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 1

Flowchart for RNA sequencing of urine samples and kidney allograft biopsy specimens.

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Flowchart for RNA sequencing of urine samples and kidney allograft biops...
Urine samples and kidney allograft biopsy specimens were selected from the Weill Cornell biorepository to include 3 major diagnostic categories: Banff Category 1, normal biopsy or nonspecific changes (designated in this report as No Rejection); Banff Category 2, antibody-mediated changes (AMR); or Banff Category 4, T cell–mediated rejection (TCMR). Total RNA was isolated from urinary cells and from the kidney allograft biopsies, and the quantity, purity, and integrity of the isolated RNA were determined. TrueSeq sample preparation kit v2 was used to prepare individual cDNA libraries, and RNA sequencing was performed using Illumina sequencer; the sequence read data were stored in FASTQ format. Sequenced reads were aligned to the human reference genome GRCh38/hg38 using STAR aligner. Aligned reads were quantified against the reference annotation to obtain fragments per kilobase per million (FPKM) and raw counts using CuffLinks (v2.2.1) and HTSeq, respectively. Among the 70 urine specimens selected for RNA sequencing, sequence reads from 13 were excluded for downstream analysis based on less than 20% alignment to the human reference genome GRCh38/hg38 and RNA integrity number (RIN) lower than 2 (RNA quality thresholds). Sequencing data from the remaining 57 urine specimens from 53 kidney allograft recipients (patients) with biopsies classified as No Rejection biopsy (n = 27 biopsies from 25 patients), AMR (n = 8 biopsies from 8 patients), or TCMR (n = 22 biopsies from 20 patients) were included in downstream data analysis. RIN and sequence reads from all 49 kidney allograft biopsies (No Rejection biopsies, n = 20 biopsies from 20 patients), AMR (n = 17 biopsies from 17 patients), or TCMR (n = 12 biopsies from 12 patients) from 49 kidney allograft recipients met RNA quality thresholds and were included in downstream data analysis. Among the urine and biopsy samples included in data analysis, 11 were paired samples (i.e., urine and biopsy were from the same kidney allograft recipient).