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 was generated by introduc- tific). D/D-solubilizer (Clontech/Takara) was used to induce ing two gRNAs via PCR and Gibson assembly into the pCFD4 endoplasmic reticulum (ER) release at a concentration of plasmid (Addgene; #49411). The DNA was injected into flies 5 mM, and 100 mg/ml of cycloheximide was added during re- expressing phiC31 integrase under vasa promoter with an attP lease to prevent further protein synthesis. Staining of nephrin landing site in 51C by the fly facility of the Department of arriving at the cell surface was carried out with anti-HA anti- Genetics at the University of Cambridge (Cambridge, UK). bodies (Covance) conjugated to Cy5 that were applied to the RNAi crosses were raised at 30°C. Flies expressing gRNAs apical or basolateral side of live cells. and a Cas9 variant were raised at 25°C as higher temperatures For immunofluorescence, cells were fixed with 4% formal- induced unspecific toxicity by Cas9 expression alone (data not dehyde for 15 minutes at room temperature. After excising shown). filters with a scalpel, they were mounted in DABCO-medium Fluorescent tracer uptake in nephrocytes was performed as and imaged using Nikon A1R confocal microscope. Quantifi- previously described.61 Briefly, nephrocytes were dissected in cation of cell surface arrival of nephrin was done as described PBS and incubated with FITC-albumin (Sigma) for 30 seconds. previously, with a custom-written MATLAB program.56,57 After a fixation step of 5 minutes in 8% paraformaldehyde cells were rinsed in PBS and exposed to Hoechst 33342 (1:1000) for Immunoblotting, Immunoprecipitation, Pull-Down 20 seconds and mounted in Roti-Mount (Carl Roth). Cells Assay, and Immunofluorescence Staining were imaged using a Zeiss LSM 880 laser scanning microscope. Immunoblotting, immunoprecipitation, and immunofluores- Quantitation of fluorescent tracer uptake was performed with cence staining were performed as described previously.58 ImageJ software. The results are expressed as a ratio to a control Briefly, HEK293Twere lysed and precleared using rec-Protein experiment with GFP RNAi that was done in parallel. A-Sepharose 4B Conjugate (Life Technologies) overnight. For immunohistochemistry, nephrocytes were dissected, Then, equal amounts of protein were incubated with EZview fixed for 20 minutes in PBS containing 4% paraformaldehyde, Red Anti-c-Myc Affinity Gel (Sigma-Aldrich). Coimmuno- andstainedaccordingto the standardprocedure. The following precipitation experiments were performed in three indepen- primary antibodies were used: rabbit anti-sns62 (1:500, gift dent experiments. For chloroquine treatment, cells were from S. Abmayr) and guinea pig anti-Kirre63 (1:200, gift from exposed to culture medium containing 80 mM chloroquine S. Abmayr). For imaging, a Zeiss LSM 880 laser scanning mi- for 24 hours. Immunoblotting was performed using mouse croscope was used. Image processing was done by ImageJ and anti-TBC1D8B (SC-376637; Santa Cruz Biotechnology), rabbit GIMP software. anti-RAB11 (#5589; Cell Signaling Technology), mouse anti– For transmission electron microscopy nephrocytes were c-Myc (sc-40; Santa Cruz Biotechnology), rabbit anti–c-Myc dissected and fixed in 4% formaldehyde and 0.5% glutaralde- (sc-789; Santa Cruz Biotechnology), mouse anti-GFP (sc- hyde in 0.1 M cacodylate buffer at pH 7.4. Transmission elec- 9996; Santa Cruz Biotechnology), rabbit anti-GAPDH (14C10/ tron microscopy was carried out using standard techniques. 2118; Cell Signaling Technology), mouse anti-actin (ab20272; Abcam), rabbit anti-LC3B (2775; Cell Signaling Technology), Statistical Analyses and rabbit anti-GFP (sc-8334; Santa Cruz Biotechnology). Paired t test was used to determine the statistical significance Immunofluorescence of TBC1D8B was performed with between two interventions. ANOVA followed by Dunnett’s correc- rabbit anti-TBC1D8B (ab121780; Abcam) or mouse anti- tion (unless otherwise indicated) was used for multiple compari- TBC1D8B (SC-376637). Other antibodies used were rabbit sons (GraphPad Prism software). Asterisks indicate significance as anti-RAB11 (#5589S; Cell Signaling Technology), and mouse follows: *P,0.05; **P,0.01; ***P,0.001. A statistically significant anti-Myc (9E10; Developmental Studies Hybridoma Bank). difference was defined as P,0.05. Error bars indicate SD. Fluorescence images were obtained with a Zeiss LSM 880 laser scanning microscope, including the application of Airyscan technology for super-resolution microscopy. RESULTS

Drosophila Studies Novel Mutations in TBC1D8B Cause SNRS The Drosophila melanogaster stable RNAi stocks Tbc1d8b The known monogenic causes of SRNS explain only a fraction RNAi (#32929) and Rab11 RNAi (#42709) were obtained of idiopathic cases. We performed WES to discover novel mu- from the Bloomington Drosophila Stock Center (BDSC). Pros- tations. After excluding relevant mutations in known SRNS pero-GAL459 or Dorothy-GAL4 (# 6903; BDSC) with or without genes, we identified hemizygous mutations of the gene TBC1 tub-GAL80ts (#7018; BDSC) were used to control expression in Domain Family Member 8B (TBC1D8B)infive individuals garland cell nephrocytes. RAB11 overexpression was achieved from five different families with nephrotic syndrome (Figure 1, using wild-type GFP-Rab11 (# 8506; BDSC). CRISPR/Cas9- A–F, Supplemental Figure 1, A–G, Table 1). All patients were mediated loss of function was generated in modification of male, and the available information was compatible with an an established protocol,60 using Dorothy-GAL4 to direct X-linked inheritance. One heterozygous female conductor

2340 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH

A ATG TAA

1 2 3 4 516 71819 10 112 13 415 16 71819 20 21

3,363 bp

TBC1D8B GRAM GRAM TBC

aa:1 155 388 485 694 1120

A2563 J12190/18 A3803 B3482 B931 c.190C>T c.1030C>T c.1316T>G c.1383G>A c.2338A>T p.Arg64Cys p.Arg344* p.Phe439Cys p.Trp461* p.Thr780Ser

BCDA2563_21 TBC1D8B A3803_21 TBC1D8B B3482_21 TBC1D8B c.190C>T; p.Arg64Cys c.1316T>G; p. Phe439Cys c.1383G>A; p. Trp461* Phe Arg Ile Val Phe His Ser Trp Lys ref TTTC G C A T C ref G T A T T T C A C ref T C A T GGA A A TTTT G C A T C G T A T G T C A C T C AAT G A A A A2563 A3803 B3482

Phe Cys Ile Val Cys His Ser Stop Lys

EFG A2563 TBC1D8B Arg64

B931_21 TBC1D8B J12190/18_21 H. sapiens KVAPFR I LHQTP c.2338A>T; TBC1D8B c.1030C>T; p. Thr780Ser p.Arg344* M. musc. KVAPFR I LHQTP Thr Thr Lys Leu Arg Glu G. gallus KVAPFR I LHQTP X. tropicalis KVAPFR I LHQTP ref A C A A C A A A A ref C T A C G A G A G A C A T C A A A A C T A T G A G A G D. rerio KVAPFR I LHQTP B931 J12190/ C. intestinalis KVSPFR I LHQTP 18 C. elegans RPPPYR I I YDFD Thr Ser Lys Leu Stop Glu D. melanog. KPAPYR I LHQTP S. cerevisiae RDEKFRLKYKLP

