Nephrin Dissociates from Actin, and Its Expression Is Reduced in Early Experimental Membranous Nephropathy
Huaiping Yuan*,
Emiko Takeuchi*,
Gregory A. Taylor*,
Margaret McLaughlin,
Dennis Brown and
David J. Salant*
*Renal Section and Department of Medicine, Evans Biomedical Research Center, Boston University Medical Center, and Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Correspondence to Dr. David J. Salant, Renal Section, EBRC 504, Boston University Medical Center, 650 Albany St., Boston MA 02118. Phone: 617-638-7330; Fax: 617-638-7326; E-mail: djsalant{at}bu.edu
ABSTRACT. These studies examined the expression of the podocyteslit diaphragm protein nephrin and its association with actinat the onset of proteinuria in passive Heymann nephritis (PHN),a rat model of human membranous nephropathy. Four days afterimmunization, 58% of PHN rats had mild proteinuria. At thattime, most slit diaphragms were still visible on electron microscopy;however, in those locations where the deposits encroached onthe filtration slits, the slit diaphragms were either displacedor absent. On day 7, the PHN rats were severely proteinuric,and most slit diaphragms were either absent, displaced, or replacedby occluding-type junctions. Immunofluorescence microscopy withantibodies to the external and cytoplasmic domains of nephrinshowed a progressive loss of staining and a change in the distributionof nephrin from an interrupted linear pattern in normal controlsto a more dispersed and clustered pattern in PHN. In contrast,the intensity of staining for ZO-1 and CD2-associated protein(CD2AP), two other proteins that are located on the cytoplasmicface of the slit diaphragm, was undiminished. Immunogold electronmicroscopy confirmed the progressive disappearance of nephrinfrom podocyte foot processes and retention of CD2AP. Glomeruliand glomerular cell membranes were extracted sequentially withTriton X-100, followed by DNase I or potassium iodide to depolymerizeactin. Western blot analysis of the extracts showed a progressivedecline of total nephrin on days 4 and 7 of PHN as well as areduction in the actin-associated fraction. These findings showthat nephrin partly dissociates from actin at the onset of podocyteinjury in PHN. This is accompanied by a progressive loss ofnephrin from the podocyte foot processes and prominent changesin the morphology of the slit diaphragms. These events may underliethe loss of podocyte barrier function in membranous nephropathy.
Membranous nephropathy (MN) is an autoimmune disease characterizedby subepithelial immune deposits, effacement of podocyte footprocesses, and expansion of the glomerular basement membrane.The primary clinical manifestation is proteinuria, which isoften associated with nephrotic syndrome. Although the targetantigen/s responsible for human MN are unknown, it is likelythat they reside on the basal plasma membrane of the podocytefoot processes, as in Heymann nephritis, a rat model of MN (1,2).Podocyte injury and proteinuria in the rat model are mediatedby complement C5b-9 (3,4), and it is likely that a similar processis at play in human MN (5).
During the development of proteinuria in passive Heymann nephritis(PHN), as well as in many cases of active MN, the actin cytoskeletonis condensed at the base of the effaced podocyte foot processes(6). In vitro studies have suggested that this is due to C5b-9–mediateddisruption of actin microfilaments and dissolution of integrin-associatedfocal adhesion complexes (7).
Given that the podocyte foot processes and the slit diaphragmsthat bridge the intervening filtration slits form the finalbarrier to macromolecular permeability, it is not surprisingthat severe podocyte injury in PHN and MN would give rise toproteinuria. Yet the actual mechanisms responsible for proteinuriain these conditions remain uncertain. Possibilities includefocal areas of detachment of foot processes from the glomerularbasement membrane or disruption of the slit diaphragms. Althoughpodocyte detachment during the acute phase of injury has notbeen entirely excluded as a cause of proteinuria, there is goodreason to examine a role for the slit diaphragm more closely.Morphological alterations in the filtration slits have beenwell described in human and experimental MN (8–10). Inaddition, several investigators have examined the integrityof the slit diaphragm in acquired proteinuric diseases, usingthe slit diaphragm protein nephrin as a marker. Thus, it hasbeen reported that nephrin mRNA levels are reduced in the glomeruliof patients with MN (11), and it has been recently reportedthat nephrin is reduced in amount and altered in distributionin patients with MN (12). In addition, Benigni et al. (13) reportedthat the glomeruli of rats with PHN show a progressive declinein nephrin mRNA and protein content from 1 wk to 8 mo afterdisease induction, which is preventable by angiotensin II blockade.At the stage at which all these observations were made, however,the disease was well established. Therefore, it is impossibleto determine whether the observed changes in nephrin were theprimary cause of proteinuria or the result of advanced podocyteinjury. This question is examined best at the onset of podocyteinjury and proteinuria, which can be predicted precisely inthe PHN model.
