TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome
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BASIC RESEARCH www.jasn.org TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome Lina L. Kampf,1 Ronen Schneider,2 Lea Gerstner,1 Roland Thünauer,3,4 Mengmeng Chen,1 Martin Helmstädter,1 Ali Amar,2 Ana C. Onuchic-Whitford,2,5 Reyner Loza Munarriz,6 Afig Berdeli,7 Dominik Müller,8 Eva Schrezenmeier,9 Klemens Budde,9 Shrikant Mane,10 Kristen M. Laricchia,11 Heidi L. Rehm ,11 Daniel G. MacArthur,11 Richard P. Lifton,10,12 Gerd Walz,1 Winfried Römer,3 Carsten Bergmann,13,14,15 Friedhelm Hildebrandt,2 and Tobias Hermle 1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT 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. JASN 30: 2338–2353, 2019. doi: https://doi.org/10.1681/ASN.2019040414 Received April 24, 2019. Accepted August 7, 2019. The glomerular filter of the kidney is three-layered, consisting of a fenestrated endothelium, the glo- L.L.K., R.S., and L.G. contributed equally to this work. merular basement membrane, and the podocytes Published online ahead of print. Publication date available at that form the slit diaphragm. Disorders of the glo- www.jasn.org. merular filter commonly involve the podocyte and Correspondence: Dr. Tobias Hermle, Renal Division, Department manifest with edema, hypoalbuminemia, and severe of Medicine, University Medical Center Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany, or Dr. Friedhelm Hildebrandt, Boston proteinuria, a clinical triad that characterizes the Children’s Hospital, Harvard Medical School, Enders 561, 300 nephrotic syndrome. Steroid-resistant nephrotic Longwood Avenue, Boston, MA 02115. E-mail: tobias.hermle@ syndrome (SRNS) commonlyentails declining renal uniklinik-freiburg.de or [email protected] function, and represents the second most frequent Copyright © 2019 by the American Society of Nephrology 2338 ISSN : 1046-6673/3012-2338 JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH cause of ESRD in patients manifesting before 25 years of age.1 Significance Statement Mutations in about 50 different genes have been identified as monogenic causes of SRNS, including proteins of the slit di- The discovery of monogenic causes of nephrotic syndrome led to aphragm complex and actin regulators, but also factors of insights about the role of podocytes and the slit diaphragm in the CoQ biosynthesis, nucleoporins, or members of the KEOPS pathogenesis of the disease. The authors describe novel mutations 10 in TBC1D8B in five families with steroid-resistant nephrotic syn- 2–43 complex. Many of these genetic causes of SRNS are rare; drome. TBC1D8B binds to active RAB11A and RAB11B. Silencing however,thediscoveryofthesesinglegenemutationsas TBC1D8B leads to upregulation of RAB11-dependent processes molecular causes of SRNS contributed significantly to the suggesting TBC1D8B inhibits RAB11. TBC1D8B also interacts and understanding of the complex pathogenesis of nephrotic colocalizes with the slit diaphragm protein nephrin. Silencing TBC1D8B Drosophila fi fl syndrome and podocyte biology. in podocyte-like nephrocytes causes mistraf cking of y nephrin. Nephrin trafficking in Drosophila requires Rab11,whereas There is mounting evidence supporting an essential role of overexpression of Rab11 causes a similar phenotype as TBC1D8B vesicular trafficking via endocytosis for the function of the silencing. These findings implicate regulation of RAB11-dependent glomerular filter.44,45 Disrupting components of the endocytic vesicular trafficking by TBC1D8B as a novel pathogenetic pathway in machinery in mouse resulted in podocyte dysfunction with nephrotic syndrome. severe proteinuria.46–48 Nephrin, an essential component of fi 49–55 the slit diaphragm, undergoes endocytic traf cking. BioCAT GmbH). Human TBC1D8B was subcloned by PCR Recently, we discovered mutations of the RAB5 interactors from cDNA representing the shorter isoform 2 (GenBank GAPVD1 and ANKFY1 as novel causes of nephrotic syndrome BC122564; GE Dharmacon). Truncation constructs were gen- fi and the rst endosomal regulators in the pathogenesis of erated by PCR. Primers are shown in Supplemental Table 1. 