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A Novel, Dual Role of CCN3 in Experimental Glomerulonephritis

Pro-Angiogenic and Antimesangioproliferative Effects
      In contrast to factors that promote mesangial cell proliferation, little is known about their endogenous inhibitors. During experimental mesangioproliferative nephritis, expression of the glomerular CCN3 (nephroblastoma overexpressed gene [NOV]) gene is reduced before the proliferative phase and increased in glomeruli and serum when mesangial cell proliferation subsides. To further elucidate its role in mesangioproliferative glomerulonephritis, CCN3 systemically was overexpressed by muscle electroporation in healthy or nephritic rats. This increased CCN3 serum concentrations more than threefold for up to 56 days. At day 5 after disease induction, CCN3-transfected rats showed an increase in glomerular endothelial area and in mRNA levels of the pro-angiogenic factors vascular endothelial growth factor and PDGF-C. At day 7, CCN3 overexpression decreased mesangial cell proliferation, including expression of α-smooth muscle actin and matrix accumulation of fibronectin and type IV collagen. In progressive nephritis (day 56), overexpression of CCN3 resulted in decreased albuminuria, glomerulosclerosis, and reduced cortical collagen type I accumulation. In healthy rat kidneys, overexpression of CCN3 induced no morphologic changes but regulated glomerular gene transcripts (reduced transcription of PDGF-B, PDGF-D, PDGF-receptor–β, and fibronectin, and increased PDGF-receptor–α and PDGF-C mRNA). These data identify a dual role for CCN3 in experimental glomerulonephritis with pro-angiogenic and antimesangioproliferative effects. Manipulation of CCN3 may represent a novel approach to help repair glomerular endothelial damage and mesangioproliferative changes.
      Diabetic nephropathy and glomerulonephritides, in particular the most common type IgA nephropathy, account for >50% of end-stage renal disease cases in most Western countries. Both diabetic nephropathy and the majority of progressive glomerulonephritides are characterized histologically by glomerular mesangial cell proliferation and/or matrix accumulation.
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      The progression of renal disease.
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      Mesangial cell turnover: effect of heparin and peptide growth factors.
      Ample evidence links the PDGF system to these processes and the roles of PDGF-B and PDGF-D chains in mediating mesangioproliferative changes are particularly well established.
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      Multiple roles for platelet-derived growth factor in renal disease.
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      Role of platelet-derived growth factor in glomerular disease.
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      A new look at platelet-derived growth factor in renal disease.
      Inhibition of PDGF-BB or PDGF-DD in mesangioproliferative nephritis can prevent renal failure, glomerulosclerosis, and tubulointerstitial fibrosis.
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      Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers.
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      Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis.
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      A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.
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      • Floege J.
      Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis.
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      • Arrol S.
      • Ostendorf T.
      • Floege J.
      PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.
      Although there is abundant information on promitogenic and profibrotic mediators in the glomerulus, little is known about endogenous factors that might counteract and terminate these processes. In a cDNA array, we recently detected a prominent down-regulation of CCN3 (also known as nephroblastoma overexpressed gene, NOV) in PDGF-B– or PDGF-D–stimulated mesangial cells.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.
      We subsequently identified CCN3 as an endogenous inhibitor of mesangial cell growth in vitro and a modulator of PDGF-induced mitogenesis in mesangioproliferative disease.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.
      CCN3 belongs to the CCN protein family (cysteine-rich, angiogenic inducer 61 [Cyr61], connective tissue growth factor [CTGF], NOV), all of which are involved in the regulation of cell proliferation, migration, attachment, and differentiation.
      • Perbal B.
      NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
      In the developing human kidney, CCN3 mRNA and/or protein is expressed in the mesonephric and paramesonephric ducts as well as in the stromal and other tubular cells.
      • Perbal B.
      NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
      • Kocialkowski S.
      • Yeger H.
      • Kingdom J.
      • Perbal B.
      • Schofield P.N.
      Expression of the human NOV gene in first trimester fetal tissues.
      In the adult rat kidney, CCN3 localized to arterial smooth muscle cells, the medullary interstitium, and podocytes.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.
      • Kocialkowski S.
      • Yeger H.
      • Kingdom J.
      • Perbal B.
      • Schofield P.N.
      Expression of the human NOV gene in first trimester fetal tissues.
      Given the inhibition of mesangial cell proliferation by CCN3 in vitro and the regulated CCN3 expression during anti-Thy1.1 nephritis, a rat model of mesangioproliferative glomerulonephritis, we asked whether CCN3 could be manipulated therapeutically. For this, we overexpressed CCN3 in skeletal muscle by electroporation and studied the effects of systemic CCN3 increases on the acute and chronic course of mesangioproliferative anti-Thy1.1 glomerulonephritis.

