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From the Departments of Medical Biochemistry and Biophysics* and Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; and the Institute of Biotechnology, University of Helsinki, Helsinki, Finland
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Abstract |
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Congenital nephrotic syndrome of the Finnish type (CNF or NPHS1, MIM 256300) is a rare, but one of the most severe forms of genetic kidney disorders. Development of the clinical syndrome in CNF is closely correlated with major structural and morphological changes in podocyte foot processes and absence of the SD.3,9 The nephrin gene (NPHS1), mutated in CNF, encodes a type-1 transmembrane glycoprotein belonging to the immunoglobulin superfamily (IgSF).4,10 The mature polypeptide consists of 1241 amino acid residues with a calculated molecular mass of 132,532. However, because of posttranslational glycosylation, the mature polypeptide migrates as a 180- to 200-kd protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).11 Nephrin consists of eight extracellular immunoglobulin-like domains followed by a fibronectin type III domain, a short transmembrane part, and a cytoplasmic C-terminal domain. In the kidney, nephrin expression is restricted to the glomerular podocytes, and by immunoelectron microscopy nephrin has been localized to the SD area.5-7 The central role of nephrin in the formation and function of the SD has been demonstrated in CNF patients12 and in nephrin-deficient mice,13 as both cases lead to a lack of the SD and massive proteinuria. Nephrotic syndrome has also been reported in mice lacking CD2AP, an adapter actin-binding protein,14 and in patients with mutations in the NPHS2 gene-encoding podocin,15 a new member of the stomatin family with hairpin-like membrane-integrated protein topology. Also, inactivation of the novel SD proteins NEPH1 and FAT results in the development of congenital nephrotic syndrome and neonatal death, respectively.16,17
It has recently been demonstrated that both CD2AP and podocin interact directly with the intracellular C-terminal part of nephrin and, together with nephrin, can be isolated as a raft-associated component of the podocyte SD.18-20 Based on its specific location in the SD and the fact that nephrin is a member of the IgSF, a hypothetical model has been proposed that nephrin molecules emerging from adjacent foot processes interact with each other in a homophilic manner and form the backbone of the SD structure;8 however, evidence for such interactions has, thus far, not been presented.
In the present study, we have shown by using a surface plasmon resonance technology that the extracellular part of soluble nephrin exhibits homophilic interactions. We also demonstrated that cells transfected with full-length nephrin cDNA aggregate to form cell-cell junctions with a spacing of similar width as that of native SD. Furthermore, the cellular aggregates can be dissociated with antibodies against the extracellular domain of nephrin. The results suggest that nephrin molecules could also in vivo, contribute to the SD filter structure through homophilic interactions.
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Materials and Methods |
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The construction of the full-length cDNA clone encoding the human nephrin has been reported earlier.21 For expression of Fc-fused nephrin chimera (NphFc), a cDNA clone encoding the entire extracellular part of human nephrin was cloned into a modified mammalian expression vector pCR-3 (Invitrogen, Carlsbad, CA) with a SalI/NotI cDNA cassette encoding the Fc part (the hinge, CH2, and CH3 domains) of the human IgG1 (the pCR3Fc vector was kindly provided by Dr. Pascal Schneider, Institute of Biochemistry, University of Lausanne, Switzerland). Using polymerase chain reaction (PCR) and 3'-end primer (FNSalI 5'-AGTCAGTCGACCCCCGAGGGTCCT-3'), complementary to the sequence encoding the last four amino acid residues (GPSG) in the fibronectin type III domain of nephrin, followed by a SalI site (underlined), and an upstream primer, UP1 5'-AAGGTTGTGAGTCTGACCCCAC-3', was used to amplify a short 3'-end fragment. The 3'-end fragment was cleaved with AccI and SalI to generate the AccI/SalI 3'-end fragment. A large EcoRI/AccI fragment corresponding to the middle part of nephrin cDNA together with the HindIII/EcoR1 5'-end fragment (see the full-length construct) and the AccI/SalI 3'-end fragment were ligated into the HindIII/SalI-cleaved pCR3Fc vector to generate a construct (pCR3NphFc) encoding the entire extracellular nephrin followed by the Fc part of human IgG1. Finally, the ligated fragments were checked by sequencing.