Nephrin is a member of the Ig superfamily of transmembrane celladhesion molecules (14). It is the protein that is mutated incertain forms of congenital nephrotic syndrome and is knownto reside in the slit diaphragm (14,15). CD2-associated protein(CD2AP) is a ubiquitous adapter that appears to link Ig superfamilymembrane proteins to the actin cytoskeleton (16). It is essentialfor normal glomerular permeability and has been found to bindthe cytoplasmic tail of nephrin when it is expressed in a heterologouscell system (17). We have also shown that nephrin is associatedwith the podocyte actin cytoskeleton (18). Therefore, consideringthat the actin cytoskeleton is disrupted by complement-mediatedpodocyte injury (7), we examined the glomeruli of rats withPHN to determine whether the expression of nephrin and its associationwith actin are altered at the onset of proteinuria.
Experimental Protocol
Sheep anti-Fx1A was prepared as described elsewhere (19). PHNwas induced in 180 to 200 g adult male Sprague Dawley rats (CharlesRiver Laboratories, Wilmington, MA) by two intravenous injectionsof anti-Fx1A, 0.25 and 0.5 ml, on successive days. Controlswere injected with an equal amount of normal sheep globulin.Urine was collected from all rats overnight in individual metabolismcages on days 3 to 4 and 6 to 7 after the first injection andanalyzed for protein by a sulfosalicylic acid method, as describedelsewhere (19). On days 4 and 7, the kidneys were removed underanesthesia. A 3- to 4-mm coronal slice of cortex was embeddedin Tissue-Tek O.C.T. Compound (Sakura, Torrance, CA) and snap-frozenat -80°C without prior fixation for immunofluorescence.The rest was frozen immediately in liquid nitrogen and storedat -80°C until used for glomerular isolation (vide infra).The kidneys of three additional control and PHN rats were perfusionfixed in situ with periodate-lysine-paraformaldehyde (20) ondays 4 and 7 for ultrastructural studies.
Antibodies and Reagents
Rabbit antibody to the complete cytoplasmic domain of mousenephrin (21) was a gift from Dr. Larry Holzman (University ofMichigan, Ann Arbor, MI) and was used for Western blots. A rabbitanti-nephrin antibody was produced by immunization with a 21–aminoacid peptide (DRD TRS STV STA EVD PNY YSC) from the C terminusof rat nephrin (Alpha Diagnostics, San Antonio, TX). This peptideis part of the cytoplasmic tail of rat nephrin as deduced fromits cDNA sequence (22), with the addition of a terminal cysteineto facilitate conjugation to keyhole limpet hemocyanin. It isconserved between rats and mice and has low homology to otherknown proteins. At dilutions up to 1:5000, this antibody identifieda double band at 185 kD on Western blot analysis of an extractof rat glomeruli. No other bands were present. This antibodywas used for immunofluorescence studies. A monoclonal antibodythat identifies an epitope in the extracellular domain of ratnephrin, mAb 5–1-6 (23), was kindly supplied by Drs. HiroshiKawachi and Fujio Shimizu (Niigata University, Niigata, Japan).Rabbit anti-CD2AP provided by Dr. Andrey Shaw (Washington UniversitySchool of Medicine, St. Louis, MO) was used for Western blots,and rabbit anti-CD2AP from Santa Cruz Biotechnology, Inc. (SC9137;Santa Cruz, CA) was used for immunofluorescence. Rabbit anti–ZO-1was from Zymed Laboratories, Inc. (South San Francisco, CA).Rabbit anti-rat actin (A2066) and secondary antibodies—goatanti-rabbit IgG–horseradish peroxidase (A8275) and fluorescein-conjugatedgoat anti-rabbit IgG (F0382)—were purchased from Sigma-Aldrich(St. Louis, MO). CY3-conjugated goat anti-rabbit IgG (AP132C),donkey anti-mouse IgG (AP129C), and rabbit anti-sheep IgG (AP147C)were from Chemicon (Temecula, CA). Chemicals and reagents, includingDNase I (EC 3.1.21.1, from bovine pancreas, D4263) were fromSigma-Aldrich unless stated otherwise.
Isolation of Glomeruli
Glomeruli were isolated from individual rat kidneys by differentialsieving (19) that used phosphate-buffered saline (PBS, 10 mMphosphate buffer [pH 7.4] and 100 mM NaCl) with a cocktail ofprotein inhibitors (PI) (1 mM phenylmethylsulfonyl fluoride,5 µg/ml soybean trypsin inhibitor, 4 mM N-ethylmaleimide,and 5 mM benzamidine hydrochloride).
