Kidney Week

Abstract: SA-PO619

Transgenic Human Nephrin in Drosophila Nephrocytes Facilitates Variant Analysis

Session Information

• Genetic Kidney Diseases: Models, Mechanisms, and Therapies
  October 26, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Wolff, Julia Melina, University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Julia Melina Wolff

• Lang, Konrad, University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Konrad Lang, MD

• Milosavljevic, Julian, University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Julian Milosavljevic, MD

• Chen, Mengmeng, University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Mengmeng Chen, MD

• Helmstädter, Martin, University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Martin Helmstädter

• Bahar, Sami, An-Najah National University, Faculty of Medicine and Health Sciences, Department of Medicine, Nablus, Nablus Governerate, Palestine, State of

Sami Bahar, MD

• Ulbrich, Maximilian H., University of Freiburg, Faculty of Medicine, Department of Anatomy and Cell Biology, Department of Neuroanatomy, Freiburg, Baden-Württemberg, Germany

Maximilian H. Ulbrich

• Hermle, Tobias F., University Medical Center Freiburg Department of Medicine, Divison of Nephrology, Freiburg, Baden-Württemberg, Germany

Tobias F. Hermle, MD

Background

Nephrin, the key structural protein of the slit diaphragm, is encoded by NPHS1. Pathogenic variants in this gene are the primary cause of congenital nephrotic syndrome. About 400 variants have been described but functional characterization is curtailed by the lack of slit diaphragm formation within in vitro models. A molecularly conserved and accessible slit diaphragm can be found in the Drosophila nephrocyte model.

Methods

Human nephrin was transgenically expressed in Drosophila nephrocytes in combination with silencing of the nephrin ortholog sns. Nephrocytes were phenotypically characterized by immunofluorescence, tracer studies, and transmission electron microscopy including immunogold labeling. Automated image annotation was employed to quantify distinct staining patterns. A novel nephrin variant was identified in a patient with congenital nephrotic syndrome using whole exome sequencing. Transgenes reflecting this novel and another variant of NPHS1, previously identified as deleterious, were generated for functional analysis in Drosophila.

Results

Transgenic human nephrin assembled into a complex linear architecture in nephrocytes after silencing of Sns. This suggests lateral clustering of nephrin into a macromolecular configuration within the podocyte-like cells. Transgenic nephrin colocalized with the endogenous slit diaphragm protein Pyd, ortholog of ZO-1, suggesting a hybrid multi-protein complex. Upon transgenic co-expression of murine Neph1, nephrin similarly recruited its binding partner. However, the linear nephrin exhibited an atypical, tubular ultrastructure and did not adequately direct actin remodeling, underscoring the need for further indispensable co-factors.
The linear nephrin assembly was disrupted in transgenes reflecting canonical patient variant S366R as well as novel variant V1241G, which affects the nephrin C-terminus. Automated annotation of Pyd staining patterns provided a quantitative read-out for investigation of these patient-derived variants.

Conclusion

Transgenesis of NPHS1 in nephrocytes is a viable approach for investigation of basic steps of slit diaphragm formation and functional characterization of patient variants in NPHS1.

Funding

• Government Support – Non-U.S.