H I J B931 A3803 B931 TBC1D8B Phe439 TBC1D8B Thr780

H. sapiens TEALMTVFHPQNLE H. sapiens IQTLEETTKQNVLR M. musc. TEALMTVFHPQNLE M. musc. IQTLEETTKQNVLR X. tropicalis TEALMTVFHPQDAE G. gallus IQTLETTTKQNVLR D. rerio TEALMNVFHPHDAE X. tropicalis IQTLETTTRQNVVR D. rerio I QTLEDTTKQNVLR

Figure 1. WES identifies recessive mutations in TBC1D8B in five families with nephrotic syndrome. (A) Schematic of TBC1D8B cDNA with the corresponding protein, including its functional domains. Arrows indicate the position of five hemizygous mutations of TBC1D8B that were identified by WES in individuals with nephrotic syndrome from five families (A2563, A3803, B3482, B931, and J12190/18). (B–F) Shown are Sanger chromatograms of the respective regions of TBC1D8B in which the individual mutation of each of the five patients is located. (G) Alignment of TBC1D8B amino acid sequences for Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalis, Danio rerio, Ciona intestinalis, Caenorhabditis elegans, Drosophila melanogaster,andSaccharomyces cerevisiae demon- strates conservation of the residue Arginine 64. (H) Alignment of amino acid sequence of TBC1D8B for H. sapiens, M. musculus, X. tropicalis,andD. rerio, demonstrates conservation of the residue Leucine Phenylalanine 439. (I) Alignment of amino acid sequence of TBC1D8B for H. sapiens, M. musculus, G. gallus, X. tropicalis,andD. rerio indicates conservation of the residue Threonine 780. (J) Renal histology (periodic acid–Schiff base staining) of patient B931_21 with the Thr780Ser mutation of TBC1D8B shows segmental glomerulosclerosis.

JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2341 BASIC RESEARCH www.jasn.org

exhibited borderline proteinuria (B931, Figure 1, E and I, Supplemental Figure 1, B and C). Such information was not available for the other families. In two families (J12190/18 and Renal Biopsy B3482), we detected mutations introducing premature stop ND Mesangioprolif. GN codons (c.1030C.T, p.Arg344* and c.1383G.A, p.Trp461*, respectively), while three families showed hemizygous mis- sense mutations (c.2338A.T, p.Thr780Ser, c.1316T.G,

Renal . Function p.Phe439Cys, and c.190C T, p.Arg64Cys). The altered amino acid residues are well conserved in evolution (Figure 1, G–I, Table 1). Nephrotic syndrome was described as steroid- 4 ND 7 Normal FSGS 9 Normal FSGS yr

18 ESRD resistant in all but one patient (A2563). Extrarenal symptoms Onset Age at were not reported in any of these patients. The histology was (proteinuria), described as FSGS in three cases (B931, A2563, A3803, Figure 1J, Table 1) and mesangioproliferative GN (MesPGN) in one case to ND (J12190/18). No biopsy result was available for B3482. Sup- Steroids Response Resistant Sensitive Resistant 13 Mild reduction FSGS Resistant fi fi Saccharomyces cerevisiae ; ND, no data; Mesangioprolif. GN, porting the signi cance of our ndings, mutations of ., TBC1D8B were also reported most recently in two families with SRNS and X-linked inheritance.64 Parental Consanguinity S.c ;M,male; TBC1D8B Specifically Binds Active RAB11A and RAB11B and the Mutations from Patients with Ethnic Origin Nephrotic Syndrome Affect Interaction with Danio rerio ., Endogenous RAB11 D.r M German No Sex M Turkish Yes M Hispanic No TBC1D8B is a member of a family of more than 40 TBC- proteins that share the eponymous Tre-2-Bub2-Cdc16

– 65 (het/ hemi) (TBC) domain. This domain commonly confers a func- gnomAD tional role as a GTPase-activating protein (GAP) for specific Rab-GTPases, the master regulators of vesicular trafficking –– –– 1/0 2/1 4/2 M German No 1/1 0/1 M Pakistan Yes (het/ ExAC hemi) including endocytosis. TBC1D9B, closely related to TBC1D8B, has been described as a GAP specific for RAB11A66 that plays a role in endocytic recycling, autophagy, and exocytosis. We per- formed coimmunoprecipitation assays, comparing the binding S.c. D.r. D.r.

Amino of TBC1D8B to RAB11A, the early endosomal RAB5A, and the to Species

Acid Conserved late endosomal RAB7A. We observed strongest binding toward RAB11A (Figure 2A, quantitation Figure 2B). Rab proteins cycle between an active GTP-bound and an inactive GDP-bound state. GAP proteins promote the conversion to the inactive GDP- bound state through catalyzing GTP hydrolysis, demonstrating ––– – ––– – specific binding to the active form (Figure 2C). To evaluate such specific binding, we generated constitutively active and domi- nant negative variants of three Rab proteins that are involved in endocytic recycling: RAB4A (Q72L and S27N), RAB11A (Q70L ve families with nephrotic syndrome. fi and S25N), and RAB11B (S25N and Q70L). We performed Zygosity Exon PPH2 SIFT MT coimmunoprecipitation, using these variants together with TBC1D8B, and quantified the relative band density of

Acid the respective dominant negative and constitutively active Amino Change Rab protein. We consistently observed a binding ratio .1(active/ A p.Trp461* Hemi 9 T p.Arg344* Hemi 6 T p.Thr780Ser Hemi 15 0,98 Del DC inactive Rab) for both RAB11A and RAB11B, but not RAB4A G p.Phe439Cys Hemi 8 0.87 Del DC T p.Arg64Cys Hemi 2 1 Del DC . . . . . (Figure 2D, quantitation Figure 2E). We introduced TBC1D8B

Change mutations discovered in patients with nephrotic syndrome Nucleotide (W461*, F439C, T780S, and R64C) into the corresponding Mutations of TBC1D8B in amino acid residues of the murine full-length Tbc1d8b (W460*, F438C, T779S, and R64C). The mutation p.Arg344*

Family was not investigated in light of the early premature stop codon Individual J12190/18 _21 c.1030 C B3482_21 c.1383 G A2563_21A3803_21 c.190 C c.1316 T B931_21 c.2338 A fi Mesangioproliferative GN. PPH2, PolyPhen-2 prediction score (http://genetics.bwh.harvard.edu/pph2/);Consortium database SIFT, (http://exac.broadinstitute.org); Sorting Het, Tolerant heterozygous; Hemi, From hemizygous; Intolerant Del, prediction deleteriousness; score DC, (http://sift.jcvi.org/); disease causing; MT, mutation taster (http://www.mutationtaster.org/); ExAC, Exome Aggregation Table 1. TBC1D8B preceding Trp461. Analyzing the binding af nity to

2342 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH

A B C 4 *** kDa *** TBC/ GFP-RAB11GFP-RAB5GFP-RAB7 GFP-RAB11GFP-RAB5GFP-RAB7Blot: ns GTP 60 3 GAP 60 RAB GFP-RAB 2 active 50 α GFP proteins 50 - + 1 inactive Myc-TBC1D8B Lysates IP: α Myc 80 80 (shorter RAB 0 α Myc isoform) Density [RAB/TBC1D8B] GDP RAB11 RAB5 RAB7

DE

const. act. dom.neg. const. act. dom.neg. const. act. dom.neg. const. act. dom.neg. const. act. dom.neg. const. act. dom.neg. A 4.0

3.0 kDa kDa GFP-RAB11AGFP-RAB11AGFP-RAB11BGFP-RAB11BGFP-RAB4AGFP-RAB4A GFP-RAB11AGFP-RAB11AGFP-RAB11BGFP-RAB11BGFP-RAB4GFP-RAB4ABlot: 60 ††† ** * 60 GFP-RAB 2.0 50 proteins 50 α GFP 1.0

80 80 Myc- 0 IP: α Myc TBC1D8B Density [const.act./dom.neg.]

