The secreted micropeptide C4orf48 enhances renal fibrosis via an RNA-binding mechanism
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
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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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 6

Cf48 LNA administration reduces renal fibrosis and inflammation in mouse CKD models.

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Cf48 LNA administration reduces renal fibrosis and inflammation in mouse...
(A) Mice received 10 mg/kg control LNA (CTL LNA), 10 mg/kg Cf48 LNA1, 10 mg/kg Cf48 LNA2, or 5 mg/kg Cf48 LNA1 + 5 mg/kg Cf48 LNA2 combined treatment on days 1 and 4 after completing UUO surgery, with mice euthanized on day 7. Sham-operated mice were controls. Western blot of Cf48 expression in day 7 UUO and sham groups. (B–G) Day 7 UUO with 5 mg/kg Cf48 LNA1 + 5 mg/kg Cf48 LNA2 (termed Cf48 LNA) or CTL LNA treatment, with sham controls. (B) Western blot of kidney Cf48, collagen I (Col1A1), and Acta2 expression, with quantification of blots (C). (D) Masson’s trichrome staining, with quantification of interstitial collagen staining (E). (F) Immunostaining of F4/80+ macrophages, with quantification (G). (H–P) Nos3–/– mice were treated weekly with Cf48 LNA or CTL LNA from 3 weeks after STZ (or buffer administration) until being euthanized on week 8. (H) Glycated hemoglobin A1c levels (HbA1c). (I) Fasting blood glucose levels. (J) Urinary albumin-to-creatinine levels. (K) Serum creatinine levels. (L) Serum cystatin C levels. (M) PAS staining of kidney sections. (N) Immunofluorescent staining of collagen IV and Acta2. (O) Quantification of percentages of glomerular collagen IV staining. (P) RT-qPCR analysis of kidney Ccl2 mRNA levels. Data are expressed as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparisons test. NS, not significant. Scale bars: 100 μm (D), 50 μm (F and M), and 25 μm (N).