Autophagy is a lysosomal degradation and recycling pathway that controls the quality and quantity of cytoplasmic material. Autophagy requires the formation of a double-membrane vacuole called the autophagosome that sequesters proteins and other cytoplasmic components to be delivered into the lysosome. Autophagy plays crucial roles in cellular and tissue homeostasis, and dysfunctional autophagy has been linked to human pathologies including kidney diseases. Autophagy is now recognized to have non-cell autonomous functions via the secretion of cytosolic molecules sequestered in autophagosomes or autophagosome-like structures. By controlling the release of a large panel of molecules (e.g., cytokines, amino acids, nucleotides), non-cell-autonomous autophagy (NCAA) mediates communication between cells within the same organ and between organs. Recently, studies have paved the way for research into the function of NCAA in human physiology and pathophysiology; however, little is known about the role of NCAA in kidney physiology. In 2016, we demonstrated that shear stress generated by urinary fluid flow induces a primary cilia-dependent autophagy in kidney epithelial cells and that this induction of autophagy influences kidney epithelium homeostasis by regulating cell volume. We now propose to investigate the contribution of non-cell autonomous autophagy to kidney physiology. We will focus our attention on nucleotides (such as ATP and its metabolites) as these extracellular messengers are known to participate in the control of renal electrolyte tubular transport. This hypothesis is based on our preliminary results and on published data showing that inhibition of autophagy blocks ATP secretion under different stress conditions. This proposal is organized in three specific aims: (1) We will determine whether NCAA controls kidney epithelial cell size upon shear stress. We will first decipher the molecular machinery required for the release of ATP and identify the purinergic receptors responsible for cell size regulation upon shear stress. Next we will determine whether this autophagic-dependent secretion of ATP affects cell volume in autocrine or/and paracrine dependent manners. (2) We will study how NCAA regulates kidney cell size in vivo using mouse and zebrafish models incompetent for autophagy. For this purpose we will analyze the accumulation of ATP-positive vesicles in kidney epithelial cells of chloroquine-treated mice. We will also determine the sizes of distal tubular and collecting duct cells in mice in which autophagy is conditionally impaired in the proximal tubules (PEPCK-Cre Atg7fl/fl mice). Finally, in zebrafish we will track the induction of autophagy and the release of ATP in the tubular pronephros during physiological urinary fluid flow (at 48 hours post fertilization) using transgenic LC3 (CMV:GFPLC3) zebrafish to enable monitoring of autophagy; Atg5-deficient larvae will be used as a negative control. (3) Finally, we will determine whether NCAA controls another aspect of kidney physiology by regulating its endocrine role. We will focus our attention on the release of renin because adenosine (produced by extracellular breakdown of ATP, which is secreted by cells from the macula densa) inhibits the release of renin from the juxtaglomerular cells. In conclusion, this proposal will determine how NCAA affects kidney physiology. We expect to show that NCAA regulates kidney cell size in cultured cells and in vivo. We also anticipate that NCAA will prove to be a mediator of crosstalk within cells in the same organ and also between different organs, which will represent a conceptual breakthrough in the autophagy and the physiology fields.