Investigation of the role of extracellular vesicles in induction of nephrogenesis using a flexible model of nephrogenesis

In embryonic mouse kidney, ureteric bud (UB) and metanephric mesenchyme (MM) bidirectionally interact to form nephrons. Signaling molecules are involved in this interaction, including Wnt proteins and growth factors. How the UB and MM are interacting via signaling molecules is not completely understood. We investigated signaling via extracellular vesicles (EVs) in nephrogenesis. EVs play role in cellcell communication exchanging proteins and nucleic acids to perform functions in organisms. However, there is very little data on EV role in embryonic development. Embryonic day (E) 11.5 kidney UB and MM have limited ability for proliferation, in addition to the very low number of primary cells obtained from kidney. Such limitations obstruct studying EVs in induction of nephrogenesis. We developed a UB derived cell line (UB cell line) based flexible model of nephrogenesis permitting expandable cell culturing, and characterization, tracking and blocking of EVs. Due to such advantages, our model outweighs in vivo, ex vivo and stem cell models for EV research. UB cell line aggregation with E11.5 MM cells generated segmented nephrons. UB cell line expresses several but not all markers of E11.5 UB. Our study showed that EVs are secreted by both UB and MM during nephrogenesis. UB cell line derived EVs were characterized by size and markers expression (CD63, TSG101, CD9 and CD81). Proteomics data revealed that UB cell line EVs carry large number of proteins involved in nephrogenesis related signaling pathways. Our data showed that UB cell line expresses Wnt7b to induce nephrons in MM cells. According to our preliminary data Wnt7b is secreted both as a component of EVs and in a soluble form in supernatant. Also, Wnt11 was detected in the EV-like particles secreted by the UB cells in E11.5 kidney. Palmitoylated GFP tagged EVs from UB cell line were secreted into nephron formation zone during nephrogenesis. UB cell line EVs did not induce formation of nephrons in MM cells but contributed to the survival and nephrogenesis competency of MM cells. EV secretion was inhibited by the knockdown of RalA and RalB gene expression in UB cell line with shRNAs, which impaired nephrogenesis. However, impaired nephrogenesis was partially rescued by the addition of EVs. To sum up, this study established a flexible model of nephrogenesis that solved the limitations of existing models for EV research. EVs were found to be integral part of nephrogenesis process.

Keywords: nephrogenesis, UB, MM, Wnt, EVs, UB cell line, RalA, RalB