Lysates (7.5 %) (wt/ shorter 60 60 α Myc RAB4A isoform) RAB11A RAB11B

FG* * WT R64C T779S F438C W460 WT R64C T779S F438C W460 * ns

Mock-MycMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8b kDa Mock-MycMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8bMyc-Tbc1d8bBlot: ** kDa ] 2.0 ** 25 25 RAB11 (endog.) wt/mutant 1.5 α RAB11 Myc- 150 150 Tbc1d8b (wt/mut) 1.0

IP: α Myc 0.5 Lysates (7.5 %) α Myc Density GFP-RAB [endog.

RAB11/Myc-Tbc1d8b 0 * Myc- wt W461* Tbc1d8b R64C 50 50 T779S F438C W460

Figure 2. TBC1D8B specifically binds to active forms of RAB11A and RAB11B and patient derived mutations of TBC1D8B affect binding to endogenous RAB11. (A) Upon overexpression and coimmunoprecipitation with anti-Myc antibody in HEK293T cells, GFP-tagged RAB11A precipitates with Myc-TBC1D8B whereas GFP-tagged RAB5A and RAB7A show weaker binding. (B) Quantitation of densities from (A) depicts a significantly weaker affinity of GFP-RAB5A and GFP-RAB7A toward Myc-TBC1D8B compared with GFP-RAB11A. Densitometry results from (A) are expressed as ratio of Rab protein/TBC1D8B (n=4, P,0.001 for RAB11 versus RAB5, P,0.001 for RAB11 versus RAB7 and P.0.5 for RAB5 versus RAB7). Sidak post hoc analysis was used to correct for multiple com- parisons.). (C) Schematic showing a Rab protein shuttling between active (GTP-bound) and inactive (GDP-bound) states. GAP or TBC proteins predominantly bind to the active Rab protein (GTP-bound) to catalyze GTP hydrolysis, promoting the inactive state. (D) Overexpression and coimmunoprecipitation of GFP-tagged RAB11A, RAB11B, and RAB4A dominant negative (dom. neg.) and con- stitutively active (const. act.) constructs together with Myc-TBC1D8B. RAB11A and RAB11B constructs each interact with TBC1D8B. † The constitutively active forms ( ) show a stronger affinity toward TBC1D8B than the dominant negative forms (*). Binding affinity of RAB4 constructs is weaker. (E) Quantitation of density from precipitates analogous to (D) normalized to respectively precipitated TBC1D8B protein shown as a ratio of the respective constitutively active form and the dominant negative form. Ratios are consistently .1 for RAB11A and RAB11B but not RAB4, indicating stronger binding of the active Rab protein for RAB11. (F) Murine Tbc1d8b cDNA constructs that reflect the mutations from patients with nephrotic syndrome exhibit reduced binding affinity toward endogenous RAB11, except Tbc1d8bF438C (after correcting for the amount of TBC1D8B). The RAB11 antibody cannot discriminate between en- dogenous RAB11A and RAB11B. Please note that the mildly reduced interaction for the mutation F439C was not confirmed in other experiments [see quantitation in (G)]. (G) Quantitation of densities from (F) confirms a significantly reduced affinity of Tbc1d8b mutants to GFP-RAB11B except F438C. Densitometry results from (F) were expressed as endogenous RAB11/Tbc1d8bwild-type/mutant (n=3–5, P,0.01 for R64C, P,0.01 for T779S, P.0.05 for F438C, *P,0.05 for W460). endogenous RAB11 via coimmunoprecipitation in HEK cells, Silencing of TBC1D8B Upregulates RAB11-Dependent we observed a significant reduction for mutant proteins, except Autophagy and Exocytosis Suggesting a Function as Tbc1d8bF438C (Figure 2F, quantitation Figure 2G). Mutations GAP for RAB11 of TBC1D8B that cause nephrotic syndrome thus affect the To evaluate endogenous expression of TBC1D8B in podocytes interaction with endogenous RAB11. and HEK293T cells, we performed immunoblotting, and used

JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2343 BASIC RESEARCH www.jasn.org transient siRNA transfection and CRISPR/Cas9-mediated loss Promotion of autophagosome maturation is an established of function.67 Podocytes and HEK293T cells exhibited endoge- cellular function of RAB11.68–70 Toconfirm a role of TBC1D8B nous expression of the long, full-length isoform of TBC1D8B that as an RAB11-GAP, we studied basal autophagy upon silencing was reduced upon both loss-of-function strategies (Figure 3A, of TBC1D8B in HEK293T cells and podocytes. Using LC3B/ Supplemental Figure 2C). To study the subcellular localization GAPDH-ratio as a read-out, immunoblotting indicated an in- of endogenous TBC1D8B protein, we tested the conditional crease of autophagy in both cell lines (Figure 3, B and C, Sup- CRISPR/Cas9-mediated loss of function in individual cells plemental Figure 2D). To evaluate autophagic flux, we tested marked by GFP.67 We did not observe a consistent reduction the effect of lysosomal inhibition via chloroquine. The differ- of the antibody signal in cells with TBC1D8B loss of function, ence between control and chloroquine treatment was more using two available antibodies (Supplemental Figure 3, A–C). pronounced upon silencing of TBC1D8B, suggesting a higher Overexpression constructs of TBC1D8B, including murine autophagic flux (Figure 3D). As autophagy might be induced Tbc1d8b constructs representing the patient mutations, predom- independent of RAB11, we tested an effect of TBC1D8B inantly localized to the cytosol (Supplemental Figure 3, D–I), but siRNA on exocytosis, another cellular process that requires did not demonstrate overt colocalization with overexpressed or RAB11 function71 without direct connection to autophagy. endogenous RAB11 protein (Supplemental Figure 2, A–B’’). The To study basal exocyst activity, we generated a secretory localization of the endogenous protein remains unclear. form of GFP by introducing an IFNA2-derived signal

A Podocyte HEK293T BC TBC1D8B TBC1D8B Podocyte HEK293T TBC1D8B TBC1D8B Podocyte 1.5 ** kDa control siRNA-1siRNA-2 kDa control siRNA-1siRNA-2 const.act * 160 150 1.0 control siRNA-1siRNA-2 RAB11B controlsiRNA-1siRNA-2 110 100 kDa kDa 15 15 0.5 anti-LC3B anti-LC3B anti-TBC1D8B anti-TBC1D8B

50 Density [LC3B/GADH] 50 0

37 37 37 control siRNA-1 siRNA-2 anti-GAPDH

37 anti-actin anti-GAPDH

MW: 128 kDa MW: 128 kDa anti-GAPDH

D E F Control Chloroquine treatment treatment const.act. ** 2.5 * 2.0 control siRNA-1 siRNA-2 control siRNA-1siRNA-2 Control siRNATBC1D8B-siRNA-1TBC1D8B-siRNA-2Myc-RAB11 kDa kDa 1.5 GFP 15 30 1.0 (secreted) Density anti- LC3B 0.5 GFP [knockdowns/control] 0 GFP 37 30 (lysate) anti- GAPDH

