Urinary cell transcriptomics and acute rejection in human kidney allografts
Akanksha Verma, … , Olivier Elemento, Manikkam Suthanthiran
Akanksha Verma, … , Olivier Elemento, Manikkam Suthanthiran
Published February 27, 2020
Citation Information: JCI Insight. 2020;5(4):e131552. https://doi.org/10.1172/jci.insight.131552.
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Clinical Research and Public Health Transplantation

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.

Authors

Akanksha Verma, Thangamani Muthukumar, Hua Yang, Michelle Lubetzky, Michael F. Cassidy, John R. Lee, Darshana M. Dadhania, Catherine Snopkowski, Divya Shankaranarayanan, Steven P. Salvatore, Vijay K. Sharma, Jenny Z. Xiang, Iwijn De Vlaminck, Surya V. Seshan, Franco B. Mueller, Karsten Suhre, Olivier Elemento, Manikkam Suthanthiran

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Figure 5

Cell type enrichment analysis using xCell.

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Cell type enrichment analysis using xCell. Cell type enrichment analysis...
Cell type enrichment analysis of the RNA sequencing data determined using xCell, a bioinformatics tool that generates cell type enrichment scores based on gene expression data for 64 immune and stromal cell types. (A) The x axis depicts the xCell enrichment score, and the y axis lists 26 of the 64 cell types that were differentially enriched (FDR < 0.1, Wilcoxon test with Benjamini-Hochberg correction) in TCMR urine versus No Rejection urine. Twenty-two of the 26 cell types were positively enriched, and 4 were negatively enriched in TCMR urine versus No Rejection urine. Box plots of the immune score (composite score of immune cell types) and the microenvironment score (composite scores of immune cell types and stromal cell types) are also shown. (B) Box plots depict the RT-qPCR validation of differential abundance of several mRNAs contributing to the higher immune score in TCMR urine versus No Rejection urine. mRNA abundance was quantified using customized RT-qPCR assays and shown as log2 copies per μg of total RNA in the urinary cells on the y axis. Number of No Rejection urine samples and the number of TCMR urine samples are shown in parenthesis. P values of pairwise group comparisons are based on the Mann-Whitney U test. (C) Cell type enrichment scores of TCMR biopsy compared with the cell type enrichment scores of TCMR urine. (D) Cell type enrichment scores of AMR biopsy compared with the cell type enrichment scores of AMR urine. In C and D, the y axis lists only differentially enriched cell types (FDR-adjusted P < 0.1, Wilcoxon test with Benjamini-Hochberg correction), and the x axis shows the –log10 P values. The blue bars represent cell types significantly enriched in the kidney allograft biopsies compared with urinary cells (higher score in the biopsies), and yellow bars represent cell types significantly enriched in the urinary cells compared with the kidney allograft (higher score in urinary cells). Stromal cell types were enriched in biopsy compared with urine, and immune cell types were enriched in urine compared with biopsy.