Detergent Extraction of Glomeruli and Depolymerization of Actin with DNase I
The isolated glomeruli from each animal were extracted on icefor 30 min with extraction buffer composed of 0.5% Triton (Tx-100)in 20 mM phosphate buffer (pH 6.2), 10 mM NaCl, and 1.5 mM MgCl2.The Tx-insoluble material was removed by centrifugation at 15,000x g at 4°C for 10 min and then incubated with 100 µg/mlDNase I in PBS at 37°C for 30 min to depolymerize F-actin(24). The soluble proteins released by DNase I were separatedfrom the DNase I–insoluble fraction by centrifugationat 15,000 x g at 4°C for 10 min. The protein concentrationof the Tx-100 extracts was measured with the BCA protein assay(Pierce Inc., Rockford, IL). The volume of each sample was adjustedso that 30 µg of the Tx-100 extracts and equivalent volumesof the DNase I–soluble fractions were analyzed by Westernblotting.
Preparation of Cell Membranes
Cell membranes were prepared by use of a modification of themethod of Lockwich et al. (25). Glomeruli were pooled from threerats, and 1-ml aliquots of each pool were frozen overnight inlysis buffer (100 mM Tris-HCl [pH 8.0], 1 mM MgCl2, and PI),thawed, and homogenized on ice with a Sonifier Cell Disrupter(S250A, Branson Ultrasonics Corp., Danbury, CT) at output 6and duty cycle 50% for 3 x 10 bursts with 10-s intervals. A0.4-ml aliquot of each homogenate was diluted in an equal volumeof 2x homogenizing buffer (0.5 M sucrose, 20 mM Tris-Hepes [pH7.4], 2 mM dithiothreitol, and 2x PI) and centrifuged at 3000x g for 15 min at 4°C to remove cellular debris. The supernatantswere ultracentrifuged at 50,000 x g for 30 min at 4°C toseparate glomerular cell membranes (pellet) from cytosolic proteins(supernatant). The cell membranes were resuspended in the samebuffer, and both preparations were stored at -80°C.
Extraction of Glomerular Cell Membranes
Glomerular membranes were extracted sequentially as follows.Aliquots of 60 µl from each pool were thawed on ice, sonicated,and incubated for 30 min at 4°C with 1% Tx-100 in 88 µlof 20 mM phosphate (pH 6.2), 10 mM NaCl, 1.5 mM MgCl2, and PI.The Tx-soluble extract was separated from the residual insolublemembranes by centrifugation at 50,000 x g for 30 min at 4°C.The pellet was then reextracted for 30 min at 4°C in 1%Tx-100 extraction buffer with the addition of 1 M potassiumiodide (KI) to depolymerize F-actin (25,26). The supernatantscontaining proteins released by KI were separated by centrifugationat 145,000 x g for 30 min at 4°C, and the residual pelletswere suspended by sonication in an equal volume of PBS. Allsamples were stored at -80°C. The protein concentrationof the Tx-100 extracts was measured, and the volume of eachsample was adjusted so that 16 µg of the Tx-100 extractsand equivalent volumes of the cytosolic and KI-soluble fractionsand insoluble pellets were analyzed by Western blotting.
Western Blot Analyses
Samples were boiled for 5 min in sodium dodecyl sulfate samplebuffer that contained 2% dithiothreitol, centrifuged, and equalamounts loaded onto 4% to 20% sodium dodecyl sulfate polyacrylamidegels (Ready Gel Tris-HCl, Bio-Rad Laboratories, Hercules, CA).Proteins were transferred to nitrocellulose membranes (OsmonicsInc., Westborough, MA), blocked with 6% milk in Tris-bufferedsaline with Tween 20 (50 mM Tris (pH 7.6), 150 mM NaCl, and0.2% Tween 20) and immunoblotted with rabbit anti-mouse nephrin(1:3000) and goat anti-rabbit IgG–horseradish peroxidase(1:5000) with Tris-buffered saline with Tween 20 washes betweenantibodies. Immunoreactive proteins were identified by enhancedchemiluminescence (SuperSignal, Pierce Inc.) and autoradiography.Sequential immunoblotting of the same membranes with rabbitanti-CD2AP (1:500) and anti-actin (1:500) was facilitated bythe different sizes of the three proteins of interest and thespecificity of the antibodies. Each immunoreactive band wasscanned from the autoradiographs into Adobe Photoshop 5.0 (AdobeSystems Inc.), and the densities were measured with NIH Image1.61. The nephrin and CD2AP band densities were normalized byuse of the corresponding actin bands.