Control siRNA

TBC1D8B-siRNA-1TBC1D8B-siRNA-2

Figure 3. Silencing TBC1D8B increases autophagy and exocytosis, suggesting a role as an RAB11-GAP. (A) Transient, siRNA-mediated silencing of TBC1D8B in human immortalized podocytes and HEK293T cells is demonstrated by immunoblot. This indicates endog- enous expression of TBC1D8B in both cell lines and confirms the efficiency of the siRNAs. (B) Immunoblotting with anti-LC3B reveals an increase of basal autophagy in podocytes and HEK293T cells upon silencing TBC1D8B. Anti-GAPDH signal serves as loading control. (C) Quantitation of densities from podocyte data in (B) confirms a significant increase of the signal from anti-LC3B expressed as a ratio to the loading control (n=3–4, P,0.05 for siRNA-1 and P,0.01 for siRNA-2). (D) Immunoblotting with anti-LC3B indicates a higher difference between control treatment and chloroquine exposure for TBC1D8B siRNAs compared with control siRNA. Anti-GAPDH signal serves as loading control. Representative blot from three consecutive experiments is shown. (E) Western blot reveals increased delivery of GFP to the supernatant (above) upon siRNA-mediated silencing of TBC1D8B and overexpression of constitutively active RAB11B in HEK293T while the amount of intracellular GFP (lysates) is comparable. (F) Quantitation of densities from (E) confirms a significant increase of the GFP secretion for two independent siRNAs directed against TBC1D8B. Density results are expressed as ratio of GFP signal in the supernatant to GFP within the lysates (n=4–5, P,0.05 for siRNA-1 and P,0.01 for siRNA-2).

2344 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH peptide into the N terminus of GFP. Upon transient trans- of ER release (Figure 4H, upper panels), indicating success- fection, HEK293T cells constitutively secreted GFP into the ful ER retention. Upon exposure to D/D solubilizer, we ob- surrounding medium (Supplemental Figure 2D). Both, served trafficking of nephrin toward the apical cell surface overexpression of constitutively active RAB11B and silenc- over time (Figure 4H, lower panels). This suggests that ing of TBC1D8B increased secretion of GFP (Figure 3E, nephrin travels initially to the apical surface. Using this quantitation Figure 3F, Supplemental Figure 2, E and F). novel assay for nephrin trafficking, we studied the effect of Thus, basal activity of both autophagy and exocytosis were overexpressed Tbc1d8b within this system. Interestingly, increased upon knockdown of TBC1D8B. Taken together, overexpression of murine full-length Tbc1d8b reduced the these findings suggest a disinhibition of endogenous RAB11 activity being well compatible with a function of apical delivery of nephrin compatible with an inhibition fi TBC1D8B as an RAB11-specificGAP. of RAB11-dependent traf cking, but one of the patient- derived mutations failed to have a similar effect in these cells TBC1D8B Interacts with the Slit Diaphragm Protein (Figure 4I, quantitation Figure 4J). These findings indicate Nephrin and Regulates its Trafficking that TBC1D8B regulates nephrin trafficking and support The slit diaphragm protein nephrin is subject to endocytic traf- a pathogenetic role of Rab11-dependent vesicular traffick- ficking.72 We hypothesized that TBC1D8B plays a role in vesicu- ing of nephrin in nephrotic syndrome. Basolateral delivery lar trafficking of nephrin as the underlying pathogenesis for of nephrin was minimal in all conditions (Supplemental glomerular dysfunction. Performing coimmunoprecipitation Figure 4, E and F). experiments in HEK293T cells, we found that Myc-nephrin precipitated with human GFP-TBC1D8B (Figure 4A) and mu- Loss of Function of the Drosophila Ortholog of rine GFP-Tbc1d8b (Supplemental Figure 4A). Conversely, we ob- TBC1D8B Impairs Nephrocytes and Results in served that the GFP-tagged intracellular domain (ICD) of Mistrafficking of Fly Nephrin nephrin precipitated with Myc-TBC1D8B (Supplemental Tovalidate a role of TBC1D8B in nephrin trafficking in vivo,we Figure 4B). The N-terminal half of the nephrin ICD was suf- used the Drosophila model that harbors the podocyte-like ficient to bind Myc-TBC1D8B, whereas the C-terminal ICD nephrocytes. Within these cells, the ortholog of nephrin forms failed to bind (Figure 4B). Truncation mapping thus identifies autocellular slit diaphragms across membrane invaginations. an interacting domain of 76 amino acids on the nephrin Nephrocytes have been established as a model for monogenic C-terminal tail (Figure 4C). We evaluated binding of mutant forms of nephrotic syndrome.61 By sequence analysis we iden- Tbc1d8b by coimmunoprecipitation and observed a re- tified the uncharacterized Drosophila gene CG7324 as the duced binding to Myc-nephrin for all mutants (Figure 4D, ortholog of human TBC1D8B (Figure 5A). This gene encodes a quantitation Figure 4E). The mutations of TBC1D8B that protein of 1256 amino acids that contains two GRAM domains cause nephrotic syndrome thus impair the interaction with and one TBC domain like the human TBC1D8B. We introduce nephrin. In podocytes, overexpressed GFP-TBC1D8B the term Tbc1d8b for the Drosophila ortholog CG7324. and Myc-nephrin colocalized completely within vesicles Tostudy loss of function of Tbc1d8b within this invertebrate (Figure 4, F–F’’). We confirmed colocalization of both pro- podocyte model, we expressed Tbc1d8b RNAi within nephro- teins in HEK cells and partially in MDCK cells (Supple- cytes using prospero-GAL4. Studying the uptake of FITC- mental Figure 4, C–D’’). albumin, an established read-out of nephrocyte function,61 To study biosynthetic trafficking of nephrin directly, we observed a significant functional impairment of nephro- we utilized an approach on the basis of conditional aggregation cytes compared with control conditions (Figure 5B, quantita- domains (CAD) that has been used for other proteins.56,57 tion Figure 5C). To confirm this finding, we generated a Adapting this approach for nephrin, we introduced CADs to conditional CRISPR/Cas9-mediated loss of Tbc1d8b in neph- the N terminus of nephrin (pCAD4-HA-nephrin-mCherry, rocytes. Combining nephrocyte-restricted Cas9- expression Figure 4G). CADs are expected to induce retention of newly by Dorothy-GAL4 with stable ubiquitous expression of a tan- synthesized protein within the ER by formation of aggregates. dem Tbc1d8b gRNA, we noted a reduction of nephrocyte func- Such aggregates can be dispersed by adding a membrane tion that slightly exceeded the effect of the RNAi (Figure 5B, permeable small molecule (D/D solubilizer), which entails quantitation Figure 5C). We stained the slit diaphragm pro- rapid, synchronized ER release of the protein and consecutive teins Sns (ortholog of nephrin) and Kirre (ortholog of removal of the CAD in the Golgi. The presence of two tags NEPH1) in nephrocytes expressing Tbc1d8b RNAi, and ob- (HA-tag and mCherry) eventually permits separate detection served protrusions of these proteins from the surface in a fine of nephrin at the surface by extracellular staining (HA) and the linear pattern that also occurred solitarily (insets, Supplemen- total protein (mCherry, Figure 4G). We chose MDCK cells tal Figure 5, A–B’’). These protrusions were absent in control that form an epithelial monolayer with apico-basal polarity nephrocytes (compare Figure 5, F–F’’ and D–D’’). With a for this assay because these cells had previously been used for lower penetrance, we further observed patches lacking slit di- this approach.56,57 Studying nephrin trafficking this way, we aphragms entirely upon expression of Tbc1d8b RNAi (Supple- observed no nephrin on the cell surface before the induction mental Figure 5, A–B’’). Typically, the classic fingerprint-like

JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2345 BASIC RESEARCH www.jasn.org

A B C

1084-11601160-1241 1084-11601160-1241 nephrin kDa GFP-TBC1D8BMock-GFP kDa GFP-TBC1D8BMock-GFPBlot: 150 150 Full length ECD TM ICD GFP- aa:1-1241 + 100 100 TBC1D8B kDa Mock-GFPGFP-nephrinGFP-nephrin kDa Mock-GFPGFP-nephrinGFP-nephrinBlot: ICD 50 50 GFP-ICD ICD + truncations aa:1084-1241 37 37 N-terminal ICD

α GFP Mock- N-terminal ICD α GFP aa:1084-1160 + 25 25 GFP IP: α Myc IP: α Myc 100 100 Myc- C-terminal ICD Lysates (7.5 %) Lysates (7.5 %) 37 75 75 C-terminal ICD 37 TBC1D8B aa:1160-1241 -

Mock- α Myc GFP Sufficient for interaction: Myc- LWQRR L RRL AEGIS EKT E AGSE EDRVRNEYEESQWTGE 150 150 nephrin R DTQSSTVST TEAE P YYRSL RD F S PQ LPP TQEEVSY SR α Myc

D * * E

WT F438C W460 T779S R64C WT F438C W460 T779S R64C *** 1.5 *** / *** ***

kDa GFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8b kDa GFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8bGFP-Tbc1d8bBlot: 1.0 150 wt/mutant 150 GFP-Tbc1d8b 75 (wild-type/ mutant) 0.5

75 α GFP 75 Density [GFP-Tbc1d8b Myc-nephrin]

IP: α Myc 0 Myc- 150 Lysates (7.5 %) 150 nephrin Wt * α Myc R64C T779S F438C W460

Tbc1d8b Tbc1d8b Tbc1d8b Tbc1d8b Tbc1d8b FF’F’’ podocytes

10 µm GFP-TBC1D8B Myc-nephrin merge

G extracellular HA- H surface nephrin (HA) total nephrin (mCherry) merge CADHA nephrin mCherry staining

removal of CAD-domain 10 µm in Golgi 0 h release 1 h release

no release: addition of D/D solubilizer:

expression + ER retention ER-release + trafficking 3 h release

I surface nephrin total nephrin GFP J (HA) (mCherry) (Tbc1d8b/ control) merge 0.8 ns * 0.6 (GFP) Control 10 µm 0.4

0.2 wild-type Tbc1d8b-

0.0

Fluorescence intensity [extracellular/total] -0.2 T779S

Tbc1d8b- P) 79S wild-type T7 Control (GF 8b d8b Tbc1d Tbc1 3h release

Figure 4. The N-terminal cytosolic domain of nephrin interacts with TBC1D8B and both proteins co-localize in cultured cell lines. (A) Upon overexpression in HEK293T cells and coimmunoprecipitation, GFP-tagged TBC1D8B precipitates with Myc-tagged nephrin. (B) Upon overexpression in HEK293T cells and coimmunoprecipitation, the GFP-tagged N-terminal section of the ICD of nephrin (aa 1084–1160) precipitates with Myc-tagged TBC1D8B, whereas the C-terminal section of the ICD (aa 1160–1241) shows no

2346 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH staining pattern of slit diaphragm proteins on the surface was expression using the temperature-sensitive Gal80ts that suppresses preserved upon expression of Tbc1d8b RNAi, whereas indi- GAL4 at nonpermissive temperatures. Acute Rab11 silencing re- vidual lines appeared brighter (compare Figure 5, panels E sulted in localized loss of slit diaphragms (Figure 6, A–A’’) and and G). The fine linear protrusions of slit diaphragm protein shortened lines of slit diaphragm protein on the surface (Fig- fromthesurfacewerevisiblesubcorticallywhenTbc1d8b ure 6, B–B’’), whereas nephrocytes from animals raised at RNAi was expressed but absent from subcortical nonpermissive temperatures showed regular slit diaphragms control sections (compare insets Figure 5, E and G). To (Supplemental Figure 5, C–C’’). Prolonged expression of understand the ultrastructural basis of the protrusions of Rab11 RNAi caused extensive loss of slit diaphragm protein slit diaphragm protein from the membrane, we performed fromthemembrane(Figure6,C–C’’)withappearanceof electron microscopy and observed the formation of addi- puncta of nephrin on the surface (insets Figure 6, D–D’’) tional, ectopic slit diaphragms deeper within the membrane and clusters of Kirre protein between nephrocytes in absence invaginations (Figure 5I, control Figure 5H). These ectopic of flynephrin(Figure6,D’–D’’). To study a mild gain of slit diaphragms obviously corresponded to our observations function similar to the effect of a defective Rab11-GAP, we using immunofluorescence (Figure 5, F–G’’). To confirm our overexpressed GFP-tagged Drosophila Rab11 in nephrocytes findings in an independent loss-of-function approach, we and observed appearance of ectopic vesicles of flynephrinand stained Sns/Kirre in nephrocytes with CRISPR/Cas9-medi- fine protrusions of fly nephrin from the membrane (Figure 6, ated loss of Tbc1d8b compared with control conditions E–E’’). Thus, overexpression of Rab11 partially phenocopies (Cas9 expression without gRNA, Figure 5, K–L’’). Upon loss of Tbc1d8b. However, overexpression of wild-type Rab11 loss of Tbc1d8b, we observed a phenotype that was more produced an overtly milder phenotype than lack of Tbc1d8b pronounced but matched our findings using the Tbc1d8b (compare Figure 5F’ to Figure 6E’). This may be explained RNAi (Figure 5, J and M–N’’).Themoreseverephenotype by additional Rab11-independent functions of Tbc1d8b,low upon CRISPR/Cas9-mediated loss of function compared efficiency of the exogenous wild-type Rab11 expression, or with RNAi expression matched the stronger reduction of divergent compensatory regulation. Taken together, these FITC-albumin endocytosis and suggested an incomplete findings indicate that slit diaphragm formation in nephro- knockdown using RNAi. Taken together, these findings indi- cytes requires Rab11 and they are compatible with a function cate mistrafficking of fly nephrin with mislocalization of slit of Tbc1d8b as an Rab11-GAP in Drosophila. diaphragms upon loss of Drosophila Tbc1d8b.

