Abstract
Abstract. Nephrin is a central component of the glomerular podocyte slit diaphragm and is essential for the normal renal filtration process. This study describes the complete structure of the mouse nephrin gene, which was shown to be homologous to the human gene, the major difference being 30 exons in the mouse gene as opposed to 29 in human. The complete primary structure of mouse and rat nephrins was also determined. The sequence identity between the mouse and rat proteins was shown to be 93%, while both rodent proteins have only about 83% sequence identity with human nephrin. The availability of the three mammalian sequences is significant for the interpretation of sequence variants and mutations in the nephrin gene in patients with congenital nephrotic syndrome. In situ hybridization analyses of whole mouse embryos and tissues revealed high expression of nephrin in kidney glomeruli and, surprisingly, an intense and highly restricted expression in a set of cells in hindbrain and spinal cord. No expression was observed elsewhere. This expression pattern may explain occasionally occurring neural symptoms caused by inactivating mutations in the nephrin gene in patients with congenital nephrotic syndrome.
The renal filtration barrier, in which ultrafiltration of plasma occurs, consists of the glomerular basement membrane (GBM) located between the fenestrated endothelial cell and epithelial podocyte layers, as well as the slit diaphragm located between the podocyte foot processes (1). During embryonic development, epithelial cells becoming glomerular podocytes differentiate from the metanephric mesenchyme at the S-shaped body stage, when the podocyte layer is first seen at day 13 (E13) of mouse embryonic development (2). Although a wealth of knowledge has accumulated on the structure and properties of GBM components (3,4), the filtration role of the podocyte cell layer, particularly that of the slit diaphragm, has long been unclear. Only a couple of proteins have been localized to the slit diaphragm region, one of which was a 51-kD antigen recognized by monoclonal antibody 5-1-6 (5,6). The antigen epitopes recognized by the antibody are unknown, however. Another protein that has been localized to the region is the α--isoform of the intracellular tight junction protein zonula occludens-1, located at sites where the slit diaphragm meets the lateral cell membrane of the foot processes of podocytes (7,8).
We have recently identified a novel protein, termed nephrin (9), and shown its specific localization to the slit diaphragm (10). The nephrin gene is mutated in congenital nephrotic syndrome (NPHS1) (9,11), a disease characterized by massive proteinuria and edema (12). Based on Northern and in situ hybridization, nephrin has been shown to be expressed by the glomerular podocytes (9). Using immunoelectron microscopy, we recently localized nephrin to the slit diaphragm where nephrin is surmised to form a zipper-like isoporous filter structure (10) similar to that proposed by Rodewald and Karnovsky based on transmission electron microscopic observations (13). Because nephrin resides in the slit diaphragm, and mutations in the gene cause severe nephrotic syndrome, this protein must have a crucial role in the filtration mechanism of the glomeruli and may participate more generally in the pathogenesis of proteinuria and renal failure.
To further characterize nephrin and facilitate studies on its involvement in renal physiology and disease, we have now determined the primary structure of both mouse and rat nephrin based on cDNA sequences, determined the structure of the mouse gene, and studied its expression pattern both by Northern and in situ hybridization analyses during mouse embryogenesis.
