Evans Biomedical Research Center, Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118
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ABSTRACT |
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Nephrin is an Ig-like transmembrane
protein. It is a major component of the podocyte slit diaphragm and is
essential for maintaining normal glomerular permeability.
CD2-associated protein (CD2AP) is also necessary for normal glomerular
permeability and is a putative nephrin adapter molecule. Here, we
document that nephrin and CD2AP are linked to the actin cytoskeleton.
As detected by Western blot analysis, nephrin and CD2AP were both
insoluble when cell membranes from normal rat glomeruli were extracted
with 0.5% Triton X-100 (TX-100) at 4°C in the presence of divalent
cations, but they were solubilized when the extraction included
potassium iodide (KI) to depolymerize F-actin. In addition, a small
fraction of the solubilized nephrin and CD2AP was recovered in the
low-density fractions of OptiPrep flotation gradients, which indicates
that a portion of nephrin, possibly associated with CD2AP, resides in a
cholesterol- or sphingolipid-rich region of the plasma membrane. Immunofluorescent staining of unfixed sections of normal rat kidney for
nephrin, CD2AP, and F-actin was unaltered by treatment with TX-100 but
was greatly diminished by addition of KI. Nephrin staining was slightly
reduced by cholesterol depletion with methyl--cyclodextrin in the
presence of TX-100 but was nearly absent after addition of KI. These
results document that nephrin anchors the slit diaphragm to the actin
cytoskeleton, possibly by linkage to CD2AP, and that nephrin traverses
a relatively cholesterol-poor region of the podocyte plasma membrane.
In addition, a small pool of actin-associated nephrin and CD2AP resides
in lipid rafts, possibly in the cholesterol-rich apical region of the
podocyte-foot processes.
glomerulus; kidney; CD2-associated protein; proteinuria; membrane lipids; lipid rafts; detergent-resistant membranes
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INTRODUCTION |
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IT IS 27 YEARS SINCE Rodewald and Karnovsky's (19) classic description of the slit diaphragm as a zipper-like membrane with a central rodlike structure connected to neighboring podocyte-foot processes by horizontal bars that border rectangular pores, but only very recently has information on its function, composition, and possible molecular structure begun to emerge. Thus it is now generally accepted that the slit diaphragm is the final barrier to plasma protein filtration, and several proteins that contribute to its composition have been identified.
Nephrin, a member of the Ig-superfamily (Ig-SF) of transmembrane cell adhesion molecules, is a major structural component of the slit diaphragm (20). It is mutated in certain forms of congenital nephrotic syndrome (11, 20), and it is also the target of mAb 5-1-6, a nephritogenic monoclonal antibody that binds to an epitope of nephrin in the slit diaphragm of rats (10, 16, 27). CD2-associated protein (CD2AP), a ubiquitous adapter that appears to link Ig-SF membrane proteins to the actin cytoskeleton (3), is essential for normal glomerular permeability and was found to bind to the cytoplasmic tail of nephrin when expressed in a heterologous cell system (24). Other proteins, including P-cadherin (18), the protocadherin, FAT (8), podocin (7), and zonula occludens-1 (23), may also contribute to the slit diaphragm and its connection to the cytoskeleton; however, the molecular interactions that govern the assembly of these various subunits into a functional filter remain largely unknown.
We have found that substantial amounts of nephrin and CD2AP remain insoluble after glomerular cell membranes are extracted with low concentrations of nonionic detergents in the cold. This might signify that the proteins are complexed with the cytoskeleton or that nephrin resides in so-called lipid rafts, a fraction of membrane lipids that are rich in cholesterol and sphingolipids and are relatively insoluble in nonionic detergents at 4°C (14, 26). In fact, these are not mutually exclusive possibilities, and there is reason to believe that nephrin may be linked to actin and reside in lipid rafts (26, 27). Although many lipid raft-associated proteins are anchored to the outer plasma membrane by glycosyl-phosphatidylinositol or to the inner leaflet by acylation, palmitoylation, or direct association with cholesterol, there are several examples of raft-associated transmembrane proteins (25). Indeed, the situation with nephrin may be analogous to CD2, another Ig-SF protein that resides in lipid rafts and is linked to the T cell cytoskeleton by CD2AP (3, 30).
