| BACKGROUNDGlomerulus filtration barrier is composed of glomerular endothelial cells (GeCs), the glomerular basement membrane (GBM), and podocytes. Proteinuria occurs when the permeability of the glomerular barrier is increased. Proteinuria is a cardinal sign and a prognostic marker of kidney disease[1].Disfunction of podocyte exits in many human kidney diseases and Kidney disease animal models of macroalbuminuria. Effacement and fusion of the podocyte foot processes is a common and most important feature of podocyte morphologic alteration. As a result,podocyte has always being the focus in studying the mechanism of proteinuria in recent years [2]. Cytoskeleton reorganization of podocyte will cause foot processes fusion and proteinuria[3].uPAR(urokinase-type plasminogen activator receptor)is a glycosylphosphatidy linositol (GPI)-anchored glycoprotein and has important roles in wound healing, inflammation,tumor invasion and metastasis[4].In2008,Wei C etc. found that in the individuals with glomerular diseases, the puromycin aminonucleoside nep hrosis(PAN) model and the LPS model of transient proteinuria, the high expres sion of uPAR, together with activation ofβ3integrin in podocyte is required f or the increased podocyte motility and proteinuria[5]. In2011,Wei C etc.found that soluble uPAR may involved in the pathogenesis of FSGS[6]. From this, i t can be seen that uPAR and emerge of proteinuria may be closely relatedciclosporin is an immunosuppressive drug used to treat various proteinuric diseases[7]. The immunosuppressive effect of ciclosporin results from inhibition of signaling by the transcription factor NFAT in T cells, and this action has also been believed to mediate ciclosporin’s antiproteinuric effect. However, Faul et al.[8]showed that Ciclosporin blocked cathepsin-L-mediated proteolysis of the actin-organizing protein synaptopodin caused by calcineurin and reduced proteinuria. It happens that there is a similar case. The study of STEFANIDIS et al. showed that podocyte is a target in the management of the nephrotic syndrome caused by WTl mutations with cyclosporin A[9].So ciclosporin has a direct antiproteinuric effect on podocytes.NFAT(nuclear factor of activated T cells)was first found in T cell as a nuclear factor which can binds to the promoter of the human IL-2gene. NFATc1is a member of NFAT family.Increases in cytoplasmic Ca2+concentration induce NFAT dephosphorylation and NFAT translocation to the nucleus where it binds to cis regulatory elements of target genes[12]. NFATc1transcriptional activity is modulated by cytoplasmic Ca2+concentration through various Ca2+associated signaling pathways [13]. NFAT family has played an important part in osteoporosis[14]> psoriasis[15]> myocardial hypertrophy[16] and so on.In2010, Wang et al. suggested that conditional NFATc1activation in podoyctes is sufficient to induce proteinuria and glomerulus sclerosis in mice. The results of our first part are as follows. First, Cyclosporin can reduce proteinuria of mouse induced by LPS and of rat for5/6nephrectomy.Second, cyclosporin can inhibit the activation of β3integrin in both animal models.Third, cyclosporin can inhibit the activation of β3integrin and the expression of uPAR in podocytes in vitro.We wonder how cyclosporine can reduce proteinuria by inhibit the expression of uPAR.We know that cyclosporine can reduce proteinuria by inhibit the function of Calcineurin and NFATc1transcriptional activity is modulated by Calcineurin. It is possible, however, that the suppression of NFAT signaling in podocytes also plays a role in the antiproteinuric effect of CsA. We first observed the effect of CsA on the expression of uPAR andβ3integrin in mouse proteinuria model induced by LPS and rats proteinuria model of5/6nephrectomy. we also experimented of LPS inducing podocyte in vitro, to see the effects of NFATc1on uPAR-β3integrin signaling by designing and synthesising NFATc1siRNA, Promoting and inhibiting NFATc1activation. We preliminarily investigated the role of CsA on nonimmune anti-proteinuric effect.METHODSPart one To observe the effect of CsA on the expression of uPAR and β3integrin in mouse proteinuria model induced by