Materials and Methods
Isolation and Characterization of Genomic Mouse Bacterial Artificial Chromosome Clones
Genomic mouse bacterial artificial chromosome (BAC) clones containing the nephrin gene were isolated using 5′-ACG TGA GGG CCG AGC GGA CAT G-3′ and 5′-CCA CAT AGT CCA GCC ACT TGA-3′ primers (GenomeSystems) that are specific for amyloid β precursor-like protein 1 (APLP1), whose gene is located immediately adjacent to the nephrin gene (9,14). Approximately 50 μg of BAC clone DNA was digested with EcoRI and BamHI restriction enzymes, and the resulting fragments were run in a 1% agarose gel and Southern-blotted to a Biodyne B nylon filter (Pall Corp., Lund, Sweden) (15). The filter was hybridized at 42°C with human nephrin cDNA, first with a fragment comprising exons 2 to 3, and then with a fragment containing exons 26 to 29. These probes were radioactively labeled with [α-32P]dCTP (3000 Ci/mmol) (Amersham Pharmacia Biotech), using random priming (Life Technologies). Positive fragments, 2.4-kb EcoRI (positive for exons 2 to 3) and 7.2-kb BamHI (positive for exons 26 to 29), were purified with the Gel Extraction kit (Qiagen, Chatsworth, CA) and subcloned into the pBluescript SK(+) vector. The plasmids were isolated using the QIAprep spin miniprepkit (Qiagen) and sequenced on an ABI 310 Genetic Analyzer (Perkin Elmer). For identification of exons, the sequences were compared with those of the human nephrin cDNA sequence using the FASTA program. This comparison yielded exons 1 to 4 from an EcoRI fragment and exons 24 to 30 from a BamHI fragment of the genomic BAC clone.
Cloning of Mouse Nephrin cDNA
Mouse nephrin exon-specific primers were designed from genomic sequence data obtained from the subcloned BAC clone fragments. Two primer pairs were used for PCR to amplify the cDNA from mouse kidney QUICK-Clone cDNA (Clontech, Palo Alto, CA) with Advantage cDNA polymerase mix (Clontech): m(forw)1 from the 5′ untranslated region (UTR) 5′-GAC AGC AAC AAA CAA GCT GCT GG-3′ together with m(rev)1 primer from exon 29 5′-TCA CAC CAG ATG TCC CCT CAG C-3′; and the same m(forw)1 primer together with m(rev)2 primer from the 3′UTR region 5′-GGA AAC AGG TGT CGT GAA GAG TC-3′. The PCR products were agarose-gel purified and subcloned into the pCR-Script SK(+) vector (Stratagene, La Jolla, CA). Two clones from each amplification were sequenced from both strands using either ABI 310 or 377 Genetic Analyzers. The sequences were compared to each other using the FASTA program, and nonmatching sequence segments were reamplified using Vent DNA polymerase (New England Biolabs, Beverly, MA) from mouse kidney QUICK-Clone cDNA in two rounds. The PCR fragments were agarose-gel purified and sequenced directly from both strands. The final mouse cDNA sequence was determined by comparing the sequences to that of the gene. Furthermore, the 5′ region of the cDNA was amplified (rapid amplification of cDNA ends [5′ RACE]) in two separate reaction sets in two rounds from a mouse kidney Marathon-Ready cDNA (Clontech) with the Advantage cDNA polymerase mix. In the first round, AP1 primer was used together with exon 2-specific m(rev)3 primer 5′-CAG AAG CAG CCC ATC CTT AGC-3′ and m(rev)4 primer 5′-CAG GTA ACT GTG CTT CCT GCC TC-3′. The second, nested amplification round was performed with AP2 primer together with exon 2-specific m(rev)3b primer 5′-CAG ATA GAG CCC AGA AGC CTC G-3′ and m(rev)3 primer that is upstream to the m(rev)4 primer, respectively. The nested PCR round products were agarose gel-purified and subcloned into pCR-Script SK(+) vector. Three subclones were sequenced from both strands.
