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Objective To investigate the role of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway in Dexamethasone (DEX) inhibiting podocytes apoptosis which was induced by Puromycin (PAN).Methods Mouse glomerular podocytes were cultured in vitro,and were divided into control group,dimethyl sulfoxide (DMSO) group,PAN group,DEX group,and LY294002 (inhibitor of PI3 K) group.The mRNA expression of CD2-associated protein (CD2AP) was measured by using real time fluorescent quantitative polymerase chain reaction,and intracellular distribution was detected by using indirect immunofluorescence staining.Co localization of CD2AP and p85 was detected by using confocal fluorescence microscopy.The expressions of Akt,phosphorylated (p)-Akt,glycogen synthase kinase-3β (GSK3 β) and phosphorylated (p)-GSK3β were evaluated by using Western blot.Results The expressions of CD2AP mRNA in PAN group at each time point (8 h,24 h,48 h) (1.11 ± 0.16,0.78 ±0.09,0.56 ± 0.43) were significantly lower than those in the control group (1.90 ± 0.26,2.09 ± 0.12,2.28 ±0.95),and the differences were statistically significant (all P ＜ 0.05);CD2AP distributed in foot process with uniform filament and discontinuous coarse particle around perinuclear;CD2AP and p85 distributed in cell membrane and cytoplasm evenly in control group,but accumulated in nuclei in the PAN group.The expressions of CD2AP mRNA in DEX group at each time point (8 h,24 h,48 h) (1.53 ± 0.14,2.15 ± 0.27,2.13 ± 0.15) were significantly higher than those in the PAN group,and the differences were also statistically significant (all P ＜ 0.05);the distribution density and range of CD2AP were greater than those in the PAN group,and the accumulation with p85 in nuclei decreased obviously.The expressions of p-Akt and p-GSK3β were inhibited by PAN in a dose-dependent manner (P ＜0.05).The expressions of p-Akt and p-GSK3 β were lowest after PAN stimulated at 15 min and 30 min respectively.However,the expressions of p-Akt and p-GSK3 β increased depending on the concentration of DEX (P ＜ 0.05).In addition,the expressions of p-Akt and p-GSK3 β could be blocked by LY294002 (P ＜ 0.01).Conclusion DEX can protect podocytes and inhibit podocytes apoptosis through stabilizing the expression and distribution of CD2AP.The stale expression of PI3K/Akt signaling pathway is the key factor in DEX protecting podocytes.
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microRNAs play an important regulative role in body's growth and development,and the development of the disease process.Much microRNAs can maintain normal kidney function and regulate kidney pathological process,the miR-30a has extensive effect on kidney development and progression of renal diseases.In this review,a brief overview on the role of miR-30a in renal pathology is presented.
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Objective To verify the targeting regulatory relationship between microRNA -939 (miR -939) and CD2 -associated protein (CD2AP).Methods The online RegRNA software was used to predict the human CD2AP promoter for potential binding sites complementary to miR -939.HEK -293T cells were cotransfected with hu-man CD2AP promoter plasmid pGL3 -2K and microRNA negative control (miR -NC)or miR -939 mimics,and the relative luciferase activity(RLA)was detected at 24 h post -transfection.HEK -293T cells were transfected with miR -NC or miR -939 mimics for 48 h,and the CD2AP mRNA expression level was detected by adopting reverse tran-script and real -time fluorescence quantification -PCR,while the CD2AP protein expression level was detected by using Western blot.Results (1 )There were 2 miR -939 binding sites at CD2AP promoter region,located at -468 to -491 and -654 to -677 upstream of initiation codon ATG (marked as +1 )relatively.(2)At 30 nmol/L,50 nmol/L,the RLA in miR -NC group and miR -939 group were 6.81 ±0.88 vs 6.07 ±2.24,5.88 ±1 .44 vs 3.94 ± 0.79 relatively,and there were no significant differences between the 2 groups (t =3.04,2.06,all P >0.05),while the RLA between the 2 groups were 5.58 ±0.58 vs 3.29 ±0.64 at 1 00 nmol/L,and the difference was significant between the 2 groups(t =4.07,P <0.05).(3)At 30 nmol/L,50 nmol/L and 100 nmol/L,the relative CD2AP mRNA expression in miR -NC group and miR -939 group were 1 .00 ±0.01 vs 0.80 ±0.08,1 .00 ±0.00 vs 0.80 ±0.1 3 and 1 .00 ± 0.00 vs 0.72 ±0.07 relatively,while the CD2AP mRNA expression was decreased by 20% -30% at each concentration level,and there were significant differences between the 2 groups (t =4.44,2.93,6.84,all P <0.05).(4)At 50 nmol/L, the relative CD2AP protein expression in miR -NC group and miR -939 group were 0.48 ±0.09 vs 0.19 ±0.12,and the CD2AP protein expression was decreased,and the difference was significant (t =3.36,P <0.05).Conclusions CD2AP is the target gene of miR -939,and miR -939 can down -regulate the expression of CD2AP both in mRNA and protein levels by targeting its promoter region,which indicates that miR -939 may mediate the podocyte injury.
