BackgroundLittle is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease.MethodsIntegrated droplet– and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney.ResultsA single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair.ConclusionsComplementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Short running title: Myeloid cell heterogeneity in kidney injury and repair Corresponding authors: Bryan Conway (Bryan.Conway@ed.ac.uk), Eoin O'Sullivan (Eoin.osullivan@ed.ac.uk), Tamir Chandra (Tamir.Chandra@ed.ac.uk) Laura Denby (Laura.Denby@ed.ac.uk) Word Count = 4810 Number of figures = 8 2 AbstractThe kidney has a limited capacity to repair following injury, however, the endogenous reparative pathways are not well understood. Here we employ integrated droplet-and platebased scRNA-seq in the murine reversible unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single cell level during renal injury and resolution of fibrosis.We generate a comprehensive catalogue of the changes induced during injury and repair, revealing significant myeloid cell heterogeneity, which would not have been identifiable by conventional flow cytometry. We identify new markers for the myeloid populations within the kidney as well as identification of novel subsets including an Arg1 + monocyte population specific to early injury and a Mmp12 + macrophage subset exclusive to repair. Finally, using paired blood exchange to track circulating immune cells, we confirm that monocytes are recruited to the kidney early after injury and are the source of Ccr2 + macrophages that accumulate in late injury. Our data demonstrate the utility of complementary technologies to identify novel myeloid subtypes that may represent therapeutic targets to inhibit progression or promote regression of kidney disease. Methods Animal modelsAll protocols and surgical procedures were approved by the Animal Ethics Committee, University of Edinburgh. Animal experiments were conducted in accordance with the Animals Scientific Procedures Act UK 1986, under Home Office project licenses 70/8093 and 70/8867. Reversible unilateral ureteric obstruction model (R-UUO)The R-UUO model was performed as previously described (33). Briefly, 8 week old male C57BL/6JOlaHsd mice (Enviago) underwent laparotomy and the left ureter was isolated and the distal portion was ligated twice with 6/O black braided silk suture close to the bladder. In

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