Immunofluorescence Microscopy
Four-micron cryosections of control and day 4 and 7 PHN kidneyswere transferred to Superfrost/Plus slides (Fisher Scientific,Pittsburgh, PA), washed with cold PBS, blocked with 1% bovineserum albumin in PBS, and stained with rabbit anti-rat nephrin(1:640), rabbit anti-ZO-1 (1:100), rabbit anti-CD2AP (1:400),or mAb 5–1-6 (1:100), followed by CY3-conjugated goatanti-rabbit IgG (1:500) or donkey anti-mouse IgG (1:800). Thesections were examined by epifluorescent microscopy that useda Nikon 40x Plan Apo oil-immersion lens. The images were capturedwith a Spot CCD camera (Diagnostic Instruments, Inc., SterlingHeights, MI) and exported into Adobe Photoshop. All exposuresettings were kept constant for each group of kidneys. Fluorescenceintensity was measured by outlining the perimeter of 5 to 7glomeruli in each section and reading the luminosity from theHistogram command in the "Image" pull-down menu in Adobe Photoshop.Calibration of the CCD exposure time assured that the settingschosen were in the linear range and well below saturation.
Transmission Electron Microscopy
Perfusion-fixed, 1-mm3 blocks of cortex were postfixed with1% osmium in 0.1 M cacodylate buffer for 1 h, dehydrated ingraded ethanols, embedded in Epon, sectioned, and stained withuranyl acetate and lead citrate for transmission electron microscopy.Ultrathin sections were examined and photographed with a PhilipsCM10 electron microscope (Philips Electronic Instruments Co.,Mahwah, NJ).
Immunoelectron Microscopy
The cortex of perfusion-fixed kidneys was cut into 1-mm3 blocks,washed with PBS, dehydrated in graded ethanols (30%, 50%, and70%), and embedded in LR White resin (catalog number 14381,Electron Microscopy Sciences, Ft. Washington, PA). Ultrathinsections were transferred to Formvar-coated nickel grids, blockedwith 1% bovine serum albumin and 1% normal goat serum in PBS,incubated with anti-nephrin (1:100) or anti-CD2AP (1:50) inDAKO antibody diluent (catalog number S3022, DAKO Corporation,Carpinteria, CA) followed by goat anti-rabbit IgG coupled to10-nm colloidal gold (catalog number 15731-1, Ted Pella Inc.,Redding, CA). The sections were postfixed with 1% glutaraldehyde,contrasted with uranyl acetate, and examined with a Philip CM10electron microscope.
Statistical Analyses
Analysis of variance and paired t test were performed with MicrosoftExcel 2001.
Proteinuria, Electron Microscopy, and Immunofluorescence
Preliminary immunofluorscence studies of day 7 PHN kidneys withmAb 5–1-6 showed a remarkable redistribution of the stainingpattern (Figure 1). This antibody identifies an extracellularepitope of nephrin (23) and normally decorates the slit diaphragmin an interrupted linear pattern (Figure 1). In contrast, thestaining of day 7 PHN glomeruli was more dispersed and granular(Figure 1). The evolution of these changes was examined in detailin the experiments described below.
Figure 1. Representative immunofluorescent micrographs of glomeruli from control rats and those with passive Heymann nephritis (PHN) on day 7. Cryosections were stained with mAb 5–1-6, which detects an epitope on the external domain of nephrin. Control glomeruli illustrate the normal interrupted linear staining pattern of the slit diaphragms with this antibody. The staining of PHN glomeruli is more dispersed and granular. Original magnification, x400.
Urine protein excretion was measured on overnight collectionsfrom day 3 to 4 and from day 6 to 7 after the first dose ofnormal sheep globulin or anti-Fx1A in control and PHN rats,respectively. Day 4 is the earliest time point at which proteinuriais detectable and represents the onset of complement-mediatedheterologous phase injury (27,28). Urine protein excretion waselevated above control values in 58% of PHN rats on day 4, and90% had severe proteinuria on day 7 (Figure 2). Transmissionelectron microscopy showed many subepithelial electron-densedeposits on day 4 with effacement of overlying podocyte footprocesses. In several locations, the immune deposits encroachedon the filtration slits, and in these locations the slit diaphragmswere displaced or not visible (Figure 3). Immunofluorescenceat this time with an antibody to the cytoplasmic domain of nephrinshowed a change in the distribution of nephrin in PHN kidneysfrom an interrupted linear pattern, as seen in controls injectedwith normal sheep globulin, to a more granular appearance (Figure 4A).On day 7, electron microscopy of PHN kidneys showed extensiveand severe foot process effacement overlying large subepithelialdeposits, accompanied by more pronounced displacement and disruptionof slit diaphragms (Figure 3). In some locations, the filtrationslits were replaced by occluding-type junctions and, in others,"ladder-like" structures were present (Figure 3). Early basementmembrane "spike" formation was present in some locations onday 7 (Figure 3). Immunostaining for nephrin on day 7 was evenmore attenuated and clustered, and the overall intensity ofstaining was reduced (Figure 4A). Quantitative analysis revealeda significant reduction in the intensity of nephrin fluorescencein PHN glomeruli on days 4 and 7 (Figure 4B). In contrast tonephrin, immunostaining for ZO-1 was unchanged in PHN in comparisonto controls on day 4 or 7 (Figure 4A). The immunofluorescencefor CD2AP was also undiminished in PHN, but the staining appearedto be more condensed on both days 4 and 7, whereas it was morediffusely distributed in controls (Figure 4A). Cross-reactivityof the CY3-conjugated anti-rabbit IgG secondary antibody withsheep and rat IgG in the glomerular immune deposits was excludedby the absence of staining when the primary rabbit anti-nephrin,anti–ZO-1, or anti-CD2AP were excluded (not shown).
