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From the Division of Bacteriology and Immunology,*
Haartman Institute, University of Helsinki, Helsinki, Finland; and the
Division of Ultrastructural Pathology and Cell
Biology,
Institute of Clinical Pathology,
University of Vienna, Vienna, Austria
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3ß1 integrin.6
The distinct structure of podocytes
with primary and secondary foot processes interlinked by filtration
slits appears to provide additional means for dynamic shape changes and
regulation of functions. The composition and exact role of the
filtration slits, however, remain to be determined. The structural
complexity of podocytes is the characteristic feature of an intact
glomerular filtration barrier, as contrasted with the flattening,
retraction, and fusion of foot processes in human and experimental
diseases with proteinuria.1,7
Thus, various lines of
evidence point to the interdependence of podocyte structure, function,
and attachment for the filtration barrier. Kestilä et al8 recently identified NPHS1, the causative gene of Finnish type congenital nephrotic syndrome (CNF), whose protein product, nephrin, appears to be a transmembrane protein with multiple immunoglobulin-like domains. In situ hybridization results indicated that the nephrin gene is expressed exclusively in podocytes during glomerulogenesis. Furthermore, the results of Kestilä et al showed that the nephrin gene is expressed within the kidney but not in other tissues.8 The localization of nephrin protein within the kidney or in other tissues is not known.
Here we report our studies of the expression of nephrin mRNA in the normal human kidney as well as in 28 CNF kidney samples. We used nephrin-specific antipeptide antibodies to localize nephrin within the kidney and to study its association with other proteins of the filtration slit area including occludin and ZO-1. In immunoelectron microscopy, nephrin was preferentially localized in the filtration slit area although some reactivity was also seen on the surface of podocytes. Furthermore, our results revealed a major splicing variant of nephrin that lacks the entire transmembrane domain.
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Materials and Methods |
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Renal tissues of CNF patients (n = 28) were obtained at nephrectomies performed by an established treatment protocol as earlier described.9 Diagnosis of CNF was done based on the typical clinical picture at birth (placental weight >40% of the weight of the newborn, edema, and massive proteinuria), exclusion of other types of congenital nephroses, and later the typical pathology at nephrectomy.10,11 All procedures were approved by the ethics committee of the Helsinki University Central Hospital.
The CNF kidneys at nephrectomy were perfused with Ringer’s solution, and glomeruli were rapidly isolated as described earlier12 and immediately processed for RNA isolation.12,13 Also samples of CNF cortical kidney tissue were processed for RNA isolation, immunohistochemistry, and electron microscopy as previously described.12,14,15
For normal controls, cadaver kidneys (n = 5; age of donors, 12–48 years) unsuitable for transplantation for vascular anatomical reasons (Department of Surgery, University of Helsinki) or the normal poles of kidneys removed because of Wilms’ tumor (n = 2; ages, 3 and 5 years) were used.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Cloning and Sequencing
RNA samples from isolated glomerular fractions or from cortical kidney were used as a starting material in RT-PCR analysis as earlier described.16 The following primers fully covering the transmembrane area were used for human nephrin: sense primer 5'-CCC ATC ACT ACC CCA GGT CT corresponding to nucleotides 3094–3113 (amino acids (aa) 1033–1039) and antisense primer 5'-CTC TGT TGT GCT GAC CGT G corresponding to nucleotides 3384–3402 (aa 1130–1136). For PCR, total RNA from cortical kidney or from isolated glomeruli was DNase treated (DNase RQ1; Promega, Madison, WI) and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) as previously described.16 cDNAs were amplified by using AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) on an MJ Research thermal cycler (PTC-200; MJ Research Inc., Watertown, MA). The PCR product was analyzed by agarose gel electrophoresis and ethidium bromide staining, purified (Magic PCR Preps DNA purification and separation kit, Promega), and sequenced with gene-specific primers (ABIPrism 310, Perkin-Elmer Applied Biosystems, Foster City, CA) as previously described.16
Some of the gel-purified PCR products obtained were cloned17 to a pGEM-T vector (Promega). For each PCR product, 3 to 6 colonies from a single transformation were cultured. Plasmid DNA was isolated,16 and clones were sequenced and screened for homology with database sequences using the BLAST search algorithm obtained via Internet (www.ncbi.nlm.nih.gov/BLAST) from the National Center for Biotechnology (Washington, DC).
