Seq-ing the Truth

BACKGROUND: Arteriovenous fistula (AVF) non-maturation remains common. Although attention has historically focused on post-operative processes, evidence suggests that pre-anastomotic vessel wall signaling, at AVF creation, contributes to subsequent AVF outcomes. To integrate quantitative baseline biochemistry, and vein histology with single-nucleus RNA sequencing (snRNA-seq) from intraoperative tissue and relate structural, cellular, and molecular features to subsequent AVF maturation. METHODS: In a prospective single-centre observational cohort, 17 consecutive patients undergoing first AVF creation contributed venous tissue for snRNA-seq. Maturation was adjudicated using clinical and/or ultrasound criteria. snRNA-seq used donor-level pseudobulk differential expression within annotated cell types with pre-ranked Hallmark pathway enrichment; serum IL-6 (where available) was included. RESULTS: Nine AVFs matured and eight failed to mature. Histologic intima-media thickness did not differ by outcome. An intact endothelial layer was more frequent in matured AVFs but did not reach significance (78% vs 25%; P=0.06). Serum IL-6 did not differ (P=0.69). Brachial artery flow at assessment was markedly higher in matured AVFs (mean 973 vs 146 mL/min; p<0.001). Endothelial cells from failed-to-mature AVFs showed strong enrichment of interferon-α/γ responses and TNFα/NF-κB signalling (FDR<0.001), with additional enrichment of mTORC1 and MYC targets. In vascular smooth muscle cells, failed-to-mature AVFs showed depletion of oxidative phosphorylation, MYC/E2F targets, and translation/ribosome programs, alongside enrichment of TNFα/NF-κB signalling. CONCLUSIONS: Baseline vessel-wall transcriptomes were associated with endothelial inflammatory priming and reduced vascular smooth muscle bioenergetic/proliferative programs in AVFs that subsequently failed to mature. This may inform early risk stratification.