Synaptic Vesicle Protein 2B Is Expressed in Podocyte, and Its Expression Is Altered in Proteinuric Glomeruli
Naoko Miyauchi*,
Akira Saito*,
Tamaki Karasawa*,
Yutaka Harita*,
Koichi Suzuki*,
Hiroko Koike*,
Gi Dong Han*,,
Fujio Shimizu* and
Hiroshi Kawachi*
* Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; and Department of Food Science and Technology, Yeungnam University, Gyeongsan, Republic of Korea
Address correspondence to: Dr. Hiroshi Kawachi, Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Niigata, 951-8510, Japan. Phone: +81-25-227-2160; Fax: +81-25-227-0770; kawachi{at}med.niigata-u.ac.jp
Received for publication December 14, 2005.
Accepted for publication July 18, 2006.
Synaptic vesicle protein 2B (SV2B) was identified by the subtractionhybridization technique as a molecule of which mRNA expressionwas decreased in puromycin aminonucleoside (PAN) nephropathyby glomerular cDNA subtraction assay. The expression of SV2Bwas detected in glomerular lysate with Western blot analysis.Dual-labeling immunofluorescence studies with glomerular cellmarkers demonstrated that SV2B is expressed in glomerular visceralepithelial cells (podocytes). The expression of SV2B is detectedalso in cultured podocyte and in human kidney section as podocyticpattern. The decrease of SV2B mRNA was already detected beforethe onset of proteinuria in PAN nephropathy. The mRNA expressionof SV2B clearly is altered not only in PAN nephropathy but alsoin another proteinuric state that is caused by an antibody againstnephrin, a functional molecule of the slit diaphragm. The decreasedintensity in SV2B staining was already detected before the peakof proteinuria in both models with immunofluorescence study.A reduced amount of SV2B was detected in both models also withWestern blot analysis. CD2AP, another functional molecule ofthe slit diaphragm, was observed in cytoplasm, including theprocesses area of the cultured podocyte, and when the podocytewas treated with small interfering RNA for SV2B, CD2AP stainingat the process area was not detected. These results suggestthat SV2B is a functional molecule of podocyte, and SV2B mayplay a role in the expression and proper localization of CD2AP.
The clarification of the pathogenic mechanism of proteinuriais one of the most important themes in the nephrology field.It is becoming clear that disorder of the glomerular visceralepithelial cell (podocyte) causes proteinuria and results innephrotic syndrome. Recent studies have focused on the roleof the slit diaphragm, located between the foot processes ofthe podocytes, in maintaining the barrier function of the glomerularcapillary wall (1–7). We and others reported previouslythat nephrin and podocin are localized at the slit diaphragmand that their expressions clearly are altered already at theearly phase of puromycin aminonucleoside (PAN) nephropathy,an experimental model of minimal-change nephrotic syndrome (8–10).These findings indicate that these molecules are critical componentsof the slit diaphragm and that alteration of their expressionis involved in the development of proteinuria. However, theprecise mechanism of the development of proteinuria in PAN nephropathyis not well understood yet.
In this study, we aimed to identify the molecules that are involvedin the development of proteinuria. Because it is conceivablethat the molecules whose expression decreased before the onsetof proteinuria may have been involved in the development ofproteinuria, we intended to purify the molecules whose expressionswere downregulated 24 h after PAN injection, using cDNA subtractivehybridization techniques. We identified 28 genes that were downregulated,and eight molecules of them were confirmed to be expressed inglomeruli and cultured podocyte. Among them, we focused on synapticvesicle protein 2B (SV2B) in our study. SV2B is an isoform ofSV2, which is a glycosylated synaptic vesicle membrane proteinthat comprises 12 transmembrane regions (11,12). SV2 is knownto participate in the regulation of calcium-mediated synaptictransmission and to play a role in vesicle trafficking by bindingto other cell surface proteins (13,14). Although SV2 originallywas reported to be expressed specifically in the central nervoussystem, recent studies have shown that it also is distributedin other organs (15–17). Hayashi et al. (18) reportedthat SV2B is expressed in the microvesicles of pinealocytes,which are rich in process terminals. It has been pointed outthat neurons and podocytes have common characteristics. Bothare terminally differentiated cells and have similar shapesthat are characterized by processes (19,20). Both of them alsodisplay cell type–specific intercellular contact: Synapsesin neurons and the slit diaphragm in podocytes. Furthermore,functionally important molecules in the podocyte, such as nephrinand podocin, have been found to be shared specifically withthe neuron (9,10,21). Recently, Rastaldi et al. (22) reportedthat synaptic vesicle molecule Rab3A and its specific effector,rabphilin-3a, are expressed in the podocyte and that they areinvolved in vesicle trafficking. Cormont et al. (23) reportedthat Rab4, another molecule of the Rab family, interacted withCD2AP and might play a role in its trafficking. It has beenreported that CD2AP also is expressed in the podocyte and thatCD2AP is involved in maintaining the barrier function of theslit diaphragm by interacting with nephrin (24–29). Italso has been reported that the expression of CD2AP is decreasedin cultured podocyte that is treated with PAN (30,31). All ofthe properties of these synaptic vesicle–associated moleculesprompted us to consider that SV2B might be associated with theslit diaphragm.
