescent protein (GFP) in testes; (3) These electroporated male mice were mated with wild-type females after 35 days and their progenies (F1) were identified by PCR. Furthermore, we devided F1 mice into 3 groups based on identification:ANGPTL3 transgenetic mice (AT), ANGPTL3 RNAi mice (AR) and wild-type mice (WT). We sacrificed 5 mice (age:6 weeks) in every group randomly and measured their weight, serum biochemistry and 24h urine protein excretion. Glumerular structure was observed by electron microscopy. We measured expression of ANGPTL3 in serum, liver and kidney cortex by ELISA, Real-time PCR, Western blot and IHC; (4) TUNEL was used to assess apoptosis in glomerulus. We also measured expression of LXR, integrin-?3, nephrin, podocin, Wilm tumor protein 1 (WT1), vascular endothelium growth factor (VEGF) and synaptopdin in kidney cortex. Results (1) After 1,3 and 6 months, testes of FO mice had transfected spermatogonial cells,, but its trend was toward reduced. After 12 months, there were no GFP expression in testes; (2) We identified a total of 298 F1 mice by PCR, in which the total positive transgenetic mice were 134 (44.9%), AT were 50.0%(66/132) and AR were 40.9%(68/166). Eleven Balb/C (55.0%) and ten KM (100%) FO mice had their offspring; the positive rates of Balb/C and KM Fl mice identified with transgenetic by PCR were 40.0% and 60.3%, respectively; (3) The levels of ANGPTL3 in serum: AT (32.6±2.0ug/ml)>WT (25.2±1.7ug/ml)> AR (21.0±2.2ug/ml) (P<0.05). The mRNA and protein expression of ANGPTL3 in liver and kidney cortex were significant increased in AT than that of AR (all.P<0.05). According to ANGPTL3 content in kidney cortex, up-regulated rate of AT was 65.5% and knockdown rate of AR was 31.2%; (4) Compared with AR and WT, AT had significantly higher triglyceride level in blood and lower body weight (all P<0.05). There were no difference in 24h urine protein excretion, serum cholesterol level, glomerular structure and cell apoptosis among AT, AR and WT. However, expression of integrin-?3, podocin, VEGF and synaptopdin were significantly increased and nephrin was significantly decreased in AT renal cortex than that of AR and WT.Summary We succeed in making transgenetic mice models with the increased and decreased expression of ANGPTL3 in glomeruli using transgenetic technology via permanent integration of genes in repopulating spermatogonial cells in vivo. The positive rate of transgenic generation was more than 45%. Up-regulation 65.5% and knockdown 31.2% of ANGPTL3 did not change renal structure and function in mice, but the former induced the increased expression of integrin-?3, podocin, synaptopdin and VEGF, and the decreased expression of nephrin in renal cortex.Part II Influence of regulation of expression of ANGPTL3 on renal structure and function in Adriamycin-induced nephrotic mice Objectives To observe the influence of regulation of expression of ANGPTL3 on renal structure and function in Adriamycin-induced nephrotic mice and investigate the possible mechanism.Methods We intravenously injected Adriamycin 10.5mg/kg in 7 AT, WT and AR male mice with 6 weeks of age, named as:A-AT, A-WT and A-AR, respectively.24-hour urine was separately collected on 1,2,4 and 6 weeks. Mice were sacrificed after 6 weeks and kidney specimens and serum samples were collected; (2) the detected indicators and methods were the same as part II.Results (1) After adriamycin injection,24-hour urine protein excretion of A-AT, A-WT and A-AR all began to increase at 2 weeks, and in which A-AR was lower than A-WT at 2 weeks and A-AT was significantly higher than that of A-AR at 2 weeks and 6 weeks (P=0.026 and 0.035, respectively); Sixth weeks later, serum triglyceride levels of A-AT, A-WT and A-AR were all significantly higher than that of no-adriamycin-injected control mice, in which A-AT was significantly higher than A-AR (P=0.028). Cholesterol levels of A-AT also was significantly higher than that of AT (P=0.039). Compared with A-AR, A-WT and no-adriamycin-injected control mice, their cholesterol level had no significant difference; (2) Under electron microscope, A-AT, A-WT and A-AR were all found generous foot process fusion in glomerulus, in which A-AT had more extensive fusion and larger number of microvilli than that of A-AR and A-WT; (3) TUNEL showed that A-AT, A-WT and A-AR had more apoptosis in glomerulus than no-adriamycin-injected control mice, in which A-AT was significantly higher than that of A-AR and A-WT; (4) ANGPTL3 levels in serum of A-AT was significantly higher than that of A-AR, A-WT and AT (P=0.038 and 0.011, respectively); compared with A-WT, mRNA expression of ANGPTL3 expression in A-AT renal cortical was increased and A-AR was decreased; (5) compared with A-WT and A-WT, mRNA expression of integrin-?3, synaptopdin and VEGF in A-AT renal cortical were increased, nephrin and WT1 were decreased, and LXR was of no difference. Summary The up-regulation of ANGPTL3 can induce the more severe renal injury and higher levels of triglyceride and cholesterol in blood in Adriamycin-induced nephrotic mice. With Adriamycin injury, the higher expression of ANGPTL3 can lead to the more apoptosis and promote the expression of synaptopdin, VEGF and intergrin-B3, and decreased the expression of nephrin and WT1 in mice glomerulus. There were no differences in renal structure, function and podocytes molecules between AR (ANGPTL3 was knocked down 31.2%) and WT after Adriamycin injury.Conclusion1. Transgenetic technology via permanent integration of genes in repopulating spermatogonial cells in vivo is feasible and effective. The positive rate of transgenic generation was more than 45%. According to ANGPTL3 content in kidney cortex, up-regulated rate of AT was 65.5% and knockdown rate of AR was 31.2%. The simpler the structure of plasmids, the higher the transgenic positive rate of progeny is. Reproductive ability and positive rate of transgenic mice of progeny are related to the different strains of animals:KM mice were higher than that of Balb/C mice.2. The up-regulated expression of ANGPTL3 can cause increased triglycerides level, weight loss and altered expression of other podocyte proteins, including integrin-?3, podocin, synaptopdin, VEGF and nephrin, which may be mediated through its receptor intergrin-?3.3. After Adriamycin-induced injury, the increased ANGPTL3 expression can cause more severe podocyte injury. It maybe is because that the increased expression of ANGPTL3 leads to the higher serum triglyceride and cholesterol levels, glomerular apoptosis and expression of VEGF, intergrin-?3, and decreased expression of nephrin and WT1 in glomeruli.4. After Adriamycin-induced injury, the decreased ANGPTL3 expression can delay the occurrence of proteinuria. There were no differences in renal structure and podocytes molecules between AR and WT after Adriamycin injury, which maybe is because of the lower knockdown rate of ANGPTL3 in AR mice. |
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