| Background and objectPrimary nephrotic syndrome (PNS) is a common glomerular disease among chidren, accounting for about 90% of children nephrotic syndrome (NS) Hyperlipidemia and high lipoprotein hematic disease is one of the most important pathophysiological characteristics of PNS. Studies show that the possible mechanisms of chronic kidney disease are maybe oxidative stress, endoplasmic reticulum stress, inflammation and so on.Excess lipid depositing in kidney will cause renal dysfunction. But, the mechanism of lipid-induced renal damaging is not clear yet.In recent years, studies have shown that oxidized low density lipoprotein (ox-LDL) could injury renal mesangial cells, endothelial cells, and so as sertoli cells. In a lot of glomerular diseases, such as diabetic nephropathy (DN), minimal change disease nephrotic syndrome (MCN), focal segmental glomerular sclerosis (FSGS) and membranous nephropathy (MN), sertoli cell is the main damaged part and early damage index. Sertoli cells are highly differentiated renal inherent cells, which cover the surface of glomerular basement membrane (GBM) through the foot processes. When the podocyte was damaged, it will result in the generation of proteinuria. The mechanism that ox-LDL causes podocyte damaging is unclear.CXCL16, a kind of chemokines, which was found in recent years. It converges scavenger receptor function and inflammatory chemokine functions in one. It is unique in CXC chemokines family, which plays an important role in many aspects, such as the immune reaction and inflammation. Early research about CXCL16 focused on atherosclerosis, it includes membrane bound and soluble CXCL16. Recently, study found that CXCL16 participated in a variety of kidney disease and its development. Early studies of this issue showed in the process of adriamycin nephropathy development, CXCL16 may as a scavenger receptor of ox-LDL, helping cells absorb ox-LDL’6’. However, the machanism how ox-LDL induces podocytes damage through CXCL16 is not clear yet in PNS.Cell detachment and apoptosis are the two main reasons which are known for reducing the number of sertoli cells. Integrin-β1 is an important adhesion molecule on GBM, it plays an important role in sertoli cell detachment, cell losing and signal transduction inside and outside the cell. When the expression of integrin-(31 is reducing, it may contribute to sertoli cell fall off and result in glomerular sclerosis. Chen et al reported sertoli cells lossing accompany with integrin-β1 expession reducing. When we talke about apoptosis, ROS is the the hot research topic recently. Sertoli cells are terminal differentiated, which own a number of mitochondria to maintain its energy demand. When the mitochondria is damaged, it must cause podocytes dysfunction. Coupled with cell injury, permeability of mitochondrial membrane increasing, can cause cell apoptosis, resulting in ROS production after electronic lost of oxidation respiratory chain. Mitochondrial damage and the generation of ROS can further cause podocyte injury and the formation of a large number of ROS, lead to a vicious cycle. Now, there is no report about whether CXCL16 as scavenger receptor of ox-LDL results in the change of integrin-β1 and ROS in PNS.So, this experiment aimed to build a podocyte model of CXCL16 gene silence, observating the expression changes of integrin-β1 and ROS, exploring the role of CXCL16 in podocytes damage with ox-LDL treatment. We explored whether CXCL16 gene silence could protect podocytes when treated with ox-LDL.MethodsThe mouse podocytes were transfected with CXCL16 gene short hairpin RNA (shRNA) lentivirus vector and negative control lentivirus vector. The expression of CXCL16 protein and mRNA were determined by Western blotting and Real-time PCR. The podocytes vitality was detected by CCK8 assay. The mouse podocytes (cell line) were divided into the following four groups:negative control group(NC group), ox-LDL group, CXCL16-shRNA group, ox-LDL+CXCL16-shRNA group. The total cholesterol was detected by cholorimetric detection to measure intracelluar lipid accumulations. The expression of CXCL16 and integrin-β1 were detected by Western blotting. The expression of ROS was detected by ROS Deep Red Dye.Results1.Effect of entivirus transfectionGreen fluorescence can be obviously seen after transfection with MOI= 50, cells in good condition, so, the optimal MOI is 50. To maintain cell infection in the process of cell reproduction, puromycindi hydrochloride (1μg/ml)was added into culture medium. Cells with green fluorescence were account for more than 90%.2.The rate of CXCL16 gene silencingThe expression of CXCL16 mRNA was inhibited about 70% and CXCL16 protein was inhibited about 60% of CXCL16-shRNA podocytes compared to the negative control(Z=2.61, P<0.05).3.Podocytes vitalityCCK8 revealed no significant difference between NC group and CXCL16-shRNA group of podocytes vitality(Z=0.369, P>0.05).4.1ntracellular total ox-LDL cholesterolNC group and CXCL16-shRNA group were both treated with ox-LDL about 0h、 24h、48h、72h. Treated with ox-LDL for 24h-72h, total cholesterol levels increased. After 72h, total cholesterol levels increased significantly. The total cholesterol level of ox-LDL group was higher than that of NC, CXCL16 shRNA, CXCL16 shRNA+ ox-LDL group, the difference was statistically significant (P< 0.05).5.Expressions of CXCL16 and integrin-β1 with ox-LDL treatmentAfter treated with ox-LDL for 48h, the expression of CXCL16 increased (F=19.24, P<0.05)and integrin-β1 decreased(F=54.15, P<0.001). Treated 72h, the expression of integrin-β1 decreased both in ox-LDL group and CXCL16-shRNA group, also, the level of integrin-β1 in ox-LDL group is lower than that of NC, CXCL16 shRNA, CXCL16 shRNA+ox-LDL group, the difference was statistically significant (F=51.12, P<0.001).6.1ntracellular ROSThe production of ROS was detected by fluorescence intensity. ROS in NC, CXCL16-shRNA, ox-LDL+CXCL16 shRNA group were higher than that in ox-LDL group, difference was statistically significant (F= 307.57, P<0.001).Conclusion1. When MOI=50 could obtain satisfactory result of virus transfection, it suggested that the gene silencing model could be made with MOI=50 for 40-48h.2. Compared with negative control, the expression of CXCL16 protein significant reduced in CXCL16-shRNA group than NC group. It suggested that we successfully made the model.3. Compared with negative control, after gene silence, the podocytes vitality was the same with the NC group, so, we could further discussed the difference between NC group and CXCL16-shRNA group damage when treated with ox-LDL.4. With ox-LDL treatment, lipid deposition of ox-LDL group was significantly higher than that of ox-LDL+CXCL16-shRNA group, suggested that lipid deposition reduced after CXCL16 gene silence, CXCL16 was the scavenger receptor of ox-LDL5. Treated with ox-LDL, the expression of ROS was increased and integrin-β1 decreased, suggested the way that ox-LDL damaged podocytes.6. After CXCL16 gene silence, ROS integrin-β1 decreasing, integrin-β1 increasing, it suggested CXCL16 gene silence could alleviate podocytes injury. |
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