Fig. 5.
Transplantation of TUDCA-treated CKD-hMSCs protects against kidney fibrosis via upregulation of PrPCin the mouse model of adenine-induced CKD. (A) Treatment TH-1 cells with or without P-cresol cultured alone for 24 h, or co-cultured with Healthy-hMSCs, CKD-hMSCs, TUDCA-treated CKD-hMSCs (TUDCA+CKD-hMSC), TUDCA-treated CKD-hMSCs pretreated with si-PRNP (si-PRNP+CKD-hMSC), or TUDCA-treated CKD-hMSCs pretreated with si-Scr (si-Scr+CKD-hMSC) using culture inserts for 48 h, after analysis of the proliferation of TH-1 cells (n = 3 sample/group). Values represent the mean ± SEM. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. control, ##p < 0.01 vs. P-cresol group, $p < 0.05, and $$p < 0.01 vs. Healthy-hMSC group, && p < 0.01 vs. CKD-hMSC group, AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (B) Treatment of TH-1 cells with or without P-cresol cultured alone or with Healthy-hMSC group, CKD-hMSC group, TUDCA+CKD-hMSC group, si-PRNP+CKD-hMSC group, or si-Scr+CKD-hMSC group using culture inserts; we measured PrPC expression only in lysates of TH-1 by ELISA. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. control, ##p < 0.01 vs. P-cresol group, $p < 0.05, and $$p < 0.01 vs. Healthy-hMSC group, && p < 0.01 vs. CKD-hMSC group, AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (C) The positive signals of MitoSOX were counted by FACS analysis with staining after treatment of TH-1 cells with or without P-cresol cultured alone, or with Healthy-hMSC group, CKD-hMSC group, TUDCA+CKD-hMSC group, si-PRNP+CKD-hMSC group, or si-Scr+CKD-hMSC group. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. control, ##p < 0.01 vs. P-cresol group, $p < 0.05, and $$p < 0.01 vs. Healthy-hMSC group, && p < 0.01 vs. CKD-hMSC group, AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (D, E) Complex Ⅰ and complex Ⅳ activities were analyzed after treatment of TH-1 cells with or without P-cresol cultured alone, or with Healthy-hMSC group, CKD-hMSC group, TUDCA+CKD-hMSC group, si-PRNP+CKD-hMSC group, or si-Scr+CKD-hMSC group. Values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs. control, ##p < 0.01 vs. P-cresol group, $p < 0.05, and $$p < 0.01 vs. Healthy-hMSC group, && p < 0.01 vs. CKD-hMSC group, AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (F) The scheme of the mouse model of CKD. The CKD model mice were fed 0.25% adenine for 1 week, and then we euthanized some mice in each group for validating the CKD model by measurement of BUN and creatinine. Each CKD mouse was injected with PBS or with Healthy-hMSCs, CKD-hMSCs, TUDCA-treated CKD-hMSCs (TUDCA+CKD-hMSC), TUDCA-treated CKD-hMSCs pretreated with si-PRNP (si-PRNP+CKD-hMSC), or TUDCA-treated CKD-hMSCs pretreated with si-Scr (si-Scr+CKD-hMSC) at 0, 5, and 10 days via a tail vein (n = 10 mice/group). After 25 days, measurement of BUN, creatinine, or PrPC was carried out. (G–I) At 25 days after discontinued feeding of adenine, we measured BUN, creatinine, and PrPC in each mouse group in their serum samples (100 μL). Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. Normal, #p < 0.05, and ##p < 0.01 vs. PBS, $p < 0.05 and $$p < 0.01 vs. Healthy-hMSC group, & p < 0.05 and && p < 0.01 vs. CKD-hMSC group, A p < 0.05 and AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (J and K) Regeneration of kidney tissue in CKD was confirmed by H&E staining (J) or Masson's trichrome (K). Scale bar = 100 μm, 400 μm. (L, M) The level of IL-10 or TNF-α in each serum sample was analyzed by ELISA. Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. Normal, #p < 0.05, and ##p < 0.01 vs. PBS, $p < 0.05 and $$p < 0.01 vs. Healthy-hMSC group, & p < 0.05 and && p < 0.01 vs. CKD-hMSC group, A p < 0.05 and AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test). (N) After feeding adenine, macrophage marker (CD11b; red) was analyzed by immunohistochemistry. (O) Values represent the mean ± SEM. *p < 0.05, and **p < 0.01 vs. Normal, #p < 0.05, and ##p < 0.01 vs. PBS, $p < 0.05 and $$p < 0.01 vs. Healthy-hMSC group, & p < 0.05 and && p < 0.01 vs. CKD-hMSC group, A p < 0.05 and AA p < 0.01 vs. si-PRNP+CKD-hMSC group. (One-way ANOVA, using Tukey's post-hoc test).