PRDM16 acts as a therapeutic downstream target of TGF-β signaling in chronic kidney disease
Qian Yuan, Ben Tang, Yuting Zhu, Chao Wan, Yaru Xie, Yajuan Xie, Cheng Wan, Hua Su, Youhua Liu, Chun Zhang
Qian Yuan, Ben Tang, Yuting Zhu, Chao Wan, Yaru Xie, Yajuan Xie, Cheng Wan, Hua Su, Youhua Liu, Chun Zhang
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Research Article Metabolism Nephrology

PRDM16 acts as a therapeutic downstream target of TGF-β signaling in chronic kidney disease

Abstract

Transforming growth factor β (TGF-β) signaling is the master modulator of renal fibrosis. However, targeting drugs have failed to prevent the progression of chronic kidney disease (CKD) in clinical trials due to the extensive biological regulation of TGF-β signaling. It is necessary to investigate the precise downstream mechanisms of TGF-β signaling that regulate renal fibrosis. In this study, we found that PR-domain containing 16 (PRDM16) expression in human renal tubular epithelial cells was markedly reduced by TGF-β. Mechanistically, activated Smad3 induced by TGF-β interacted with the cofactor H-Ras and bound to the promoter of PRDM16, downregulating its transcription. Tubular-specific knockout of Prdm16 promoted renal fibrosis in models of unilateral ureteral occlusion (UUO) and unilateral ischemia-reperfusion injury (UIRI) by exacerbating mitochondrial dysfunction. In vitro, PRDM16 blocked TGF-β–induced mitochondrial injury and lipid deposition by upregulating peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Delivery of the exogenous PRDM16 gene preserved renal function and ameliorated the progression of renal fibrosis by protecting mitochondrial function. We report PRDM16 as a potential downstream target of TGF-β signaling that attenuates renal fibrosis by safeguarding tubular mitochondrial function.

Authors

Qian Yuan, Ben Tang, Yuting Zhu, Chao Wan, Yaru Xie, Yajuan Xie, Cheng Wan, Hua Su, Youhua Liu, Chun Zhang

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Figure 2

TGF-β transcriptionally downregulated PRDM16 in an H-Ras/p-Smad3–dependent way.

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TGF-β transcriptionally downregulated PRDM16 in an H-Ras/p-Smad3–depende...
(A and B) Representative Western blotting (A) (n = 4 or 5) and quantification of PRDM16 (B) in HK-2 cells exposed to 5 ng/ml TGF-β for 24 hours; (C) Relative mRNA level of PRDM16 in HK-2 cells (n = 3); (D and E) Western blotting (D) (n = 4) and quantification of PRDM16 (E) in primary mouse tubular epithelial cells exposed to TGF-β for 24 hours; (F) PRDM16 mRNA levels in primary tubular epithelial cells (n = 3); (G–I) The protein lysates of HK-2 cells provided with TGF-β and SIS3 (5 μmol/L) for 24 hours were used. Western blotting and quantification (G and H), and mRNA level (I) (n = 3). were shown; (J) The predicted motif of the Smad3 DNA-binding domain from jaspar.genereg.net and primers were designed to target the latent binding site of Smad3 at the PRDM16 promoter. (K) Representative ChIP-PCR images of HK-2 cells exposed to 5 ng/ml TGF-β or vehicle for 45 minutes, the experiment was repeated 3 times. (L) Schematic diagram of DNA pull-down; (M) The differential proteins in DNA pull-down complex shared 5 proteins with the Wound healing GO set (GO: 0042060); (N) Protein-protein interaction network of H-Ras and Smad3 according to the STRING database; (O) The mass spectrum of H-Ras; (P) Coimmunoprecipitation of H-Ras and p-Smad3 in HK-2 cells exposed to TGF-β for 45 minutes; (Q) Relative mRNA levels of H-Ras in HK-2 cells (n = 3) transfected with H-Ras siRNA1 (H-Ras-si1), H-Ras siRNA2 (H-Ras-si2), or Scramble; (R–U) HK-2 cells provided with TGF-β, and H-Ras siRNA1 (R and S) or siRNA2 (T and U) transfection for 24 hours. Representative Western blotting (n = 3) and quantification of PRDM16, PGC-1α, and fibronectin were shown. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; ††P < 0.01, †††P < 0.001. Asterisks indicate comparison to sham or control group. Crosses indicate comparison to TGF-β or UUO/UIRI/FA groups. Two-tailed Student’s unpaired t test analysis (B, C, E, F, and Q), One-way ANOVA followed by Tukey’s post test (H, I, S, and U).