      Materials and Methods

      Animal Experiments

      All studies were approved by the local Institutional Review Board. Male Wistar rats weighing 180 g (Charles River, Sülzfeld, Germany) were used for this study. Animals were held in rooms with constant temperature and humidity, with 12:12-hour light cycles, and had ad libitum access to drinking water (ozone-treated and acidified) and standard rat chow.
      Anti-Thy1.1 mesangial proliferative glomerulonephritis was induced in all rats by intravenous injection of 1 mg/kg anti-Thy1.1 monoclonal antibody (clone OX-7; European Collection of Animal Cell Cultures, Salisbury, England). To study the kinetics of CCN3 serum concentrations during acute anti-Thy1.1 nephritis, 54 rats were sacrificed and serum samples were obtained at 4 hours and at 1, 2, 4, 7, 9, 14, 21, and 28 days after disease induction (n = 6 each). Healthy rats injected with PBS served as controls (n = 6).
      To study the effects of CCN3 overexpression on the course of anti-Thy1.1 nephritis, disease was induced in 28 rats. Muscle electroporation was performed 16 hours after disease induction as described.
      • Molnar M.J.
      • Gilbert R.
      • Lu Y.
      • Liu A.B.
      • Guo A.
      • Larochelle N.
      • Orlopp K.
      • Lochmuller H.
      • Petrof B.J.
      • Nalbantoglu J.
      • Karpati G.
      Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles.
      • Muramatsu T.
      • Arakawa S.
      • Fukazawa K.
      • Fujiwara Y.
      • Yoshida T.
      • Sasaki R.
      • Masuda S.
      • Park H.M.
      In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression.
      In brief, under short inhalation anesthesia with isoflurane (Abbott, Wiesbaden, Germany) the left hind limb was shaved and the gastrocnemius muscle was injected with 150 U/animal of hyaluronidase type IV (Sigma-Aldrich, Deisenhofen, Germany). Two hours later, the rats were shortly anesthetized (isoflurane) and 500 μg expression vector encoding either human CCN3 cDNA (n = 14) or LacZ (n = 14) driven by the cytomegalovirus-promotor were injected into the primed gastrocnemius muscle. Five minutes later, the muscle was exposed to transcutaneous electroporation stimulation using 8 low-voltage pulses of 175 V and 20 ms at 1-Hz frequency using the Electro Square Porator ECM 830 (BTX; Genetronics, San Diego, CA) with circular paddle electrodes. The rats were sacrificed at day 5 (n = 6 each) or day 7 (n = 8 each) after disease induction.
      Progressive glomerulonephritis was induced in 32 rats by uninephrectomy followed by intravenous injection of 1 mg/kg anti-Thy1.1 antibody at day 0. Muscle electroporation was performed 16 hours after disease induction (n = 16 each) as described earlier. Renal function was controlled by measurement of proteinuria and serum creatinine in urine and serum samples, respectively, taken at days 6 and 56. During the experiment, 9 rats (6 in the LacZ group and 3 in the CCN3 group) died as a result of the anesthesia.
      In an additional experiment, 28 healthy Wistar rats were electroporated as described earlier with expression vectors encoding for CCN3 (n = 14) or LacZ (n = 14) and the rats were sacrificed at day 5 (n = 6 each) or day 7 (n = 8 each).
      One day before sacrifice, systolic blood pressure was measured by tail cuff plethysmography, using a programmed sphygmomanometer (Softron Co., Tokyo, Japan). In addition, 5-bromo-2′-deoxyuridine (BrdU, 100 mg/kg) was injected intraperitoneally in all rats. Subsequently, all rats were placed in metabolic cages for 24-hour stool-free urine collections.
      After sacrifice of the various groups, renal tissue for histology, immunohistochemistry, and RNA analyses were harvested and processed immediately. From the remaining cortical tissue, glomeruli were isolated by differential sieving as described.
      • Ostendorf T.
      • van Roeyen C.R.
      • Peterson J.D.
      • Kunter U.
      • Eitner F.
      • Hamad A.J.
      • Chan G.
      • Jia X.C.
      • Macaluso J.
      • Gazit-Bornstein G.
      • Keyt B.A.
      • Lichenstein H.S.
      • LaRochelle W.J.
      • Floege J.
      A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.
      Serum and urine biochemistries were assessed by an autoanalyzer (Vitros 250; Ortho Clinical Diagnostics, Neckargemuend, Germany).

