Single-cell spatial transcriptomics reveal intraglomerular cell activation and ligand-receptor relationships in chronic, active antibody mediated rejection.

Abstract

Chronic active antibody-mediated rejection (CAMR) is a leading cause of late kidney allograft dysfunction, characterized by transplant glomerulopathy involving glomerular endothelial cells (GEC), natural killer (NK) cells, and macrophages. Mechanistic understanding remains limited due to the inability of bulk and single-cell RNA techniques to capture spatial molecular interactions. Using spatial transcriptomics at single-cell resolution (CosMx Spatial Molecular Imager), we analyzed formalin fixed paraffin embedded kidney biopsies with CAMR and compared with controls without rejection. Cell typing, clustering, and dimensionality reduction identified 36 reference cell types, while selectively localizing GEC subtypes, NK cells, and macrophages in glomeruli. Cellular differential gene expression (DGE), cell proximity, and cell-to-cell ligand-receptor analyses elucidated candidate CAMR molecular mechanisms. Spatially resolved single-cell transcriptomics revealed upregulation of intraglomerular NK cell and macrophage genes in CAMR related to cytotoxicity, IgG Fc receptor and non-self-recognition. GEC subtypes developed distinctive transcript phenotypes that included upregulated genes related to complement protection, the MHC target of donor specific antibodies and IFNγ pathway and downregulation of sialyltransferase involved in protective glycocalyx synthesis and vascular integrity. Proximity of NK cells and macrophages with GEC revealed several potential ligand receptor interactions previously unappreciated, including GEC IL33→NK cell IL1RL1 and GEC HLA-DQA1→Macrophage FCGR3A, implicating NK cell and macrophage activation in endothelial injury. High-resolution spatial transcriptomics provided novel and confirmatory insights into CAMR pathogenesis, highlighting cell-specific activation states, molecular interactions, and potential mechanistic pathways involving GECs, NK cells, and macrophages. Our findings advance understanding of CAMR and identify molecular targets for further investigation.