Urinary cell transcriptomics and acute rejection in human kidney allografts

Abstract

BACKGROUND. RNA sequencing (RNA-Seq) is a molecular tool to analyze global transcriptional changes, deduce pathogenic mechanisms, and discover biomarkers. We performed RNA-Seq to investigate gene expression and biological pathways in urinary cells and kidney allograft biopsies during an acute rejection episode and to determine whether urinary cell gene expression patterns are enriched for biopsy transcriptional profiles. METHODS.We performed RNA-Seq of 57 urine samples collected from 53 kidney allograft recipients (patients) with biopsies classified as acute T cell-mediated rejection (TCMR; n = 22), antibody-mediated rejection (AMR; n = 8), or normal/nonspecific changes (No Rejection; n = 27). We also performed RNA-Seq of 49 kidney allograft biopsies from 49 recipients with biopsies classified as TCMR (n = 12), AMR (n = 17), or No Rejection (n = 20). We analyzed RNA-Seq data for differential gene expression, biological pathways, and gene set enrichment across diagnoses and across biospecimens. RESULTS.We identified unique and shared gene signatures associated with biological pathways during an episode of TCMR or AMR compared with No Rejection. Gene Set Enrichment Analysis demonstrated enrichment for TCMR biopsy signature and AMR biopsy signature in TCMR urine and AMR urine, irrespective of whether the biopsy and urine were from the same or different patients. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of immune cell types in urinary cells compared with biopsies.CONCLUSIONS. RNA-Seq of urinary cells and biopsies, in addition to identifying enriched gene signatures and pathways associated with TCMR or AMR, revealed genomic changes between TCMR and AMR, as well as between allograft biopsies and urinary cells.

Urinary Cell Transcriptome Profiling and Identification of ITM2A, SLAMF6, and IKZF3 as Biomarkers of Acute Rejection in Human Kidney Allografts

“…As recently reported, 18 whole transcriptome profiling of urinary cells by RNA-seq and differential gene expression analysis identified 180 differentially expressed genes in urine matched to TCMR biopsies compared with urine matched to NR biopsies using log 2 FC ≥2 and FDR <0.05 as the thresholds for differential gene expression. At identical thresholds, 544 differentially expressed genes were identified in urine matched to AMR biopsies compared with urine matched to NR biopsies.…”

“…Among the genes over expressed in urine matched TCMR biopsies or AMR biopsies versus urine matched to NR biopsies, 127 genes formed an overlapping set of genes that were shared between the urine matched to TCMR biopsies and urine matched to AMR biopsies. 18 This set of 127 shared genes was then ranked by log 2 FC >2 for TCMR urine versus NR urine to identify the most highly expressed genes. Of highly expressed genes, ITM2A, SLAMF6, and IKZF3 were selected for their high level of expression, novelty, and mechanistic relevance to rejection.…”

“…ITM2A, however, was uniquely enriched in TCMR and AMR urine but not in AMR or TCMR biopsy. 18 The reason for the enrichment in urinary cells and not in kidneys may reside in their differential expression in lymphoid cells compared with kidney parenchymal cells.…”

FOXP3 mRNA profile prognostic of T cell mediated rejection and human kidney allograft survival

“…This is less of a concern in the light of RNA sequencing demonstrating that kidney allograft biopsy transcriptional signatures are enriched in urinary cells (28).…”

Urinary Biomarkers for Diagnosis and Prediction of Acute Kidney Allograft Rejection: A Systematic Review

“…Barabadi [30] AR (27) ALL-B (normal, 45; CAN, 19) FOXP3 mRNA expression was significantly higher in AR (p < 0.001) Mockler [31] * TCMR (5; borderline, 3) STA-B There was no significant association between 6 months post-transplant CCL2 and TCMR changes (p = 0.46) Gandolfini [36] TCMR (22) ALL-B (normal, 19) CXCL9 > 200 pg/mL in TCMR, 100-200 in dysfunction graft, and < 100 pg/mL in stable graft (p < 0.01) Domenico [38] AR 23ALL-B (ATN, 18; normal, 8) mirRNA 142-3p was significantly higher in AR compared to stable graft (p < 0.001); not compared to ATN (p = 0.079) Lee [39] AR (8) STA (8); DYS-B (ATN, 8; other, 4)…”