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J Am Soc Nephrol 14:1154-1163, 2003
© 2003 American Society of Nephrology

Angiogenic Protein Cyr61 is Expressed by Podocytes in Anti-Thy-1 Glomerulonephritis

Kazutomo Sawai, Kiyoshi Mori, Masashi Mukoyama, Akira Sugawara, Takayoshi Suganami, Masao Koshikawa, Kensei Yahata, Hisashi Makino, Tetsuya Nagae, Yuriko Fujinaga, Hideki Yokoi, Tetsuro Yoshioka, Akihiro Yoshimoto, Issei Tanaka and Kazuwa Nakao

Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Dr. Kiyoshi Mori, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 81-75-751-4286; Fax: 81-75-771-9452;


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Dynamic recovery of glomerular structure occurs after severe glomerular damage in anti-Thy-1 glomerulonephritis (Thy-1 GN), but its mechanism remains to be investigated. To identify candidate genes possibly involved in glomerular reconstruction, screening was performed for genes that are specifically expressed by podocytes and are upregulated in glomeruli of Thy-1 GN. Among them, cysteine-rich protein 61 (Cyr61 or CCN1), a soluble angiogenic protein belonging to the CCN family, was identified. By Northern blot analysis, Cyr61 mRNA was markedly upregulated in glomeruli of Thy-1 GN from day 3 through day 7, when mesangial cell migration was most prominent. By in situ hybridization and immunohistochemistry, Cyr61 mRNA and protein were expressed by proximal straight tubules and afferent and efferent arterioles in normal rat kidneys and were intensely upregulated at podocytes in Thy-1 GN. Platelet-derived growth factor–BB (PDGF-BB) and transforming growth factor–{beta}1 (TGF-{beta}1), of which the gene expression in the glomeruli of Thy-1 GN was upregulated in similar time course as Cyr61, induced Cyr61 mRNA expression in cultured podocytes. Furthermore, supernatant of Cyr61-overexpressing cells inhibited PDGF-induced mesangial cell migration. In conclusion, it is shown that Cyr61 is strongly upregulated at podocytes in Thy-1 GN possibly by PDGF and TGF-{beta}. Cyr61 may be involved in glomerular remodeling as a factor secreted from podocytes to inhibit mesangial cell migration. E-mail: keyem@kuhp.kyoto-u.ac.jp


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The glomeruli of the kidney seem to possess an ability to repair their own structure, because resolution of glomerular sclerotic lesions occurs in diabetic patients after pancreas transplantation (1). Rat anti-Thy-1 glomerulonephritis (Thy-1 GN) is one of the best-studied reversible models of glomerulonephropathy (2–5). In this model, mesangiolysis leads to severe destruction of glomerular structure, characterized by so-called "ballooning lesions." Glomerular reconstruction begins with migration of mesangial cells from vascular poles and angiogenesis by immature endothelial cells (2,3). During the course of glomerular remodeling, various soluble factors have been identified to be expressed by mesangial cells, such as platelet-derived growth factor (PDGF), basic fibroblast growth factor, and endothelin (5). However, most of them enhance mesangial cell proliferation and potentially exacerbate glomerulonephritis. On the other hand, vascular endothelial growth factor, secreted by mesangial cells, podocytes, and infiltrating leukocytes, enhances the recovery by stimulating endothelial cell proliferation (6,7). Other soluble factors may also be involved in this complex process.

A characteristic feature of Thy-1 GN is that podocytes are kept almost intact throughout the course. Preceding minor podocyte injury to this model leads to irreversible mesangial alteration (8), demonstrating the importance of podocytes for the glomerular recovery in this model. These findings suggest that podocytes contribute to maintenance and recovery of the glomerular structure by counteracting the hydrostatic force on the glomerular filtration barrier mechanically. Furthermore, it is also possible that podocytes may regulate mesangial migration, proliferation, or matrix accumulation by secreting undetermined factors and that these factors might play a role in glomerular remodeling during Thy-1 GN. Therefore, to identify new candidate genes possibly involved in the reconstruction of damaged glomeruli, we screened for genes whose expressions are podocyte-specific and are upregulated in the glomeruli of Thy-1 GN. Using the suppressive subtractive hybridization method, we identified several such genes, one of which was cysteine-rich protein 61 (Cyr61 or CCN1).