In this study, we demonstrated that SV2B is expressed in thepodocyte and that its expression decreases not only in PAN nephropathybut also in the proteinuric model induced by the antibody againstnephrin, a functional molecule of the slit diaphragm. We alsodemonstrated that CD2AP distribution clearly is altered in culturedpodocyte that is treated with small interfering RNA (siRNA)for SV2B, suggesting that SV2B plays a role in the expressionof podocyte functional proteins such as CD2AP.
Animal
All experiments of animals in this study were conducted in accordancewith the National Institutes of Health Guide for the Care andUse of Laboratory Animals. Procedures for this study were approvedby the Animal Committee at Niigata University School of Medicine,and all animals were treated according to the guidelines foranimal experimentation of Niigata University. Specific pathogen-free7-wk-old female Wistar rats (Charles River Japan, Kanagawa,Japan) that weighing approximately 200 g were used for the materials.
Podocyte Cell Culture and SV2B-Expressing Cell
Mouse cultured podocyte was donated by Dr. Peter Mundel (AlbertEinstein College of Medicine, Bronx, NY). Cultivation of conditionallyimmortalized mouse podocytes was conducted as reported previously(32). In brief, podocytes were maintained in RPMI 1640 medium(Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10%FBS (Life Technologies, Grand Island, NY). For propagation ofpodocytes, cells first were cultivated at 33°C and maintainedfor 1 wk at 37°C to induce differentiation.
COS-7 cells were cultured in Eagle’s MEM (Nissui Pharmaceutical,Tokyo, Japan) with 10% FBS. COS-7 cells were transfected withexpression vector pcDNA3.1/His with an insert of full-lengthcDNA of SV2B by chloroquine method.
Antibodies
Rabbit anti-SV2B and mouse anti-Rab3A antibodies were purchasedfrom Synaptic Systems (Goettingen, Germany). Goat anti-CD2APwas purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Mouse anti–RECA-1, mouse anti-synaptopodin, rabbit anti–-actinantibodies, a rhodamine-phalloidin, and an acridine orange werepurchased from Serotec (Oxford, UK), PROGEN (Heidelberg, Germany),Sigma (St. Louis, MO), Cytoskeleton (Denver, CO), and Polysciences(Warrington, PA), respectively. Rabbit anti-CD2AP, mouse monoclonalanti-nephrin antibody (ANA; mAb 5-1-6), and mouse monoclonalanti-Thy1.1 antigen antibody (mAb 1-22-3) were prepared as describedpreviously (29,33,34). Normal rabbit serum was used as a negativecontrol. Secondary antibodies that were used for immunofluorescence(IF) study were as follows: FITC-conjugated swine anti-rabbitIgG (DAKO, Glostrup, Denmark), TRITC-conjugated goat anti-mouseIgG1, TRITC-conjugated goat anti-mouse IgG3 (Southern BiotechnologyAssociates, Birmingham, AL), and FITC- and TRITC-conjugateddonkey anti-goat IgG (Protos Immunoresearch, San Francisco,CA). Alkaline phosphatase–conjugated goat anti-rabbitIgG (Biosource International, Tago Immunologicals, Camarillo,CA) was used for Western blot analysis.
Subtractive Hybridization
Ten rats received intravenous injection of 100 mg/kg body wtPAN (Sigma). Preparation of cDNA from rat glomerular mRNA wasaccording to the method described previously (9,10). Subtractivehybridization using cDNA from glomerular RNA of normal ratsas tester and that from rats with PAN nephropathy (24 h afterinduction) as driver was performed with PCR-Select cDNA subtractionKit (Clontech, Palo Alto, CA), and the basic procedures wereaccording to the manufacturer’s protocol. Amplified productwas subcloned into the plasmid vector pCRII-TOPO (TOPO TA CloningKit Dual Promoter; Invitrogen, Carlsbad, CA) and sequenced.
Expression of Synaptic Vesicle–Associated Proteins in Kidney and Other Organs
Cerebrum, cerebellum, medulla oblongata, kidney, thyroid, thymus,heart, lungs, spleen, liver, pancreas, stomach, small intestine,colon, adrenal glands, uterus, ovary, testis, urinary bladder,skin, skeletal muscles, and placenta (18.5 gestational days)were removed from rats. Total RNA was extracted from these organsand from kidney cortex, kidney medulla, and glomeruli, and theirmRNA expression was analyzed with reverse transcription–PCR(RT-PCR) study.
Expression of SV2B in Normal Rat Glomeruli and Cerebrum, Normal Human Glomeruli, and Cultured Cell
We performed RT-PCR using the forward primer with start codonand the reverse primer just upstream of the stop codon to analyzewhether the full-length coding sequence (CDS) of SV2B mRNA wasexpressed in glomeruli. The sequences of all primers that wereused in this study are shown in Table 1.
