The glomerular filter of the kidney is three-layered, consisting of a fenestrated endothelium, the glomerular basement membrane, and the podocytes that form the slit diaphragm. Disorders of the glomerular filter commonly involve the podocyte and manifest with edema, hypoalbuminemia, and severe proteinuria, a clinical triad that characterizes the nephrotic syndrome . Steroid-resistant nephrotic syndrome (SRNS) commonly entails declining renal function, and represents the second most frequent cause of ESRD in patients manifesting before 25 years of age.1 10 biosynthesis, nucleoporins, or members of the KEOPS complex.2–43 nephrotic syndrome and podocyte biology.
There is mounting evidence supporting an essential role of vesicular trafficking via endocytosis for the function of the glomerular filter.44 , 45 46–48 49–55 GAPVD1 and ANKFY1 as novel causes of nephrotic syndrome and the first endosomal regulators in the pathogenesis of human nephrotic syndrome .41 TBC1D8B , discovered by whole-exome sequencing (WES) in five individuals with nephrotic syndrome . Our analysis in vitro and in the Drosophila model supports a novel role of RAB11-dependent vesicular trafficking in the pathogenesis of the nephrotic syndrome .
Methods
Study Approval
Approval for human subjects research was obtained from the University of Michigan, University of Freiburg and the Boston Children’s Hospital Institutional Review Boards. All participants or their guardians provided written informed consent.
Study Participants
After obtaining informed consent, clinical data and blood samples were collected from individuals with nephrotic syndrome . Clinical data were acquired using an established questionnaire. The diagnosis of nephrotic syndrome was made by (pediatric) nephrologists, on the basis of standardized clinical and renal histologic criteria. Renal biopsy specimens were evaluated by renal pathologists.
Homozygosity Mapping, Whole-Exome Resequencing, and Mutation Calling
Homozygosity mapping, whole-exome resequencing, and mutation calling were performed as described previously.23
Plasmids, siRNAs, Cell Culture, and Transfection
Murine full-length Tbc1d8b cDNA was subcloned by PCR from full-length murine cDNA (GenBANK BC147581.1; BioCAT GmbH). Human TBC1D8B was subcloned by PCR from cDNA representing the shorter isoform 2 (GenBank BC122564; GE Dharmacon). Truncation constructs were generated by PCR. Primers are shown in Supplemental Table 1
The following expression vectors were used: pRK5-N-Myc, pCDNA6.2-C-GFP, and pCDNA6.2-N-GFP. The mutations identified in individuals with nephrotic syndrome were introduced into the cDNA constructs by site-directed mutagenesis via Gibson assembly (New England Biolabs). The following constructs were obtained from Addgene: pSpCas9(BB)-2A-GFP (PX458) (#48138), pX459V2.0-eSpCas9 (#108292), GFP-RAB11 WT (#12674), and GFP-RAB7 (#61803) NPHS1 and RAB5 cDNA constructs have been described elsewhere.41
The TBC1D8B -specific and control siRNAs were purchased from GE Dharmacon.
Overexpression experiments were performed in HEK293T cells or immortalized human podocytes that were a gift from Dr. Moin Saleem (University of Bristol, Bristol, UK).
HEK293T cells were maintained in DMEM, supplemented with 10% FBS, 50 IU/ml penicillin, and 50 μ g/ml streptomycin. Podocytes were maintained in RPMI plus GlutaMAX-I (Gibco) supplemented with 10% FBS, 50 IU/ml penicillin/50 μ g/ml streptomycin, and insulin-transferrin-selenium X.
Plasmids and siRNAs were transfected into HEK293T cells at 37°C or podocytes grown at the permissive temperature of 33°C using Lipofectamine 2000 (Invitrogen) or polyethyleneimine (Polyplus-transfection or Sigma).
Nephrin Trafficking Assay
MDCK strain II cells (wt MDCK) were a gift from Enrique Rodriguez-Boulan (Weill Cornell Medical College, NY) and were maintained in DMEM supplemented with 5% FCSFCS, at 37°C and 5% CO2 in 10 cm culture dishes, and passaged every 2–3 days.
For growing polarized monolayers, cells were cultured on Transwell filters (#3401; from Corning Costar) for 4 days. Transfections with a plasmid encoding a nephrin construct (pCAD4-HA-nephrin-mCherry) comprising 4× conditional aggregation domains (CADs), furin cleaving site, HA-tag, nephrin, and mCherry (pCAD4-HA-nephrin-mCherry) were carried out using lipofectamine 2000 (Thermo Fisher Scientific). D/D-solubilizer (Clontech/Takara) was used to induce endoplasmic reticulum (ER) release at a concentration of 5 μ M, and 100 µ g/ml of cycloheximide was added during release to prevent further protein synthesis. Staining of nephrin arriving at the cell surface was carried out with anti-HA antibodies (Covance) conjugated to Cy5 that were applied to the apical or basolateral side of live cells.
For immunofluorescence, cells were fixed with 4% formaldehyde for 15 minutes at room temperature. After excising filters with a scalpel, they were mounted in DABCO-medium and imaged using Nikon A1R confocal microscope. Quantification of cell surface arrival of nephrin was done as described previously, with a custom-written MATLAB program.56 , 57
Immunoblotting, Immunoprecipitation, Pull-Down Assay, and Immunofluorescence Staining
Immunoblotting, immunoprecipitation, and immunofluorescence staining were performed as described previously.58 µ M chloroquine for 24 hours. Immunoblotting was performed using mouse anti-TBC1D8B (SC-376637; Santa Cruz Biotechnology), rabbit anti-RAB11 (#5589; Cell Signaling Technology), mouse anti–c-Myc (sc-40; Santa Cruz Biotechnology), rabbit anti–c-Myc (sc-789; Santa Cruz Biotechnology), mouse anti-GFP (sc-9996; Santa Cruz Biotechnology), rabbit anti-GAPDH (14C10/2118; Cell Signaling Technology), mouse anti-actin (ab20272; Abcam), rabbit anti-LC3B (2775; Cell Signaling Technology), and rabbit anti-GFP (sc-8334; Santa Cruz Biotechnology).
