Spatial transcriptomics maps complement-driven cell-to-cell interactions during acute kidney injury

Understanding the mechanisms that drive (acute kidney injury (AKI) and subsequent tissue repair is crucial for developing novel therapeutic strategies to halt inflammation and prevent tissue scarring. Intracellular and locally secreted complement components are increasingly recognised as modulators of tissue injury and repair, yet the spatial niches in which they operate within the kidney remain undefined. Standard single-cell RNA-seq detects extra-hepatic complement transcription but loses positional context, obscuring micro-domains where complement mediates crosstalk among parenchymal, stromal, and immune cells during acute kidney injury (AKI). Formalin-fixed paraffin-embedded kidney sections from mouse models of ischaemia–reperfusion injury (IRI), unilateral ureteral obstruction (UUO), and sham surgery underwent 10x Visium HD and Xenium in-situ–hybridisation spatial transcriptomics. Data were pre-processed, normalised, and clustered, followed by differential-expression, pathway-enrichment, cell-neighbourhood, and ligand–receptor interaction analyses. Key transcripts and proteins were validated by RNAscope, RT-qPCR, immunoblotting, and confocal microscopy. Spatial maps revealed a loss of healthy S1/S2 proximal tubules and expansion of VCAM1 + injured tubules in both IRI and UUO compared with shams. Injured tubules showed robust up-regulation of C3 and Cfb , while stromal cells and macrophage clusters selectively increased other complement components. Cell-neighbourhood analysis identified discrete loci where C3-high tubules and C3ar1-high macrophages were spatially adjacent. Ligand-receptor modelling confirmed significant C3-C3AR1 and integrin-mediated signalling within these niches. RNAscope and confocal imaging validated C3 protein localisation to injured tubules and C3AR1 to nearby macrophages. High-resolution spatial transcriptomics uncovers compartmentalised complement expression and signalling hotspots in AKI, linking injured proximal tubules with infiltrating macrophages through local C3 production. These findings provide a tissue atlas for targeting intrinsic complement pathways and will guide ongoing translation to human kidney biopsies. This abstract has been submitted by the PI on behalf of an ECR.