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Kidney Week

Abstract: TH-PO794

TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Kampf, Lina Luise, Medical Center - University of Freiburg, Freiburg, Germany
  • Schneider, Ronen, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Gerstner, Lea, Medical Center - University of Freiburg, Freiburg, Germany
  • Chen, Mengmeng, Medical Center - University of Freiburg, Freiburg, Germany
  • Bergmann, Carsten, Medical Center- University of Freiburg, Freiburg, Germany
  • Hildebrandt, Friedhelm, Boston Children's Hospital/Harvard Medical School, Boston, Massachusetts, United States
  • Hermle, Tobias F., Medical Center - University of Freiburg, Freiburg, Germany

Group or Team Name

  • Hermle lab
Background

Steroid-resistant nephrotic syndrome (NS) frequently underlies CKD. Mutations in about 50 genes were identified as monogenic causes of NS and these genes rendered significant insight into podocyte biology. We previously reported mutations in TBC1D8B in two families with NS. TBC1D8B harbors a TBC domain, that commonly confers a functional role as a GTPase-activating protein (GAP) for specific Rab-GTPases. GAPs promote the inactive state of their Rab protein. However, the function of TBC1D8B and its pathogenetic role remained unclear.

Methods

To identify additional mutations of TBC1D8B, we performed whole-exome sequencing (WES). We analyzed the functional role of TBC1D8B and its mutations in vitro and conducted studies in podocyte-like Drosophila nephrocytes.

Results

We identified one hemizygous missense mutation (c.1316T>G, p.Phe439Cys) and two hemizygous nonsense mutations (c.1030C>T, p.Arg344* and c.1383G>A, p.Trp461*) in patients with NS. To explore the function of TBC1D8B and its target Rab proteins, we performed co-immunoprecipitation assays (coIP). We observed strongest interaction between RAB11 and TBC1D8B and specificity for the active form. Overexpressed murine Tbc1d8b bearing patient-derived mutations exhibited lower affinity for endogenous RAB11 than wild type protein. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocyst activity, two cellular functions that are independently regulated by RAB11. This suggests disinhibition of endogenous RAB11 and thus a regulatory role of TBC1D8B. CoIP assays further indicated interaction between TBC1D8B and the slit diaphragm protein nephrin. Both proteins co-localized in cultured podocytes. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of the Drosophila ortholog of nephrin. Expression of Rab11-RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane while RAB11 overexpression revealed a phenotypic overlap to Tbc1d8b loss-of-function.

Conclusion

Novel mutations in TBC1D8B are monogenic causes of NS and our data indicate that TBC1D8B serves as a GAP protein for RAB11. TBC1D8B interacts with nephrin, being required for its trafficking. This connects RAB11-dependent vesicular trafficking to the pathogenesis of human NS.

Funding

  • Other NIH Support