Immunofluorescence of TBC1D8B was performed with rabbit anti-TBC1D8B (ab121780; Abcam) or mouse anti-TBC1D8B (SC-376637). Other antibodies used were rabbit anti-RAB11 (#5589S; Cell Signaling Technology), and mouse anti-Myc (9E10; Developmental Studies Hybridoma Bank).
Fluorescence images were obtained with a Zeiss LSM 880 laser scanning microscope, including the application of Airyscan technology for super-resolution microscopy.
Drosophila StudiesThe Drosophila melanogaster stable RNAi stocks Tbc1d8b RNAi (#32929) and Rab11 RNAi (#42709) were obtained from the Bloomington Drosophila Stock Center (BDSC). Prospero -GAL459 Dorothy -GAL4 (# 6903; BDSC) with or without tub-GAL80ts (#7018; BDSC) were used to control expression in garland cell nephrocytes. RAB11 overexpression was achieved using wild-type GFP-Rab11 (# 8506; BDSC). CRISPR/Cas9-mediated loss of function was generated in modification of an established protocol,60 Dorothy -GAL4 to direct Cas9-expression (#58985) in nephrocytes. The CRISPR gRNA construct targeting CG7324 was generated by introducing two gRNAs via PCR and Gibson assembly into the pCFD4 plasmid (Addgene; #49411). The DNA was injected into flies expressing phiC31 integrase under vasa promoter with an attP landing site in 51C by the fly facility of the Department of Genetics at the University of Cambridge (Cambridge, UK). RNAi crosses were raised at 30°C. Flies expressing gRNAs and a Cas9 variant were raised at 25°C as higher temperatures induced unspecific toxicity by Cas9 expression alone (data not shown).
Fluorescent tracer uptake in nephrocytes was performed as previously described.61
For immunohistochemistry, nephrocytes were dissected, fixed for 20 minutes in PBS containing 4% paraformaldehyde, and stained according to the standard procedure. The following primary antibodies were used: rabbit anti-sns62 63
For transmission electron microscopy nephrocytes were dissected and fixed in 4% formaldehyde and 0.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4. Transmission electron microscopy was carried out using standard techniques.
Statistical Analyses
Paired t test was used to determine the statistical significance between two interventions. ANOVA followed by Dunnett’s correction (unless otherwise indicated) was used for multiple comparisons (GraphPad Prism software). Asterisks indicate significance as follows: *P <0.05; **P <0.01; ***P <0.001. A statistically significant difference was defined as P <0.05. Error bars indicate SD.
Results
Novel Mutations in TBC1D8B Cause SNRS
The known monogenic causes of SRNS explain only a fraction of idiopathic cases. We performed WES to discover novel mutations. After excluding relevant mutations in known SRNS genes, we identified hemizygous mutations of the gene TBC1 Domain Family Member 8B (TBC1D8B ) in five individuals from five different families with nephrotic syndrome (Figure 1, A–F , Supplemental Figure 1, A–G Table 1 ). All patients were male, and the available information was compatible with an X-linked inheritance. One heterozygous female conductor exhibited borderline proteinuria (B931, Figure 1, E and I , Supplemental Figure 1, B and C Figure 1, G–I , Table 1 ). Nephrotic syndrome was described as steroid-resistant in all but one patient (A2563). Extrarenal symptoms were not reported in any of these patients. The histology was described as FSGS in three cases (B931, A2563, A3803, Figure 1J , Table 1 ) and mesangioproliferative GN (MesPGN) in one case (J12190/18). No biopsy result was available for B3482. Supporting the significance of our findings, mutations of TBC1D8B were also reported most recently in two families with SRNS and X-linked inheritance.64
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 , and Saccharomyces cerevisiae demonstrates conservation of the residue Arginine 64. (H) Alignment of amino acid sequence of TBC1D8B for H. sapiens , M. musculus , X. tropicalis , and D. 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 , and D. 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.
Table 1. -
Mutations of TBC1D8B in five families with
nephrotic syndrome .
Family Individual
Nucleotide Change
Amino Acid Change
Zygosity
Exon
PPH2
SIFT
MT
Amino Acid Conserved to Species
ExAC (het/hemi)
gnomAD (het/hemi)
Sex
Ethnic Origin
Parental Consanguinity
Response to Steroids
Age at Onset (proteinuria), yr
Renal Function
Renal Biopsy
TBC1D8B
B931_21
c.2338 A>T
p.Thr780Ser
Hemi
15
0,98
Del
DC
D.r.
–
–
M
Hispanic
No
Resistant
9
Normal
FSGS
A2563_21
c.190 C>T
p.Arg64Cys
Hemi
2
1
Del
DC
S.c.
1/0
–
M
Turkish
Yes
Sensitive
7
Normal
FSGS
A3803_21
c.1316 T>G
p.Phe439Cys
Hemi
8
0.87
Del
DC
D.r.