LPS and rats proteinuria model of5/6nephrectomy. Both of the animal models were established by our group and frozen sections of kidney tissue were preserved in the freezer at-80℃. Evaluation the expression of uPAR and β3integrin through confocal microscope.Part two Culturing, identification and dividing subgroup of podocyte1. Mouse podocytes cultureConditionally immortalized mouse podocyte friendly present as a gift by Professor Danesh of Baylor Medical College. Podocyte should be cultured on the plates or dishes coated with collagen â… . Mother podocyte should be incubated at33℃ in5%CO2, with10%FBS RPMI1640(containing20-100U·mL-1INF-gamma) to pass, and those podocytes were grown under "growth permissive" condition.Then podocytes were cultured at37℃in5%CO2,with10%FBS DMEM (without INF-gamma)for differentiation. For10-14days, the cells became differentiated and acquired a quiescent phenotype, being ready to experiment.2. Identification and morphology observation of differentiated podocyteWe photographed the growing podocyte cultured in RPMI1640with INF-gamma at33℃and the differentiated podocyte cultured in DMEM without INF-gamma at37℃for10-14days, observing the variance of podocytes morphology in different situation.Cells Which expressed synaptopodin were differentiated and quiescent phenotype podocytes.3. The groups of differentiated podocytes were treated as follows1) Control group:The podocytes were cultured with the same volume DMSO2) LPS group:The podocytes were cultured with LPS (50mg·L-1) for24hours;3) NFATc1-siRNA group:The podocytes were cultured with NFATcl-siRNA (20nMã€50nMã€80nM) for48hours;4) LPS+NFATcl siRNA(50nM) group:The podocytes were cultured with LPS (50mg·L-1) for24hours and NFATc1-siRNA (50nM) for48ho5) LPS+CsA(0.25) group:The podocytes were cultured with LPS (50mg·L-1)and CsA (0.25mg·L-1) for24hours:6) LPS+CsA(0.5) group:The podocytes were cultured with LPS (50mg·L-1)and CsA (0.5mg·L-1) for24hours;7) LPS+CsA(1) group:The podocytes were cultured with LPS (50mg·L-1)and CsA (1mg·L-1) for24hours; 8) Ionomycin(500nM) group:The podocytes were cultured with Ionomycin(500nM) for1hours;9) Ionomycin(1uM) group:The podocytes were cultured with Ionomycin(1uM) for1hours;10) Ionomycin(2uM) group:The podocytes were cultured with Ionomycin(2uM) for1hours;11)LPS+11R-VIVIT(10nM) group:The podocytes were cultured with LPS (50mg·L-1)and11R-VIVIT(10nM) for24hours;12)LPS+11R-VIVIT(100nM) group:The podocytes were cultured with LPS (50mg·L-1)and11R-VIVIT(100nM) for24hours;13)LPS+11R-VIVIT(1000nM) group:The podocytes were cultured with LPS (50mg·L-1)and11R-VIVIT(1000nM) for24hours.Part three To evaluate the the expession of uPARã€expression and activation of beta3-integrin.To investigate the expression and activation of beta3-integrin and the expession of uPAR by Immunofluorescence, Flow cytometry and quantitate-PCR.Part four To detect the motility of different groups of differentiated podocytesWound healing assay:Six-well plate was coated with collagen I, with stretched preparation sheets on it, then on which podocyt were cultured. Observing the injure podocyte migration in different groups after treatment by Immunofluorescence.Part five Statistical AnalysesAll measurement data were expressed as mean±SD. Statistical analyses were conducted with SPSS13.0for Windows, comparing continuous variables of groups used One-way ANOVA,LSD method was used as multiple comparison for homoscedasticity, and Dunnett’s T3method was used as multiple comparison for heterogeneity of variance.Significance was difined as P<0.05. RESULTS一ã€CsA could inhibit the expression of uPAR but not the expression of beta3-integrin of podocyte in mouse proteinuria model induced by LPS and rats proteinuria model of5/6nephrectomy(一) CsA could inhibit the expression of uPAR of podocyte in mouse proteinuria model induced by LPS and rats proteinuria model of5/6nephrectomy: Observed by confocal microscop, the specific skeleton protein-Synaptopodin mainly expressed in renal podocyte and uPAR generally expressed in renal tubule and glomerulus in Con and Sham group. But fluorescence intensity was higher and the expression of uPAR protein increased in LPS and NTX group, which was localized in podocytes. as indicated by colabeling with synaptopodin, a marker of this cell type. In contrast, we found that, when treated with CsA, the fluorescence intensity was lowered and the expression of uPAR expression was downregulated in LPS+CsA and NTX+CsA group.