      Renal Morphology and Immunohistochemical Analysis

      Tissues for light microscopy and immunoperoxidase staining were fixed in methyl Carnoy's solution and embedded in paraffin. Sections (4 μm) were stained with PAS reagent and counterstained with hematoxylin (PAS staining). Glomerulosclerosis and tubulointerstitial damage was graded on a scale as follows: 0, no glomeruli/cortical area affected, 1, >0% to ≤25% glomeruli/cortical area affected; 2, >25% to ≤50% glomeruli/cortical area affected; 3, >50% to ≤75% glomeruli/cortical area affected; and 4, >75% to ≤100% of glomeruli/cortical area affected.
      In the PAS-stained sections, the total number of mitoses in 50 consecutive glomerular cross-sections was evaluated. Immunohistochemical analyses were performed following a previously published protocol in a blinded manner.
      • Eitner F.
      • Ostendorf T.
      • Van Roeyen C.
      • Kitahara M.
      • Li X.
      • Aase K.
      • Grone H.J.
      • Eriksson U.
      • Floege J.
      Expression of a novel PDGF isoform PDGF-C, in normal and diseased rat kidney.
      Renal tissues were stained using the following antibodies: rabbit polyclonal anti-rat fibronectin (AB 1954; Chemicon, Hampshire, United Kingdom), goat polyclonal anti-human collagen type IV (SouthernBiotec, Birmingham, AL), mouse monoclonal anti-human α-smooth muscle actin (clone 1A4; Dako, Hamburg, Germany), mouse monoclonal anti-rat CD68 (clone ED-1; Serotec, Oxford, United Kingdom), mouse monoclonal anti-human proliferating cell nuclear antigen (PCNA) (PC-10; Oncogene, San Diego, CA), mouse monoclonal anti-rat epithelial aminopeptidase P (clone JG12; BenderMedSystems, Vienna, Austria), mouse monoclonal anti-BrdU (clone Bu-1; Amersham, Freiburg, Germany), mouse monoclonal anti-human desmin (clone D33; Dako), and polyclonal anti-CCN3 antibody (K19M, described by Kyurkchiev et al
      • Kyurkchiev S.
      • Yeger H.
      • Bleau A.M.
      • Perbal B.
      Potential cellular conformations of the CCN3(NOV) protein.
      ). Negative controls for the immunohistochemical procedures consisted of substitution of the primary antibody with nonimmune IgG.
      For PCNA, BrdU, and CD68 staining, positively stained cells in 50 randomly selected glomeruli were counted. For the quantification of positively stained glomerular areas of JG12, α-smooth muscle actin (α-SMA), collagen type IV, and fibronectin, 50 glomeruli were evaluated by computer-based morphometry using the analysis v3.1 software (Soft imaging System GmbH, Münster, Germany) as described previously.
      • Boor P.
      • Konieczny A.
      • Villa L.
      • Kunter U.
      • van Roeyen C.R.
      • LaRochelle W.J.
      • Smithson G.
      • Arrol S.
      • Ostendorf T.
      • Floege J.
      PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.
      In all analyses, the investigator was unaware of the origin of the slides.

