The Role And Regulation Of Comlement Factor B In Autosomal Dominant Polycystic Kidney Disease

Objective:This study was to explore the mechanism of CFB overexpression in autosomal dominant polycystic kidney disease(ADPKD)and to investigate whether polycystin-1regulates CFB expression.Methods:1.Western blot and Immunohistochemistry was performed on human and rat cystic kidney tissues to detect the expression of p-JAK2,p-STAT1,CFB and PC1-CTT.2.To determine whether STAT1 regulates CFB expression in renal epithelial cells,we over-expressed STAT1 plasmids and used the STAT1 inhibitor fludarabine in UCL93 immortalized human renal epithelial cells.Western blot was performed to detect the expression of p-STAT1 and CFB.3.STAT1 gene,CFB promoter(CFB-pr)and renilla were co-transfected in renal epithelial cells and were assayed for luciferase activity.Empty vector,CFB-pr and renilla co-transfected cells were used as control.4.To confirm the direct association of the STAT1 protein with human CFB promoter,we performed chromatin immunoprecipitation(ChIP)assays.5.To test whether PC1-CTT regulates CFB expression,a soluble PC1-CTT was over-expressed in UCL93 renal epithelial cells.To next tested whether the PC1-CTT induced expression of CFB requires STAT1,PC1-CTT transfected renal epithelial cells were treated with fludarabine.Western blot was performed to detect PC1-CTT,p-STAT1 and CFB.6.To investigate whether the STAT1 response element is needed for PC1-CTT mediated transactivation of CFB promoter,CFB-pr and a STAT1 binding site Mutant CFB promoter(CFB-pr-Mut)and renilla were co-transfected in renal epithelial cells.luciferase activity were assayed.7.To further investigate the mechanism of STAT1 regulation of CFB expression,PC1-CTT plasmid was co-transfected with two different dominant-negative(DN)STAT1plasmids(DN1-STAT1 with Y701 F mutation or DN2-STAT1 with S727 A mutation).luciferase activity were assayed.8.To test whether the NF-?B pathway partly mediates PC1-CTT induced CFB expression,we first treat renal epithelial cells with the NF-?B inhibitor QNZ.Second,PC1-CTT plasmids was overexpressed in UCL93 cells and then treated withNF-?B inhibitor QNZ.Western blot was used to detect the p50/p65 NF-?B and CFB.Third,CFB promoter activity was tested after co-transfected with PC1-CTT plasmids and treated with QNZ.9.To study whether CFB triggers macrophage M2 phenotype differentiation,bioactive human CFB was used to treat RAW264.7 mouse macrophage cells.Western blot and RT-PCR were performed to analyze macrophage differentiation.10.CFB antibody was included in conditioned medium that was generated from renal epithelial cells transfected with PC1-CTT(PC1-CTT-CM)to treat RAW264.7 cells.Western blot and RT-PCR were performed to analyze macrophage differentiation.11.To explore the role of STAT1 in renal epithelial cell induced M2 macrophage differentiation,we generated conditioned medium from renal epithelial cells which were treated with STAT1 specific inhibitor fludarabine to treat RAW264.7 cells.Western blot was performed to analyze macrophage differentiation.12.Conditioned medium that was generated from renal epithelial cells transfected with PC1-CTT and treated with STAT1 inhibitor to treat RAW264.7 cells.Western blot was performed to analyze macrophage differentiation.Results:1.CFB was up-regulated in human and rat cystic kidney tissues which was correlated with increased p-JAK2,p-STAT1 and PC1 C-terminal tail(PC1-CTT).2.PC1-CTT is capable of activating CFB transcription through the promoter element in the region-219 to-210 which contains the STAT1 binding site.3.NF-?B partly mediates PC1-CTT induced CFB expression.4.PC1-CTT induced macrophage activation into an M2 phenotype is mediated by STAT1 and CFB.Conclusion:This tudy reveals possible mechanisms of CFB up-regulation in ADPKD and a novel role of PC1-CTT on ADPKD associated inflammation.Furthermore this studysuggests that targeting suppressing CFB expression may be a new strategy to prevent inflammation in the kidney of patients with ADPKD.