The animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center (IACUC protocol no. 12-033 to T.O.). The AB strain of zebrafish was maintained at 28.5°C under a 14 h light/10 h dark cycle. Embryos were maintained at 28.5°C in 0.5X E2 egg medium.

Full-length zebrafish nephrin cDNA that was subcloned into the pCR-BluntII-TOPO vector was a kind gift from Dr Iain Drummond (9). Full-length zebrafish podocin cDNA was obtained by performing RT-PCR on the total RNA isolated from 4 days post-fertilization (dpf) embryos using RNAqueous-4PCR kit (Life Technologies, Carlsbad, CA, USA) and subsequently performing nested PCR. RT-PCR was performed using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Life Technologies) and nested PCR using Phusion High-Fidelity DNA Polymerase (ThermoScientific, Waltham, MA, USA). The primer sets used were: RT-PCR, forward: 5′-ATC TGC ACT GGC CTC CTG ATA-3′ and reverse: 5′-ATG CGA AGG AAA TCC GTC AAC-3′ and nested PCR, forward: 5′-CAC CAG AGG ACA CTT CAC AAC A-3′ and reverse 5′-CAG CCA ATA ATC AGT ACA GTC TTG AAA-3′. Podocin cDNA was subcloned into pCR-BluntII-TOPO and verified by DNA sequencing.

In situ hybridization was conducted as previously described (1–3,9). In brief, the pCR-BluntII-TOPO- nephrin and -podocin digested with XhoI were used as a template for anti-sense RNA probe. The probe was synthesized with an SP6 RNA polymerase (New England BioLabs, Ipswich, MA, USA) and DIG-RNA labeling kit (Roche Diagnostics, Mannheim, Germany). Embryos were fixed in 4% paraformaldehyde (PFA), 0.1% Tween-20 in PBS for 2 h at room temperature (RT), altered to 100% methanol and stored at −20°C. Whole-mount in situ hybridization was performed as previously described (28). Following color development, the samples were dehydrated with a graded series of methanol and embedded in JB-4 resin (Polysciences, Inc., Warrington, PA, USA). Ten micron sections were sliced using an RN2255 microtome (Leica Microsystems, Wetzlar, Germany) and counter-stained with special eosin II (BBC Biochemical, Mount Vernon, WA, USA). After mounting in Poly-Mount (Polysciences, Warrington, PA, USA), the stained sections were photographed on a Provis AX-70 microscope (Olympus, Tokyo, Japan) equipped with a RETIGA EXi digital camera (QImaging, Surrey, Canada).

A polyclonal anti-zebrafish Nephrin antibody was prepared as previously described (3,21). Rabbit polyclonal anti-zebrafish Podocin antibody was raised in rabbits using the amino-terminal peptide VKLQEPHKRKE (amino acids 43–53) coupled to KLH. The antiserum was affinity-purified against the immunizing peptide (Covance, Denver, PA, USA).

Proteins were extracted from 1 to 5 dpf zebrafish. Prior to protein extraction, the yolk ball was removed from 4 dpf zebrafish larvae, as a large amount of yolk-derived proteins occasionally affects SDS-PAGE and immunoblot analysis (29). Deyolked larvae were homogenized and solubilized in the protein extraction buffer (1% NP-40, 150 mM NaCl, 50 mM KI, 1 mM EDTA and 10% glycerol in 50 mM HEPES, pH 7.4). The homogenate was centrifuged at 11,300 × g for 10 min and the supernatant was collected as the lysate. Each lysate containing 50 μg of protein was subjected to SDS-PAGE (Mini-PROTEAN TGX Gel, 4–15% gradient; Bio-Rad Laboratories, Hercules, CA, USA) and separated proteins were transferred onto a PVDF membrane (Immobilon Transfer Membrane; Millipore, Billerica, MA, USA). Each membrane was incubated with the primary antibody for 2 h at RT and then with an anti-HRP-conjugated secondary antibody (working dilution 1:5,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at RT. Dilutions of primary antibodies were 1:2,500 for anti-Nephrin, 1:5,000 for anti-Podocin and 1:5,000 for anti-α-tubulin.