–
–
M
German
No
Resistant
13
Mild reduction
FSGS
J12190/18 _21
c.1030 C>T
p.Arg344*
Hemi
6
–
–
–
–
2/1
4/2
M
German
No
ND
18
ESRD
Mesangioprolif. GN
B3482_21
c.1383 G>A
p.Trp461*
Hemi
9
–
–
–
–
1/1
0/1
M
Pakistan
Yes
Resistant
4
ND
ND
PPH2, PolyPhen-2 prediction score (
http://genetics.bwh.harvard.edu/pph2/ ); SIFT, Sorting Tolerant From Intolerant prediction score (
http://sift.jcvi.org/ ); MT, mutation taster (
http://www.mutationtaster.org/ ); ExAC, Exome Aggregation Consortium database (
http://exac.broadinstitute.org ); Het, heterozygous; Hemi, hemizygous; Del, deleteriousness; DC, disease causing;
D.r .,
Danio rerio ; M, male;
S.c .,
Saccharomyces cerevisiae ; ND, no data; Mesangioprolif. GN, Mesangioproliferative GN.
TBC1D8B Specifically Binds Active RAB11A and RAB11B and the Mutations from Patients with Nephrotic Syndrome Affect Interaction with Endogenous RAB11TBC1D8B is a member of a family of more than 40 TBC-proteins that share the eponymous Tre-2-Bub2-Cdc16 (TBC) domain.65 66 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 dominant negative variants of three Rab proteins that are involved in endocytic recycling: RAB4A (Q72L and S27N), RAB11A (Q70L and S25N), and RAB11B (S25N and Q70L). We performed coimmunoprecipitation, using these variants together with TBC1D8B, and quantified the relative band density of the respective dominant negative and constitutively active Rab protein. We consistently observed a binding ratio >1 (active/inactive Rab) for both RAB11A and RAB11B, but not RAB4A (Figure 2D , quantitation Figure 2E ). We introduced TBC1D8B mutations discovered in patients with nephrotic syndrome (W461*, F439C, T780S, and R64C) into the corresponding amino acid residues of the murine full-length Tbc1d8b (W460*, F438C, T779S, and R64C). The mutation p.Arg344* was not investigated in light of the early premature stop codon preceding Trp461. Analyzing the binding affinity to endogenous RAB11 via coimmunoprecipitation in HEK cells, we observed a significant reduction for mutant proteins, except Tbc1d8bF438C (Figure 2F , quantitation Figure 2G ). Mutations of TBC1D8B that cause nephrotic syndrome thus affect the interaction with endogenous RAB11.
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 comparisons.). (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 constitutively 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 endogenous 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).
Silencing of TBC1D8B Upregulates RAB11-Dependent Autophagy and Exocytosis Suggesting a Function as GAP for RAB11
To evaluate endogenous expression of TBC1D8B in podocytes and HEK293T cells, we performed immunoblotting, and used transient siRNA transfection and CRISPR/Cas9-mediated loss of function.67 Figure 3A , Supplemental Figure 2C 67 TBC1D8B loss of function, using two available antibodies (Supplemental Figure 3, A–C TBC1D8B , including murine Tbc1d8b constructs representing the patient mutations, predominantly localized to the cytosol (Supplemental Figure 3, D–I Supplemental Figure 2, A–B’’
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 endogenous 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).
Promotion of autophagosome maturation is an established cellular function of RAB11.68–70 TBC1D8B as an RAB11-GAP, we studied basal autophagy upon silencing of TBC1D8B in HEK293T cells and podocytes. Using LC3B/GAPDH-ratio as a read-out, immunoblotting indicated an increase of autophagy in both cell lines (Figure 3, B and C , Supplemental Figure 2D via chloroquine. The difference between control and chloroquine treatment was more pronounced upon silencing of TBC1D8B , suggesting a higher autophagic flux (Figure 3D ). As autophagy might be induced independent of RAB11, we tested an effect of TBC1D8B siRNA on exocytosis, another cellular process that requires RAB11 function71 IFNA2 -derived signal peptide into the N terminus of GFP. Upon transient transfection, HEK293T cells constitutively secreted GFP into the surrounding medium (Supplemental Figure 2D RAB11B and silencing of TBC1D8B increased secretion of GFP (Figure 3E , quantitation Figure 3F , Supplemental Figure 2, E and F TBC1D8B . Taken together, these findings suggest a disinhibition of endogenous RAB11 activity being well compatible with a function of TBC1D8B as an RAB11-specific GAP.
TBC1D8B Interacts with the Slit Diaphragm Protein Nephrin and Regulates its TraffickingThe slit diaphragm protein nephrin is subject to endocytic trafficking.72 Figure 4A ) and murine GFP-Tbc1d8b (Supplemental Figure 4A Supplemental Figure 4B Figure 4B ). Truncation mapping thus identifies an interacting domain of 76 amino acids on the nephrin C-terminal tail (Figure 4C ). We evaluated binding of mutant Tbc1d8b by coimmunoprecipitation and observed a reduced binding to Myc-nephrin for all mutants (Figure 4D , quantitation Figure 4E ). The mutations of TBC1D8B that cause nephrotic syndrome thus impair the interaction with nephrin. In podocytes, overexpressed GFP-TBC1D8B and Myc-nephrin colocalized completely within vesicles (Figure 4, F–F’’ ). We confirmed colocalization of both proteins in HEK cells and partially in MDCK cells (Supplemental Figure 4, C–D’’
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 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. Densitometry 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 µ m. (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 terminus, 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 µ m. (I) MDCK cells coexpressing 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 µ m. (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).
