PRDM16 acts as a homeostasis regulation factor to suppress the transition of AKI to CKD via upregulation of eukaryotic initiation factor 6

Fig. 1. Expression of PRDM16 exposed to TGF-β1 in BUMPT cells and the kidneys of UUO mice and individuals with OB. A-B The mRNA levels of PRDM16, as determined by RT-qPCR, in BUMPT cells with and without TGF-β stimulation, along with the renal tissue of UUO mice. C-D WB of PRDM16 and β-Tubulin in BUMPT cells subjected to TGF-β1 treatment or left untreated, as well as in kidney samples from UUO mice. E–F The grayscale analysis (GSY) between them. H Immunofluorescence examination to visualize PRDM16 expression and distribution in BUMPT cells. J Relative fluorescence intensity. Quantification of the PRDM16 -positive cells. Original magnification × 600. Scale bar: 20 µM. Data are denoted as the means ± SD (n = 6). #p < 0.05, vs. control cohort

Fig. 2. PRDM16 facilitated the TGF-β1-induced expression of fibronectin collagen I and collagen III in BUMPT cells. BUMPT cells underwent transfection with DOX and PRDM16 siRNA, then exposed to 5 ng/ml TGF-β1 or left untreated for 24 h. A WB of PRDM16, FN, HA, collagen I, collagen III, and β-Tubulin in BUMPT cells with or without TGF-β1 and DOX treatment. B-E The GSY between them. F WB of PRDM16, FN, collagen I, collagen III, and β-Tubulin in BUMPT cells with or without TGF-β1 and PRDM16 siRNA treatment. G-J The GSY between them. Representative WB from each cohort (n = 6). Data are denoted as mean ± SD (n = 6). *p < 0.05, TGF-β with Scramble cohort vs. Saline with Scramble cohort; #p < 0.05, TGF-β with DOX cohort or TGF-β with PRDM16 siRNA cohort vs. TGF-β with Scramble cohort

Fig. 3. PRDM16 facilitated the TGF-β1-induced fibrosis in BUMPT cells via eIF6. BUMPT cells underwent transfection with DOX and PRDM16 siRNA, then exposed to 5 ng/ml TGF-β1 or left untreated for 24 h. A Bioinformatic analysis revealed that the promoter region of the murine eIF6 gene had a putative PRDM16 binding motif. B ChIP assays for PRDM16 were executed utilizing chromatin procured from BUMPT cells. DNA precipitates were amplified using primers flanking the putative PRDM16 binding regions. C eIf6 mRNA levels in BUMPT cells with or without TGF-β treatment were evaluated by RT-qPCR. D eIf6 and β-Tubulin protein expression in BUMPT cells with or without TGF-β1 treatment was examined by WB. E The GSY between them. F eIf6 mRNA expression in BUMPT cells subjected to TGF-β or DOX treatment or left untreated was quantified by RT-qPCR. G WB of eIf6 & PRDM16 and β-Tubulin in BUMPT cells subjected to TGF-β treatment, DOX administration, or left untreated. H The GSY between them. I eIf6 mRNA levels in BUMPT cells subjected to TGF-β treatment, PRDM16 siRNA transfection, or left untreated were quantified by RT-qPCR. J WB of eIf6 & PRDM16 and β-Tubulin in BUMPT cells exposed to TGF-β, PRDM16 siRNA,…

Fig. 4. eIF6 modulates SP1 via the Wnt/β-catenin signaling cascade, consequently diminishing the TGF-β1-induced production of collagen I, collagen III, and fibronectin. BUMPT cells underwent transfection with eIf6 plasmid and siRNA, then exposed to 5 ng/ml TGF-β1 or left untreated for 24 h. A WB of eIf6, FN, collagen I, collagen III, and β-Tubulin in BUMPT cells with or without TGF-β1 and eIf6 plasmid treatment. B The GSY between them. C WB of eIf6, FN, collagen I, collagen III, and β-Tubulin in BUMPT cells with or without TGF-β1 and eIf6 siRNA treatment. D The GSY between them. E WB of SP1, Wnt, β-catenin, p-β-catenin, and β-Tubulin in BUMPT cells with or without TGF-β1 and eIf6 plasmid treatment. F The GSY between them. G WB of SP1, Wnt, β-catenin, p-β-catenin, and β-Tubulin in BUMPT cells with or without TGF-β1 and eIf6 siRNA treatment. H The GSY between them Data are denoted as mean ± SD (n = 6). *p < 0.05, TGF-β with Scramble cohort vs. Saline with Scramble cohort; #p < 0.05, TGF-β with eIf6 plasmid cohort or TGF-β with eIf6 siRNA cohort vs. TGF-β with Scramble cohort

