Layer-Specific Transcriptomics Changes from Native to Arteriovenous Fistula Vein in Patients on Dialysis

Background: The molecular mechanism of hemodialysis arteriovenous fistula (AVF) vein remodeling may be specific to its layers (intima, media, and adventitia) but is poorly understood. To address this gap, we studied layer-specific transcriptomics in veins of two-stage AVF creation. Methods: Vein samples were collected during the initial creation of brachial artery-basilic vein AVF (1st stage) or the 2nd stage vein transposition surgery 6 weeks later. Veins were sectioned in OCT and tissues from each layer were separately collected by laser microdissection. Following Takara’s SMARTer Stranded Total RNA-Seq Pico Input protocol, libraries were constructed and sequenced in NovaSeq. The sequencing reads were processed by fastp, aligned to genome GENCODE 45 by STAR and gene-level counts were estimated by RSEM. Layer-specific differentially expressed genes between 1st and 2nd stage were detected by edgeR and further identified the enriched pathways using Qiagen IPA. Results: 47 (24 1st stage) veins were collected from 27 patients. 13,522 protein-coding genes that had a raw count of at least 10 in more than 70% of samples were included in analysis. Compared to 1st stage, the 2nd-stage samples showed disproportional upregulation of gene expression with at least 2-fold changes and FDR<0.05 in all layers of intima (511/831), media (260/296), and adventitia (385/535). Among the 3 layers, Intima had the largest number of differentially expressed genes, followed by adventitia and media. Pathway analysis showed that all layers had activated extracellular matrix organization, collagen biosynthesis and modifying enzymes, and assembly of collagen fibrils and other multimeric structures. Cell migration, growth, and proliferation were inferred to be increased while apoptosis was inferred to be decreased in all layers. Angiotensinogen (AGT), TGFB1, TNF, and IL1B were the common key upstream regulators in all three layers. Interestingly, intima shows the least degree of activation of extracellular matrix and p38 MAPK pathway and no increase of cell viability, as compared to the other two layers. Conclusion: Similarity and difference of transcriptome changes were found in the 3 layers of the vein following AVF creation. This study provides new data of layer-specific mechanism underlying AVF adaptation that can be used to develop layer-specific therapies to improve AVF remodeling. Funding: NIDDK Support