Abstract
Background: Arteriovenous fistula (AVF) is the preferred hemodialysis access for individuals with end-stage renal disease (ESRD); however, approximately 40% of AVFs fail to mature for hemodialysis without intervention. Despite this significant failure rate, our understanding of the cellular and molecular mechanisms underlying post-anastomosis vein remodeling remains limited. In this study, we present a detailed cellular atlas capturing the temporal changes in the mouse jugular vein following AVF anastomosis at the single-cell level.
Methods: Nephrectomy (Nx) was performed on male and female AGE C57BL/6 mice. AVF was created 21 days post-Nx by anastomosing the right jugular vein to the adjacent carotid artery. Animals were euthanized at 3, 10, and 21 days post-AVF for bulk RNA-seq analysis, and at 7 and 30 days for scRNA-seq analysis. The contralateral jugular vein served as a control.
Results: Bulk RNA-seq analysis revealed that, acutely (day 3), AVF formation significantly increased the expression of genes related to leukocyte activation (e.g., Cd44 and Itgam), alongside a decrease in genes associated with vascular smooth muscle contraction (e.g., Myh11, Cnn1, and Tagln). By days 10 and 21, there was a notable upregulation of genes involved in extracellular matrix organization (e.g., Lox, Fn1, and Eln). scRNA-seq analysis confirmed an increase in myeloid cells, particularly profibrotic macrophages (identified by high expression of Spp1 and Lgas3) and neutrophils, coupled with a reduction in endothelial cells, T-cells, and Schwann cells. Remarkably, in the steady state, 82% of fibroblasts in the vein remained inactive, with only 4.7% displaying an activated state (characterized by high expression of Fap, Postn, and Acta2). However, by day 7 post-AVF, activated fibroblasts constituted ~50% of the fibroblast population. Transcription factor enrichment analysis identified Smad3 as the top enriched factor in fibroblast clusters post-AVF. Further intercellular communication analysis demonstrated that, at 7 and 30 days post-AVF, profibrotic macrophages were the primary source of TGFβ ligands (canonical activators of Smad3) acting on activated fibroblasts.
Conclusion: AVF formation induces significant changes in the vein cellular composition, particularly by increasing profibrotic macrophages, which in turn activate fibroblasts through the TGFβ/Smad3 signaling pathway.