Cyr61 is a secreted, heparin-binding extracellular matrix-binding protein belonging to the CCN family, which also includes connective tissue growth factor (CTGF or CCN2), nephroblastoma overexpressed (Nov or CCN3), and Wnt-induced secreted proteins-1, -2, and -3 (WISP-1, 2, 3 or CCN-4, 5, 6, respectively) (9–11).

Cyr61 stimulates migration and proliferation of vascular endothelial cells and fibroblasts in culture and induces neovascularization in rat corneas (12–14). To date, Cyr61 expression or its role in the kidney remains unknown.

In this study, we revealed that Cyr61 is upregulated in the glomeruli of Thy-1 GN. We investigated the sites of Cyr61 expression in normal rat kidneys and in Thy-1 GN and its regulation in cultured podocytes. Furthermore, we studied the effect of Cyr61 on mesangial cell migration and proliferation. Our data suggest that Cyr61 expressed in podocytes may be involved in glomerular remodeling during Thy-1 GN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
An immortalized mouse podocyte cell line, MPC5, was a kind gift from Dr. Peter Mundel, Albert Einstein College of Medicine, Bronx, New York (15). These cells proliferate when cultured with 10 U/ml murine interferon-{gamma} (INF-{gamma}; Life Technologies, Gaithersburg, MD) at 33°C (permissive condition), whereas they halt growing and begin to differentiate to express podocyte-specific genes such as synaptopodin when cultured without INF-{gamma} at 37°C (nonpermissive condition). Podocytes were cultured with RPMI 1640 medium (Nihonseiyaku, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; Cansera International, Ontario, Canada), 10 U/ml penicillin, and 10 µg/ml streptomycin (ICN Biomedicals, Costa Mesa, CA) on dishes coated with 100 µg/ml type I collagen (Cellgen IPC-03; KOKEN, Tokyo, Japan). Before each experiment, cells were differentiated under nonpermissive condition on type I collagen-coated dishes for 2 wk without passage and cultured with RPMI 1640 containing 0.5% bovine serum albumin (BSA; Sigma, St. Louis, MO) for 24 h, until stimulation with 5 ng/ml human transforming growth factor–{beta}1 (TGF-{beta}1) or 10 ng/ml human PDGF-BB (R&D Systems, Minneapolis, MN). Cells were used between passages 15 and 20 (15).

Mesangial cells were established from glomeruli of 4 to 6-wk-old Sprague-Dawley rats as described previously (16). Mesangial cells were cultured with Dulbecco Modified Eagle Medium (DMEM; Invitrogen, Carlsbad, CA) with 10% FCS and antibiotics and used between passages 7 and 10.

Generation of Podocyte-Specific cDNA Library
To generate a cDNA library of podocyte-specific genes (PSG), we applied the suppressive subtractive hybridization procedure between cDNA pools derived from differentiated mouse podocytes and from C57BL/6 mouse whole kidney using PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, CA). Total RNA from each sample was extracted by TRIZOL Reagent (Invitrogen), and poly(A)+ mRNA was isolated using PolyATtract mRNA Isolation System IV (Promega, Madison, WI). To confirm podocyte-specific expression of each cDNA, we performed differential screening by reverse Northern blot analysis. cDNA fragments were blotted in quadruplicate on four sets of nylon membranes (GeneScreen Plus; NEN Life Science Products, Boston, MA) and hybridized with four different 32P-labaled cDNA pools using PCR-Select Differential Screening Kit (Clontech): podocyte (Pod) subtracted with whole kidney (WK) (Pod - WK); WK - Pod; Pod unsubtracted; and WK unsubtracted. The blots were exposed to BAS-III imaging plate (Fuji, Tokyo, Japan).

Generation of Anti-Thy-1 Glomerulonephritis
All animal experiments were conducted in accordance with our institutional guidelines for animal research. Mouse monoclonal antibody against rat Thy-1 (CD90) antigen (CL005A; Cedarlane, Ontario, Canada) (17) was washed and concentrated in phosphate-buffered saline (PBS) using dialysis membrane (Slide-A-Lyser; Pierce, Rockford, IL) for 16 h at 4°C. Glomerulonephritis was induced in Wistar rats (150 to 200 g) by intravenous administration of 1.5 mg/kg anti-Thy-1 antibody diluted in 0.7 ml PBS from tail vein. The rats were killed after antibody administration for histologic examination of kidney tissues and for isolation of glomeruli to extract total RNA. For light microscopic study, kidney tissues were fixed with Dubosq-Brazil solution for 12 h at 4°C and embedded in paraffin.

