Fibrosis is a common histological manifestation of chronic kidney disease (CKD), characterized by epithelial atrophy, accumulation of myofibroblasts, collagen, and immune cells. The degree of fibrosis predicts kidney function decline regardless of disease etiology. While the kidney can fully regenerate and repair following acute kidney injury (AKI), it is believed that a maladaptive injury response plays a key role in fibrosis development. Over the past 15 years, as part of this grant, our lab has made remarkable progress in systematically dissecting adaptive and maladaptive regeneration, and demonstrated the role of Notch signaling in the injury and repair process. During the first decade of this grant, we demonstrated that transient Notch activation in progenitor cells is essential for reparative regeneration. In contrast, during fibrosis, Notch expression remains elevated in tubule cells, hindering terminal differentiation through metabolic reprogramming. Single cell studies have discovered an emergence of new PT cell subpopulation (named differently by various groups), including failed repair, maladaptive, repairing, injured (iPT), and profibrotic PT (pPT) in diseased kidneys. Experiments indicate that iPT/pPT cells secrete various chemokines, such as IL-34, which play a role in attracting macrophages, and their role in kidney fibrosis and disease has been extensively characterized. Our team has shown that iPT/pPT cells secrete CXCL1, attracting basophils and orchestrating fibrosis by inviting Th17 cells. Computational analysis highlighted strong enrichment for NFKB in iPT/pPT cells. Since NFKB is known regulator not only proinflammatory cytokines such as IL34, CXCL1, CCL2, but also key survival genes, activation of NFKB could create a circuit that may explain the emergence of iPT/pPT and the development of fibrosis. Aim 1: We will systematically characterize iPT/pPT cells in mouse and rat kidney disease models, as well as in patient samples. Specifically, we aim to identify iPT/pPT subtypes, conserved, species- and disease-specific markers, and driver transcriptional programs. We will define the spatial iPT/pPT niche and investigate their cell- cell interactions. Aim 2: To understand essential molecular changes underlying the emergence of iPT/pPT, first, we will characterize their emergence using an engineered CRISPR-Cas9 mouse line with simultaneous readout of lineage histories and gene expression profiles at single-cell resolution. We will validate the role of mitochondrial damage and cytokine treatment by studying isolated tubule cells. Aim 3: We will delineate the contribution of NFKB activation to iPT/pPT differentiation, chemokine secretion, survival, and fibrosis development by using mice with genetic deletion of pathway components.