Objectives/Goals: Kidney transplant rejection is a leading cause of graft failure. Diagnosis is dependent on allograft biopsy that 1: focuses on immune infiltrate and 2: does not inform treatment or recovery potential. Using single-cell transcriptomic technologies, we assess rejection’s impact on kidney cells and begin to define outcome-affecting interactions. Methods/Study Population: Single-cell RNA sequencing was performed on 25 kidney biopsies from 15 separate patients who had undergone acute cellular rejection. Cell typing was performed using canonical genes, and differential gene expression profiling was performed in R with Seurat. The proportion of cell types present at different time points and grades of rejection was determined. We focused first on renal parenchymal cell types and their gene expression changes with rejection grade and treatment of rejection. The spatial localization of these cell states was determined with spatial transcriptomics using the Xenium Prime 5K pan-human tissue panel on 11 kidney biopsy sections. After annotation, spatial-informed niches were identified to investigate cellular changes associated with injury. Results/Anticipated Results: From the single-cell data, we were able to identify several renal parenchymal cell states. Focusing on the proximal tubule (PT) cells as the most numerous cell type in the renal cortex, we identified healthy, injured, and damaged cell states based on expression of tubular injury markers, genes associated with epithelial to mesenchymal transition, and expression of interferon-response genes. The proportion of PT cells in an injured state increased with rejection severity and decreased with rejection treatment. Utilizing the spatial transcriptomic data, we were able to identify both healthy and injured PT states and define their localization with immune cell types and histologic injury patterns. We found varied transcriptomic niches and that niches with injured PT cells were enriched with immune cells. Discussion/Significance of Impact: PT cells exhibit patterns of injury in response to rejection and when injured cells exist in spatial niches containing immune cells that are contributing or responding to injury. Understanding these interactions will further define the injury process caused by rejection and guide development of personalized rejection treatment.
Caldwell et al. (Wed,) studied this question.