UPAR-mediated Podocyte Injury:A Discovery Of An Antiproteinuric UPAR Inhibitor&NFAT-uPAR Mediated Podocyte Injury

BACKGROUNDProteinuria is a cardinal sign of kidney disease, but also a risk factor of faster progression of kidney insufficiency. A series of large-scale clinical study confirmed that the amount and duration of proteinuria were closely related to the prognosis of kidney disease directly. As one of the inherent cell of renal glomerulus, podocyte attached to the lateral of the glomerular basement membrane(GBM), constituted the final and the most important glomerular filtration barrier together with GBM and vascular endothelial cell. As the last barrier of glomerulus hemofiltration, Podocyte almost involves in all glomerulus diseases correlating to proteinuia, and play a key role in the progression of proteinuric kidney diseases. As podocyte has became the key cell for the study of proteinuria, it is important to clarify the molecular mechanism of podocyte injury.At present, the studies of key molecules of podocyte barrier mainly focus on:①Complex areas of slit diaphragms(SDs), including nephrin, podocin, CD2AP, ZO-1;②The apical mumbrane zone of podocyte, for example, Podocalyxin and GLEPP-1;③The "basal-group" which touching to Glomerular basement membrane, including integrin and dystroglycan;④Cytoskeletal proteins of podocyte, such as synaptopodin, α-actinin-4, F-actin. Even though the recognition for podocyte structure has made significant progress by studying on monogenic and heritage of hydremic nephritis, the recognition of the injury process of podocyte has not yet made a significant breakthrough. So there is a lack of cell-specific anti-proteinuric therapeutics, now.Recently, the study (Nat Med,2008) found that in the individuals with glomerular diseases, the puromycin aminonucleoside nephrosis(PAN) model and the LPS model of transient proteinuria, the high expression of uPAR in podocyte is required for the increased podocyte motility and proteinuria. uPAR is a glycosylphosphatidylinositol (GPI)-anchored protein the has been shown to be a proteinase receptor for urokinase but has also been involved in nonprotelytic pathways, mainly through its ability to form signaling complexes with other transmembrane proteins such as intrgrins, caveolin and G-protein-coupled receptors. uPAR has important roles in wound healing, inflammation and stem cell mobilization, tumor invasion and metastasis. Further research found that uPAR-deficient mice(plaur-/-mice) are protected from proteinuria in response to LPS and most notably, when uPAR is reconstituted, PLAUR-/-mice develop heavy proteinuria after LPS injection. These data strongly suggest that uPAR is required for the development of proteinuria. So uPAR in podocyte play a key role of the molecular mechanism of proteinuria.Amiloride, a potassium-sparing diuretic, works by directly blocking the epithelial sodium channel (ENaC). Some researches found that in the colon cancer cell line, amiloride blocked its uPAR mRNA and protein synthesis, as to decrease the motility of the cell. And could amiloride inhibit uPAR induction in podocytes and thus decrease the motility of podocytes? We hypothesize that, in the injuried podocytes and proteinuria animal models, amiloride could inhibit uPAR induction in podocytes and thus attenuate proteinuria in kidney diseases. In order to validate the hypothesis above, we established two proteinuria animal models of the LPS model of transient proteinuria and of5/6nephrectomy rats, to view the effects of amiloride on proteinuria and on uPAR expression in vivo, and we also experimented of LPS inducing podocyte in vitro to see the effects of amiloride on uPAR expression and on podocyte migration. We investigated the role of amiloride on anti-proteinuric effect in kidney disease, expectly, to provid evidences for illuminating anti-proteinuric mechanism of amiloride in kidney disease. However, in tempt to open new avenues for the development of antiproteinuric drugs which will be cell-specific therapies in podocytes.Cyclosporine A is an immunosuppressive drug used to treat various proteinuric diseases. The immunosuppressive effect of cyclosporine A results from inhibition of signaling by the transcription factor NFAT in T cells, and this action has also been believed to mediate cyclosporine A’s antiproteinuric effect. However, Faul et al. showed that cyclosporine A 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 WT1mutations with cyclosporine A. So cyclosporine A 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. NFATc1transcriptional activity is modulated by cytoplasmic Ca2+concentration through various Ca2+associated signaling pathways. NFAT family has played an important part in osteoporosis, psoriasis, myocardial hypertrophy and so