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From the Biocenter and Department of Biochemistry,*
University of Oulu, Oulu, Finland; the Hospital for Children and
Adolescents,
University of Helsinki, Pediatric
Nephrology and Transplantation, Helsinki, Finland; the
Department of Bacteriology and
Immunology,
Haartman Institute, University of
Helsinki, Helsinki, Finland; the Electron Microscopy
Unit,§
Institute of Biotechnology, University
of Helsinki, Helsinki, Finland; the Department of Obstetrics and
Gynecology, University of Helsinki, Helsinki,
Finland; the Department of Pediatrics,||
Washington
University, St. Louis, Missouri; and the Division of Matrix
Biology,**
Department of Medical Biochemistry and
Biophysics, Karolinska Institute, Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The slit diaphragm is a filamentous structure spanning the slit pore between the adjacent podocyte foot processes.8 Recent findings indicate that the slit diaphragm is an essential structure in the glomerular filtration barrier for restricting the passage of plasma proteins into urine.9,10 Based on electron microscopy, an isoporous, zipper-like structure for the slit diaphragm has been suggested.11 A model for nephrin assembly into this isoporous filter was recently presented.4,9 The molecular composition of the slit diaphragm is, however, still largely unknown. The tight junction protein, zonula occludens-1 (ZO-1), is found at the cytoplasmic side of the slit diaphragm.12,13 The CD2-associated protein (CD2AP) is present in the podocyte foot processes, and it probably anchors nephrin to podocytes.14 In addition, P-cadherin was recently localized at the slit diaphragm.15
The slit diaphragm has been thought to originate from the subapical junctional complexes of immature visceral epithelial cells (primordial podocytes).16 During glomerulogenesis, these junctions migrate along the lateral cell margins toward the basal surface to form mature slit diaphragms.16 Based on the presence of the tight junction protein ZO-1 at the insertion site of the slit diaphragm, it has been assumed that the slit diaphragm represents a modified tight junction.12 On the other hand, Reiser et al recently provided evidence that the slit diaphragm could be a P-cadherin-based adherens junction.15
Here we studied the developmental expression of nephrin in human fetal kidneys and compared it to that of ZO-1 and P-cadherin. We also evaluated the morphogenesis of podocyte filtration slits in normal and NPHS1 kidneys lacking the nephrin molecule. The cytochemical and morphological data obtained indicate that nephrin is crucial for the final development of the slit diaphragm.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue samples were collected at autopsy from human fetuses at 14,
16, 22, and 23 weeks of gestation obtained through
prostaglandin-induced abortions due to anencephaly, gastroschisis,
trisomy 18, and trisomy 21 (Department of Obstetrics and Gynecology,
University of Helsinki). In these disorders, abnormalities have not
been detected in chromosomes 15, 16, and 19, where the genes for ZO-1,
P-cadherin, and nephrin, respectively, are located.3,17,18
The autopsies were performed within a few hours after the abortion.
These fetuses showed no macroscopic or histological abnormalities in
kidneys or urinary tract. In addition, renal samples from two fetuses
at 17 and 19 weeks of gestation aborted due to NPHS1 suspicion were
collected. Both showed elevated levels of amniotic fluid
-fetoprotein and normal ultrasonography features. Mutation analysis
of NPHS1 was performed as described earlier.3
Adult control samples came from cadaver kidneys unsuitable for
transplantation because of vascular abnormalities (from the IV
Department of Surgery, University of Helsinki). Considering the
suboptimal fixation conditions for specimen preparation (ie, autopsy
samples obtained from abortions), we used only tissue blocks with
adequate structural preservation for labeling.
