Integrated single-cell and spatial transcriptomic analyses identify a unique subset of pro-fibrotic macrophages in kidney fibrosis

INTRODUCTION: Acute kidney injury (AKI) is a severe clinical syndrome associated with high morbidity and mortality, and it possesses high risk of progression to chronic kidney disease (CKD). Renal infiltration of inflammatory cells, particularly macrophages, plays a key role in driving the progression from AKI to CKD. METHODS: We performed single-cell RNA sequencing and spatial transcriptomics analyses on mouse kidney tissues after unilateral ischemia-reperfusion injury (UIRI) at multiple time points (sham, 12 h, 1 day, 4 days, and 10 days post-UIRI). Immunofluorescence, flow cytometry, Western blotting, co-immunoprecipitation, surface plasmon resonance and immunohistochemical staining were applied to investigate protein expression and function. RESULTS: Six distinct mononuclear phagocyte (MNP) subpopulations were identified and each of them exhibited a unique set of gene expression patterns. In particular, we uncovered an injury-induced, spatially distinct macrophage subset with specific profibrotic properties, named as pro-fibrotic macrophages (PFMs). During the progression of AKI to CKD, PFMs secreted thrombospondin-1 (THBS1), which interacted with and activated LDL receptor-related protein 1 (LRP1) on adjacent interstitial fibroblasts, thereby promoting fibroblast activation and proliferation. In cultured kidney fibroblasts, THBS1 induced fibroblast activation and matrix production, and blockade or knockdown of LRP1 abolished this effect. Furthermore, THBS1 activated a cascade of LRP1 downstream signaling mediators including focal adhesion kinase, protein kinase B, and extracellular signal-regulated kinase 1 and 2. THBS1 also promoted TGF-β1 activation in a LRP1-dependent manner in fibroblasts. In humans, urinary THBS1 levels predicted the rate of CKD progression. CONCLUSION: These studies identify a unique subset of pro-fibrotic macrophages that drive fibroblast activation through THBS1/LRP1 signaling in a spatially confined setting. Our findings suggest that PFMs-derived THBS1 contributes to the progression from AKI to CKD.