The Role Of ANGPTL4 In Diabetic Nephropathy And Its Mechanism

Objective Diabetic nephropathy(DN) is characterized as increased microalbuminuria and progressive declining in glomerular filtration rate(GFR). Studies indicated that the control of the excretion of albuminuria is closely linked to renal prognosis, and podocytes injury play a vital role in the progress of albuminuria. Therefore, it is of special significance to research the podocytes as the research object for diabetic nephropathy. Angiopoietin- like protein 4( ANGPTL4) is a secreted protein which is affiliated to the angiogenesis regulation protein superfamily, and it plays an important role in the oxidative metabolism, angiogenesis, cell differentiation and tumor metastasis. Recent research has discovered that podocytes-secreted glomerular ANGPTL4 is upregulated in certain podocytes diseases. Furthermore, AN GPTL4 transgenic rats exhibit podocytes injury and high levels of albuminuria, which indicates that ANGPTL4 induces albuminuria in organisms with certain types of glomerulonephropathy. However, the detailed effect of ANGPTL4 on podocytes injury and further mechanism leading to a lbuminuria specific is still unclear. In this study, through animal experiments and cell-based experiments of ANGPTL4 in the condition of high-glucose, we try to explore roles and possible mechanism of ANGPTL4 in the regulation of podocytes cytoskeleton and function, thus to make possibility for ANGPTL4 targeted therapy.Methods Animal research STZ induced type 2 diabetic rats model. Select normal control rats(n = 10), DM rats(n=5) at 12 weeks, detect the levels of serum glucose, 24 hours urine albumin, observe the histological changes of glomerular, location of AN GPTL4 and detect ANGPTL4 and its related protein expression changes. Cell research(1) Human podocytes were divided into two groups: normal glucose group(5.6 mmol / L glucose), high glucose group(30 mmol / L glucose), to continue observed for 48 h, application of phalloidin to detect cytoskeleton, observe the effect of high glucose concentration to cytoskeleton in podocytes. Collect protein and RNA, then detect their expression.(2) Podocytes were cultured in 30mmol/L glucose medium, to continue observed for 72 h and detect the change of AN GPTL4 protein expression at 6h, 12 h, 24 h, 48 h and 72 h.(3)Apply human recombined ANGPTL4 protein 0, 10, 50, 100 and 1000 ng/ml into culture medium, select appropriate concentration of human recombined ANGPTL4 protein by PI/Hoechst and active caspase 3/pro-caspase 3 ratios. After cell synchronization, podocytes were treated by Ig G control, anti- ANGPTL4 neutralizing antibody, anti- integrinα3β1 neutralizing antibody for 2 h, then cultured by high glucose or human recombinant ANGPTL4 protein 100 ng/ml. Western blotting was used to detect ANGPTL4, active caspase 3 and pro-caspase 3 protein expression changes after treatment. Cytoskeleton was observed by rhodamine phalloidin.Results Animal research(1) As the data shows in SD rats, blood glucose and 24 hours urinary albumin in diabetic group are significantly higher than control group(P <0.001), kidney weight / body weight was also higher than that in the control group, the difference was statistically significant(P <0.001).(2) HE staining results showed that the glomerular volume of rats with type 2 diabetic nephropathy increased and mesangial cell and matrix proliferated. Immunohistochemistry showed glomerular fibronectin(FN) of the diabetic group was significantly higher than the control group, the difference was statistically significant(P <0.001). Electron microscopy showed that diabetes group rat glomerular foot process was more disorder, with widening and effacement of the podocytes foot process. Compared with normal control group rats, glomerular basement membrane was significantly thickened(P<0.05).(3) Immunofluorescence showed WT-1 and Synaptopodin in diabetic glomerular was significantly less than normal control group.(4) Immunofluorescence showed ANGPTL4 was expressed in glomerular podocytes and increased in diabetic rats(P<0.05). Western blotting analysis showed that compared with control group, ANGPTL4 protein expression significantly increased(P< 0.05), while FAK, MAPK were activated in kidney of diabetic rats. Cell research(1) After 48 h cultured in 30mmol/L glucose medium, ANGPTL4 m RNA expression of human podocytes were increased(P<0.05) while AN GPTL4 protein expression were decreased(P<0.05), in addition, the cytoskeleton of podocytes were visibly damaged with increased apoptosis.(2) Podocytes were cultured in 30mmol/L glucose medium, to continue observed for 72 h. In 24 hours, ANGPTL4 protein expression of podocytes were increased as time on(P<0.05). After 24 h, ANGPTL4 protein detected in podocytes was significantly decreased(P<0.05) while detected secreted AN GPTL4 was increased(P<0.05).(3) The apoptosis percentage of podocytes increased as the concentration of human recombined ANGPTL4 protein increased(P<0.05). Human recombined ANGPTL4 100ng/ml protein can lead podocytes to cytoskeleton injury and apoptosis(expressing as active caspase 3/pro-caspase 3 ratio increased)(P<0.05). Anti- ANGPTL4 neutralizing antibody would reduce cytoskeleton injury and decrease active caspase 3/pro-caspase 3 ratios(P<0.05) induced by 30mmol/L glucose, while anti- integrinα3β1 neutralizing antibody show the similar effect on podocytes cultured with human recombined ANGPTL4(P<0.05).Conclusion(1) STZ induced diabetic rats appeared podocytes injury and ANGPTL4 upregulated, while podocytes cultured in high glucose medium appeared ANGPTL4 upregulated, cytoskeleton injury and increased apoptosis.(2) High glucose damaged podocytes cytoskeleton and induced apoptosis by ANGPTL4.(3) ANGPTL4 effect on podocytes by integrin. Anti-ANGPTL4 and anti- integrinα3β1 neutralizing antibody can antagonize the effect of high glucose on podocytes, providing a new target for diabetic nephropathy therapy.