      Real-Time Quantitative RT-PCR

      Total RNA was isolated from isolated glomeruli using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Quantification of the RNA content and sample purity, cDNA synthesis, and real-time quantitative RT-PCR were assessed and performed, respectively, as described.
      • Ostendorf T.
      • van Roeyen C.R.
      • Peterson J.D.
      • Kunter U.
      • Eitner F.
      • Hamad A.J.
      • Chan G.
      • Jia X.C.
      • Macaluso J.
      • Gazit-Bornstein G.
      • Keyt B.A.
      • Lichenstein H.S.
      • LaRochelle W.J.
      • Floege J.
      A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis.
      All quantitative data from the real-time RT-PCR were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control and calculated using the ΔΔCT-method. Primer sequences are listed in Table 1. GAPDH cDNA amplification was used as an internal standard.
      Table 1Primers for Quantitative Real-Time RT-PCR
      GeneForward primerReverse primerProbe
      SYBR Green (quantitative PCR core kit for SYBR green I) (Eurogentec, Seraing, Belgium)
      GAPDH5′-ACAAGATGGTGAAGGTCGGTG-3′5′-AGAAGGCAGCCCTGGTAACC-3′
      MCP-15′-CCAGATGCAGTTAATGCCCC-3′5′-TCTCCAGCCGACTCATTGG-3′
      RANTES5′-ACTCCCTGCTGCTTTGCCT-3′5′-GTGTAAAAATACTCCTTCACGTGGG-3′
      TaqMan probe (qPCR core kit) (Eurogentec, Seraing, Belgium)
      GAPDH5′-ACAAGATGGTGAAGGTCGGTG-3′5′-AGAAGGCAGCCCTGGTAACC-3′5′-CGGATTTGGCCGTATCGGACGC-3′
      PDGF-A5′-TTCTTGATCTGGCCCCCAT-3′5′-TTGACGCTGCTGGTGTTACAG-3′5′-CAGTGCAGCGCTTCACCTCCACA-3′
      PDGF-B5′-GCAAGACGCGTACAGAGGTG-3′5′-GAAGTTGGCATTGGTGCGA-3′5′-TCCAGATCTCGCGGAACCTCATCG-3′
      PDGF-C5′-CAGCAAGTTGCAGCTCTCCA-3′5′-GACAACTCTCTCATGCCGGG-3′5′-CGACAAGGAGCAGAACGGAGTGCAA-3′
      PDGF-D5′-ATCGGGACACTTTTGCGACT-3′5′-GTGCCTGTCACCCGAATGTT-3′5′-TTGCGCAATGCCAACCTCAGGAG-3′
      PDGFR-α5′-GCCACGAAAGAGGTCAAGGA-3′5′-GCCTGATCTGGACGAAGCC-3′5′-TGAAGACAGTCACCATTTCTGTTCACGAGAA-3′
      PDGFR-β5′-AATGACCACGGCGATGAGA-3′5′-TCTTCCAGTGTTTCCAGCAGC-3′5′-CATCAACGTTACTGTGATCGAAAATGGCTATG-3′
      Col1a15′-GAAGGCAACAGTCGATTCACC-3′5′-GACTGTCTTGCCCCAAGTTCC-3′5′-ACAGCACGCTTGTGGATGGCTGC-3′
      Fibronectin5′-AAACAGGTCTGGACTCCCCA-3′5′-CAGAATGCTCGGCGTGATG-3′5′-TCTTCTGATGTCACCGCCAACTCATTCA-3′
      CCN35′-CTACAGAGTGGAGCGCGTGTT-3′5′-GGAAGATTCCTGTTGGTGACCC-3′5′-AAGAGCTGTGGAATGGGCTTGTCCAC-3′
      VEGF5′-GTACCTCCACCATGCCAAGTG-3′5′-ATGGGCTTTCTGCTCCCCT-3′5′-CCCAGGCTGCACCCACGACAG-3′
      GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP-1, monocyte chemotactic protein-1; RANTES, regulated upon activation, normally T-expressed and presumably secreted.