In this study, we examined the detergent-insoluble fraction of glomerular cell membranes to determine whether nephrin and CD2AP are associated with the actin cytoskeleton and/or lipid rafts. We found that nephrin and CD2AP became detergent soluble when conditions favored the depolymerization of filamentous (F)-actin. In addition, after release from the actin cytoskeleton, a fraction of nephrin became buoyant in density gradients, which suggests that it also is associated with lipid rafts.
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EXPERIMENTAL PROCEDURES |
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Animals, antibodies, and reagents.
Rabbit antibody to the complete cytoplasmic domain of mouse nephrin
(6) was a gift from Dr. Larry Holzman (University of Michigan, Ann Arbor, MI) and was used for all Western blots. A rabbit
anti-nephrin antibody was produced by immunization with a 21-amino acid
peptide (DRD TRS STV STA EVD PNY YSC) from the COOH terminus of rat
nephrin (Alpha Diagnostics, San Antonio, TX). This peptide is part of
the cytoplasmic tail of rat nephrin as deduced from its cDNA sequence
(9), with the addition of a terminal cysteine to
facilitate conjugation to keyhole limpet hemocyanin. It is conserved
between rats and mice and has low homology to other known proteins. At
dilutions up to 1:5,000, this antibody identified a double band at 185 kDa on Western blot analysis of an extract of rat glomeruli. No other
bands were present. This antibody was used for all immunofluorescence
studies. Rabbit anti-CD2AP was a gift from Dr. Andrey Shaw (Washington
University School of Medicine, St. Louis, MO). Sheep anti-CD59 was from
Dr. Richard Quigg (University of Chicago, Chicago, IL). Anti-caveolin-1 mAb 2297 was from BD Transduction Laboratories (San Diego, CA). Rabbit
anti-rat actin (A2066) and secondary antibodies, goat anti-rabbit IgG-horseradish peroxidase (IgG-HRP; A8275), goat anti-mouse IgG-HRP (A9309), and FITC-conjugated goat anti-rabbit IgG (F0382) were purchased from Sigma-Aldrich (St. Louis, MO). CY3-conjugated goat anti-rabbit IgG (AP132C) and rabbit anti-sheep IgG (AP147C) were from
Chemicon (Temecula, CA). Chemicals and reagents, including methyl--cyclodextrin (M
CD; C4555), were from Sigma-Aldrich unless otherwise stated.
Isolation of glomeruli and preparation of cell membranes.
Glomeruli were isolated from the kidneys of 50 normal adult
Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) or 200 frozen Sprague-Dawley rat kidneys (Pel-Freez, Rogers, AR) by
differential sieving (21) using PBS (10 mM phosphate
buffer, pH 7.4, and 100 mM NaCl) with a cocktail of protease inhibitors (PI; 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor, 4 mM N-ethylmaleimide, and 5 mM benzamidine
hydrochloride). Cell membranes were prepared by using a modification of
the method of Lockwich et al. (13). Glomeruli were
homogenized on ice with a Sonifier cell disrupter (S250A, Branson
Ultrasonics, Danbury, CT) at an output of six and a 50% duty cycle for
3 × 10 bursts with 10-s intervals. The homogenate was diluted in
sucrose buffer containing 0.25 M sucrose, 10 mM Tris-HEPES (pH 7.4),
and PI and centrifuged at 3,000 g for 15 min at 4°C. The
supernatant was centrifuged at 50,000 g for 30 min at 4°C.
The pellet containing glomerular membranes was suspended in the same
buffer and stored at 80°C. The supernatant of the 50,000 g centrifugation containing cytosolic proteins was stored
separately at
80°C.
