#3633 DIRECT EXOSOME TRANSFECTION WITH FLUORESCENTLY LABELED SMALL RNAS IS A USEFUL TOOL FOR EXOSOMAL CARGO TRAFFICKING AND RNAI IN CULTURED PODOCYTES

Abstract Background and Aims Podocytes, highly-specialized renal epithelial cells, display the outer aspect of glomerular filtration barrier. Changes their complex 3D morphology with interdigitating foot processes are leading cause for 80% chronic kidney disease (CKD) cases. During injury development podocytes release increased amounts extracellular vesicles like exosomes disease-specific small RNA cargo composition. Exosomes referred to as important means cell-cell communication an interesting tool understanding intercellular in CKD pathogenesis. It is not clear if could serve delivery system specific RNAs potential therapeutic approach. To address this, we investigated isolated exosomes, directly transfected fluorescently labeled RNAs, suitable exosomal trafficking functional cultured podocytes. Method We from cultured, murine them Cy3-labeled siRNAs miRNAs using ExoFect siRNA/miRNA transfection kit. The were characterized by transmission electron microscopy Western blot. incubated exosome uptake was observed time- localization dependently confocal laser-scanning microscopy. Isolated also filamin A (FlnA)-siRNAs pre-miR-21 co-incubated Transfection- knockdown efficiency confirmed RT-qPCR, blot immunofluorescence microscopy, respectively. Results displayed a typical shape size 20 nm revealed This consistent after transfection. They exhibited marker proteins CD9 TSG101 before show that take up RNAs. observe increasing amount intracellularly compared decreasing periphery over 1 week treatment indicating time-dependent Transfection FlnA-siRNAs led decrease FlnA-expression co-cultured Podocytes pre-miR-21-transfected 338-fold upregulation miR-21 RT-qPCR. Conclusion Here direct useful Additionally, proved into can be functionally used RNAi might novel strategy regulating protein miRNA expression