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.

Background/Aims: Emerging evidence suggests that circular RNAs (circRNAs) play a crucial role in kidney disease regulation. However, their functional significance in chronic kidney disease (CKD) remains poorly understood. Methods: In this study, circRNAs were identified by RNA sequencing in two CKD mouse models, including unilateral ureteral obstruction (UUO) and anti-glomerular basement membrane (anti-GBM) glomerulonephritis. RNase-R treatment and Sanger sequencing were used to confirm circular structure. Circ-RNF216-shRNA was used to establish stable KD mTEC cell lines. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect circ-RNF216 expression. In situ hybridization was used to find the expression and localization of circ-RNF216 in kidney. Transwell assay was performed to assess cell migration. RNA sequencing was performed to characterize the mRNA expression profiles and identify the pathways affected by circ-RNF216 knockdown in mTECs. Results: We identified 1589 circRNAs in 2 CKD mouse models. Circ-RNF216 expression was up-regulated in UUO models. Functional analyses revealed that circ-RNF216 regulates renal fibrosis through modulation of the TGF-β1/Smad3 pathway. Knocking down circ-RNF216 in mouse tubular epithelial cells led to significant suppression of migratory capacity and fibrosis. RNA sequencing showed that circ-RNF216 knockdown altered mRNA expression, with differential genes mainly enriched in the transforming growth factor-β receptor superfamily signaling pathway. Conclusion: Our findings highlight circ-RNF216 as a novel regulatory factor in CKD-related renal fibrosis, thus broadening our understanding of circRNA involvement in kidney disease pathogenesis. These results suggest that circ-RNF216 may serve as a promising target for therapeutic strategies aimed at preserving renal function.