      Measurement of CCN3 Concentration in Serum

      Transgenic CCN3 serum concentrations were measured using the human NOV/CCN3 DuoSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. For Western blot analysis the serum samples were purified and analyzed with a rabbit polyclonal anti-CCN3 antibody as described.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.

      In Vitro Experiments

      Conditionally immortalized human glomerular endothelial cells (CiGEnCs) were obtained and cultured as described in detail previously.
      • Satchell S.C.
      • Tasman C.H.
      • Singh A.
      • Ni L.
      • Geelen J.
      • von Ruhland C.J.
      • O'Hare M.J.
      • Saleem M.A.
      • van den Heuvel L.P.
      • Mathieson P.W.
      Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.
      • Boor P.
      • van Roeyen C.R.
      • Kunter U.
      • Villa L.
      • Bucher E.
      • Hohenstein B.
      • Hugo C.P.
      • Eriksson U.
      • Satchell S.C.
      • Mathieson P.W.
      • Eitner F.
      • Floege J.
      • Ostendorf T.
      PDGF-C mediates glomerular capillary repair.
      Proliferation assays were performed in unstimulated cells or CiGEnCs stimulated with recombinant CCN3 (R&D Systems) in basal and full medium using a BrdU incorporation assay according to the manufacturer's instructions (Roche, Mannheim, Germany). The vascular endothelial growth factor (VEGF) aptamer was described previously
      • Ostendorf T.
      • Kunter U.
      • Eitner F.
      • Loos A.
      • Regele H.
      • Kerjaschki D.
      • Henninger D.D.
      • Janjic N.
      • Floege J.
      VEGF(165) mediates glomerular endothelial repair.
      and used at a concentration of 1 μg/mL.

      Western Blot Analysis

      Glomeruli were isolated by differential sieving, lysed in Triton X-100 lysis buffer, and treated with ultrasound. The soluble cell lysates were separated and blotted as described
      • van Roeyen C.R.
      • Ostendorf T.
      • Denecke B.
      • Bokemeyer D.
      • Behrmann I.
      • Strutz F.
      • Lichenstein H.S.
      • LaRochelle W.J.
      • Pena C.E.
      • Chaudhuri A.
      • Floege J.
      Biological responses to PDGF-BB versus PDGF-DD in human mesangial cells.
      and probed with polyclonal antibodies against PDGF-receptor–β (PDGFR-β) (Santa Cruz Biotechnology, Santa Cruz, CA), pSMAD1/5/8 (Cell Signaling Technology, Danvers, MA), phosphorylated Sma- and Mad-related protein, homolog of Drosophila (pSmad2) (Cell Signaling Technology), Smad2 (Cell Signaling Technology), CCN2 (Santa Cruz Biotechnology), β-actin (Sigma, Taufkirchen, Germany), and GAPDH (Santa Cruz Biotechnology). The primary antibodies (diluted 1:1000) were detected using horseradish peroxidase–conjugated antibodies visualized by the Amersham ECL system after intensive washing of the membrane.

      Statistical Analysis

      All values were expressed as means ± SD. To analyze the data, the two-tailed Mann-Whitney U-test or one-way analysis of variance with Scheffé post hoc analysis were used where appropriate. For comparison of serum CCN3 levels during the course of anti-Thy1.1 with healthy animals, one-way analysis of variance with the Dunnett post hoc test was used. For comparison of serum CCN3 levels with parameters of renal damage, the Spearman correlation coefficients (rs) were calculated. Statistical significance was defined as a P value < 0.05.