on. In2010, Wang et al. suggested that conditional NFATcl activation in podoyctes is sufficient to induce proteinuria and glomerulus sclerosis in mice.The results of our first part are as follows. First, cyclosporine A 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, cyclosporine A can inhibit the activation of β3integrin and the expression of uPAR in podocytes in vitro.We wonder how cyclosporine A can reduce proteinuria by inhibit the expression of uPAR.We know that cyclosporine A can reduce proteinuria by inhibit the function of Calcineurin and NFATcl transcriptional 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 cyclosporine A. We first observed the effect of cyclosporine A on the expression of uPAR andp3integrin 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 amiloride on the expression of uPAR in podocytes.1. Mouse podocytes cultureConditionally immortalized mouse podocyte was friendly presented as a gift by Professor Danesh of Baylor Medical College. Podocyte should be cultured on the plates or dishes coated with collagen I. 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 follow(1)Control group:The podocytes were cultured without any treatments;(2)LPS group:The differentiated podocytes were cultured with LPS (50mg·L-1) for24hours; (3)LPS+amiloride group:The podocytes were cultured with LPS (50mg·L-1) and amiloride (0.5mg·L-1) for24hours.4. To observe the effect of amiloride on the expression of uPAR mRNA in podocytes:To extract the total RNA of the groups of podocytes, so detecting the expression of plaur mRNA by real-time PCR.5. To observe the effect of amiloride on the expression of uPAR protein in podocytes:(1)To detect the expression of uPAR protein in podocytes of the different groups by Flow cytometry;(2)To detect the expression of uPAR protein in podocytes of the different groups(con, LPS, LPS+amiloride) by Immunofluorescence;Part two To detect the motility of different groups of differentiated podocytes1. Transwell migration assayThe podocyte were dyed by Methylrosanilinium Chloride, then Photographsed by inverted phase contrast microscope, then podocyte migriation of different groups were observed.2. Wound healing assaySix-well plate was coated with collagen Ⅰ, with stretched preparation sheets on it, then on which podocyte were cultured and treated.After24hours, podocytes were scraped by tip and observing the injure podocyte migration in different groups.Part three To established two proteinuria models (the LPS mouse model of transient proteinuria and proteinuria model of5/6nephrectomy rats), observing the effect of amiloride on proteinuria.1. Construction of the LPS mouse model of transient proteinuriaEighteen C57BL/6male mouses were randomly divided into three groups:normal control group(Con), LPS induced group (LPS) and amiloride treated group(LPS+amiloride), there are six mouse in each group. We injected C57BL/6mice intraperitoneally with200μg LPS in a total volume of500μl. Controls received the same volume of sterile LPS-free saline. For amiloride treatment,we gavage LPS-injected mice with saline alone and amiloride once daily (10mg/kg/day) 2days before LPS injection and1day after LPS injection. We collected the mouse urine for24hours after LPS injection. All were executed at the third day.2. Construction of5/6nephrectomy rat modelFourty-three male Sprague-Dawley rats were randomly divided into three groups: sham operated group(Sham, n=14),5/6nephrectomy model group(NTX, n=14) and5/6nephrectomy with amiloride treated group (NTX+amiloride, n=15). NTX+amiloride group were given amiloride of Smg.kg-1.d-1through intragastric administration after one week of5/6nephrectomy. NTX group and Sham group, respectivly, were given the same volume vehicle through intragastric administration after one week of5/6nephrectomy and sham operation.3. Evaluation of urinary protein in different groupsThe24hours urine of mouses model induced proteinuria by LPS were collected from the second day and were detected for the concentration of protein, then the mouses were executed. The24hours urine in5/6nephrectomy rats proteinuria model were remained at the second week,fourth week, eighth week and twelfth week, detecting the proteinuria and observing the renal tissue morphology on different time point.4. Evaluation of the expression of synaptopodin and uPAR of renal tissue in two proteinuria animal models htrough confocal microscope.5. To extract the total RNA of renal tissue of two animal models, so detecting the expression of plaur mRNA of different groups by real-time PCR.Part four 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 five In vitro podocyte study1. The groups of differentiated podocytes were treated as follows1) Control group:The podocytes were cultured with the same volume DMSO 2) LPS group:The