#2556 Exploring exosome-mediated effects on renal pathology in Anderson Fabry disease

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Abstract Background and Aims Despite substantial progress in understanding the molecular basis of genetic lysosomal storage diseases, the systemic mechanisms linking lysosomal defects in specific cells to widespread organ damage remain unclear, particularly in Anderson-Fabry disease (AFD). This project introduces a novel perspective by investigating the role of exosomes as mediators of intercellular communication and contributors to the spread of pathology in AFD. The aim of this study is to explore exosome-mediated renal cell damage mechanisms in AFD progression. Methods Urinary exosomes were isolated from adult patients with AFD and from age- and sex-matched healthy controls by ultracentrifugation. Quality and quantity were checked by cytofluorimetry using p63 and p81 markers. Renal cells (HEK293) were incubated with the same amounts of exosomes for 48 and 72 hours and, then, autophagic flux and endoplasmic reticulum stress were assessed by western blot analysis. Cell respiration was monitored using the SeaHorse system (Agilent Technologies). Results Incubating healthy renal cells (HEK293) with exosomes isolated from the urine of AFD patients (male: 3; female: 4) caused a disruption of autophagic flux after 72 hours. This was evidenced by an increase in the LC3-II/LC3-I ratio (A.U. at 48 h: 0.68 ± 0.19; at 72 h: 1.14 ± 0.42, P < 0.05), a marker of autophagy, along with the lack of p62 degradation (A.U. at 48 h: 0.07 ± 0.03; at 72 h: 0.05 ± 0.04, P > 0.05), a protein typically degraded during properly functioning autophagy. No significant effects were observed when HEK293 cells were incubated with exosomes derived from the urine of healthy volunteers (male: 3; female: 2) (LC3-II/LC3-I ratio: A.U. at 48 h: 1 ± 0.23, at 72 h: 1 ± 0.38, P > 0.05; p62: A.U. at 48 h: 0.05 ± 0.03, at 72 h: 0.04 ± 0.02, P > 0.05). No differences were observed in other cellular processes (e.g. cell respiration and endoplasmic reticulum stress) after 24, 48, and 72 hours of incubation with exosomes from either experimental group. Conclusion These findings demonstrate that exosomes derived from AFD patients disrupt normal autophagic processes, providing a potential novel mechanism for the pathogenesis of the disease. Furthermore, the effect of exosomes on autophagic flux appears to be specific, as no alterations were detected in other cellular processes (e.g. cell respiration or endoplasmic reticulum stress). This study offers novel insights into the potential role of exosomes in AFD progression, challenging the traditional understanding of this disease as a primarily localized disorder characterized by substrate accumulation confined to cells expressing the genetically altered enzyme. Instead, it suggests that the systemic, multi-organ manifestations of AFD may arise, at least in part, from the active dissemination of toxic metabolites and signaling molecules via exosomes.

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