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Page 49 Ryan et al. J Transl Genet Genom. 2025;9:48-61 https://dx.doi.org/10.20517/jtgg.2024.87

podocytes. Lysosomal proteins were enriched, with a significant increase in cathepsin B (P < 0.001) and a decrease
in lipase A (P < 0.01). Furthermore, the dysregulation of proteins involved in cell cycle regulation and growth
signaling pathways, such as polo-like kinase 1 (PLK1; P < 0.0001) and proto-oncogene tyrosine-protein kinase Src
(SRC; P < 0.01), suggested broader impacts on cellular processes. Temporal live-cell imaging revealed a significant
increase in lysosome number in day 20 FD podocytes compared to day 10 FD podocytes and controls (P < 0.01).

Conclusions: These findings collectively suggest that FD podocytes undergo progressive lysosomal impairment,
which may contribute to cellular dysfunction and disease progression. These proof-of-concept findings lay a
foundation for future research on targeted FD therapies using high-throughput screening and advanced analytical
techniques.
Keywords: Fabry disease, lysosomes, podocytes, induced pluripotent stem cells, proteomics, live-cell imaging

INTRODUCTION
Fabry disease (FD) is the most common X-linked lysosomal storage disorder and is caused by pathogenic
[1,2]
variants in the GLA gene, leading to a deficiency in lysosomal α-galactosidase A (α-Gal A) . This enzyme
deficiency results in the progressive accumulation of globotriaosylceramide (Gb3), especially in kidney
podocytes, contributing to FD nephropathy, proteinuria, and chronic kidney disease (CKD). Although
therapies such as enzyme replacement therapy (ERT) can reduce Gb3 levels, the reversal of kidney damage
remains incomplete, particularly in podocytes, which exhibit slower and less complete Gb3 clearance
[3-5]
compared to other renal cell types .
Lysosomal dysfunction is a hallmark of FD, driven by Gb3 accumulation due to α-Gal A deficiency.
Accumulation of globotriaosylsphingosine (lysoGb3), deacetylated Gb3, is thought to exacerbate these
processes, driving fibrosis and inflammatory responses and further impairing lysosomal function in FD
[6-8]
nephropathy . While it is well understood that this buildup disrupts normal lysosomal function and leads
to podocyte injury, many aspects of the cellular consequences of lysosomal dysfunction remain unclear. Gb3
[9]
accumulation has been shown to impair autophagy and promote ferroptosis , and increase podocyte
[10]
stress, leading to cell depletion [4,11-14] , yet the precise signaling pathways that contribute to lysosomal
dysfunction and progressive organ damage are not yet fully understood.
To better understand these mechanisms, induced pluripotent stem cell (iPSC)-derived podocytes offer a
promising alternative to primary podocytes, which are difficult to maintain in culture. These iPSC-based
models can replicate patient-specific genetic and phenotypic characteristics, offering a promising platform
for understanding the molecular mechanisms underlying FD and for therapeutic screening, as we have
[15]
previously reported in podocytes and cardiomyocytes . We now provide evidence of the proteomic and
[10]
cellular changes occurring in FD podocytes, which collectively highlights the significant impact of lysosomal
dysfunction on podocyte homeostasis. iPSCs were derived from FD patients and compared to controls,
which were then differentiated into kidney podocytes as previously reported [10,16-18] .

This study represents the first to comprehensively characterize the proteomic and temporal changes in
lysosomal dynamics in human iPSC-derived podocytes from an individual with FD carrying the
p.Met284Thr GLA variant. Using advanced proteomic analysis and automated live-cell imaging, we provide
insights into the lysosomal abnormalities that may drive disease progression in podocytes. This model offers
a human-relevant system for investigating FD mechanisms, with implications for developing more targeted
therapies aimed at ameliorating lysosomal dysfunction in FD nephropathy.