(二) CsA had no effect on the expression of beta3-integrin of podocyte in mouse proteinuria model induced by LPS and rats proteinuria model of5/6nephrectomy:Observed by confocal microscop, the specific skeleton protein-Synaptopodin mainly expressed in renal podocyte and beta3-integrin generally expressed in renal tubule and glomerulus in Con and Sham group.compared with Con and Sham group,the beta3-integrin expression in LPS and NTX group had Little Change,so were LPS+CsA and NTX+CsA group.二ã€The result of identification and observing morphocytology of podocyte. Podocyte cultured in33℃,1640medium containing IFN-γ,was seen a morphous as cobblestone or branch, not expressing synaptopodin and being growth podocyte.When it was cultured10-14days in37℃without IFN-y, then it matured with cell body greaten, divided into primary and secondary foot process and expressed synaptopodinas differentiated podocyte.三ã€CsA could inhibit the expression of uPAR but not the expression of beta3-integrin of podocyte in podocytes in vitro(一) CsA could inhibit the expression of uPAR in podocytes in vitro: Observed by confocal microscop, compared to LPS group, the fluorescence intense were lower in Con group and LPS+CsA group. And there were the same fluorescence intense between Con group and LPS+CsA group. Flow cytometry was used to detect the protein expression of uPAR in podocytes, uPAR expression in LPS group was significantly higher than in Con group (P=0.002), with statistical significance. when treated with CsA, uPAR expression in LPS+CsA(0.5)(P=0.002) and LPS+CsA(1)(P=0.002) group were lower than in LPS group, with statistical significance.(二) CsA had no effect on the expression of beta3-integrin in podocytes in vitro: Flow cytometry was used to detect the protein expression of beta3-integrin in podocytes,there were no difference between con groupã€LPS groupã€LPS+CsA(0.25) groupã€LPS+CsA(0.5) group and LPS+CsA(1) group, with no statistical significance(allP>0.05).å››ã€NFATcl knockdown by podocyte transfection with NFATcl siRNA could inhibit the expression of uPAR, suppress β3integrin activation but not its surface expression in podocytes in vitro(一) NFATcl knockdown could inhibit the expression of uPAR in podocytes in vitro:The mRNA expression of Plaur was detected by quantitate-PCR, Plaur mRNA in LPS group was significantly higher than in Con group (P=0.000), with statistical significance. when treated with NFATcl-siRNA, Plaur mRNA in LPS+NFATcl-siRNA(50nM) group were lower than in LPS group (P=0.000), with statistical significance. Flow cytometry was used to detect the protein expression of uPAR in podocytes, uPAR expression in LPS group was significantly higher than in Con group (P=0.019), with statistical significance. when treated with NFATc1-siRNA, uPAR expression in LPS+NFATcl-siRNA(50nM) group were lower than in LPS group (P=0.045), with statistical significance.(二) NFATc1knockdown could suppress β3integrin activation but not its surface expression in podocytes in vitro:The mRNA expression of gene Itgb3was detected by quantitate-PCR. There were no significantly statistical significance of beta3-integrin mRNA expression between Con group, LPS group and LPS+NFATc1-siRNA(50nM) group (all p>0.05). Flow cytometry was used to detect the protein expression of beta3-integrin in podocytes,there were no difference between con group, LPS group and LPS+NFATcl-siRNA(50nM) group (all p>0.05). Flow cytometry was used to detect the expression of activated beta3-integrin in podocytes, activated beta3-integrin expression in LPS group was significantly higher than in Con group (P=0.000), with statistical significance. when treated with NFATc1-siRNA, activated beta3-integrin expression in LPS+NFATcl-siRNA(50nM) group were lower than in LPS group (P=0.000), with statistical significance.