The cDNA clone encoding the soluble histidine-tagged nephrin (NphHis) was generated from the pCR3NphFc construct. Two synthetic oligonucleotides, SalIHis 5'-T CGACCATCATCACCATCACCATTGA-3' with cohesive end SalI site (underlined) and a stop codon (italics and underlined), and NotIHis 5'-GGCCTCAATGGTGATGGTGATGATGG-3' with a cohesive end NotI site (underlined), were used to construct a SalI/6His/NotI-linker fragment with complementary overlapping 3'-ends encoding six histidine residues and 5'-cohesive ends corresponding to SalI and NotI sites, respectively. After annealing, the linker was inserted into the pCR3NphFc construct cleaved with SalI and NotI. The final construct (pCR3NphHis) encoded the entire extracellular portion of nephrin followed by a 6-His tag and a stop codon.
Cell Lines, Culturing Conditions, and Generation of Stable Transfected Cell Lines
All cell culture media, supplements (GibcoBRL, Grand Island, NY) and plates/flasks (Nunc, Naperville, IL) were supplied by Life Technologies Inc. The conditionally immortalized mouse podocyte cell line (MCP-5, here termed IMP) was kindly provided by Dr. Peter Mundel (Albert Einstein College of Medicine, Bronx, NY). The cells were maintained and propagated at 33°C in RPMI 1640 medium containing 10 U/ml interferon-
(Sigma, St. Louis, MO) and 10% fetal bovine serum as described.22
For differentiation the cells were cultured at 37°C in medium without the interferon-. When needed, cells were cultured on type I collagen (collagen-A; Biochrom KG, Berlin, Germany) for differentiation. The human embryonic kidney cell line QBI293A (Qbiogene, Carlsbad, CA), derived from the HEK293 cell line, was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Transfections of both cell lines were performed by lipofectin (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer’s protocol. Stable clones were selected for 2 weeks in medium containing 1 mg/ml of geneticin and were then cloned at that stage.
Expression and Purification of Recombinant Proteins
Stable HEK293 cell clones expressing the chimeric NphFc and NphHis recombinant proteins were cultured in triple flasks (Nunc). The cell clone expressing chimeric NphFc was cultured in Dulbecco’s modified Eagle’s medium containing fetal bovine serum to reduce co-purification of serum IgGs with the recombinant nephrin. After 3 days of confluency, the medium was harvested and phenylmethyl sulfonyl fluoride was added to a concentration of 2 mmol/L before filtration through a 0.22-µm membrane. One liter of the harvested medium was loaded circulating onto a 1-ml HiTrap protein A-Sepharose column (Amersham Biosciences, Uppsala, Sweden) equilibrated with phosphate-buffered saline (PBS) in a cold room overnight. The column was connected to a fast performance liquid chromatography (FPLC) system (
KTApurifier system; Amersham Biosciences), washed with 10 ml of PBS and the proteins were eluted with 50 mmol/L of citrate-NaOH, pH 3.0. The eluent was neutralized with 1 mol/L Tris-HCl buffer, pH 8.0, and dialyzed against a 20-mmol/L Tris-HCl buffer, pH 7.5. Proteins were then loaded onto an ion-exchange chromatography column (Mono Q HR 5/5, Amersham Biosciences). The NphFc recombinant protein was eluted from the column with a linear 0- to 1-mol/L NaCl-gradient (20-column volume) in 20 mmol/L of Tris-HCl, pH 7.5, and desalted using Sephadex G-50 NICK columns (Amersham Biosciences) equilibrated with 20 mmol/L of Tris-HCl, pH 7.5.
For purification of His-tagged NphHis protein, one liter of the harvested medium was loaded onto a DEAE-Sepharose column using an KTApurifier system and proteins were eluted with 20 mmol/L of Tris-HCl and 1 mol/L of NaCl, pH 7.5. The eluate was loaded onto a Ni-NTA agarose column (Qiagen, Valencia, CA) equilibrated with the washing buffer (20 mmol/L Tris-HCl, 0.5 mol/L NaCl, 10% glycerol, 0.5% Tween 20, pH 8.0). The column was washed with at least 30-column volumes before elution with a stepwise gradient of imidazole 0 to 200 mmol/L in the washing buffer. Collected fractions were analyzed by SDS-PAGE and fractions with the highest amount of NphHis (eluted at 50 to 100 mmol/L of imidazole) were pooled, dialyzed against 20 mmol/L Tris-HCl, pH 7.5, and loaded onto a Mono Q HR 5/5 column. Proteins were eluted with a linear 0- to 1-mol/L NaCl gradient (40-column volume) in 20 mmol/L of Tris-HCl buffer, pH 7.5. The eluate was desalted on Sephadex G-50 NICK columns equilibrated with 20 mmol/L of Tris-HCl, pH 7.5, aliquoted, and frozen at -20°C. The purity of the recombinant proteins was determined by SDS-PAGE and silver staining.