Library Screening for Genes Upregulated in Anti-Thy-1 Glomerulonephritis and Nucleotide Sequencing
PSG were blotted identically on four different nylon membranes (GeneScreen Plus) and hybridized with four different 32P-labeled cDNA pools derived from glomeruli of Thy-1 GN at days 0, 1, 3, and 5 using PCR-Select Differential Screening Kit. In this screening, mouse cDNAs blotted on membranes were hybridized with rat cDNA probes, because we expected that most, if not all, cDNAs have enough sequence homologies for cross-hybridization. The nucleotide sequences of cDNAs whose expression levels were increased in Thy-1 GN were determined by BigDye Terminator (Applied Biosystems, Foster City, CA) and ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Northern Blot Analyses
Northern blot analysis was performed as described (18). In brief, total RNA (30 µg in each lane) was electrophoresed on 1.0% agarose gels and transferred to nylon membranes (GeneScreen Plus). The cDNA fragments of rat Cyr61 (see below), rat CTGF (nucleotides 1221 to 1803, GenBank accession number AF120275, generated by reverse transcription [RT]-PCR) (19), rat PDGF-B (nucleotides 38 to 666, GenBank accession number Z14117, generated by RT-PCR) (20), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Clontech) were used as probes. The membranes were hybridized with [32P]dCTP-labeled probes, and the blots were exposed to BAS-III imaging plate. The amount of RNA loaded in each lane was normalized for 28S ribosomal RNA or GAPDH.

In Situ Hybridization Analyses
Rat Cyr61 cDNA fragment was cloned by RT-PCR with sense primer 5'-tgcgcgccacaatgagctccagca-3' and antisense primer 5'-cccaggagacctttagtccctgaa-3' (nucleotides 175 to 1337, GenBank accession number AF218568) (21) using total RNA from Wistar rat kidneys. Sense and antisense [35S]CTP-labeled cRNAs were generated from the rat Cyr61 cDNA ligated in pGEM-T easy vector (Promega) using T7 and SP6 RNA polymerases (Promega). In situ hybridization analysis was performed as described previously (22). In brief, 10-µm cryosections of rat kidneys were mounted on poly-L-lysine-coated slides, fixed with paraformaldehyde, acetylated, and hybridized with the cRNA probes. Slides were washed, dehydrated, and apposed to Hyperfilm {beta}-max films (Amersham, Buckinghamshire, UK) for 10 d or dipped into autoradiographic emulsion (NTB-2, Eastman Kodak, Rochester, NY) and exposed for 6 wk and counterstained with hematoxylin and eosin.

Immunohistochemical Analyses
Deparaffinized 3-µm kidney sections were treated with microwave heating (5 min twice in 10 mM citrate buffer, pH 7.4). Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide for 15 min at room temperature. Goat anti-human Cyr61 antibody (sc-8561; Santa Cruz Biotechnology, Santa Cruz, CA) was diluted 1:300 in PBS containing 1% BSA (1% BSA/PBS) and was incubated for 1 h at room temperature. After washes with 1% BSA/PBS, the sections were incubated with biotinylated secondary antibody (sc-2347, biotin-conjugated bovine anti-goat Ig; Santa Cruz Biotechnology) diluted 1:100 in 1% BSA/PBS for 30 min at room temperature. The sections were further processed with avidin-biotin-peroxidase complex kit (Vector, Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride (Kanto Chemical, Tokyo, Japan), and counterstained with hematoxylin and coverslipped. Nonimmune goat serum was used as negative control. To further confirm the specificity of the signals, anti-Cyr61 antibody was preincubated overnight at 4°C with blocking peptide (sc-8561 P, Santa Cruz Biotechnology) at the five-times higher concentration (weight/volume) than the antibody concentration before incubation with the sections.

