Epithelial-Immune-Stromal Interactions Define Divergent Repair and Fibrosis Pathways After Acute Kidney Injury in Human Renal Transplants

Acute kidney injury (AKI) is a major cause of early graft dysfunction after kidney transplantation, particularly in recipients of high-risk donor kidneys prone to ischemia-reperfusion injury. However, the cellular mechanisms dictating whether injury resolves or progresses to fibrosis remain unclear. This study combines single-nucleus RNA sequencing and imaging mass cytometry (IMC) analysis of human kidney allograft biopsies collected within eight weeks posttransplant, stratified by long-term functional outcomes. Grafts that recovered function were enriched in regenerative proximal tubular (PT) cells co-expressing PROM1, CD24, and injury markers, consistent with scattered tubular cells (STCs). In contrast, non-recovering grafts contained a unique subpopulation of transitional proximal tubule cells (tPT4) characterized by dedifferentiation, loss of epithelial identity, and acquisition of fibroblast-like features. Fibroblast trajectory analysis revealed a profibrotic lineage, progressing from stromal progenitors to myofibroblasts, exclusive to nonrecovery grafts. Immune profiling showed divergent macrophage (M{Phi}) polarization, with reparative M{Phi}2 cells and regulatory dendritic cell (DC)-like signatures in recovering grafts, versus inflammatory M{Phi}1 and pro-fibrotic DCs in non-recovery. IMC confirmed spatial colocalization of injured tubules, activated fibroblasts, and immune cells in fibrotic regions, validated in an independent cohort. Functional assays demonstrated that ischemic epithelial injury activated monocyte-derived M{Phi}s with mixed inflammatory/reparative profiles and induced fibroblast-related gene expression, while PAX8 knockdown impaired epithelial proliferation and promoted pro-inflammatory signaling. These findings reveal epithelial cell plasticity as a central driver of divergent repair outcomes following renal transplant AKI and highlight epithelial-immune-stromal crosstalk as a therapeutic target to promote recovery and prevent chronic graft injury. One Sentence SummarySingle-cell and spatial mapping of human kidney transplants reveal regenerative and fibrotic cell programs across tubular, immune, and stromal compartments that determine whether acute injury resolves or progresses to chronic allograft injury.