ABSTRACT: Renal-resident macrophages (RMs) are essential regulators of kidney homeostasis and repair, yet the mechanisms governing RM niche regeneration after acute depletion remain poorly defined. To the end, we employed an inducible hCD59 intermedilysin (ILY) ablation system to achieve rapid and specific depletion, and subsequent replenishment of RMs, followed by longitudinal scRNA-seq analysis of kidneys at baseline and days 1, 3, and 7 post-ablations. RM ablation triggered a rapid and sustained upregulation of Cx3cl1, predominantly in proximal tubular epithelial cells (PTC1/PTC2), establishing a persistent chemotactic niche signal that coincided with macrophage repopulation. Regenerating RMs transitioned from inflammatory/stress-associated states toward metabolically active and proliferative phenotypes enriched in glycolysis, oxidative phosphorylation, MYC, and cell-cycle programs, with attenuation of canonical inflammatory pathways. Cell–cell communication analysis revealed an early burst of intercellular signaling at day 1, followed by progressive normalization, with fibronectin (Fn1), osteopontin (Spp1), chemokine (Ccl), and amyloid precursor protein (App) axes emerging as key mediators of niche restoration. Transcriptional network analysis identified a conserved regulatory module (Tfe3, Mitf, Hif1a, Myc, Gabpa, Rcor1) coordinating macrophage differentiation and regenerative programming, linking metabolic adaptation to lineage reconstitution. Sub-clustering revealed five dynamically shifting RM subsets with distinct inflammatory, remodeling, proliferative, and surveillance states, reflecting a hierarchical regeneration process. Functional validation using clodronate-mediated depletion in Spp1 (Opn)-deficient mice demonstrated impaired macrophage repopulation, establishing osteopontin as a critical regulator of RM regeneration. Together, these data define a coordinated epithelial–immune circuit in which CX3CL1-driven chemotaxis, Spp1-dependent signaling, and a core transcriptional network orchestrate macrophage niche reconstitution and kidney repair following acute immune cell ablation.