Table 1. PCR primers that were used in this studya
Western blot analysis was performed basically according to themethod reported previously (9,10,29). In brief, normal or proteinuricrat glomeruli, normal rat cerebrum, and cells were solubilizedwith RIPA buffer (0.1% SDS, 1% sodium deoxycholate, 1% TritonX-100, 150 mmol/L NaCl, and 10 mmol/L EDTA in 25 mmol/L Tris-HCl[pH 7.2]) or SDS-PAGE sample buffer (2% SDS, 10% glycerol, and5% mercaptoethanol in 62.5 mmol/L Tris-HCl [pH 6.8]) with proteaseinhibitors. Normal rat glomeruli also was solubilized sequentiallywith 1% Triton X-100 and RIPA buffer and separated into TritonX-100–soluble fraction (fraction 1), Triton X-100–insoluble/RIPA-solublefraction (fraction 2), and RIPA-insoluble fractions (fraction3). RIPA-insoluble fraction was solubilized with SDS-PAGE samplebuffer. The concentration of the materials was measured by thebicinchoninic acid method (Pierce Chemical, Rockford, IL), and20 µg of these samples was subjected to SDS-PAGE with10% acrylamide gel and transferred to a polyvinylidene fluoridetransfer membrane (Pall Corp., Pensacola, FL). After blockingwith bovine skim milk, the strips of membrane were exposed torabbit anti-rat SV2B or rabbit anti-CD2AP, rabbit anti-rat -actinantibodies, or normal rabbit serum. They then were washed andincubated with alkaline phosphatase–conjugated goat anti-rabbitIgG. The reaction was developed with an alkaline phosphatasechromogen kit (Biomedica, Foster City, CA).
IF studies were performed basically according to the methodreported previously (9,10,29). The 3-µm-thick frozen sectionsof normal rat and human or cells that were cultured on a glasscoverslip were fixed with acetone for 1 min and incubated withprimary antibodies. The dual-labeling IF for SV2B was carriedout with anti-nephrin, anti-synaptopodin, anti–RECA-1,anti-Thy1.1, and anti-Rab3A antibodies.
Expression of SV2B in Proteinuric Models
A total of 18 rats received intravenous injection s of 100 mg/kgbody wt PAN or 15 mg/rat ANA (mAb 5-1-6), respectively. ForPCR analysis and IF study, kidney materials of three rats eachwere removed at 1 h (PAN and ANA nephropathy), 24 h (PAN andANA nephropathy), 5 d (ANA nephropathy), and 10 d (PAN nephropathy)after injection. Urine samples were collected, and their proteinconcentration was measured by colorimetric assay with a Bio-RadProtein Assay Reagent (Bio-Rad, Hercules, CA). The average valueof the proteinuria was 4.5 (24 h) and 238.7 mg/d (10 d) of PANnephropathy and 46.6 (24 h) and 215.8 mg/d (5 d) of ANA nephropathy.
A portion of right kidney was used for IF study. GlomerularRNA was prepared from the kidneys that were pooled from threerats. The IF staining of SV2B was semiquantified basically accordingto the method described by Macconi et al. (35) (score 0, completelyabsent; 1, signal covering 0 to 25% of the glomerular tuft area;2, 25 to 50%; 3, 50 to 75%; and 4, 75 to 100%). A score wasassigned to each glomerulus, and >30 glomeruli of each ratwere analyzed. The data are shown as a ratio (%) relative tothe total number of glomeruli scored and are expressed as mean± SD of three rats.
For Western blot analysis, three other rats each received aninjection of PAN or ANA, and the kidney materials were removedon day 10 or day 5, respectively. Glomeruli that were preparedfrom the kidneys from three rats in each group were solubilizedwith SDS-PAGE sample buffer. The concentration of the materialswas measured by the bicinchoninic acid method (Pierce Chemical),and 20 µg/lane of the materials was used for Western blotanalysis. The experiments were performed three times, and theband intensity was determined by image analysis using Bio Doc-ItSystem (UVP, Inc., Upland, CA). All results were corrected forthe amount of protein in the sample by dividing by the intensityof the internal control -actin. The columns represent mean valuesof three examinations, and the bars show SD.
RT-PCR
Semiquantitative RT-PCR was performed basically according tothe method described previously (9,10), using the primers listedin Table 1. The band intensity was determined by image analysisusing Bio Doc-It System (UVP, Inc.). All results were correctedfor the amount of mRNA in the sample by dividing by the intensityof the internal control glyceraldehyde-3-phosphate dehydrogenase.
Real-time RT-PCR was performed as described previously (29).The reactions and runs for all samples were performed threetimes. For preparation of standard material, PCR products ofSV2B and glyceraldehyde-3-phosphate dehydrogenase were subclonedinto the plasmid vector pCRII-TOPO, and the plasmids were dilutedfrom 1 x 102 copies to 1 x 109 copies to generate calibrationcurves, which were based on the linear relationship betweenthe crossing point cycle values and the logarithm of the startingcopy number.