Overexpression of Cyr61 and Western Blot Analyses
COS-7 cells were cultured with DMEM supplemented with 10% FCS and antibiotics. Full-length mouse Cyr61 cDNA was generated by RT-PCR using total RNA from C57BL/6 mouse kidneys and the following primers: sense 5'-tgcgcgccacaatgagctccagca-3' and antisense 5'-ttagtccctgaacttgtggatgtc-3' (nucleotides 179 to 1329, GenBank accession number M32490) (23). The Cyr61 cDNA was first TA-cloned into pGEM-T easy vector and transferred into EcoRI restriction site of expression vector pCXN2, a derivative of pCAGGS (24). COS-7 cells were transfected with Cyr61-pCXN2 or mock-pCXN2 cDNA using Lipofectamine Plus Reagent (Invitrogen). Media were changed 3 h after transfection, harvested 69 h later, and kept -20°C until they were used for functional assay (see below). At 72 h after transfection, cells were lysed on ice in solution that contained 20 mM Tris-HCl (pH 7.5), 12 mM-glycerophosphate, 0.1 M ethylene-glycol-bis(-aminoethyl ether)-N,N'-tetraacetic acid, 1 mM pyrophosphate, 5 mM NaF, 5 mg/ml aprotinin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 (Nacalai Tesque, Kyoto, Japan), and 1 mM sodium orthovanadate (Sigma) (25). The lysate was centrifuged at 15,000 rpm for 20 min at 4°C, and the supernatant was mixed with Laemmli sample buffer. Samples were separated by 12.5% SDS-PAGE in reducing conditions and electrophoretically transferred onto Immobilon polyvinylidine difluoride filters (Millipore, Bedford, MA). The filters were incubated with anti-Cyr61 antibody diltuted 1:1000 in Block Ace (Snow Brand Milk Products, Sapporo, Japan) for 2 h at room temperature and were developed with horseradish peroxidase (HRP)–conjugated donkey anti-goat IgG (sc-2020, Santa Cruz Biotechnology) and chemiluminescence kit (ECL, Amersham).

Cell Migration Assay
Migration of mesangial cells was analyzed by modified Boyden chamber method using 96-well chemotaxis chambers (AB96, Neuro Probe, Gaithersburg, MD) (26). Polycarbonate filters (8-µm pore size, PFD8, Neuro Probe) coated with 20 ng/ml poly-L-lysine (Sigma) for 24 h at 25°C were placed in the middle of the chambers, and the number of cells that moved from the upper chambers to the lower chambers was counted. Mesangial cells suspended in supernatant of mock-transfected COS-7 cells (1 x 105 cells/100 µl per well) were placed in the top chambers. In the lower chambers, 30-µl supernatant of Cyr61-transfected or mock-transfected COS-7 cells supplemented with or without PDGF-BB was placed. The chambers were incubated in humidified air with 5% CO2 for 3 h at 37°C. The cells remaining on the upper surfaces of the filters were scraped off, and the cells that had migrated to the lower surfaces were fixed in methanol and stained with 0.5% Coomassie Brilliant Blue R 250 (Nacalai Tesque) in 50% methanol, 40% water, and 10% acetic acid. For each well, 5 high-power field photographs (magnification, x400) were taken to calculate the mean number of cells migrated per high-power field.

Cell Proliferation Assay
Mesangial cells were plated on 24-well plates at 2 x 104 cells/well, grown for 24 h, and incubated with DMEM containing 0.2% FCS for the following 24 h. Cells were then treated with media containing supernatant of Cyr61-transfected or mock-transfected COS-7 cells diluted 1:1 by fresh DMEM containing 10% FCS, with or without 10 ng/ml PDGF-BB. [3H]-thymidine (3 µCi/ml, Amersham) was added simultaneously with the above-described media. After 48 h of incubation, cells were washed with PBS and fixed with 10% TCA (Nacalai Tesque). DNA was dissolved in 0.25 N NaOH, and incorporated thymidine was counted in liquid scintillation counter (Aloka, Tokyo, Japan) as described previously (25).

Statistical Analyses
Results are given as means ± SEM. Mann-Whitney U test was used to compare unpaired two-group means. The differences were evaluated with a Stat View software package (Abacus Concepts Inc., Berkeley, CA), and those with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of Podocyte-Specific cDNA Library
To construct a cDNA library of PSG, subtraction of cultured mouse Pod cDNA pool with mouse WK cDNA pool was performed (Pod - WK), and each of 1500 subtracted cDNA clones was placed in grids to make four identical sets of PSG array. To confirm specific expression of PSG, we performed reverse Northern blot analyses using 32P-labeled Pod - WK, WK - Pod, Pod, and WK cDNA probes, respectively. Representative results showed that thrombospondin-1 and ceruloplasmin are expressed intensely and specifically in cultured podocytes, whereas fibronectin is expressed abundantly in whole kidney (Figure 1). By this screening, 150 clones of highly podocyte-specific genes were isolated.



