Megalin-mediated siRNA uptake in kidney proximal tubule cells

Small interfering RNA (siRNA) is a clinically validated therapeutic modality, which silences gene expression via RNA interference (RNAi). Systemically delivered siRNA molecules are excreted primarily via the kidneys, where they accumulate in the renal cortex. Proximal epithelial cells within the tubule are thus an attractive target cell for utilizing RNAi, where the primary mode of entry is likely endocytic uptake. Here we report using a well-differentiated, opossum kidney (OK) proximal tubule cell (PTEC) line as a model system to mimic proximal tubule biology and investigate siRNA uptake. Fluorescently tagged siRNA were employed to observed uptake, which was observed in both a time and concentration dependent manner occurring predominantly from the apical surface of OK cells. Live cell imaging revealed significant colocalization of siRNAs with LysoTracker, consistent with endocytic uptake and delivery to lysosomes. We investigated the role of key PTEC solute recovery receptors, megalin and cubilin. siRNA uptake was inhibited by the megalin-binding protein RAP; and was independently reduced in Lrp2 knock-out (KO) cell lines. This work suggests that PTECs employ megalin to facilitate uptake of siRNA via endocytosis. Additionally, we observed colocalization of siRNA molecules and megalin in mouse kidneys from in vivo studies. Elucidating the mechanism(s) for siRNA uptake in the proximal tubule aids in the development of RNAi renal therapeutics and, more broadly, the understanding of class effects associated with siRNA nephrotoxicity. Additional work is focused on intracellular traffcking and megalin mediated siRNA uptake in mice. Project funded by Judo Bio. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.