      Results

      Muscle Electroporation Results in Systemic Overexpression of CCN3

      We first assessed temporal changes of serum levels of endogenous CCN3 in rats with acute anti-Thy1.1 nephritis. This revealed a significant increase from day 4 to 14 with a maximum on day 7 after disease induction (Figure 1).
      Figure thumbnail gr1
      Figure 1CCN3 serum concentrations during the time-course of anti-Thy1.1 mesangioproliferative nephritis. Rats were euthanized at 4 hours and at days 1, 2, 4, 7, 9, 14, 21, and 28 after disease induction (n = 6 each). The serum concentrations of CCN3 were measured by ELISA. Data are means ± SD. *P < 0.05 (analysis of variance with Dunnett post hoc test) versus PBS-treated healthy rats.
      Based on the earlier-described time information and the natural course of acute anti-Thy1.1 nephritis, we performed a muscle electroporation with a cytomegalovirus promoter-driven vector overexpressing human CCN3 or LacZ as a control at 16 hours after disease induction. This not only avoids interference of the CCN3 overexpression with the disease induction phase but also should serve to amplify the peak of the endogenous CCN3 overproduction on day 7 after disease induction. After muscle electroporation both LacZ activity and the transgenic CCN3 protein were detectable in skeletal muscle on days 5 and 7 after disease induction (Figure 2A). In parallel, serum levels of CCN3 increased 2.8-fold on day 5 and >8.4-fold on day 7 (Figure 2B) as measured with the anti-human–CCN3 ELISA, which detects both the endogenous rat as well as the transgenic human CCN3 proteins. In addition to the full-length CCN3 protein, a truncated CCN3 fragment could be detected by Western blot analysis of sera (Figure 2C). A similar truncated CCN3 fragment has been described in different biological fluids.
      • Perbal B.
      NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
      • Lazar N.
      • Manara C.
      • Navarro S.
      • Bleau A.M.
      • Llombart-Bosch A.
      • Scotlandi K.
      • Planque N.
      • Perbal B.
      Domain-specific CCN3 antibodies as unique tools for structural and functional studies.
      Figure thumbnail gr2
      Figure 2Overexpression of CCN3 in muscle and in the circulation. A: Transgenic protein expression could be detected in the transfected muscle of nephritic rats at days 5 and 7 after disease induction by immunohistochemical staining of CCN3 or by X-Gal staining. B: The secreted transgenic CCN3 protein (full length and truncated) could be detected by Western blot analysis in serum of healthy rats transfected with the CCN3 expression vector. Arrows indicate the full-length and truncated CCN3 protein detected in serum of CCN3-overexpressing rats. C: By using ELISA, serum samples of nephric and healthy rats showed increased CCN3 concentrations in comparison with control treated rats. Data are means ± SD relative to the mean of control transfected rats per each time point. *P < 0.05 versus LacZ control rats.

      CCN3 Overexpression Augments Early Glomerular Capillary Repair on Day 5 of Acute Anti-Thy1.1 Nephritis