Detergent extraction of glomerular membranes. Glomerular membranes were thawed on ice and 60-µl aliquots were incubated for 30 min at 4 or 37°C in either 50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA and PI (TNE), or 20 mM phosphate, pH 7.2, 10 mM NaCl, 1.5 mM MgCl2, and PI with 1% Triton X-100 (TX-100; vol/vol). In some cases, 1 M potassium iodide (KI) was included to depolymerize F-actin (1, 13). The extracts were cleared by centrifugation at 148,000 g for 60 min at 4°C. After the detergent-soluble supernatants were removed, the pellets were suspended in equal volumes of the same buffers. The extracts and pellets of the membrane fraction as well as the cytosolic fraction were analyzed by Western blotting.
OptiPrep flotation gradients. OptiPrep flotation gradients were prepared by a modification of established methods (13, 22). Thirty-microliter aliquots of glomerular membranes were extracted in 250 µl of 1% TX-100 with or without 1 M KI at 4 or 37°C in TNE buffer for 30 min. Each sample was mixed with 500 µl of 60% (40% final concentration) OptiPrep (Nycomed Pharma, Oslo, Norway) overlayered with 1.2 ml of 30% OptiPrep and 250 µl of the same buffer. Samples were centrifuged at 60,000 g for 2 h at 4°C, and six gradient fractions of 360 µl were collected from the top to the bottom. Proteins were recovered by methanol precipitation (29) and analyzed by Western blotting.
Western blot analysis. Samples were boiled in SDS sample buffer containing dithiothreitol for 5 min and centrifuged, and equal volumes were loaded onto 4-20% SDS polyacrylamide gels (Ready Gel Tris · HCl, Bio-Rad Laboratories, Hercules, CA). Proteins were transferred to nitrocellulose membranes (Osmonics, Westborough, MA), blocked with 6% milk in Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl) with 0.2% Tween 20 (TBST) and immunoblotted with rabbit anti-mouse nephrin (1:3,000) and goat anti-rabbit IgG-HRP (1:5,000) with TBST washes between antibodies. Immunoreactive proteins were identified by enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL) and autoradiography. Sequential immunoblotting of the same membranes with rabbit anti-CD2AP (1:500), anti-actin (1:500), and anti-caveolin-1 (1:500) was facilitated by the different sizes of the three proteins of interest and the specificity of the antibodies. Autoradiographs were scanned into Adobe Photoshop 4.01 (Adobe Systems, Mountainview, CA), and densitometry was measured with Image software (version 1.61, National Institutes of Health, Bethesda, MD).
Immunofluorescence microscopy of detergent-extracted and
cholesterol-depleted kidney sections.
Kidneys were removed from anesthetized, normal Sprague-Dawley rats,
sliced into 3- to 4-mm coronal sections, embedded in Tissue-Tek OCT
Compound (Sakura, Torrance, CA) and snap-frozen at 80°C without prior fixation. Four-micron cryosections were transferred to
Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA), washed with
cold PBS, and treated as follows. Sections were incubated with 1%
TX-100 in PBS at 4 or 37°C for 30 min with or without 1 M KI.
Cholesterol depletion was performed by incubation with M
CD in PBS at
37°C. In some cases, M
CD was followed by extraction with 1%
TX-100 in 25 mM HEPES, pH 7.5, and 150 mM NaCl with or without 1 M KI. The sections were subsequently fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, blocked with 1% BSA in PBS,
and stained with rabbit anti-rat nephrin (1:640) or rabbit anti-CD2AP
(1:400), followed by CY3-conjugated goat anti-rabbit IgG (1:500).
