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Regular Articles |
From the Department of Medicine and Department of Anatomy and
Structural Biology,*
Albert Einstein College of Medicine,
Bronx, New York; the Department of Anatomy and Cell
Biology,
University of Heidelberg, Heidelberg,
Germany; and the Department of Bacteriology and
Immunology,
University of Helsinki,
Helsinki, Finland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The importance of the filtration barrier is reflected by a number of human diseases in which the filtration barrier function is disrupted resulting in the loss of plasma proteins into the urine. Patients with persistent proteinuria ultimately develop a nephrotic syndrome with a variety of symptoms including edema, hypoalbuminemia, and hyperlipidemia.4
Because of the lack of precise molecular targets, the filtration barrier, and the slit diaphragm in particular, has not been easy to approach experimentally. This has, however, changed remarkably by the identification of NPHS1 as the gene responsible for the massive proteinuria in patients with congenital nephrotic syndrome of the Finnish type.5 NPHS1 encodes for nephrin, a transmembrane protein of the immunoglobulin superfamily, expressed particularly in glomerular podocytes. It was subsequently localized primarily to the slit diaphragm by immunoelectron microscopy.6-8 Together with the fact that congenital nephrotic syndrome of the Finnish type patients lacking functional nephrin fail to develop intact slit diaphragms, these findings have led to the conclusion that nephrin is a key component of the slit diaphragm.9
The function of nephrin at the slit diaphragm is so far not very well understood. Based on its structure, nephrin has been proposed to oligomerize in a homophilic manner between neighboring foot processes.7 Furthermore, the existence of tyrosine residues in the intracellular domain implicates a role of nephrin in signal transduction events. But experimental data are lacking. Likewise the involvement of nephrin in mechanisms leading to foot process effacement, the stereotypical morphology in proteinuric states, has not been addressed in a detailed manner so far. In rat models, these pathological events can be induced very rapidly. For instance, perfusion of the kidney with polycations such as protamine sulfate can induce foot process effacement including the conversion of slit diaphragms into tight junctions and proteinuria within minutes.10 The slit diaphragms have therefore been proposed to represent very dynamic structures within the podocyte plasma membranes.11,12
As dynamic assemblies in cell membranes, the so-called lipid rafts have gained great significance in recent years.13-15 Lipid rafts are enriched in glycosphingolipids, cholesterol, GPI-anchored proteins, and a variety of signaling molecules. By compartmentalizing cell membranes, they function in a variety of cell biological processes, such as exocytosis, endocytosis, signal transduction, and cell adhesion.
In this study, we addressed the question whether the slit diaphragm as a specialized membrane domain may be organized by lipid rafts. To ensure in vivo relevance of the data, whole rat kidneys or isolated rat glomeruli from normal control rats and from rats with foot process effacement induced by intravenous 27A IgG injection,16 were used in all experiments.
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Materials and Methods |
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In all experiments, female Sprague-Dawley rats (weighing 120 to 170 g) were used. They were provided by the Animal Facilities of the European Molecular Biology Laboratory, Heidelberg, Germany; the University of Heidelberg, Heidelberg, Germany; and the University of Helsinki, Helsinki, Finland. All experimental studies were approved by the respective ethical committees.
Preparation of Total Glomerular Membranes and TX-100 Flotation Experiments from Isolated Rat Glomeruli
Glomeruli from kidneys taken from eight rats were isolated as
previously described.16
After pelleting, they were taken
up in 3 ml of ice-cold homogenization buffer (20 mmol/L Tris-HCl, pH
7.4, 50 mmol/L NaCl plus the proteinase inhibitors chymostatin,
leupeptin, anti-pain, and pepstatin), resuspended 20 times with a
22-gauge needle and homogenized in a Dounce homogenizer on ice. Pure
glomerular membranes were prepared from the homogenate by density
gradient centrifugation as described.17
After
centrifugation for 4 hours (24,000 rpm, 4°C, SW40 rotor; Beckman,
Fullerton, CA), the light membrane fraction (500 µl) from the
5%/30% interface from the 5%/30%/40% Optiprep (Nycomed, Oslo,
Norway) step gradient was collected and used for detergent extraction.
