Kidney fibrosis is the hallmark of chronic kidney disease progression, however, currently no antifibrotic therapies exist. This is largely because the origin, functional heterogeneity and regulation of scar-forming cells during human kidney fibrosis remains poorly understood. Here, using single cell RNA-seq, we profiled the transcriptomes of proximal tubule and non-proximal tubule cells in healthy and fibrotic human kidneys to map the entire human kidney in an unbiased approach. This enabled mapping of all matrix-producing cells at high resolution, revealing distinct subpopulations of pericytes and fibroblasts as the major cellular sources of scar forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single cell RNA-seq and ATAC-seq experiments in mice, and spatial transcriptomics in human kidney fibrosis to functionally interrogate these findings, shedding new light on the origin, heterogeneity and differentiation of human kidney myofibroblasts and their fibroblast and pericyte precursors at unprecedented resolution. Finally, we used this strategy to facilitate target discovery, identifying Nkd2 as a myofibroblast-specific target in human kidney fibrosis.

Acute kidney injury (AKI) is morphologically characterized by a synchronized plasma membrane rupture of cells in a specific section of a nephron, referred to as acute tubular necrosis (ATN). Whereas the involvement of necroptosis is well characterized, genetic evidence supporting the contribution of ferroptosis is lacking. Here, we demonstrate that the loss of ferroptosis suppressor protein 1 (Fsp1) or the targeted manipulation of the active center of the selenoprotein glutathione peroxidase 4 (Gpx4cys/-) sensitize kidneys to tubular ferroptosis, resulting in a unique morphological pattern of tubular necrosis. Given the unmet medical need to clinically inhibit AKI, we generated a combined small molecule inhibitor (Nec-1f) that simultaneously targets receptor interacting protein kinase 1 (RIPK1) and ferroptosis in cell lines, in freshly isolated primary kidney tubules and in mouse models of cardiac transplantation and of AKI and improved survival in models of ischemia-reperfusion injury. Based on genetic and pharmacological evidence, we conclude that GPX4 dysfunction hypersensitizes mice to ATN during AKI. Additionally, we introduce Nec-1f, a solid inhibitor of RIPK1 and weak inhibitor of ferroptosis.

Macrophage migration-inhibitory factor (MIF) is an upstream regulator of innate immunity and a potential molecular link between inflammation and cancer. The unusual structural homology between MIF and certain tautomerases, which includes both a conserved substrate-binding pocket and a catalytic N-terminal proline (Pro1), has fueled speculation that an enzymatic reaction underlies MIF's biologic function. To address the functional role of the MIF tautomerase activity in vivo, we created a knock-in mouse in which the endogenous mif gene was replaced by one encoding a tautomerase-null, Pro13Gly1 MIF protein (P1G-MIF). While P1G-MIF is completely inactive catalytically, it maintains significant, albeit reduced, binding to its cell surface receptor (CD74) and to the intracellular binding protein JAB1/CSN5. P1G-MIF knock-in mice (mif P1G/P1G ) and cells derived from these mice show a phenotype in assays of growth control and tumor induction that is intermediate between those of the wild type (mif ؉/؉ ) and complete MIF deficiency (mif ؊/؊ ). These data provide genetic evidence that MIF's intrinsic tautomerase activity is dispensable for this cytokine's growth-regulatory properties and support a role for the N-terminal region in protein-protein interactions.Macrophage migration-inhibitory factor (MIF) is a widely expressed cytokine and upstream regulator of the immune response (23). Immunoneutralization and genetic knockout studies have established a central position for MIF in the host response to infection and tissue invasion (5, 9, 15). MIF's importance in human disease also has been revealed by the association of high-expression MIF alleles with clinical severity of different autoimmune disorders (18).An important role for MIF in tumorigenesis and in the contribution of inflammation to cancer development also has been proposed (7,20). Different tumor types express high levels of MIF, and clinical studies have shown that MIF production correlates with tumor aggressiveness and metastatic potential (1,22,27). Studies using genetically engineered MIFdeficient cells and mice show that MIF contributes to the development of the malignant phenotype by several mechanisms, including enhancement of cell cycle progression by sustained mitogen-activated protein kinase (MAPK) activation (28, 30), decreased proteasomal protein degradation (33) leading to altered expression of key cell cycle-regulatory proteins (15,21,35), and tumor promotion by neoangiogenesis (10, 48). Importantly, MIF also inhibits the proapoptotic and cell cycleregulatory function of the p53 tumor suppressor, thereby allowing for the accumulation of oncogenic mutations (20, 32). MIF's role in tumor progression additionally is supported by human genetic studies, and a recent report has described an association between high-expression MIF alleles and incidence of prostate cancer, which is a tumor type in which recurrent inflammation is considered to have an etiologic role (27).Information regarding MIF structure and function has emerged only in the last few year...

A major drawback of single-cell ATAC-seq (scATAC-seq) is its sparsity, i.e., open chromatin regions with no reads due to loss of DNA material during the scATAC-seq protocol. Here, we propose scOpen, a computational method based on regularized non-negative matrix factorization for imputing and quantifying the open chromatin status of regulatory regions from sparse scATAC-seq experiments. We show that scOpen improves crucial downstream analysis steps of scATAC-seq data as clustering, visualization, cis-regulatory DNA interactions, and delineation of regulatory features. We demonstrate the power of scOpen to dissect regulatory changes in the development of fibrosis in the kidney. This identifies a role of Runx1 and target genes by promoting fibroblast to myofibroblast differentiation driving kidney fibrosis.

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