Fig. 5. eIF6 affects the expression of TGF-β、CTGF and NLRP3 via the Wnt/β-catenin/SP1 pathway.The eIf6 and SP1 siRNAs were introduced into BUMPT cells, which were subsequently exposed to 5 ng/ml TGF-β1 or left untreated for 24 h. A Bioinformatic analysis revealed that the promoter region of murine CTGF & TGF-β1 & NLRP3 gene existed a putative SP1 binding motif. B ChIP assays for SP1 were executed with chromatin procured from BUMPT cells. DNA obtained through precipitation was amplified utilizing oligonucleotides encompassing the putative SP1 binding sites for CTGF, TGF-β1, and NLRP3. C WB of eIf6, Wnt, β-catenin, SP1, and β-Tubulin in BUMPT cells subjected to TGF-β1 treatment, eIf6 siRNA, and SP1 siRNA, or left untreated. D The GSY between them. Data are denoted as mean ± SD (n = 6). *p < 0.05, TGF-β1 with Scramble cohort or TGF-β1 with eIf6 siRNA cohort or TGF-β1 with SP1 siRNA cohort vs. Saline with Scramble cohort

Fig. 6. Inhibition of PRDM16 can reverse the anti-fibrotic effect of eIF6. The eIf6 plasmid and siRNA were introduced into BUMPT cells, which were subsequently exposed to 5 ng/ml TGF-β1 or left untreated for 24 h. A WB of PRDM16, eIf6, FN, collagen I, collagen III, and β-Tubulin in BUMPT cells subjected to TGF-β1 treatment, eIf6 plasmid transfection, and PRDM16 siRNA knockdown. B The GSY between them. Data are denoted as mean ± SD. *p < 0.05, TGF-β with Scramble cohort or TGF-β with PRDM16 siRNA cohort vs. Saline with Scramble cohort. C WB of eIf6, Wnt, β-catenin, SP1, and β-Tubulin in BUMPT cells subjected to TGF-β1 treatment, eIf6 plasmid transfection, and PRDM16 siRNA knockdown. D The GSY between them. E WB of TGF-β1, CTGF, NLRP3, IL-1β, and β-Tubulin in BUMPT cells subjected to TGF-β1 treatment, eIf6 plasmid transfection, and PRDM16 siRNA knockdown. F The GSY between them. Data are denoted as the means ± SD (n = 6). *p < 0.05, TGF-β with eIf6 plasmid cohort or TGF-β with eIf6 siRNA cohort vs. TGF-β with Scramble cohort

Fig. 7. PRDM16-KO attenuates tubulointerstitial fibrosis in UUO mice. A Hematoxylin–eosin-stained and Representative Masson’s trichrome-stained paraffin-embedded mouse kidney sections. B Semiquantitative scores of tubulointerstitial fibrosis in the kidney cortex. C Immunohistochemical staining of FN, collagen I, collagen III, α-SMA, and F4/80. D Quantification analysis of FN, collagen I, collagen III and α-SMA staining. Original magnification × 400. Scar Bar: 100 µM. Data are denoted as the means ± SD (n = 6). *p < 0.05, UUO with PRDM16-WT cohort vs. sham with PRDM16-WT cohort; #p < 0.05, UUO with PRDM16-KO vs. UUO with PRDM16-WT cohort

Fig. 8. PRDM16-KO aggravates the expression of FN, Collagen I, collagen III, and α-SMA in UUO mice via/eIF6/Wnt/β-catenin/SP1-axis. A WB of FN, collagen I, collagen III, and α-SMA and β-Tubulin in the cortex of the kidney. B The GSY between them. C WB of eIF6, Wnt, p-β-catenin, β-catenin, SP1, and β-Tubulin in the cortex of the kidney. D The GSY between them. E WB of TGF-β1, CTGF, NLRP3 IL-1β, and β-Tubulin in the cortex of the kidney. F The GSY between them. Data are denoted as the means ± SD (n = 6). *p < 0.05, UUO with PRDM16-WT cohort vs. sham with PRDM16-WT cohort; #p < 0.05, UUO with PRDM16-KO vs. UUO with PRDM16-WT cohort

Fig. 9. PRDM16-KI alleviated tubulointerstitial fibrosis in UUO mice. A Hematoxylin–eosin-stained and Representative Masson’s trichrome-stained paraffin-embedded mouse kidney sections. B Semiquantitative scores of tubulointerstitial fibrosis in the kidney cortex. C Immunohistochemical staining of FN, collagen I, collagen III, α-SMA, and F4/80. D Quantification analysis of FN, collagen I, collagen III,and α-SMA staining. Original magnification × 400. Scar Bar:100 µM. Data are denoted as the means ± SD (n = 6). *p < 0.05, UUO with PRDM16-WT cohort vs. sham with PRDM16-WT cohort; #p < 0.05, UUO with PRDM16-KI vs. UUO with PRDM16-WT cohort

Fig. 10. PRDM16-KI attenuates the expression of FN, Collagen I, collagen III, and α-SMA in UUO mice via/eIF6/Wnt/β-catenin/SP1-axis. A WB of FN, collagen I, collagen III, and α-SMA and β-Tubulin in the cortex of the kidney. B The GSY between them. C WB of eIF6, Wnt, p-β-catenin, β-catenin, SP1, and β-Tubulin in the cortex of the kidney. D The GSY between them. E WB of TGF-β1, CTGF, NLRP3 IL-1β, and β-Tubulin in the cortex of the kidney. F The GSY between them. Data are denoted as the means ± SD (n = 6). *p < 0.05, UUO with PRDM16-WT cohort vs. sham with PRDM16-WT cohort; #p < 0.05, UUO with PRDM16-KI vs. UUO with PRDM16-WT cohort