Relevant sections were also stained either with phalloidin-FITC (1:100)
to confirm that KI was effective in depolymerizing F-actin or with
sheep anti-CD59 (1:24) to confirm that M
CD was effective in
depleting the tissues of lipid raft-associated proteins
(5). As a control to ensure that KI did not destroy nephrin immunoreactivity, some sections were prefixed with
paraformaldehyde before treatment with KI. Antibody incubations were at
either room temperature for 1 h or at 4°C overnight. The
sections were examined by epifluorescent microscopy by using a Nikon
40× Plan Apo oil-immersion lens. The images were captured with a Spot
charge-coupled device camera (Diagnostic Instruments, Sterling Heights,
MI) and exported into Adobe Photoshop. All exposure settings were kept constant for each primary antibody. Fluorescence intensity was measured
by outlining the perimeter of eight glomeruli in each section and
reading the luminosity from the Histogram command in the Image
"pull-down" menu in Adobe Photoshop. Precalibration of the
charge-coupled device exposure time ensured that the settings chosen
were in the linear range and well below saturation. Analysis of
variance and Scheffé's F-test were examined with
StatView 512+.
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RESULTS |
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Nephrin and CD2AP are associated with the detergent-insoluble
fraction of glomerular cell membranes.
Nephrin was insoluble when glomerular cell membranes were extracted
with 1% TX-100 at 4°C in the presence of the divalent cation
Mg2+ and was detected almost exclusively in the
TX-insoluble pellet (Fig. 1A,
lanes 1 and 2). This is consistent with linkage to the cytoskeleton and/or association with lipid rafts. Because lipid rafts
are liquid at 37°C, one would expect nephrin to become soluble in 1%
TX-100 at 37°C if this was the only explanation for its insolubility
at 4°C. As shown in Fig. 1A (lanes 3 and 4),
nephrin remained insoluble in TX-100 at 37°C. Nephrin was partly
solubilized by TX-100 at 4°C in the absence of Mg2+ (Fig.
1A, lanes 5 and 6), a condition known to favor
the depolymerization of F-actin. These findings suggest that nephrin is
anchored to the cytoskeleton, but they do not exclude the possibility
that it is also incorporated into lipid rafts.
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Nephrin and CD2AP partition with low-density fractions of
TX-100-treated glomerular cell membranes after depolymerization of
actin.
In addition to their insolubility in low concentrations of TX-100 at
4°C and solubility at 37°C, lipid raft-associated proteins float in
the low-density fractions of OptiPrep gradients (22). In
contrast, detergent-soluble and actin-associated proteins are found in
the high-density fractions at the bottom of the gradients. Nephrin and
CD2AP were mostly located in high-density fractions after treatment of
glomerular cell membranes with 1% TX-100 at 4°C (Fig.
3A). As expected, nephrin and
CD2AP were exclusively located in the high-density fractions after
treatment with TX-100 at 37°C in the absence of KI (Fig.
3B). In contrast, when actin was depolymerized with KI, both
nephrin and CD2AP were found in low- as well as high-density fractions
after treatment with TX-100 at 4°C (Fig. 3C). Caveolin-1,
a marker of lipid rafts, was also detected in the low-density fractions
(Fig. 3C). Notably, nephrin, CD2AP, and caveolin-1 shifted
toward the high-density fractions after incubation in TX-100 at 37°C
with KI (Fig. 3D). It is noteworthy that a small amount of
actin also floated into the low-density fractions with nephrin and
CD2AP after TX-100 treatment at 4°C with KI (Fig. 3C) and
shifted toward high-density fractions at 37°C (Fig. 3D).
These results indicate that a fraction of cell membrane nephrin is
associated with detergent-insoluble lipids and that this fraction is
also bound to actin. In addition, a portion of actin-bound CD2AP is
also associated with lipid rafts, possibly as part of a complex with
nephrin.
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Depolymerizarion of F-actin and extraction of cholesterol alter
nephrin and CD2AP solubility and distribution in tissue sections.
Unfixed cryosections of normal rat kidney were treated with KI to
depolymerize F-actin and with MCD to deplete cell membranes of
cholesterol before staining for nephrin, CD2AP, actin, and CD59.
