Introduction: Chronic allograft dysfunction (CAD), characterized by interstitial fibrosis and tubular atrophy (IFTA), is the main cause of late graft loss in kidney transplantation. This study dissects the molecular and cellular heterogeneity of the human kidney stroma and immune cells driving CAD.

Methods: Using single-nuclei RNA sequencing, 41,893 nuclei were evaluated from 8 rare kidney allograft biopsies (6 CAD and 3 normal patients). These needle biopsies were collected on average at ≥15-months posttransplanation and were stored in the cryopreservative, RNAlater. Following, integrative analysis was applied including pathway and enrichment analysis, pseudotime trajectories, and ligand receptor analyses. XY chromosome linked gene expression analysis was also used to determine the origin of both immune and nonimmune cells in sex-mismatch transplants.

Results: Two states of fibrosis were discovered (low and high ECM) that differ based on significant alterations in kidney subclusters, immune cell proportions and phenotypes, and intercellular communication. Pseudotime trajectories revealed a mixed tubule cluster, a key intermediate of injured proximal tubular cells, that transitioned to an activated fibroblast cluster also enriched in myofibroblasts markers. Such differentiation of proximal tubular cells was the main driver of fibrosis. Unique to high ECM, MT1 performed replicative repair evidenced by dedifferentiation and nephrogenic transcriptional signatures. Conversely, low ECM did not show a strong replicative repair signature. Differences in immune cell populations highlighted the dynamic nature of immune responses, some of which, promoted severe metabolic dysfunction and limited repair in low ECM or perpetual tissue regeneration and sustained injury in high ECM. Low ECM was characterized by increased dendritic cells (cDCs, pDCs), mast cells, and monocytes (MO1, MO2) whereas high ECM was characterized by increased B cells, T cells (CD8+ T cells, NKs, and Tregs), and plasma cells. Moreover, 65 ligand-receptor pairs between kidney cells and donor-derived macrophages (previously unidentified after several years post-transplantation) played a novel role in injury propagation.

Conclusion: This work improves our understanding of targetable cell types and regulatory mechanisms involved in tissue repair. Interventions aimed at graft fibrogenesis likely require a more targeted approach based on the unique molecular pathways characterizing each condition.