Silencing Of CXCL12 Performs A Protective Effect On C5b-9-induced Injury In Dodocytes

Membranous nephropathy(MN),which accounts for 20-35%of primarynephrotic syndrome,is the leading cause of adult nephrotic syndrome.It seriously affect the quality of life of the patients.Although membranous nephropathy is a mild,spontaneous remission in some cases,40%of patients with membranous nephropathy eventually develop into end-stage renal disease.The characteristic pathological changes of membranous nephropathy are thickening of the basement membrane caused by podocyte injury and deposition of immune complexes.Podocytes are a layer of terminally differentiated cells outside the glomerular basement membrane that are targets of immune complex-mediated and non-immune-induced kidney damage.Although the etiology of membranous nephropathy is not well understood,it is generally thought that it is an autoantibody that forms an immune complex with the target antigen deposited under the epithelium,which in turn activates complement,causing podocyte damage and proteinuria.The chemokine C-X-C motif ligand 12(CXCL12)is a member of the chemokine family and exerts a biological role through the chemokine receptor CXCR4(C-X—C chemokinereceptor 4).It is reported that CXCL12 plays a role in kidney-related diseases.Sayyed et al found that blocking CXCL12 inhibits macrophage aggregation in diabetic nephropathy.Balabanian et al found that CXCL12 is involved in the progression of lupus nephritis,and that anti-CXCL12 can inhibit the progression of glomerulonephritis.Although CXCL12 is also expressed in podocytes,its possible biological role in the development of membranous nephropathy has not been reported yet.The signal transducers and transcription activator 3(STAT3)is a signal transduction factor and an activator of the transcriptional protein family.Its biological functions are diverse,including cell proliferation,differentiation,survival,and angiogenesis.STAT3 can be activated by many cytokines such as tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6).The STAT3 signaling pathway has been reported to be activated in rat and human glomerulonephritis.In collapsed glomerular disease caused by HIV infection,activated STAT3 can promote podocyte proliferation and dedifferentiation.In addition,when CXCL12 changes,its receptor CXCR4 activates the STAT3/JAK signaling pathway accordingly.P-STAT3 expression was significantly increased in CXCL12-treated breast cancer cells,suggesting that P-STAT3 may be a downstream factor of CXCL12.C5b-9 is a polymeric membrane attack complex.C5b-6 forms a C5b-9 complex with C7,C8 and C9.It can insert into the phospholipid bilayer of the cell membrane,forming channels or leaks in the cell membrane,leading to the death of the non-nucleated cellsand the cell damage of nucleated cells such as podocytes.C5b-9 attacks podocytes throughtransforming growth factor-?(TGF-?),Angiotensin II(Angll)orOxidant ROS to cause podocyte injury,which in turn causes the development and progression of membranous nephropathy,which ultimately leads to end-stage renal disease.In our study,we measured the level of CXCL12 in serum and urine of patients with membranous nephropathy by ELISA.we found that the level of CXCL12 is increase.That means,in membranous nephropathy,CXCL12 may be a useful diagnostic biomarker.Next we induced podocyte injury with three different concentrations of C5b-9.We found that C5b-9 inhibits podocyte proliferation in a concentration-time dependent manner.Therefore,we finally selected 0.4 ?g/mL of C5b-9 to induce podocyte injury.In this model of podocyte injury,we observed inhibition of CXCL12 by CXCL12 SiRNA can reduce podocyteapoptosis,at the same time,CXCR4/P-STAT3/caspase 3 and TNF-a/IL-6 also decreased.Other than that,the STAT3 phosphorylation inhibitor AG490 and the antagonist of CXCL12berberinealso have protective effects on CXCL12-treated podocytes.Therefore,we draw conclusions that CXCL12 plays an important role in membranous nephropathy.It has the potential to become a new diagnostic target and even to treat kidney-related diseases.Part ? CXCL12 detection of blood and urine in patients with idiopathic membranous nephropathyObjective:To understand the levels of blood and urine CXCL12 in patients with idiopathic membranous nephropathy.Methods:1 Blood and urine samples from 90 patients with idiopathic membranous nephropathy and 90 healthy controls were collected;2 The concentration of blood and urine CXCL12 was measured by ELISA.Results:The concentration of CXCL12 in blood and urine of patients with membranous nephropathy was significantly higher than that of healthy controls(P<0.05).Conclusion:CXCL12 may be involved in the development of membranous nephropathy.Part ?:Establishment of a model of podocyte injury induced by C5b-9 complex and its effect on CXCL12/CXCR4 and downstream factorsObjectives:1 To establish a podocyte injury model using C5b-9 complex;2 Find the most suitable concentration of C5b-9 complex;3 To observe the effect of C5b-9 complex on CXCL12/CXCR4 and downstream factors in podocytes.Methods:1 podocytes culture;2 cultured