For light microscopy, the tissue samples were snap frozen in cold isopentane cooled in liquid nitrogen, and fixed in 3.5% paraformaldehyde in phosphate buffer (0.1 mol/L, pH 7.3). In situ hybridization and immunoperoxidase studies were also performed on paraffin-embedded samples that had been fixed in 10% formalin in phosphate buffer (0.1 mol/L, pH 7.3). For immunoelectron microscopy, tissue blocks were cut into 1-mm3 pieces and immersed in fixative (3.5% paraformaldehyde supplemented with 0.02–0.1% glutaraldehyde). Samples for morphological evaluation in electron microscopy were fixed in 2.5% glutaraldehyde followed by 1% OsO4.
This study was approved by the ethical committees of the Department of Obstetrics and Gynecology and The Hospital for Children and Adolescents of the University of Helsinki.
In Situ Hybridization
The probes were synthesized by subcloning a cDNA fragment of 287
bp corresponding to exon 10 in human NPHS1 gene into
pBluescript, and antisense and sense RNAs produced using T3 and T7 RNA
polymerases, respectively.3
Exon 10 encodes for the fourth
extracellular immunoglobulin (Ig) domain of nephrin (Figure 1)
. Paraffin-embedded sections (10 µm)
were deparaffinized in xylene, rehydrated in decreasing alcohol series,
and treated with proteinase-K (Sigma Chemical, St. Louis, MO) before
hybridization. Thereafter both the cryosections and paraffin-embedded
sections were subjected to in situ hybridization, as
previously described,19
with some modifications. Briefly,
the tissue sections were washed in phosphate buffered saline (PBS),
acetylated, dehydrated, and then incubated with 1.2 x
106
of 33P-labeled
antisense and sense riboprobes (1000 Ci/mmol; Amersham, Arlington
Heights, IL) in a total volume of 80 µl at 60°C for 18 hours. After
washes with standard saline citrate, RNA digestion, and dehydration,
the sections were dipped in NTB2 nuclear emulsion (Kodak, Rochester,
NY) and exposed in the dark at 4°C for 2 weeks. After developing, the
sections were counterstained with hematoxylin and eosin. Microscopy was
performed with standard Leica DM RX light microscope (Wetzlar,
Germany).
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Three types of anti-nephrin antibodies were used: i) polyclonal
rabbit antibodies against the extracellular part (pAb 1), ii)
polyclonal rabbit antibodies against the intracellular part (pAb 2),
and iii) a pool of three mouse monoclonal antibodies against the
extracellular domains of nephrin (mAb 1, 2, and 3). The locations of
the nephrin epitopes of these antibodies are shown in Figure 1
.
The preparation of pAb 1 directed against the two terminal extracellular Ig domains of nephrin (amino acids 22–240) has been described earlier.4 The antigen for pAb 2 against the intracellular part of nephrin was prepared as follows. First, the intracellular portion of nephrin was expressed as six-histidine-tagged mouse dihyrofolate reductase fusion protein in Escherichia coli using QIAexpressionist expression system (Qiagen, Hilden, Germany). Then, a DNA fragment coding for amino acids 1084–1241 of human nephrin was amplified by polymerase chain reaction and cloned into vector PUC18 with Sureclone ligation kit (Pharmacia, Uppsala, Sweden). The fragment was cleaved from the vector using cleavage sites for BgIII and HindIII synthesized into polymerase chain reaction primers. The cleaved fragment was ligated into BgIII/HindIII-digested vector pQE-40. This expression plasmid was transformed into E. coli strain XL-1 blue. The fusion protein was expressed at high levels only as insoluble inclusion bodies. Protein purification was initiated by lysing bacterial cells by brief sonication and pelleting the inclusion bodies at 10,000 x g for 30 minutes. The pellet was washed with 0.2% sodium deoxycholate in Tris-buffered saline and recentrifugated. Insoluble proteins were solubilized and the fusion protein was purified in 8 mol/L urea using affinity chromatography. The purified intracellular nephrin was used to raise polyclonal antibodies in three New Zealand White rabbits using standard protocols (SVA, Uppsala, Sweden). Antisera titers were measured in enzyme-linked immunosorbent assay using the antigen as capture reagent.
