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Abstract: PO1687

Characterization of a Novel FSGS-Associated ACTN4 Mutation in Drosophila melanogaster

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Odenthal, Johanna, Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
  • Beck, Bodo B., Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
  • Schermer, Bernhard, Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
  • Benzing, Thomas, Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
  • Brinkkoetter, Paul T., Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
  • Bartram, Malte P., Uniklinik Koln, Koln, Nordrhein-Westfalen, Germany
Background

Decisive for podocyte morphology and homeostasis during health and disease is a specialized and highly regulated organization of the actin cytoskeleton. In this context, the actin cross-linking protein Alpha-actinin4 (ACTN4) has been shown to play a crucial role in podocyte architecture and function. Mutations in the ACTN4 gene are associated with focal segmental glomerulosclerosis (FSGS). Here, performing gene panel sequencing in a pediatric patient presenting with steroid resistant nephrotic syndrome and FSGS, a de novo, potentially disease causing variant of ACTN4 was identified, which was previously undescribed and not found in available genome or exome databases. Our aim is to elucidate the pathogenic potential of this variant for podocytes and FSGS progression.

Methods

To elucidate pathogenic effects of the newly identified ACTN4 variant, we employed the genetic toolbox of Drosophila. The fly holds podocyte-equivalent cells called nephrocytes, which are responsible for filtration and detoxification of the hemolymph. Cell-specific genetic manipulation enabled us to analyze RNAi-mediated knockdown of Actinin, the single fly homolog, in nephrocytes and its impact on cell morphology and function. Rescue experiments with the novel human ACTN4 variant will now give indication about possible pathogenic consequences of the mutation when compared to wildtype as well as previously described disease-associated variants of ACTN4.

Results

Knockdown of Drosophila Actinin in nephrocytes leads to severe functional defects, as filtration capacity is diminished by up to 50%. Morphologically, mislocalization of the ZO-1 homolog Polychaetoid was observed as well as overall reduction of nephrocyte diaphragms. First rescue experiments with wildtype human ACTN4 led to partial rescue of functional and morphological phenotypes observed upon Actinin knockdown.

Conclusion

Our results underline the importance of Actinin for nephrocyte biology. Capacity of wildtype human ACTN4 in rescuing the knockdown associated phenotypes indicates the model’s suitability. Further experiments will be performed to elucidate the pathogenicity of the novel ACTN4 variant also in comparison to previously described pathogenic mutations.