Loss of Function of Rab11 Impairs Trafficking of Slit DISCUSSION Diaphragm Proteins in Nephrocytes Our findings suggest a functional role of RAB11 for trafficking Here, we report the identification of five mutations of the gene of nephrin. To evaluate such a role of Drosophila Rab11 for TBC1D8B in patients with nephrotic syndrome from five dif- trafficking of slit diaphragm proteins in nephrocytes, we si- ferent families. Analyzing interaction of TBC1D8B with con- lenced Rab11 acutely by modifying the GAL4-dependent stitutively active RAB11A and RAB11B compared with their interaction. (C) A schematic of truncation constructs of nephrin indicates their ability to interact with TBC1D8B by “+” (interaction) or by “–” (lack of interaction). The amino acid sequence of the interacting domain is noted below. (D) Murine Tbc1d8b cDNA constructs that reflect the mutations from patients with nephrotic syndrome exhibit reduced binding affinity toward Myc-nephrin (correcting for the amount of Myc-nephrin). (E) Quantitation of densities from (D) confirms a significantly reduced affinity of Tbc1d8b mutants to Myc-nephrin. Den- sitometry results are expressed as Tbc1d8bwild-type/mutant/Myc-nephrin (n=5, P,0.001 for F438C, P,0.001 for R64C, *P,0.001 for W460, and P,0.001 for T779S). (F) Overexpressed GFP-nephrin and Myc-TBC1D8B (human) colocalize in immortalized human podocytes in vesicles (see also enlarged insets). Scale bar represents 10 mm. (G) Schematic of the principle of the nephrin trafficking assay and the nephrin cDNA construct (pCAD4-HA-nephrin-mCherry) that was applied. CADs and an HA-tag are introduced into the nephrin N ter- minus, whereas an mCherry-tag resides in the C terminus. Without induced ER release, the expressed nephrin forms CAD-mediated clusters and is retained within the ER (left). Addition of the membrane-permeable D/D solubilizer causes dispersal of the clusters causing a synchronized ER release. Although the CADs are removed by furin cleavage in the Golgi, the double-tagged nephrin continues trafficking. Protein reaching the apical surface can be visualized by extracellular HA-staining, as nephrin is a type 1 transmembrane protein. (H) Before an ER release is induced, an MDCK cell transfected with pCAD4-HA-nephrin-mCherry [described in (F)] lacks extracellular HA signal, whereas the C-terminal mCherry indicates a perinuclear localization (upper panel). There is no indication of leakiness of the desired ER retention. Upon induction of ER release extracellular HA-staining becomes positive, indicating an increasing amount of tagged nephrin protein is subject to trafficking toward the apical surface (middle and lower panels). Scale bar represents 10 mm. (I) MDCK cells coex- pressing pCAD4-HA-nephrin-mCherry either with GFP (control, upper panel), wild-type GFP-Tbc1d8b (middle panel), or the mutant Tbc1d8b-T779S (derived from patient with SRNS, lower panel). Apical delivery of pCAD4-HA-nephrin-mCherry is impaired by expression of wild-type Tbc1d8b but not the mutant protein. Scale bar represents 10 mm. (J) Quantitation of results from (I). Shown is the fluorescence intensity from the extracellular HA-staining in ratio to total nephrin (mCherry). Overexpression of wild-type Tbc1d8b but not mutant Tbc1d8b significantly reduces apical delivery of nephrin (.50 cells per group; P,0.05 for Tbc1d8b wild-type and P.0.05 for T779S).

JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2347 BASIC RESEARCH www.jasn.org

A BC1.5 ** *** TBC1D8 (human) Control Tbc1d8b-RNAi 1.0 GRAM TBC (pros-GAL4/+) 0.5 Fluorescence 1120aa intensity [ratio] 0.0 CG7324 (Drosophila) Control Tbc1d8b-gRNA/ (Dot>Cas9/+) Dot>Cas9 ol (GAL4)

GRAM TBC FITC-albumin Contr Tbc1d8Controlb-RNAi (Cas9) c1d8b-gRNA/Cas9 1256aa Tb DD’D’’

Sns (nephrin) Kirre (NEPH1) merge EE’E’’surfacesub-cortical surfacesub-cortical surface sub-cortical pros-GAL4/+

Sns (nephrin) Kirre (NEPH1) merge F F’ F’’

Sns (nephrin) Kirre (NEPH1) merge Gsurface G’ surface G’’ surface Tbc1d8b -RNAi

subcortical subcortical subcortical Sns (nephrin) Kirre (NEPH1) merge H I J

Dot>Cas9/ 200 nm pros>control 200 nm pros>Tbc1d8b-RNAi 200 nm Tbc1d8b-gRNA KK’’K’

Sns (nephrin) Kirre (NEPH1) merge LL’’sub-cortical L’ sub-cortical sub-cortical Dot>Cas9 /+

Sns (nephrin) Kirre (NEPH1) merge MM’’M’

Sns (nephrin) Kirre (NEPH1) merge NN’’sub-cortical N’ sub-cortical sub-cortical Dot>Cas9/Tbc1d8b -gRNA

Sns (nephrin) Kirre (NEPH1) merge

Figure 5. Silencing the Drosophila ortholog of TBC1D8B in nephrocytes affects slit diaphragm localization and nephrocyte function. (A) Amino acid sequence of human TBC1D8B is 29% identical to the Drosophila ortholog CG7324, which shares the two GRAM domains and the TBC domain. Scale bar represents 5 mm throughout the figure. (B) Shown is FITC-albumin endocytosis after exposure for 30 seconds as an established assay of nephrocyte function. Silencing the TBC1D8B ortholog by RNAi using prospero-GAL4 (upper panels) or a conditional CRISPR/Cas9-mediated approach (lower panels) in nephrocytes significantly reduces uptake of FITC-albumin compared with the respective controls. Nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (C) Quantitation of

2348 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH dominant negative variants, we observed specific binding for mechanistic link between Rab11 regulation and glomerular the active, GTP-bound forms of RAB11A and RAB11B. Loss of dysfunction. TBC1D8B increased basal autophagy, autophagic flux, and Surprisingly, the two truncating mutations (p.Arg344* exocyst activity, suggesting a disinhibition of endogenous and p.Trp461*) were observed hemizygously in two or one RAB11 function and supporting a role of TBC1D8B as a individuals within the gnomAD control population re- GAP protein for RAB11. We demonstrate TBC1D8B is an spectively. The incomplete penetrance of these nonsense interaction partner of the slit diaphragm protein nephrin mutations may be explained by a genetic compensation re- and both overexpressed proteins colocalize within different sponse.73,74 The compensatory upregulation of another immortalized cell lines. Mutations derived from patients RAB11-GAP might only be induced by a complete loss with nephrotic syndrome impair binding of TBC1D8B to en- of function of TBC1D8B but not in missense mutations. dogenous RAB11 and nephrin. Overexpression of TBC1D8B Patients with mutations of TBC1D8B typically presented affected trafficking of nephrin in vitro.Studyingthelossof with FSGS in renal biopsy, while MesPGN was described Tbc1d8b in the Drosophila model, we observed impaired fly in one case. MesPGN, a glomerular damage pattern, fre- nephrin trafficking within the podocyte-like nephrocytes quently showed progression to FSGS in repeat biopsies of in vivo. Silencing Rab11 in nephrocytes resulted in appearance children with SRNS.75 of slit diaphragm proteins independent from each other, sug- TBC1D8B binds to the active form of RAB11A and RAB11B gesting that the protein complex may be formed during indicating that TBC1D8B functions as a GAP for both RAB11-dependent transport. variants of RAB11. Our data implicate RAB11-dependent ve- Our findingsare corroborated bya most recent report in two sicular trafficking in the pathogenesis of nephrotic syndrome, families64 and the discovery of mutations of TBC1D8B in and delineate a role of TBC1D8B for trafficking of the slit di- five unrelated families with nephrotic syndrome conclusively aphragm protein nephrin as the mechanistic basis for ne- defines TBC1D8B as a novel monogenic cause of nephrotic phrotic syndrome associated with TBC1D8B mutations. We syndrome. Our results in nephrocytes confirm a role of previously implied regulators of RAB5 in nephrotic syn- TBC1D8B for glomerular function that was most recently re- drome.41 This Rab protein functions at early endosomes, ported in fish.64 Beyond that, the Drosophila model facilitated while RAB11 regulates endocytic recycling and exocytosis. precise study of the subcellular localization of flynephrin Localized cycling between exocytosis and endocytosis may upon loss of function of Tbc1d8b and allowed acute silencing provide plasticity and robustness for the glomerular filter po- of Rab11. Thus, we could show a role of Tbc1d8b and Rab11 tentially similar to the mechanisms at neuronal synapses.76 for flynephrintrafficking in vivo to identify a possible A direct role of autophagy within the pathogenesis of