Figure 2. Urine protein excretion in control and PHN rats on days 4 and 7. Mild proteinuria was present in 7 of 12 PHN rats on day 4. The differences in the mean values between the controls and PHN rats was significant at both times (analysis of variance [ANOVA]). Values are mean ± SEM. n = 12/group on day 4 and 8/group on day 7.
Figure 3. Representative transmission electron micrographs of glomeruli from control and PHN rats selected randomly on days 4 or 7. (A) Control rat glomerulus on day 4 shows normal podocyte foot process (Ep) morphology with slit diaphragms visible in most filtration slits. An arrow indicates one filtration slit in which the slit diaphragm is not visible. (B) PHN rat glomerulus on day 4 demonstrates subepithelial electron dense deposits (*) and focally effaced foot processes. In several locations, the deposits encroach on the filtration slits, and in these locations the slit diaphragms are displaced (fine arrows) or absent (bold arrows). (C) PHN glomerulus on day 7 shows growth of the immune deposits. The foot processes are distorted and more extensively effaced. Normal slit diaphragms are rarely visible, and those that are present are displaced (fine arrows). In some places, the filtration slits are narrowed and replaced by occluding-type junctions or "ladder-like" structures (bold arrows). In others, the slit diaphragms are not visible (). Similar observations were made in the glomeruli of two other rats from each group. Magnification, x27,600.
Figure 4. (A) Representative immunofluorescent micrographs of glomeruli from control and PHN rats on days 4 and 7 stained for nephrin, ZO-1, and CD2-associated protein (CD2AP). Staining with an antibody to the cytoplasmic domain of nephrin shows a progressive change in the distribution of nephrin from an interrupted linear pattern in controls, to a less abundant and more dispersed granular appearance on days 4 and 7 of PHN. In contrast, the intensity of staining for ZO-1 and CD2AP is undiminished in PHN, although CD2AP appears to be more condensed on both days 4 and 7, whereas it is more diffusely distributed in controls. (B) Quantification of the intensity of fluorescent staining for nephrin shows a progressive and significant decline in PHN glomeruli on days 4 and 7 (ANOVA). Mean ± SEM. n = 5 to 7/group. Original magnification, x400.
Immunogold electron microscopy with an antibody to the cytoplasmicdomain of nephrin revealed gold particles on the cytoplasmicface of podocyte foot processes in many filtration slits ofcontrol glomeruli (Figure 5). In contrast, day 4 PHN glomerulicontained fewer gold particles in their normal location (Figure 5).Where present, they were associated with preserved, albeitdisplaced, slit diaphragms. On day 7, however, there was analmost complete absence of gold particles from filtration slits,including those that were devoid of visible slit diaphragmsand those that appeared occluded (Figure 5). Gold particleswere rarely, but not consistently, seen on the apical plasmamembrane and in subepithelial immune deposits but not in lysosomes(Figure 5). In keeping with the findings from immunofluorescence,there did not appear to be a difference between control andPHN glomeruli in the number of CD2AP-bound gold particles inthe foot processes, although in some places they appeared tobe less intimately associated with the plasma membrane borderingfiltration slits (Figure 6).
Figure 5. Representative immunogold electron micrographs of glomeruli from (A) control and (B) PHN day 4 and (C) PHN day 7 rats stained for the cytoplasmic domain of nephrin. Gold particles are seen labeling nephrin on the cytoplasmic face of podocyte foot processes (Ep) bordering several filtration slits () in the control glomerulus. Progressively fewer gold particles are visible in PHN days 4 and 7 glomeruli, especially in those filtration slits that contain deposits (*). Magnification, x39,700.
Figure 6. Representative immunogold electron micrographs of glomeruli from (A) control and (B) PHN day 4 and (C) PHN day 7 rats stained for CD2AP. Gold particles labeling CD2AP are seen in the foot processes of control and PHN day 4 and 7 glomeruli, despite the presence of immune deposits(*). Magnification, x39,700.
