PROJECT SUMMARY Extracellular vesicles (EV) transport and deliver signaling molecules to distant cells to support metabolic processes, kidney development and homeostasis. Deregulation of EV signals are associated with metabolic and chronic kidney disease. EV cargo inhibit glucose transporters, leading to insulin resistance. In addition, EV stimulate profibrotic factors to drive kidney fibrosis in diabetic patients. Furthermore, EV are implicated in the pathogenesis of polycystic kidney disease. Despite the growing realization that EV play a vital role in kidney disease, our basic knowledge of the molecular principles that control EV formation and cargo loading remains fundamentally limited. Identifying and targeting molecules that control EV biogenesis in predictive animal models has the potential to lead to novel and synergizing therapeutics for the benefit of kidney disease patients. EV release is an evolutionary conserved process from algae to flies to humans, suggesting that broadly generalizable molecular requirements for this process exist. Drosophila melanogaster has been invaluable in the discovery of conserved signaling dynamics relevant to human diseases, including nephropathies. To begin to explore the molecular mechanism of EV release and cargo loading, we have developed an in vivo EV labeling system in Drosophila epithelial tissues. This system permits the visualization of EV secretion steps in live imaging approaches. Importantly, we have adapted this system for EV proteomic studies aimed at identifying novel regulators of EV biogenesis and cargo loading. Further, the system is amenable to targeted genetic mutant analyses, permitting robust functional validation of putative EV regulators. In this exploratory R21 grant, we are proposing to use our systems to identify novel EV regulators. Validated regulators will form the basis of a follow- up R01 grant focused on delineating the underlying mechanisms using tissue culture and murine models of chronic kidney disease.