To study biosynthetic trafficking of nephrin directly, we utilized an approach on the basis of conditional aggregation domains (CAD) that has been used for other proteins.56 , 57 Figure 4G ). CADs are expected to induce retention of newly synthesized protein within the ER by formation of aggregates. Such aggregates can be dispersed by adding a membrane permeable small molecule (D/D solubilizer), which entails rapid, synchronized ER release of the protein and consecutive removal of the CAD in the Golgi. The presence of two tags (HA-tag and mCherry) eventually permits separate detection of nephrin at the surface by extracellular staining (HA) and the total protein (mCherry, Figure 4G ). We chose MDCK cells that form an epithelial monolayer with apico-basal polarity for this assay because these cells had previously been used for this approach.56 , 57 Figure 4H , upper panels), indicating successful ER retention. Upon exposure to D/D solubilizer, we observed trafficking of nephrin toward the apical cell surface over time (Figure 4H , lower panels). This suggests that nephrin travels initially to the apical surface. Using this novel assay for nephrin trafficking, we studied the effect of overexpressed Tbc1d8b within this system. Interestingly, overexpression of murine full-length Tbc1d8b reduced the apical delivery of nephrin compatible with an inhibition of RAB11-dependent trafficking, but one of the patient-derived mutations failed to have a similar effect in these cells (Figure 4I , quantitation Figure 4J ). These findings indicate that TBC1D8B regulates nephrin trafficking and support a pathogenetic role of Rab11-dependent vesicular trafficking of nephrin in nephrotic syndrome . Basolateral delivery of nephrin was minimal in all conditions (Supplemental Figure 4, E and F
Loss of Function of the Drosophila Ortholog of TBC1D8B Impairs Nephrocytes and Results in Mistrafficking of Fly Nephrin
To validate a role of TBC1D8B in nephrin trafficking in vivo , we used the Drosophila model that harbors the podocyte-like nephrocytes. Within these cells, the ortholog of nephrin forms autocellular slit diaphragms across membrane invaginations. Nephrocytes have been established as a model for monogenic forms of nephrotic syndrome .61 Drosophila gene CG7324 as the ortholog of human TBC1D8B (Figure 5A ). This gene encodes a protein of 1256 amino acids that contains two GRAM domains and one TBC domain like the human TBC1D8B. We introduce the term Tbc1d8b for the Drosophila ortholog CG7324 .
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 µm 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 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 µ m 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.
To study loss of function of Tbc1d8b within this invertebrate podocyte model, we expressed Tbc1d8b RNAi within nephrocytes using prospero -GAL4. Studying the uptake of FITC-albumin, an established read-out of nephrocyte function,61 Figure 5B , quantitation Figure 5C ). To confirm this finding, we generated a conditional CRISPR/Cas9-mediated loss of Tbc1d8b in nephrocytes. Combining nephrocyte-restricted Cas9 - expression by Dorothy -GAL4 with stable ubiquitous expression of a tandem Tbc1d8b gRNA, we noted a reduction of nephrocyte function that slightly exceeded the effect of the RNAi (Figure 5B , quantitation Figure 5C ). We stained the slit diaphragm proteins Sns (ortholog of nephrin) and Kirre (ortholog of NEPH1) in nephrocytes expressing Tbc1d8b RNAi, and observed protrusions of these proteins from the surface in a fine linear pattern that also occurred solitarily (insets, Supplemental Figure 5, A–B’’ Figure 5, F–F’’ and D–D’’ ). With a lower penetrance, we further observed patches lacking slit diaphragms entirely upon expression of Tbc1d8b RNAi (Supplemental Figure 5, A–B’’ Tbc1d8b RNAi , whereas individual lines appeared brighter (compare Figure 5, panels E and G ). The fine linear protrusions of slit diaphragm protein from the surface were visible subcortically when Tbc1d8b RNAi was expressed but absent from subcortical control sections (compare insets Figure 5, E and G ). To understand the ultrastructural basis of the protrusions of slit diaphragm protein from the membrane, we performed electron microscopy and observed the formation of additional, ectopic slit diaphragms deeper within the membrane invaginations (Figure 5I , control Figure 5H ). These ectopic slit diaphragms obviously corresponded to our observations using immunofluorescence (Figure 5, F–G’’ ). To confirm our findings in an independent loss-of-function approach, we stained Sns/Kirre in nephrocytes with CRISPR/Cas9-mediated loss of Tbc1d8b compared with control conditions (Cas9 expression without gRNA, Figure 5, K–L’’ ). Upon loss of Tbc1d8b , we observed a phenotype that was more pronounced but matched our findings using the Tbc1d8b RNAi (Figure 5, J and M–N’’ ). The more severe phenotype upon CRISPR/Cas9-mediated loss of function compared with RNAi expression matched the stronger reduction of FITC-albumin endocytosis and suggested an incomplete knockdown using RNAi. Taken together, these findings indicate mistrafficking of fly nephrin with mislocalization of slit diaphragms upon loss of Drosophila Tbc1d8b .
Loss of Function of Rab11 Impairs Trafficking of Slit Diaphragm Proteins in Nephrocytes
Our findings suggest a functional role of RAB11 for trafficking of nephrin. To evaluate such a role of Drosophila Rab11 for trafficking of slit diaphragm proteins in nephrocytes, we silenced Rab11 acutely by modifying the GAL4-dependent expression using the temperature-sensitive Gal80ts that suppresses GAL4 at nonpermissive temperatures. Acute Rab11 silencing resulted in localized loss of slit diaphragms (Figure 6, A–A’’ ) and shortened lines of slit diaphragm protein on the surface (Figure 6, B–B’’ ), whereas nephrocytes from animals raised at nonpermissive temperatures showed regular slit diaphragms (Supplemental Figure 5, C–C’’ Rab11 RNAi caused extensive loss of slit diaphragm protein from the membrane (Figure 6, C–C’’ ) with appearance of puncta of nephrin on the surface (insets Figure 6, D–D’’ ) and clusters of Kirre protein between nephrocytes in absence of fly nephrin (Figure 6, D’–D’’ ). To study a mild gain of function similar to the effect of a defective Rab11-GAP, we overexpressed GFP-tagged Drosophila Rab11 in nephrocytes and observed appearance of ectopic vesicles of fly nephrin and fine protrusions of fly nephrin from the membrane (Figure 6, E–E’’ ). Thus, overexpression of Rab11 partially phenocopies loss of Tbc1d8b. However, overexpression of wild-type Rab11 produced an overtly milder phenotype than lack of Tbc1d8b (compare Figure 5F’ to Figure 6E’ ). This may be explained by additional Rab11-independent functions of Tbc1d8b , low efficiency of the exogenous wild-type Rab11 expression, or divergent compensatory regulation. Taken together, these findings indicate that slit diaphragm formation in nephrocytes requires Rab11 and they are compatible with a function of Tbc1d8b as an Rab11-GAP in Drosophila .
