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.