RNA Silencing Analysis
The siRNA sequences that targeted SV2B (GenBank accession no.AF372834) were synthesized by iGENE (Tsukuba, Japan): The siRNAhad 25 nucleotides in length, corresponding to positions 69to 93 of the SV2B open reading frame. Before transfection, podocyteswere cultured to a density of 70 to 80% at 37°C, and thenthey were transfected with the siRNA using a Trans IT-TKO transfectionreagent (Mirus, Madison, WI). Nonspecific nucleotide sequencetransfected cells were used as negative control. Cells wereharvested 24 h after siRNA treatment for RT-PCR, Western blotanalysis, and IF analysis. Dual-labeling IF of CD2AP was performedwith anti-SV2B antibody, rhodamine-phalloidin, and acridineorange. Rhodamine-phalloidin was used to detect actin fiber,and acridine orange was used to detect nucleus.
Molecules Identified with cDNA Subtractive Hybridization
Twenty-eight genes were identified with the subtraction hybridizationtechniques, and eight molecules of them were confirmed to beexpressed in glomeruli and cultured podocyte. These podocyte-associatedmolecules are summarized in Table 2.
Table 2. Podocyte-associated molecules that were identified with subtractive hybridization
Glomerular mRNA Expression of SV2B Was Decreased at 24 H of PAN Nephropathy
An approximately 300-bp band whose nucleotide sequence showed100% identity with that of rat SV2B was detected in the subtractedcDNA (glomerular cDNA of PAN nephropathy rats [24 h after induction]from that of normal controls). To analyze whether the full-lengthCDS of SV2B is expressed in glomeruli, we performed RT-PCR withthe primers shown in Table 1. An approximately 2050-bp bandwas detected in glomeruli and cerebrum. The size of the bandwas compatible with that of reported CDS of SV2B (Figure 1B).
Figure 1. (A) mRNA expression of synaptic vesicle protein 2B (SV2B), SV2A, and Rab3A in several organs. SV2B expression was detected in the glomeruli by reverse transcription–PCR (RT-PCR). SV2B mRNA expression also was detected in the whole kidney, kidney cortex, kidney medulla, central nervous system (cerebrum, cerebellum, and medulla oblongata), lungs, digestive tract (stomach, small intestine, and colon), genital organs (uterus, ovary, and testis), skin, and placenta. SV2B was not detected in secretory organs (thyroid, pancreas, and adrenal glands), thymus, heart, spleen, liver, urinary bladder, or skeletal muscles. In contrast, mRNA expression of SV2A and Rab3A was detected in all organs examined. (B) Full-length coding sequence (CDS) of SV2B expression in glomeruli and cerebrum. An approximately 2050-bp band was detected in glomeruli and cerebrum. The size of the band was compatible with that of reported CDS of SV2B.
SV2B Expression Is More Restricted than SV2A or Rab3A Expression
SV2B mRNA expression was detected in the glomeruli and alsoin the central nervous system, lungs, digestive system, skin,and placenta (Figure 1A). SV2B mRNA expression was not detectedin secretory organs thyroid, pancreas, and adrenal glands. Incontrast, the expression of SV2A and Rab3A was detected in allorgans examined.
SV2B Is Expressed in the Glomerular Podocyte
A positive band of approximately 80 kD was detected in rat glomerularand cerebrum lysates that were solubilized with RIPA bufferby Western blot analysis with anti-SV2B antibody (Figure 2A,lanes 1 and 5). The intensity of the band clearly was lowerin the stripe that was stained with anti-SV2B antibody thatwas preabsorbed with the peptide that was used for immunization(Figure 2A, lane 3). No change was observed with anti-SV2B antibodythat was preabsorbed with the irrelevant peptide (Figure 2A,lane 4). No band was detected with normal rabbit serum (Figure 2A,lanes 2 and 6). Clear staining of anti-SV2B antibody was detectedin COS-7 cells that were transfected with pcDNA3.1/His withan insert of full-length cDNA of rat SV2B (Figure 2B, a), althoughthe staining that was detected in COS-7 cells that were transfectedwith vector without insert was faint (Figure 2B, d). Negativestaining was detected in COS-7 cells that were transfected withthe insert with normal rabbit serum (Figure 2B, b) or with anti-SV2Bantibody that was preabsorbed with the peptide that was usedfor immunization (Figure 2B, c). Clear staining of SV2B wasobserved along the glomerular capillary loop (Figure 2C, a).SV2B staining also was detected in distal tubular epithelialcell. No positive staining was detected with normal rabbit serum(Figure 2C, b) and anti-SV2B antibody that was preabsorbed withthe peptide that was used for immunization (Figure 2C, c). SV2Bstaining also was observed along the glomerular capillary loopin human kidney section (Figure 2D, a). The staining patternwas very similar to that in rat sections. SV2B staining wasdetected in cultured podocytes (Figure 2E, a). Positive stainingof SV2B was detected in cytoplasm, including processes of culturedpodocyte. No positive staining was detected with normal rabbitserum (Figure 2E, b). SV2B staining clearly was apart from thatof RECA-1, the endothelial cell marker, or Thy1.1, the mesangialcell marker (Figure 3A, b and c, arrows). A dual-labeling IFstudy with podocyte markers in rat kidney sections revealedthat major parts of the SV2B staining were co-stained with nephrinand synaptopodin (Figure 3A, a and d, arrowheads), and someportions of SV2B staining were co-stained with Rab3A (Figure 3C).
