The Effect And Regulation Of CIP4on Expression Of E-cadherin And Occludin In Renal Tubular Epithelia

Renal interstitial fibrosis is an inevitable outcome of almost all kinds of chronic kidney diseases (CKD). Epithelial-mesenchymal transition (EMT) is considered as one of the most important-events leading to fibrosis. During the process of EMT, the biomarkers of epithelia, such as E-cadherin and occludin, the expression of which is lost. E-cadherin, form the 'adhesive complex' with β-catenin together, which is crucial to the cell-cell adhesion. β-catenin is a good candidate to regulate the expression of E-cadherin, β-catenin translocation to the nucleus, then activate the promoter of Snail gene family, the repressor of E-cadhein. Occludin is a component of tight junction, which maintains the polarity of epithelia, is regulated by the polarity protein Par6. Evidences show that transforming growth factor-betal (TGF-β1) is an important cytokine during fibrogenesis, to regulate its function, TGF-β1utilizes multiple signaling pathways by canonical TGF-β/Smad pathway components'cross talks'with other signaling pathways, namely non-Smad pathways, these include various branches of MAP kinase pathways, phosphatidylinositol-3-kinase (PI3K)/AKT pathways, ERK pathways and Rho-like small GTPase signaling pathways. Cdc42-interacting protein4(CIP4), a member of RhoGTPase family, is a downstream effector of Cdc42, and participates the modulation of cytoskeletal protein actin. CIP4is crucial to maintain the normal morphology of cell.In our study, we demonstrated that in both animal models,5/6nephrectomized rats and unilateral unreteral obstructed mouse, CIP4expression was upregulated comparing with sham operated group, and was distributed in renal tubule-interstitial area, mainly at the baso-lateral sides of epithelia, the exact location of cell-cell junctions. CIP4upregulation was companied by downregulation of E-cadherin and occludin. We treated NRK52E cells with TGF-β1to set up a EMT model in vitro, the expression of CIP4was found increased accompaning with the decreased expression of E-cadherin and occludin, which was consistent with the results of in vivo experiments. Over-expression of CIP4without the stimulation of TGF-β1, the related proteins above were show the similar change in protein expression. Also we found, in both TGF-β1treated cells and CIP4over-expression cells, CIP4interacted with β-catenin, the expression of β-catenin had no difference among those groups in the whole cell lystaes. However, the expression of β-catenin in nuclear protein had been elevated in TGF-β1treated and CIP4over-expression cell groups, which was reduced when CIP4was silenced by the specific siRNA, meanwhile, E-cadherin was upregulated in CIP4siRNA-transfected cells. Furthermore, β-catenin knockdown by speeific siRNA while CIP4was over-expressed, E-cadherm expression had no change. On the other hand, in both TGF-β1treated cells and CIP4over-expressed cells, Par6expression had no differences comparing with control group, however, the interaction between CIP4and Par6was observed in these two groups, but not in control group. All above, we conclude that CIP4may repress the expression of E-cadherin by promoting β-catenin translocate to the nucleus, also, CIP4may regulate the expression of occluding by interacting with Par6. Part1The expression and distribution of CIP4in renal fibrotic modelsObjective To investigate the distribution and expression change of CIP4in5/6nephrectomized SD rats and unilateral ureteral obstructive (UUO) Balb/c mouse.Methods The renal fibrotic animal models were induced by5/6subtotal nephrectomy in SD rats and left ureteral obstruction in Balb/c mouse. The serum was collected in SD rats to detect the change of serum creatine (Scr) and blood urea nitrogen (BUN). In both models, the severity of renal fibrosis was detected by Masson staining. Immunohistochemical staining was used to detected the expression and distribution of CIP4. The protein expression of CIP4, β-catenin, E-cadherin. Occludin, Par6and α-SMA were defected by western blotting.Results In the model of5/6nephrectomized rats, the value of Scr and BUN were higher than those in sham operated rats(P<0.05). Masson staining showed sclerotic glomorulus, renal tubular atrophy and epithelial drop. CIP4expression was upregulated, and was mainly distributed in basolateral side of epithlia. In