Heterogeneous gene expression during early arteriovenous fistula remodeling suggests that downregulation of metabolism predicts adaptive venous remodeling
Background: Although clinical outcome of arteriovenous fistula (AVF) as a preferred vascular access for hemodialysis has been inadequate, biological mechanisms of AVF maturation and failure is still poorly understood. Since our mouse AVF model recapitulates human AVF maturation and partial failure, we hypothesized that high throughput RNA sequencing (RNA-seq) analysis would identify early changes in gene expression that might predict AVF maturation and failure. Method: Aortocaval fistula creation (AVF group) or sham operation (sham group) was performed with C57BL/6 mice (10 wk). Ultrasound was performed to confirm the AVF patency and to measure hemodynamics. Venous limbs were collected on postoperative day 7 and total RNA was extracted. After Poly-A mRNA libraries were constructed, RNA-seq was performed. Differentially expressed genes (DEG) were identified, and subsequent enrichment analyses of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed with bioinformatics techniques. Results: 1057 DEG were identified between the AVF and sham groups. Genes in metabolic pathways were significantly downregulated in the AVF. Since gene expression patterns among the AVF group were significantly heterogenous, the AVF group was divided into 2 groups ('normal' AVF group; nAVF and 'outliers' group; OUT), and subgroup analyses were performed. Although physiological data of body weight change and hemodynamics were not different between the nAVF and OUT groups, 413 DEG were identified between them. Enrichment analyses showed significant overrepresentation of metabolism, immunity, and coagulation in the OUT group. Comparing the nAVF group with the sham group, 403 DEG were identified. Enrichment analyses showed increase of gene expression related to cell cycle, extracellular matrix, and immunity. Comparing these results with previously published data, the gene expression patterns of the nAVF group was consistent with AVF maturation and adaptive remodeling, whereas the OUT group showed attenuated difference, suggesting the heterogeneity of the AVF group reflects early gene expression changes in the process of AVF maturation and failure. Significant sex differences were not observed in the AVF group. Conclusions: A small subset of AVF undergoes aberrant biological processes in the early phase including diminished ECM remodeling, activated coagulation and upregulated metabolism, that could lead to fistula failure. Detection of these processes might be a viable translational strategy to predict fistula failure and reduce patient morbidity. Overall design: All animal experiments were performed in compliance with federal guidelines and with approval from the Institutional Animal Care and Use Committee of Yale University.  10-week-old wild-type female and male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Infrarenal aortocaval fistulae were created. Briefly, inhaled 2 - 2.5 % isoflurane in 1.0 L/min 02 was used for general anesthesia, and extended-release buprenorphine (Ethiqa XR; North Brunswick, NJ, USA) was administered subcutaneously with a dosage of 3.25 mg/kg body weight for intraoperative and postoperative analgesia. The abdominal aorta and the inferior vena cava (IVC) were exposed with a midline laparotomy; the bowels were gently retracted. After the vessels were clamped at an immediately caudal level of the origins of the left renal artery and vein, a fistula between the vessels was created by using a 25-gauge needle to puncture the aorta and IVC, from a left lateral side of the aorta into a lumen of the IVC at a level cranial to the aortic bifurcation. Hemostasis of the puncture site on the lateral aortic wall was achieved by a gentle compression with a surrounding retroperitoneal tissue. A pulsatile bright-red blood flow visible intraoperatively through the IVC wall was considered as a primary technical success of the AVF creation. Sham-operated mice underwent all the same steps except the needle puncture.   After euthanasia and circulatory flushing with normal saline, the IVC was harvested on postoperative day 7. The range of the extracted IVC from mice with an AVF was from just above the fistula opening through just below the origin of the left renal vein. The tissues were immediately submerged in RNAprotect Tissue Reagent (QIAGEN, Hilden, Germany) and incubated overnight at 4°C, then stored at -20°C without the reagent. Total RNA was extracted using RNeasy Mini Kit with DNase I (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. Total RNA quality and concentration were estimated with A260/A280 and A260/A230 ratios measured using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA).  Preparation and sequencing of poly-A mRNA sequencing libraries were performed by the Yale Center for Genome Analysis (YCGA). RNA integrity was estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with RNA integrity number (RIN) values of = 8.0 were used for analysis.  Poly-A mRNA was purified from approximately 200 ng of total RNA and the stranded mRNA sequencing libraries were constructed using KAPA mRNA HyperPrep Kit for Illumina sequencing (Roche, Basel, Switzerland). Indexed libraries were quantified by qRT-PCR using KAPA Library Quantification Kits (Roche, Basal, Switzerland) and insert size distribution was determined using an Agilent 2100 Bioanalyzer. Samples with a yield of = 0.5 ng/µL and a size distribution of 150 - 300 bp were sequenced. Sample concentrations were normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell (Illumina, San Diego, CA, USA) at a concentration that yielded 25 million passing filter clusters per sample. Samples were sequenced using 101 bp paired-end sequencing on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Data generated during sequencing runs were simultaneously transferred to the YCGA high-performance computing cluster.  The FASTQ files of raw sequencing reads were imported into a computing cluster based on Red Hat Linux computing system and analyzed. For the purpose of quality control, poly A and poly T sequences, Illumina adaptor sequences and low-quality sequences in the reads were deleted using PRINSEQ v0.20.4 and Trimmomatic v0.39 software. The trimmed sequencing data were aligned to the GRCm39 reference genome with the gene annotation information  (Ensembl release 106) and expression values for each gene were counted using STAR v2.7.7a and RSEM v1.3.3 software.