Generation and characterization of the mouse monoclonal anti-nephrin
antibodies (mAb 1, 2, and 3) is described in detail elsewhere
(Ruotsalainen V, Putaala H, Tryggvason K, manuscript in
preparation). These antibodies were derived from a mouse
immunized with a recombinant nephrin produced in human embryonic kidney
A293 cell line. The antigen contained the whole extracellular portion
of the molecule (amino acids 1–1059 fused to a sequence of six
histidines). The epitopes of the three antibodies were characterized.
They recognize the second and eighth extracellular Ig domains and the
fibronectin domain, as shown in Figure 1
. A pool of these antibodies
was used in immunohistochemistry.
The specificity of the anti-nephrin antibodies was confirmed by Western blotting. All preparations (pAb 1, pAb 2, and the pool of mAb 1, 2, and 3) gave an immunoreactive band at the size of nephrin (180 kd) when glomerular extract was used as an antigen (data shown elsewhere).4,20
Rabbit polyclonal antibody, directed against the cytoplasmic slit diaphragm protein ZO-1 was purchased from Zymed Laboratories (San Francisco, CA). Mouse monoclonal anti-P-cadherin antibodies were purchased from Transduction Laboratories (Lexington, KY).
The polyclonal antibodies against the extracellular part of nephrin (pAb 1) were used exclusively for immunoelectron microscopy. The polyclonal antibodies against the intracellular part of nephrin (pAb 2), as well as ZO-1 antibodies, were used in immunoperoxidase and immunofluorescence stainings and in immunoelectron microscopy. The pool of three monoclonal anti-nephrin antibodies (mAb 1, 2, and 3), as well as P-cadherin antibodies, were used only in immunoperoxidase and immunofluorescence stainings.
Immunoperoxidase Staining
Cryosections and deparaffinized sections (3 µm) were incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity, followed by microwave treatment exclusively for paraffin sections in 10 mmol/L citric acid for 10 minutes to improve antibody penetration. Primary antibodies were diluted to PBS and incubated for 2 to 3 hours at room temperature. Amplification of the primary antibody reaction was achieved by incubating the sections with biotinylated anti-rabbit-IgGs and anti-mouse-IgGs (Vector Laboratories, Burlingame, CA) for 30 minutes. This was followed by a complex of avidin and biotinylated peroxidase (Vector Laboratories) for 30 minutes. Each incubation was followed by three 5-minute washes in PBS. The binding was visualized using diaminobenzidine substrate (Dako Corp., Carpinteria, CA), and the tissues were counterstained with Harris’s hematoxylin. Controls included replacement of the primary antibodies with PBS or nonimmune rabbit or mouse IgGs. Microscopic observations were carried out with a standard Leica DM RX light microscope.
Double Immunofluorescence Staining
The free aldehyde groups from cryosections (5 µm) were blocked with glycine-PBS and nonspecific binding sites with 3% bovine serum albumin (BSA) and 1% gelatin in PBS (blocking solution). The antibodies were diluted in the blocking solution and incubations were performed sequentially. The monoclonal anti-nephrin antibodies were incubated overnight at 4°C, and anti-ZO-1 antibodies 2 to 3 hours at room temperature. P-cadherin antibodies were incubated overnight at 4°C, followed by polyclonal anti-nephrin antibodies for 2 to 3 hours at room temperature. Cyanine- (Cy2, Cy3) and rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were incubated for 2 hours. Each incubation was followed by three 5-minute washes with PBS; finally, sections were mounted in Mowiol (Calbiochem, La Jolla, CA). For controls, sections were incubated in PBS or nonimmune rabbit or mouse IgGs instead of a primary antibody. The sections were examined either with a standard Leica DM RX light microscope or Leica TCS NT confocal laser scanning microscope.