For this, 250 µl was extracted by addition of 250 µl of 0.4%
TX-100 in homogenization buffer on ice for 30 minutes. The TX-100
extract was then split into two TLS-55 tubes (Beckman), adjusted to
40% Optiprep, overlaid with 1.25 ml 30% and 200 µl 0% Optiprep
containing 0.2% TX-100 and centrifuged for 2 hours (55,000 rpm, 4°C,
TLS 55 rotor; Beckman). Six fractions (each 350 µl) were collected
from top to the bottom. For further pelleting experiments, the
fractions were spun in a TLA-100 ultracentrifuge (Beckman) in a TLA-100
rotor for 20 minutes at 80,000 rpm at 4°C (Figure 1B)
, or the fractions were precipitated
directly with 10% trichloroacetic acid and taken up in sample
buffer. Equal volumes were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by
Western blotting. The same protocol was also used for cultured mouse
podocytes18
transiently transfected with the Fugene-6
transfection method (Roche, Mannheim, Germany) with a cDNA construct
containing rat nephrin.19
Cholesterol depletion was
performed by pretreating the total glomerular membranes with 0.2%
saponin (Sigma, St. Louis, MO) for 10 minutes on ice before TX-100
extraction.20
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The first [detergent-resistant membrane (DRM)] containing fraction and the fifth (soluble) fractions of the TX-100 flotation gradients were adjusted to 0.4% SDS and loaded onto a linear 5 to 30% sucrose gradient in homogenization buffer containing 0.2% TX-100 and 0.4% SDS.21 After centrifugation in a SW60 rotor (Beckman) for 16 hours at 40,000 rpm, 500-µl fractions were collected from the top. For Western blot analysis, fractions were precipitated with trichloroacetic acid, resuspended in sample buffer, and separated by SDS-PAGE.
Endoglucosidase H (Endo H) Digestion
One hundred fifty µl of incubation buffer (50 mmol/L phosphate buffer, pH 5 to 6) was added to either 50 µl of the soluble gradient fraction or 50 µl of total glomerular membranes and boiled for 5 minutes. After cooling down to room temperature, 300 µl of 0.75% Nonidet P-40 was added, boiled again for 1 minute, 100 mU Endo H (Roche) was added to the cooled sample and incubated overnight at 37°C in the presence of chymostatin, leupeptin, anti-pain, and pepstatin. Digested proteins were trichloroacetic acid-precipitated with trichloroacetic acid and analyzed by SDS-PAGE and subsequent Western blotting.
Immunoblotting
The following primary antibodies were used: rabbit polyclonal
anti-rat nephrin antiserum with or without Protein A purification as
described previously,19
rabbit polyclonal anti-human ZO-1
(Zymed Laboratories, San Francisco, CA), rabbit polyclonal anti-mouse
CD2AP (courtesy of Dr. Andrey Shaw, Washington University, St. Louis,
MO), rabbit polyclonal anti-human podocalyxin,22
rabbit
polyclonal anti-rat 
3-integrin (Chemicon Int., Temecula, CA), rabbit
polyclonal anti-human caveolin 1 (Santa Cruz Biotechnology, San Diego,
CA), mouse monoclonal anti-human transferrin receptor (Zymed
Laboratories), and mouse monoclonal anti-phosphotyrosine antibody PY20
(Transduction Laboratories, Lexington, KY). All antibodies have been
shown to react with the respective rat proteins. The horseradish
peroxidase-conjugated secondary antibodies were against rabbit and
mouse (Bio-Rad Laboratories, Hercules, CA). Proteins separated by
SDS-PAGE were transferred to nitrocellulose filters using a semi-dry
blotting system (Amersham Pharmacia, Uppsala, Sweden) and after
blocking and incubation with the respective antibodies detected by
enhanced chemiluminescence (Amersham Pharmacia).
In Vivo Injection of Antibodies, Immunohistochemistry, and Immunoprecipitation
Three mg of either the mouse monoclonal 27A IgG3 or control mouse IgG3 (Sigma Chemical Co.) were intravenously injected into rats via the tail vein as described previously.23 At 1 hour after injection, kidneys were removed and processed for immunohistochemistry, immunoprecipitation, or immunoelectron microscopy.
For immunohistochemistry, small pieces of the kidneys were frozen in liquid nitrogen and stored at -70°C until used. Tissues were cut at 4 µm and processed for the antibody stainings as previously described.24 27A IgG3 bound to the surface of podocytes was detected with rhodamine-conjugated IgG (Dynatech, Denkendorf, Germany).
