Macrophage Niche Reconstitution Reveals Dynamic Transcriptional and Communication Networks Renal Macrophage-Epithelial Communication

Background Renal-resident macrophages (RMs) are essential regulators of kidney homeostasis and repair, yet the cellular and molecular mechanisms governing RM niche regeneration after acute depletion remain poorly defined. How epithelial-immune interactions coordinate RM repopulation is particularly unclear. Methods We employed an inducible hCD59 intermedilysin (ILY) ablation system to achieve rapid and specific depletion, and subsequent replenishment of RMs, followed by longitudinal single-cell RNA sequencing (scRNA-seq) of kidneys at baseline and days 1, 3, and 7 post-ablation. Integrated transcriptomic, pathway, transcription factor, and cell-cell communication analyses were combined with functional validation using clodronate-mediated macrophage depletion in Spp1 (Opn)-deficient mice. Results Acute ILY-mediated ablation resulted in rapid and selective RM depletion, followed by robust regeneration reaching ~ 75% of baseline by day 7. scRNA-seq faithfully captured RM loss and recovery and revealed a sustained epithelial-derived chemotactic response, with proximal tubule epithelial cells identified as the dominant source of CX3CL1 driving RM recruitment and maintenance. Regenerating macrophages adopted a transient injury-adaptive transcriptional program characterized by metabolic activation, proliferation, and stress-response pathways, with relative attenuation of canonical inflammatory signaling. Cell-cell communication analysis identified macrophages as dominant signaling hubs, coordinating immune and epithelial responses through temporally regulated Spp1, Fn1, Ccl, and App-mediated networks. Functional studies demonstrated that Spp1/osteopontin is required for efficient RM regeneration following depletion. SCENIC–STRING analysis connected 18 upregulated transcription factors (TFs) to IL-1 signaling, myeloid differentiation, and tissue remodeling, indicating a coordinated transcriptional program driving RM regeneration. Sub-clustering uncovered five RM subsets and ten proximal tubule cell states with dynamic, time-dependent shifts, revealing a hierarchical macrophage-epithelial communication program that orchestrates niche restoration and tubular repair. Conclusion Our study defines RM regeneration as a transcriptionally regulated, communication-driven process orchestrated by epithelial-derived chemokines, macrophage metabolic reprogramming, and subtype-specific signaling networks. These findings revealed a hierarchical macrophage-epithelial communication program coordinating RM niche restoration and tubular repair.