Ambra1 in exosomes secreted by HK-2 cells damaged by supersaturated oxalate induce mitophagy and autophagy-ferroptosis in normal HK-2 cells to participate in the occurrence of kidney stones

Abstract

Injury to the renal tubular epithelium has emerged as a leading factor underlying the formation of kidney stones. Indeed, epithelial cell damage contributes to the adherence and aggregation of crystals, thereby accelerating the formation of renal stones. Meanwhile, exosomes play an instrumental role in cellular communication, including DNA, RNA, mRNA, etc. In this study, homogenous cells were treated with exosomes derived from damaged cells in an attempt to establish “positive feedback” of cell damage, and the desired results were achieved. To begin, a serum-free medium and supersaturated concentrations of oxalate were added to the HK-2 cell line, and then exosomes were isolated from the two groups for analysis and comparison, and the autophagy-related gene Ambra1 (autophagy and beclin-1 regulator 1) was detected. Subsequently, normal HK-2 cells were treated with exosomes, and the related indexes of autophagy, ferroptosis and mitophagy were determined. Thereafter, Ambra1 was knocked down in exosome-derived HK-2 cells, resulting in the down-regulation of Ambra1 expression in exosomes produced by HK-2 cells following oxalate intervention. Thereafter, the ability of exosomes to stimulate autophagy, mitophagy and ferroptosis was re-evaluated in HK-2 cells after Ambra1 knockdown. The results corroborated that exosomes secreted by oxalate-treated HK-2 can directly elevate autophagy, ferroptosis and mitophagy levels in normal cells, and this effect was significantly mitigated following Ambra1 knockdown within exosomes. Meanwhile, exosomes-induced autophagy and ferroptosis were alleviated after knockdown of beclin-1 in recipient HK-2 cells. These results further suggest that beclin-1 plays a critical role in the process of exosome-induced autophagy-ferroptosis.