Immunohistochemical analysis was performed as previously described (3). Larvae were fixed with Dent’s fixative (20% DMSO in methanol) overnight at 4°C. Fixed samples were rehydrated with a graded series of methanol and washed with PBS containing 0.5% Triton X-100 (PBSTx). For antigen retrieval, the samples were heated in Antigen Retrieval Reagent Universal (R&D Systems, Minneapolis, MN, USA) for 15 min at 95°C on a heat block. Subsequently, the samples were blocked with the incubation solution (PBSTx containing 10% normal goat serum and 1% DMSO) for 2 h at RT and incubated with the primary antibody (working dilution 1:100) in the incubation solution for 12 h at 4°C. After washing with PBSTx, the samples were incubated with Alexa-Fluor546-conjugated goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Laboratories) and diluted with the incubation solution (1:1,000) for 2 h at RT. Stained samples were dehydrated with a graded series of methanol, embedded in JB-4 resin (Polysciences, Inc.) and sliced into 10-μm sections with a RN2255 microtome. The sections were photographed with an FV-1000 confocal laser scanning microscope (Olympus).

cDNA corresponding to zebrafish podocin was generated using RT-PCR, digested with BamHI and XbaI, subcloned into pEV3S-Flag vector and verified by DNA sequencing. This cDNA was used to generate the R150Q mutation, which was also cloned into the pEV3S-Flag vector and verified by DNA sequencing. Human embryonic kidney 293T cells, which were grown to ~25% confluency in 60-mm dishes, were transiently transfected with 2 μg Flag-Podocin and 7 μg pBluescript KS⁺ using the calcium phosphate coprecipitation method (30). Following 12-h incubation, the precipitate was removed by washing twice with 2 ml PBS and the cells were incubated for an additional 36 h in DMEM supplemented with 10% fetal bovine serum at 37°C. Thereafter, the cells were lysed by boiling in Laemmli buffer and the insoluble material was removed by centrifugation (31). The resultant protein lysate was subjected to SDS-PAGE and the separated proteins were transferred to a PVDF membrane (Immobilon; Millipore) and processed for immunoblotting with the indicated antibodies (32).

MOs were designed for the splice blocking of nephrin-exon 25 (nephrinMOex25: 5′-TGC ACC AAC ACG ACT CAC CTC TGC TC-3′) and of podocin-exon 3 (podocinMOex3: 5′-TGT AGT CAC TTT TGC AGA CCT GGG CT-3′) (Gene Tools LLC, Philomath, OR, USA). The two morpholinos target the splice acceptor site. The morpholinos were diluted with the injection solution containing 100 mM KCl and 10 mM HEPES (pH 7.6) and were injected at a final concentration of 0.15 mM (nephrinMOex25) and 0.25 mM (podocinMOex3) into one- or two-cell stage embryos using a Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA). The injected volume was ~4.6 nl. Splice blocking was verified using RT-PCR and nested PCR. Total RNA was isolated from five embryos with an RNAqueous-4PCR kit (Life Technologies). RT-PCR was performed using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Life Technologies) followed by nested PCR using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The primer sets used were: forward, 5′-GGC AGG ATC TGC AAG CTA CAT-3 and 5′-CTC AGG GCC TTC AGG GT GAG-3′ (RT-PCR for nephrin); 5′-CTC CTG AAC CCA TTC ATC TGC-3′ and 5′-CTC ATA GAC GCT GCT GTC AGG-3′ (nested PCR for nephrin); 5′-GAT GCT TCC TGC GGA GAT AGA-3′ and 5′-TTC CTG TCC AGC AAA ATG TCA-3′ (RT-PCR for podocin) and 5′-TCA TCT CTA GCA GCA CGG TTG-3′ and 5′-TCT GGA ATG CTA GCG AAG GAG-3′ (nested PCR for podocin). Altered PCR products were subcloned into pCR-BluntII-TOPO and sequenced.

