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Abstract: FR-PO325

Trafficking and Localization of Disease-Causing Podocin Mutants in Human Podocytes

Session Information

Category: Genetic Diseases of the Kidneys

  • 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Lu, Pei-Chen, University of Bristol, Bristol, United Kingdom
  • Welsh, Gavin Iain, University of Bristol, Bristol, Bristol, United Kingdom
  • Saleem, Moin, University of Bristol, Bristol, Bristol, United Kingdom

Group or Team Name

  • Renal group, University of Bristol
Background

Mutations in the slit diaphragm protein podocin result in the commonest form of monogenic steroid-resistant nephrotic syndrome (SRNS). Podocin localizes to the podocyte slit diaphragm, which is crucial for the filtration process. Most disease-causing mutations of podocin cause it to be trafficked incorrectly intracellularly and this results in aberrant formation/function of the slit diaphragm complex. We have developed human podocyte cell lines expressing several disease-causing mutants of podocin to further understand the trafficking, intracellular localization and protein binding partners of this protein in both health and disease.

Methods

Wild type and R138Q, R238S, G92C, V180M, and R291W disease-causing mutants of podocin were tagged with Myc and stably expressed in conditionally immortalized human. Intracellular trafficking and localization of the tagged podocin constructs was determined by high resolution confocal and total internal reflection fluorescence (TIRF) microscopy. Sucrose density gradient centrifugation was used to study lipid raft localization. Proteomic analysis of podocin interacting proteins was carried out using mass spectrometry of co-immunoprecipitated proteins.

Results

Confocal and TIRF microscopy (Fig 1) showed that all the mutant proteins, except for the R138Q mutant, localized to the plasma membrane of podocytes. However, only the wild type and R238S protein were found to be associated with lipid raft structures. Mass spectrometry analysis of podocin binding proteins has identified several novel interactors including trafficking proteins and regulators of cell adhesion. Interestingly these protein/protein interactions are altered differentially by the podocin mutants.

Conclusion

Defining how disease-causing mutations alter the localization and protein binding partners of podocin will lead to a greater understanding of the pathogenesis of Nephrotic Syndrome.

Figure 1. podocin in the total internal reflection fluorescence (TIRF) imaging. The wild type podocin-myc is in green and caveolin-1 is in red, and merged parts is in yellow.

Funding

  • Private Foundation Support