Figure 1. Podocytes in Fabry disease show persistent lysosomal dysfunction and damage despite enzyme replacement therapy.

(A) Transmission electron microscopy (TEM) comparison of foot processes between control and Fabry kidney biopsy before and after ERT with many foot processes widened in Fabry biopsies both untreated and after ERT. Asterisks show Gb3 inclusions in podocytes. Original magnification, ×52,800. (B) Significant decrease of podocyte Gb3 inclusions after ERT but persistence of increased foot process width. (C) Schematic overview of GLA-knockout (KO) podocyte generation by CRISPR/Cas9 genome editing. (D) Western blots show a complete absence of GLA expression in several GLA-KO clones. (E) Abolished GLA activity in 2 KO clones compared with WT cells. (F) Mass spectrometry analysis confirms the accumulation of Gb3-C24-0 isoform in KO cells, normalized upon 96 hours of α-galactosidase therapy (n = 3). (G) TEM shows zebra bodies exclusively in GLA-KO clones (red arrowheads). While WT cells depict a normal ultrastructure, aGAL-treated KO cells have remnant vacuoles (green arrowheads) without zebra bodies. Scale bars: 1 μm. (H) Lysosomal visualization using LAMP1 staining in differentiated WT and KO cells reveals an increased number and size (arrowheads) of lysosomes in the GLA-KO cells. Scale bar: 10 μm. (I) Quantification of lysosomal area (n = 14), pH (n = 12), and ROS production (n = 8). (J) Seahorse XFp experiments confirm normal mitochondrial function in KO cells (n = 8). OCR, oxygen consumption rate. (K) Mitochondrial import receptor subunit (TOM20) staining in WT and KO cells is equally abundant and normally distributed. TEM images confirm a normal mitochondrial ultrastructure in KO cells. Scale bars: 10 μm in immunofluorescence, 500 nm in electron microscopy. Violin plots indicate median (red) and upper and lower quartile (blue). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey’s multiple-comparison test.