Benchmarking urinary cell transcriptomes for noninvasive differentiation of BK polyomavirus–associated nephropathy from T cell–mediated rejection
Franco B. Mueller, Carol Li, Darshana M. Dadhania, Surya V. Seshan, Thalia Salinas, Vijay K. Sharma, Jenny Z. Xiang, Hans H. Hirsch, Thangamani Muthukumar, Manikkam Suthanthiran
Franco B. Mueller, Carol Li, Darshana M. Dadhania, Surya V. Seshan, Thalia Salinas, Vijay K. Sharma, Jenny Z. Xiang, Hans H. Hirsch, Thangamani Muthukumar, Manikkam Suthanthiran
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Research Article Immunology Nephrology

Benchmarking urinary cell transcriptomes for noninvasive differentiation of BK polyomavirus–associated nephropathy from T cell–mediated rejection

Abstract

BK polyomavirus–associated nephropathy (BKVN) adversely impacts kidney allograft survival and often mimics acute T cell–mediated rejection (TCMR), confounding diagnosis and management. To address this conundrum, we performed unbiased RNA sequencing of urinary cells matched to biopsies classified as BKVN with intragraft inflammation (BKVN-P), BKVN without inflammation (BKVN-N), TCMR, or no rejection (NR). BKVN-N displayed dominant host DNA replication, cell cycle, and repair programs, while BKVN-P samples exhibited expansive innate immune activation, antigen presentation, chemokine upregulation, and epithelial injury. Both BKVN subtypes shared signatures of T cell exhaustion and mature and tolerogenic dendritic cell activation but differed in immune orientation — Th1 predominance in BKVN-N versus Treg and CD8 enrichment in BKVN-P. Compared with TCMR samples, BKVN-P lacked robust TCR/CD28 signaling and was enriched for viral and innate modules; BKVN-N lacked alloimmune activation. B cell exhaustion characterized BKVN-N, while BKVN-P displayed robust B cell activation with metabolic downregulation. A ratiometric urinary cell biomarker, CXCL10 mRNA/CD3E mRNA, distinguished both BKVN subtypes from TCMR with diagnostic accuracy, replicated by quantitative reverse transcription PCR for clinical translation, and confirmed in an independent cohort. These findings demonstrate the utility of urinary cell transcriptomics for resolving viral injury from alloimmunity, enabling precision diagnostics and targeted immunomodulation in kidney transplantation.

Authors

Franco B. Mueller, Carol Li, Darshana M. Dadhania, Surya V. Seshan, Thalia Salinas, Vijay K. Sharma, Jenny Z. Xiang, Hans H. Hirsch, Thangamani Muthukumar, Manikkam Suthanthiran

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Figure 1

Workflow for RNA-seq of urine samples matched to kidney allograft biopsies.

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Workflow for RNA-seq of urine samples matched to kidney allograft biopsi...
Urine samples matched to kidney allograft biopsies were selected from the Weill Cornell biorepository to include BK polyomavirus–associated nephropathy (BKVN) with intragraft inflammation (BKVN-P); BKVN without intragraft inflammation (BKVN-N); Banff category 4, acute T cell–mediated rejection (TCMR); or Banff category 1, normal biopsy or nonspecific changes (designated as no rejection). Urine was centrifuged, and total RNA was isolated from the sedimented urinary cell pellet. RNA quantity, purity, and integrity were assessed, and ribosomal RNA (rRNA) was depleted. cDNA libraries were prepared using the TruSeq Sample Preparation kit v2 (Illumina), followed by RNA-seq on an Illumina platform. Sequence reads were stored in FASTQ format and aligned to the human reference genome GRCh38/hg38 using the STAR aligner. Gene-level quantification was performed using CuffLinks (v2.2.1) and HTSeq to obtain raw counts and fragments per kilobase of transcript per million mapped reads (FPKM) values, which were subsequently converted to transcripts per million (TPM). Genes with counts per million greater than 1 in at least 2 samples were retained. Differential gene expression analysis was conducted using robust statistical methods at the individual gene level, gene set level, and pathway level (KEGG and Reactome) to address the biological questions listed in the bottom panel of the figure.