PROJECT SUMMARY/ABSTRACT The iron overload disorder hereditary hemochromatosis is an endocrine liver disease that results from an inability to produce sufficient amounts of hepcidin, the iron-regulatory hormone produced by the liver. In hemochromatosis, increased absorption of dietary iron leads to the appearance of plasma non-transferrin-bound iron (NTBI), which is taken up by various tissues and cells leading to tissue iron overload and related pathology. Plasma NTBI is also commonly seen in the hematologic disease beta-thalassemia major, an inherited blood disorder that requires regular blood transfusions, which over time result in iron overload. Although NTBI is the major contributor to tissue iron loading, our understanding of the molecular mechanisms that mediate NTBI uptake is incomplete. The primary long-term objective of this proposal is to define the proteins that transport iron into various tissues and cells, particularly those affected by iron-overload related pathology. Our central hypothesis is that the membrane transport proteins SLC39A14 (ZIP14) and (ZIP8) participate in iron homeostasis and NTBI uptake. In the previous funding period, we generated a variety of conditional and double-knockout mouse models, which enabled us to find that SLC39A14 is required for iron loading of the heart in a mouse model of hemochromatosis and that SLC39A8 participates in systemic iron recycling, iron uptake by the lung, and iron accumulation by the kidney and brain. We also demonstrated that reducing SLC39A14 expression (pharmacologically or genetically) combined with iron chelation is more effective than iron chelation alone in decreasing hepatic, cardiac, and total body iron in a mouse model of hemochromatosis. The current project will investigate the efficacy of pharmacological approaches to reduce tissue-specific SLC39A14 expression in mouse models of beta thalassemia and hemochromatosis. Another substantial focus will be to characterize the molecular mechanisms of iron uptake and accumulation by the kidney, as renal iron accumulation and associated adverse effects have been documented in a variety of disorders, including acute kidney injury, chronic kidney disease, and diabetic nephropathy.