Hematoxylin and eosin staining was conducted as previously described (32). In brief, larvae were fixed with histology fixative (1.5% glutaraldehyde, 4% PFA, 3% sucrose in 0.1 M phosphate buffer, pH 7.3) overnight at 4°C, dehydrated with a graded series of methanol and embedded in JB-4 resin. Four micron sections were sliced and stained with Harris hematoxylin and special eosin II (BBC Biochemical, Mount Vernon, WA, USA). The stained sections were photographed with a Provis AX-70 microscope equipped with a RETIGA EXi digital camera (QImaging).

Transmission electron microscopy was conducted as previously described (3). Larvae were fixed with histology fixative overnight at 4°C. The samples were immersed in 1% OsO₄ in 0.1 M phosphate buffer for 1 h, dehydrated with a graded series of ethanol and then embedded in Epon-Araldite resin (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin silver-gold sections were produced with an ultra 45° diamond knife (Diatome, Biel, Switzerland) and were transferred to copper grids (50 mesh, Nisshin EM) with a carbon-coated Formvar membrane. The sections were stained with uranyl acetate and lead citrate and photographed on a H-7600 transmission electron microscope (Hitachi High Technologies Inc., Schaumburg, IL, USA) equipped with a Kodak 2K × 2K digital camera.

cDNA encoding the intracellular region of zebrafish Nephrin (amino acids 1,065–1,242) was generated by performing PCR on the total RNA from 2 dpf zebrafish larvae as a template, subcloned into pCS3⁺-6Myc vector and verified by DNA sequencing. Generation of Flag-Podocin vector was mentioned earlier. Each 1 μg of indicated Flag-Podocin and 6Myc-Nephrin-C vectors were transfected together with 7 μg pBluescript KS⁺ into 293T cells essentially as previously described (31). Following 36 h incubation, the cells were lysed in 600 μl 50 mM Tris (pH 7.4), 50 mM NaF, 150 mM NaCl, 0.2 mM DTT, 0.5% NP-40, 1 mM PMSF, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 1 μg/ml pepstatin A and 0.2 mM Na₃VO₄. Cell lysates were processed as described earlier utilizing the anti-Flag M2 monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) for the immunoprecipitation (34). Immunoprecipitated complexes were resolved by SDS-PAGE and co-precipitated proteins were detected by anti-Myc immuno-blot analysis (35).

The human NEPHRIN and PODOCIN cDNAs were kind gifts from Dr Lawrence Holzman. PCR was used to introduce a non-sense mutation into human NEPHRIN cDNA causing R1109X and a single base substitution into human PODOCIN cDNA causing R138Q. The following primer sets were used for the generation of mutated cDNA: for NEPHRIN mutant: HNEPHRIN-forward: 5′-GTC AAA GCT TAT GGC CCT GGG GAC GAC GCT CAG and -reverse: 5′-AAG GAT AGC GGC CGC CTA CAC CAG ATG TCC CCT CAG CTC GAA G; HNE- PHRIN-1109X-forward: 5′-GGG TCG GAA GAG GAC TAG GTC AGG AAC GAA TAT GAG GAG AGC C-3′ and -reverse: 5′-GGC TCT CCT CAT ATT CGT TCC TGA CCT AGT CCT CTT CCG ACC CTG CC-3′; for PODOCIN mutant: HPODOCIN-forward: 5′-GTC AAA GCT TAC CAT GGA GAG GAG GGC GCG GAG CTC CTC CAG-3′ and -reverse: 5′-AAG GAT AGC GGC CGC CTA TAA CAT GGG AGA GTC TTT CTT TTT AGG-3′, HPODOCIN-138Q-forward: 5′-AGA GTA ATT ATA TTC CAG CTG GGA CAT CTG CTT CCT GGA AGA G-3′ and -reverse: 5′-CAG ATG TCC CAG CTG GAA TAT AAT TAC TCT TTC ATA CTC TTG TAC AAC C-3′. The PCR products were subcloned into pCR-BluntII-TOPO and verified by DNA sequencing. These vectors were digested with restriction enzymes and used as templates for the synthesis of capped RNA. Capped RNA was synthesized using the mMESSAGE mMACHINE kit (Life Technologies). Synthesized capped RNA (~0.64 pg for human wild-type and mutated NEPHRIN and ~32 pg for human wild-type and mutated PODOCIN) was injected into one-cell stage embryos along with morpholinos (0.15 mM nephrinMOex25 or 0.25 mM podocinMOex3).

