Abstract
Synaptic vesicle protein 2B (SV2B) was identified by the subtraction hybridization technique as a molecule of which mRNA expression was decreased in puromycin aminonucleoside (PAN) nephropathy by glomerular cDNA subtraction assay. The expression of SV2B was detected in glomerular lysate with Western blot analysis. Dual-labeling immunofluorescence studies with glomerular cell markers demonstrated that SV2B is expressed in glomerular visceral epithelial cells (podocytes). The expression of SV2B is detected also in cultured podocyte and in human kidney section as podocytic pattern. The decrease of SV2B mRNA was already detected before the onset of proteinuria in PAN nephropathy. The mRNA expression of SV2B clearly is altered not only in PAN nephropathy but also in another proteinuric state that is caused by an antibody against nephrin, a functional molecule of the slit diaphragm. The decreased intensity in SV2B staining was already detected before the peak of proteinuria in both models with immunofluorescence study. A reduced amount of SV2B was detected in both models also with Western blot analysis. CD2AP, another functional molecule of the slit diaphragm, was observed in cytoplasm, including the processes area of the cultured podocyte, and when the podocyte was treated with small interfering RNA for SV2B, CD2AP staining at the process area was not detected. These results suggest that SV2B is a functional molecule of podocyte, and SV2B may play a role in the expression and proper localization of CD2AP.
The clarification of the pathogenic mechanism of proteinuria is one of the most important themes in the nephrology field. It is becoming clear that disorder of the glomerular visceral epithelial cell (podocyte) causes proteinuria and results in nephrotic syndrome. Recent studies have focused on the role of the slit diaphragm, located between the foot processes of the podocytes, in maintaining the barrier function of the glomerular capillary wall (1–7). We and others reported previously that nephrin and podocin are localized at the slit diaphragm and that their expressions clearly are altered already at the early phase of puromycin aminonucleoside (PAN) nephropathy, an experimental model of minimal-change nephrotic syndrome (8–10). These findings indicate that these molecules are critical components of the slit diaphragm and that alteration of their expression is involved in the development of proteinuria. However, the precise mechanism of the development of proteinuria in PAN nephropathy is not well understood yet.
In this study, we aimed to identify the molecules that are involved in the development of proteinuria. Because it is conceivable that the molecules whose expression decreased before the onset of proteinuria may have been involved in the development of proteinuria, we intended to purify the molecules whose expressions were downregulated 24 h after PAN injection, using cDNA subtractive hybridization techniques. We identified 28 genes that were downregulated, and eight molecules of them were confirmed to be expressed in glomeruli and cultured podocyte. Among them, we focused on synaptic vesicle protein 2B (SV2B) in our study. SV2B is an isoform of SV2, which is a glycosylated synaptic vesicle membrane protein that comprises 12 transmembrane regions (11,12). SV2 is known to participate in the regulation of calcium-mediated synaptic transmission and to play a role in vesicle trafficking by binding to other cell surface proteins (13,14). Although SV2 originally was reported to be expressed specifically in the central nervous system, recent studies have shown that it also is distributed in other organs (15–17). Hayashi et al. (18) reported that SV2B is expressed in the microvesicles of pinealocytes, which are rich in process terminals. It has been pointed out that neurons and podocytes have common characteristics. Both are terminally differentiated cells and have similar shapes that are characterized by processes (19,20). Both of them also display cell type–specific intercellular contact: Synapses in neurons and the slit diaphragm in podocytes. Furthermore, functionally important molecules in the podocyte, such as nephrin and podocin, have been found to be shared specifically with the neuron (9,10,21). Recently, Rastaldi et al. (22) reported that synaptic vesicle molecule Rab3A and its specific effector, rabphilin-3a, are expressed in the podocyte and that they are involved in vesicle trafficking. Cormont et al. (23) reported that Rab4, another molecule of the Rab family, interacted with CD2AP and might play a role in its trafficking. It has been reported that CD2AP also is expressed in the podocyte and that CD2AP is involved in maintaining the barrier function of the slit diaphragm by interacting with nephrin (24–29). It also has been reported that the expression of CD2AP is decreased in cultured podocyte that is treated with PAN (30,31). All of the properties of these synaptic vesicle–associated molecules prompted us to consider that SV2B might be associated with the slit diaphragm.
In this study, we demonstrated that SV2B is expressed in the podocyte and that its expression decreases not only in PAN nephropathy but also in the proteinuric model induced by the antibody against nephrin, a functional molecule of the slit diaphragm. We also demonstrated that CD2AP distribution clearly is altered in cultured podocyte that is treated with small interfering RNA (siRNA) for SV2B, suggesting that SV2B plays a role in the expression of podocyte functional proteins such as CD2AP.
