*Purpose: An accurate, noninvasive assay based on urinary gene expression profiles would be a powerful tool for clinical management of kidney transplant patients. We have been using the Nanostring platform to investigate gene expression changes during acute rejection, with a pre-designed codeset (PanCancer Immunology) that enables quantification of 795 transcripts in a single sample.

*Methods: To investigate whether gene probes absent from the codeset would indicate more robust biomarkers of rejection, we performed RNA sequencing of paired urine and biopsy RNA samples from thirteen kidney transplant recipients enrolled in either the Clinical Trials in Organ Transplantation (CTOT)-19 study or the Northwestern University Comprehensive Transplant Center’s renal biorepository. Eight control subjects had stable graft function and no histological evidence of rejection, and five subjects were diagnosed with Tcell-mediated acute rejection. The biopsy samples were either stored in RNAlater (7 subjects; RNAL) or formalin fixed (6 subjects, FFPE).

*Results: We used hierarchical clustering of the RNAseq data to assess how well expression levels of the genes in the Nanostring codeset could distinguish control samples from those with rejection. For each sample type (urine, RNAL, FFPE) we compared clustering with the Nanostring genes to that of the most highly differently expressed genes (DEGs) in that sample type. In both RNAL and FFPE tissue, the Nanostring genes and the other DEGs clearly distinguished rejection and control samples. In urine, however, the Nanostring gene set misclassified half of the rejection samples. Only the DEGs correctly divided the samples. Of all the genes either up- or down-regulated at the time of rejection fewer than ten percent were common to the urine and tissue samples. The most highly upregulated pathways during rejection in tissue reflect an activated immune system, including IL-15 production, Baff and April signaling, T and B cell development, and CD28 signaling in T helper cells. The majority of upregulated pathways during rejection in urine are related to mitochondrial injury, DNA repair, regulation of transcription, and apoptosis.

*Conclusions: These data highlight the differences in the transcriptome of biopsy and urine RNA and suggest potential novel biomarker targets in urine for incorporation into the Nanostring codeset.

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