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 µ m 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 fly nephrin vesicles and fine linear protrusions of slit diaphragm protein similar to the observations with Tbc1d8b RNAi (arrow in enlarged inset).
Discussion
Here, we report the identification of five mutations of the gene TBC1D8B in patients with nephrotic syndrome from five different families. Analyzing interaction of TBC1D8B with constitutively active RAB11A and RAB11B compared with their dominant negative variants, we observed specific binding for the active, GTP-bound forms of RAB11A and RAB11B. Loss of TBC1D8B increased basal autophagy, autophagic flux, and exocyst activity, suggesting a disinhibition of endogenous RAB11 function and supporting a role of TBC1D8B as a GAP protein for RAB11. We demonstrate TBC1D8B is an interaction partner of the slit diaphragm protein nephrin and both overexpressed proteins colocalize within different immortalized cell lines. Mutations derived from patients with nephrotic syndrome impair binding of TBC1D8B to endogenous RAB11 and nephrin. Overexpression of TBC1D8B affected trafficking of nephrin in vitro . Studying the loss of Tbc1d8b in the Drosophila model, we observed impaired fly nephrin trafficking within the podocyte-like nephrocytes in vivo . Silencing Rab11 in nephrocytes resulted in appearance of slit diaphragm proteins independent from each other, suggesting that the protein complex may be formed during RAB11-dependent transport.
Our findings are corroborated by a most recent report in two families64 TBC1D8B in five unrelated families with nephrotic syndrome conclusively defines TBC1D8B as a novel monogenic cause of nephrotic syndrome . Our results in nephrocytes confirm a role of TBC1D8B for glomerular function that was most recently reported in fish.64 Drosophila model facilitated precise study of the subcellular localization of fly nephrin upon loss of function of Tbc1d8b and allowed acute silencing of Rab11 . Thus, we could show a role of Tbc1d8b and Rab11 for fly nephrin trafficking in vivo to identify a possible mechanistic link between Rab11 regulation and glomerular dysfunction.
Surprisingly, the two truncating mutations (p.Arg344* and p.Trp461*) were observed hemizygously in two or one individuals within the gnomAD control population respectively. The incomplete penetrance of these nonsense mutations may be explained by a genetic compensation response.73 , 74 TBC1D8B but not in missense mutations. Patients with mutations of TBC1D8B typically presented with FSGS in renal biopsy, while MesPGN was described in one case. MesPGN, a glomerular damage pattern, frequently showed progression to FSGS in repeat biopsies of children with SRNS.75
TBC1D8B binds to the active form of RAB11A and RAB11B indicating that TBC1D8B functions as a GAP for both variants of RAB11. Our data implicate RAB11-dependent vesicular trafficking in the pathogenesis of nephrotic syndrome , and delineate a role of TBC1D8B for trafficking of the slit diaphragm protein nephrin as the mechanistic basis for nephrotic syndrome associated with TBC1D8B mutations. We previously implied regulators of RAB5 in nephrotic syndrome .41 76 nephrotic syndrome associated with TBC1D8B is conceivable but will require further investigation.
Trafficking of nephrin in podocytes in vivo may deviate from our observations in MDCK cells, but it is possible that nephrin is delivered initially to the apical surface before eventually being stabilized within the slit diaphragm complex at the apico-basal border. Nephrin trafficking does not seem to occur constitutively, but as a regulated process. Together with our findings in Drosophila , our data support such a regulatory role for TBC1D8B. The mutations of TBC1D8B caused no extrarenal manifestations, suggesting that tight regulation of nephrin trafficking by TBC1D8B/RAB11 is required for the establishment and maintenance of the glomerular filter, whereas (compensatory) function of other RAB11-GAPs may suffice in other tissues.
In conclusion, our findings define TBC1D8B as an RAB11-GAP and together with a previous report64 nephrotic syndrome . This supports RAB11-dependent trafficking of nephrin as a novel pathogenetic mechanism.
Disclosures
Dr. Bergmann is an employee of Bioscientia/Sonic Healthcare and holds a part-time faculty appointment at the University of Freiburg. Dr. Hildebrandt is a cofounder of Goldfinch-Bio and receives royalties from Claritas. Dr. MacArthur reports personal fees from Goldfinch Bio, outside the submitted work. All of the remaining authors have nothing to disclose.
Funding
This research was supported by grants from the Deutsche Forschungsgemeinschaft National Institutes of Health National Human Genome Research Institute National Eye Institute National Heart, Lung, and Blood Institute Deutsche Forschungsgemeinschaft
Dr. Bergmann, Dr. Hermle, Dr. Laricchia, Dr. Lifton, Dr. MacArthur, Dr. Mane, Dr. Onuchic-Whitford, Dr. Rehm, and Dr. Schneider generated total genome linkage analysis, performed exome capture and massively parallel sequencing, and performed whole-exome evaluation and mutation analysis. Ms. Chen, Ms. Gerstner, Dr. Hermle, Ms. Kampf, Dr. Römer, Dr. Schneider, and Dr. Thünauer performed cDNA cloning, protein purification, tracer endocytosis and immunofluorescence, and subcellular localization studies in cell lines by confocal microscopy. Ms. Chen, Ms. Gerstner, Dr. Hermle, Ms. Kampf, and Dr. Schneider performed coimmunoprecipitation and the experiments in Drosophila . Dr. Helmstädter performed electron microscopy. Dr. Amar, Dr. Berdeli, Dr. Hermle, Dr. Hildebrandt, Dr. Loza Munarriz, Dr. Müller, and Dr. Walz recruited patients and gathered detailed clinical information for the study. Dr. Hermle conceived of and directed the study with support from Dr. Hildebrandt. Dr. Hermle wrote the manuscript. The manuscript was critically reviewed by all the authors.
