Podocyte slit-diaphragm protein nephrin is linked to the actin cytoskeleton

Huaiping Yuan, Emiko Takeuchi, and David J. Salant

Evans Biomedical Research Center, Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-beta -cyclodextrin (Mbeta 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 Mbeta CD in PBS at 37°C. In some cases, Mbeta 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 Mbeta 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+.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Representative Western blots demonstrating association of nephrin and CD2-associated protein (CD2AP) with actin. Glomerular cell membranes were extracted with 1% Triton X-100 (TX-100): at 4°C (A; lanes 1 and 2) or 37°C (lanes 3 and 4) in the presence (lanes 1-4) or absence (lanes 5 and 6) of MgCl2; or at 4°C in the presence of MgCl2 (B; lanes 1-4). Potassium iodide (KI) was included to depolymerize actin (B; lanes 3 and 4). The TX-100-soluble fraction (S) and -insoluble pellet (P) were separated by centrifugation and resolved on 4-20% SDS-PAGE under reducing conditions.

Because CD2AP contains an actin-binding domain (3) and has been shown to bind to the cytoplasmic tail of nephrin (24), we examined whether both proteins could be released from the TX-insoluble pellet by depolymerizing actin. This was achieved by including KI in the extraction buffer (Fig. 1B). Nephrin, CD2AP, and actin were largely insoluble at 4°C in TX-100 in the presence of 1.5 mM MgCl2 (Fig. 1B, lanes 1 and 2). Addition of 1 M KI to the extraction buffer at 4°C partly depolymerized actin and solubilized nephrin, CD2AP, and actin (Fig. 1B, lanes 3 and 4). It is evident that the amount of protein loaded in lanes 3 and 4 of Fig. 1B is greater than in lanes 1 and 2 despite equal-sized aliquots of cell membrane in the starting material. This is because a substantial amount of the TX-insoluble pellet (in the absence of KI) remains insoluble after boiling in SDS sample buffer, whereas most of the sample is solubilized when actin is depolymerized first with KI. Therefore it is impossible to make an accurate quantitative comparison between the extractions with and without KI. Nonetheless, lane 1 of Fig. 1B did not contain any visible bands of nephrin or CD2AP, even after prolonged exposure. The residual nephrin in the detergent-insoluble pellets in lane 6 of Fig. 1A and lane 4 of Fig. 1B might be due to incomplete depolymerization of actin, or it might represent nephrin in lipid rafts. Similar results to those shown in Fig. 1B were obtained by depolymerizing actin with DNase I from bovine pancreas or with 3 mM ATP (not shown).

Sequential fractionation and extraction of glomerular lysates was performed to determine the relative proportions of nephrin in various cellular compartments. The experiment was performed in triplicate, and the amount of sample added to each lane was adjusted to maintain proportionality with the starting material. Sufficient protein was loaded to permit densitometric quantitation of each fraction. As shown in Fig. 2, there is a significant cytoplasmic pool of nephrin (19 ± 1.5%), but the major fraction of nephrin is membrane associated (81 ± 1.5%) and largely insoluble in 1% TX-100 at 4°C (52 ± 2.0%). In accord with the results shown in Fig. 1, 28 ± 2.1% of the membrane fraction remained in the insoluble pellet after extraction with TX-100 and KI. This fraction of nephrin could not be solubilized by further extraction with TX-100 and KI at 4°C (not shown), which suggests that it resides in a detergent-resistant membrane fraction (lipid raft).


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Fig. 2.   Representative Western blot of a sequentially fractionated and extracted glomerular lysate showing the relative distribution of nephrin in cytosolic and membrane fractions. A glomerular lysate was separated into cytosolic and membrane fractions. The cell membranes were extracted with 1% TX-100 at 4°C and separated into TX-100-soluble (TX) and -insoluble fractions. The insoluble fraction was further extracted with 1% TX-100 at 4°C in the presence of KI and separated by centrifugation into a soluble fraction (S) and insoluble pellet (P). The fractions were resolved on 5% SDS-PAGE under reducing conditions. Three different glomerular lysates were analyzed in identical fashion.