Materials and Methods
Animal
All experiments of animals in this study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Procedures for this study were approved by the Animal Committee at Niigata University School of Medicine, and all animals were treated according to the guidelines for animal experimentation of Niigata University. Specific pathogen-free 7-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 (Albert Einstein College of Medicine, Bronx, NY). Cultivation of conditionally immortalized 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 of podocytes, cells first were cultivated at 33°C and maintained for 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 with expression vector pcDNA3.1/His with an insert of full-length cDNA of SV2B by chloroquine method.
Antibodies
Rabbit anti-SV2B and mouse anti-Rab3A antibodies were purchased from Synaptic Systems (Goettingen, Germany). Goat anti-CD2AP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti–RECA-1, mouse anti-synaptopodin, rabbit anti–β-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.
Subtractive Hybridization
Ten rats received intravenous injection of 100 mg/kg body wt PAN (Sigma). Preparation of cDNA from rat glomerular mRNA was according to the method described previously (9,10). Subtractive hybridization using cDNA from glomerular RNA of normal rats as tester and that from rats with PAN nephropathy (24 h after induction) as driver was performed with PCR-Select cDNA subtraction Kit (Clontech, Palo Alto, CA), and the basic procedures were according to the manufacturer’s protocol. Amplified product was subcloned into the plasmid vector pCRII-TOPO (TOPO TA Cloning Kit 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 organs and from kidney cortex, kidney medulla, and glomeruli, and their mRNA 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 codon and the reverse primer just upstream of the stop codon to analyze whether the full-length coding sequence (CDS) of SV2B mRNA was expressed in glomeruli. The sequences of all primers that were used in this study are shown in Table 1.
PCR primers that were used in this studya
Western blot analysis was performed basically according to the method reported previously (9,10,29). In brief, normal or proteinuric rat glomeruli, normal rat cerebrum, and cells were solubilized with RIPA buffer (0.1% SDS, 1% sodium deoxycholate, 1% Triton X-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, and 5% mercaptoethanol in 62.5 mmol/L Tris-HCl [pH 6.8]) with protease inhibitors. Normal rat glomeruli also was solubilized sequentially with 1% Triton X-100 and RIPA buffer and separated into Triton X-100–soluble fraction (fraction 1), Triton X-100–insoluble/RIPA-soluble fraction (fraction 2), and RIPA-insoluble fractions (fraction 3). RIPA-insoluble fraction was solubilized with SDS-PAGE sample buffer. The concentration of the materials was measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and 20 μg of these samples was subjected to SDS-PAGE with 10% acrylamide gel and transferred to a polyvinylidene fluoride transfer membrane (Pall Corp., Pensacola, FL). After blocking with bovine skim milk, the strips of membrane were exposed to rabbit anti-rat SV2B or rabbit anti-CD2AP, rabbit anti-rat β-actin antibodies, or normal rabbit serum. They then were washed and incubated with alkaline phosphatase–conjugated goat anti-rabbit IgG. The reaction was developed with an alkaline phosphatase chromogen kit (Biomedica, Foster City, CA).
IF studies were performed basically according to the method reported previously (9,10,29). The 3-μm-thick frozen sections of normal rat and human or cells that were cultured on a glass coverslip were fixed with acetone for 1 min and incubated with primary antibodies. The dual-labeling IF for SV2B was carried out 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/kg body wt PAN or 15 mg/rat ANA (mAb 5-1-6), respectively. For PCR analysis and IF study, kidney materials of three rats each were removed at 1 h (PAN and ANA nephropathy), 24 h (PAN and ANA nephropathy), 5 d (ANA nephropathy), and 10 d (PAN nephropathy) after injection. Urine samples were collected, and their protein concentration was measured by colorimetric assay with a Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA). The average value of the proteinuria was 4.5 (24 h) and 238.7 mg/d (10 d) of PAN nephropathy 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. Glomerular RNA was prepared from the kidneys that were pooled from three rats. The IF staining of SV2B was semiquantified basically according to the method described by Macconi et al. (35) (score 0, completely absent; 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 was assigned to each glomerulus, and >30 glomeruli of each rat were analyzed. The data are shown as a ratio (%) relative to the total number of glomeruli scored and are expressed as mean ± SD of three rats.