Cloning of Rat Nephrin cDNA
To obtain the rat nephrin cDNA, a rat kidney λgt10 cDNA library (Clontech) was screened using a human nephrin cDNA fragment comprising exons 3 to 29 as a probe, according to the manufacturer's instructions. Three positive primary plaques were obtained and, after secondary screening, three positive-phage DNA were isolated using a lambda mini kit (Qiagen), digested with EcoRI and subcloned into pBluescript SK(+). Three clones, 3.32, 5.92, and 6.21, were sequenced from both strands and the sequences were analyzed with the FASTA program. A missing segment between clones 3.32 and 5.92 was amplified using Vent DNA polymerase from the rat kidney λgt10 cDNA library using two primer pairs: r(forw)1 5′-CAT CCT GGC CAA CTC GTC CG-3′ together with r(rev)1 5′-GGA GTA GGC TGA TCC ACC TG-3′; and r(forw)2 5′-CAG GCG ACG CCT TGA ACT TG-3′ together with r(rev)2 5′-GCA AAT CGG ACG ACA AGA CG-3′. The fragments were agarose-gel purified and subcloned into the pCR-Script SK(+) vector. Two clones from each reaction were sequenced from both strands. To obtain the missing 5′ end, two sets of amplification reactions were performed from the rat kidney Marathon-Ready cDNA (Clontech) using Vent DNA polymerase (5′ RACE). First, a round using primer pair AP1 together with r(rev)3 5′-AGC ACG ATC TCC TTC CAG GC-3′ was carried out, followed by a second nested round using primer pair AP2, together with r(rev)4 5′-GAA GCC TGG CAT CTT CGG G-3′. The resulting PCR fragments were agarose gel-purified and sequenced directly from both strands.
Analysis of cDNA and Amino Acid Sequences
The final cDNA and amino acid sequences were compared with the human ones using the FASTA and BLAST programs. Alignment of amino acid sequences was performed with the PileUp program (Genetics Computer Group, Madison, WI), and signal peptide prediction with the SPScan program (Wisconsin Package version 10.0, Genetics Computer Group). Protein patterns were searched using the PROSITE protein pattern search tool (16).
Determination of Exon-Intron Boundaries of the Mouse Nephrin Gene
To determine the exon-intron boundaries, exon-specific primers were designed and intervening intron sequences were PCR amplified from BAC clones using HotStarTaq DNA polymerase (Qiagen). The fragments were size-determined and agarose-gel purified. The exonintron boundaries were sequenced from both strands either from the amplified fragment or from the subcloned BAC-fragments described above. The smallest introns were sequenced entirely. For intron 23, the BAC Southern nylon filter was hybridized at 65°C with a mouse nephrin cDNA fragment comprising exons 23 to 24. A positive 8.7-kb EcoRI fragment was agarose gel-purified, subcloned into pBluescript SK(+), and sequenced with exons 23 and 24-specific primers to find the exon-intron junction. A region with high GC content in the boundary of exon 23 and intron 23 was sequenced until a suitable site for PCR primer annealing was found. The rest of intron 23 was PCR-amplified with intron 23-specific primers.
Northern Analysis of Mouse Nephrin Expression
By using multiple-tissue Northern analysis, poly(A)+ RNA from eight adult mouse tissues and 7-, 11-, 15-, and 17-d-old mouse embryos were studied (Clontech). Hybridizations were carried out at 65°C using a PCR-amplified fragment comprising exons 23 to 29 as probe, as described above. The Northern blots were also probed with similarly labeled human β actin cDNA to compare the loading in each lane.
In Situ Hybridization
For in situ hybridization, a probe from the 3′ end of the gene was amplified from a mouse kidney QUICK-Clone cDNA using exon 29 specific m(forw)2 primer 5′-GAC CCC TAT GAC CTT CGC TG-3′, together with a 3′UTR-specific m(rev)5 primer 5′-CAG ATG TCA GCT GGA GTC TTC-3′. The amplification was performed using Advantage cDNA polymerase mix. The resulting fragment was subcloned into the pCR-Script SK(+) vector and sequenced from both strands. The 219-bp sense BamHI and antisense NotI 35S-labeled cRNA probes were transcribed, and in situ hybridization was performed essentially as described by Wilkinson and Green (17) with some modifications. Briefly, E11 to newborn whole mouse embryos and isolated kidneys were fixed in 4% paraformaldehyde and processed for paraffin histology. Sections at 7 μm were cut, treated in prehybridization solutions as described (17), and finally hybridized overnight at +65°C. After high stringency washes at +65°C, the sections were dehydrated, air-dried, dipped into NTB-2 emulsion (Eastman Kodak), and exposed at +4°C in the dark for 10 to 14 d. The sections were developed in D19 (Eastman Kodak), fixed in sodium fixative (Eastman Kodak), counterstained in hematoxylin (Shandon, Pittsburgh, PA), and mounted. Sections were analyzed and photographed with Olympus Provis AX microscope equipped with a charge-coupled device camera (Photometrics Ltd.). The dark-field images were inverted, stained red, and combined with the bright-field images in the Adobe PhotoShop 4.0 program.