Increased Nephrin Solubility in PHN
After extraction with Tx-100 at 4°C, a substantial fractionof nephrin remains insoluble and can be released by furtherextraction with agents that depolymerize F-actin. This suggeststhat nephrin is associated with the actin cytoskeleton. In addition,CD2AP, a known adapter protein with actin-binding properties,associates with nephrin (17). To determine whether the associationof nephrin with the actin cytoskeleton is altered during thedevelopment of PHN, glomeruli were isolated from the kidneysof days 4 and 7 PHN and control rats and extracted sequentiallywith 1% Tx-100 at 4°C, followed by extraction of the Tx-insolublefraction with DNase I, which is known to depolymerize F-actin(24). The extracts were analyzed by Western blotting for nephrin,CD2AP, and actin. As shown in Figure 7, a substantial amountof nephrin and almost all CD2AP was associated with actin incontrol glomeruli. In contrast, in PHN glomeruli, most of thenephrin was Tx soluble, and very little was found in the actin-associatedfraction, whereas CD2AP remained unchanged.
Figure 7. Representative Western blot demonstrates the association of nephrin and CD2AP with actin in control glomeruli and reduction in the amount of actin-associated nephrin in days 4 and 7 PHN glomeruli. Glomeruli were isolated from the kidneys of days 4 and 7 control and PHN rats and extracted sequentially with 1% Tx-100 (Tx) at 4°C, followed by extraction of the Tx-insoluble fraction with DNase I, which is known to depolymerize F-actin. The Tx- and DNase I–soluble fractions were resolved by 4% to 20% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) under reducing conditions, transferred to nitrocellulose membranes, and immunoblotted sequentially with rabbit antibodies to nephrin, CD2AP, and actin.
Two sets of experiments were done to quantify the nephrin contentof glomeruli and its association with the actin cytoskeleton.In the first, glomeruli were isolated from the kidneys of sixPHN and control rats on days 4 and 7 and extracted sequentiallywith 1% Tx-100 and DNase I, as described above. The Tx-100 andDNase I extracts from day 4 PHN and control and day 7 PHN andcontrol rats were selected randomly and run on six separategels, transferred to nitrocellulose membranes, and immunoblottedfor nephrin, CD2AP, and actin. Densitometric readings of theWestern blots from each membrane were compared by pairwise analysisto control for completeness of transfer and film exposure time.As shown in Figure 8, the ratio of actin-associated (DNase I–soluble)to Tx-100–soluble nephrin was 0.48 on day 4 and 0.61 onday 7 in controls (the difference between day 4 and day 7 controlswas NS by paired t test) and was significantly reduced to 0.28on day 4 and 0.30 on day 7 in PHN glomeruli. There was no significantdifference in CD2AP and actin levels in control and PHN glomeruliat either time point (not shown).
Figure 8. Quantitative Western blot analysis of control and PHN glomeruli reveals a reduction in actin-associated nephrin in PHN. (A) Glomeruli were isolated from the kidneys of individual days 4 and 7 control and PHN rats and extracted and analyzed as described in the legend to Figure 6. (B) Densitometry of the individual bands indicates that the ratio of actin-associated (DNase I–soluble) to detergent-extractable (Tx-100–soluble) nephrin is significantly reduced in whole glomeruli from days 4 and 7 PHN rats (paired t test). Mean ± SEM. n = 6/group.
In the second set of experiments, we further fractionated theglomeruli to obtain membrane-associated nephrin and corticalactin. In these studies, F-actin was depolymerized with KI sothat all extractions could be done at 4°C. Because the amountsof cell membrane that can be isolated from the glomeruli ofindividual rats is too small for analysis, we pooled the glomerulifrom three rats to give three different sets of glomeruli fromnine controls and nine day 4 and nine day 7 PHN rats. Four sampleswere analyzed from each pool as shown in Figure 9A. These included(1) the supernatant remaining after ultracentrifugation to preparethe membrane fraction, (2) the Tx-100–soluble extractof the membrane fraction, (3) the KI-soluble fraction of theTx-insoluble membrane fraction, and (4) the KI-insoluble pellet.Because KI solubilization is carried out in the presence of1% Tx-100, we established that Tx-100 alone did not extractany additional nephrin from the Tx-insoluble fraction. The majorfindings from this analysis were that total nephrin was significantlyreduced in PHN glomeruli (Figure 9B) and that the amount ofactin-associated (Tx + KI–soluble) nephrin in the membranefraction was relatively decreased in PHN (Figure 9C). As expected,the amount of Tx-soluble nephrin in the membrane fraction wasreciprocally increased (densitometry not shown). In addition,the residual nephrin in the Tx + KI–insoluble pellet wasreduced in PHN membranes (densitometry not shown). These changesin PHN were present at 4 d and were more severe at 7 d, in keepingwith the immunofluorescent findings. Immunoblots for actin onthe same membranes showed that KI was effective in solubilizingactin and that the changes in nephrin could not be explainedby differences in sample loading.
