Repair of the kidney tubules after an acute insult requires the clearance of dead cells and proliferative repopulation of the tubules by those cells that survive the initial insult. Our data obtained by tracking individual tubules using high resolution multiphoton Z-stack images on days 1 and 3 after ischemia-reperfusion injury confirms that the S3 segment of the proximal tubule is the site of most tubular cell loss and intraluminal cast formation, and that epithelial proliferation and cast clearance dominate the histologic findings in the outer stripe of the renal medulla from days 2-5 after injury. During this exact time, macrophages (termed mononuclear phagocytes (MNPs) for uniformity) progressively accumulate in the injured kidney and are specifically induced to express reparative genes beginning on day 2-3 after ischemia reperfusion injury (IRI). We now show that one of those pro-repair markers, arginase 1 (Arg1), is selectively induced only in MNPs in the outer stripe directly adjacent to the injured S3 segment. General depletion of reparative MNPs using liposomal clodronate or selective knock-out of MNP Arg1 expression using LysM-Cre;Arg1fl/fl mice reduces both proliferative S3 repair and cast clearance by the injured kidney, with increased mortality, higher BUN and creatinine, and worse tubule repair in the MNP Arg1 null mice. Single cell RNA sequencing performed on cells isolated on day 3 after IRI or sham operation demonstrates that these Arg1+ cells make up a distinct subset of MNP that selectively express the homing receptors Ccr2 and CD74, while injured PT cells express the cognate ligands Ccl2 and Mif. The specific induction of Arg1 in the OS MNP, combined with the ability of these cells to promote proliferative tubule repair and cast clearance, has led us to hypothesize that the injured OS forms a niche for recruiting and selectively activating MNP to facilitate S3 repair. In this proposal we will use an in vitro OS mimetic to define the critical cellular components and secreted factors that promote MNP homing and reparative activation in the OS, identify a pharmacologic approach to recreate this reparative activation, and then use this pharmacologic activation of reparative MNP as well as imaging mass cytometry and immunofluorescence (IF) analysis to validate these in vitro findings in the mouse kidney after IRI (SA 1). We will then pursue the mechanism by which reparative macrophages induce epithelial proliferation and cast clearance using a combination of candidate factor screening, scRNA-seq and proteomic analysis to identify the MNP-secreted factors that stimulate tubule cell proliferation, as well as the role of reparative MNP in detoxifying metabolites of PT cell amino acid catabolism and promoting cast degradation at the S3-thin descending limb junction (SA 2). The combined output of this proposal will provide mechanistic understanding of how MNPs promote both reparative proliferation and cast clearance, and we will use thi...