Study On Physiological Functions Of CD2-associated Protein In Podocytes

Objective To study the distribution of CD2-associated protein (CD2AP) in normal renal cell lines and its interaction with nephrin and F-actin in podocytes.Methods The HMC and HK-2 were cultured in DMEM. Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640. The expressions of CD2AP and nephrin in podocytes were examined by RT-PCR and Western blot. The distribution of CD2AP in HMC, HK-2, differentiated and undifferentiated podocytes was observed by laser scanning confocal microscope. The colocalizations of CD2AP with nephrin and F-actin in undifferentiated podocytes were also detected.Results CD2AP was distributed within the cytoplasm and perinulcear region of HK-2 and undifferentiated podocytes, but was absent in HMC cells. Its distribution profile changed and presented as peripheral accumulation when podocytes were put into differentiation-permissive conditions. CD2AP colocalized with nephrin and F-actin in podocytes.Conclusion CD2AP can be detected in epithelial-originated renal cells. The alteration of distribution profile of CD2AP indicates it may participate in the process of podocytes differentiation and be involved in the regulation of slit diaphragm and cytoskeleton. Objective To study the changes of biological characters and the potential role of CD2AP in podocyte differentiation.Methods Conditionally immortalized murine podocyte cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions and shifted to 37℃non-permissive conditions to induce differentiation. Podocytes morphology changes, growth curve, proliferation ability and cell cycle were measured respectively in undifferentiated and differentiated podocytes. The expressions of CD2AP,α-actinin, nephrin and cytoskeletal proteins (i.e. F-actin and tubulin) were detected by laser scanning confocal microscope. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to 37℃non-permissive conditions. Simultaneously, untransfected cells were taken as differentiation control. The changes of cell proliferation and the expression of synaptopodin were examined respectively.Results In differentiated podocytes, cell morphology changed along with secondary foot process formation and reproductive activity descended. Cells in G0/G1 period accumulated and in S, G2/M period decreased. The expressions of CD2AP, nephrin andα-actinin were elevated in differentiated podocytes. The distribution profiles of podocyte associated proteins and cytoskeletal proteins apparently altered. After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth arrested. Synaptopodin, the differentiation podocyte marker, were apparently down-regulated and cells showed delayed to differentiate.Conclusion These results demonstrated that podocyte differentiation was accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in it. Objective To study the effect of CD2-associated protein (CD2AP) in podocyte proliferation and cell division.Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. After labeling with red fluorescent dye PKH-26, podocytes were transfected with CD2AP small interfering RNA (siRNA) using transfection reagent Metafectene. Transfection efficiency was measured by flow cytometer 24h later and inhibition effect of CD2AP siRNA was determined by RT-PCR and Western blot 48h after transfection. Proliferation index and cell cycle of podocytes were examined by flow cytometer 72h later. Microbule of podocyte was detected by Oregon Green? 488-conjugated paclitaxel. The ratio of binuclear and polynuclear podocytes were counted under laser scanning confocal microscope (LSCM).Results The transfection efficiency of CD2AP siRNA is 66.27%. 48h after transfection with specific siRNA, the expressions of CD2AP mRNA and protein were down-regulated by 57% and 39% respectively. The podocyte proliferation index apparently descended (p<0.05) and the cells in the phase of G2/M accumulated significantly when detected at 72h (p<0.05). With the help of LSCM, some podocytes that could not separate after M-period were easily visible. The ratio of binuclear and polynuclear podocytes in CD2AP siRNA transfeced group was markedly higher than that in control group (p<0.05).Conclusion Lowered CD2AP expression may play a crucial role in podocytes injury during the pathogenesis of proteinuria by interfering cell separation after mitosis and podocytes proliferation. Objective To study the effect of CD2-associated protein (CD2AP) in podocyte adhesion and extension ability and explore its possible mechanism.Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and scrambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagenⅣcoated plates. The relative cell adhesion and cell area were examined 90min later. Apoptotic rates of CD2AP siRNA transfected podocytes and different PAN concentrations incubated podocytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confocal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot.Results The relative cell adhesion and cell area of CD2AP siRNA transfected podocytes were apparently less than that of control group(P<0.05). The apoptotic rate in CD2AP siRNA transfected podocytes was significantly higher than control podocytes(P<0.05). 100μg/ml PAN could markedly induce podocytes to apoptosis and impaired cell adhesion ability(P<0.05). Nevertheless, no obvious difference was founed to cell body spreading(P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level were conspicuous descended to some degree(P<0.05).Conclusion CD2AP depletion facilitated podocytes to apoptosis and impaired the cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness that caused by CD2AP depletion might be responsible for the decline of cell adhesion and spreading. Objective To observe the proteinuria endocytosis of podocytes and the part the CD2AP plays in it.Methods In this study, conditionally immortalized murine podocytes cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions. FITC conjugated bovine serum albumin (BSA) was prepared to mimic the podocytes floating in Bowman's capsule under proteinuria circumstance in vivo. The endocytosis of BSA in podocytes was investigated by flow cytometer and laser scanning confocal microscope (LSCM). The co-localization of CD2AP and FITC-BSA was detected during endocytosis. CD2AP expression profile was measured by Western blot.Results The results demonstrated that podocytes had endocytosis ability to albumin even under physiological threshold (0.05mg/ml). Endocytosis curve of FITC-BSA with time was made at large concentration (1mg/ml) and exhibited an"S"shape. A dynamic equilibrium of endocytosis was observed at 2h or so. Co-localization of CD2AP and FITC-BSA was conformed by LSCM in podocytes. CD2AP protein expression was markedly up-regulated 24h later. Apoptosis podocytes and ultrastructure changes induced by BSA were observed.Conclusion Beyond acting as the filtration barrier component, podocytes also exert its endocytosis to proteinuria in microalbuminuria and macroalbuminuria individual. CD2AP is involved in the urine protein endocytosis of podocytes.