#3633 DIRECT EXOSOME TRANSFECTION WITH FLUORESCENTLY LABELED SMALL RNAS IS A USEFUL TOOL FOR EXOSOMAL CARGO TRAFFICKING AND RNAI IN CULTURED PODOCYTES
Authors: Lange Tim , Schüter Rabea , Endlich Nicole 
Published: June 2023

Abstract

Background and Aims
Podocytes, highly-specialized renal epithelial cells, display the outer aspect of the glomerular filtration barrier. Changes of their complex 3D morphology with interdigitating foot processes are the leading cause for 80% of chronic kidney disease (CKD) cases. During injury development podocytes release increased amounts of extracellular vesicles like exosomes with disease-specific small RNA cargo composition. Exosomes are referred to as important means of cell-cell communication and display an interesting tool for understanding intercellular communication in CKD pathogenesis. It is not clear if exosomes could serve as delivery system for specific small RNAs to podocytes as potential therapeutic approach. To address this, we investigated if isolated exosomes, directly transfected with fluorescently labeled small RNAs, are suitable for exosomal cargo trafficking and for functional delivery of small RNAs in cultured podocytes.

Method
We isolated exosomes from cultured, murine podocytes and transfected them directly with Cy3-labeled siRNAs and miRNAs using ExoFect siRNA/miRNA transfection kit. The transfected exosomes were characterized by transmission electron microscopy and Western blot. The fluorescently labeled exosomes were incubated with cultured, murine podocytes and exosome cargo uptake was observed time- and localization dependently by confocal laser-scanning microscopy. Isolated exosomes were also transfected with filamin A (FlnA)-siRNAs and pre-miR-21 and were co-incubated with cultured, murine podocytes. Transfection- and knockdown efficiency were confirmed by RT-qPCR, Western blot and immunofluorescence microscopy, respectively.

Results
The isolated exosomes displayed a typical shape and size of 20 nm revealed by transmission electron microscopy. This was also consistent after transfection. They also exhibited exosomal marker proteins CD9 and TSG101 before and after transfection. We could show that cultured, murine podocytes take up fluorescently labeled exosomal RNAs. We could observe an increasing amount of fluorescently labeled RNA intracellularly compared to a decreasing amount of fluorescently labeled RNAs in the periphery over 1 week of treatment indicating a time-dependent exosome uptake by podocytes. Transfection of exosomes with FlnA-siRNAs led to a decrease of FlnA-expression in podocytes co-cultured with transfected exosomes as revealed by immunofluorescence microscopy and Western blot. Podocytes incubated with pre-miR-21-transfected exosomes exhibited a 338-fold upregulation of miR-21 in RT-qPCR.

Conclusion
Here we show that direct transfection of exosomes with fluorescently labeled small RNAs is a useful tool for exosome cargo trafficking in podocytes. Additionally, we proved that small RNAs transfected into exosomes can be functionally used for RNAi in cultured podocytes. This might be a novel strategy for regulating protein and miRNA expression in cultured podocytes.