| Objective:This study aims to verify the differential expression of actin nucleator Cordon-bleu（Cobl）in autosomal dominant polycystic kidney disease（ADPKD）,and to investigate the potential mechanism of Cobl on primary ciliary structure and function.Thus,it can further enrich the pathogenic hypothesis of ADPKD caused by abnormal primary cilia,and provide the theory basis for clinical new targets of ADPKD.Methods:1.Biological validation of the differential expression of Cobl and the abnormal structure and function of primary cilia in ADPKD.（1）The differential expression of Cobl in normal renal tubular epithelial cell（RCTEC）,polycystic kidney cyst lining epithelial cell（WT9-12）,pkd1-/-and pkd1+/+ mice’s’ kidneys were detected by western blot and real-time PCR assays.（2）Using immunohistochemical method to observe the location of Cobl in RCTEC,WT9-12,pkd1-/-and pkd1+/+ mice’s’ kidney.（3）Using confocal laser scanning to observe the structural differences of primary cilia between RCTEC and WT9-12.（4）The differential expression of KIF3 A and IFT88 between RCTEC and WT9-12 were detected by real-time PCR assays.2.The impact to basal body migration and structure and function of primary cilia under the circumstance of actin cytoskeleton disordered.（1）The structural differences of actin cytoskeleton between RCTEC and WT9-12,pkd1-/-and pkd1+/+ mice’s’ kidney were observed by immunofluorescence method.（2）Using immunohistochemical method to observe the changes of actin cytoskeleton,basal body’s location and the primary ciliary structure after treating these cells with cytochalasin B which inhibits actin filament polymerization.（3）Using scanning electron microscope（SEM）to observe the changes of cellular shape and ciliary structure.3.The impact to structure and function of primary cilia when regulates the expression of Cobl in normal renal tubular epithelial cells.（1）Using western blot and real-time PCR methods to test the changes in geneexpression of Cobl,Pacsin 2 and KIF3 A in RCTECs after overexpression and downregulation of Cobl by gene transfection technology.（2）The changes of actin cytoskeleton,basal body and primary ciliary length after overexpression and downregulation of Cobl in RCTECs were observed by confocal laser scanning.（3）Using MTT assay to quantify the cell proliferation activity after overexpression and downregulation of Cobl in RCTECs.（4）The changes of intracellular calcium after overexpression and downregulation of Cobl in RCTECs were tested by flow cytometry method.（5）Directional cell migration after overexpression and downregulation of Cobl in RCTECs was measured by making a scratch-wound and healing assay.Results:1.Cobl is highly expressed in WT9-12 and pkd1-/-mice’s kidney when compared with RCTEC and pkd1+/+,respectively.2.Cobl shows enriched expression in the apical domain of renal tubular epithelial cells of pkd1-/-mice.3.The length of primary cilia in WT9-12 is shorter than RCTEC,and the expression of KIF3 A and IFT88 are decreased in WT9-12.4.WT9-12 and pkd1-/-mice show with actin cytoskeleton disordered when compared with RCTEC and pkd1+/+,respectively.5.Cytochalasin B inhibits actin cytoskeletal dynamics and primary ciliary elongation in RCTECs.6.The length of primary cilia and the amount of F-actin increased after overexpression of Cobl in RCTECs.7.In RCTECs,the reduction in Cobl coincides with reduction in the amount of F-actin and the length of primary cilia.8.Cell proliferation activity decline after overexpression of Cobl in RCTECs,while it is enhanced after downregulation of Cobl in the same kind of cells9.After overexpression and downregulation of Cobl in RCTECs,the intracellular calcium concentrations are increased and decreased,respectively.10.Cells after overexpression of Cobl in RCTECs appears to detach from the monolayer and migrate faster than control group,while which downregulation of Cobl orientate randomly at the edge of wound closure.Conclusion:According to the results of our study,we can find that actin nucleator Cordon-bleu（Cobl）is highly expressed in polycystic kidney cyst lining epithelial cell and pkd1-/-mice’s kidney,and it also shows the polycystic kidney cyst lining epithelial cell with shorter primary cilia than the normal renal tubular epithelial cell.Abnormal expression of Cobl can lead to actin cytoskeleton depolymerize,consequently inhibit basal body migration and primary ciliary elongation in renal tubular epithelial cell.When overexpression of Cobl can rescue the ciliary structure and function.Thus,it indicates Cobl may represent a molecular activity that connects developmental patterning signals with actin cytoskeletal dynamics to support morphogenesis of primary cilia. |
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