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 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.