We are grateful to the families and study individuals for their contribution. We thank the Yale Center for Mendelian Genomics and the Broad Center for Mendelian Genomics for whole exome sequencing analysis. We thank Dr. R. Nitschke, Life Imaging Centre, University of Freiburg, for help with confocal microscopy. We thank Severine Kayser for technical assistance with electron microscopy. We thank the Developmental Studies Hybridoma Bank for antibodies.
Supplemental Material
This article contains the following supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019040414/-/DCSupplemental
Supplemental Figure 1 TBC1D8B .
Supplemental Figure 2
Supplemental Figure 3 TBC1D8B .
Supplemental Figure 4
Supplemental Figure 5 Drosophila .
Supplemental Table 1
References
1. Smith JM, Stablein DM, Munoz R, Hebert D, McDonald RA: Contributions of the transplant registry: The 2006 annual report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). Pediatr Transplant 11: 366–373, 200717493215
2. Bierzynska A, Soderquest K, Dean P, Colby E, Rollason R, Jones C, et al.; NephroS; UK study of
Nephrotic Syndrome :
MAGI2 mutations cause congenital
nephrotic syndrome . J Am Soc Nephrol 28: 1614–1621, 201727932480
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. J Clin Invest 127: 912–928, 201728165339
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 primary microcephaly. Nat Genet 49: 1529–1538, 201728805828
5. Kestilä M, Lenkkeri U, Männikkö M, Lamerdin J, McCready P, Putaala H, et al.: Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital
nephrotic syndrome . Mol Cell 1: 575–582, 19989660941
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, 200717713465
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 Hum Genet 96: 153–161, 201525557779
8. Gee HY, Sadowski CE, Aggarwal PK, Porath JD, Yakulov TA, Schueler M, et al.: FAT1 mutations cause a glomerulotubular nephropathy. Nat Commun 7: 10822, 201626905694
9. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, et al.: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant
nephrotic syndrome . Nat Genet 24: 349–354, 200010742096
10. Ozaltin F, Li B, Rauhauser A, An SW, Soylemezoglu O, Gonul II, et al.: DGKE variants cause a glomerular microangiopathy that mimics membranoproliferative GN. J Am Soc Nephrol 24: 377–384, 201323274426
11. Ozaltin F, Ibsirlioglu T, Taskiran EZ, Baydar DE, Kaymaz F, Buyukcelik M, et al.; PodoNet Consortium: Disruption of PTPRO causes childhood-onset
nephrotic syndrome . Am J Hum Genet 89: 139–147, 201121722858
12. Has C, Spartà G, Kiritsi D, Weibel L, Moeller A, Vega-Warner V, et al.: Integrin α3 mutations with kidney, lung, and skin disease. N Engl J Med 366: 1508–1514, 201222512483
13. Kambham N, Tanji N, Seigle RL, Markowitz GS, Pulkkinen L, Uitto J, et al.: Congenital focal segmental glomerulosclerosis associated with beta4 integrin mutation and epidermolysis bullosa. Am J Kidney Dis 36: 190–196, 200010873890
14. Zenker M, Aigner T, Wendler O, Tralau T, Müntefering H, Fenski R, et al.: Human laminin beta2 deficiency causes congenital nephrosis with mesangial sclerosis and distinct eye abnormalities. Hum Mol Genet 13: 2625–2632, 200415367484
15. Ashraf S, Gee HY, Woerner S, Xie LX, Vega-Warner V, Lovric S, et al.: ADCK4 mutations promote steroid-resistant
nephrotic syndrome through CoQ10 biosynthesis disruption. J Clin Invest 123: 5179–5189, 201324270420
16. Diomedi-Camassei F, Di Giandomenico S, Santorelli FM, Caridi G, Piemonte F, Montini G, et al.: COQ2 nephropathy: A newly described inherited mitochondriopathy with primary renal involvement. J Am Soc Nephrol 18: 2773–2780, 200717855635
17. Heeringa SF, Chernin G, Chaki M, Zhou W, Sloan AJ, Ji Z, et al.: COQ6 mutations in human patients produce
nephrotic syndrome with sensorineural deafness. J Clin Invest 121: 2013–2024, 201121540551
18. López LC, Schuelke M, Quinzii CM, Kanki T, Rodenburg RJ, Naini A, et al.: Leigh syndrome with nephropathy and CoQ10 deficiency due to decaprenyl diphosphate synthase subunit 2 (PDSS2) mutations. Am J Hum Genet 79: 1125–1129, 200617186472
19. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, et al.: Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis. Nat Genet 24: 251–256, 200010700177
20. Gbadegesin RA, Hall G, Adeyemo A, Hanke N, Tossidou I, Burchette J, et al.: Mutations in the gene that encodes the F-actin binding protein anillin cause FSGS. J Am Soc Nephrol 25: 1991–2002, 201424676636
21. Akilesh S, Suleiman H, Yu H, Stander MC, Lavin P, Gbadegesin R, et al.: Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J Clin Invest 121: 4127–4137, 201121911940
22. Gupta IR, Baldwin C, Auguste D, Ha KC, El Andalousi J, Fahiminiya S, et al.: ARHGDIA: A novel gene implicated in
nephrotic syndrome . J Med Genet 50: 330–338, 201323434736
23. Gee HY, Saisawat P, Ashraf S, Hurd TW, Vega-Warner V, Fang H, et al.: ARHGDIA mutations cause
