Urinary cell transcriptomics and acute rejection in human kidney allografts
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
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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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 4

Kidney allograft biopsy gene signatures are enriched in urinary cells.

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Kidney allograft biopsy gene signatures are enriched in urinary cells. K...
Kidney allograft biopsy signatures derived by RNA sequencing of 49 biopsies from 49 patients. (A) Gene Set Enrichment Analysis was performed to compare biopsy gene signatures with urinary cell gene expression patterns. (B) TCMR biopsy up- or downregulated signatures were enriched in urinary cells (both FDR <0.001). The ranked list of genes in the biopsy (x axis) and the enrichment score (ES) (y axis) are shown. A positive ES indicates that the top-ranked genes in the biopsy are enriched in urinary cells. The top portion of the plot shows the ES (green line). The ES for this gene set is the score at the peak of the plot. The middle portion shows where urinary cell genes appear in the ranked list of biopsy genes. The bottom portion shows the value of the ranking metric moving down the list of ranked genes and goes from positive (correlation with TCMR) to negative (correlation with No Rejection). The normalized enrichment score (NES) accounts for differences in gene set size and correlations between the urine and biopsy gene sets. The FDR is the estimated probability that a gene set with a given NES represents a false-positive finding. Enrichment of top differentially expressed upregulated (Positive NES) and downregulated (Negative NES) biopsy signatures in urine are shown. (C) AMR biopsy upregulated and downregulated signatures were enriched in urinary cells (both FDR< 0.001). Positive NES and Negative NES biopsy signatures in urine are shown. (D) Venn diagram depicts top differentially expressed genes in TCMR biopsy versus No Rejection biopsy and TCMR urine versus No Rejection urine; 75 genes were shared between the TCMR biopsy and TCMR urine. (E) Venn diagram depicts top differentially expressed genes in AMR biopsy versus No Rejection biopsy and in AMR urine versus No Rejection urine; 187 genes were shared between the AMR biopsy and AMR urine.