The secreted micropeptide C4orf48 enhances renal fibrosis via an RNA-binding mechanism
Jiayi Yang, … , David J. Nikolic-Paterson, Xueqing Yu
Jiayi Yang, … , David J. Nikolic-Paterson, Xueqing Yu
Published April 16, 2024
Citation Information: J Clin Invest. 2024;134(10):e178392. https://doi.org/10.1172/JCI178392.
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Research Article Nephrology

The secreted micropeptide C4orf48 enhances renal fibrosis via an RNA-binding mechanism

Abstract

Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-β1–induced fibrotic responses in renal fibroblasts and epithelial cells independently of Smad3 phosphorylation. Cellular uptake of Cf48 and its profibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation–sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3′UTR of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis that operates as an RNA-binding peptide to promote the production of extracellular matrix.

Authors

Jiayi Yang, Hongjie Zhuang, Jinhua Li, Ana B. Nunez-Nescolarde, Ning Luo, Huiting Chen, Andy Li, Xinli Qu, Qing Wang, Jinjin Fan, Xiaoyan Bai, Zhiming Ye, Bing Gu, Yue Meng, Xingyuan Zhang, Di Wu, Youyang Sia, Xiaoyun Jiang, Wei Chen, Alexander N. Combes, David J. Nikolic-Paterson, Xueqing Yu

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Figure 8

Cf48 is an mRNA-binding protein that regulates mRNA stability.

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Cf48 is an mRNA-binding protein that regulates mRNA stability. (A) Volca...
(A) Volcano plot of changes in protein abundance on day 28 of folic acid-induced nephropathy (FAN) in WT or Cf48-KO kidneys. Average protein expression ratio of 3 replicates (log2 transformed) between FA KO and FA WT. Different treatment groups were plotted against P values obtained by 2-tailed Student’s t test (–log10 transformed). The cutoffs of P = 0.05 and 2-fold change are marked by blue and red dots, respectively. (B) RT-qPCR analysis of Serpine1 mRNA levels in day 28 FAN or buffer-treated control WT or Cf48-KO kidneys. Data are expressed as mean ± SD. ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test. (C) Western blot (WB) analysis of Serpine1, Ccn2, and collagen I (Col1A1) protein levels in day 28 FAN and control WT and KO kidneys. (D and E) NRK49F cells were transduced with retroviral vector PMSCV-Cf48-IRES-GFP or PMSCV-IRES-GFP. GFP-positive cells were isolated by FACS and then cultured with or without TGF-β1 for 2 days for WB analysis (D), or cultured with and without CoCl2 for 6 hours and Serpine1 mRNA levels were assessed by RT-qPCR (E). (F) NRK49F cells were cultured with or without rCf48 with or without CoCl2 for 6 hours and Serpine1 mRNA levels were assessed by RT-qPCR. (G) Nucleotide sequence of Serpine1 mRNA from position 2926–3060 with potential binding sites and their mutated versions shown. (H) RNA electrophoretic mobility shift assay (RNA-EMSA) analysis of binding interactions between biotin-labeled Serpine1 probes and the rCf48 peptide. Lanes are the following: 1, bio-labeled WT (WT-bio) probe; 2, mutant I-bio; 3, mutant II-bio; 4, WT-bio + rCf48; 5, mutant I-bio + rCf48; 6, mutant II-bio + rCf48; 7, WT-bio + excess WT + rCf48; 8, WT-bio + excess mutant I + rCf48; and 9, WT-bio + excess mutant II + rCf48. (I) NRK49F cells were stimulated with TGF-β1 for 6 hours, and then actinomycin D was added and decay of Serpine1 mRNA in the presence or absence of rCf48 was measured by RT-qPCR. RT-qPCR demonstrated levels of Serpine1 mRNA after TGF-β1 with or without rCf48 treatment. ****P < 0.0001 by unpaired, 2-tailed Student’s t test. (J) Upper panel: RIP and RT-qPCR identified rCf48-binding mRNAs, Serpine1 and Ccn2, while TGF-β1 increased the binding of Cf48 to Serpine1 and Ccn2 mRNAs in NRK49F cells. Data are expressed as mean ± SD. *P < 0.05; ****P < 0.0001 by 2-tailed Student’s t test. Lower panel: End products of RIP–RT-qPCR for Serpine1 and Ccn2 were visualized in agarose gels. (K) Nucleotide sequence of Acta2 mRNA from position 1580–1711 with the potential Cf48 binding site, and its mutation. (L) RNA-EMSA for interactions between Acta2 probes and the rCf48 peptide. Lanes are the following: 1, biotin-labeled WT (WT-bio) probe; 2, mutant-bio; 3, WT-bio + rCf48; 4, mutant-bio + rCf48; 5, WT-bio + excess WT + rCf48; and 6, WT-bio + excess mutant + rCf48.