Single-Cell Transcriptomic Landscape Reveals an IFN-Primed Vascular Smooth Muscle Cell State Associated with Neutrophil Extracellular Traps in CKD

Vascular calcification (VC) is a life-threatening cardiovascular complication in chronic kidney disease (CKD), largely driven by phenotypic switching of vascular smooth muscle cells (VSMCs). Although microenvironmental cues are recognized contributors to VSMC phenotypic remodeling, the cellular landscape and spatial organization of these interactions in CKD-associated medial calcification remain incompletely characterized. Here, we performed single-cell RNA sequencing (scRNA-seq) combined with spatial validation to characterize the cellular landscape of calcified aortas in a 5/6 nephrectomy with high-phosphorus diet mouse model of CKD. Pseudotime trajectory inference suggested a branching transcriptional trajectory in which contractile VSMCs were associated with an IFN (interferon)-primed intermediate state, followed by inferred branching toward Sca1⁺ precursor-like and osteo-chondrogenic VSMC-associated programs. This IFN-primed transcriptional state is characterized by a prominent Type I IFN transcriptional program accompanied by enrichment of membrane sensing and receptor-mediated endocytic pathways. Notably, we identified an accumulation of neutrophils with transcriptional signatures consistent with neutrophil extracellular traps (NETs) formation within the diseased vascular microenvironment. Spatial and transcriptomic analyses suggest a potential interaction between extracellular NET structures and VSMCs, which may contribute to activation of interferon signaling programs. Furthermore, in vitro experiments demonstrated that NETs exacerbate phosphate-induced VSMC osteogenic differentiation via the activation of downstream IFN signaling. Together, these findings provide a single-cell–resolved characterization of CKD-associated VC and support a potential contribution of NET-associated inflammatory signaling to IFN-primed VSMC remodeling during VC.