nephrotic syndrome via defective RHO GTPase signaling. J Clin Invest 123: 3243–3253, 201323867502
24. Brown EJ, Schlöndorff JS, Becker DJ, Tsukaguchi H, Tonna SJ, Uscinski AL, et al.: Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis. Nat Genet 42: 72–76, 201020023659
25. Gee HY, Zhang F, Ashraf S, Kohl S, Sadowski CE, Vega-Warner V, et al.: KANK deficiency leads to podocyte dysfunction and
nephrotic syndrome . J Clin Invest 125: 2375–2384, 201525961457
26. Mele C, Iatropoulos P, Donadelli R, Calabria A, Maranta R, Cassis P, et al.; PodoNet Consortium: MYO1E mutations and childhood familial focal segmental glomerulosclerosis. N Engl J Med 365: 295–306, 201121756023
27. Heath KE, Campos-Barros A, Toren A, Rozenfeld-Granot G, Carlsson LE, Savige J, et al.: Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes. Am J Hum Genet 69: 1033–1045, 200111590545
28. Ovunc B, Otto EA, Vega-Warner V, Saisawat P, Ashraf S, Ramaswami G, et al.: Exome sequencing reveals cubilin mutation as a single-gene cause of proteinuria. J Am Soc Nephrol 22: 1815–1820, 201121903995
29. Berkovic SF, Dibbens LM, Oshlack A, Silver JD, Katerelos M, Vears DF, et al.: Array-based gene discovery with three unrelated subjects shows SCARB2/LIMP-2 deficiency causes myoclonus epilepsy and glomerulosclerosis. Am J Hum Genet 82: 673–684, 200818308289
30. Boerkoel CF, Takashima H, John J, Yan J, Stankiewicz P, Rosenbarker L, et al.: Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia. Nat Genet 30: 215–220, 200211799392
31. Vollrath D, Jaramillo-Babb VL, Clough MV, McIntosh I, Scott KM, Lichter PR, et al.: Loss-of-function mutations in the LIM-homeodomain gene, LMX1B, in nail-patella syndrome. Hum Mol Genet 7: 1091–1098, 19989618165
32. Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, Nürnberg G, et al.: Positional cloning uncovers mutations in PLCE1 responsible for a
nephrotic syndrome variant that may be reversible. Nat Genet 38: 1397–1405, 200617086182
33. Gee HY, Otto EA, Hurd TW, Ashraf S, Chaki M, Cluckey A, et al.: Whole-exome resequencing distinguishes cystic kidney diseases from phenocopies in renal ciliopathies. Kidney Int 85: 880–887, 201424257694
34. Colin E, Huynh Cong E, Mollet G, Guichet A, Gribouval O, Arrondel C, et al.: Loss-of-function mutations in WDR73 are responsible for microcephaly and steroid-resistant
nephrotic syndrome : Galloway-Mowat syndrome. Am J Hum Genet 95: 637–648, 201425466283
35. Sethi S, Fervenza FC, Zhang Y, Smith RJ: Secondary focal and segmental glomerulosclerosis associated with single-nucleotide polymorphisms in the genes encoding complement factor H and C3. Am J Kidney Dis 60: 316–321, 201222594991
36. Jeanpierre C, Denamur E, Henry I, Cabanis MO, Luce S, Cécille A, et al.: Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database. Am J Hum Genet 62: 824–833, 19989529364
37. Yasukawa T, Suzuki T, Ueda T, Ohta S, Watanabe K: Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. J Biol Chem 275: 4251–4257, 200010660592
38. Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, et al.: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 308: 1801–1804, 200515879175
39. Rao J, Ashraf S, Tan W, van der Ven AT, Gee HY, Braun DA, et al.: Advillin acts upstream of phospholipase C ϵ1 in steroid-resistant
nephrotic syndrome . J Clin Invest 127: 4257–4269, 201729058690
40. Ashraf S, Kudo H, Rao J, Kikuchi A, Widmeier E, Lawson JA, et al.: Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment. Nat Commun 9: 1960, 201829773874
41. Hermle T, Schneider R, Schapiro D, Braun DA, van der Ven AT, Warejko JK, et al.:
GAPVD1 and
ANKFY1 mutations implicate RAB5 regulation in
nephrotic syndrome . J Am Soc Nephrol 29: 2123–2138, 201829959197
42. Braun DA, Lovric S, Schapiro D, Schneider R, Marquez J, Asif M, et al.: Mutations in multiple components of the nuclear pore complex cause
nephrotic syndrome . J Clin Invest 128: 4313–4328, 201830179222
43. Braun DA, Warejko JK, Ashraf S, Tan W, Daga A, Schneider R, et al.: Genetic variants in the LAMA5 gene in pediatric
nephrotic syndrome . Nephrol Dial Transplant 34: 485–493, 201929534211
44. Swiatecka-Urban A: Endocytic trafficking at the mature podocyte slit diaphragm. Front Pediatr 5: 32, 201728286744
45. Martin CE, Jones N: Nephrin signaling in the podocyte: An updated view of signal regulation at the slit diaphragm and beyond. Front Endocrinol (Lausanne) 9: 302, 201829922234
46. Harris DP, Vogel P, Wims M, Moberg K, Humphries J, Jhaver KG, et al.: Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function. Mol Cell Biol 31: 63–80, 201120974805
47. Bechtel W, Helmstädter M, Balica J, Hartleben B, Kiefer B, Hrnjic F, et al.: Vps34 deficiency reveals the importance of endocytosis for podocyte homeostasis. J Am Soc Nephrol 24: 727–743, 201323492732
48. Soda K, Balkin DM, Ferguson SM, Paradise S, Milosevic I, Giovedi S, et al.: Role of dynamin, synaptojanin, and endophilin in podocyte foot processes. J Clin Invest 122: 4401–4411, 201223187129