Results
Isolation and Characterization of the Mouse Nephrin Gene
Hybridization of several mouse cDNA and genomic libraries with human cDNA or genomic clones did not yield any mouse nephrin clones. Therefore, isolation of the nephrin gene locus was attempted by isolating BAC clones containing the putative neighboring APLP1 gene. Human chromosome 19q and the proximal portion of mouse chromosome 7 have been shown to be highly syntenic (18,19) (Mouse Genome Database, Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, ME; Internet: http://www.informatics.jax.org). The number and order of genes as well as their spacing are fairly similar between the two chromosomes, the main difference being reversed centromeric-telomeric orientation on the mouse and human chromosomes (18). Three APLP1-positive BAC clones were found to hybridize with human nephrin cDNA probes.
Southern hybridization of the APLP1-positive BAC clones with different fragments of the human nephrin cDNA demonstrated that the entire mouse nephrin gene was present in two of the three clones obtained. Two restriction fragments of 2.4 and 7.2 kb hybridizing with the human cDNA were sequenced and shown to contain sequences corresponding to exons 1 to 4 and 24 to 29 in the human gene, respectively. Primers based on these exon sequences were then used for amplification of the mouse nephrin cDNA from pooled kidney mRNA, and the full-length cDNA could be compiled and used to determine the exon structure.
The mouse nephrin gene spans about 27 kb (Figure 1). To determine the exon-intron boundaries of the gene, exon-specific primers were used to amplify the intervening intron sequences from BAC clones except for intron 23, which was cloned separately (see Materials and Methods). Unlike human NPHS1, which has 29 exons, the mouse gene has 30 exons, the last exon encoding only for the major part of the 3′ UTR. The exon sizes range from 25 to 216 bp (Figure 2) and are similar between human and mouse, except for exons 3, 8, 13, and 28, which differ by 1 to 6 bp (11). The codon for translation initiator methionine is in the first exon, and the translational stop codon TGA is in exon 29.
Schematic structure of the mouse nephrin gene. Exons are indicated by black rectangles; introns and flanking are indicated by rectangles with diagonal lines. Exons are numbered. 5′ and 3′ untranslated regions are marked with hatched boxes, the sizes of which are unknown. The scale at the bottom is in kilobases (kb).
Exon-intron boundaries and sizes of exons and introns in the mouse nephrin gene. Intron sequences are shown with lowercase letters. Exon sequences are depicted by uppercase letters, with the one-letter amino acid residues shown below. Exon and intron sizes are given in base pairs. Introns marked with an asterisk were size-determined by agarose gel electrophoresis of PCR products, except for intron 23, which was partially sequenced.
In human, an unusual donor site starting with GC instead of the GT exists at exon 23, which encodes part of the fibronectin type III module (11). This turned out to be the case at mouse exon 23 as well. All other acceptor (AG) and donor (GT) sites are conventional.
Determination of Mouse and Rat Nephrin cDNA Sequences
The coding sequence of the full-length mouse cDNA was shown to be 3768 nucleotides. The codon for the first possible initiator methionine was more 5′ than that of the human sequence (Figure 3). This was verified using a 5′ RACE reaction, as well as by analyzing the sequence of the 5′ end of the gene (Figure 2). The analyzed genomic exon sequences of the mouse gene were all found in the sequenced mouse cDNA clones.