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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Fig. 3.   Representative Western blots demonstrating that a fraction of nephrin and CD2AP is recovered in low-density fractions of OptiPrep flotation gradients after release from the actin cytoskeleton. Glomerular cell membranes were extracted with 1% TX-100 at 4°C (A and C) or 37°C (B and D) in the absence (A and B) or presence (C and D) of KI and applied to 0-40% OptiPrep gradients without further separation. Fractions 1-6 represent low (0%) to high (40%) density. Each fraction was resolved on 4-20% SDS-PAGE under reducing conditions.

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 Mbeta CD to deplete cell membranes of cholesterol before staining for nephrin, CD2AP, actin, and CD59. Untreated with KI, TX-100- and Mbeta 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 Mbeta 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 Mbeta 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 Mbeta 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 Mbeta 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 Mbeta 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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Fig. 4.   Representative micrographs documenting that KI depolymerizes F-actin and that methyl-beta -cyclodextrin (Mbeta CD) extracts the raft-associated glycosyl-phosphatidylinositol-linked protein CD59. Unfixed sections of normal rat kidney were treated at 4°C with TX-100 (A and E), TX-100+KI (C and D), and Mbeta CD (B and F) and stained with phalloidin-FITC (A-C) or anti-CD59 (E and F). A and B: F-actin is seen in glomerular capillaries (arrows) and proximal tubular brush borders (arrowheads). KI completely abolished staining for F-actin (C); the arrowheads indicate the glomerulus seen by phase-contrast microscopy (D), and the arrow (C) points to autofluorescence of the elastic lamina of an arteriole (D). CD59 is diffusely present in glomeruli and tubular cells (E) and is almost completely eliminated by cholesterol depletion with Mbeta CD (F). Original magnification, ×400.



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Fig. 5.   Representative micrographs of unfixed normal kidney sections stained for nephrin (A-D) and CD2AP (E and F) after treatment with TX-100 alone (A and E), TX-100+KI (B and F), TX-100+Mbeta CD (C), and TX-100+Mbeta CD+KI (D). Staining for nephrin and CD2AP is unaffected by TX-100 alone (A and E) but is almost completely eliminated by addition of KI (B and F). Nephrin staining intensity is decreased slightly by TX-100+Mbeta CD (C) but is substantially reduced by addition of KI (D). Original magnification, ×400.

The results of IF were further analyzed quantitatively. Compared with PBS alone, TX-100 produced a slight, but significant, decrease in the relative luminosity of nephrin (35.1 ± 5.6 vs. 28.7 ± 5.4, P < 0.05) but had no effect on CD2AP. The effects of temperature and F-actin depolymerization on the TX-100 solubility of nephrin and CD2AP are shown in Table 1. In the presence of TX-100, there was a modest but significant decline in the staining intensity of nephrin at 37°C compared with the values at 4°C. TX-100+KI at 4°C considerably reduced the intensity of nephrin staining, and there was a further decline to background levels when the TX-100+KI extraction was carried out at 37°C. In contrast, staining for CD2AP was greatly reduced by KI, but the incubation temperature had no significant effect. Table 2 shows the effects of cholesterol depletion with Mbeta CD. In the presence of TX-100, Mbeta CD induced a small but significant reduction in nephrin staining compared with TX-100 alone. However, the addition of Mbeta CD+TX-100+KI substantially reduced the intensity of nephrin staining. CD2AP staining was unaffected by Mbeta CD but was markedly reduced by addition of KI.

                              
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Table 1.   Quantitative immunofluorescence for nephrin and CD2AP: effects of temperature and F-actin depolymerization with KI on TX-100 solubility


                              
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Table 2.   Quantitative immunofluorescence for nephrin and CD2AP: effect of cholesterol depletion with Mbeta CD


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Mbeta CD in the absence or presence of KI. Whereas Mbeta CD+TX-100 had only a small but significant effect on the intensity of nephrin staining (Fig. 5C and Table 2), incubation with Mbeta 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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