For immunoprecipitation, glomeruli were isolated and homogenized as described above, but in the presence of 0.5 mmol/L of sodium vanadate (Sigma) to block tyrosine phosphatases. Two hundred fifty µl of total glomerular membranes from the 27A-treated and the control-treated kidneys containing equal amounts of proteins were detergent-extracted by the addition of 250 µl of homogenization buffer containing 0.2% TX-100 and 0.5 mmol/L vanadate. Then, they were immunoprecipitated with anti-nephrin antibody overnight at 4°C. Protein A-Sepharose 4B beads (Zymed) were added and incubated for 1 hour at 4°C. The beads were washed three times with the same buffer and resuspended in sample buffer. Immunoblot analysis was performed as above.
For the immunoprecipitation with the 27A IgG3 to isolate rafts from total glomerular membranes or from TX-100 flotation gradient fractions, the same protocol was used.
Electron Microscopy
For unlabeled transmission electron microscopy, isolated rat glomeruli were extracted with 0.2% TX-100 for 30 minutes on ice with or without pretreatment with saponin. After pelleting (10,000 rpm at 4°C), the glomeruli were fixed with 2% glutaraldehyde in phosphate-buffered saline (PBS) for 1 hour at room temperature. After washing with PBS, a modified postfixation and staining technique was used, which minimizes treatment with OsO4 and uses tannic acid as a contrast agent, followed by subsequent dehydration of the specimens in cold acetone.25 All tissues were then embedded in Epon 812 according to standard procedures.
For immunogold labeling, the isolated rat glomeruli were fixed with 2% paraformaldehyde in PBS for 30 minutes at room temperature or rat kidneys were perfusion-fixed in situ. Ultrathin frozen sections were prepared from fixed tissue and then transferred to nickel grids as described previously.24 The anti-nephrin rabbit polyclonal antibody was used at a dilution of 1:100 and goat anti-rabbit IgG coupled to 10-nm colloidal gold (Biocell, Marburg, Germany) at 1:100. The sections were postfixed with 2% glutaraldehyde for 10 minutes and contrasted with OsO4, uranyl acetate, and polyvinyl alcohol. All ultrathin sections were observed under a Philips EM 301 transmission electron microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine which podocyte proteins associate with lipid raft microdomains, rafts were isolated from total rat glomerular membranes based on their insolubility in TX-100 and low buoyant density in Optiprep gradients. In these gradients, raft-associated proteins float to the top as DRM fractions, whereas detergent-soluble proteins or detergent-insoluble protein complexes associated with the cytoskeleton are found in the heavy bottom fractions.
Six fractions of the gradient were collected from the top to the bottom
and analyzed by Western blotting. The slit diaphragm protein nephrin
was found to a significant extent in the DRM fractions, but also in the
heavy fractions, as shown in Figure 1A
. The effect of TX-100 was
further tested by subsequent pelleting of the DRM and the heavy
fractions. Only the DRM-associated nephrin was pelletable (Figure 1B)
.
Peripheral membrane proteins such as CD2AP and ZO-1 that are located to
the intracellular aspect of the slit diaphragm26,27
were
also present in the DRM fractions but not in the same amounts as
nephrin. Especially in the case of CD2AP, the amount of protein found
in the DRM fractions varied between different preparations. Podocalyxin
and 3-integrin, transmembrane podocyte proteins with no relation to
the slit diaphragm, were mostly found in the heavy fractions. The
quality of the gradients was confirmed by the proper localization of
the raft marker protein caveolin-1 and the nonraft marker protein
transferrin receptor,17
respectively (Figure 1A)
.
To further characterize the raft association of nephrin, we
performed the same experiments after pretreating rat glomerular
membranes with saponin. Saponin is a cholesterol-depleting agent, which
destabilizes DRM structures rendering them sensitive to detergents such
as TX-100.20
Under these conditions, nephrin was released
from the DRM fractions (Figure 1C)
. These results show that nephrin
associates with rafts in a cholesterol-dependent manner.
