Spatial Transcriptomics Maps Complement-Driven Local Cell-to-Cell Interactions During Kidney Injury

Background: Complement components are increasingly recognized as key modulators of acute kidney injury (AKI) and tissue repair. While single-cell RNA-seq reveals extrahepatic complement transcription, it lacks spatial context, limiting insight into microenvironments where complement mediates interactions among epithelial, stromal, and immune cells. Methods: FFPE kidney sections from murine models of ischemia–reperfusion injury (IRI), unilateral ureteral obstruction (UUO), and sham surgery were analyzed using 10x Visium HD and Xenium spatial transcriptomics. Data were processed for clustering, differential expression, pathway enrichment, spatial proximity, and ligand–receptor analyses. Findings were validated by RNAscope, RT-qPCR, immunoblotting, and confocal imaging. Results: Spatial transcriptomic analysis revealed a marked loss of healthy S1/S2 proximal tubules and a concomitant expansion of VCAM1+ injured tubular segments in both IRI and UUO models compared to sham controls. Injured proximal tubules exhibited robust upregulation of complement C3 and Cfb, whereas stromal and macrophages demonstrated enrichment of other complement factors. Cell neighborhood analysis uncovered discrete anatomical niches where C3-high proximal tubular cells were spatially juxtaposed with C3ar1-expressing macrophages, suggesting localized ligand-receptor interactions. Additional Ligand-receptor modeling identified C3-high injured tubules also neighbored stromal cells with specific gene expression patterns. These interactions were corroborated by RNAscope and confocal microscopy, which confirmed complement expression localized to injured tubules, adjacent innate immune, and stromal cells. Bin 2 cell-based spatial segmentation of cells at 2-micron resolution assisted in identifying subtypes of immune and stromal cells, interacting with injured tubular cells. Conclusion: High-resolution spatial mapping reveals distinct complement signaling niches in AKI, with C3-producing injured tubules engaging macrophages and stromal cells in their immediate environment through specific receptor-ligand pairs. This spatial atlas highlights therapeutic targets in intrinsic complement pathways and supports similar efforts in human kidney disease. Funding: NIDDK Support