Leflunomide Inhibited Smad Pathway Of High Glucose Induced Apoptosis In Podocytes

BackgroundDiabetic nephropathy is the most common and serious chronic complications. Although the pathogenesis of diabetic nephropathy has not yet entirely clear, but a large number of studies have shown that podocyte injury and diabetic nephropathy is closely related to the formation and development. TGF-?/smad signaling pathway in the pathogenesis of diabetic nephropathy plays an important role, research shows that: High glucose can activate TGF-? system, the smads protein phosphorylation-mediated TGF-? signaling from the cell membrane into the nucleus to induce enough apoptosis, promoting glomerulosclerosis. Leflunomide as a novel immunosuppressive agent, widely used in recent years, the treatment of kidney disease. Studies have reported that in vitro mesangial cells, leflunomide partially blocked the activation of TGF-β/Smad signaling pathway, and leflunomide in human glomerular podocyte is available through the pathway had not yet play a role in further studies. In this study, high glucose stimulated through adequate smad pathway of apoptosis and to study the relationship between the leflunomide can inhibit the smad signal transduction pathway to inhibit podocyte apoptosis, for the treatment of diabetic nephropathy and to provide more theoretical experimental basis.Methords1. Subcultured after human glomerular podocytes purposes under the different experimental groups:⑴normal glucose control group: D-glucose, 10mmol / L;⑵hypertonic control group: 20 mmol / L mannitol + D-glucose 10mmol / L;⑶high glucose group: D-glucose, 30mmol / L;⑷leflunomide treatment group: the final concentration of 20μmol / L of leflunomide + D-glucose, 30mmol / L;⑸inhibitor Staurosporine Group: final concentration To 5umol / L of Staurosporine + D-glucose, 30mmol / L. All experiments were 5-10 with a foot cell generation.2. Using western blotting method to detect the smad2 / 3 protein and p-smad2 / 3 protein expression was detected by flow cytometry at different time points podocyte apoptosis.3. Observe the effect of high glucose p-smad2 / 3 protein expression and apoptosis rate of foot and observe the p-smad2 / 3 inhibitor Staurosporine and active leflunomide metabolite A771726 p on the high glucose -smad2 / 3 protein expression and podocyte apoptosis. Results⑴Between the groups total smad2 / 3 is no significant difference in protein expression. Control group, normal glucose and hypertonic podocyte only trace p-smad2 / 3 protein expression, high glucose group at 24h, podocyte smad2 / 3 protein phosphorylation, begins to activate, 72h peak, 72h protein expression began to decline after . 72h in the expression of high glucose peak point, the leflunomide group and p-smad2 / 3 inhibitor Staurosporine group of p-smad2 / 3 expression was significantly reduced in both leflunomide group, p-smad2 / 3 protein compared with high glucose was significantly (P <0.05), its role was a certain time-dependent relationship.⑵the cells were cultured 24h, 48h, 72h, 96h after the detection of apoptosis by flow cytometry. Compared with the control group, each group the percentage of apoptosis 24h stimulation did not change significantly. after 48h with high glucose, apoptosis significantly increased the percentage of the beginning (P <0.05), and increased with time. Compared with the high glucose group, leflunomide group and the inhibitor group was significantly decreased percentage of apoptosis, in which leflunomide group of high glucose decreased (24±2.3)% (P <0.05). In conclusion, high glucose stimulated podocytes p-smad2 / 3 protein increased, and increased podocyte apoptosis; and high glucose group, after adding inhibitors and leflunomide, podocytes p-smad2 / 3 protein were reduced, and the reduction of podocyte apoptosis.ConclusionLeflunomide is partially blocked high glucose-induced podocyte smad pathway of apoptosis, reduce the degree of hardening of the kidney, which may be associated with reduced expression of TGF-βprevented thereby preventing activation of smad protein induced caspase family apoptosis, thereby delaying the progress of diabetic nephropathy.