Comparison of mouse, rat, and human nephrin at amino acid level sequences. The sequence alignment was performed with the PileUp program. The signal peptide cleavage site is marked by an arrow. Ig motifs are boxed and the fibronectin type III-like domain is boxed with a hatched line. The transmembrane domain is underlined with a solid line. Conserved cysteine residues are shaded, unconserved residues are shown in bold, and conserved N-glycosylation sites are indicated with a triangle (▾). Conserved tyrosine residues in the intracellular domain are indicated with a dot (•), and two possible protein kinase C phosphorylation sites with the consensus sequence Ser/Tyr-X-Arg/Lys are marked with a star (★).
The sequence of the rat nephrin cDNA was determined from three cDNA clones, 3.32 containing bases 270-1819, 5.92 containing bases 2526-3840, and 6.21 containing bases 2730-3971 (not shown). The missing parts were amplified either from the same cDNA library or with 5′ RACE. The full-length rat cDNA contained 3756 bp. Two codons for methionine are present in the 5′ end, the first located at the same site as that in the mouse cDNA and the other 12 bp downstream of the codon for the initiator methionine in the human cDNA. Because the nucleotides around the upstream methionine codon better fit the Kozak's consensus sequence (20) for translation initiation than the downstream one, the upstream codon is probably used for the initiator methionine. This is also supported by the high identity (82%) in nucleotide sequence between the mouse and rat from the first rat methionine codon to the second.
Comparison of cDNA-Deduced Mouse, Rat, and Human Nephrins
While the mouse and rat amino acid sequences show 93% identity, both the mouse and rat amino acid sequences show 83% identity with the overlapping areas of the human nephrin sequence. The most notable difference is at the amino termini, where the mouse and rat proteins are 14 residues longer than in human (Figure 3). Mouse nephrin has 1256 amino acid residues with a predicted molecular weight of 136 kD without posttranslational modifications, whereas rat nephrin has 1252 residues with a predicted molecular weight of 136 kD. Human nephrin has previously been shown to contain 1241 residues, with a molecular weight of 135 kD (9).
The signal peptide cleavage site in human nephrin is located between residues 22 and 23, whereas it is predicted to be between residues 36 and 37 in mouse and rat. All 10 potential N-glycosylation sites are conserved between the three species (Figure 3). Of the seven serine-glycine (SG) doublets that are potential heparan sulfate attachment sites, only two are conserved between the three species.
Similar to Ig motifs in other proteins (21), all eight Ig motifs in nephrin of the three species contain two conserved cysteines that form disulfide bonds within the motif. In addition, the mouse and rat nephrin molecules contain three cysteine residues that are conserved between the species and, depending on the species, one or two unconserved residues. The conserved residues are one in the first Ig motif, one in the spacer domain, and one in the transmembrane domain. The unconserved residues include one cysteine in Ig motif 4 in the mouse, one each in Ig motifs 2 and 4 in the rat, and one residue in the fibronectin III domain in human nephrin. Furthermore, both the mouse signal peptide and the rat intracellular part have one cysteine residue each (Figure 3).
The intracellular domain in human has nine tyrosine residues that are tentative phosphorylation sites for intracellular signaling, while the corresponding domains of mouse and rat contain 10 and eight residues, respectively. Six of those tyrosine residues are conserved between the three species. However, the sequences around the tyrosines are completely conserved between the three species only in the case of three of the residues. According to the numbering of mouse nephrin, these are tyrosines 1128, 1208, and 1232. Tyrosine phosphorylation of nephrin is still an open question, so the naming of any specific interactors is purely speculative. Yet, comparison to the optimal recognition motifs for SH2 domains from different proteins reveals that if tyrosine 1208 is phosphorylated, a binding site for an adapter protein Nck might be generated. This motif pYDEV, and the one containing tyrosine 1232, pYDQV, may also provide a binding site for tyrosine kinases of the Src family (22,23). Furthermore, two potential conserved protein kinase C phosphorylation sites with the consensus sequence Ser/Tyr-X-Arg/Lys (Figure 3) were detected by PROSITE protein pattern search tool (16).