Figure 2. (A) Western blot analysis of glomerular (lanes 1 through 4) and cerebrum (lanes 5 and 6) lysates. A positive band of approximately 80 kD was detected with anti-SV2B antibody (lanes 1 and 5). The intensity of the band clearly was lower in the stripe that was stained with anti-SV2B antibody that was preabsorbed with the peptide that was used for immunization (lane 3). No change was observed with anti-SV2B antibody that was preabsorbed with the irrelevant peptide (lane 4). No band was detected with normal rabbit serum (lanes 2 and 6). (B) Immunofluorescence (IF) finding of anti-SV2B antibody in COS-7 cells. Clear staining of anti-SV2B antibody was detected in COS-7 cells that were transfected with pcDNA3.1/His with an insert of full-length cDNA of rat SV2B (a), although the staining that was detected in COS-7 cells that were transfected with vector without insert was faint (d). Negative staining was detected in COS-7 cells that were transfected with the insert with normal rabbit serum (b) or with anti-SV2B antibody that was preabsorbed with the peptide that was used for immunization (c). (C) IF finding of SV2B in rat kidney section. The SV2B staining was observed as a continuous granular pattern along the glomerular capillary loop (a). No positive staining was detected with normal rabbit serum or anti-SV2B antibody that was preabsorbed with the peptide that was used for immunization (b and c). (D) IF finding of SV2B in human kidney section. Positive staining of SV2B also was observed along the glomerular capillary loop in human kidney section (a). No positive staining was detected with normal rabbit serum (b). (E) IF finding of SV2B in cultured podocyte. Positive staining of SV2B was observed in cultured podocytes (a). No positive signal was detected with normal rabbit serum (b).
Figure 3. (A) Dual-labeling IF of SV2B with glomerular cell markers. Dual-labeling IF results with cell markers in rat kidney sections are shown. Anti-nephrin and anti-synaptopodin antibodies were used as the podocyte markers. Anti–RECA-1 and anti-Thy1.1 antibodies were used as the endothelial cell marker and the mesangial cell marker, respectively. SV2B was stained green, and the markers were stained red. SV2B staining clearly was apart from that of RECA-1 and Thy1.1 (b and c, arrows). A dual-labeling IF study with podocyte markers in rat kidney sections revealed that major parts of the SV2B staining were co-localized with nephrin and synaptopodin staining (yellow; a and d, arrowheads). (B) Western blot analysis of sequentially solubilized glomerular lysates. A clear band was detected in Triton X-100–soluble fraction (Fx 1), and a weak one was observed in Triton X-100–insoluble/RIPA-soluble fraction (Fx 2). (C) Dual labeling finding of SV2B and a vesicle marker Rab3A. Some portions of SV2B (a) were co-stained with Rab3A (b, arrowheads); c shows a merged image of a and b. Magnification, x400 in A and C.
To analyze further the subcellular localization of SV2B, weperformed Western blot analysis with sequentially solubilizedglomerular lysates (Figure 3B). Most of SV2B was detected infraction 1 (78.4 ± 13.6%). A small amount of SV2B wasdetected in fraction 2 (16.0 ± 4.0%).
SV2B Expression Is Altered in Proteinuric States
The kinetics of SV2B mRNA expression in PAN and ANA nephropathyare shown in Figure 4. We confirmed that glomerular SV2B mRNAexpression was decreased at 24 h of PAN nephropathy (58.6 ±19.7%). SV2B mRNA expression had already decreased at 1 h ofPAN nephropathy (20.7 ± 17.5%). The expression of SV2BmRNA recovered to normal range on day 10 (125.1 ± 16.5%),when proteinuria peaked in this model. A decrease in the expressionof SV2B mRNA also was detected in ANA nephropathy (1 h, 97.9± 26.5; 24 h, 10.9 ± 1.1%). In ANA nephropathy,SV2B mRNA expression still was low on day 5 (63.0 ± 13.8%),when proteinuria peaked. Real-time RT-PCR analysis also demonstratedthat SV2B mRNA expression clearly decreased at the early phasesof both models (Figure 4). Positive IF signal of SV2B was loweredat the early phase of both models and clearly was lowered atthe peak of proteinuria of both models (Figure 5A). Westernblot analysis with glomerular lysate that was solubilized withSDS-PAGE sample buffer showed that the amount of SV2B clearlyis reduced in both models (day 10 of PAN, 57.1 ± 17.4%;day 5 of ANA, 34.3 ± 26.0% to normal; Figure 5B).