Immunoelectron Microscopy
Immunoelectron microscopy was performed on heat-cured LR-white resin sections (London Resin, Besingstoke, UK) as described earlier.4 Briefly, thin sections were collected on Pioloform-carbon-coated nickel grids (Agar Scientific, Stansted, UK) and incubated in the primary antibodies diluted in blocking solution. After incubation in diluted 5- or 10-nm gold-conjugated secondary antibodies in 0.3% BSA-PBS, the sections were post-stained in 1% uranyl acetate. Double labeling for nephrin (visualized by 10 nm gold goat anti-rabbit IgG) and ZO-1 (visualized by 5 nm gold goat anti-rabbit IgG) was performed sequentially on different sides of thin resin sections, as described earlier.21 For this, thin sections were collected into uncoated grids. Thus, any possible interaction between the two sets of labeling reactions were avoided. Post-staining with 1% uranyl acetate was performed on only one side of the grid. Controls included incubation of grids after the same procedure but incubating with a nonimmune rabbit IgG as a primary antibody. The sections were examined with a Jeol 1200 EX electron microscope (Tokyo, Japan) at 60 kV. Note that the fixation and embedding protocol used yielded satisfactory labeling density but very low image contrast. Therefore, intracellular structures were hardly visible, except for the nucleus.
Morphological Electron Microscopy
Electron microscopy was performed according to standard procedures (fixation in 2.5% glutaraldehyde, followed by OsO4 and Epon embedding). Thin sections were post-stained with 1% uranyl acetate and lead. The sections were examined with a Jeol 1200 EX electron microscope at 60kV.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nephrin mRNA was first detected in late S-shaped bodies as studied
by in situ hybridization of 13- to 23-week human fetal
kidney samples (Figure 2, C and D)
. The
signal was localized to the area next to vascular cleft. Earlier stages
of glomerulogenesis (vesicle, comma-shaped, and early S-shaped bodies)
remained negative for nephrin mRNA (Figure 2, A and B)
. All glomeruli
of capillary loop and maturing stages were strongly positive for
nephrin mRNA. The signal was most intense at early capillary loop stage
(Figure 2, E and F)
. In situ hybridization experiments with
nephrin sense riboprobe, used as controls, revealed only background
staining (data not shown).
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. At capillary
loop stage, nephrin was present along the basal margins and also as
discrete lines between the developing podocytes (Figure 3B)
. Moreover,
intensely stained dots could be detected at lateral surfaces of
adjacent developing podocytes (Figure 3C)
. These were found in 36%
(25/70) of the analyzed capillary stage glomeruli. In maturing stage
glomeruli, nephrin reactivity was present as a strong continuous line
at the basal margin of podocytes (Figure 3D)
. Nonimmune rabbit and
mouse sera, used as controls, did not show glomerular reactivity
(Figure 3E)
.
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.
In more mature glomeruli, nephrin was concentrated at the slit
diaphragm area (Figure 4, E and F)
. Earlier stages, ie, S-shaped
bodies, could not be identified in these samples.
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The two fetuses aborted due to NPHS1 suspicion were homozygous for
Fin-major mutation. This mutation is a 2-bp deletion in exon 2, causing
a frameshift and a translational stop at the end of exon 2, leading to
a truncated nephrin protein of only 90 amino acids (Figure 1)
.3
Although nephrin mRNA was detected in these
kidneys (Figure 2, G and H)
, immunostaining with both polyclonal
antisera failed to show nephrin reactivity (Figure 5, A and B)
.
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. However,
junctions with ladder-like structures were missing in capillary stage
glomeruli of both NPHS1 kidneys. In total, 12 (5 and 7) capillary stage
glomeruli were analyzed in two kidneys. In four normal fetal kidneys,
the analysis of 12 capillary stage glomeruli (3 glomeruli/kidney)
revealed 9 junctions with ladder-like structures (1, 2, 3, and 3
junctions/kidney).
