Supplementary Methods and Figure Legends

Extracellular vesicles (EVs) contain RNA, protein, lipids, and other molecules from their parental cell type, which led to the hypothesis that urine-derived stem cell EVs (USC-EVS) could have potent regenerative effects in the setting of acute kidney injury (AKI). We tested this hypothesis in two different in vitro AKI models and an in vivo AKI model. To test USC-EVs ability to recover human kidney cells from nephrotoxicity, HK-2 human proximal tubular epithelial cell line and human kidney organoids derived from induced pluripotent stem cells were treated with 5 μM cisplatin for 48 h followed by 10 μg USC-EVs for 48 h. We next also tested USC-EVs in the unilateral nephrectomy followed by ischemia reperfusion model (UNIRI) AKI model to explore systemic effects in animals. USC-EV treatment of human kidney cells resulted in increased cellular proliferation (p ≤ 0.0001), decreased cytotoxicity (p < 0.0001), and lowered oxidative stress (p ≤ 0.01) as compared to cisplatin alone groups. In mouse models, we found improved pathology as well as reduced kidney injury marker KIM-1 expression in the USC-EV treated group as compared to the UNIRI alone group. Analysis of USC-EV miRNA-targeting pathways associated with epithelial cell proliferation, migration, and positive regulation of MAPK signaling pathway. As predicted by this analysis, we found increased phosphorylation of the MAPK downstream target ERK1/2 in USC-EV-treated HK-2 cells. This study suggests that much of the reported benefit of USCs is due to their EVs and supports further development of USC-EVs as a potential next-generation therapeutic for AKI.