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  		Figure 1. Screening for genes specifically expressed in cultured podocytes (Pod). Pod-derived cDNAs subtracted with whole kidney (WK)–derived cDNAs (Pod - WK) were blotted in quadruplicate and hybridized with four kinds of cDNAs obtained from Pod - WK, WK - Pod, Pod, and WK, respectively. Clones 1 and 2 were expressed more intensely in Pod than in WK, while expression of clone 3 was more abundant in WK.

 
Histologic Examination of Anti-Thy-1 Glomerulonephritis
We next examined the time course of histologic alteration during Thy-1 GN with light microscopy (data not shown). In the present study, we used anti-Thy-1 antibody at the concentration sufficient to cause almost complete elimination of mesangial cells at day 1. At day 3 of Thy-1 GN, most of mesangial cells were located at vascular poles, suggesting that mesangial cells were migrating into glomerular tufts (2). They reached the periphery of most glomerular tufts at day 7. Glomerular microaneurysms appeared at day 3, progressing to ballooning lesion at day 5, which indicated that severe destruction of the capillary tufts occurred. At day 10, microaneurysms were observed in only few glomeruli. At day 21, mesangial cell and matrix accumulation were still seen, but they subsided at day 28.

Identification of Cyr61 as PSG Upregulated in the Glomeruli of Anti-Thy-1 Glomerulonephritis
To isolate PSG possibly involved in glomerular reconstruction, we examined which PSG were upregulated in the glomeruli of Thy-1 GN. We carried out second set of reverse Northern blot analyses by radiolabeling glomerular cDNA of Thy-1 GN at days 0, 1, 3, and 5. Of 150 PSG clones, we identified 40 to be upregulated, and we determined their nucleotide sequences. One of them was a cDNA fragment of Cyr61 (nucleotides 1290 to 1568, GenBank accession number M32490) (23), which is known as a soluble angiogenic protein associated with extracellular matrix (9,10).

Cyr61 Expression in Normal Kidney and in Anti-Thy-1 Glomerulonephritis
In Thy-1 GN, glomerular gene expression of Cyr61 was low at day 0, increased significantly from day 1 through day 7, reaching the peak at day 5 by 4.5-fold, and gradually decreased toward day 10 by Northern blot analysis (Figure 2). The gene expression of CTGF, another member of the CCN family, was similar to Cyr61, peaking at day 3. We also examined the gene expression of growth factors that have been implicated in Thy-1 GN (5) and found that gene expression of PDGF-B and TGF-{beta}1 in glomeruli was also induced maximally around days 3 to 5.



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  		Figure 2. Gene expression of Cyr61, connective tissue growth factor (CTGF), platelet-derived growth factor-B (PDGF-B), and transforming growth factor–{beta}1 (TGF-{beta}1) in glomeruli of anti-Thy-1 glomerulonephritis. Cyr61, CTGF, PDGF-B and TGF-{beta}1 expressions were all induced maximally around days 3 to 5 by Northern blot analysis. The graphs on the bottom show relative gene expression level of each gene normalized for ethidium bromide staining intensity of 28S ribosomal RNA. The level at day 0 was arbitrarily defined as 1. * P < 0.05 as compared with day 0; n = 3.

 
To examine the location of Cyr61 gene expression in normal kidney and in Thy-1 GN, in situ hybridization was performed with 35S-labeled cRNA probe (Figure 3). At autoradiograph, moderate hybridizing signals were observed in outer stripe of outer medulla in normal kidney. At day 5 of Thy-1 GN, strong signals were seen in dot pattern in cortex. At photomicrograph, these signals in Thy-1 GN were observed intensely in glomeruli, consistently with the findings by Northern blot analysis (Figure 2).



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  		Figure 3. Localization of Cyr61 gene expression in normal kidney and in anti-Thy-1 glomerulonephritis by in situ hybridization. Antisense (A, B, D, and E) or sense (C and F) Cyr61 cRNA probe was hybridized with sections from normal kidney (A and D) and Thy-1 GN (B, C, E, and F), and autoradiograph (A through C, x4) and photomicrograph with counterstaining by hematoxylin and eosin (D through F, x400) were carried out. In normal kidney, Cyr61 gene was expressed predominantly in outer stripe of outer medulla (A); in Thy-1 GN at day 5, it was induced markedly in glomeruli (B and E, arrow). No specific signals were seen in sections hybridized with the sense probe (C and F).