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. The slit pores between the
foot processes were of various sizes. The filamentous structure of the
slit diaphragm, however, was completely missing in 100 filtration slits
analyzed (Figure 7, B and C)
. This is in sharp contrast to what is seen
in normal fetal kidneys, where these filaments were present in over
60% (71/110) of the mature filtration slits studied (Figure 7, D and E)
.
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In normal fetal kidney, ZO-1 was found in visceral and parietal
epithelial cells (Bowman’s capsule) of glomeruli, as well as in
tubular epithelial cells (Figure 8)
. The
expression of ZO-1 in NPHS1 kidneys lacking nephrin was similar to that
of normal fetal kidneys (Figures 5, 6, and 8)
. During glomerulogenesis,
ZO-1 was first detected in late S-shaped glomeruli, colocalizing with
nephrin (Figure 8C)
. At capillary loop stage, ZO-1 colocalized with
nephrin along the basal margins and also between the developing
podocytes (Figure 8D)
. Abundant localization of ZO-1 to cell junctions
at the lateral margins of developing podocytes was evident in
immunoperoxidase staining (Figure 5D)
. The intense dots at junctions of
developing podocytes were observed in over 70% (36/51) of the
capillary loop stage glomeruli.
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. Double-labeling immunoelectron microscopy revealed
colocalization of nephrin and ZO-1 in junctions with the ladder-like
structures (Figure 4, C and D)
.
Monoclonal anti-P-cadherin antibodies gave strong reactivity against
ureteric buds and developing tubuli (Figure 8F)
. Weaker expression of
P-cadherin was detected in developing glomeruli (Figure 5, F
-I, and
Figure 8F
). The expression of P-cadherin in NPHS1 kidney was similar to
that of normal fetal kidney (Figure 5, F
-I). P-cadherin was first seen
at vesicle stage, staining the apical margins of the developing
epithelial cells (Figure 5F)
. In the late S-shaped bodies, P-cadherin
colocalized with nephrin and ZO-1 (Figure 5G)
. At capillary loop stage,
staining for P-cadherin was observed at the basal margins, and to some
extent, also between the developing podocytes (Figure 5H)
. However,
spot-like staining between developing podocytes, as seen in nephrin and
ZO-1 staining, was not observed (Figure 8G)
. In mature stage glomeruli,
intensity for P-cadherin staining was reduced in immunoperoxidase
staining, giving a linear staining pattern along the glomerular
basement membrane (Figure 5I)
. In immunofluorescence staining,
P-cadherin was not detected in mature stage glomeruli (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glomerulogenesis is morphologically divided into four stages: the vesicle, S-shaped body, capillary loop, and maturing stages.16,24 S-shaped glomerulus is the earliest stage at which the presumptive podocytes can be identified.25 At this stage, the developing podocytes are connected at the apical-lateral cell membrane interface by junctional complexes, which subsequently are believed to form the slit diaphragms. The junctions migrate down the cell membrane and reach their most basal location near the glomerular basement membrane during the capillary loop stage. After interdigitation of basolateral surfaces of adjoining cells, the slit diaphragms are formed in maturing stage glomeruli. These electron microscopic findings are supported by data showing that the tight junction protein, ZO-1, first appears in apically located junctions and then migrates down to the level of slit diaphragm.12
The developmental expression of nephrin in rat and mouse has been reported.6,26 The mAb 5–1-6, recognizing an epitope of the extracellular domain of rat nephrin,27 detected nephrin in the S-shaped bodies.26 According to this study, nephrin was present below the tight junctions on the basal and lateral surfaces of the epithelial cells in capillary loop stage glomeruli. Later, the reactivity concentrated at the filtration slits. Kawachi et al26 suggested that nephrin and ZO-1 would reach the slit diaphragm from opposite directions. In mouse, nephrin first appeared in the early capillary loop stage as studied by immunofluorescence staining.6 Using immunoelectron microscopy, nephrin was observed on or near the intercellular junctions between the forming foot processes of the epithelial cells. In contrast, labeling was not found on lateral and apical surfaces above these regions or on basal surfaces facing the underlying glomerular basement membrane.6
