To further enhance the long-term outcome of underlying recipients and allografts, it is necessary to illuminate the pathomechanism. Results suggested that BAFF-R was significantly upregulated upon IFN- stimulation, and enhancement of BAFF signaling promoted cell survival and reduced their apoptotic rate, while simultaneously reducing the epithelial phenotype and promoting the interstitial transformation of cells. Furthermore, Pin1 was significantly increased, along with the upregulation of BAFF signaling in the RTECs, and participated in interstitial transformation induced by BAFF signaling. Collectively, the present results elucidate the potential mechanism of loss of normal function of RTECs under long-term high dose of BAFF stimulation provides a potential therapeutic target for renal interstitial fibrosis, and underlining mechanisms of shortening of long-term outcomes of kidney allografts via augmenting of BAFF signaling. test, with em P /em 0.05 considered statistically significant. Results Identification of primary mouse RTECs The primary cells we obtained had a typical polygonal cobblestone-like morphology, with tight junctions among them, with good refraction and relatively large sizes (Fig. ?(Fig.11 em A /em ). Open in a separate window Physique 1. Identification of mice primary renal tubular epithelial cells (RTECs). ( em A /em ) The cells displayed a typical polygonal cobblestone-like morphology, with good refraction and a relatively large cell size (400). ( em B /em ) Immunofluorescence staining revealed that this rate ST 2825 of CK-18 positivity in the primary cells was 90%. ( em C /em ) The ability of Na+ transfer was also tested through an immunofluorescence assay. Intracellular fluorescence intensity gradually increased with time. ( em D /em ) The growth curve of primary RTECs. The cells took about 4 d to overgrow the flask and continued to grow well for nearly 10 d. ( em E /em ) BAFF-R expression was detected by flow cytometry every 2 d. BAFF-R was stably expressed on primary RTECs during the initial 14 incubation days, and then it went down. ( em F /em ) CK-18-positive population (%) through the initial 16 incubation SMARCA4 days, stained by immunofluorescence assays. It was shown that CK-18 was stably expressed for about 14 d, and then it went down. CK-18 was particularly expressed on epithelial cells, potentially serving as a biomarker of primary RTECs. Immunofluorescence staining revealed that this rate of CK-18 positivity in the primary cells was 90% (Fig. ?(Fig.11 em B /em ). Ion transport function is usually another important function of epithelial cells. We investigated the Na+ transferability through immunofluorescence. We observed a gradual increase in the intracellular fluorescence intensity with time (Fig. ?(Fig.11 em C /em ). To observe the growth status of primary RTECs, we counted the cell number every 2 d. The results suggested that cells could grow well for about 14 d (3 generations) (Fig. ?(Fig.11 em D /em ). And BAFF-R ST 2825 expression was stably on primary RTECs through the initial 3 generations, and then it went down (Fig. ?(Fig.11 em E /em ). Moreover, the expression rate of CK-18 was basically consistent with BAFF-R (Fig. ?(Fig.11 em F /em ). BAFF-R was significantly upregulated on primary RTECs after IFN- stimulation FCM assays revealed a significant increase in the mean fluorescence intensity (MFI) of BAFF-R on RTECs after IFN- stimulation (4166.25913.91 vs 3602.75162.47.58, respectively; em P /em 0.05); however, the expression of BAFF, BCMA, and TACI did not vary significantly (1027.4098.5 vs 1066.40143.42, 2392.32178.21 vs 2543.23209.18, 1629.84197.35 vs 1737.64338.05, respectively; em P /em 0.05) (Fig. ?(Fig.22). Open in a separate window Physique 2. BAFF-R was significantly upregulated on primary renal tubular epithelial cells (RTECs) after IFN- stimulation. Flow cytometry ST 2825 assays indicated that this mean fluorescence intensity of BAFF-R on RTECs significantly increased after IFN- stimulation (* em P /em 0.05). However, the expression of BAFF, BCMA, and TACI expression did not vary significantly ( em P /em 0.05). BAFF upregulation promoted proliferation and inhibited apoptosis in primary RTECs The MTS assay revealed that, compared with blank control group (cont) and BAFF-R-Fc alone treatment group, 5 ng/mL and 20 ng/mL rBAFF significantly promote proliferation in primary RTECs ( em P /em 0.05), but the effect was not dose-dependent. Pre-treatment with BAFF-R-Fc fusion protein significantly.