      Nephritic rats overexpressing LacZ or CCN3 in muscle remained normotensive and showed albuminuria and a minor decrease in renal function on day 5 with no significant differences between the two groups (data not shown).
      The most prominent finding in CCN3-overexpressing nephritic rats on day 5 as compared with the LacZ group was a significantly increased glomerular endothelial cell area, as assessed by immunostaining with the JG12 antibody (Figure 3A). In parallel, a nonsignificant trend toward an increased number of proliferating PCNA/JG12 double-positive endothelial cells was observed in the CCN3 group (Figure 3A). CCN3 overexpression also led to glomerular mRNA overexpression of the two pro-angiogenic factors VEGF and PDGF-C (Figure 3B).
      Figure thumbnail gr3
      Figure 3Systemic overexpression of CCN3 induces angiogenesis. A: The overexpressed CCN3 protein induces an increased glomerular endothelial, JG12-positive area, and a trend toward increased glomerular endothelial cell proliferation as identified by double-immunostaining for PCNA (blue) and JG12 (brown). Data are means ± SD of n = 6 rats, each. B: The increased glomerular endothelial area is accompanied by glomerular mRNA overexpression of the pro-angiogenic factors VEGF and PDGF-C, as measured by real-time RT-PCR (n = 6 rats, each). A reduced mRNA expression of CCN3 and PDGFR-β could be observed by real-time RT-PCR in glomeruli of CCN3 in comparison with LacZ-overexpressing rats. Data are means ± SD relative to the mean of control transfected rats (n = 6, each). *P < 0.05 versus LacZ control rats. C: The percentage of CCN3-positive glomerular area is not changed by CCN3 overexpression as identified by immunostaining.
      At the transcriptional level, the overexpressed CCN3 down-regulated glomerular CCN3 mRNA expression, whereas the CCN3-positive glomerular area was not altered (Figure 3, B and C). Glomerular matrix protein expression of fibronectin, and types I and IV collagen, was unchanged at the mRNA and/or protein level in CCN3 compared with LacZ-transfected nephritic rats (data not shown).
      In nephritic rats, on day 5, CCN3 versus LacZ overexpression did not affect overall glomerular cell proliferation (ie, the number of glomerular mitoses or PCNA-positive cells), mesangial cell proliferation (ie, PCNA/α-SMA double-positive cells), mesangial cell activation (as assessed by their de novo expression of α-SMA), or the number of infiltrating glomerular ED-1–positive macrophages (data not shown). However, there was significantly reduced expression of glomerular PDGFR-β chain mRNA in the CCN3 group (Figure 3B).

      Recombinant CCN3 Protein Affects the Proliferation of Glomerular Endothelial Cells in Vitro

      CiGEnCs were stimulated with recombinant CCN3 protein under permissive (33°C, undifferentiated endothelial cells) and nonpermissive (37°C, differentiated glomerular endothelial cells) conditions in vitro. Under permissive conditions, incubation with CCN3 did not lead to a significantly increased cell proliferation when cells were growing in basal medium, but it potently induced proliferation of cells growing in full medium (Figure 4A). Next, we analyzed the proliferation of CiGEnCs stimulated with conditioned media from CiGEnCs and human mesangial cells that had been stimulated with CCN3. Conditioned medium from endothelial but not mesangial cells induced a significant increase in proliferating differentiated CiGEnCs (Figure 4B), suggesting that CCN3 induces an autocrine release of pro-angiogenic mediators in CiGEnCs. By using a VEGF-specific aptamer
      • Ostendorf T.
      • Kunter U.
      • Eitner F.
      • Loos A.
      • Regele H.
      • Kerjaschki D.
      • Henninger D.D.
      • Janjic N.
      • Floege J.
      VEGF(165) mediates glomerular endothelial repair.
      we excluded that the latter included VEGF (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr4
      Figure 4Effects of CCN3 in glomerular endothelial cells in vitro. A: CCN3 alone did not induce CiGEnC proliferation using permissive (33°C) or nonpermissive temperatures (37°C). When added to full medium, CCN3 induced marked proliferation of undifferentiated CiGEnC. This effect was not observed in differentiated cells. B: Conditioned medium from CCN3-stimulated endothelial cells but not from CCN3-stimulated mesangial cells induced proliferation of CiGEnC at nonpermissive conditions. *P < 0.05 versus basal medium; P < 0.05 versus full medium; P < 0.01 versus full medium.