The Endoplasmic Reticulum (ER) Form of Nephrin Does Not Associate with Rafts
In all of the flotation gradients, nephrin showed an interesting
band pattern. In the heavy fractions, a double band was seen in
contrast to a single band in the DRM fractions. A similar double band
in the heavy fractions of a detergent flotation gradient prepared from
Madin-Darby canine kidney cells has been reported previously for the
raft protein prominin.28
In the case of prominin, the
upper band represented the cell-surface form and the lower band the ER
form of the protein, as determined by Endo H treatment. This enzyme
only cleaves N-linked sugar residues synthesized in the ER whereas more
complex N-linked sugar residues added in later compartments of the
secretory pathway are Endo H-resistant. As lipid rafts first assemble
at the level of the Golgi apparatus, the final site of
glycosphingolipid synthesis, Endo H-sensitive proteins are usually not
present in rafts. Because multiple potential N-glycosylation sites are
present in the nephrin sequence,5
we treated the double
band-containing heavy fraction with Endo H. A significant
band shift was seen for the lower band, whereas only a small band shift
occurred for the upper band (Figure 2A)
.
From these results we conclude that the lower band of the doublet found
in different species with different antibodies,6-8,29
represents the Endo H-sensitive ER form of nephrin. This form is
detergent-soluble supposedly because it has not yet been exposed to
rafts.
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It has been suggested that nephrin ectodomains oligomerize through
a homophilic head-to-head assembly at the plasma membrane level of the
slit diaphragm.7
To address the question whether it really
is the slit diaphragm-associated nephrin that partitions into rafts, we
tested whether the raft-associated fraction of nephrin is oligomeric.
Sedimentation velocity centrifugation in a linear sucrose gradient in
the presence of 0.2% TX-100 and 0.4% SDS was used, which separates
protein complexes into several size classes.21
When DRM
fractions and heavy fractions from TX-100 flotation gradients were
analyzed in this manner, nephrin from the DRM fraction sedimented to
the pellet of the gradient corresponding to a protein complex size of
>600 kd (Figure 2B)
. With regard to its resistance against SDS, it can
be assumed that the complex is held together by strong protein-protein
interactions. In contrast, nephrin from the heavy fractions was found
in the middle of the gradient containing protein complexes with
approximately the size of monomeric nephrin (
180 kd).
The Slit Diaphragm Itself Is Detergent-Resistant in a Cholesterol-Dependent Manner
Next, we wanted to show morphologically that the slit
diaphragm itself behaves similarly as nephrin toward detergents such as
TX-100. For this purpose, we extracted isolated rat glomeruli with
0.2% TX-100 for 30 minutes at 4°C, with or without
pretreatment with saponin, as we had been treating total glomerular
membranes earlier. The extracted tissue was then processed for
transmission electron microscopy. As has been shown
previously,1,26,29,30
the slit diaphragms exhibited a
remarkable resistance against TX-100 treatment. Although most podocyte
foot processes detached from the glomerular basement membrane and most
areas on the podocyte plasma membrane were extracted, the slit
diaphragms remained mostly intact (Figure 3B)
. The situation dramatically changed
after saponin pretreatment, which caused a disruption of the slit
diaphragm and overall foot process architecture (Figure 3D)
. Consistent
with a recent study,29
nephrin was still present at the
slit diaphragm of TX-100-extracted rat glomeruli as revealed by
immunoelectron microscopy (Figure 3C)
. We suggest that the
nephrin-based slit diaphragm is TX-100 resistant in a
cholesterol-dependent manner.
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Another biochemical method for the isolation of rafts is
co-immunoprecipitation with anti-ganglioside antibodies.31
Gangliosides are sialic acid-containing glycosphingolipids and, as
such, important components of lipid rafts. For the isolation of rafts
the buffer conditions were chosen that left the rafts intact. Using the
27A antibody against the podocyte-specific 9-O-acetyl
GD332
and a cold TX-100-containing buffer, it was possible
to co-immunoprecipitate the surface form of nephrin (Figure 4A)
. 27A co-immunoprecipitated also other
raft proteins such as caveolin-1, but not nonraft proteins such as the
transferrin receptor (Figure 4A)
. Consistent with these data,
immunogold-labeling of the foot processes with the 27A antibody showed
occasional labeling of the slit diaphragm but also elsewhere in the
podocyte plasma membrane (data not shown). To test whether the
interaction of nephrin and 9-O-acetyl GD3 takes place in
rafts, the DRM fraction and the heavy fraction of the TX-100 flotation
gradient were co-immunoprecipitated with the 27A antibody. Nephrin was
only present in the precipitates from the DRM fraction and cholesterol
depletion disrupted the interaction between nephrin and
9-O-acetyl GD3. After saponin treatment, neither in the DRM
nor in the heavy fraction was nephrin co-immunoprecipitated by the 27A
antibody (Figure 4B)
. Hence, the interaction between nephrin and
9-O-acetyl GD3 is raft-dependent.
