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Ryan et al. J Transl Genet Genom. 2025;9:48-61 https://dx.doi.org/10.20517/jtgg.2024.87 Page 58

On day 10, FD podocytes displayed fewer LAMP-1+ lysosomes compared to day 20 FD podocytes, with a
significant increase in lysosomal accumulation that was evident over time [Figure 5G]. Quantification of
lysosomal number per cell [Figure 5H] showed a significant increase in FD podocytes at day 20 compared to
controls (P < 0.05), indicating progressive lysosomal changes following the differentiation period. Using the
plasma membrane-CFP transfection construct [Figure 5E], cellular area was quantified in conjunction with
the number of LAMP-1+ lysosomes [Figure 5F]. When normalized to the total cellular area, significant
increases in the number of LAMP-1+ vesicles were observed in day 20 FD podocytes compared to both day
10 and day 20 control podocytes (Figure 5I; P < 0.0001). A significant increase in LAMP-1+ vesicles as a
proportion of cellular area was also observed in day 20 FD podocytes compared to day 10 FD podocytes
(Figure 5I; P < 0.01).

DISCUSSION
Our study combines proteomics and advanced live-cell imaging to assess lysosomal dysfunction in FD using
patient-derived iPSC-podocytes. Proteomic analysis identified a significant dysregulation of lysosomal
proteins involved in the glycosphingolipid pathway, which aligns with the established dysfunction of
lysosomal Gb3 storage in FD progression. Automated live-cell imaging revealed a time-dependent increase
in LAMP-1+ lysosomes in FD podocytes, demonstrating a progressive accumulation in lysosomal number.
When normalized to cell area, there was a significant increase in lysosomal number in day 20 cultured FD
podocytes compared to day 10 podocytes and controls, indicating persistent lysosomal stress and cellular
dysfunction.

Elevated plasma Gb3 and lysoGb3 are well-established hallmarks of FD , and although lysosomal Gb3
[6-8]
storage is a key factor in disease manifestation, the formation of pathogenic lysoGb3 likely plays a
significant role in the onset and progression of symptoms [19,20] . In our study, proteomic analysis identified
significant dysregulation of proteins involved in lysosomal storage and lipid degradation, supporting
previous studies that link Gb3 accumulation to disrupted lysosomal function, leading to cellular damage
and multi-organ pathology [5,15,21] . This accumulation has been linked to reactive oxygen species production
[21]
and mitochondrial impairment in podocytes, further damaging the tubular epithelium and leading to
subsequent podocyte damage, which extends to the tubular epithelium . Although inhibiting Notch1
[19]
shows promise in mitigating these effects , current treatments, including ERT, do not fully reverse the
[22]
underlying pathology [4,5,23] . Even after prolonged ERT treatment, the reversal of lysosomal dysfunction in
podocytes remains insufficient, as shown in the persistence of structural damage and with new therapeutic
targets, such as α-synuclein (SNCA) . Delaleu et al. demonstrated that early ERT only partially reverts gene
[5]
expression patterns associated with FD nephropathy, indicating that significant pathways, including those
[4]
related to transporter activity, remain dysregulated or re-emerge despite treatment .
The differentiation of podocytes from patient-derived iPSCs offers direct insights into the disease
mechanisms within affected cells, complementing proteomic studies using FD patient plasma that have
highlighted inflammatory and angiogenesis-related proteins [13,14,24] . In this study, we observed a significant
dysregulation of lysosomal proteins, consistent with previous research in cardiomyocytes and podocytes,
which are the most affected cell types in FD [1,12,25] . The downregulation of the main effector protein, GLA,
aligns with the expected disease phenotype [5,15,26] . In contrast, the upregulation of enzymes such as GBA and
GALC suggests a de novo compensatory response to manage lipid accumulation, as evidenced by clinical
trials showing substrate reduction therapy (SRT) efficacy in reducing Gb3 and lysoGb3 levels [27,28] . These
findings collectively demonstrate that FD podocytes exhibit widespread proteomic alterations, particularly
in pathways related to lysosomal function and glycolipid metabolism, which may contribute to disease
pathology and the cellular phenotype observed in FD nephropathy.