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. 2021 Jun;57(6):649-659.
doi: 10.1007/s11626-021-00598-y. Epub 2021 Jun 14.

BAFF signaling drives interstitial transformation of mouse renal tubular epithelial cells in a Pin1-dependent manner

Haiyan Xu  1 , Dan Song  1 , Renfang Xu  1 , Xiaozhou He  2
Affiliations

BAFF signaling drives interstitial transformation of mouse renal tubular epithelial cells in a Pin1-dependent manner

Haiyan Xu et al. In Vitro Cell Dev Biol Anim. 2021 Jun.

Abstract

Aberrant expression of B cell-activating factor belonging to TNF superfamily (BAFF) and its receptors results in abnormal biological activities in hematopoietic and non-hematopoietic cells and is closely associated with the occurrence and development of various diseases. However, the biological significance and potential mechanisms underlying BAFF signaling in renal tubular epithelial cells (RTECs) remain unknown. This study aimed to investigate the biological role of BAFF signaling in RTECs. Mice primary RTECs were applied. The proliferation status and apoptotic rates were examined by MTS assay and flow cytometry, respectively. The expression of BAFF and its receptors was analyzed via flow cytometry and sodium ion transport function, and cytokeratin-18 expression was detected through immunofluorescence staining. In addition, Pin1 was knocked down via siRNA and its expression was assessed through reverse transcription PCR. Lastly, western blotting was performed to analyze E-cadherin, ɑ-SMA, and Pin1 expression. Results suggested that BAFF-R was significantly upregulated upon IFN-γ 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.

Keywords: BAFF; Interstitial transformation; Pin1; Renal tubular epithelial cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Identification of mice primary renal tubular epithelial cells (RTECs). (A) The cells displayed a typical polygonal cobblestone-like morphology, with good refraction and a relatively large cell size (400×). (B) Immunofluorescence staining revealed that the rate of CK-18 positivity in the primary cells was >90%. (C) The ability of Na+ transfer was also tested through an immunofluorescence assay. Intracellular fluorescence intensity gradually increased with time. (D) 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. (E) 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. (F) CK-18-positive population (%) through the initial 16 incubation days, stained by immunofluorescence assays. It was shown that CK-18 was stably expressed for about 14 d, and then it went down.
Figure 2.
Figure 2.
BAFF-R was significantly upregulated on primary renal tubular epithelial cells (RTECs) after IFN-γ stimulation. Flow cytometry assays indicated that the mean fluorescence intensity of BAFF-R on RTECs significantly increased after IFN-γ stimulation (*P<0.05). However, the expression of BAFF, BCMA, and TACI expression did not vary significantly (P>0.05).
Figure 3.
Figure 3.
Upregulation of BAFF signaling promotes proliferation and decreases apoptosis in primary renal tubular epithelial cells (RTECs). (A) MTS assay indicated that 5 ng/mL and 20 ng/mL rBAFF significantly promoted proliferation in primary RTECs (*P<0.05, respectively), but the effect was not dose-dependent; pre-treatment with BAFF-R-Fc fusion protein significantly mitigated the effect of rBAFF on RTECs, compared to 5 ng/mL rBAFF treatment (*P<0.05). (B) rBAFF stimulation significantly inhibited apoptosis in RTECs, compared to the control groups (*P<0.05). Furthermore, pre-treatment with BAFF-R-Fc fusion protein significantly reduced the effect of rBAFF on RTECs (*P<0.05).
Figure 4.
Figure 4.
Upregulation of BAFF signaling decreased the epithelial characteristics of primary renal tubular epithelial cells (RTECs). (A) BAFF upregulation reduced CK-18 expression, compared to the control cells (*P<0.05); however, pre-treatment with BAFF-R-Fc fusion protein significantly mitigated the effect of BAFF on CK-18 expression, compared to rBAFF treatment (*P<0.05). (B) BAFF upregulation significantly decreased the transferability of Na+ in RTECs (*P<0.05). Furthermore, pre-treatment with BAFF-R-Fc fusion protein significantly rescued Na+ transferability in RTECs, which had initially decreased because of rBAFF stimulation (*P<0.05).
Figure 5.
Figure 5.
Upregulation of BAFF signaling stimulated interstitial transformation in primary renal tubular epithelial cells (RTECs). (A) The original western blot. The experimental groups were rBAFF stimulation, BAFF-R-Fc protein treatment and rBAFF stimulation group, and control group. E-cadherin (E-cad), β-catenin (β-cat), and ɑ-SMA were detected, and β-actin was considered the internal reference. (B, C, D) The results of grayscale scanning indicated that rBAFF significantly downregulated E-cadherin but significantly upregulated ɑ-SMA, and pre-treatment of BAFF-R-Fc protein mitigated the effect of rBAFF on primary RTECs (*P<0.05). No significant effect of rBAFF and/or BAFF-R-Fc protein treatment was observed.
Figure 6.
Figure 6.
Pin1 was upregulated with the upregulation of BAFF signaling. (A) The original images of western blot. The experimental groups were the same as those indicated in Fig. 5. (B) The grayscale scanning results indicate that rBAFF stimulation significantly upregulated the expression of Pin1 (*P<0.05; **P<0.01).
Figure 7.
Figure 7.
Pin1 is involved in interstitial transformation during BAFF signaling. (A) The original images of western blot. The effect of Pin1 siRNA and the control siRNA treatment were observed, in combination with rBAFF and BAFF-R-Fc protein treatment groups. (B) The grayscale scanning results suggest that Pin1 siRNA knocked down Pin1 mRNA (**P<0.01), and no similar effect of control siRNA was observed (*P<0.05; **P<0.01). (C) Pin1 siRNA treatment reversed the effect of rBAFF stimulation of downregulating E-cadherin (*P<0.05). (D) Pin1 siRNA treatment reversed the effect of rBAFF stimulation of upregulating ɑ-SMA (*P<0.05; **P<0.01).
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