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Intravenous injection of the 27A IgG3 into
rats leads to severe morphological changes of podocytes including
effacement-like changes of foot processes and filtration
slits.23
As described before,23
injection of
the antibody induced swelling of the foot processes and obliteration of
the slit diaphragms within 1 hour. The binding of 27A
IgG3 to the podocytes was confirmed by
immunocytochemistry (Figure 5A)
. By
immunogold-labeling we detected the dislocation of nephrin to the
apical pole of the narrowed filtrations slits (Figure 5D)
. In addition,
the intravenous injection of 27A IgG3 into rats
induced tyrosine phosphorylation of nephrin after 1 hour (Figure 5E)
.
Injection of control mouse IgG3 did not cause
tyrosine phosphorylation of nephrin (Figure 5E)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we provide evidence for the association of nephrin with signaling domains on the plasma membrane called lipid rafts in an oligomerized form suggesting slit diaphragm localization. Ultrastructural studies revealed that the slit diaphragm is detergent-resistant in a cholesterol-dependent manner. Furthermore, we immunoisolated nephrin-containing rafts with an antibody against the podocyte-specific 9-O-acetyl GD3 ganglioside. Finally, we showed that the in vivo injection of this antibody not only induced morphological changes of the filtration slits but also apical dislocation and tyrosine phosphorylation of nephrin.
The assembly of nephrin complex commences when the podocyte precursors transdifferentiate from typical polarized epithelial cells of the S-shaped body to more mature mesenchymal cells of the capillary loop stage.8,29 The hallmark of these events is the downward migration of apical tight junctions along with their conversion into slit diaphragms.1,26 A retrograde conversion is observed in pathological situations in humans extensively studied with the use of rat experimental models.4,10-12,16,33-35 In particular in the protamine sulfate and the puromycin aminonucleoside models, these events were shown to depend on energy, calcium, and most importantly, signaling events including tyrosine phosphorylation.11
In the bi-directional modulation of slit diaphragms to tight
junctions, raft involvement may be important. A recent report proposed
a role for lipid rafts in the organization of tight junctions and the
regulation of paracellular permeability.36
DRM fractions
of TX-100 flotation gradients from an intestinal epithelial cell line
were shown to contain major pools of ZO-1 and occludin. In contrast to
Madin-Darby canine kidney cells,36
ZO-1 that localized to
the slit diaphragms and tight junctions of podocytes was not found to
be as enriched in the DRM fraction in the present study. Similar to
CD2AP, ZO-1 may partly disassociate from the nephrin complex under the
detergent conditions used here. This might also explain why nephrin in
the heavy fractions of our gradients was shown to be soluble in TX-100
despite its suggested linkage to the cytoskeleton via CD2AP (Figure 1B)
.27,37
A large number of signaling events have been shown to occur in the lipid raft environment.15 Lipid rafts serve as building blocks for the establishment of signal transduction platforms favored by many kinases, in particular doubly acylated kinases of the src family.15 A recurring theme in the building up of these platforms is the oligomerization of proteins. The T-cell receptor is a well-studied example for this.38 Stimulation of T-cell receptor by antigen-presenting cells triggers its oligomerization. This process is accompanied by the recruitment of the T-cell receptor into raft microdomains leading to the engagement into higher order protein complexes. These complexes contain co-stimulatory molecules as well as multiple kinases and their phosphorylation substrates. Whether the functional role of nephrin at the slit diaphragm has similarities with the dual role of co-stimulatory molecules at the immunological synapse in severing cell-cell contacts and transducing signals remains to be explored. Given its structure, nephrin, in fact, has been suggested to play such a dual role.7 As a member of the Ig superfamily, like most of the co-stimulatory molecules, the extracellular part of nephrin contains a number of modules important for cell adhesion function. The intracellular part contains eight tyrosine residues potentially involved in signaling.