Design of Synthetic Peptides
Sequence-specific intracellular (aa 1101–1126) and extracellular (aa 1039–1056) oligopeptides were selected over the human nephrin sequence (GenBank accession number AF035835) by using the PredictProtein program obtained via Internet (www.embl-heidelberg.de/predictprotein/predictprotein.html) from the European Molecular Biology Laboratory (Heidelberg, Germany). These peptides showed no homology to other known protein sequences and were synthesized and purified at a local peptide synthesis unit (Haartman Institute, University of Helsinki).
Antipeptide Antibodies
For immunizations the peptides were coupled to a multiple
antigenic peptide-polylysine matrix18,19
and injected into
two rabbits each. The first immunization was with 500 µg of peptide
in Freund’s complete adjuvant (Difco Laboratories, Detroit, MI), and
two booster immunizations with 300 µg each in Freund’s incomplete
adjuvant 4 weeks after the previous immunization. Peptide-specific
fractions were immunoaffinity-purified on CNBr-Sepharose (Pharmacia,
Uppsala, Sweden) coupled to the corresponding linear peptides. The
specificity of the antisera was tested by immunofluorescence on kidney
sections with and without free peptide competition (Figure 1A)
, by immunoblotting of glomerular
extracts (Figure 7)
and precipitation of a full-length nephrin in an
in vivo transcription and translation assay with the
specific antibodies (see below).
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For SDS-PAGE, the detergent extracts of isolated human glomeruli were suspended in the Laemmli sample buffer, boiled for 5 minutes, and run under reducing conditions using 8% gels and a Protean Minigel electrophoresis system (Bio-Rad Laboratories, Richmond, CA) as previously described.20 The separated proteins were transferred to nitrocellulose sheets for Western blotting with a Novablot semidry blotting apparatus (Pharmacia). After blocking with 3% bovine serum albumin, the nitrocellulose strips were incubated with the respective antibodies, washed thoroughly, and further incubated with anti-rabbit IgG coupled to horseradish peroxidase. After washing, the bound antibodies were detected by using the ECL Western blotting kit (Amersham Pharmacia Biotech, Uppsala, Sweden).20
Immunofluorescence and Immunoelectron Microscopy
Samples from normal human cortical tissue were prepared for immunofluorescence as described earlier.12,16 Briefly, frozen cortical tissues were cut at 4 µm, fixed in acetone at -20°C for 10 minutes, and reacted with the antipeptide antibodies (1.1 mg/ml; used at 1:100 dilution in phosphate-buffered saline) for 1 hour. Fluorescein isothiocyanate-anti-rabbit IgG (Boehringer Mannheim, Mannheim, Germany) was used for second antibodies. As a control, the primary antibodies were either omitted or replaced by irrelevant antipeptide antibodies. An additional control included preincubation of the antibody with a dilution series of the oligopeptide used as the original immunogen. Postembedding electron microscopy was done as previously described,18,21 using CNF and normal cortical kidney samples fixed in freshly prepared 4% formaldehyde in phosphate buffered saline, embedded in Lowicryl K4M (Chemische Werke LOW1, Waldkraiburg, Germany), and further incubated with the rabbit antinephrin antibodies (50 µg/ml) and the respective 10-nm gold conjugate (1:50).
In Vitro Transcription/Translation
In vitro transcription/translation of the full-length nephrin sequence under the T3 promoter of pBK-cytomegalovirus (Promega) was performed according to the manufacturer’s instructions with a TNT T3-coupled reticulocyte lysate system (Promega), a single-tube modification of rabbit reticulosyte lysate translations.22 During translation nehprin was labeled with [35S]methionine (NEN Life Science Inc., Boston, MA). Intracellular antibodies (see above) and [35S]labeled nehprin were incubated without or with increasing amounts (0.1, 1, and 10 µg) of intracellular peptide overnight at 4°C. Immunocomplexes were collected with protein A-Sepharose (Zymed Laboratories Inc., San Francisco, CA) by incubating immunocomplexes and protein A-Sepharose for 45 minutes at 4°C. After incubation the immunocomplexes were washed eight times with washing buffer (150 mmol/L NaCl, 20 mmol/L Tris, pH 7.6, 0.15% Tween 20, 0.1% bovine serum albumin, 0.02% sodium azide), and the radioactivity of precipitates was measured in scillation liquid OptiPhase SuperMix (EG&G Wallac, Turku, Finland) with 1450 MicroBeta Trilux liquid scintillation & luminescence counter (EG&G Wallac).