For Western blot analysis, three other rats each received an injection of PAN or ANA, and the kidney materials were removed on day 10 or day 5, respectively. Glomeruli that were prepared from the kidneys from three rats in each group were solubilized with SDS-PAGE sample buffer. The concentration of the materials was measured by the bicinchoninic acid method (Pierce Chemical), and 20 μg/lane of the materials was used for Western blot analysis. The experiments were performed three times, and the band intensity was determined by image analysis using Bio Doc-It System (UVP, Inc., Upland, CA). All results were corrected for the amount of protein in the sample by dividing by the intensity of the internal control β-actin. The columns represent mean values of three examinations, and the bars show SD.
RT-PCR
Semiquantitative RT-PCR was performed basically according to the method described previously (9,10), using the primers listed in Table 1. The band intensity was determined by image analysis using Bio Doc-It System (UVP, Inc.). All results were corrected for the amount of mRNA in the sample by dividing by the intensity of 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 three times. For preparation of standard material, PCR products of SV2B and glyceraldehyde-3-phosphate dehydrogenase were subcloned into the plasmid vector pCRII-TOPO, and the plasmids were diluted from 1 × 102 copies to 1 × 109 copies to generate calibration curves, which were based on the linear relationship between the crossing point cycle values and the logarithm of the starting copy number.
RNA Silencing Analysis
The siRNA sequences that targeted SV2B (GenBank accession no. AF372834) were synthesized by iGENE (Tsukuba, Japan): The siRNA had 25 nucleotides in length, corresponding to positions 69 to 93 of the SV2B open reading frame. Before transfection, podocytes were cultured to a density of 70 to 80% at 37°C, and then they were transfected with the siRNA using a Trans IT-TKO transfection reagent (Mirus, Madison, WI). Nonspecific nucleotide sequence transfected cells were used as negative control. Cells were harvested 24 h after siRNA treatment for RT-PCR, Western blot analysis, and IF analysis. Dual-labeling IF of CD2AP was performed with anti-SV2B antibody, rhodamine-phalloidin, and acridine orange. Rhodamine-phalloidin was used to detect actin fiber, and acridine orange was used to detect nucleus.
Results
Molecules Identified with cDNA Subtractive Hybridization
Twenty-eight genes were identified with the subtraction hybridization techniques, and eight molecules of them were confirmed to be expressed in glomeruli and cultured podocyte. These podocyte-associated molecules are summarized in 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 showed 100% identity with that of rat SV2B was detected in the subtracted cDNA (glomerular cDNA of PAN nephropathy rats [24 h after induction] from that of normal controls). To analyze whether the full-length CDS of SV2B is expressed in glomeruli, we performed RT-PCR with the primers shown in Table 1. 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 (Figure 1B).
(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 also in the central nervous system, lungs, digestive system, skin, and placenta (Figure 1A). SV2B mRNA expression was not detected in secretory organs thyroid, pancreas, and adrenal glands. In contrast, the expression of SV2A and Rab3A was detected in all organs examined.
SV2B Is Expressed in the Glomerular Podocyte
A positive band of approximately 80 kD was detected in rat glomerular and cerebrum lysates that were solubilized with RIPA buffer by Western blot analysis with anti-SV2B antibody (Figure 2A, 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 (Figure 2A, lane 3). No change was observed with anti-SV2B antibody that 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 detected in COS-7 cells that were transfected with pcDNA3.1/His with an insert of full-length cDNA of rat SV2B (Figure 2B, a), although the staining that was detected in COS-7 cells that were transfected with vector without insert was faint (Figure 2B, d). Negative staining was detected in COS-7 cells that were transfected with the insert with normal rabbit serum (Figure 2B, b) or with anti-SV2B antibody that was preabsorbed with the peptide that was used for immunization (Figure 2B, c). Clear staining of SV2B was observed along the glomerular capillary loop (Figure 2C, a). SV2B staining also was detected in distal tubular epithelial cell. No positive staining was detected with normal rabbit serum (Figure 2C, b) and anti-SV2B antibody that was preabsorbed with the peptide that was used for immunization (Figure 2C, c). SV2B staining also was observed along the glomerular capillary loop in human kidney section (Figure 2D, a). The staining pattern was very similar to that in rat sections. SV2B staining was detected in cultured podocytes (Figure 2E, a). Positive staining of SV2B was detected in cytoplasm, including processes of cultured podocyte. No positive staining was detected with normal rabbit serum (Figure 2E, b). SV2B staining clearly was apart from that of RECA-1, the endothelial cell marker, or Thy1.1, the mesangial cell marker (Figure 3A, 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-stained with nephrin and synaptopodin (Figure 3A, a and d, arrowheads), and some portions of SV2B staining were co-stained with Rab3A (Figure 3C).
(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).