Developmental Expression of Mouse Nephrin
The overall expression pattern of mouse nephrin was studied by Northern analysis, using RNA from whole mouse embryos of different ages and adult mouse tissues. No signals above background could be observed with poly(A)+ RNA from four different aged mouse embryos (not shown). With poly(A)+ RNA from eight adult mouse tissues, the analysis revealed a signal only with RNA from kidney (Figure 4). RNA from heart, brain, spleen, lung, liver, skeletal muscle, and testis were all negative.
Northern analysis of mouse nephrin expression with mRNA from eight adult mouse tissues and whole mouse embryos. Mouse nephrin cDNA fragment comprising exons 23 to 29 was used as a probe. The tissues studied are marked above the filter, and molecular size markers (kb) are shown to the sides of the filters. The arrow indicates the observed band.
Because Northern analyses may not reveal the presence of transcripts with highly restricted expression, we carried out in situ hybridization on whole mouse embryos and isolated metanephric and mesonephric kidneys. At E11, when the metanephric differentiation is initiated, no signal could be detected from the mesenchymal cells of the presumptive kidney (Figure 5C). However, nephrin expression was seen in the E11 mesonephric kidney, where cranial tubules with podocyte-like structures revealed strong expression, while the mesonephric mesenchyme and less differentiated caudal tubules remained negative (Figure 5, A and B). A highly specific expression was observed in the podocytes of the developing kidney beginning from the E13 mouse embryos (Figure 5, E and F). Early S-shaped bodies, as well as the metanephric mesenchyme, remained negative at this stage. Surprisingly, high expression of nephrin mRNA was observed in the hindbrain and spinal cord (Figure 6A). At E11 in the brain, the expression was limited to a subset of neurons in the hindbrain area and continued from there dorsally, confined to the mantle layer neurons of the spinal cord (Figure 6B). A similar expression pattern was observed at E13 (Figure 6C). From E13 to E17, nephrin mRNA was detected in neuroepithelium of cerebellum primordium at the roof of the fourth ventricle, but the epithelial cells of developing choroid plexus, which are involved in the formation of cerebrospinal fluid, remained negative. Expression was not observed in any other tissues of the embryo.
In situ hybridization analysis of nephrin expression during mouse kidney development. (A and B) Embryonic day 11 (E11) mouse mesonephric (Me) kidney. Nephrin mRNA is seen in the podocyte layer of the cranial (Cr) mesonephric nephron. The caudal (Ca) tubules remain negative. (C) Mouse metanephric kidney at E11. Metanephric mesenchyme surrounding the ureteric bud (ub) does not express nephrin mRNA at this stage. Caudal mesonephric tubule (arrow), genital ridge (gr), and wolffian duct (wd). (D) Early nephrones of E13 kidney cortex. Presumptive podocytes (*) at the S-shaped bodies are still negative. Ureteric buds (ub) are marked with arrows. (E) Nephrin mRNA is first detected at E13 glomerular structures. Early podocytes from the S-shaped body (*) are negative. (F) Glomerular podocytes from newborn kidney show high expression of nephrin mRNA. The cortical undifferentiated structures are negative. Bars: 150 μm in A and B; 100 μm in C; 60 μm in D; 80 μm in E and F.
Nephrin mRNA expression in the developing mouse nervous system. (A) At E11 hindbrain, nephrin mRNA is highly expressed in a subset of neurons both in the upper cerebellar plate and in the opposite side of the fourth ventricle, in the myelencephalon. (B) In the neural tube at the lumbar area of the same embryo, the expression is in the dorsal mantle layer. (C) At E13, the expression in brain is concentrated to the neuroepithelium of the cerebellar primordium on the roof of the fourth ventricle. Some expression is also seen at the floor of the ventricle, but the choroid plexus (arrow) is negative. Bars: 250 μm in A and B; 150 μm in C.
