Our studies advance knowledge about the cellular and molecular mechanisms that underlie the pathogenesis of anemia associated with chronic kidney disease (CKD), also called renal anemia. CKD represents a major public health burden worldwide and is associated with high cardiovascular morbidity and mortality. In the United States the prevalence of CKD in the general population has been estimated to range between 10 and 15% and is even higher in veterans. Anemia is a classic manifestation of advanced CKD and results from (erythropoietin) EPO deficiency and dyregulated iron homeostasis. The current treatment of renal anemia consists of EPO replacement therapy with recombinant versions of EPO, typically administered in conjunction with intravenous iron. Although recombinant EPO is effective in treating renal anemia, clinical studies have raised significant concerns regarding its cardiovascular safety profile, providing a strong incentive for the development of new therapeutic approaches. EPO deficiency results from the diminished ability of diseased kidneys to produce adequate amounts of the glycoprotein hormone EPO in response to anemia or hypoxia. EPO is essential for red blood production and is produced in the kidney by perivascular fibroblasts and pericytes. In these kidney interstitial cells, oxygen- dependent prolyl 4-hydroxylase domain (PHD) dioxygenases (PHDs) function as the oxygen-sensors that control EPO synthesis by regulating hypoxia-inducible factor (HIF) 2 activity. The inability to activate HIF2 leads to EPO deficiency as shown by our laboratory. Perivascular fibroblasts and pericytes give also rise to myofibroblasts, which promote kidney fibrosis through the enhanced production of collagen and other matrix molecules. Thus, kidney fibrosis and the development of EPO deficiency are directly linked. Despite their importance in erythropoiesis and pathogenesis of kidney fibrosis, very little is known about the metabolic characteristics of renal interstitial cells. In particular, the role of mitochondria in interstitial cell differentiation and function is unclear and has not been investigated. Under this grant, we hypothesize that mitochondria play a central role in the regulation of renal interstitial cell differentiation, HIF2 oxygen sensing and pathogenesis of EPO deficiency in CKD. The application uses genetic mouse models in conjunction with state-of-the-art high resolution 3D imaging of mitochondria, metabolic flux analysis and single cell transcriptomics to investigate a) the role of mitochondrial depletion in renal interstitial cell differentiation and metabolism, b) the role of mitochondria in the regulation of the HIF2/PHD/EPO axis and hypoxia responses in renal interstitial cells, and c) the role of the mitochondrial electron transport chain in the development of EPO deficiency and renal anemia.