Effect Of B Cell-activating Factor (BAFF)/BAFFReceptor(BAFF-R) Signal On Mouse Renal Tubular Epithelial Cells And The Potential Mechanism

Part Ⅰ Objective:To establish a method for isolation and culture of mouse primaryrenal tubular epithelial cells(RTECs)with high purityfor futhure study.Methods: The renal cortex was isolated from C57BL/6 mice(4 to 10 weeks old)which weredigested by the collagenase,then filtered with the sieve.The obtained cells were incubated with DMEM/F12 medium containing 10% fetal bovine serum(FBS),1%diabody,1% Insulin-Transferrin-Selenium-X(ITS-X)and 50 nmo L/L hydrocortisone.Cytokeratin-18(CK-18)detection and fluorescein sodium transport experiments were applied to identify the RTECs.Results:Thecultured cells would fuse into a single and closely linkedlayer,with typical polygonal pebble-likeand good refraction.CK-18 detectionresults showed that the cells were stain by yellow-green fluorescence above 90%;Fluorescein sodium transport assays showed that the intracellular fluorescence intensity increased with the prolongation of culture,which suggested that the cells had the ion transport ability of RTECs.Conclusion: In this study,we set up a simple,reproducible mouse primary RTECs culture method,which could obtain high purifiedprimary RTECs.Part Ⅱ Objective :To investigate the expression of B cell activating factor(BAFF)and BAFF receptor(BAFF-R),and the effect on mouse primary RTECs.Methods: After stimulated by 500U/m L IFN-γ for 48 h,the expression of BAFF and BAFF-R on RTECs were detected by FCM.After treated with recombinant mouse BAFF and/or blockade BAFF-R-Fc fusion protein were applied to stimulate RTECs,the transport ability of fluorescein sodium and CK-18 expression of RTECs were detected,respectively;The proliferation ability and apoptosis rates of RTECs were tested by MTS and FCM method,respectively;Data were analyzed by SPSS17.0,and P< 0.05 was considered to be statistically significant.Results: BAFF-R expression on RTECstreated by IFN-γsignificantly up-regulated,compared with control group(4166.250±913.914 vs 3602.750±875.584,P < 0.05).Fluorescein sodium transport experiment showed the average light density of BAFF stimulation group was significantly lower than that of BAFF-R-Fc+BAFF group(0.011±0.003 vs 0.020±0.007,P< 0.05),and control group(0.011±0.003 vs 0.016±0.006,P< 0.05);CK-18 detectionresults showed the average light density of BAFF stimulation group was significantly lower than that of BAFF-R-Fc+BAFF group(0.032±0.005 vs 0.043±0.007,P < 0.05),and control group(0.032±0.005 vs 0.041±0.008,P< 0.05);Cell proliferation assay results showed that,OD value of 5ng/m L and 20ng/m L BAFF group were significantly higher than that of control group(1.532±0.058 vs 1.473±0.045;1.509±0.056 vs 1.473±0.045,P< 0.05,respectively),and OD value of 5ng/m L BAFF group were all significantly higher than that of BAFF-R-Fc+BAFF group(1.532±0.058 vs 1.477±0.050,P< 0.05);Apoptosis test results showed that the apoptosis rate of BAFF-treated group was significantly lower than that of BAFF-R-Fc+BAFF group(39.850%±8.544% vs 42.950%±8.330%,P< 0.05),and control group(39.850%±8.544% vs 44.467%±7.642%,P< 0.05).Conclusion:Enhancement of BAFF signaling could promote the proliferation and inhibit apoptosis of RTECs,but down-regulate the epithelial cell characteristics and ion transport ability of RTECs.Part Ⅲ Objective :To investigate the effect of BAFF signal enhancement on interstitial transformation of RTECs and its potential mechanism.Methods:After treated with recombinant mouse BAFF and/or blockade BAFF-R-Fc fusion protein for 48 h,the total protein of cells were extracted,and the expression of E-Cadherin,α-SMA,β-catenin and Pin1 protein was detected by Western blot;Mouse RTECs were transiently transfected with Pin1 si RNA,then of Pin1 m RNA level was detected by RT-PCR after 48 h,and the expression of Pin1 protein was detected by Western blot after 72h;After treated with recombinant mouse BAFF and/or blockade BAFF-R-Fc for 48 h,the expression of α-SMA,E-cadherin and Pin1 protein in RTECstransfected with Pin1 si RNA or controlsi RNA were detected by Western blot;The protein bands were analyzed by Image J analysis software;Data were analyzed by SPSS17.0,and P< 0.05 was considered to be statistically significant.Results: The expression of α-SMA and Pin1 was up-regulated and the expression of E-Cadherin wasdown regulated in RTECs in recombinant BAFF-stimulated group,and the difference was statistically significant(P< 0.05)compared with the blank control group and BAFF-R-Fc+BAFF stimulation group;The expression of β-catenin protein was not changed significantly,and there was no significant difference between the blank control group and BAFF-R-Fc + BAFF group(P> 0.05).General PCR and Western blot results showed that compared with the control si RNA group and the blank control group,the expression of Pin1 m RNA was down-regulated by about 80% and the expression of Pin1 protein was significantly decreased in mouse RTECs treated by Pin1 si RNA instantaneous transfection.Compared with control si RNA + BAFF group,the expression of E-cadherin protein was significantly up-regulated and the expression of α-SMA protein was significantly down-regulatedin Pin1 si RNA + BAFF stimulation group,and the difference was all statistically significant(P <0.05).Conclusion:BAFF signaling can promote the transformation of RTECs to interstitial cells by upregulating the Pin1 protein.