TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome
Authors: Kampf Lina L. , Schneider Ronen , Gerstner Lea , Thünauer Roland , Chen Mengmeng , Helmstädter Martin , Amar Ali , Onuchic-Whitford Ana C. , Loza Munarriz Reyner , Berdeli Afig , Müller Dominik , Schrezenmeier Eva , Budde Klemens , Mane Shrikant , Laricchia Kristen M. , Rehm Heidi L. , MacArthur Daniel G. , Lifton Richard P. , Walz Gerd , Römer Winfried , Bergmann Carsten , Hildebrandt Friedhelm , Hermle Tobias 
Published: November 2019

Significance Statement
The discovery of monogenic causes of nephrotic syndrome led to insights about the role of podocytes and the slit diaphragm in the pathogenesis of the disease. The authors describe novel mutations in TBC1D8B in five families with steroid-resistant nephrotic syndrome. TBC1D8B binds to active RAB11A and RAB11B. Silencing TBC1D8B leads to upregulation of RAB11-dependent processes suggesting TBC1D8B inhibits RAB11. TBC1D8B also interacts and colocalizes with the slit diaphragm protein nephrin. Silencing TBC1D8B in podocyte-like Drosophila nephrocytes causes mistrafficking of fly nephrin. Nephrin trafficking in Drosophila requires Rab11, whereas overexpression of Rab11 causes a similar phenotype as TBC1D8B silencing. These findings implicate regulation of RAB11-dependent vesicular trafficking by TBC1D8B as a novel pathogenetic pathway in nephrotic syndrome.

Background
Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.

Methods
We used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes.

Results
We identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function.

Conclusions
Novel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.