podocytes were cultured with LPS (50mg·L-1) for24hours;3) NFATcl-siRNA group:The podocytes were cultured with NFATcl-siRNA (20nM、50nM、80nM) for48hours;4) LPS+NFATc1siRNA(50nM) group:The podocytes were cultured with LPS (50mg·L-1) for24hours and NFATcl-siRNA (50nM) for48hours;5) 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(luM) 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.2. Effect of NFAT manipulation on the the expession of uPAR 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 six Effect of NFAT manipulation on the motility of different groups of differentiated podocytes Wound 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 seven 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.RESULTS1. Identification of differentiated podocytes.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-γ, then it matured with cell body greaten, divided into primary and secondary foot process and expressed synaptopodinas differentiated podocyte.2. The effect of amiloride on the expression of uPAR in podocytes.1) The effect of amiloride on the expression of uPAR mRNA in podocytes:The mRNA expression of gene Plaur were detected by real-time quantitative PCR. Compared to the expression of uPAR mRNA of LPS group, there was downregulated in Con group(2.41±0.32vs1.00±0.00, P=0.024) and LPS+amiloride group (2.41±0.32vs1.33±0.21,98.33%CI0.41-2.15), with significantly statistical significance.2) The expression of uPAR protein in podocytes of different groups:As expected, compared to LPS group, the fluorescence intense were lower in Con group and LPS+amiloride group. And there were the same fluorescence intense between Con group and LPS+amiloride group. We found low expression of uPAR protein in Con group. In contrast, LPS group had a significant increase in uPAR cell surface expression expression [(50.74±6.78)%vs (10.34±3.25)%, P<0.05]. Of note, we found that, when treated with amiloride, LPS+amiloride group showed a reduced uPAR cell surface expression expression [(50.74±6.78)%vs (29.97±10.26)%, P<0.05]2. The effect of amiloride on podocyte motility1) Transwell migration assayThe average number of migrating podocytes were higher in LPS group (324.25±14.97) than Con group (248.75±10.11) and LPS+amiloride group (274.5±8.35), with significantly statistical significance(all P<0.05).2) Wound healing assayIn order to analyze podocyte molitiy directly, we used Wound healing assay to detect the repairing ability of injury podocyte. We found that the average number of podocytes was higher in LPS group(42.6±6.88) than Con group (15±4.74) and LPS+amiloride group(18.4±6.35), with significantly statistical significance(all P<0.05), but there were no significantly statistical significance between Con group and LPS+amiloride group (PP>0.05).3. Amiloride could reduce the proteinuria of the LPS mouse model of transient proteinuria and proteinuria model of5/6nephrectomy rats1) Proteinuria in diffrent groups of the LPS mouse model of transient proteinuria.Comparing with Con group, the24h urine protein of LPS group was significantly increased [(4.12±1.06) mg vs (1.35±0.68) mg; P<0.05]. In contrast, proteinuria in LPS mice treated with amiloride was significantly lower than in LPS group[(4.12±1.06) mg vs (1.99±0.96) mg; P<0.05]. And there were no statistical significance between LPS+amiloride group and Con group(P>0.05).2) The change of proteinuria in diffrent groups of proteinuria model of5/6nephrectomy rats.At second week, compared with Con group, the24h urine protein of NTX group was not significantly increased [(47.50±28.05) mg vs (14.28±3.8) mg,P>0.05]. And there was distinguished statistical significance about24-hour urine protein between NTX+amiloride group and NTX group [(51.56±21.03) mg vs (47.50±28.05) mg, P<0.05]. At fourth week, compared to NTX group, the24h urine protein was no distinguished statistical significance in Sham group[(50.09±22.34) mg vs (21.77±5.29) mg, P>0.05] and NTX+amiloride group[(50.09±22.34) mg vs (33.52±10.11) mg, P>0.05]. At eighth week, comparing with NTX group,24-hour urine protein was lowered in Sham group[(162.39±27.62) mg vs (19.38±8.26) mg, P<0.05] and NTX+amiloride group[(162.39±27.62) mg vs (109.22±31.26) mg, P<0.05], with statistical significance. The same situation was also observed at the twelfth week, comparing with NTX group,24-hour urine protein decreased in Sham group[(188.31±29.82) mg vs (21.32±8.59) mg, P<0.05] and NTX+amiloride group[(188.31±29.82) mg vs (121.37±31.14) mg, P<0.05], with statistical significance.3) Amiloride could inhibit the expression of uPAR protein of podocyte both in the LPS mouse model of transient proteinuria and proteinuria model of5/6nephrectomy ratsObserved by confocal microscop, the specific skeleton protein-Synaptopodin mainly expressed in renal podocyte and