Figure 4. Kinetics of SV2B mRNA expression in puromycin aminonucleoside (PAN) and anti-nephrin antibody (ANA) nephropathy. (A) The mRNA expression of SV2B was semiquantified by RT-PCR. The band intensity was determined by image analysis. Ratios of the densitometric signals of SV2B and the internal control (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were analyzed. Representative agarose gel electrophoretic patterns from one of the three experiments are shown at the bottom, and the top data are shown as ratios relative to normal rat findings and are expressed as mean ± SD of three independent experiments. SV2B mRNA expression markedly decreased in both models before the peak of proteinuria. In PAN nephropathy, SV2B mRNA expression had already decreased 1 h after induction (20.7 ± 17.5%), before proteinuria occurred. In ANA nephropathy, SV2B mRNA expression decreased 24 h after induction (10.9 ± 1.1%) and still was low on day 5 (63.0 ± 13.8%), at the peak of proteinuria. (B) Decrease of SV2B mRNA expression at the early phase of both models was confirmed by real-time RT-PCR. The top panels show the fluorescence intensity of the RT-PCR of SV2B and GAPDH. The x axis shows the number of PCR cycles, and the y axis shows the fluorescence intensity. The lines of the graphs are normal (a), 1 h (b), and 24 h (c). The threshold was set at 30 U of fluorescence intensity. The copy numbers were determined using the calibration curves. The PCR reactions and runs were performed three times per sample, and the data are shown as mean ± SD. The expression of SV2B to 108 copies of GAPDH in normal glomeruli was 12,799 ± 2184 copies; this value was calculated with the external standard curves for SV2B; (y = –0.286x + 10.779; r2 = 0.999) and GAPDH (y = –0.3x + 12.04; r2 = 1). The expression of SV2B to 108 copies of GAPDH decreased in PAN (1 h, 4350 ± 497; 24 h, 1971 ± 822 copies) and ANA nephropathy (1 h, 8958 ± 3374; 24 h, 327 ± 233 copies). The ratios to GAPDH were as follows: PAN: 1 h, 34.3 ± 3.1%; 24 h, 16.1 ± 8.1; ANA: 1 h, 68.7 ± 17.3; 24 h, 2.4 ± 1.6.
Figure 5. Expression of SV2B in PAN and ANA nephropathy. (A) The alterations of SV2B expression in pathogenic rat glomeruli were evaluated as described in Materials and Methods. Representative glomeruli showing score 0 to 4 are shown at the top. Data are shown as mean ± SD (n = 3 rats for each group) on the left. Representative IF findings are shown on the right. The intensity of a positive signal was lowered at the early phase of both models and clearly was lowered at the peak of proteinuria of both models. (B) Western blot analysis with glomerular lysate solubilized with SDS-PAGE sample buffer showed that the amount of SV2B clearly was lowered in both models (day 10 of PAN, 57.1 ± 17.4%: day 5 of ANA, 34.3 ± 26.0% to normal). Magnifications: x200 in a, c, e, g, and I; x400 in b, d, f, h, and j.
Localization of CD2AP Is Altered in SV2B Knockdown Cultured Podocytes
Next, to analyze the relationship between SV2B and the slitdiaphragm components, we examined the expression and the localizationof CD2AP in cultured podocytes that were treated with SV2B siRNA.The expression of SV2B clearly decreased in the material thatwas treated with SV2B siRNA (mRNA level 14.6 ± 12.6%[Figure 6A]; protein level 47.7 ± 7.0% [Figure 6B]).Although CD2AP staining was detected in cytoplasm, includingthe process area, in the cells that were treated with negativecontrol siRNA (Figure 6C, c, e, and g), CD2AP staining at theprocess area was not observed in the cells that were treatedwith SV2B siRNA (Figure 6C, d, f, and h).
Figure 6. Gene silencing of SV2B in the cultured podocytes. Differentiated mouse podocyte was cultured for 7 d and treated with small interfering RNA (siRNA) for SV2B for 24 h. (A) The mRNA expression of SV2B and CD2AP was semiquantified by RT-PCR. GAPDH was analyzed as an internal control. The data are shown as a ratio (%) relative to the control siRNA-treated cells and are expressed as mean ± SD of three independent experiments. The mRNA expression of SV2B clearly was lowered in the material that was treated with siRNA for SV2B (14.6 ± 12.6%). The mRNA expression of CD2AP was not lowered (123.7 ± 0.5%). (B) Western blot analysis with cell lysate that was solubilized with SDS-PAGE sample buffer. The data are shown as a ratio (%) relative to the control siRNA-treated cells and are expressed as mean ± SD of three independent experiments. The intensity of the band of SV2B clearly was lowered in the material that was treated with siRNA for SV2B (47.7 ±+ 7.0%), whereas the intensity of the band of CD2AP was not lowered (110.0 ± 3.4%). (C) IF finding of the cells that were treated with control siRNA (a, c, e, and g) and with SV2B siRNA (b, d, f, and h). SV2B was stained green in a, b, c, and d. CD2AP was stained green in e and f. CD2AP was stained red in c, d, g, and h. Actin fiber was stained red with rhodamine-phalloidin in e and f. Nucleus was stained green with acridine orange in g and h. SV2B staining in the control material was detected in cytoplasm (a), whereas SV2B staining in the material that was treated with SV2B siRNA clearly was lowered (b). Dual-labeling IF finding of CD2AP with SV2B showed that CD2AP staining was detected in cytoplasm, including the process area (arrowheads), in the cells that were treated with negative control siRNA (c), but the staining at the process area was not detected in the cells that were treated with SV2B siRNA (d). Dual-labeling IF finding of CD2AP with phalloidin or acridine orange showed that CD2AP staining at the process area was not observed in the cells that were treated with SV2B siRNA (f and h). No change was detected in staining of actin (e and f) and nucleus (g and h). AO, acridine orange. Magnification, x400.
