During the progression of most chronic kidney diseases (CKD) podocytes and glomerular endothelial cells (GEC) are irreversibly damaged. Injury to these cells or changes within the composition of the glomerular basement membrane lead to alterations of the structure and function of the glomerular filtration barrier (GFB). Re- establishing GFB function by stimulating endogenous repair mechanisms could slow kidney disease progression. Data presented in this proposal show that extracellular vesicles derived from human amniotic fluid stem cells (hAFSC-EVs) are renoprotective in vivo in our animal model of CKD, Alport Syndrome (AS). No side effects or stimulation of an immune response occurred. These EVs can activate repair mechanisms in glomerular cells by cargo transfer. Specifically, they modulate the levels of miR-93 in both podocytes and GEC. miR-93 plays a key role in CKD since changes in its expression level are associated with the development of renal damage and fibrosis; specifically, miR-93 expression is decreased in mouse and human AS glomeruli and urine samples and hEVs can restore miR-93 levels to normal. We hypothesize that hEVs can re-establish glomerular function by preventing further glomerular injury, thus minimizing renal disease progression. Using transgenic Alport mice, EVs of human origin, and human AS biopsies combined with an innovative spatial transcriptomics approach, we will study the EV mechanism of action with specific focus on the ability of the EV/miR-93 axis to “re-program” cellular signaling networks that regulate glomerular cell biology. We also aim to evaluate the disease modifying capability of hEVs at different AS stages (Aim 1). Finally, we aim to study the molecular signaling pathway changes within the glomerulus during disease progression with specific focus on GEC and to validate these data in human AS biopsies (Aim 2). Successful completion of this proposal will provide novel insights into the key factors critical for maintenance of glomerular structure and function. Importantly, this knowledge would likely be applicable to other forms of CKD and possibly will facilitate the discovery of new therapeutic agents tailored specifically to minimize glomerular damage.