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 2

Differential gene expression analysis.

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Differential gene expression analysis. Total RNA isolated from 57 urine ...
Total RNA isolated from 57 urine specimens collected at the time of 57-kidney allograft biopsies from 53 kidney allograft recipients (22 urine specimens from 20 patients with 22 TCMR biopsies, 8 urine specimens from 8 patients with 8 AMR biopsies, and 27 No Rejection urine specimens from 25 patients with 27 No Rejection biopsies) were RNA sequenced. Differential gene expression analysis was performed using the limma package in R. (A and B) Volcano plots display differences in urinary cell gene expression between TCMR urine and No Rejection urine (A) and between AMR urine and No Rejection urine (B). The x axis depicts the log2 fold change (FC) in gene expression and the y axis is the –log10 P value. A positive FC (red dots) denotes increased expression in TCMR urine or AMR urine versus No Rejection urine. A negative FC (blue dots) denotes increased expression in No rejection urine versus TCMR urine or AMR urine. With log2 FC ≥ 1 and FDR-adjusted P < 0.1 as the thresholds for differential expression, 4702 genes (27% of total genes) were differentially expressed in TCMR urine versus No Rejection urine, and 6516 genes (41%) were differentially expressed in AMR urine versus No Rejection urine. Supplemental Table 3 lists the differentially expressed genes. A total of 180 genes (1.04%) were differentially expressed in TCMR urine versus No Rejection urine, and 534 genes (3.40%) were differentially expressed in AMR urine versus No Rejection urine with log2FC ≥ 2 and FDR-adjusted P < 0.05 as the thresholds for differential expression.