We also found in this study that the oligomerized form of nephrin associates with lipid rafts. We do not know at present whether the oligomerization occurs within the raft or is a prerequisite for being recruited into the raft. The latter is supported by in vitro studies using cultured podocytes (data not shown). When overexpressing rat nephrin in these cells we were not able to detect raft association of nephrin. In these cells, nephrin was not found in protein complexes but only in a monomeric form.
Another interesting question is whether the oligomerization takes place only once early in development with the formation of the slit diaphragms by the maturing podocytes.1 Alternatively, nephrin oligomers could assemble and disassemble continuously as a means of regeneration and cleansing of the glomerular filter.
The dynamic assembly and disassembly of nephrin oligomers would require control of activity and energy, which could occur via tyrosine phosphorylation of nephrin as detected after binding of 27A IgG3 to the surface of podocytes. We assume that the 27A antibody effect lies in the cross-linking of raft components as reported for other anti-ganglioside antibodies or other antibodies against raft components.31,39 The cross-linking could induce oligomerization of nephrin regulated by kinases with affinity for nephrin. The signaling effect of the 27A antibody has been confirmed previously using cultured human peripheral blood mononuclear cells as a model in which the src family kinase, syk, was found to become activated.40
The pathological changes of podocyte morphology induced by the anti-9-O-acetyl GD3 antibody are difficult to interpret. Because the injected rats did not develop significant proteinuria,23 the relation of the effacement seen after binding of the antibody and the effacement produced by the toxic agents protamine sulfate and puromycin aminonucleoside or in human glomerulopathies remains to be established. In any case, apical dislocation similar to that of nephrin after injection of 27A is also seen for other junction proteins such as ZO-1 in PAN or protamine sulfate nephrosis.12 Therefore, it is possible that the manipulation of this raft microdomain, by antibody binding or by charge, represents a general mechanism for foot process effacement. As exampled by congenital nephrotic syndrome of the Finnish type, genetic mutations of protein components within this microdomain lead to similar effects.9 Podocin is another example for a protein that seems to be indispensable for the function of the slit diaphragm. Podocin is a recently identified protein of the raft-associated stomatin family41 whose gene NPHS2 is mutated in families with autosomal-recessive steroid-resistant nephrotic syndrome.42 Interestingly, podocin localizes to the slit diaphragm and associates with the nephrin-containing raft (K Schwarz, M Simons, J Reiser, W Kriz, LB Holzman, AS Shaw, and P Mundel, submitted for publication). It will be interesting to see what other molecules, eg, kinases, can be found in this specialized microdomain and how their function and organization is affected in different pathological situations.
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Acknowledgements |
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Footnotes |
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Supported by the European Union grant "Quality of Life and Management of Living Resources," a grant from the University of Helsinki, and a grant from the Deutsche Forschungsgemeinschaft (SFB352). M. S. was a recipient of an EMBO short-term fellowship.
P.M. and H.H. contributed equally to this work.
Accepted for publication June 11, 2001.
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B. Smeets, H. B. P. M. Dijkman, N. A. J. M. te Loeke, J. P. H. F. van Son, E. J. Steenbergen, K. J. M. Assmann, J. F. M. Wetzels, and P. J. T. A. Groenen Podocyte changes upon induction of albuminuria in Thy-1.1 transgenic mice Nephrol. Dial. Transplant., December 1, 2003; 18(12): 2524 - 2533. [Abstract] [Full Text] [PDF]
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T. B. Huber, B. Hartleben, J. Kim, M. Schmidts, B. Schermer, A. Keil, L. Egger, R. L. Lecha, C. Borner, H. Pavenstadt, et al. Nephrin and CD2AP Associate with Phosphoinositide 3-OH Kinase and Stimulate AKT-Dependent Signaling Mol. Cell. Biol., July 15, 2003; 23(14): 4917 - 4928. [Abstract] [Full Text] [PDF]