Sample Preparation and Western Blot Analysis
Cell lysates were prepared by washing the cells twice before addition of hot SDS sample buffer (63 mmol/L Tris-HCl, 2% SDS, 10% glycerol, 0.1 mol/L dithiothreitol, pH 6.8). The cell lysates were collected with a rubber scraper and passed through 26-gauge needles and then transferred into Eppendorf tubes, boiled, and centrifuged for 10 minutes before loading. Human glomeruli were isolated by differential sieving through 400-µm and 200-µm mesh brass sieves and washed extensively with ice-cold PBS including protease inhibitors. Isolated glomeruli were collected by centrifugation at 10,000 x g and resuspended in PBS at a concentration of 20,000 glomeruli per ml. Fractions of the isolated glomeruli were lysed in equal volumes of hot 2x SDS sample buffer and centrifuged to remove unsolubilized material. To remove the carbohydrate moieties on nephrin, a N-glycosidase F deglycosylation kit was used according to the manufacturer protocol (catalog no. 1836552, Roche). All samples were subjected to SDS-PAGE and the proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated with affinity-purified pAb1 antibodies (0.2 µg/ml), washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies. The immunoreactivity was detected by a chemiluminescent kit (Life Science Products) according to the manufacturer’s instructions. The prestained molecular standard used in the SDS-PAGE was from Bio-Rad (catalog number 161-0372; Precision Protein Standards). Protein measurements were performed using a protein assay according to the manufacturer’s protocol (Bio-Rad, Hercules, CA).
Surface Plasmon Resonance
Protein interaction analysis based on surface plasmon resonance technology was performed using a Biacore 2000 optical biosensor (Biacore AB, Uppsala, Sweden). Proteins were immobilized on carboxymethylated dextran surfaces of research-grade (CM5 sensor chips) using amine-coupling chemistry. Flow cells were activated with a 1:1 mixture of 0.1 mol/L N-hydroxysuccinimide and 0.4 mol/L 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide at a flow rate of 20 µl/min at 25°C. All proteins were chromatographed on a Sephadex G-25 column equilibrated in running buffer, HBS-P [10 mmol/L HEPES, 0.15 mol/L NaCl, 0.005% surfactant P20 (Biacore), pH 7.4], containing 3.4 mmol/L ethylenediaminetetraacetic acid (EDTA) before use. The ligands, NphHis, NphFc, and human IgG1 (DAKO, Glostrup, Denmark), were diluted in immobilization buffer (10 mmol/L malate buffer, pH 6.0) to a final concentration of 10 µg/ml and immobilized in equal molar ratios, resulting in immobilization densities of 5000:5000:2500 response units for NphHis:NphFc:IgG1, respectively. After immobilization, the surfaces were blocked with 1 mol/L of ethanolamine, pH 8.0, followed by extensive wash with regeneration buffer (HBS-P containing 3.4 mmol/L EDTA and 1 mol/L NaCl). Recorded interactions were performed at 25°C with proteins diluted in HBS-P containing either 3.4 mmol/L EDTA or 1 mmol/L Ca2+. Binding of analytes to the immobilized ligands were measured in resonance units, response unit (1000 response units = 1 ng/mm2 bound protein). Binding data were analyzed as previously described,23 using an algorithm for calculation of association and dissociation rate constants that corrected for mass transport effects.
Immunofluorescence Staining and Antibody Treatment
Rabbit polyclonal antibodies (pAb1) against the two first Ig-like domains (amino acids 22 to 240) of human nephrin have been described.5,12 For immunofluorescence staining, cells were cultured on glass coverslips (coated with type I collagen), then washed with PBS and fixed with 2% formaldehyde and 0.1% glutaraldehyde in PBS for 30 minutes. Cells were then washed twice with PBS blocked with 2% bovine serum albumin and 1% casein hydrolysate in PBS for 1 hour at room temperature. The cells were incubated with affinity-purified rabbit anti-nephrin antibodies pAb1 (10 µg/ml) for 1 hour at room temperature. After three washes, the cells were treated for 30 minutes with a secondary antibody (fluorescein isothiocyanate-conjugated swine anti-rabbit IgG, DAKO). Actin cytoskeleton was stained with rhodamine-phalloidin. The cells were finally washed three times, mounted, and examined by a DMRB Leica microscope and photographed with a digital camera (Hamamatsu C4742-95, Bridgewater, NJ).