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Objective To observe the effect of puromycin aminonucleoside(PAN) on the expressions of CD2associated protein (CD2AP) and phosphatidylinositol 3-kinase (PI3 K) p85 in induced podocytes,and to explore the significance of abnormal expression of CD2AP in apoptosis of podocytes in vitro.Methods Mouse podocytes were injected into the control group and the PAN treatment group (PAN group).The cells were cultured for 8 h,24 h and 48 h respectively.The podocyte morphology was observed by phase-contrast microscope.The apoptosis ratio of podocytes was determined with flow cytometry.The mRNA expression of CD2AP was detected by real time fluorescent quantitative polymerase chain reaction.The protein expressions of CD2AP and PI3K p85 were detected by Western blot,respectively.The distributions and the relationships between CD2AP and PI3K p85 in the podocytes were detected by confocal fluorescence microscopy.Results PAN treatment led to significant shrinkage of podocytes with decreasing distribution tendency,podocyte foot process retraction as well as effacement and loss of cell contact.Compared with the control group,the apoptosis rate was increased at 24 h and 48 h[(7.52 ± 1.35)％,(17.09 ±2.53)％ vs (4.32 ±0.81％,(6.81 ± 1.34)％] in PAN group and the differences were significant (t =4.98,8.79,all P ＜ 0.05) ;CD2AP mRNA expression tended to decrease at each time point,and the differences were significant (0.34 ± 0.12,0.46 ±0.21,0.47 ± 0.10 vs 0.62 ± 0.23,0.97 ± 0.14,0.96 ± 0.23),and there were statistical differences (t =2.64,4.95,4.79,all P ＜ 0.05) ;CD2AP and PI3K p85 protein expressions tended to decrease at 24 h (0.36 ± 0.12,0.61 ± 0.20 vs 0.64 ±0.23,0.97 ±0.31) and 48 h(0.27 ± 0.15,0.48 ± 0.20 vs 0.51 ± 0.20,0.84 ± 0.35),and the differences were significant(t =2.64,2.31,2.35,2.40,all P ＜ 0.05).In the control group,the distribution of CD2AP was uniformly filamentary structure in cytoplasm and nucleus of podocytes,after PAN treatment the distribution of CD2AP changed to discontinuous coarse granular concentrated in the perinuclear.Immunocyte fluorescence showed that the distribution and the relationships between CD2AP and PI3K p85 in the podocytes became abnormal.Conclusions When the apoptosis of podocytes was induced by PAN,the expression and distribution of CD2AP were abnormal.CD2AP may play an important role in the survival of podocytes though the PI3K signaling pathway.
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Objective To identify the important regulatory elements in the promoter of human CD2 associated protein(CD2AP) by conserved sequence analysis among different species and luciferase functional detection. Methods The promoter sequences of CD2AP from different species were analyzed by BLAST. Plasmids containing different length of deletion mutations of human CD2AP promoter were constructed. Pro-moter activities were tested in 3 kinds of cells from different species by luciferase analysis and were tested in HEK-293 cells treated with all-trans-retinoic acid. Results Homologous sequence comparison in CD2AP promoter area among human, cattle and pig showed that putative specific protein 1 (Sp1) sites and down-stream promoter element (DPE) were highly evolutional]y conserved. Progressive deletion luciferase analysis of DNA fragments revealed similar promoter activity style among 3 different cell lines from 3 different spe-cies, HEK-293, BHK-21 and Vero cells. One basic promoter activity located within 500 bp upstream of ATG. Fragments of further upstream 100 bp or more had drastically 10 times increased promoter activity. Two putative Sp1 sites were in this 100 bp region. All-trans-retinoic acid decreased the luciferase activity of CD2AP promoter. Conclusion Putative Spl sites and DPE have important functions in the promoter activity of CD2AP.
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Objective To study the effects of CD2-associated protein (CD2AP) on podocyte adhesion and extension ability and to 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 serambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen IV coated plates. The relative cell adhesion and cell area were examined 90 min later. Apoptotic rates of CD2AP siRNA transfected podoeytes and different PAN concentrations incubated podoeytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confoeal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative ceil adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[(41.72±6.07)% vs (64.46±8.53)%, P<0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [(5.73±0.61)% vs (3.26±0.45)%, P<0.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability (P<0.05). Nevertheless, no significant difference was found in 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 was conspicuously descended to some degree (P<0.05). Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may he partly responsible for the decline of cell adhesion and spreading.
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AIM: To investigate the role of CD2AP and F-actin in the pathogenesis of puromycin aminonucleoside nephrosis in rats. METHODS: Puromycin aminonucleoside nephrosis was induced by single intraperitoneal injection of puromycin aminonucleoside (PAN). Renal tissues were studied at 3, 7, 10 and 20 days after PAN injection by means of immunohistochemistry, RT-PCR, Western blotting and fluorescence. RESULTS: At day 3, CD2AP expression in podocytes began to decrease, and significantly decreased at day 7 and 10 (P