Untreated with KI, TX-100- and M
CD-treated sections exhibited intense fluorescence for F-actin in glomerular peripheral capillary loops and mesangium as well as tubular brush borders when
stained with phalloidin-FITC (Fig. 4,
A and B). Inclusion of KI in the incubation
buffer abolished phalloidin-FITC staining (Fig. 4, C and
D), which indicates that F-actin was effectively
depolymerized. CD59, a glycosyl-phosphatidylinositol-linked membrane
protein that is known to be present on podocytes, was used as a control for lipid raft-associated proteins (5). The glomeruli of
TX-100-treated sections stained brightly for CD59, and this was largely
eliminated after cholesterol extraction with M
CD (Fig. 4,
E and F). Untreated and TX-100-treated sections
demonstrated bright peripheral capillary loop staining for nephrin in
an interrupted linear pattern with a polyclonal antibody to the
cytoplasmic tail (Fig. 5A).
This staining was markedly diminished by treatment with TX-100+KI (Fig. 5B). Treatment with M
CD+TX-100 slightly reduced the
intensity but did not alter the pattern of staining for nephrin (Fig.
5C). In contrast, depletion of cholesterol with M
CD
followed by extraction with TX-100+KI substantially reduced the
staining intensity and pattern of nephrin (Fig. 5D).
Treatment with TX-100 had no effect on the staining of glomeruli with
anti-CD2AP (Fig. 5E), but the addition of KI greatly reduced
the intensity of staining for CD2AP (Fig. 5F). When the
kidney sections were prefixed with paraformaldehyde and then treated
with KI, staining for nephrin and CD2AP was preserved (not shown),
which indicates that the KI-induced loss of staining in unfixed tissues
was not simply due to altered immunoreactivity. These results further
demonstrate that nephrin and CD2AP are resistant to extraction by
TX-100 at 4°C unless F-actin is depolymerized. The results with
M
CD indicate that cholesterol depletion effectively solubilizes
CD59, a known raft-associated protein, but cholesterol depletion alone
has only a small effect on nephrin. Moreover, the ability to extract
nephrin and CD2AP with TX-100 and KI in the absence of M
CD is
consistent with the results shown in Fig. 3C and suggests
that only a small fraction of membrane-associated nephrin is situated
in lipid rafts.
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DISCUSSION |
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Although the role of the slit diaphragm in regulating glomerular permeability is now recognized, its precise structure and composition have not been fully established. Among the key questions that remain is how the slit diaphragm is attached to the podocyte foot process and anchored in place. This is relevant because the slit diaphragm may be disrupted or dislocated in proteinuric diseases (2, 12, 17). In addition, nephrin shifts from its usual interrupted linear pattern on immunofluorescence to a clustered granular pattern during the development of proteinuria induced with mAb 5-1-6, a monoclonal antibody that identifies an epitope on the external domain of nephrin (9, 27). This feature, together with the presence of putative phosphorylation sites on the internal domain of nephrin, suggests that it may form part of a signaling complex that can respond to external stimuli. In support of this was the recent demonstration that nephrin may exist in specialized cell membrane domains called lipid rafts, which are known to be hosts to a number of cell signaling molecules that cluster when cross-linked by ligand or antibody (26). Furthermore, in cotransfection experiments, Huber et al. (7) showed that nephrin phosphorylation and signaling are facilitated by binding to podocin, a putative membrane-anchored and raft-associated protein that is mutated in late-onset congenital nephritic syndrome.
It has been proposed that the cytoplasmic tail of nephrin, like other transmembrane Ig-SF members, is anchored to the actin cytoskeleton of podocyte foot processes, whereas the external, highly glycosylated, Ig-like domain forms the slit diaphragm and regulates glomerular permeability (28). The finding that CD2AP binds the COOH terminus of nephrin supports this view, because CD2AP is known to posses an actin-binding region and has been shown to link CD2 to the cytoskeleton in lymphocytes (3). However, to date there has been no evidence that nephrin is bound to actin directly or by means of an adapter such as CD2AP. Nephrin itself has no predictable binding domains for actin or other adapter proteins, but its relative detergent insolubility and the fact that we found actin peptides in anti-nephrin immunoprecipitates (27) further suggest that such a link exists.