For experiments with antibody treatment, wild-type HEK293 and IMP cells and cells transfected with full-length nephrin were cultured on glass coverslips coated with type I collagen for 4 to 7 days. Thereafter, the medium was replaced with prewarmed fresh medium containing 1% fetal calf serum and affinity-purified pAb1 in a range of 0 to 40 µg/ml. An equal amount of preimmune rabbit IgG was used in a parallel experiment as negative control. Cells were incubated at 37°C for 1 hour and then either photographed immediately using a phase contrast microscope or fixed and stained for nephrin as described above.
Electron Microscopy (EM) and Electron Tomography
For immuno-EM, nephrin-expressing HEK293 cells were fixed with 3.5% paraformaldehyde alone or with different concentrations (0.01 to 0.1%) of glutaraldehyde, processed and immunostained as described.5,24 In short, LR-White thin sections on gold-nickel grids were incubated with primary antibodies in blocking solution and washed. After incubation with 5- or 10-nm gold-conjugated secondary antibodies, the sections were poststained in 1% uranyl acetate. Controls included use of nonimmune rabbit IgG as primary antibody. A Jeol 1200 EX electron microscope was used for examining the sections.
For electron tomography, the immunoresin sections were prepared as for EM but immuno-marked only with 5-nm-gold and the grids were treated with 10-nm-gold protein A (Amersham Biosciences) for alignment purposes in image reconstruction. The electron tomography was performed essentially as described earlier24,25 using a Philips CEM 200 FEG transmission electron microscopy. Automatic low-dose tilt series were recorded with a slow-scan camera (2048 x 2048 CCD chip, pixel size 14 µm; TVIPS GmbH, Gauting, Germany) and using the EMMENU software. Images were recorded at 1 or 2° tilt intervals (-65 to + 60°, 26,700x, final pixel size of 5.24 Å). The total dose on the immunoresin sections was below 30 e-/Å.2 Geometrical image alignment was performed using gold markers (error usually under 1 pixel). Image refinement was done using the COMET technique.24 The reconstructions were visualized by isodensity contouring as surface rendered or wire-frame representation with the program XTV25 or by volume rendering with the program BOB (Ken Chin-Purcell, Minnesota Supercomputer Center Inc.).
RNA Isolation, Primer Design, and Reverse Transcriptase (RT)-PCR
Total RNA was isolated from wild-type and nephrin-transfected HEK293 cells using an RNeasy kit according to the manufacturer’s protocol (Qiagen). Forward and reverse primers were designed to examine the expression levels of human genes encoding the SD components, CD2AP, FAT1, NEPH1, nephrin, podocin, and P-cadherin in the cultured cells. For expression levels of human CD2AP gene (GI:11321633), forward primer (5'-ggcatgggaatgtagcaagt-3', in exon 3) and reverse primer (5'-tctccaaatccaattcctcg-3', in exon 6) were designed to amplify a 433-bp PCR-fragment. For the gene encoding human FAT1 (GI:1107686), forward primer (5'-ggacccgctacggctttctt-3', in exon 26) and reverse primer (5'-gctttcccgggcactgtatg-3', in exon 27) were designed to amplify a 420-bp fragment corresponding to the cytoplasmic part of the molecule. Forward primer (5'-gagaggaccaactcaggcag-3', in exon 10) and reverse primer (5'-gctctcggttcactgtctt-3', in exon 12) to human NEPH1 (GI:14572520) were chosen to amplify a 310-bp PCR fragment. For the human nephrin gene, NPHS1 (GI:4758821), forward primer (5'-gtgaacgagggctcccagc-3', in exon 5) and reverse primer (5'-gcagtccatccatgactgtc-3', covering the splice junction between exon 9 and 10) were designed to amplify a PCR product of 620 bp. To detect expression of human NPHS2 gene (GI:25137568)-encoding podocin, the forward primer (5'-ggttgaccttcgtctccaaa-3', exon 4) and reverse primer (5'-gaatctcagctgccatcctc-3', exon 8) were designed to amplify a 443-bp PCR fragment. For human P-cadherin gene (GI:14589890), forward primer (5'-ggcacgggaacccttctgcta-3', in exon 13) and reverse primer (5'-cgccatagtagaagacgttgt-3', in exon 15) were designed to amplify a 520-bp PCR fragment. RT-PCR was applied using a one-step RT-PCR kit, according to the manufacturer’s protocol (Titanium One-Step RT-PCR kit; Clontech, Palo Alto, CA). For each reaction, 600 ng of total RNA was used. To synthesize the first cDNA strand, the thermocycler was set to 50°C for 1 hour followed by a denaturizing step at 94°C for 5 minutes. To amplify gene-specific expression, 35 cycles of 94°C for 5 minutes, 58°C for 30 seconds, and 68°C for 1 minute were repeated followed by an additional extension cycle at 68°C for 2 minutes. The PCR reactions were analyzed by gel electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and photographed under UV light.