To identify the functional molecules that are involved in regulatingthe barrier function of the glomerular capillary wall, we performeda subtraction hybridization assay with the cDNA of normal andinjured glomeruli. We found the synaptic vesicle–associatedprotein SV2B is expressed in glomerular podocyte and that theexpression of SV2B clearly decreased in the injured podocyte.
Synaptic vesicles mediate membrane trafficking at the presynaptictermination. Neurotransmitters in synaptic vesicle are releasedby exocytosis according to the following mechanism: The vesiclesdock at the plasma membrane and undergo a maturation step, termedpriming, then the influx of calcium promotes the fusion of thevesicle with the plasma membrane (36). The cycle of the synapticvesicle resembles the mechanism of membrane trafficking thatgenerally is observed in several types of cells. SV2B is understoodto play a role in vesicle trafficking by binding to cell-surfaceproteins. We detected the expression of full-length cDNA ofSV2B in glomerular RNA (Figure 1B) and also detected the samesize band of SV2B, with Western blot analysis, in glomerularand cerebrum lysates (Figure 2A). In this study, we observedthat SV2B mRNA was expressed in several organs and tissues,including glomeruli, lungs, and digestive system (Figure 1A).However, no expression was detected in secretory organs, thymus,heart, spleen, or liver (Figure 1A). Conversely, another synapticvesicle–associated molecule, Rab3A, and another isoformof SV2, SV2A, were ubiquitously expressed in general organsand tissues (Figure 1A). Because Rab proteins confer specificityto vesicles, these results indicate that "synaptic vesicle–likevesicles" function in trafficking and secretion in general tissues.Some recent studies reported that SV2B plays an important rolein maintaining tissue-specific function (15–18). The tissue-specificexpression of SV2B suggests that SV2B plays a role in formingthe tissue-specific structure.
Next, we analyzed the localization of SV2B in glomeruli usinga specific antibody. The antibody reacted with a protein massof approximately 80 kD in glomerular and cerebrum lysates. Thespecificity for SV2B of this antibody was confirmed by absorptionassay with the peptide that was used for immunization (Figure 2A).The specificity also was confirmed by IF with kidney sections(Figure 2C) and SV2B-transfected COS-7 cells (Figure 2B). SV2Bwas detected as podocyte pattern along capillary loop in ratand human kidney (Figure 2, C, a, and D, a). To analyze furtherthe localization of SV2B, we performed dual-labeling IF studywith glomerular cell markers. SV2B was localized very closeto some of podocyte markers. The expression of SV2B also wasdetected in differentiated conditioned cultured podocyte (Figure 2E).All of these observations showed that podocyte possesses SV2B.A dual-labeling IF study with podocyte markers revealed thatmajor parts of the SV2B staining were co-stained with nephrinand synaptopodin (Figure 3A, a and d, arrowheads). These co-localizationstudies further suggest that SV2B is present in the podocytes,although the exact subcellular localization has not been established.Most of SV2B was detected in Triton X-100–soluble fractionof glomerular lysate (Figure 3B), which suggested that SV2Bmight be a membrane-associated molecule.
Then we analyzed the kinetics of the expression of SV2B in PANnephropathy, an experimental model of minimal-change nephroticsyndrome. We showed here that SV2B mRNA expression already decreased1 h after PAN injection. The decreased expression of mRNA stillwas detected at 24 h of PAN nephropathy, but the expressionwas normalized by day 10 at the peak of proteinuria. The observationssuggest that the decrease in SV2B expression is not merely theoutcome of proteinuria but also has an etiologic significance.It is very interesting that mRNA expression of SV2B is alreadydramatically downregulated 1 h after PAN administration andwas recovered at the peak of proteinuria. Although the initiationmechanism of PAN nephropathy is uncertain, it has been reportedthat PAN treatment induces significant early changes in reactiveoxygen species in podocyte (37). The downregulation of SV2Bmight be mediated by reactive oxygen species. We also analyzedthe kinetics of SV2B mRNA expression in ANA nephropathy. Proteinuriain this model results from the molecular rearrangement of theslit diaphragm as a result of the binding of the ANA to nephrin,a functional molecule of the slit diaphragm. A decrease in SV2BmRNA expression also was detected in this proteinuric model.Decreased SV2B expression, not only on the mRNA level (Figure 4)but also on the protein level (Figure 5), was detected in bothproteinuric states. Immunostaining of SV2B was already alteredin the early phase of diseases (Figure 5A, c, d, g, and h) andclearly reduced at the peak of proteinuria in both models (Figure 5A,e, f, i, and j). Western blot analysis showed that the amountof SV2B clearly was decreased at the peak of proteinuria inboth models (Figure 5B). All of these findings indicate thatSV2B is a functional molecule in podocytes. Janz et al. (11)showed that SV2B knockout mice were normal for up to 2.5 yr.The result means that the decreased expression of SV2B doesnot always lead to lethal changes. Although the reason thatSV2B knockout mice were normal is uncertain, one possibilityis that other molecules compensate for the function of SV2B.