 
Precise sites of Cyr61 expression were further studied by immunohistochemistry with antibody against Cyr61 (Figure 4). In normal kidney, Cyr61 protein was expressed in outer stripe of outer medulla, which corresponded to brush border membrane-positive proximal straight tubules (S3 segment). At high-power field, intense expression of Cyr61 protein was also observed in some of afferent and efferent arterioles in normal kidney, but the protein was not expressed in larger vessels. At day 5 of Thy-1 GN, Cyr61 expression was also detected in glomeruli. The signals were observed on outer surfaces of glomerular basement membrane (GBM), which appeared to be podocytes. Anti-Cyr61 antibody specifically recognized Cyr61 protein (40 kD) expressed in COS-7 cells by Western blot analysis. Specificity of the antibody binding in immunohistochemistry was further confirmed by disappearance of the signals when the antibody was preabsorbed with blocking peptide (data not shown) or when nonimmune serum was used as primary antibody. The expression of Cyr61 mRNA and protein in glomeruli and podocytes increased in Thy-1 GN, whereas expression in tubules and arterioles did not seem to change in Thy-1 GN.



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  		Figure 4. Immunohistochemical analyses of Cyr61 protein expression in normal kidney and in anti-Thy-1 glomerulonephritis. In normal kidney (A) and in Thy-1 GN at day 5 (B), Cyr61 protein was expressed in outer stripe of outer medulla. The analysis of high-power field at the border between outer and inner stripes (C and D) revealed that the signals were confined to proximal straight tubules (C) with brush border membranes, which were visualized as pink by periodic acid-Schiff (PAS) staining in the adjacent section (D, black arrow). Cyr61 protein was also expressed intensely in some, but not all, afferent and efferent arterioles in normal kidney (E). The protein was not expressed in larger vessels such as interlobular arteries (G, white arrow). In Thy-1 GN at day 5, Cyr61 expression was also seen in podocytes (F, arrowhead), which were present outside of glomerular basement membrane. Incubation of sections with nonimmune serum as primary antibody gave no signals in proximal straight tubules (I), arterioles or podocytes in Thy-1 GN (H). By Western blot analysis (J), anti-Cyr61 antibody specifically recognized a 40-kD protein in lysate of Cyr61-transfected COS-7 cells (lane 2, arrow), but not in that of mock-transfected cells (lane 1). Magnifications: x40 in A, B, and I; x200 in C, D, and G; x400 in E, F, and H.

 
Cyr61 Induction by PDGF-BB and TGF-{beta}1 in Cultured Podocytes
To explore the regulatory mechanism of Cyr61 expression in podocytes, cultured podocytes were treated with PDGF-BB or TGF-{beta}1, because we found that PDGF-B, TGF-{beta}1, and Cyr61 genes were coordinately upregulated in Thy-1 GN. Treatment of podocytes with 10 ng/ml PDGF-BB upregulated Cyr61 gene expression significantly at 10 min by 1.2-fold and at 24 h by 1.5-fold (Figure 5). Addition of 5 ng/ml TGF-{beta}1 to podocytes induced Cyr61 expression significantly at 1 h by 2.2-fold and 24 h by 2.1-fold (Figure 6). We also observed that PDGF-BB and TGF-{beta}1 caused upregulation of CTGF gene expression in podocytes significantly at 10 min to 24 h and at 1 to 24 h, respectively.



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  		Figure 5. PDGF-BB-induced Cyr61 and CTGF gene expression in cultured podocytes. PDGF-BB was added to podocytes and the time course of Cyr61 and CTGF gene expression was examined by Northern blot analyses. The graphs on the bottom show relative gene expression level of each gene normalized for GAPDH expression. The level in untreated podocytes (control) was arbitrarily defined as 1. * P < 0.05 as compared with control; n = 4.

 


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  		Figure 6. TGF-{beta}1–induced Cyr61 and CTGF gene expression in cultured podocytes. * P < 0.05 as compared with control; n = 3 to 5.

 
Inhibition of PDGF-Induced Mesangial Cell Migration by Supernatant of Cyr61-Overexpressing Cells
To examine the functional role of Cyr61 upregulation in podocytes during Thy-1GN, migration of mesangial cells in supernatant of Cyr61-transfected or mock-transfected COS-7 cells was studied using modified Boyden chambers (Figure 7). In the absence of PDGF-BB, supernatant of Cyr61-transfected COS-7 cells had no significant effect on mesangial cell migration compared with that of mock-transfected COS-7 cells. With addition of 10 ng/ml PDGF-BB, cell migration in mock-transfected supernatant was enhanced by 2.4-fold. On the other hand, conditioned media from Cyr61-overexpressing cells significantly suppressed PDGF-induced mesangial cell migration by 24%.



