Podocyte injury and loss are central drivers of chronic kidney disease (CKD) and contribute to most glomerular diseases. A recurrent molecular change during glomerular injury is upregulation of miR-21, which suppresses protective target genes such as PTEN. Anti‑miR-21 strategies have shown efficacy in preclinical models of Alport syndrome and diabetic nephropathy, but clinical translation of free anti‑miR oligonucleotides may be limited by insufficient uptake into glomeruli and podocytes. The current study tests whether loading locked nucleic acid anti‑miR-21 (LNA-21) into podocyte-derived exosomes improves intracellular delivery and functional inhibition of miR-21.
Exosomes were isolated from immortalized murine SVI podocytes and directly loaded with fluorescently labeled LNA anti‑miR-21 (LNA-21) or a control anti‑miR. Exosome integrity after loading was evaluated by multiple assays: transmission electron microscopy to examine morphology, dynamic light scattering to assess particle size and number, and Western blot analysis for established exosome markers CD9 and TSG101. The authors report that exosome number, morphology, and marker expression remained unchanged after LNA loading, indicating that the loading procedure preserved exosome integrity.
Uptake efficiency and intracellular delivery were assessed using confocal laser-scanning microscopy with time-series imaging from 2 to 48 hours, alongside conventional flow cytometry and imaging flow cytometry. Time-series imaging demonstrated progressive intracellular accumulation of exosome-delivered LNA-21 cargo between 2 and 48 hours. In contrast, free LNA-21 showed no detectable uptake in these imaging experiments. Flow cytometry and imaging flow cytometry quantified efficient intracellular delivery mediated exclusively by exosomes, with an overall intracellular delivery rate reported at approximately 76.6%.
Functional inhibition of miR-21 was measured by TaqMan RT-qPCR in three experimental systems: differentiated immortalized SVI podocytes, primary murine podocytes, and puromycin aminonucleoside (PAN)-injured primary glomeruli. Exosome-mediated delivery of LNA-21 induced potent miR-21 suppression in all systems: a 99% reduction in immortalized podocytes, a 97% reduction in primary podocytes, and a 97% reduction in PAN-injured glomeruli (all reported p < 0.05). By contrast, free LNA-21 did not produce significant suppression in these models, indicating that the exosome vehicle was required to achieve target engagement in podocytes and injured glomeruli.
To evaluate functional consequences of miR-21 inhibition, PTEN protein expression—an established direct target of miR-21—was examined by Western blot and immunofluorescence. Restoration of PTEN protein was observed only following exosome-mediated LNA delivery. These data indicate that exosome-mediated delivery not only reduces miR-21 levels but also enables recovery of a downstream protective protein, consistent with effective functional inhibition in podocytes.
Transfection experiments with scrambled control oligonucleotides were performed to confirm sequence specificity. The reported results indicate that the observed miR-21 suppression and PTEN restoration are sequence-specific effects of the LNA-21 cargo delivered by exosomes, rather than nonspecific consequences of the delivery process.
The study demonstrates that exosome-mediated delivery of LNA anti‑miR-21 overcomes the limited cellular uptake observed with free oligonucleotides and enables robust miR-21 inhibition and restoration of PTEN protein in multiple podocyte-relevant models. Exosome number, morphology, and marker expression were preserved after cargo loading. Imaging and flow cytometry data support efficient intracellular delivery via exosomes, and functional assays show marked target suppression and downstream protein recovery. These findings provide mechanistic proof-of-principle that exosome-based miR inhibitor delivery can improve podocyte-targeted engagement and may represent a potential strategy to enhance therapeutic delivery in CKD.
Limitations and notes: this work is reported in a preprint and has not been peer-reviewed. Detailed experimental parameters, dosing, and in vivo data were not reported in the provided source abstract. Competing interests declared by the authors include a company affiliation and a related patent filing.