Immunoprecipitation
For immunoprecipitation the glomerular lysate (1 mg/ml) in radioimmunoprecipitation assay buffer was incubated with rabbit or mouse antibodies against occludin (Zymed Laboratories) or ZO-1 (Zymed), using 10 µg of IgG/200 µl of glomerular lysate at 4°C overnight. Immune complexes were collected with protein-A-Sepharose (Pharmacia LKB Biotechnology), washed, and processed for immunoblotting with antinephrin antibodies as described above.
Mutation Analysis
Analysis for the Finmajor (in exon 2) and Finminor (exon 26) mutations in the only patient sample out of 28 studied that showed immunoreactivity with the antinephrin antibodies was done as described.8 Briefly, after DNA isolation,17 the respective exon areas were amplified by PCR using AmpliTaq DNA polymerase (Perkin Elmer) and the following conditions: initial denaturation at 94°C for 12 minutes, 30 PCR cycles (95°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute), followed by final elongation at 72°C for 8 minutes. The primer sequences for exon 2 were 5'-GAG AAA GCC AGA CAG ACG CAG-3' and 5'-AGC TTC CGC TGG TGG CT-3'. For exon 26 the primer sequences were 5'-CTC GGG GAG ACC CAC CC-3' and 5'-CCT GAT GCT AAC GGC AGG GC-3'. The sequencing was done as described above, using the ABIPrism (Perkin-Elmer).
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. However, a
finely dotted linear reactivity giving a preferentially epithelial-like
staining pattern could be seen (Figure 1B)
. No appreciable reactivity
of the underlying basement membrane, endothelia, mesangium, or tubuli
could be observed (see Figure 2
for electron micrographs).
Antibodies against the extracellular nephrin domain showed a closely
similar glomerular reactivity pattern. All but one of 28 CNF samples
studied failed to show reactivity in glomeruli with the extra- and
intracellular nephrin-specific antibodies. Preimmune serum gave no
reactivity (Figure 1C)
, and preincubation of the antiserum with the
respective peptide before tissue staining also decreased the reactivity
to negligible levels (data not shown). Further evidence of antibody
specificity was obtained by the specific immunoprecipitation and
immunoblotting reactions (see Figure 6
). Furthermore, results from
in vitro transcription and translation of full-length
nephrin sequences followed by immunoprecipitation with the
intracellular antibodies showed specific precipitation that could be
prevented by the respective peptides (Table 1)
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-D). However, some immunogold particles were also seen in the plasma
membrane of podocytes, preferentially in the vicinity of the filtration
slits but also in clusters at the apical surface (see Figures 2 and 3
). In the CNF kidney samples, some
nephrin-specific gold particles were seen at the flattened apical
surface of podocytes in association with microvilli (Figure 3, A
-C) and
rarely at the intercellular junctional areas (Figure 3A)
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(Figure 4)
with a calculated 131-kd
molecular mass of the protein core (full-length nephrin,
135 kd). The authenticity of these PCR products was verified by
sequencing both products directly or after cloning in an appropriate
vector. The sequence of nephrin- was identical with that of nephrin
except that exon 24 (nucleotides 3167–3286) was missing (Figure 5)
. This exon includes the putative
transmembrane region of nephrin (nucleotides 3178–3258).
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. For nephrin, a broad band at 200 kd
was seen, possibly also containing the nephrin- variant. However, no
specific reaction product with antinephrin antibodies could be seen in
the material first precipitated with antioccludin or anti-ZO-1
antibodies. Neither of these proteins was detected when
immunoprecipitation was done first with antinephrin antibodies.
Mutation analysis by direct sequencing of exon 2 (for
Finmajor) and exon 26 (for
Finminor) of the sample, showing antibody
reactivity within glomeruli, was negative for both
Finmajor and Finminor
mutations (Figure 7)
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Discussion |
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Congenital nephrotic syndromes are rare pediatric kidney diseases
manifesting at or soon after birth with massive proteinuria. One of the
best characterized congenital nephrotic syndromes is CNF with
treatment-resistant proteinuria but no symptoms from other
tissues.10,11
Thus, CNF is considered a unique human model
of disease to study the mechanisms maintaining the glomerular
filtration barrier. Prenatal diagnosis of CNF can be made on the basis
of extremely high -fetoprotein in maternal serum or in amniotic
fluid at the early second trimester of pregnancy.23
In
light of the present results, it is interesting that the centripetal
morphological maturation of the glomeruli and their filtration slits
occurs early during fetal development,24
whereas little is
known of the respective functional maturation of the glomerular filter.