uPAR generally expressed in renal tubule and glomerulus in Con group. But fluorescence intensity was higher and the expression of uPAR protein increased in LPS 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 amiloride, the fluorescence intensity was lowered and the expression of uPAR expression was downregulated in LPS+amiloride group.Also in the proteinuria model of5/6nephrectomy rats, the fluorescence intensity was higher and the expression of the uPAR protein increased in the NTX group, which was localized in podocytes, as indicated by colabeling with synaptopodin, a marker of this cell type; compared with NTX group, the fluorescence intensity was lowered and the expression of the uPAR protein downregulated in Sham group and NTX+amiloride group.4) Amiloride could inhibit the expression of uPAR mRNA both in the LPS mouse model of transient proteinuria and proteinuria model of5/6nephrectomy ratsIn the LPS mouse model of transient proteinuria, as expected, comparing with Con group, the expression of uPAR mRNA of LPS group was significantly increased [(2.12±0.35) vs (1.04±0.14), P<0.05]. In contrast, the expression of uPAR mRNA in LPS mice treated with amiloride was significantly lower than in LPS group[(2.12±0.35) vs (1.30±0.22), P<0.05].In the proteinuria model of5/6nephrectomy rats, comparing with Sham group, the expression of uPAR mRNA of NTX group was significantly increased [(9.74±1.44) vs (1.01±0.13), P<0.05]. In contrast, the expression of uPAR mRNA in NTX rats treated with amiloride was significantly lower than in NTX group[(9.74±1.44) v (5.01±1.36), P<0.05].4. 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 proteinoria model of5/6nephrectomy1)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.2)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.5. CsA could inhibit the expression of uPAR but not the expression of beta3-integrin of podocyte in podocytes in vitro1)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.2)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 group、LPS group、LPS+CsA(0.25) group、LPS+CsA(0.5) group and LPS+CsA(1) group, with no statistical significance(P>0.05).6. NFATc1knockdown by podocyte transfection with NFATc1siRNA could inhibit the expression of uPAR, suppress β3integrin activation but not its surface expression in podocytes in vitro1)NFATc1knockdown 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).2)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+NFATcl-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).7. Ionomycin(NFAT activator)could increase the expression of uPAR, increase β3integrin activation but not its surface expression in podocytes in vitroIonomycin(NFAT activator)could increase the expression of uPAR in podocytes in vitro:The mRNA expression of Plaur was detected by quantitate-PCR, Plaur mRNA in Ionomycin group was significantly higher than in Con group(.P<0.05). Flow cytometry was used to detect the protein expression of uPAR in podocytes, uPAR expression in Ionomycin(500nM) group,lonomycin(1uM) group and Ionomycin(2uM) group was significantly higher than in Con group (all P=0.000) with statistical significance.8.Ionomycin(NFATcl activator)could increase 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, Ionomycin(500nM) group,Ionomycin(luM) group and lonomycin(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 group、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.9.11R-VIVIT(NFAT inhibitor) could inhibit the expression of uPAR, suppress β3integrin activation but not its surface expression in podocytes in vitro1)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, 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, 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.2)11R-VIVIT(NFATc1inhibitor) 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, 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 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 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)10. Effect of NFAT manipulation on podocyte motilityThe average number of cells was higher in LPS group and Ionomycin(luM) 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.CONCLUSIONS1. Amiloride can inhibit the induction of uPAR expression in podocytes by blocking uPAR mRNA and protein synthesis.2. Amiloride decreases podocyte motility, thus stabilizing podocyte and preventing it from injury.3. Amiloride could reduce the proteinuria of the LPS mouse model of transient proteinuria and proteinuria model of5/6nephrectomy rats by inhibiting the induction of uPAR expression.4. CsA could inhibit the expression of uPAR, suppress beta3integrin activation but not its surface expression of podocyte in vivo and vitro.5. NFAT mediates uPAR-beta3integrin signaling, which may be the mechanism for CsA’s anti-proteinuric effect.