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  		Figure 7. Effects of culture media of Cyr61-overexpressing cells on PDGF-BB-induced mesangial cell migration. Mesangial cells plated in modified Boyden chambers were incubated for 3 h with supernatant of Cyr61-transfected or mock-transfected COS-7 cells, which was supplemented with or without PDGF-BB. In the absence of PDGF-BB, supernatant of Cyr61-transfected COS-7 cells had no effects on cell migration compared with that of mock-transfected COS-7 cells (A). In culture media from mock-transfected cells, addition of 10 ng/ml PDGF-BB significantly increased mesangial cell migration (B and D). Supernatant of Cyr61-overexpressing cells significantly suppressed the chemotaxis in the presence of PDGF-BB (C and D). * P < 0.05; ns, not statistically significant; HPF, high-power field; n = 4 to 8. Magnification, x400 in B through D.

 
No Effects of Supernatant of Cyr61-Overexpressing Cells on Mesangial Cell Proliferation
We further examined whether culture media from Cyr61-transfected cells affect proliferation of mesangial cells (Figure 8). PDGF-BB significantly increased mesangial cell numbers by twofold after 48 h, but addition of supernatant of Cyr61-transfceted cells had no significant effects on cell numbers both in the absence and presence of PDGF and on PDGF-induced DNA synthesis.



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  		Figure 8. Effects of culture media of Cyr61-overexpressing cells on mesangial cell proliferation. Mesangial cells were incubated with media containing supernatant of Cyr61-transfected or mock-transfected COS-7 cells, together with or without 10 ng/ml PDGF-BB. The cell numbers after 24 h and 48 h were not significantly different between the treatments with Cyr61-conditioned and control media, both in the absence and presence of PDGF-BB (A). Supernatant of Cyr61-overexpressing cells also did not significantly affect PDGF-BB-induced mitogenesis as judged by [3H]-thymidine incorporation after 48 h (B). ns, not statistically significant; n = 6 to 12.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we generated a podocyte-specific cDNA library and investigated the expression levels of the cDNAs during Thy-1 GN, a reversible model of glomerular disease, and identified that gene expression of soluble angiogenic factor Cyr61 was highly induced in the glomeruli of Thy-1 GN. We revealed for the first time, at least to our knowledge, that Cyr61 gene and protein expression was specifically induced in podocytes in Thy-1 GN and that physiologic sites of Cyr61 expression were proximal straight tubules and afferent and efferent arterioles. PDGF-B and TGF-{beta}1 were upregulated in similar time course as Cyr61 in Thy-1 GN, and they induced Cyr61 gene expression in cultured podocytes, suggesting that they are inducers of podocyte Cyr61 expression in vivo. Furthermore, culture media of Cyr61-transfected cells inhibited mesangial cell migration induced by PDGF-BB, suggesting that Cyr61 may be involved in glomerular remodeling in Thy-1 GN.

In glomeruli of Thy-1 GN, Cyr61 gene expression was markedly upregulated from day 3 through day 7, when mesangial cell migration was most active, as shown by our histologic analysis, which was consistent with previous reports (2–5). Furthermore, neovascularization, characterized with immature endothelial cell proliferation, is also reported to take place in a period involving days 3 to 7 (2,17). Cyr61 exerts various actions through interaction with cell surface integrin heterodimer complexes (13,27–29). Cyr61 induces endothelial cell migration through integrin {alpha}v{beta}3 and stimulates neovascularization in rat cornea (12). In fibroblasts, Cyr61 induces cell migration through {alpha}v{beta}5 and proliferation through {alpha}v{beta}3 (13). Mesangial cells also express {alpha}v{beta}3 and {alpha}v{beta}5 integrins (30); therefore, an angiogenic factor Cyr61, which has high affinity to extracellular matrix (31), may be secreted by podocytes, bound to GBM, and act upon endothelial and mesangial cells to modulate migration and proliferation during remodeling of glomerular structure in Thy-1 GN.

