Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking

SIMPLE SUMMARY: Extracellular vesicles shed from primary cilia may be involved in renal cystogenesis. The disruption of the Pkd1 gene in our cell culture system increased the production of EVs in a similar way that occurs when the Tsc2 gene is disrupted. Disruption of the primary cilia depresses EV production, and this may be the reason that the combined Kif3A/Pkd1 mutant mouse has a less severe phenotype than the Pkd1 mutant alone. We initiated studies aimed at understanding the renal trafficking of renally-derived EVs and found that single gene disruptions can alter the EV kinetics based on dye tracking studies. These results raise the possibility that EV features, such as cargo, dose, tissue half-life, and targeting, may be involved in the disease process, and these features may also be fertile targets for diagnostic, prognostic, and therapeutic investigation. ABSTRACT: Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.