*Purpose: Transcriptome-based clustering of urinary cells in kidney transplant recipients may help identify cell-type specific injury and develop cell-type specific biomarkers for the noninvasive assessment of human kidney allografts.

*Methods: Single cell RNA-sequencing (scRNA-seq) was performed on urinary cells obtained at the time of allograft biopsy from kidney transplant recipients with biopsies classified as acute T cell mediated rejection [TCMR], chronic active antibody mediated rejection [ABMR], or normal/no rejection [Normal]. Droplet-based 10x Chromium platform (10x Genomics) was used to capture the individual urinary cells in emulsion, followed by cDNA synthesis, sequencing, and data analysis.

*Results: We obtained 3947 high quality cellular transcriptomes from the urine samples matched to TCMR biopsy, ABMR biopsy, or Normal biopsy. Cell-type assignment following uniform manifold approximation and projection (UMAP)-based visualization of urinary single cells from the patient with Normal/No-Rejection biopsy (Panel A), acute TCMR biopsy (Panel B), or ABMR biopsy (Panel C) are shown in Figure 1. Urine samples matched to the TCMR biopsy was exemplified by increased macrophages, dendritic cells, T cells, and NK cells whereas renal tubular epithelial cells were dominant in urine matched to normal biopsy. Further resolution of immune cells revealed sub-clusters of dendritic cells (Figure 2).

*Conclusions: We have demonstrated the feasibility of transcriptome analysis of individual cells in urine samples matched to human kidney allograft biopsies. To our knowledge, this is the first report of urinary cell scRNA-seq in kidney transplant recipients. Our study has deciphered the complex cellular landscape of kidney allograft rejection and provides opportunity to interrogate molecular events at a hitherto unattained level of resolution.

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