The studies reported here clearly document that nephrin is bound to actin. Thus nephrin can be released from the detergent-insoluble, actin-containing pellet of glomerular cell membranes under conditions that are known to depolymerize F-actin. These include KI and exclusion of divalent cations, as well as DNase I and ATP (not shown). These same conditions solubilized CD2AP from the cytoskeletal pellet. Thus Fig. 1 shows that nephrin and CD2AP were both solubilized together with actin by KI. This was further demonstrated in tissue sections. Whereas low concentrations of TX-100 alone had no effect on nephrin or CD2AP staining (Fig. 5, A and E), the addition of KI completely abolished F-actin (Fig. 4C) and rendered both nephrin and CD2AP highly extractable with TX-100 (Fig. 5, B and F). Although these studies provide strong evidence that nephrin and CD2AP are associated with glomerular cell actin, it remains uncertain whether CD2AP acts as a scaffolding protein to link nephrin to actin. It remains possible that nephrin attaches independently to CD2AP and actin or that some other adapter is responsible for the nephrin-actin association.
Our results also confirm that at least a portion of membrane-associated
nephrin exists in lipid rafts. The most compelling evidence of this is
shown in Fig. 3, which shows that nephrin was retrieved in low-density,
caveolin-containing fractions of an OptiPrep gradient. However, this
occurred only after it was released from the actin cytoskeleton with KI
at 4°C (compare A and C in Fig. 3), which
indicates that the raft-associated fraction of cell membrane nephrin is
also linked to the cytoskeleton. The phase shift in membrane lipids
induced by raising the extraction temperature to 37°C caused nephrin
to be recovered predominantly in high-density fractions even in the
presence of KI (Fig. 3D), another feature of raft-associated
proteins. It is interesting to note that CD2AP and a small amount of
actin were also recovered from low-density fractions in the presence of
KI at 4°C (Fig. 3C). This suggests the possibility that
nephrin, CD2AP, and monomeric actin floated as a lipid raft-associated
complex after release from polymeric actin. However, we cannot exclude
the possibility that CD2AP itself is attached to the endoplasmic
leaflet of lipid raft domains by acylation or palmitoylation or that it
is bound to some other raft-associated transmembrane protein.
Additional evidence that raft-associated nephrin is linked to actin was
obtained by examining tissue sections that had been depleted of
cholesterol with MCD in the absence or presence of KI. Whereas
M
CD+TX-100 had only a small but significant effect on the intensity
of nephrin staining (Fig. 5C and Table 2), incubation with
M
CD+KI almost completely abolished staining for nephrin (Fig.
5D).
Our findings show that nephrin in normal rat kidney is attached to the cytoskeleton and that the bulk of it traverses a relatively cholesterol-poor, detergent-soluble region of the podocyte plasma membrane. This region probably includes the attachment site of the slit diaphragm. This is consistent with the findings of Orci et al. (15), who showed, by using filipin labeling and freeze fracture electron microscopy, that there is an abrupt fall in the cholesterol content of the podocyte plasma membrane at the level of the slit diaphragm, with the apical membrane having a high level and the basal membrane being relatively depleted of cholesterol. We suggest that a small pool of nephrin normally resides in lipid rafts in the apical membrane adjacent to the slit diaphragm and that the clustering of nephrin seen when proteinuria is induced with mAb 5-1-6 (9) may represent a shift into this pool as nephrin is released from its attachment to actin. This would be in keeping with the observation of Fujigaki et al. (4), who showed, by immunogold electron microscopy, that injected mAb 5-1-6 was localized at the filtration slits at 2 h and by 12 h had moved onto the apical plasma cell membrane of the foot processes, where it formed patch- or caplike clusters. The subsequent fate of the nephrin-mAb 5-1-6 complexes remains uncertain. Our previous studies suggest that endocytosis into lysosomes is one route of disposal (10), but it is possible that the complex may be shed from the plasma membrane or that nephrin may shift back into the slit diaphragm as the antibody dissociates and permeability recovers.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. John H. Schwartz for thoughtful suggestions and review of this manuscript and to Gregory A. Taylor for technical help.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30932.