It should be noted that the expression of SV2B decreased notonly in PAN nephropathy but also in ANA nephropathy, which suggeststhat SV2B might be related to the slit diaphragm. It is becomingwidely accepted that slit diaphragm dysfunction is involvedin the development of proteinuria in several kinds of glomerulardisease (8–10,28,29,38–44), and some novel moleculeshave been identified as slit diaphragm components in the pastseveral years (24,45–47). However, the questions of howslit diaphragm molecules are arranged and how the proper arrangementis maintained are yet to be clarified. We previously reportedthat nephrin first appears as a plasma membrane protein on thebasal and lateral sides below the junctional site of the presumptivepodocyte at the S-shaped body stage in rats (48). With the developmentof the interdigitating foot processes, nephrin becomes concentratedin the slit pores between the foot processes. We also reportedthat nephrin was redistributed and disappeared entirely fromthe cell surface when injected with ANA (49). During recovery,nephrin reappeared on the cell surface as proteinuria subsided.Very recently, two reports showed that phosphorylated nephrinbinds Nck adaptor proteins and that this Nck–nephrin interactionis required for nephrin-dependent actin reorganization (50,51).Tryggvason et al. (52) described in their review that afterinjury of the slit diaphragm, nephrin molecules become clustered,which induces their phosphorylation, Nck association, and actinpolymerization. It is conceivable that Nck–nephrin interactionis involved in the repairing process in ANA-induced nephropathy.CD2AP is reported to have a critical role in maintaining thebarrier function of the slit diaphragm by enabling nephrin tointeract with actin cytoskeleton. It is understood that nephrin–CD2APinteraction, which is a phosphorylation-independent contact,may be important for the maintenance of the slit diaphragm structurein normal matured glomeruli (52). Li et al. (25) reported thatCD2AP is expressed in various tissues and has a general rolein maintaining specialized subcellular architecture. Recently,Welsch et al. (53) reported that CD2AP was linked to endosomeof podocyte and was involved in endosomal trafficking via regulationof actin assembly on vesicles. We observed that CD2AP was expressedin cytoplasm, including the process area, in the differentiatedconditioned cultured podocytes (Figure 6C, c, e, and g), andwhen the podocyte was treated with SV2B siRNA, the CD2AP stainingat the process area was not observed (Figure 6C, d, f, and h).The result indicates that SV2B plays a role in the expressionand the proper localization of CD2AP.
This study resulted in three novel findings: (1) SV2B is expressedin glomeruli in a podocyte-like distribution, (2) SV2B expressionin glomeruli is decreased in proteinuric states that are causedby podocyte injuries, and (3) SV2B plays a role in the expressionand the proper localization of CD2AP. On the basis of theseresults, we propose that SV2B is one of the important moleculesmaintaining podocyte function. Further investigations into thefunction of SV2B and the vesicle trafficking system in the podocytewill enable the establishment of a novel selective therapy forproteinuria.
Acknowledgments
This work was supported by Grant-Aids for Scientific Research(B) (13557084 and 14370317 to H.Ka. and 15390268 to F.S.) fromthe Ministry of Education, Culture, Sports, Science and Technologyof Japan.
A portion of this study was presented at the annual meetingof the American Society of Nephrology; October 29 through November1, 2004; St. Louis, MO; and was published in abstract form (JAm Soc Nephrol 15: 238A, 2004).
We thank Mutsumi Kayaba and Chiharu Nagasawa for excellent technicalassistance. We thank Dr. Tetsuo Morioka (Department of CellularPhysiology, Institute of Nephrology, Niigata University) forhelpful advice.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-actin antibodies, a rhodamine-phalloidin, and an acridine orange were purchased from Serotec (Oxford, UK), PROGEN (Heidelberg, Germany), Sigma (St. Louis, MO), Cytoskeleton (Denver, CO), and Polysciences (Warrington, PA), respectively. Rabbit anti-CD2AP, mouse monoclonal anti-nephrin antibody (ANA; mAb 5-1-6), and mouse monoclonal anti-Thy1.1 antigen antibody (mAb 1-22-3) were prepared as described previously (29,33,34). Normal rabbit serum was used as a negative control. Secondary antibodies that were used for immunofluorescence (IF) study were as follows: FITC-conjugated swine anti-rabbit IgG (DAKO, Glostrup, Denmark), TRITC-conjugated goat anti-mouse IgG1, TRITC-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates, Birmingham, AL), and FITC- and TRITC-conjugated donkey anti-goat IgG (Protos Immunoresearch, San Francisco, CA). Alkaline phosphatase–conjugated goat anti-rabbit IgG (Biosource International, Tago Immunologicals, Camarillo, CA) was used for Western blot analysis. 