results from (B) in ratio to a control experiment performed in parallel (n=5–7 per genotype, P,0.01 for Tbc1d8b-RNAi and P,0.001 for Tbc1d8b-gRNA/Cas9). Sidak post hoc analysis was used to correct for multiple comparisons. (D–D’’) Equatorial cross section of a control garland cell nephrocyte (pros-GAL4/+) costained for the nephrin ortholog Sns (green) and the KIRREL/NEPH1 ortholog Kirre (red). Slit diaphragm proteins localize at the cell periphery in a fine line. Inset depicts a magnification from the area marked by a white box, and regular spaced dots corresponding to individual slit diaphragms are discernible. Sns and Kirre are restricted to the plasma membrane. Scale bar represents 5 mm throughout the figure, unless otherwise indicated. (E–E’’) Surface section from control garland cell nephrocytes reveals the typical fingerprint-like staining pattern. Inset depicts subcortical section of the same cell with absence of slit diaphragm protein. (F–F’’) Equatorial cross section of garland cell nephrocyte expressing Tbc1d8b RNAi shows appearance of fine linear protrusions of slit diaphragm protein from the membrane toward the interior of the cell (arrowheads, see also magnified inset). (G–G’’) Surface section from garland cell nephrocyte expressing Tbc1d8b RNAi reveals the typical fingerprint-like staining pattern but individual slits seem brighter. Inset depicts subcortical section of the same cell with appearance of fine lines of slit diaphragm protein (arrowheads). (H) Electron microscopy (EM) image from a section through the surface of a control nephrocyte reveals regular slit diaphragms (black arrowheads) bridging the membrane invaginations called labyrinthine channels. Scale bar represents 200 nm. (I) EM image from a section through the surface of a garland cell nephrocyte expressing Tbc1d8b RNAi demonstrates regular slit diaphragms on the surface (black arrowheads) but also formation of additional, ectopic slit diaphragms deeper within the cell (red arrowheads). Scale bar represents 200 nm. (J) EM image from a section through the surface of a garland cell nephrocyte with CRISPR/Cas9-mediated loss of Tbc1d8b confirms the findings with Tbc1d8b RNAi. Scale bar represents 200 nm. (K–K’’) Equatorial cross section of a control garland cell nephrocyte (Dot.Cas9/+) costained for the nephrin ortholog Sns (green) and the KIRREL/NEPH1 ortholog Kirre (red) reveals a regular staining pattern. (L–L’’) Surface section from control garland cell nephrocytes (Dot.Cas9/+) reveals the typical fingerprint-like staining pattern. Inset shows subcortical section with absence of slit diaphragm protein. (M–M’’) Equatorial cross section of garland cell nephrocyte with conditional CRISPR/Cas9- mediated loss of Tbc1d8b demonstrates appearance of extensive linear and circular lines of slit diaphragms protruding from the surface but also without apparent connection to the membrane (see also magnified inset). Individual cells lack all slit diaphragm protein on a segment of the membrane (arrowhead). (N–N’’) Surface section from garland cell nephrocyte with conditional CRISPR/Cas9-mediated loss of Tbc1d8b reveals a reduction of lines of slit diaphragm proteins with irregular spacing and gaps (arrowheads). Inset depicts subcortical section of the same cell with appearance of extensive linear and circular lines of slit diaphragm proteins.

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AAknockdown 2 d ’Aknockdown 2 d ’’knockdown 2 d

Sns (nephrin) Kirre (NEPH1) merge BBknockdown 2 d ’Bknockdown 2 d ’’knockdown 2 d

Sns (nephrin) Kirre (NEPH1) merge > Rab11 -RNAi ts CCknockdown 4 d ’Cknockdown 4 d ’’knockdown 4 d Dot-GAL4; GAL80

Sns (nephrin) Kirre (NEPH1) merge

DDknockdown 4 d ’Dknockdown 4 d ’’knockdown 4 d

Sns (nephrin) Kirre (NEPH1) merge E E’ E’’ pros>RAB11-GFP RAB11-GFP Sns (nephrin) merge

Figure 6. Rab11 is required for slit diaphragm formation in Drosophila.(A–A’’) Equatorial cross-section of garland cell nephrocyte acutely expressing Rab11 RNAi after shift to permissive temperature (30°C) for 2 days. Cells are costained for the nephrin ortholog Sns (green) and the KIRREL/NEPH1 ortholog Kirre (red), revealing localized gaps of slit diaphragm protein on the cell surface (arrowhead, see also magnified insets with rarefied puncta corresponding to misspaced slit diaphragms). Scale bar represents 5 mm throughout the figure unless otherwise indicated. (B–B’’) Surface section of same cell as (A) shows localized thinning and shorter lines of slit diaphragm proteins (see also magnified inset). (C–C’’) Equatorial cross section of garland cell nephrocyte expressing Rab11 RNAi for 4 days after shift to permissive temperature (30°C). Cells are costained for the nephrin ortholog Sns (green) and the KIRREL/NEPH1 ortholog Kirre (red), revealing extensive loss of slit diaphragms from the membrane (arrowhead, see also magnified insets with rarefied puncta corresponding to misspaced slit diaphragms). (D–D’’) Surface section of same background and staining as (C) reveals local clustering of Kirre without presence of Sns (arrowhead) and localized thinning and shorter lines of slit diaphragm protein (see also magnified inset). Lines of slit diaphragm proteins become shortened increasingly to become puncta or miss entirely (see also enlarged inset). (E–E’’) Shown is an equatorial cross section of a garland cell nephrocyte expressing wild-type Rab11-GFP that localizes in a fine vesicular pattern particularly at the periphery of the cell. Staining the ortholog of nephrin (red) demonstrates appearance of additional flynephrin vesicles and fine linear protrusions of slit diaphragm protein similar to the observations with Tbc1d8b RNAi (arrow in enlarged inset). nephrotic syndrome associated with TBC1D8B is conceivable the apico-basal border. Nephrin trafficking does not seem to but will require further investigation. occur constitutively, but as a regulated process. Together with Trafficking of nephrin in podocytes in vivo may deviate our findings in Drosophila, our data support such a regulatory from our observations in MDCK cells, but it is possible that role for TBC1D8B. The mutations of TBC1D8B caused no nephrin is delivered initially to the apical surface before even- extrarenal manifestations, suggesting that tight regulation of tually being stabilized within the slit diaphragm complex at nephrin trafficking by TBC1D8B/RAB11 is required for the