In the present work, we found that human nephrin was first expressed in the late S-shaped bodies simultaneously with ZO-1. Interestingly, the signal intensity for nephrin mRNA was strongest in the developing podocytes at the capillary loop stage, before the appearance of foot processes or slit diaphragms. At this stage, nephrin was mostly localized at the basal margin of the developing podocytes, but also on the lateral surfaces as strings and dots. This pattern resembled that of ZO-1. However, in double immunofluorescence staining, the reactivities for nephrin and ZO-1 did not show complete overlapping. The expression of ZO-1 was more abundant at the cellular junctions (the dots) as compared to nephrin. This was also seen in immunoelectron microcopy where ZO-1 localized to junctional complexes as previously reported.12
An interesting finding in immunoelectron microscopy was that both nephrin and ZO-1 were localized at junctions with the ladder-like structures, which are commonly found between the developing podocytes in capillary loop stage glomeruli.16 These structures typically disappear before the appearance of the slit diaphragms in maturing stage glomeruli. The ladder-like structures have also been found in aminonucleoside nephrosis and immune complex-mediated nephritis in animals.28,29 In the nephrotic model, the ladder-like structures were suggested to be redundant slit diaphragms. Absence of ZO-1 in the ladder-like structures in rat nephrosis has also been reported,30 suggesting that ladder-like structures could represent something else. The relationship between these two structures is still unclear.
We found that the slit diaphragms were completely missing in the fetal NPHS1 kidneys lacking the nephrin molecule. Recently, we found the same in kidneys of NPHS1 children nephrectomized at the age of 1 to 2 years.7 The lack of the slit diaphragm already in the fetal NPHS1 kidneys of Fin-major homozygotes strongly favors an idea that this is a primary disorder, not secondary to the long-lasting proteinuria. Interestingly, the early junctions between the developing podocytes at the capillary loop stage seemed normal in electron microscopy. ZO-1 was also normally expressed in these junctions. Thus, nephrin was clearly not needed for the early development and migration of junctional complexes at the S-shaped and capillary loop stages. On the other hand, the formation of the ladder-like structures, as well as the slit diaphragms, seemed to be dependent on the expression of nephrin. It is tempting to speculate that the ladder-like structures represent a fragmented form of tight junctions, which, in normal kidney, differentiate into mature slit diaphragm.
Although nephrin and the slit diaphragms were completely missing in the NPHS1 kidneys, the broadened and irregular podocyte foot processes remained attached to the GBM. This morphological alteration is observed also in other human nephroses and in animal models.31 The molecular basis of this phenomenon is not known. The actin-rich cytoskeleton of the foot processes plays a major role in the maintenance of the podocyte foot process structure.8 These microfilaments are associated with integrins that anchor the foot processes to the glomerular basement membrane. The expression of integrins and the glomerular basement membrane components in NPHS1 kidneys is quite normal32 and could contribute to the maintenance of the podocyte structure in NPHS1. The lateral surfaces of the podocyte foot processes are rich in negatively charged glycoproteins, which probably help to keep the foot processes apart from each other.8 The relative role of the slit diaphragm components and the cell surface glycocalyx for the normal foot process ultrastructure is not known. It is perhaps surprising that lack of nephrin, or even minor mutations in this molecule, have such dramatic effects on the foot process organization.
ZO-1 is a tight junction protein; its presence at the slit diaphragm suggests that this structure is a modified tight junction.12 Recently, however, Reiser et al suggested that the slit diaphragm is a modified adherens junction.15 They located P-cadherin at the slit diaphragm area in podocyte cultures and presented a model, where P-cadherin served as the core protein of the slit diaphragm. Homophilic interactions of P-cadherin would make the bridge between the podocytes. This is in contrast to the model of Ruotsalainen et al,4,9 suggesting that nephrin forms the porous substructure of the slit diaphragm.