49. Quack I, Rump LC, Gerke P, Walther I, Vinke T, Vonend O, et al.: beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity. Proc Natl Acad Sci U S A 103: 14110–14115, 200616968782
50. Teng B, Schroder P, Müller-Deile J, Schenk H, Staggs L, Tossidou I, et al.: CIN85 deficiency prevents nephrin endocytosis and proteinuria in diabetes. Diabetes 65: 3667–3679, 201627531950
51. Qin XS, Tsukaguchi H, Shono A, Yamamoto A, Kurihara H, Doi T: Phosphorylation of nephrin triggers its internalization by raft-mediated endocytosis. J Am Soc Nephrol 20: 2534–2545, 200919850954
52. Babayeva S, Rocque B, Aoudjit L, Zilber Y, Li J, Baldwin C, et al.: Planar cell polarity pathway regulates nephrin endocytosis in developing podocytes. J Biol Chem 288: 24035–24048, 201323824190
53. Königshausen E, Zierhut UM, Ruetze M, Potthoff SA, Stegbauer J, Woznowski M, et al.: Angiotensin II increases glomerular permeability by β-arrestin mediated nephrin endocytosis. Sci Rep 6: 39513, 201628004760
54. Quack I, Woznowski M, Potthoff SA, Palmer R, Königshausen E, Sivritas S, et al.: PKC alpha mediates beta-arrestin2-dependent nephrin endocytosis in hyperglycemia. J Biol Chem 286: 12959–12970, 201121321125
55. Tian X, Kim JJ, Monkley SM, Gotoh N, Nandez R, Soda K, et al.: Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance. J Clin Invest 124: 1098–1113, 201424531545
56. Thuenauer R, Hsu YC, Carvajal-Gonzalez JM, Deborde S, Chuang JZ, Römer W, et al.: Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates. Proc Natl Acad Sci U S A 111: 4127–4132, 201424591614
57. Stroukov W, Rösch A, Schwan C, Jeney A, Römer W, Thuenauer R: Synchronizing protein traffic to the primary cilium. Front Genet 10: 163, 201930906310
58. Braun DA, Sadowski CE, Kohl S, Lovric S, Astrinidis SA, Pabst WL, et al.: Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant
nephrotic syndrome . Nat Genet 48: 457–465, 201626878725
59. Weavers H, Prieto-Sánchez S, Grawe F, Garcia-López A, Artero R, Wilsch-Bräuninger M, et al.: The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457: 322–326, 200918971929
60. Port F, Chen HM, Lee T, Bullock SL: Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111: E2967–E2976, 201425002478
61. Hermle T, Braun DA, Helmstädter M, Huber TB, Hildebrandt F: Modeling monogenic human
nephrotic syndrome in the
Drosophila garland cell nephrocyte. J Am Soc Nephrol 28: 1521–1533, 201727932481
62. Bour BA, Chakravarti M, West JM, Abmayr SM: Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev 14: 1498–1511, 200010859168
63. Galletta BJ, Chakravarti M, Banerjee R, Abmayr SM: SNS: Adhesive properties, localization requirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech Dev 121: 1455–1468, 200415511638
64. Dorval G, Kuzmuk V, Gribouval O, Welsh GI, Bierzynska A, Schmitt A, et al.: TBC1D8B loss-of-function mutations lead to X-linked
nephrotic syndrome via defective trafficking pathways. Am J Hum Genet 104: 348–355, 201930661770
65. Fukuda M: TBC proteins: GAPs for mammalian small GTPase Rab? Biosci Rep 31: 159–168, 201121250943
66. Gallo LI, Liao Y, Ruiz WG, Clayton DR, Li M, Liu YJ, et al.: TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells. Mol Biol Cell 25: 3779–3797, 201425232007
67. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F: Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8: 2281–2308, 201324157548
68. Fader CM, Sánchez D, Furlán M, Colombo MI: Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells. Traffic 9: 230–250, 200817999726
69. Longatti A, Lamb CA, Razi M, Yoshimura S, Barr FA, Tooze SA: TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes. J Cell Biol 197: 659–675, 201222613832
70. Szatmári Z, Kis V, Lippai M, Hegedus K, Faragó T, Lorincz P, et al.: Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization. Mol Biol Cell 25: 522–531, 201424356450
71. Takahashi S, Kubo K, Waguri S, Yabashi A, Shin HW, Katoh Y, et al.: Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. J Cell Sci 125: 4049–4057, 201222685325
72. Inoue K, Ishibe S: Podocyte endocytosis in the regulation of the glomerular filtration barrier. Am J Physiol Renal Physiol 309: F398–F405, 201526084928
73. El-Brolosy MA, Kontarakis Z, Rossi A, Kuenne C, Günther S, Fukuda N, et al.: Genetic compensation triggered by mutant mRNA degradation. Nature 568: 193–197, 201930944477
74. Ma Z, Zhu P, Shi H, Guo L, Zhang Q, Chen Y, et al.: PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. Nature 568: 259–263, 201930944473
75. Trautmann A, Bodria M, Ozaltin F, Gheisari A, Melk A, Azocar M, et al.; PodoNet Consortium: Spectrum of steroid-resistant and congenital
nephrotic syndrome in children: The PodoNet registry cohort. Clin J Am Soc Nephrol 10: 592–600, 201525635037
76. Lou X: Sensing exocytosis and triggering endocytosis at synapses: Synaptic vesicle exocytosis-endocytosis coupling. Front Cell Neurosci 12: 66, 201829593500
