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 1

Identification of Gm1673 (C4orf48) as a candidate enhancer of renal fibrosis.

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Identification of Gm1673 (C4orf48) as a candidate enhancer of renal fibr...
(A) Flow chart of the screen of kidney tissue from streptozotocin-induced (STZ-induced) diabetic nephropathy (DN) in Nos3−/− mice versus age-matched Nos3−/− control (CTL) mice. Ribosome-sequencing (Ribo-seq) with bioinformatic analysis identified 471 differentially expressed RNAs as candidate extracellular molecules, including those encoding the following 4 peptides: mouse Gm1673 (C4orf48 in humans, Cf48), Apoc1, Hilpda, and Tmsb4x. Each peptide was overexpressed in NRK49F cells using a retroviral vector, and then cells underwent 48 hours with or without TGF-β1 stimulation and were analyzed for Acta2, collagen I (Col1A1), and fibronectin (Fn1) expression by Western blotting (WB). (B–D) 293T cells were transfected with an empty vector (MSCV) or Cf48-expressing vector (MSCV-Cf48). Cell-conditioned medium was collected after 48 hours and used to treat NRK49F cells with or without TGF-β1 stimulation for 48 hours. (C) WB analysis of Acta2, collagen I, and Serpine1 expression, with quantification (D). Data are expressed as mean ± SD. *P < 0.05 by 1-way ANOVA with Tukey’s multiple-comparison test. (E and F) Analysis of Cf48 expression in kidneys 6 weeks after STZ-induced DN in Nos3−/− mice versus control buffer-treated mice showing (E) RT-qPCR and (F) WB. ****P < 0.0001 by 2-tailed Student’s t test. (G and H) Confocal microscopy of Cf48 (green) and Acta2 (red) staining kidney of control Nos3–/– (G) and Nos3–/– DN (H). Arrows indicate double-labeled Acta2+Cf48+ cells. (I–K) Analysis of kidney Cf48 expression on day 7 of folic acid–induced nephropathy (FAN) showing confocal microscopy of Cf48 staining in control (CTL) (I) and FAN kidneys (J), and RT-PCR analysis of Cf48 RNA expression (K). Data are expressed as mean ± SD. **P < 0.01; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test. Scale bars: 50 μm.