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Page 59 Ryan et al. J Transl Genet Genom. 2025;9:48-61 https://dx.doi.org/10.20517/jtgg.2024.87

Beyond the glycosphingolipid pathway, we identified significant dysregulation of other lysosomal proteins,
including cathepsin B, lipase A (LIPA), and COL6A1. The upregulation of cathepsin B suggests enhanced
lysosomal protease activity, possibly as a response to accumulated substrates. Conversely, the
downregulation of LIPA and COL6A1 indicates disruptions in lipid degradation and lysosomal structural
integrity. Additionally, lysosomal membrane proteins such as LUM and Niemann-Pick disease type C2
protein (NPC2) were upregulated, suggesting compensatory cellular remodeling due to lysosomal
dysfunction. The reduced abundance of structural proteins like COL6A1 and occludin (OCLN) suggests
disruptions in lysosomal and broader cellular architecture, consistent with previous reports [5,26] . This study
provides evidence for the involvement of lysosomal proteins beyond glycosphingolipid metabolism in FD
pathology. The integration of proteomic analysis with automated live-cell imaging offers a robust platform
for further exploration of disease-specific cellular mechanisms.

The advanced image analysis used in this study is the first to quantitively visualize lysosome accumulation
in FD podocytes in real time, allowing us to capture dynamic changes in lysosomal numbers, size, and
distribution with disease progression over time of culture. This live cell imaging approach enabled a more
accurate assessment of lysosomal stress and dysfunction compared to traditional static methods, offering a
deeper understanding of the pathological mechanisms driving FD nephropathy. Using an unbiased,
automated quantification of LAMP-1+ lysosomes in FD podocytes, we observed a significant increase in the
size and number of LAMP-1+ vesicles compared to controls, reflecting heightened lysosomal stress. This
technique allowed us to track changes in real time, offering a more comprehensive understanding of how
lysosomal dysfunction evolves during podocyte differentiation due to a FD pathogenic variant. The
persistence of lysosomal stress in FD podocytes over later stages of differentiation suggests that these
podocytes continue to undergo significant dysfunction, which could contribute to ongoing podocyte injury
and the progression of FD nephropathy.

This study provides valuable insights into lysosomal dysfunction in FD podocytes; however, several
limitations must be acknowledged. First, the investigation focuses on a single case utilizing iPSC-derived
podocytes from a patient with the p.Met284Thr pathogenic variant. Future studies should include larger
cohorts of male and female patients with different GLA mutations to validate these findings and assess
variability across genetic and demographic subgroups. Second, lysosomal alterations are known to occur in
various cell types, and the extent to which the observed changes are specific to podocytes remains unclear.
Further research is required to determine whether similar lysosomal impairments and downstream cellular
disruptions occur in other affected renal cell types, such as mesangial or epithelial cells, and whether the
clinical manifestations arise from cell-specific genetic profiles or shared systemic mechanisms. Finally, while
the proteomic and imaging approaches employed here provide robust in vitro insights into lysosomal
dysfunction and its progression, translating these findings to in vivo systems and clinical settings is
necessary to fully understand their implications for disease progression and therapeutic development.

In conclusion, our study highlights the utility of iPSC-derived podocytes as a model for FD, combining
quantitative proteomics and advanced imaging to offer insights into disease pathology. The progressive
increase in lysosomal size and number reflects ongoing dysfunction, underscoring the need for therapeutic
strategies targeting lysosomal functional clearance of Gb3. The culture of iPSC-derived podocytes may not
only facilitate the identification of novel therapeutic targets but also support high-throughput screening of
potential therapies, aiming to reverse lysosomal dysfunction and improve clinical outcomes for FD patients.