Throughout zebrafish pronephric development [34 h post-fertilization (hpf) to 4 dpf], nephrin and podocin mRNA expression was detected in the glomerular primordia and compared with the glomerulus (Fig. 1A–H). The expression patterns of nephrin and podocin markedly resembled that for Wilms’ tumor 1a, which is predominantly expressed in the podocytes within the pronephric glomerulus (1).

Antibodies specific to Nephrin and to Podocin were developed to determine their expression pattern and localization. The specificity of the anti-Nephrin antibody was previously described (3). To examine the specificity of the anti-Podocin antibody, the possibility of a reaction with Flag-tagged zebrafish Podocin expressed in 293T cells was investigated. Immunoblot analysis showed that the anti-Podocin antibody was capable of detecting an ~55 kDa protein, corresponding to the Flag-tagged Podocin (Fig. 1I). These anti-Nephrin and anti-Podocin antibodies could also detect ~240 and ~40 kDa protein bands corresponding to endogenous Nephrin and Podocin, respectively (Fig. 1J).

With these antibodies, Nephrin and Podocin were immunohistochemically detected in 4 dpf larvae as two continuous tortuous lines, which were likely to be situated along the glomerular basement membrane (Fig. 1K and L). A similar tortuous linear pattern for Nephrin localization was also observed for the rodent metanephric glomerulus (36,37).

To determine the function of Nephrin and Podocin in the development of the zebrafish pronephric glomerulus, splice blocking MOs were designed for Nephrin and Podocin to target the splice acceptor sites of exon 25 for nephrin (nephrinMOex25) and exon 3 for podocin (podocinMOex3), respectively (Fig. 2A and B).

Injection of nephrinMOex25 resulted in mis-spliced mRNA as detected by an altered RT-PCR product (Fig. 3A). This altered product was ~50 bp smaller than the wild-type. Sequencing of the altered RT-PCR product revealed a deletion of exon 25 that resulted in a frame shift and non-sense translation of the subsequent exons, resulting in an almost complete loss of the intracellular domain of the Nephrin protein in nephrinMOex25 morphants (Fig. 2A). The efficacy for nephrinMOex25 was consistently recognized from 1 to 4 dpf (Fig. 3A) and the protein expression of Nephrin was reduced at 4 dpf (Fig. 3C).

Administration of podocinMOex3 also resulted in mis-spliced mRNA as detected by an altered RT-PCR product that was ~150 bp smaller than the wild-type amplicon (Fig. 3B). Sequencing of the altered RT-PCR product revealed an in-frame deletion of exons 3 and 4 (Fig. 2B). The truncated RT-PCR product was recognized from 1 to 4 dpf, although a significant expression of intact Podocin mRNA was also detected (Fig. 3B). The reduced expression of the Podocin protein was observed in the 3 dpf morphants, however, the expression was recovered at the same level as in the control at 4 dpf (Fig. 3D).

NephrinMOex25 and PodocinMOex3 morphants exhibited pericardial and yolk edema from 3 to 4 dpf (Fig. 2C–E). In addition to edema, the majority of the morphants showed body-axis curvature (Fig. 2C–E). In wild-type zebrafish, a pair of pronephric glomerular primordia merged with glomerular capillaries and mesangium at the midline to form a single glomerulus (Fig. 2F) (1). The glomerulus was smaller in size in the morphants compared with that in the wild-type larvae and contained poorly developed glomerular capillaries and mesangium (Fig. 2G and H).

In 4 dpf control larvae, regular foot processes with SD covered a large area of the urinary surface of the glomerular basement membrane (Fig. 2I). At the same stage, nephrinMOex25 morphants were associated with a great reduction in the area covered with regular foot processes. Instead of the foot processes, irregularly shaped processes covered the larger area of its urinary surface and the SD was not observed between these irregularly shaped processes (Fig. 2J). In 4 dpf podocinMOex3 morphants, the foot processes with SD covered a large area of the glomerular wall, however, the processes varied in shape and size (Fig. 2K).