41 human nephrotic syndrome. However, the functional role The following expression vectors were used: pRK5-N-Myc, of endocytosis for the slit diaphragm is still unclear. In partic- pCDNA6.2-C-GFP, and pCDNA6.2-N-GFP. The mutations ular, the mechanistic role of the various aspects of the endo- identifiedinindividualswithnephroticsyndromewereintroduced cytic pathway for slit diaphragm formation and maintenance into the cDNA constructs by site-directed mutagenesis via Gibson remains elusive. We now report hemizygous mutations of the assembly (New England Biolabs). The following constructs were endosomal regulator TBC1D8B, discovered by whole-exome obtained from Addgene: pSpCas9(BB)-2A-GFP (PX458) fi sequencing (WES) in ve individuals with nephrotic syn- (#48138), pX459V2.0-eSpCas9 (#108292), GFP-RAB11 WT drome. Our analysis in vitro and in the Drosophila model (#12674), and GFP-RAB7 (#61803) NPHS1 and RAB5 cDNA fi supports a novel role of RAB11-dependent vesicular traf ck- constructs have been described elsewhere.41 ing in the pathogenesis of the nephrotic syndrome. The TBC1D8B-specific and control siRNAs were purchased from GE Dharmacon. Overexpression experiments were performed in HEK293T METHODS cells or immortalized human podocytes that were a gift from Dr. Moin Saleem (University of Bristol, Bristol, UK). Study Approval HEK293T cells were maintained in DMEM, supplemented Approval for human subjects research was obtained from the with 10% FBS, 50 IU/ml penicillin, and 50 mg/ml streptomy- University of Michigan, University of Freiburg and the Boston cin. Podocytes were maintained in RPMI plus GlutaMAX-I Children’s Hospital Institutional Review Boards. All partici- (Gibco) supplemented with 10% FBS, 50 IU/ml penicillin/ pants or their guardians provided written informed consent. 50 mg/ml streptomycin, and insulin-transferrin-selenium X. Plasmids and siRNAs were transfected into HEK293T cells Study Participants at 37°C or podocytes grown at the permissive temperature of After obtaining informed consent, clinical data and blood samples 33°C using Lipofectamine 2000 (Invitrogen) or polyethylenei- were collected from individuals with nephrotic syndrome. Clinical mine (Polyplus-transfection or Sigma). data were acquired using an established questionnaire. The diag- nosisofnephroticsyndromewasmadeby(pediatric)nephrologists, on the basis of standardized clinical and renal histologic criteria. Nephrin Trafficking Assay Renal biopsy specimens were evaluated by renal pathologists. MDCK strain II cells (wt MDCK) were a gift from Enrique Rodriguez-Boulan (Weill Cornell Medical College, NY) and Homozygosity Mapping, Whole-Exome Resequencing, were maintained in DMEM supplemented with 5% FCSFCS, and Mutation Calling at 37°C and 5% CO2 in 10 cm culture dishes, and passaged Homozygosity mapping, whole-exome resequencing, and every 2–3 days. mutation calling were performed as described previously.23 For growing polarized monolayers, cells were cultured on Transwell filters (#3401; from Corning Costar) for 4 days. Plasmids, siRNAs, Cell Culture, and Transfection Transfections with a plasmid encoding a nephrin construct Murine full-length Tbc1d8b cDNA was subcloned by PCR (pCAD4-HA-nephrin-mCherry) comprising 43 conditional from full-length murine cDNA (GenBANK BC147581.1; aggregation domains (CADs), furin cleaving site, HA-tag, JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2339 BASIC RESEARCH www.jasn.org nephrin, and mCherry (pCAD4-HA-nephrin-mCherry) were Cas9-expression (#58985) in nephrocytes. The CRISPR carried out using lipofectamine 2000 (Thermo Fisher Scien- gRNA construct targeting CG7324