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Results |
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Three recombinant nephrin variants were produced for studies on the cellular and protein interactions for nephrin (Figure 1A)
. Full-length human nephrin expressed in HEK293 cells was shown to be 
185 kd by Western blot analysis, using a rabbit polyclonal antibody5
(pAb1) raised against the first two Ig domains of human nephrin (Figure 1B)
. The same size was obtained for recombinant nephrin expressed in IMP cells (not shown). In contrast, nephrin isolated from normal human glomeruli migrated with an apparent weight of 200 kd. The observed size difference is because of different degrees of glycosylation, as enzymatic removal of the N-linked carbohydrates with N-glycosidase F resulted in proteins with similar migration in SDS-PAGE (Figure 1C)
. When analyzed by gradient SDS-PAGE, the full-length nephrin expressed in IMP-NPH1 and HEK293 cells appeared as a closely migrating double immunoband of 185 kd (Figure 1C)
. However, after removal of all N-linked carbohydrates, a single band was detected (Figure 1C)
, indicating that the minor difference in migration is because of glycosylation. As expected, protein samples derived from glomerular extracts of a CNF patient with a Fin-major mutation4
and wild-type HEK293 cells were devoid of nephrin (Figure 1B)
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. However, without reduction, a minor fraction of NphHis also migrated as protein complexes with high molecular weights (Figure 1B)
, indicating that intermolecular disulfide bridges might be formed in vitro by the free cysteine residues present in the extracellular part. As predicted, the NphFc chimera migrated without reduction with a size corresponding to a dimer, while it migrated with the size of a monomer after reduction (Figure 1B)
. Soluble Nephrin Molecules Exhibit Homophilic Interactions
To examine the molecular interactions of nephrin molecules in solution, we used a surface plasmon resonance biosensor, which allows for monitoring of association and dissociation between biomolecules in real time, provided that one of the binding partners is covalently immobilized or captured onto a sensor surface.26
Both NphFc and NphHis were covalently immobilized on separate lanes of a CM5 sensor chip using amine-coupling chemistry (see Materials and Methods). As a background control, human IgG1, which shares an identical Fc portion with the NphFc chimera, was immobilized on a separate lane. The association and dissociation of NphHis to a surface of NphHis is shown in Figure 2, A and C
. Increased binding was detected with higher concentrations of NphHis in the fluid phase. A similar pattern of increasing interaction was seen when NphHis was flushed over immobilized NphFc (data not shown). Human IgG1 did not show any binding to either immobilized NphHis or immobilized IgG1 (Figure 2B)
, demonstrating that the observed binding of NphHis to immobilized nephrin represents a specific homophilic binding between nephrin molecules. A small, nonspecific binding was observed when NphHis was run over immobilized IgG1 (Figure 2A)
; subtraction of this binding from the nephrin-nephrin binding curve gave smooth association-dissociation binding curves (Figure 2A)
. A series of such binding curves, obtained by running different concentrations of NphHis (Figure 2C)
, were used in a global curve-fitting analysis to calculate association and dissociation rate constants for the nephrin-nephrin binding interaction. This resulted in an association rate constant ka = 2.2 x 105 mol/L-1 s-1, a dissociation rate constant kd = 0.0011 seconds-1, and an equilibrium binding constant KD = 5.0 x 10-9 mol/L, indicating that the primary nephrin-nephrin binding is of high affinity. We further studied the nature of the nephrin-nephrin homophilic binding in the presence of Ca2+. When Ca2+ at the physiological concentration of 1 mmol/L was included in the buffer, a more than twofold increase of the binding was observed, indicating that the homophilic binding of nephrin is promoted by Ca2+ ions (Figure 2D)
. Because of the complex nature of the nephrin-nephrin binding in the presence of Ca2+, we did not attempt to calculate the association-dissociation rate constants under these conditions.