To investigate whether administration of human intact mRNA rescues the morphant phenotypes, splice blocking morpholino and human wild-type mRNA were simultaneously injected into one-cell stage embryos. Human NEPHRIN and PODOCIN mRNA were detected in the 4 dpf-injected larvae by RT-PCR (Fig. 4B), indicating that the injected mRNA remained until at least four days following injection. nephrinMOex25 and podocinMOex3 morpholino-injected embryos exhibited pericardial edema, yolk edema and body-axis curvature. Moreover, the glomerulus was smaller in size compared with the wild-type larvae and contained poorly developed glomerular capillaries and mesangium (Fig. 2F–H). All these features may be rescued by ~89% by co-injection of human Nephrin and Podocin mRNAs (Fig. 4D). In addition, rescued morphants exhibited a straight body axis (Fig. 4E and F) and a well-developed pronephric glomerulus at 4 dpf (Fig. 4G and H), although slight pericardial edema was observed in the larvae injected with human PODOCIN mRNA and podocinMOex3 (Fig. 4F). These data therefore indicate that the functions of human and zebrafish Nephrin and Podocin are interchangeable.

A number of types of disease-causing mutations in Nephrin and Podocin proteins have been reported in human patients, including R1109X in Nephrin (25) and R138Q in Podocin (26). NEPHRIN-R1109X is a non-sense mutation leading to a large deletion of the intracellular domain of Nephrin and it is one of the two major disease-causing mutations in Finnish-type congenital nephrotic syndrome (24) (Fig. 4A). PODOCIN-R138Q is a missense mutation resulting in the substitution of arginine at position 138 for the uncharged amino acid glutamine and is also one of the most common disease-causing mutations in steroid-resistant nephrotic syndrome (23) (Fig. 4A).

To determine the effect of mutated mRNA on the rescue of the morphant phenotypes, nephrinMOex25 and podocinMOex3 were injected together with mutated mRNA encoding human NEPHRIN-R1109X and PODOCIN-R138Q, respectively, into one-cell stage embryos. The persistence of the two mutated mRNAs was detected in 4 dpf larvae by RT-PCR (Fig. 4B and C), but the ratio of rescued larvae was significantly lower for the mutated mRNAs compared with the wild-type mRNA (Fig. 4D). Non-rescued larvae exhibited pericardial edema, body-axis curvature and hypoplastic glomerulus, as observed in the larvae injected with the morpholino only (Fig. 4G, H, K and L). The formation of regular foot processes was affected in the larvae injected with NEPHRIN-R1109X and nephrinMOex25, as with nephrinMOex25 alone (Fig. 4M). Notably, the larvae injected with PODOCIN-R138Q and podocinMOex3 did not exhibit any regular foot processes with SD (Fig. 4N). However, larvae injected with podocinMOex3 formed foot processes to some extent (Fig. 2K), indicating that PODOCIN-R138Q exacerbated the morphant phenotype observed with podcinMOex3 alone. The phenotype was scored by morphology and altered glomerular barrier and, in contrast to human Nephrin and Podocin mRNAs, co-injection of and nephrinMOex25 and mutant NEPHRIN-R1109X or podocinMOex3 and PODOCIN-R138Q only rescued 39 and 8.1%, respectively.

To examine whether the protein properties of a mutated PODOCIN were biochemically altered, the abilities of Flag-tagged wild-type and mutated (R150Q) zebrafish full-length Podocin to bind to the Myc-tagged C-terminal intracellular domain of zebrafish Nephrin were compared. Notably, the R150Q zebrafish Podocin mutation corresponded to the disease-causing R138Q mutation in human Podocin. Consistent with previous studies for mammalian Nephrin and Podocin (17,38), binding between wild-type zebrafish Podocin and the intracellular region of Nephrin (Fig. 5) was observed. Binding between the mutated Podocin-R150Q and Nephrin (Fig. 5) was also detected. However, mutated Podocin interacted significantly more with Nephrin despite the lower expression levels of mutated Podocin compared with wild-type Podocin (Fig. 5). The latter may suggest that the mutation of Podocin makes it more prone to protein degradation.