podocyte grouping:The first group(WT):wild-type cells had no any treatmen The second group:0.05?g/mL C5b-9 treated cells The third group:0.1?g/mL C5b-9 treated cells The forth group:0.2?g/mL C5b-9 treated cells The fifth group:0.4?g/mL C5b-9 treated cells3 detection of cell proliferation activity by CCK8;4 The expression of CXCL12,CXCR4,STAT3 and P-STAT3 was detected by Western Blot method.Results:1 By CCK8 detection,the podocyte proliferation activity decreased after the C5b-9 complex acted on the podocytes;2 Podocyte proliferation activity is inhibited by C5b-9 complex in a concentration-and time-dependent manner;3 After treatment with C5b-9 complex,the expression of CXCL12,CXCR4 and P-STAT3 was significantly increased,while STAT3 was unchanged.Conclusions:1 Successfully established a model of podocyte injury induced by C5b-9 complex;2 The concentration of 0.4 ?g/mL was selected as the C5b-9 complex-induced podocyte injury model.;3 CXCL12,CXCR4,and P-STAT3 may be involved in podocyte injury induced by C5b-9 complex.Part ? CXCL12 damages podocytes by activating the CXCL12/p-STAT3 signal pathwayObjective:To further investigate the CXCL12/p-STAT3 signaling pathway by using STAT3 blockers and antagonists of CXCL12Methods:1 cultured podocyte grouping:The first group(WT):wild-type cells had no any treatment The second group:CXCL12 treated cells+AG490 The third group:CXCL12 treated cells+berberine2 detection of podocyte apoptosis by Flow cytometry;3 The expression of CXCL12,CXCR4,STAT3,P-STAT3 and caspases3 was detected by Western Blot.Results:1 Apoptosis of podocytes and expression of apoptosis-related protein caspases3 increased significantly after CXCL12 treatment;2 Expression of CXCR4?STAT3?P-STAT3 increased significantly after CXCL 12 treatment;3 AG490 significantly inhibited podocyte apoptosis and expression of apoptosis-related protein caspases3 induced by CXCL12;4 AG490 significantly inhibited the expression of CXCL 12,CXCR4 and P-STAT3 in podocytes induced by CXCL12;5 Berberine significantly inhibited podocyte apoptosis and expression of apoptosis-related protein caspases3 induced by CXCL 12;6 Berberine significantly inhibited the expression of CXCL12,CXCR4 and P-STAT3 in podocytes induced by CXCL 12.Conclusion:CXCL12 damage to podocytes by activating the CXCL12/p-STAT3 signaling pathway.Part ? Selection of SiRNA sequences and its effect on CXCL12 in podocytesObjectives:1 To observe the effect of SiRNA transfection on podocyte CXCL12;2 Select the most efficient SiRNA sequence.Methods:1 According to CXCL 12 mRNA,three different kinds of SiRNA were used to knockdown the level of CXCL12 in injured podocytes.Meanwhile,an unspecific scrambled siRNA as a negative control was applied;2 liposome-mediated SiRNA transfection;3 cultured podocyte grouping:first group(WT),wild-type cells had no any treatment;second group(NC),cells were transfected with negative control siRNA;third group(siCXCL12-1),cells were transfected with the first CXCL12 siRNA;fourth group(siCXCL12-2),cells were transfected with the second CXCL12 siRNA;fifth group(siCXCL12-3),cells were transfected with the third CXCL12 siRNA;4 Detection of CXCL12 mRNA levels by RT-PCR;5 Detection of CXCL12 expression by Western Blot method.Results:1 After transfection of SiRNA,the level of CXCL12 mRNA was significantly lower than that of the WT group and the NC group;2 After SiRNA transfection,the expression of CXCL12 protein was significantly lower than that of the WT group and the NC group.Conclusions:1 CXCL12 SiRNA can effectively inhibit the expression of CXCL12;2 According to the inhibition efficiency of CXCL12,the third sequence was selected for subsequent use.Part ? CXCL12 SiRNA protection against C5b-9 induced podocyte injury and its mechanism of actionObjectives:1 To observe the effect of CXCL12 SiRNA transfection on C5b-9-induced podocytes;2 To investigate the mechanism of CXCL12 SiRNA transfection on C5b-9-induced podocytes injury.Methods:1 cultured podocyte grouping:The first group:0.4?g/mL C5b-9 treated cells The second group:0.4?g/mL C5b-9 treated cells+negative control siRNA transfection The third group:0.4?g/mL C5b-9 treated cells+siCXCL12-3 transfection2 detection of podocyte proliferation activity by CCK8;3 detection of podocyte apoptosis by Flow cytometry;4 CXCL12,TNF-?,and IL-6 were detected by ELISA;5 The expression of CXCL12,CXCR4,STAT3,P-STAT3 and caspases3 was detected by Western Blot.Results:1 Compared with the first group and the negative control group,transfection of siCXCL12-3 significantly promoted cell proliferation;2 Compared with the first group and the negative control group,transfection of siCXCL12-3 significantly inhibited cells apoptosis;3 siCXCL12-3 transfection reduced the content of CXCL12,TNF-a and IL-6 in the cell supernatant by ELISA;4 siCXCL12-3 transfection reduced the expression of CXCL12,CXCR4,STAT3,P-STAT3,and caspases3 by Western Blot.Conclusions:1CXCL12 SiRNA has protective effect on podocyte injury induced by C5b-9;2The protective effect of CXCL12 SiRNA on C5b-9-induced podocyte injury was achieved by inhibition of CXCL12/CXCR4/P-STAT3 parthway.