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Wild-type HEK cells and stable nephrin-expressing transfectants were used to study the effects of nephrin expression on cellular behavior. Wild-type cells not expressing nephrin did not form clusters (Figure 3A)
. In phase contrast microscopy, cell clones with low levels of expression did not significantly differ from parental nontransfected cells with respect to morphology (not shown). In contrast, cell clones with high nephrin expression formed clusters of cells with aggregated morphology (Figure 3B)
. Higher magnification of such aggregated colonies showed localization of nephrin at the cell membrane, especially at the intercellular plasma membranes between nephrin-expressing cells (Figure 3C)
. Immortalized mouse podocytes transfected with a human nephrin construct also revealed an aggregated cell morphology (Figure 3E)
, while the nontransfected counterparts did not cluster (Figure 3D)
.
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. Electron tomography of such areas (Figure 4B)
provided three-dimensional visualization possibilities of the contact sites (Figure 4, C and D)
. In face view, a layer of extracellular strands was typically seen at the site of nephrin label, usually consisting of several immunogold particles (Figure 4C)
. Irregular winding pores of varying size (5 to 10 nm) penetrated the layer. Visualized in cross-section (Figure 4D)
, the layer characteristically formed a horizontal 40- to 50-nm-wide and 20- to 30-nm-thick shelf between the cell membranes. Outside the 40- to 50-nm-wide contact sites, solitary gold particles were seen on the free cell surfaces (Figure 4B)
. Gold label was not found along areas where cells formed usual 10- to 20-nm contact faces lacking visible extracellular material.
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To examine whether nephrin was functionally involved in cell-cell interaction and aggregation, we challenged transfected cells with medium containing increasing concentrations of affinity-purified rabbit IgG directed against the two first extracellular Ig-like motifs of human nephrin. The cells were cultured at 37°C on glass coverslips coated with type I collagen for 4 to 7 days. The cell culture medium was then replaced by prewarmed medium containing different concentrations of pAb1 and incubated for 1 hour. Equal concentrations of preimmune rabbit IgG were also used in a parallel experiment as negative control. The cells were then fixed and studied in immunofluorescence microscopy. Aggregated nephrin-expressing HEK293 cells (Figure 3C)
retracted from each other after addition of the antibodies (data not shown). This phenomenon was also evident in comparison of wild-type and nephrin-expressing IMP cells (Figure 5)
. Addition of preimmune serum to the nephrin-expressing IMP cells did not affect the cell clustering, while addition of the anti-nephrin antibodies resulted in dissociation of the aggregated cells. This effect was also shown to be dependent on the antibody concentration (Figure 5)
, so that with increasing antibody concentration, the transfected IMP cells retracted from each other completely and rounded up.
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In addition to nephrin, the podocyte SD region contains podocin, CD2AP, P-cadherin, FAT, and NEPH1, that are essential for maintaining a functional SD. To examine if any of those are expressed in the HEK293 cells used in this study, we examined the expression of those by RT-PCR. Gene-specific primers were designed from two exon sequences flanked by at least one intron to ensure that each amplified PCR fragment is corresponding to the size of targeted mRNA and not contaminating genomic DNA. As shown in Figure 6
, the wild-type HEK293 cells exhibit expression of CD2AP, FAT1, NEPH1, and P-cadherin, but not nephrin or podocin. In the nephrin-transfected HEK293 cells no alterations were observed in the expression of the other genes examined.