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H. Ahola, E. Heikkila, E. Astrom, M. Inagaki, I. Izawa, H. Pavenstadt, D. Kerjaschki, and H. Holthofer A Novel Protein, Densin, Expressed by Glomerular Podocytes J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1731 - 1737. [Abstract] [Full Text] [PDF]
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 R. Verma, B. Wharram, I. Kovari, R. Kunkel, D. Nihalani, K. K. Wary, R. C. Wiggins, P. Killen, and L. B. Holzman Fyn Binds to and Phosphorylates the Kidney Slit Diaphragm Component Nephrin J. Biol. Chem., May 30, 2003; 278(23): 20716 - 20723. [Abstract] [Full Text] [PDF]
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G.-M. Barletta, I. A. Kovari, R. K. Verma, D. Kerjaschki, and L. B. Holzman Nephrin and Neph1 Co-localize at the Podocyte Foot Process Intercellular Junction and Form cis Hetero-oligomers J. Biol. Chem., May 23, 2003; 278(21): 19266 - 19271. [Abstract] [Full Text] [PDF]
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T. B. Huber, M. Schmidts, P. Gerke, B. Schermer, A. Zahn, B. Hartleben, L. Sellin, G. Walz, and T. Benzing The Carboxyl Terminus of Neph Family Members Binds to the PDZ Domain Protein Zonula Occludens-1 J. Biol. Chem., April 4, 2003; 278(15): 13417 - 13421. [Abstract] [Full Text] [PDF]
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P. Gerke, T. B. Huber, L. Sellin, T. Benzing, and G. Walz Homodimerization and Heterodimerization of the Glomerular Podocyte Proteins Nephrin and NEPH1 J. Am. Soc. Nephrol., April 1, 2003; 14(4): 918 - 926. [Abstract] [Full Text] [PDF]
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J. A. Kreidberg Podocyte Differentiation and Glomerulogenesis J. Am. Soc. Nephrol., March 1, 2003; 14(3): 806 - 814. [Full Text] [PDF]
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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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P. Mundel and S. J. Shankland Podocyte Biology and Response to Injury J. Am. Soc. Nephrol., December 1, 2002; 13(12): 3005 - 3015. [Full Text] [PDF]
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M. R. Pollak Inherited Podocytopathies: FSGS and Nephrotic Syndrome from a Genetic Viewpoint J. Am. Soc. Nephrol., December 1, 2002; 13(12): 3016 - 3023. [Full Text] [PDF]
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J. Sorensson, W. Fierlbeck, T. Heider, K. Schwarz, D. S. Park, P. Mundel, M. Lisanti, and B. J. Ballermann Glomerular Endothelial Fenestrae In Vivo Are Not Formed from Caveolae J. Am. Soc. Nephrol., November 1, 2002; 13(11): 2639 - 2647. [Abstract] [Full Text] [PDF]
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T. Palmen, S. Lehtonen, A. Ora, D. Kerjaschki, C. Antignac, E. Lehtonen, and H. Holthofer Interaction of Endogenous Nephrin and CD2-Associated Protein in Mouse Epithelial M-1 Cell Line J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1766 - 1772. [Abstract] [Full Text] [PDF]
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J. Reiser, G. von Gersdorff, M. Simons, K. Schwarz, C. Faul, L. Giardino, T. Heider, M. Loos, and P. Mundel Novel concepts in understanding and management of glomerular proteinuria Nephrol. Dial. Transplant., June 1, 2002; 17(6): 951 - 955. [Full Text] [PDF]
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M. Rantanen, T. Palmen, A. Patari, H. Ahola, S. Lehtonen, E. Astrom, T. Floss, F. Vauti, W. Wurst, P. Ruiz, et al. Nephrin TRAP Mice Lack Slit Diaphragms and Show Fibrotic Glomeruli and Cystic Tubular Lesions J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1586 - 1594. [Abstract] [Full Text] [PDF]
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H. Yuan, E. Takeuchi, G. A. Taylor, M. McLaughlin, D. Brown, and D. J. Salant Nephrin Dissociates from Actin, and Its Expression Is Reduced in Early Experimental Membranous Nephropathy J. Am. Soc. Nephrol., April 1, 2002; 13(4): 946 - 956. [Abstract] [Full Text] [PDF]
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 J. H. Miner Focusing on the Glomerular Slit Diaphragm : Podocin Enters the Picture Am. J. Pathol., January 1, 2002; 160(1): 3 - 5. [Full Text] [PDF]
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 H. Yuan, E. Takeuchi, and D. J. Salant Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton Am J Physiol Renal Physiol, April 1, 2002; 282(4): F585 - F591. [Abstract] [Full Text] [PDF]
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