(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, ×400 in A and C.
To analyze further the subcellular localization of SV2B, we performed Western blot analysis with sequentially solubilized glomerular lysates (Figure 3B). Most of SV2B was detected in fraction 1 (78.4 ± 13.6%). A small amount of SV2B was detected in fraction 2 (16.0 ± 4.0%).
SV2B Expression Is Altered in Proteinuric States
The kinetics of SV2B mRNA expression in PAN and ANA nephropathy are shown in Figure 4. We confirmed that glomerular SV2B mRNA expression was decreased at 24 h of PAN nephropathy (58.6 ± 19.7%). SV2B mRNA expression had already decreased at 1 h of PAN nephropathy (20.7 ± 17.5%). The expression of SV2B mRNA recovered to normal range on day 10 (125.1 ± 16.5%), when proteinuria peaked in this model. A decrease in the expression of 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 demonstrated that SV2B mRNA expression clearly decreased at the early phases of both models (Figure 4). Positive IF signal of SV2B was lowered at the early phase of both models and clearly was lowered at the peak of proteinuria of both models (Figure 5A). Western blot analysis with glomerular lysate that was solubilized with SDS-PAGE sample buffer showed that the amount of SV2B clearly is reduced in both models (day 10 of PAN, 57.1 ± 17.4%; day 5 of ANA, 34.3 ± 26.0% to normal; Figure 5B).
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.
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: ×200 in a, c, e, g, and I; ×400 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 slit diaphragm components, we examined the expression and the localization of CD2AP in cultured podocytes that were treated with SV2B siRNA. The expression of SV2B clearly decreased in the material that was 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, including the process area, in the cells that were treated with negative control siRNA (Figure 6C, c, e, and g), CD2AP staining at the process area was not observed in the cells that were treated with SV2B siRNA (Figure 6C, d, f, and h).
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, ×400.
Discussion
To identify the functional molecules that are involved in regulating the barrier function of the glomerular capillary wall, we performed a subtraction hybridization assay with the cDNA of normal and injured glomeruli. We found the synaptic vesicle–associated protein SV2B is expressed in glomerular podocyte and that the expression of SV2B clearly decreased in the injured podocyte.
Synaptic vesicles mediate membrane trafficking at the presynaptic termination. Neurotransmitters in synaptic vesicle are released by exocytosis according to the following mechanism: The vesicles dock at the plasma membrane and undergo a maturation step, termed priming, then the influx of calcium promotes the fusion of the vesicle with the plasma membrane (36). The cycle of the synaptic vesicle resembles the mechanism of membrane trafficking that generally is observed in several types of cells. SV2B is understood to play a role in vesicle trafficking by binding to cell-surface proteins. We detected the expression of full-length cDNA of SV2B in glomerular RNA (Figure 1B) and also detected the same size band of SV2B, with Western blot analysis, in glomerular and cerebrum lysates (Figure 2A). In this study, we observed that 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 synaptic vesicle–associated molecule, Rab3A, and another isoform of SV2, SV2A, were ubiquitously expressed in general organs and tissues (Figure 1A). Because Rab proteins confer specificity to vesicles, these results indicate that “synaptic vesicle–like vesicles” function in trafficking and secretion in general tissues. Some recent studies reported that SV2B plays an important role in maintaining tissue-specific function (15–18). The tissue-specific expression of SV2B suggests that SV2B plays a role in forming the tissue-specific structure.
Next, we analyzed the localization of SV2B in glomeruli using a specific antibody. The antibody reacted with a protein mass of approximately 80 kD in glomerular and cerebrum lysates. The specificity for SV2B of this antibody was confirmed by absorption assay 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). SV2B was detected as podocyte pattern along capillary loop in rat and human kidney (Figure 2, C, a, and D, a). To analyze further the localization of SV2B, we performed dual-labeling IF study with glomerular cell markers. SV2B was localized very close to some of podocyte markers. The expression of SV2B also was detected 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 that major parts of the SV2B staining were co-stained with nephrin and synaptopodin (Figure 3A, a and d, arrowheads). These co-localization studies 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 fraction of glomerular lysate (Figure 3B), which suggested that SV2B might be a membrane-associated molecule.