In this work, strong reactivity for P-cadherin was observed in ureteric buds, tubular structures, and vesicle stage glomeruli, as reported previously.33,34 All these structures were negative for nephrin. In the S-shaped bodies and capillary stage glomeruli, nephrin, and P-cadherin colocalized to the basal margin of developing podocytes. The staining intensity for P-cadherin was reduced at these stages as compared to vesicle stage glomeruli, and, in contrast to nephrin, P-cadherin did not show dots of staining at the lateral surfaces of developing podocytes. These findings are in line with the immunofluorescence study of human and pig fetal kidneys by Tassin et al.34 The findings suggested that during the polarization process of developing podocytes, P-cadherin was not concentrated to cellular junctions in the same way as nephrin. So far, this conclusion has not been verified at the ultrastructural level, because none of the antibody preparations has given a reliable signal for P-cadherin in immunoelectron microscopy.
The major observation on P-cadherin, however, was that it was expressed normally in NPHS1 kidneys missing the slit diaphragms. This finding favors the idea that nephrin, not P-cadherin, forms the backbone of the slit diaphragm. This is supported by the fact that knockout mice lacking P-cadherin do not develop nephrosis,35 whereas mice not expressing nephrin die due to nephrosis in the first day (Putaala H, Tryggvason K, unpublished data). The fact that point mutations in NPHS1 gene cause massive proteinuria in newborn babies36 further supports the importance of nephrin.
To summarize our results, nephrin is first expressed in late S-shaped body during human glomerulogenesis. Nephrin colocalizes with ZO-1 in the ladder-like structures between differentiating podocytes before the formation of foot processes and mature slit diaphragms. In the kidneys with Fin-major/Fin-major genotype, filamentous ladder-like structures and slit diaphragms are completely missing, whereas ZO-1 and P-cadherin are expressed normally.
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Acknowledgements |
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Footnotes |
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Supported by The Sigrid Jusélius Foundation, The Ulla Hjelt Foundation, Helsinki University Central Hospital Research Fund, The Swedish Medical Research Council, Novo Nordisk Foundation, and NIH grant DK54724.
V. R. and J. P. contributed equally to this work.
Accepted for publication August 14, 2000.
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 H. Pavenstadt, W. Kriz, and M. Kretzler Cell Biology of the Glomerular Podocyte Physiol Rev, January 1, 2003; 83(1): 253 - 307. [Abstract] [Full Text] [PDF]
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M. A. Saleem, L. Ni, I. Witherden, K. Tryggvason, V. Ruotsalainen, P. Mundel, and P. W. Mathieson Co-Localization of Nephrin, Podocin, and the Actin Cytoskeleton : Evidence for a Role in Podocyte Foot Process Formation Am. J. Pathol., October 1, 2002; 161(4): 1459 - 1466. [Abstract] [Full Text] [PDF]
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K. Yan, J. Khoshnoodi, V. Ruotsalainen, and K. Tryggvason N-Linked Glycosylation Is Critical for the Plasma Membrane Localization of Nephrin J. Am. Soc. Nephrol., May 1, 2002; 13(5): 1385 - 1389. [Abstract] [Full Text] [PDF]
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S. Roselli, O. Gribouval, N. Boute, M. Sich, F. Benessy, T. Attie, M.-C. Gubler, and C. Antignac Podocin Localizes in the Kidney to the Slit Diaphragm Area Am. J. Pathol., January 1, 2002; 160(1): 131 - 139. [Abstract] [Full Text] [PDF]
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 L. Liu, S. C. Done, J. Khoshnoodi, A. Bertorello, J. Wartiovaara, P.-O. Berggren, and K. Tryggvason Defective nephrin trafficking caused by missense mutations in the NPHS1 gene: insight into the mechanisms of congenital nephrotic syndrome Hum. |