五Ionomycin(NFATc1activator)could increase the expression of uPAR, increase P3integrin activation but not its surface expression in podocytes in vitro(一) Ionomycin(NFATc1activator)could increase the expression of uPAR in podocytes in vitro:The mRNA expression of Plaur was detected by quantitate-PCR, Plaur mRNA in Ionomycin(2uM) group was significantly higher than in Con group (P=0.010), with statistical significance. Flow cytometry was used to detect the protein expression of uPAR in podocytes, uPAR expression in Ionomycin(500nM) group,Ionomycin(luM) group and Ionomycin(2uM) group was significantly higher than in Con group (all P=0.000), with statistical significance.(二)Ionomycin(NFATc1activator)could increase (33integrin activation but not its surface expression in podocytes in vitro:The mRNA expression of gene Itgb3was detected by quantitate-PCR. There were no significantly statistical significance of beta3-integrin mRNA expression between Con group, Ionomycin(500nM) group,Ionomycin(luM) group and Ionomycin(2uM) group (all p>0.05). Flow cytometry was used to detect the protein expression of beta3-integrin in podocytes,there were no difference between con groupã€Ionomycin(500nM) group,Ionomycin(luM) group and Ionomycin(2uM) group (all p>0.05). Flow cytometry was used to detect the expression of activated beta3-integrin in podocytes, activated beta3-integrin expression in Ionomycin(500nM) group,Ionomycin(luM) group and Ionomycin(2uM) group was significantly higher than in Con group (P=0.024, P=0.000, P=0.000), with statistical significance.å…11R-VIVIT(NFATc1inhibitor) could inhibit the expression of uPAR, suppress β3integrin activation but not its surface expression in podocytes in vitro(一)11R-VIVIT(NFATcl inhibitor) could inhibit the expression of uPAR in podocytes in vitro:The mRNA expression of Plaur was detected by quantitate-PCR, Plaur mRNA in LPS group was significantly higher than in Con group (P=0.014) with statistical significance. when treated with11R-VIVIT, Plaur mRNA in LPS+11R-VIVIT(100nM) group and LPS+11R-VIVIT(1000nM) group were lower than in LPS group (P=0.022, P=0.003), with statistical significance. Flow cytometry was used to detect the protein expression of uPAR in podocytes, uPAR expression in LPS group was significantly higher than in Con group (P=0.000), with statistical significance. when treated with11R-VIVIT, uPAR expression in LPS+11R-VIVIT(100nM) group and LPS+11R-VIVIT(1000nM) group were lower than in LPS group (P=0.000, P=0.000), with statistical significance.(二)11R-VIVIT(NFATc1inhibitor) could suppress (33integrin activation but not its surface expression in podocytes in vitro:The mRNA expression of gene Itgb3was detected by quantitate-PCR. There were no significantly statistical significance of beta3-integrin mRNA expression between Con group, LPS group, LPS+11R-VIVIT(10nM) group,LPS+11R-VIVIT(100nM) group and LPS+11R-VIVIT(1000nM) group (all p>0.05). Flow cytometry was used to detect the protein expression of beta3-integrin in podocytes,there were no difference between con groups LPS group XPS+11R-VIVIT(10nM) group,LPS+11R-VIVIT(100nM) group and LPS+11R-VIVIT(1000nM) group (all p>0.05). Flow cytometry was used to detect the expression of activated beta3-integrin in podocytes, activated beta3-integrin expression in LPS group was significantly higher than in Con group (P=0.01), with statistical significance, when treated with11R-VIVIT, activated beta3-integrin expression in LPS+11R-VIVIT(100nM) group and LPS+11R-VIVIT(1000nM) group were lower than in LPS group(P=0.005, P=0.011), with statistical significance.七Detect the change of podocyte motility by Wound healing assayThe average number of cells was higher in LPS group and Ionomycin(1uM) group than in Con group (P=0.000), with significantly statistical significance.Compared with LPS group, the average number of cells in LPS+siRNA(50nM) group,LPS+11R-VIVIT(100nM) group and LPS+CsA (0.5mg·L-1) group were reduced(allP=0.000), with significantly statistical significance.CONCLUSIONS1CsA could inhibit the expression of uPAR, suppress beta3integrin activation but not its surface expression of podocyte in vivo and vitro.2CsA could protect podocytes and reduce proteinuria by inhibiting NFATc1mediated uPAR-beta3integrin signaling. |
