Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.
Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.
Laboratory of Immunogenetics and Molecular Biology, Federal University of Piaui, Teresina, Brazil.
Ester M
EM
Pereira
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Anatália
A
Labilloy
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Megan L
ML
Eshbach
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Ankita
A
Roy
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Arohan R
AR
Subramanya
Laboratory of Immunogenetics and Molecular Biology, Federal University of Piaui, Teresina, Brazil; semiramis@ufpi.edu.br.
Semiramis
S
Monte
Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Guillaume
G
Labilloy
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and.
Ora A
OA
Weisz
D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000795:Fabry Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D050199:Podocytes / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction / Q000502:physiology; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism*
D000519:alpha-Galactosidase
CRISPR/Cas9; Fabry nephropathy; podocyte
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