Whether nephrin has a true functional role during kidney development
remains to be studied in detail.
Using PCR primers flanking the transmembrane domain of nephrin,
two reaction products could be observed. These PCR products were
further cloned, sequenced, and verified to be nephrin-specific.
Interestingly, the novel nephrin- variant misses the whole amino
acid sequence spanning the transmembrane domain (encoded by exon 24).
That such a splicing yields variants without the complete
transmembrane area is interesting and could lead to secretion of the
protein, with important functional consequences. Examples of such
secreted transmembrane-negative splicing variants, with biologically
important regulation of the respective receptor affecting the ligand
binding, include interleukin-6 receptor25
and T cell
receptor.26
The exact role of nephrin- in proteinuric
diseases is currently being studied. Our preliminary results from the
rat and mouse suggest that additional splicing of nephrin homologues
may also occur in these species27
at the transmembrane
domain.
Kestilä et al8 reported two major NPHS1 gene defects responsible for CNF in the Finnish population, in exon 2 (Finmajor) and exon 26 (Finminor), whereas a variety of mutations including deletions, frameshift mutations, nonsense mutations, and early stop codons were found along nephrin genes in the CNF patients from other parts of the world, leading to various clinical pictures.28 Our previous studies of CNF patients have suggested that biochemical variants of the disease can also be found. Thus, eg, the ratios of urinary glycosaminoglycan to creatinine and urinary heparan sulfate to chondroitin sulfate show variation among these patients.14,29 The respective genotypes of these patients remain to be determined. Interestingly, in our study all but one CNF patient kidney failed to show reactivity with either the extra- or intracellular nephrin antipeptide antibodies. This suggests that the amount of nephrin in these patient glomeruli is below the detection limit or that no immunoreactive nephrin protein is expressed. The latter possibility is the obvious explanation in patients homozygous for Finmajor mutations with the early stop codon in exon 28 of NPHS1, whereas CNF patients with Finmajor Finminor heterozygosity could demonstrate negligible amounts of the protein product. Interestingly, the CNF patient with glomerular reactivity with our antibody failed to show either the Finmajor or Finminor mutation in direct sequencing. Thus, this patient most probably represents other mutations in nephrin, in the NPHS1 promoter area, or even in some nephrin-associated proteins, as suggested by Lenkkeri et al.27
Our immunoelectron microscopic (IEM) results show that nephrin is distinctly located at the filtration slit area and may also be present at the apical plasma membrane of podocytes. This is in line with results of Tryggvason (personal communication). Our results with experimental models of glomerular diseases show a similar plasma membrane localization of nephrin in IEM analysis (Luimula P, manuscript in preparation).
However, nephrin was also seen in IEM analyses of some of the CNF samples studied, preferentially at the plasma membranes. This result appears controversial because, particularly in Finmajor, no protein should be expressed.28 However, as discussed above, only homozygosity in respect to Finmajor mutation leads to complete lack of nephrin, whereas the Finminor mutation found in 20% of the Finnish patients causes a nonsense mutation at exon 26 in the intracellular domain. This site is downstream, beyond the recognition site of our intracellular antibodies and, if transcribed and translated, our antibodies should normally also detect this product. Thus, the heterozygotes may produce nephrin detectable only in IEM analysis but not in indirect immunofluorescence microscopy.
Little is known of the composition and molecules of the slit membrane and especially of their interactions, although, eg, synaptopodin and ZO-1 have previously been associated at this site.30,31 Our current results suggest that, although sharing the localization at filtration slits, nephrin may not directly interact with ZO-1 or occludin, as suggested by the lack of coprecipitation of these molecules. Further double-labeling IEM studies are underway to search for the possibly interacting molecules.
Together, our results reveal the characteristic nephrin splicing and show the localization of its protein product in a key localization in the glomerulus. Thus, nephrin appears to be the first component of the podocyte slit membrane in humans with a crucial role in glomerular permeability.
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Acknowledgements |
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Footnotes |
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Supported by the Academy of Finland, a research grant from Helsinki University Hospital, the Finnish Foundation of Heart Disease, and the Sigrid Juselius Foundation.
Accepted for publication July 15, 1999.
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-fetoprotein. Lancet 1976, 7977:123-125
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