Discussion
The recent identification of nephrin as the first known molecular component of the slit diaphragm and as a protein whose mutations cause proteinuria has opened new possibilities for exploring the mechanisms of glomerular ultrafiltration and the pathogenetic mechanisms of proteinuria (9,10,11). In this regard, it is important to be able to carry out studies on nephrin and its involvement in protein filtration in experimental systems, including well established proteinuria models in mice and rats. The present work facilitates such studies by providing both nucleotide and amino acid information on mouse and rat nephrins, as well as by determining the complete structure of the mouse gene and the location of its activity during the embryonic development.
The present analyses of the mouse gene demonstrated its similarity to the human one, except for the 3′ UTR, which is encoded by two exons (exons 29 and 30) in the mouse gene as opposed to one (exon 29) in the human gene (11). The gene sequence was also important for verification of the mouse cDNA sequence, which differs particularly in the 5′ end, that is, in the signal peptide from the human gene. The presently elucidated mouse gene structure will be valuable for enabling the generation of a mouse model for congenital nephrotic syndrome, using gene targeting in embryonic stem cells.
The mouse and rat cDNA sequences determined here showed that mouse and rat nephrins have 93% sequence identity, while human nephrin gene shows a considerably lower sequence identity of 83% with the two rodent species. These sequences of nephrin from three mammalian species are of considerable value in several respects. First, the conserved residues suggest significant structural motifs and functional importance for the molecule. Second, the availability of the sequences is important not only for the interpretation of the impact of the numerous sequence variants found thus far both in patients with nephrotic syndrome and in the disease carriers, but also for the numerous homozygous and heterozygous allele variants found in the general population with no family history of nephrotic syndrome (9,11).
Several conclusions can be made from the comparison of the three primary structures of nephrin. Thus, the three conserved, apparently free cysteines in the Ig motif 1, the spacer domain, and the transmembrane domain are probably crucial for the function of the protein. This supports our hypothetical model for the zipper-like nephrin structure of the slit membrane (10), where nephrin molecules from two opposite foot processes interact in an interdigitating manner through homophilic interactions reinforced by intermolecular disulfide bonds between the two free extracellular cysteines in Ig motif 1 and the spacer domain. Furthermore, the glycoprotein nephrin has 10 conserved N-glycosylation sites, suggesting that controlled glycosylation is important for the protein.
There are six conserved tyrosine residues in the intracellular domain of nephrin. It is not yet known whether any of those serves as a phosphoacceptor site. If this turns out to be the case, one could envision several roles for the tyrosine phosphorylation. First, the phosphorylation could have a role in the activation of signal transduction pathways upon binding of the extracellular ligand(s). Interestingly, residues immediately carboxy-terminal to three of the tyrosines are completely conserved between the three species. Thus, comparison to optimal binding sequences for SH2 domains suggests that signaling molecules, such as an adaptor protein Nck and Src-family protein kinases, may be recruited to nephrin if tyrosines 1208 and 1232 are phosphorylated. Second, it is also possible that tyrosine phosphorylation of nephrin plays either a positive or negative role in the assembly of the glomerular slit diaphragm. In this regard, it is noteworthy that tyrosine phosphorylation of the intracellular domain of neurofascin, which also is a transmembrane protein of the Ig superfamily, abolishes its binding to the membrane skeletal protein ankyrin and reduces coupling of neurofascin to the cytoskeleton (24). Yet another possibility is that tyrosine phosphorylation regulates extracellular ligand specificity of nephrin, as might be the case with platelet endothelial cell adhesion molecule-1, another member of the Ig superfamily (25).
Thus far, 21 missense mutations resulting in amino acid changes, mainly in the Ig modules, have been reported in patients with congenital nephrotic syndrome (9,11). All of the mutated amino acid residues, except for two, are conserved between the three mammalian species analyzed in the present study. The two missense mutations affecting unconserved amino acid residues are Ile 173 → Asn in Ig 2 and Arg 1140 → Cys in the intracellular domain. Although not all missense mutations involve conserved amino acids, it is apparent that one can suspect individuals with allelic changes in codons for conserved amino acids to be carriers for congenital nephrotic syndrome. Thus, the three sequences can indeed have direct practical value.
