Mitochondrial dysfunction and reactive oxygen species (ROS) play critical roles in disease processes from ischemic AKI, to immune response and inflammation, diabetes, obesity, metabolic syndrome, aging, neurodegenerative and cardiovascular diseases. Therefore, elucidating mechanisms of mitochondrial dysfunction is key to understanding the pathophysiology of many disease processes. Using the ischemia/reperfusion (I/R) kidney injury model to characterize mitochondrial pathways for kidney protection, we identified stanniocalcin-1 (STC1) as a potential therapeutic target. STC1 is a mitochondrial intracrine molecule (defined as an extracellular signaling molecule that enters cells, translocates to mitochondria and alters their function). STC1 activates AMPK, which in turn upregulates uncoupling proteins (UCPs) and SIRT3, promoting mitochondrial anti-oxidant defenses. Transgenic overexpression of STC1 confers resistance to IR kidney injury. The mechanism whereby intracrines such as STC1 are translocated to mitochondria is unknown. We have identified the multi-ligand receptor megalin as a trafficking vehicle for STC1 from the cell surface to mitochondria via the retrograde early endosome-to-Golgi. Megalin KO in cultured cells decreases the expression of SIRT3, diminishes mitochondrial respiration and glycolysis, and increases susceptibility to ischemic kidney injury. This proposal will examine the hypotheses that: megalin is critical for mitochondrial biology; KO of megalin aggravates ischemic kidney injury; and mitochondrial targeting of STC1 is required for STC1-mediated cytoprotection. Aim 1A will determine the phenotype of ischemia/reperfusion kidney injury in mice with doxycycline-inducible and tubular epithelium-specific knockout of megalin. Aim 1B will characterize the mitochondrial phenotype in cultured proximal tubule cells with megalin KO, at baseline and following hypoxia/reoxygenation. Aim 2A will determine whether mitochondrial targeting of STC1 is required for STC1-mediated cytoprotection in vitro. Aim 2B will determine whether mitochondrial targeting of STC1 is required for STC1-mediated cytoprotection in vivo. Our proposed studies will elucidate the role of megalin and STC1 in mitochondrial function and may offer fundamental insights into the pathogenesis of diseases where mitochondrial dysfunction is critical.