2350 JASN JASN 30: 2338–2353, 2019 www.jasn.org BASIC RESEARCH establishment and maintenance of the glomerular filter, (UM1HG008900), the National Eye Institute, and the National Heart, Lung, whereas (compensatory) function of other RAB11-GAPs and Blood Institute. A. Onuchic-Whitford acknowledges support from the may suffice in other tissues. T32 Ruth L. Kirschstein Institutional National Research Service Award fi fi (DK007527). Dr. Schrezenmeier is participant in the BIH-Charité Clinician In conclusion, our ndings de ne TBC1D8B as an RAB11- Scientist Program funded by the Charité–Universitätsmedizin Berlin GAP and together with a previous report64 as a novel cause of and the Berlin Institute of Health. Dr. Römer acknowledges the support by hereditary nephrotic syndrome. This supports RAB11- the German Research Foundation (grant RO 4341/2-1), the Excellence Initiative dependent trafficking of nephrin as a novel pathogenetic of the German Research Foundation (EXC 294), and the Ministry of Science, mechanism. Research and the Arts of Baden-Württemberg (Az: 33-7532.20). Dr. Bergmann acknowledges support from the Deutsche Forschungsgemeinschaft Collaborative Research Centre (KIDGEM 1140) and the Federal Ministry of Education and Research (01GM1515C). ACKNOWLEDGMENTS

Dr. Bergmann, Dr. Hermle, Dr. Laricchia, Dr. Lifton, Dr. MacArthur, SUPPLEMENTAL MATERIAL Dr. Mane, Dr. Onuchic-Whitford, Dr. Rehm, and Dr. Schneider generated total genome linkage analysis, performed exome capture This article contains the following supplemental material online at and massively parallel sequencing, and performed whole-exome http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019040414/-/ evaluation and mutation analysis. Ms. Chen, Ms. Gerstner, Dr. Hermle, DCSupplemental. Ms. Kampf, Dr. Römer, Dr. Schneider, and Dr. Thünauer performed Supplemental Figure 1. Additional information concerning pa- cDNA cloning, protein purification, tracer endocytosis and immu- tients with mutations of TBC1D8B. nofluorescence, and subcellular localization studies in cell lines by Supplemental Figure 2. TBC1D8B does not colocalize with RAB11 confocal microscopy. Ms. Chen, Ms. Gerstner, Dr. Hermle, Ms. Kampf, but affects RAB11 function. and Dr. Schneider performed coimmunoprecipitation and the Supplemental Figure 3. Localization of TBC1D8B protein in cul- experiments in Drosophila. Dr. Helmstädter performed electron tured cell lines and secretory GFP upon CRISPR/Cas-mediated loss of microscopy. Dr. Amar, Dr. Berdeli, Dr. Hermle, Dr. Hildebrandt, TBC1D8B. Dr. Loza Munarriz, Dr. Müller, and Dr. Walz recruited patients and Supplemental Figure 4. TBC1D8B and nephrin trafficking. gathered detailed clinical information for the study. Dr. Hermle con- Supplemental Figure 5. Tb1d8b and Rab11 in Drosophila. ceived of and directed the study with support from Dr. Hildebrandt. Supplemental Table 1. Primer sequences. Dr. Hermle wrote the manuscript. The manuscript was critically reviewed by all the authors. REFERENCES We are grateful to the families and study individuals for their contribution. We thank the Yale Center for Mendelian Genomics 1. Smith JM, Stablein DM, Munoz R, Hebert D, McDonald RA: Contribu- and the Broad Center for Mendelian Genomics for whole exome tions of the transplant registry: The 2006 annual report of the North sequencing analysis. We thank Dr. R. Nitschke, Life Imaging Centre, American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). University of Freiburg, for help with confocal microscopy. We Pediatr Transplant 11: 366–373, 2007 thank Severine Kayser for technical assistance with electron mi- 2. Bierzynska A, Soderquest K, Dean P, Colby E, Rollason R, Jones C, et al.; croscopy. We thank the Developmental Studies Hybridoma Bank NephroS; UK study of Nephrotic Syndrome: MAGI2 mutations cause – for antibodies. congenital nephrotic syndrome. J Am Soc Nephrol 28: 1614 1621, 2017 3. Lovric S, Goncalves S, Gee HY, Oskouian B, Srinivas H, Choi WI, et al.: Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency. JClinInvest127: 912–928, 2017 DISCLOSURES 4. Braun DA Jr., Rao J, Mollet G, Schapiro D, Daugeron MC, Tan W, et al.: Mutations in KEOPS-complex genes cause nephrotic syndrome with Dr. Bergmann is an employee of Bioscientia/Sonic Healthcare and holds a primary microcephaly. Nat Genet 49: 1529–1538, 2017 part-time faculty appointment at the University of Freiburg. Dr. Hildebrandt 5. Kestilä M, Lenkkeri U, Männikkö M, Lamerdin J, McCready P, Putaala is a cofounder of Goldfinch-Bio and receives royalties from Claritas. H, et al.: Positionally cloned gene for a novel glomerular protein-- Dr. MacArthur reports personal fees from Goldfinch Bio, outside the submit- nephrin--is mutated in congenital nephrotic syndrome. Mol Cell 1: ted work. All of the remaining authors have nothing to disclose. 575–582, 1998 6. Löwik MM, Groenen PJ, Pronk I, Lilien MR, Goldschmeding R, Dijkman HB, et al.: Focal segmental glomerulosclerosis in a patient homozygous for a CD2AP mutation. Kidney Int 72: 1198–1203, 2007 FUNDING 7. Ebarasi L, Ashraf S, Bierzynska A, Gee HY, McCarthy HJ, Lovric S, et al.: Defects of CRB2 cause steroid-resistant nephrotic syndrome. Am J This research was supported by grants from the Deutsche Forschungsge- Hum Genet 96: 153–161, 2015 meinschaft to Dr. Hermle (HE 7456/3-1) and the National Institutes of Health 8. Gee HY, Sadowski CE, Aggarwal PK, Porath JD, Yakulov TA, Schueler to Dr. Hildebrandt (DK076683), and to the Yale Center for Mendelian Geno- M, et al.: FAT1 mutations cause a glomerulotubular nephropathy. Nat mics (U54HG006504). Ms. Kampf was supported by the MOTI-VATE pro- Commun 7: 10822, 2016 gram of the Medical Faculty of the University of Freiburg. The Broad CMG was 9. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, et al.: funded by a grant from the National Human Genome Research Institute NPHS2, encoding the glomerular protein podocin, is mutated in

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AFFILIATIONS

1Renal Division, Department of Medicine, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany; 2Division of Nephrology, Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts; 3Signalling Research Centres BIOSS and CIBSS and Faculty of Biology, University of Freiburg, Freiburg, Germany; 4Advanced Light and Fluorescence Microscopy Facility, Centre for Structural Systems Biology (CSSB) and University of Hamburg, Hamburg, Germany; 5Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; 6Department of Pediatrics, Universidad Peruana Cayetano Heredia, Lima, Peru; 7Department of Pediatrics, Molecular Medicine Laboratory, Ege University, Izmir, Turkey; 8Department of Pediatric Nephrology, Charité Universitätsmedizin Berlin, Berlin, Germany; 9Department of Nephrology and Medical Intensive Care, Charité - Universitätsmedizin Berlin, Berlin, Germany; 10Department of Genetics, Yale University School of Medicine, New Haven, Connecticut; 11Broad Center for Mendelian Genomics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge; 12Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, New York; 13Center for Human Genetics, Mainz, Germany; 14Center for Human Genetics, Bioscientia, Ingelheim, Germany; and 15Department of Medicine, University Hospital Freiburg, Freiburg, Germany

JASN 30: 2338–2353, 2019 TBC1D8B Mutations Cause Nephrotic Syndrome 2353