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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated
Ester M Pereira1, Anatália Labilloy2, Megan L Eshbach2
1Laboratory of Immunogenetics and Molecular Biology, Federal University of Piaui, Teresina, Brazil.
Abstract:
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.
Insights
Researchers developed new human kidney cell models for Fabry disease (FD) using CRISPR/Cas9 gene editing. These models show impaired signaling pathways, aiding Fabry nephropathy research.
Area of Science:
- Genetics and Molecular Biology
- Cell Biology
- Nephrology
Background:
- Fabry nephropathy, a complication of Fabry disease (FD), causes significant morbidity and mortality.
- FD is an X-linked lysosomal storage disorder characterized by the accumulation of globotriaosylceramide (Gb3).
- Human kidney podocytes are a key cell type affected in Fabry nephropathy.
Purpose of the Study:
- To develop novel human kidney cell models of Fabry disease using CRISPR/Cas9 genome editing.
- To investigate the molecular mechanisms and signaling pathways affected by α-galactosidase A deficiency in podocytes.
- To establish a platform for studying Fabry nephropathy pathogenesis.
Main Methods:
- CRISPR/Cas9 genome editing was used to target the GLA gene in human immortalized podocytes.
- Developed podocyte cell models with deficient α-galactosidase A activity and elevated Gb3 levels.
- Utilized high-throughput antibody arrays for phosphorylation profiling to analyze signaling pathway activation.
Main Results:
- Successfully generated human podocyte models lacking α-galactosidase A activity and exhibiting Gb3 accumulation.
- Identified significant alterations in both total protein levels and site-specific phosphorylation.
- Phosphorylation profiling revealed dysregulation in multiple signaling pathways critical for podocyte function.
Conclusions:
- The developed CRISPR/Cas9-edited podocyte models accurately represent key aspects of Fabry nephropathy.
- α-galactosidase A deficiency in podocytes leads to widespread impairments in cellular signaling pathways.
- These findings provide a foundation for further research into Fabry nephropathy mechanisms and therapeutic strategies.