Address for reprint requests and other correspondence: D. J. Salant, Renal Section, EBRC 504, Boston Univ. Medical Center, 650 Albany St., Boston, MA 02118 (E-mail: djsalant{at}bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00290.2001
Received 17 September 2001; accepted in final form 6 November 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Burtnick, LD.
Tb3+ as a luminescent probe of actin structure: effects of polymerization, KI, and the binding of deoxyribonuclease I.
Arch Biochem Biophys
216:
81-87,
1982[ISI][Medline].
2.
Doublier, S,
Ruotsalainen V,
Salvidio G,
Lupia E,
Biancone L,
Conaldi PG,
Reponen P,
Tryggvason K,
and
Camussi G.
Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.
Am J Pathol
158:
1723-1731,
2001
3.
Dustin, ML,
Olszowy MW,
Holdorf AD,
Li J,
Bromley S,
Desai N,
Widder P,
Rosenberger F,
van der Merwe PA,
Allen PM,
and
Shaw AS.
A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.
Cell
94:
667-677,
1998[ISI][Medline].
4.
Fujigaki, Y,
Morioka T,
Matsui K,
Kawachi H,
Orikasa M,
Oite T,
Shimizu F,
Batsford SR,
and
Vogt A.
Structural continuity of filtration slit (slit diaphragm) to plasma membrane of podocyte.
Kidney Int
50:
54-62,
1996[ISI][Medline].
5.
Hannan, LA,
and
Edidin M.
Traffic, polarity, and detergent solubility of a glycosylphosphatidylinositol-anchored protein after LDL-deprivation of MDCK cells.
J Cell Biol
133:
1265-1276,
1996[Abstract].
6.
Holzman, LB,
St John PL,
Kovari IA,
Verma R,
Holthofer H,
and
Abrahamson DR.
Nephrin localizes to the slit pore of the glomerular epithelial cell.
Kidney Int
56:
1481-1491,
1999[ISI][Medline].
7.
Huber, TB,
Kottgen M,
Schilling B,
Walz G,
and
Benzing T.
Interaction with podocin facilitates nephrin signaling.
J Biol Chem
276:
41543-41546,
2001
8.
Inoue, T,
Yaoita E,
Kurihara H,
Shimizu F,
Sakai T,
Kobayashi T,
Ohshiro K,
Kawachi H,
Okada H,
Suzuki H,
Kihara I,
and
Yamamoto T.
FAT is a component of glomerular slit diaphragms.
Kidney Int
59:
1003-1012,
2001[ISI][Medline].
9.
Kawachi, H,
Koike H,
Kurihara H,
Yaoita E,
Orikasa M,
Shia MA,
Sakai T,
Yamamoto T,
Salant DJ,
and
Shimizu F.
Cloning of rat nephrin: expression in developing glomeruli and in proteinuric states.
Kidney Int
57:
1949-1961,
2000[ISI][Medline].
10.
Kawachi, H,
Kurihara H,
Topham PS,
Brown D,
Shia MS,
Orikasa M,
Shimizu F,
and
Salant DJ.
Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes.
Am J Physiol Renal Physiol
273:
F984-F993,
1997
11.
Kestila, M,
Lenkkeri U,
Mannikko M,
Lamerdin J,
McCready P,
Putaala H,
Ruotsalainen V,
Morita T,
Nissinen M,
Herva R,
Kashtan CE,
Peltonen L,
Holmberg C,
Olsen A,
and
Tryggvason K.
Positionally cloned gene for a novel glomerular proteinnephrin
is mutated in congenital nephrotic syndrome.
Mol Cell
1:
575-582,
1998[ISI][Medline].
12.
Kurihara, H,
Anderson JM,
Kerjaschki D,
and
Farquhar MG.
The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1.
Am J Pathol
141:
805-816,
1992[Abstract].
13.
Lockwich, TP,
Liu X,
Singh BB,
Jadlowiec J,
Weiland S,
and
Ambudkar IS.
