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Abstract: TH-PO1068

Functional Analysis of a Novel FSGS-Associated ACTN4 Mutation in Podocytes and Drosophila Melanogaster

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology


  • Odenthal, Johanna, University Hospital Cologne, Cologne, Germany
  • Dittrich, Sebastian, University Hospital Cologne, Cologne, Germany
  • Beck, Bodo B., University of Cologne Medical Center, Cologne, Germany
  • Schermer, Bernhard, University Hospital Cologne, Cologne, Germany
  • Benzing, Thomas, University of Cologne, Köln, Germany
  • Rinschen, Markus M., The Scripps Research Institute, La Jolla, California, United States
  • Brinkkoetter, Paul T., University Hospital Cologne, Cologne, Germany
  • Bartram, Malte P., University Hospital Cologne, Cologne, Germany

The actin cytoskeleton is a central element of podocyte morphology and homeostasis during health and disease. Alpha-actinin4 (ACTN4) has been shown to play an important role in podocyte architecture and function and mutations in the ACTN4 gene are associated with focal segmental glomerulosclerosis (FSGS). Here, in a pediatric patient presenting with steroid resistant nephrotic syndrome (SRNS) and FSGS, gene panel sequencing of genes associated with rare kidney diseases identified an undescribed de novo, potentially disease-causing variant of ACTN4 that was not found in available genome or exome databases. Aim of this study was to characterize this variant and elucidate its pathogenic potential for podocyte homeostasis.


We analyzed patient-derived primary urinary cells (PUCs) as well as cultured human podocytes that express the novel ACTN4 mutant. Results were obtained using quantitative proteomic analysis as well as cell biology studies in vitro. In order to perform in vivo studies, we exploited Drosophila melanogaster genetics and characterized Actinin loss in nephrocytes, podocyte-like cells of the fruit fly. Here, rescue experiments with human ACTN4 wildtype, other previously described, pathogenic ACTN4 mutations as well as the novel variant will give further insight into its pathogenicity.


Mapping the PUC proteome, we quantified more than 3000 proteins as compared to healthy controls. PUCs of the index patient showed high abundance of DNA-damage response associated proteins, and depletion of the known ACTN4 interactor ZNF385. Cultured human podocytes overexpressing the ACTN4 mutant present with disturbed appearance and localization of the actin cytoskeleton and first experiments suggest the ACTN4 mutant to impact cell viability. Knockdown of Drosophila Actinin in nephrocytes leads to a severe functional phenotype, as cells no longer perform proper filtration. Morphologically, we could show that localization of the nephrocyte diaphragm is perturbed, suggesting false architecture of the nephrocyte.


Our results indicate that the identified novel ACTN4 mutation leads to a strong phenotype in vitro, likely making it a disease-causing mutation. The in vivo data underline the importance of actinin in nephrocyte architecture and function.