A regulatory role of podocytes on mesangial cells has been implied by recent studies. One study showed that minor podocyte injury with puromycin preceding Thy-1 GN induction results in irreversible mesangial lesions (8). Another report demonstrated that mice deficient of podocyte-specific molecule CD2-associated protein exhibit mesangial cell proliferation and mesangial matrix expansion (32). These findings seem to raise a possibility that factors secreted from podocytes may regulate mesangial cell activity. In the present study, we found that supernatant of cells transfected with Cyr61, a candidate for such podocyte-derived factors, inhibited PDGF-BB-induced mesangial cell migration, implying that podocytes may suppress migratory activity of mesangial cells when mesangial cells reach the periphery of glomerular tufts, that is GBM, and may help the cessation of glomerulonephritis. Cyr61 enhances both migration and growth factor-induced proliferation in vascular endothelial cells and fibroblasts (12–14). However, here we show that, in mesangial cells, Cyr61-conditioned media inhibited PDGF-BB-induced migration but did not affect PDGF-BB-induced proliferation. The discrepancy may be due to distinct responses in different cells or due to difference in experimental settings: use of culture media of transfected cells versus recombinant protein.

Cyr61 protein has been reported to be synthesized by serum-stimulated NIH 3T3 fibroblasts, but it is associated with extracellular matrix and cannot be detected in the conditioned media (31). When we overexpressed Cyr61 in COS-7 cells, Cyr61 protein was detected in the supernatant by Western blot analysis (data not shown), but several possibilities can still be considered concerning the mechanism for migration inhibitory effects of Cyr61. First, Cyr61 may bind directly to mesangial cells and exert its action, presumably through cell surface integrin complexes (see above). Second, Cyr61 might interact with PDGF in the media to interfere with its action, as is the case with CTGF and bone morphogenic protein 4 reported in Xenopus (33). Third, Cyr61 may alter secretion of other soluble factors such as extracellular matrix proteins (see below) from COS-7 cells, which have activity to modify mesangial cell migration.

Previous reports showed that Cyr61 is expressed in endothelial and smooth muscle cells in the developing mouse blood vessels (34,35) but only weakly in vessels of normal adult mice and humans (36). In the present study, Cyr61 was expressed in normal afferent and efferent arterioles, in which Cyr61 expression might be upregulated by strong mechanical stretch due to shear stress (37).

Cyr61 and CTGF share structural and functional similarity and belong to the CCN family (9,10), but their tissue distribution (31) and intrarenal localization is clearly different. In normal kidney, the main site of Cyr61 expression was proximal straight tubules. On the contrary, we and others have shown that neither CTGF gene (19,38) nor protein (31,39,40) are expressed in normal proximal tubules. In glomeruli of Thy-1 GN, Cyr61 protein expression was predominantly induced in podocytes, whereas CTGF gene expression is broadly upregulated in podocytes, glomerular parietal epithelial cells, mesangial cells, and periglomerular myofibroblasts (39). The spatial differences of Cyr61 and CTGF transcriptional regulation in physiologic and pathophysiologic conditions suggest that Cyr61 and CTGF play distinct roles in vivo. Furthermore, recent studies revealed functional difference between Cyr61 and CTGF; in fibroblasts CTGF upregulates collagen I and fibronectin expression (19,41), whereas Cyr61 does not upregulate fibronectin expression but rather downregulates collagen I expression (42).

In conclusion, we show that Cyr61, which exerts suppressive effects on mesangial cell migration, is strongly upregulated at podocytes in Thy-1 GN during a phase when mesangial cell migration is active. These findings raise a possibility that podocytes may participate in the reconstruction of glomeruli by secreting factors that affect mesangial cell activity. Possible autocrine and paracrine role of Cyr61 not only in podocytes but also in arterioles and tubular cells also must be investigated in future studies.


   Acknowledgments
 
The authors gratefully acknowledge Dr. Peter Mundel (Albert Einstein College of Medicine) for providing mouse podocyte cell line MPC5, Dr. Jun-ichi Miyazaki (Osaka University, Japan) for expression vector pCXN2, Dr. Akira Shimizu (Nippon Medical School, Japan) for technical suggestion to generate Thy-1 GN, and Dr. Takashi Kuwahara (Saiseikai Nakatsu Hospital, Japan) for technical advice for histologic examination. We are also grateful to J. Nakamura and A. Wada for technical assistance, and A. Sonoda and S. Doi for secretarial assistance. This work was supported in part by research grants from the Japanese Ministry of Education, Science, Sports and Culture, the Japanese Ministry of Health and Welfare, Smoking Research Foundation, and "Research for the Future" (RFTF) of Japan Society for the promotion of Science.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication October 11, 2002. Accepted for publication January 23, 2003.




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