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Discussion |
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The present surface plasmon resonance biosensor studies demonstrated that soluble nephrin molecules can participate in high-affinity homophilic interactions. The fact that this interaction is increased by Ca2+ indicates that nephrin is a Ca2+-binding protein and that the binding of Ca2+ promotes and regulates the homophilic binding properties of nephrin. The binding scenario is likely to be more complex in vivo, where other molecular components of the SD may also interact with nephrin. Beside nephrin, several other proteins have been reported to be located in the SD region.28-30 Such molecules include ZO-1, CD2AP, podocin, P-cadherin, and FAT, of which the two latter belong to the cadherin superfamily. ZO-1 has long been known for its location in the cytoplasmic vicinity of the SD, and it is most likely involved in signaling events during the modification of cell junctions between podocyte foot processes.31 CD2AP and podocin have been shown to be intracellular binding partners for nephrin.18-20,32 Recently, NEPH1, a novel homologue of nephrin, has been reported to be essential for the glomerular filtration barrier, as interruption of its gene in mice leads to foot process effacement and nephrotic syndrome.33 Like nephrin, NEPH1 is a type-1 transmembrane protein with somewhat shorter extracellular part (five Ig-like domains) and an intracellular part that interacts with podocin.17 While this article was being revised, two separate groups reported on evidence for homophilic and heterophilic interactions of nephrin and NEPH1,34,35 thus supporting the results of the present study.
The results of this study clearly demonstrated that nephrin molecules can interact with each other through their extracellular part and that this molecular interaction is not dependent on but rather promoted by Ca2+ ions. The reversibility of this interaction also indicates that the proposed disulfide bonds between the free cysteine residues of the extracellular part of nephrin are not involved in this molecular interaction.
The cell culture results provided further support for nephrin-nephrin interactions, as transfected cell lines with high expression of nephrin formed aggregated cell colonies. The observation that transfected cells with low expression of nephrin did not show aggregated cell morphology indicates that high local levels of nephrin at the plasma membrane are needed to promote tight cell-cell interaction. Importantly, it was shown that wild-type HEK293 cells express key components of the podocyte SD, ie, CD2AP, P-cadherin, FAT, and NEPH1. Yet, clustering and formation of cell-cell junctions first occurred on transfection and expression of nephrin. This implies that nephrin is crucial for junction formation. Considering that isolated nephrin molecules self-associate, such junction formation is likely to reflect homophilic binding between nephrin molecules on adjacent cells, although heterophilic binding with endogenous NEPH1 cannot be excluded. Our electron microscopic analysis revealed that the spacing between cells at the nephrin-containing contact sites had similar dimensions as podocyte slits in vivo.2 This further suggests that nephrin molecules determine the width of the SD in vivo. Electron tomography, is a relatively new imaging method that enables detailed three-dimensional structural analysis of individual cellular structures and macromolecular complexes in situ.36,37 When applied here to the nephrin-containing cell-cell junctions between transfected cells, this method revealed that these contact sites include an anti-nephrin-labeled layer of strands forming a porous structure similar to that seen in SD in vivo by conventional EM2,5-7 and by electron tomography (our unpublished results).
The results of this work also provided additional support for a direct role of nephrin in cell-cell interactions, as the cell-cell junctions observed in nephrin-expressing cells could be dissociated by anti-nephrin antibodies. Such outside-in signaling could be mediated through tyrosine phosphorylation, resulting in a rapid disruption of intercellular junctions. In fact, such nephrin-mediated outside-in signaling may have a major role in some acquired kidney diseases, especially in patients having anti-nephrin antibodies in their blood circulation,38 or in experimental nephrosis in rat models.31,39,40 Hence, the localization of nephrin at the SD is crucial for maintaining proper foot process morphology and both width and structure of the SD. The intimate relationship between the actin cytoskeleton, nephrin, CD2AP, and podocin that has recently been shown,18-20 suggests that any interference with nephrin adhesion could lead to reorganization of the cortical F-actin and withdrawal of foot processes with consequent proteinuria. The present results indicate that nephrin-expressing cells can provide a useful in vitro model for studies on the podocyte SD.
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Acknowledgements |
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Footnotes |
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Supported by the Swedish Research Council (projects 05200 and 11545), the Swedish Cancer Foundation (project 4720), Polysackaridforskning AB, the Novo Nordisk Foundation, the Sigrid Jusélius Foundation, the National Institutes of Health (grant DK-54724), and the European Union (grants BIO4-CT97-2364 and BIO4-CT96-0099).
Present address for J. K.: Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University Medical Center, S-3223 Medical Center North, 1161 21st Avenue South, Nashville, TN 37232-2372.
Accepted for publication August 15, 2003.
