Investigation of Urinary Extracellular Vesicles as Novel and Safe Therapeutics for Autosomal Recessive Polycystic Kidney Disease ( ARPKD )

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ABSTRACT Autosomal recessive polycystic kidney disease (ARPKD) is a severe inherited disorder caused primarily by mutations in PKHD1 and in a minority of cases, CYS1 . These genes encode fibrocystin and cystin, respectively. ARPKD typically manifests in infancy with enlarged kidneys, progressive cyst formation, and an estimated peri‐natal high mortality rate of 20%. Given the lack of efficient therapies and the genetic complexity of many rare diseases such as ARPKD, strategies that restore functional proteins defective in the disease may offer a disease‐modifying approach. Urinary extracellular vesicles (uEVs) are naturally secreted by renal and urinary tract cells and contain functional kidney proteins, including fibrocystin and cystin. As such, uEVs may be capable of supplementing these missing proteins and delivering them directly to diseased cells in ARPKD. To investigate the therapeutic potential of uEVs for ARPKD, we first isolated and characterized uEVs from healthy mouse urine by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting for EV markers. PCR confirmed the presence of Cys1 and Pkhd1 mRNAs in uEVs, while cellular uptake was verified by fluorescence microscopy and flow cytometry in collecting duct epithelial cells (mpkCCDc14). In vitro, uEV treatment enhanced Cys1 and Pkhd1 levels in healthy cells, and rescued Cys1 levels in Cys1 ‐deficient cells, derived from Cys1 cpk/cpk ( cpk ) mice. Upon administration in the cpk mouse model of ARPKD, uEV improved the survival rate in cpk mice. Furthermore, in utero administration of uEVs demonstrated accumulation in the fetal kidney and enhanced Cys1 level following intra‐amniotic (IA) administration, highlighting the feasibility of prenatal therapy for the most severe cases of ARPKD that are lethal in utero or within the first 24–48 h after birth. Taken together, our findings reveal that uEVs represent a promising therapeutic modality for ARPKD, capable of restoring deficient CYS1 protein levels and mitigating disease progression.

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