UUO model, Masson stainning also showed sclerotic glomorulus, interstitial fibrosis. Renal tubules were partial enlarged, partial atrophy. Western blotting of both models showed that CIP4was upregulated(3.91-fold and3.64-fold respectively) accompanied by upregulation of α-SMA and downregulation of E-cadherin and occludin (P<0.05), β-catenin and Par6showed no change in protein expression.Conclusions CIP4was found increased in protein expression in fibrotic renal tissue, and was mainly distributed in renal tubular epithelia, which indicated that CIP4was closely related with renal tissue fibrogenesis.Part2The expression and distribution of CIP4in TGF-β1induced renal tubular EMTObjectives To investigate the expression change of CIP4in murine epithelial cell line NRK52E while incubating with TGF-β1.Methods NRK52E cells were incubated with TGF-β1(10ng/ml) for72h, the morphological change was observed by light microscope. The protein expression of CIP4, β-catenin, E-cadherin, occludin, Par6and a-SMA were detected by western blotting.Results The morphological appearance of control cells were showed as cobble-stone in shape, cell-cell junction was tight. After TGF-β1treatment, cells had a large and irregular shape, cell junctions were loosen. Western blotting showed that in TGF-β1treated cells, CIP4was upregulated1.72-fold comparing with control group, together with α-SMA, reduced expression of E-cadherin and occludin were also abserved (P<0.05). The expression of β-catenin and Par6had no difference.Conclusions The results in vitro were consistence with those in vivo, which indicated that CIP4participate in the process of TGF-β1induced EMT in NRK52E cells.Part3Effect and regulation of CIP4to the expression of E-cadherinObjectives To investigate the effect of CIP4to P-catenin translocation to the nucleus, and the expression of E-cadherin.Methods The plasmid pcDNA4.0/CIP4was transient transfected into NRK52E cells by lipofectamine2000, all procedures were followed by the instructions. The colocalization of CIP4and β-catenin was detected by immunofluoresence using confocal microscope. The interaction between CIP4and β-catennin was detected by immunoprecipitation.. CIP4and β-catenin were silenced by specific siRNA respectively.Results The expression of CIP4and β-catenin were elevated in nuclear protein after TGF-β1treatment,1.82-fold and1.59-fold respectively. Under the cofocal microscope, immunofluroscence showed partial colocalization of CIP4and β-catenin at the cell membrane in control cells, the expression of both proteins were elevated and had partial colocalization in the nucleus after TGF-β1incubation. CIP4interacted with β-catenin in both groups detected by immunoprecipitation. In CIP4-transfected NRK52E cells, α-SMA was upregulated accompanied with CIP4, E-cadherin was downregulated (P<0.05), the results was similar as those in cells treated by TGF-β1only. To analyze the nuclear proteins, β-catenin expression was upregulated in CIP4over-expressed cells, E-cadherin expression was reduced in whole cell lysates\(P<0.05). After CIP4was knockdown by the specific siRNA, we observed reduced β-catenin expression in nuclear proteins, and E-cadherin expression was restored. Furthermore, after CIP4over-expression, β-catenin was silenced by siRNA, the E-cadherin expression had no difference.Conclusions CIP4may repress the expression of E-cadherin by promoting translocation of β-catenin to the nucleus. Part4Effect of CIP4on the expression of occludinObjectives To investigate the effect of CIP4over-expression on the expression of tight junction protein occludin, and the interaction between CIP4and Par6in NRK52E cells.Methods The plasmid pcDNA4.0/CIP4was transient transfected by lipofectamine2000into NRK52E cells, all the procedures were followed by the instructions. The protein expression of CIP4, Par6, α-SMA and occludin was detected by western blotting. The interaction between CIP4and Par6was detected by immunoprecipitation.Results CIP4over-expression alone without the stimulation of TGF-β1, increased expression of α-SMA and reduced expression of occludin was observed, which was consistent with the results in TGF-β1induced cells. The interaction between CIP4and Par6was detected in TGF-β1treated cells and CIP4over-expression cells, but t not in control cells.Conclusions CIP4may participate the modulation of expression occludin by interacting with Par6.