Then we analyzed the kinetics of the expression of SV2B in PAN nephropathy, an experimental model of minimal-change nephrotic syndrome. We showed here that SV2B mRNA expression already decreased 1 h after PAN injection. The decreased expression of mRNA still was detected at 24 h of PAN nephropathy, but the expression was normalized by day 10 at the peak of proteinuria. The observations suggest that the decrease in SV2B expression is not merely the outcome of proteinuria but also has an etiologic significance. It is very interesting that mRNA expression of SV2B is already dramatically downregulated 1 h after PAN administration and was recovered at the peak of proteinuria. Although the initiation mechanism of PAN nephropathy is uncertain, it has been reported that PAN treatment induces significant early changes in reactive oxygen species in podocyte (37). The downregulation of SV2B might be mediated by reactive oxygen species. We also analyzed the kinetics of SV2B mRNA expression in ANA nephropathy. Proteinuria in this model results from the molecular rearrangement of the slit diaphragm as a result of the binding of the ANA to nephrin, a functional molecule of the slit diaphragm. A decrease in SV2B mRNA 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 both proteinuric states. Immunostaining of SV2B was already altered in the early phase of diseases (Figure 5A, c, d, g, and h) and clearly reduced at the peak of proteinuria in both models (Figure 5A, e, f, i, and j). Western blot analysis showed that the amount of SV2B clearly was decreased at the peak of proteinuria in both models (Figure 5B). All of these findings indicate that SV2B 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 does not always lead to lethal changes. Although the reason that SV2B knockout mice were normal is uncertain, one possibility is that other molecules compensate for the function of SV2B.
It should be noted that the expression of SV2B decreased not only in PAN nephropathy but also in ANA nephropathy, which suggests that SV2B might be related to the slit diaphragm. It is becoming widely accepted that slit diaphragm dysfunction is involved in the development of proteinuria in several kinds of glomerular disease (8–10,28,29,38–44), and some novel molecules have been identified as slit diaphragm components in the past several years (24,45–47). However, the questions of how slit diaphragm molecules are arranged and how the proper arrangement is maintained are yet to be clarified. We previously reported that nephrin first appears as a plasma membrane protein on the basal and lateral sides below the junctional site of the presumptive podocyte at the S-shaped body stage in rats (48). With the development of the interdigitating foot processes, nephrin becomes concentrated in the slit pores between the foot processes. We also reported that nephrin was redistributed and disappeared entirely from the 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 nephrin binds Nck adaptor proteins and that this Nck–nephrin interaction is required for nephrin-dependent actin reorganization (50,51). Tryggvason et al. (52) described in their review that after injury of the slit diaphragm, nephrin molecules become clustered, which induces their phosphorylation, Nck association, and actin polymerization. It is conceivable that Nck–nephrin interaction is involved in the repairing process in ANA-induced nephropathy. CD2AP is reported to have a critical role in maintaining the barrier function of the slit diaphragm by enabling nephrin to interact with actin cytoskeleton. It is understood that nephrin–CD2AP interaction, which is a phosphorylation-independent contact, may be important for the maintenance of the slit diaphragm structure in normal matured glomeruli (52). Li et al. (25) reported that CD2AP is expressed in various tissues and has a general role in maintaining specialized subcellular architecture. Recently, Welsch et al. (53) reported that CD2AP was linked to endosome of podocyte and was involved in endosomal trafficking via regulation of actin assembly on vesicles. We observed that CD2AP was expressed in cytoplasm, including the process area, in the differentiated conditioned cultured podocytes (Figure 6C, c, e, and g), and when the podocyte was treated with SV2B siRNA, the CD2AP staining at the process area was not observed (Figure 6C, d, f, and h). The result indicates that SV2B plays a role in the expression and the proper localization of CD2AP.
Conclusion
This study resulted in three novel findings: (1) SV2B is expressed in glomeruli in a podocyte-like distribution, (2) SV2B expression in glomeruli is decreased in proteinuric states that are caused by podocyte injuries, and (3) SV2B plays a role in the expression and the proper localization of CD2AP. On the basis of these results, we propose that SV2B is one of the important molecules maintaining podocyte function. Further investigations into the function of SV2B and the vesicle trafficking system in the podocyte will enable the establishment of a novel selective therapy for proteinuria.
Acknowledgments
This work was supported by Grant-Aids for Scientific Research (B) (13557084 and 14370317 to H.Ka. and 15390268 to F.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
A portion of this study was presented at the annual meeting of the American Society of Nephrology; October 29 through November 1, 2004; St. Louis, MO; and was published in abstract form (J Am Soc Nephrol 15: 238A, 2004).
We thank Mutsumi Kayaba and Chiharu Nagasawa for excellent technical assistance. We thank Dr. Tetsuo Morioka (Department of Cellular Physiology, Institute of Nephrology, Niigata University) for helpful advice.
Footnotes
Published online ahead of print. Publication date available at www.jasn.org.
- © 2006 American Society of Nephrology
References
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