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S. E. Quaggin and J. A. Kreidberg Development of the renal glomerulus: good neighbors and good fences Development, February 15, 2008; 135(4): 609 - 620. [Abstract] [Full Text] [PDF]
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 P. Garg, R. Verma, D. Nihalani, D. B. Johnstone, and L. B. Holzman Neph1 Cooperates with Nephrin To Transduce a Signal That Induces Actin Polymerization Mol. Cell. Biol., December 15, 2007; 27(24): 8698 - 8712. [Abstract] [Full Text] [PDF]
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H. Holthofer Molecular architecture of the glomerular slit diaphragm: lessons learnt for a better understanding of disease pathogenesis Nephrol. Dial. Transplant., August 1, 2007; 22(8): 2124 - 2128. [Full Text] [PDF]
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 L. Kaufman, G. Yang, K. Hayashi, J. R. Ashby, L. Huang, M. J. Ross, M. E. Klotman, and P. E. Klotman The homophilic adhesion molecule sidekick-1 contributes to augmented podocyte aggregation in HIV-associated nephropathy FASEB J, May 1, 2007; 21(7): 1367 - 1375. [Abstract] [Full Text] [PDF]
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 K.-W. Lai, C.-L. Wei, L.-K. Tan, P.-H. Tan, G. S.C. Chiang, C. G.L. Lee, S. C. Jordan, and H.-K. Yap Overexpression of Interleukin-13 Induces Minimal-Change-Like Nephropathy in Rats J. Am. Soc. Nephrol., May 1, 2007; 18(5): 1476 - 1485. [Abstract] [Full Text] [PDF]
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 I. Quack, L. C. Rump, P. Gerke, I. Walther, T. Vinke, O. Vonend, T. Grunwald, and L. Sellin beta-Arrestin2 mediates nephrin endocytosis and impairs slit diaphragm integrity PNAS, September 19, 2006; 103(38): 14110 - 14115. [Abstract] [Full Text] [PDF]
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 K. Tryggvason, J. Patrakka, and J. Wartiovaara Hereditary proteinuria syndromes and mechanisms of proteinuria. N. Engl. J. Med., March 30, 2006; 354(13): 1387 - 1401. [Full Text] [PDF]
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G. Walz Slit or pore? A mutation of the ion channel TRPC6 causes FSGS Nephrol. Dial. Transplant., September 1, 2005; 20(9): 1777 - 1779. [Full Text] [PDF]
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S. Lehtonen, J. J. Ryan, K. Kudlicka, N. Iino, H. Zhou, and M. G. Farquhar Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and {alpha}-actinin are components of the nephrin multiprotein complex PNAS, July 12, 2005; 102(28): 9814 - 9819. [Abstract] [Full Text] [PDF]
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 M. H. Beall, F. Amidi, D. A. Gayle, S. Wang, R. Beloosesky, and M. G. Ross Placental and Fetal Membrane Nephrin and Neph1 Gene Expression: Response to Inflammation Reproductive Sciences, July 1, 2005; 12(5): 298 - 302. [Abstract] [PDF]
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B. Bussolati, M. C. Deregibus, V. Fonsato, S. Doublier, T. Spatola, S. Procida, F. Di Carlo, and G. Camussi Statins Prevent Oxidized LDL-Induced Injury of Glomerular Podocytes by Activating the Phosphatidylinositol 3-Kinase/AKT-Signaling Pathway J. Am. Soc. Nephrol., July 1, 2005; 16(7): 1936 - 1947. [Abstract] [Full Text] [PDF]
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G. Wolf, S. Chen, and F. N. Ziyadeh From the Periphery of the Glomerular Capillary Wall Toward the Center of Disease: Podocyte Injury Comes of Age in Diabetic Nephropathy Diabetes, June 1, 2005; 54(6): 1626 - 1634. [Abstract] [Full Text] [PDF]
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 K. Tryggvason and J. Wartiovaara How Does the Kidney Filter Plasma? Physiology, April 1, 2005; 20(2): 96 - 101. [Abstract] [Full Text] [PDF]
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 R. R. Foster, M. A. Saleem, P. W. Mathieson, D. O. Bates, and S. J. Harper Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes Am J Physiol Renal Physiol, January 1, 2005; 288(1): F48 - F57. [Abstract] [Full Text] [PDF]
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T. Benzing Signaling at the Slit Diaphragm J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1382 - 1391. [Full Text] [PDF]
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