Figure 9. Sequential extraction and quantitative Western blot analysis of control and PHN glomeruli reveals a progressive reduction in total glomerular nephrin and in the actin-associated (potassium iodide [KI]–soluble) fraction of cell membrane nephrin in PHN. (A) Representative Western blot of glomeruli pooled from 3 rats/group. Four samples were analyzed from each pool: (S) the supernatant remaining after ultracentrifugation to prepare the membrane fraction, (T) the Tx-100–soluble extract of the membrane fraction, (T + K) the KI-soluble fraction of the Tx-insoluble membrane fraction, and (P) the KI-insoluble membrane pellet. The fractions were resolved on 4% to 20% SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and immunoblotted sequentially with rabbit antibodies to nephrin and actin. Three similar pools were analyzed in parallel. Densitometry of the individual bands indicates that (B) total glomerular nephrin and (C) the actin-associated fraction of cell membrane nephrin are significantly reduced (ANOVA). Mean ± SEM. n = 3/group.
These studies in PHN show that the glomerular distribution ofnephrin is altered and that there is a reduction in total glomerularcontent of nephrin coincident with or before the onset of proteinuriaon day 4. In addition, the fraction of membrane-associated nephrinthat is bound to actin is diminished. These changes correspondto an alteration in podocyte morphology, including displacementand loss of slit diaphragms. The loss of slit diaphragms isaccompanied in some locations by a narrowing of filtration slitsand replacement with occluding-type junctions and ladder-likestructures. The changes in the amount and distribution of nephrinappear to be specific and are not simply a consequence of podocytedamage. Thus, the amount of CD2AP and ZO-1, two proteins locatedon the cytoplasmic face of the slit diaphragm, was not reduced.Furthermore, the association of CD2AP with actin was unaffected.If, indeed, CD2AP is an adapter that binds nephrin to actinin vivo, this latter finding suggests that the link betweennephrin and CD2AP is disrupted in PHN and that CD2AP may condensewith actin at the base of the effaced podocytes. Although thiscould explain the reduced association of nephrin with actin,it remains possible that other as-yet-unidentified adapter proteins,or even a direct actin-nephrin link, might be perturbed in PHN.
In our analysis of whole rat glomeruli, we found that the actin-associatedfraction of nephrin was substantially (and similarly) reducedon days 4 and 7 of PHN as determined by DNase I sensitivity.As seen in Figures 7 and 8, the Tx-soluble fraction of nephrinis detectable as two closely spaced bands, whereas the DNaseI–soluble band in the control lanes appears to correspondto the lower band. This raises the possibility that only thesmaller protein, said to be a splice variant that lacks thetransmembrane-spanning domain (29), is the component that isbound to actin. To examine this further, we conducted an additionalset of experiments with glomerular cell membranes and usingKI to depolymerize actin (Figure 9). The use of KI instead ofDNase I allowed us to do the extractions at 4°C. These studiesrevealed a substantial amount of nephrin in the KI-soluble (actin-associated)cell membrane fraction in control glomeruli and a progressivedecline in this component on days 4 and 7 of PHN. We can onlyspeculate that the marked reduction in actin-associated nephrinon day 7 in the cell membrane fraction (Figure 9) and the lesserreduction seen on day 7 in whole glomeruli (Figure 8) mightbe due to retention of an intracellular pool. It is also noteworthythat there was residual nephrin present in the KI-insolublecell membrane pellet (Figure 9). This may be the result of incompletedepolymerization of actin by KI or represent nephrin in a detergentresistant membrane fraction—so-called lipid rafts (18,30).This fraction was also relatively depleted in the PHN glomerularcell membranes (quantification not shown).
It is noteworthy that filtration slits are often narrowed, andoccluding-type junctions appear when nephrin and slit diaphragmsare lost (this study and reference [31]). This loss of filtrationsurface area accounts for the reduction in GFR in MN (9), butit is possible that these abnormal slits are narrowed but notcompletely occluded. If so, bulk flow would be reduced, butsubstantial protein permeation could still occur. In this regard,ultrastructural tracer studies showed that the greatest permeationof macromolecules occurred in the vicinity of immune depositsin Heymann nephritis (32), and the most prominent changes inthe slit diaphragms in this study were evident in filtrationslits that contained electron-dense immune deposits. It is alsointeresting to speculate on the explanation for the narrowedfiltration slits seen in this study and in nephrin knockoutmice (31). It has been proposed that patency of the filtrationslits is maintained by the repulsive negative charge generatedon the surface of opposing foot processes by podocalyxin (33).It is possible that the slit diaphragm may also hold adjacentfoot processes apart while forming a barrier to plasma proteinleakage.