Assembly of Trp1 in a signaling complex associated with caveolin-scaffolding lipid raft domains.
J Biol Chem
275:
11934-11942,
2000
14.
London, E,
and
Brown DA.
Insolubility of lipids in Triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts).
Biochim Biophys Acta
1508:
182-195,
2000[ISI][Medline].
15.
Orci, L,
Singh A,
Amherdt M,
Brown D,
and
Perrelet A.
Microheterogeneity of protein and sterol content in kidney podocyte membrane.
Nature
293:
646-647,
1981[ISI][Medline].
16.
Orikasa, M,
Matsui K,
Oite T,
and
Shimizu F.
Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody.
J Immunol
141:
807-814,
1988
17.
Patrakka, J,
Kestila M,
Wartiovaara J,
Ruotsalainen V,
Tissari P,
Lenkkeri U,
Mannikko M,
Visapaa I,
Holmberg C,
Rapola J,
Tryggvason K,
and
Jalanko H.
Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.
Kidney Int
58:
972-980,
2000[ISI][Medline].
18.
Reiser, J,
Kriz W,
Kretzler M,
and
Mundel P.
The glomerular slit diaphragm is a modified adherens junction.
J Am Soc Nephrol
11:
1-8,
2000
19.
Rodewald, R,
and
Karnovsky MJ.
Porous substructure of the glomerular slit diaphragm in the rat and mouse.
J Cell Biol
60:
423-433,
1974
20.
Ruotsalainen, V,
Ljungberg P,
Wartiovaara J,
Lenkkeri U,
Kestila M,
Jalanko H,
Holmberg C,
and
Tryggvason K.
Nephrin is specifically located at the slit diaphragm of glomerular podocytes.
Proc Natl Acad Sci USA
96:
7962-7967,
1999
21.
Salant, DJ,
and
Cybulsky AV.
Experimental glomerulonephritis.
Methods Enzymol
162:
421-461,
1988[ISI][Medline].
22.
Scheiffele, P,
Rietveld A,
Wilk T,
and
Simons K.
Influenza viruses select ordered lipid domains during budding from the plasma membrane.
J Biol Chem
274:
2038-2044,
1999
23.
Schnabel, E,
Anderson JM,
and
Farquhar MG.
The tight junction proten ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.
J Cell Biol
111:
1255-1263,
1990[Abstract].
24.
Shih, NY,
Li J,
Karpitskii V,
Nguyen A,
Dustin ML,
Kanagawa O,
Miner JH,
and
Shaw AS.
Congenital nephrotic syndrome in mice lacking CD2-associated protein.
Science
286:
312-315,
1999
25.
Simons, K,
and
Toomre D.
Lipid rafts and signal transduction.
Nat Rev Mol Cell Biol
1:
31-39,
2000[ISI][Medline].
26.
Simons, M,
Schwarz K,
Kriz W,
Miettinen A,
Reiser J,
Mundel P,
and
Holthofer H.
Involvement of lipid rafts in nephrin phosphorylation and organization of the glomerular slit diaphragm.
Am J Pathol
159:
1069-1077,
2001
27.
Topham, PS,
Kawachi H,
Haydar SA,
Chugh S,
Addona TA,
Charron KB,
Holzman LB,
Shia M,
Shimizu F,
and
Salant DJ.
Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin.
J Clin Invest
104:
1559-1566,
1999
28.
Tryggvason, K.
Unraveling the mechanisms of glomerular ultrafiltration: nephrin, a key component of the slit diaphragm.
J Am Soc Nephrol
10:
2440-2445,
1999
29.
Wessel, D,
and
Flugge UI.
A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.
Anal Biochem
138:
141-143,
1984[ISI][Medline].
30.
Yashiro-Ohtani, Y,
Zhou XY,
Toyo-Oka K,
Tai XG,
Park CS,
Hamaoka T,
Abe R,
Miyake K,
and
Fujiwara H.
Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.
J Immunol
164:
1251-1259,
2000