Recently, cDNA and amino acid sequences were reported for the mouse (26) and rat (27) nephrin homologues. Comparison of the amino acid sequences between the mouse sequence reported here and in that by Holzman et al. (26) reveals differences in the 5′ end or in the signal peptide region. In the sequence reported by Holzman et al. (26), the first 10 amino acids are 100% identical to the human sequence. The 5′ end mouse sequence reported here, however, is longer, and it differs significantly from the human one. Comparison of the 5′ end of the mouse sequence with that of the mouse genomic sequence showed that the sequence reported in this study is correct. Thus, the reason for the difference between the two mouse sequences is unknown. Otherwise, only two amino acids were different in the two mouse amino acid sequences: Thr763 reported here was Ala in the Holzman study, and Leu1076 was reported to be Gln in their study (26). Both Thr763 and Leu1076 are conserved in human and rat, indicating that they are also conserved in mouse. With regard to the rat sequences, we found a longer signal peptide than that reported by Ahola et al. (27). Furthermore, Ahola et al. reported that there is Asn in position 1229, whereas we found it to be Asp between all three species studied here.
Nephrin mRNA was located by in situ hybridization both to the embryonic mesonephric and metanephric kidneys. The undifferentiated metanephric mesenchyme and first epithelial structures, the comma- and early S-shaped bodies, remained negative. The first mRNA were detected in late S-shaped bodies in the definitive podocytes. A similar expression pattern was noticed in the glomerular structures of the mesonephric kidney, which reveals that the mouse cranial mesonephric nephrones (28) could be secretory. Instead, the caudal mesonephric nephrones did not express nephrin mRNA.
The presence of nephrin mRNA in a specific region of the brain and spinal cord raises the question about a role of this protein in neuronal development and function. Thus far, severe extrarenal symptoms have not been reported in NPHS1 patients who have received kidney transplants (29). However, of approximately 50 transplanted Finnish patients, about 10% have congenital neurologic symptoms, such as ataxia, not normalizing after transplantation. These symptoms cannot be explained by thrombosis or other secondary insults due to NPHS1 (Christer Holmberg, personal communication). It is quite plausible that these neurologic symptoms are related to absence of normal nephrin in brain tissue, and that this function cannot be compensated for in all individuals. Participating in guidance of neurons (30,31), axonal fasciculation (32), and growth and stabilization of synapses (33), many of the members of the Ig superfamily have been shown to possess multiple roles in neuronal differentiation. Because congenital ataxias are often associated with impaired development of the cerebellum and its connections (34), the expression of nephrin in the nervous system could indeed indicate a role in neurogenesis.
The recent evidence on interaction of the cytosolic CD2-associated protein (CD2AP) with nephrin (35) is of great interest. First and foremost, the interaction provides information on potential linkage mechanisms between nephrin and the actin cytoskeleton of podocyte foot processes (36). Furthermore, removal of this gene was shown to cause a nephrotic syndrome similar to NPHS1. However, the CD2AP-deficient mice die at 6 to 7 wk of age (35), while nephrin-deficient mice die on their first day of life (H. Putaala, unpublished information). Therefore, it is possible that nephrin has multiple associating ligands that could have a role in the filtration mechanism.
Note Added in Proof: After submitting this work, mAb 5-1-6 was shown to be directed against the extracellular domain of rat nephrin (Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shimizu F, Salant DJ. J Clin Invest 104: 1559-1566, 1999).
Acknowledgments
This work was supported in part by grants from the National Institutes of Health (DK 54724), the Sigrid Juselius Foundation, the Novo Nordisk Foundation, and Hedlund's Foundation. We thank Timo Pikkarainen for valuable comments on the manuscript and Vesa Ruotsalainen for calculating the molecular weights.
Footnotes
- © 2000 American Society of Nephrology
References
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