The immunohistological and immunochemical studies reported hereclearly establish that the amount of podocyte nephrin is substantiallydiminished in PHN, but it remains unknown whether this is dueto reduced synthesis, increased degradation, shedding from thecell surface, or a combination of these factors. Studies elsewherehave offered support for each of these possibilities. Thus,reduced nephrin mRNA expression has been documented in the glomeruliof patients with MN (11) and in rats with nephropathies inducedby purine aminonucleoside, mAb 5–1-6, and mercuric chloride,as well as in advanced PHN (13,22,34,35). We also found thatnephrin and mAb 5–1-6 undergo coordinate redistributionand that the antibody can be identified in podocyte lysosomesat the onset of proteinuria induced by mAb 5–1-6 (36),which suggests that the protein is endocytosed and degraded.Finally, it has been reported that the truncated alpha spliceform of nephrin can be detected in the urine of rats with purineaminonucleoside nephrosis (35). Although we cannot exclude thepossibility that suppressed nephrin synthesis may account forthe reduced abundance of the protein on day 4 of PHN, the substantialdecline in the fraction bound to actin suggests other possibilities.Thus, it may be that loss of anchorage to the cytoskeleton allowsmembrane-associated nephrin to redistribute and to be shed fromthe cell surface into the urinary space or to be endocytosedand targeted for degradation.
The mechanism/s by which antibody and complement alter the distributionof nephrin and its association with actin are still unknown.Several cellular processes are activated in glomerular epithelialcells by sublethal C5b-9–mediated injury, including influxand release of calcium from intracellular stores, phospholipaseactivation, adenosine triphosphate depletion, and generationof free radicals, as well as disruption of actin microfilaments(7,37–40). In addition, it has recently been reportedthat insertion of C5b-9 into the plasma membrane of culturedhuman podocytes induces the shedding of nephrin with a lossof surface expression (12). These findings need to be consideredin the light of recent observations that have shown that atleast a fraction of cell membrane nephrin resides in lipid rafts,where it can be tyrosine phosphorylated in response to a glycolipid-specificantibody (30), and that nephrin forms a signaling complex withpodocin when the two proteins are coexpressed in cultured cells(41). Although it is premature to attempt to construct an all-embracingscheme to explain our findings and all these important observations,we propose the following possible model. Rather than signalingthe podocyte via nephrin from the outside, we suggest that theinsertion of C5b-9 into the podocyte plasma membrane triggersa series of signaling events that disrupt the association ofnephrin with the cytoskeleton and its stability within the cellmembrane. Whether this involves alterations in the phosphorylationstatus of nephrin, its association with podocin and/or CD2AP,or changes in the lipid composition of the cell membrane hasyet to be established. In any event, the outcome appears tobe the dislocation or dissolution of slit diaphragms and theloss of barrier function.
In summary, these studies in experimental MN document the redistributionand loss of nephrin from glomerular podocytes and show thatthis is an early event that coincides with or even precedesthe onset of proteinuria. These changes are most likely dueto reduced anchorage of nephrin to the actin cytoskeleton, whicheither allows it to be shed from the plasma membrane or targetsit for endocytosis and degradation. The result is that the slitdiaphragms become displaced or disrupted, thereby removing thefinal barrier to plasma protein permeation. It is importantto emphasize that these changes need not involve every filtrationslit to result in massive proteinuria. In fact, complete lossof barrier function in a small fraction of filtration slitscould account for the levels of proteinuria seen in adult humanswith nephrotic syndrome from MN.
Acknowledgments
This work was supported by research Grant DK30932 (to D.J.S.)and in part by a Boston Area Diabetes Endocrinology ResearchCenter Grant DK57521 to the MGH Program in Membrane Biology,Microscopy Core Facility (to D.B. and M.M.). The authors aregrateful to Drs. Larry Holzman, Andrey Shaw, and Hiroshi Kawachifor their collegial support and willingness to provide valuablereagents for these studies.
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Received for publication October 4, 2001.
Accepted for publication December 2, 2001.
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Top
Abstract
Introduction
). Similar observations were made in the glomeruli of two other rats from each group. Magnification, x27,600.
) in the control glomerulus. Progressively fewer gold particles are visible in PHN days 4 and 7 glomeruli, especially in those filtration slits that contain deposits (*). Magnification, x39,700.