      CCN3 Overexpression Reduces Mesangioproliferative Changes on Day 7 of Acute Anti-Thy1.1 Nephritis

      On day 7 of acute anti-Thy1.1 nephritis, both groups of rats showed mild albuminuria and a minor decrease of renal function with no significant differences between both groups (data not shown).
      On day 7, overexpression of CCN3 resulted in a significant decrease of activated mesangial cells (ie, reduced glomerular staining of α-SMA) (Figure 5A). In addition, the overall glomerular cell proliferation (ie, numbers of mitotic figures and BrdU-positive cells) was reduced significantly (Figure 5A). Double immunostaining for α-SMA and BrdU confirmed that the decreased glomerular cell proliferation in CCN3-overexpressing rats was owing to reduced mesangial cell proliferation (Figure 5A). Reduced mesangial cell proliferation was accompanied by decreased glomerular accumulation of type IV collagen and fibronectin (Figure 5B).
      Figure thumbnail gr5
      Figure 5Overexpressed CCN3 leads to reduced mesangial cell proliferation and glomerular matrix accumulation. A: In CCN3-overexpressing rats the α-SMA–positive glomerular area was decreased in comparison with control rats (n = 8 each). In addition, the number of glomerular mitotic figures and of glomerular proliferating mesangial cells [ie, cells positive for both BrdU (brown) and α-SMA (blue)] was reduced after CCN3 overexpression. Arrows indicate double-positive stained proliferating mesangial cells. *P < 0.05 versus LacZ control rats. B: Overexpressed CCN3 resulted in decreased glomerular matrix deposition, as shown for collagen type IV and fibronectin. Data are means ± SD. C: PDGFR-β glomerular protein levels were decreased in CCN3-overexpressing compared with control transfected rats as shown in a Western blot analysis of glomerular lysates. GAPDH was used as loading control. D: Real-time RT-PCR analysis revealed an mRNA overexpression of monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T cell expressed and secreted (RANTES) after CCN3 overexpression. Data are means ± SD relative to the mean of control transfected rats. *P < 0.05 versus LacZ control rats.
      Overexpression of CCN3 also resulted in a diminished glomerular expression of PDGFR-β (Figure 5C) and a significant increase in glomerular regulated upon activation, normally T-expressed and presumably secreted and monocyte chemotactic protein-1 mRNA levels (Figure 5D). However, the number of ED-1–positive cells per glomerular cross-section did not differ between the two groups (data not shown).

      CCN3 Overexpression Reduces Glomerulosclerosis on Day 56 in Rats with Progressive Anti-Thy1.1 Nephritis

      Progressive anti-Thy1.1 nephritis was induced by uninephrectomy and injection of the anti-Thy1.1 antibody at day 0. Muscle electroporation was performed 16 hours later. CCN3 serum concentrations were measured at day 6 after disease induction to confirm the success of electroporation.
      At day 56, albuminuria was decreased in CCN3 in comparison with LacZ-expressing nephritic rats (Figure 6A). In addition, the CCN3 rats showed a decreased glomerulosclerosis score and a nonsignificant trend toward diminished tubulointerstitial damage (Figure 6B). This finding was accompanied by significantly decreased cortical accumulation of collagen type I in CCN3-overexpressing rats, whereas the difference in glomerular accumulation of collagen type I failed to reach significance (Figure 6C). Cortical α-SMA as well as desmin protein expression at the glomerular edge, which identifies podocyte damage, was not altered by CCN3 overexpression (data not shown).
      Figure thumbnail gr6
      Figure 6Systemic overexpression of CCN3 in progressive glomerulonephritis reduces albuminuria and glomerulosclerosis at day 56. A: In CCN3-overexpressing rats albuminuria was decreased in comparison with control rats. B: Renal damage was measured by glomerulosclerosis and the tubulointerstitial damage score. C: Accumulation of collagen type I was detected by immunohistochemistry and the positive-stained area was measured by morphometry (n = 10 LacZ control and n = 13 CCN3-overexpressing rats). *P < 0.05 CCN3 versus LacZ-overexpressing rats.
      Some of the variability in the long-term experiment may relate to differences in maintaining CCN3 levels over time. Indeed, on days 28 and 42 we found a significant correlation between serum CCN3 levels and tubulointerstitial damage (day 28: P = 0.020, rs = 0.576; day 42: P = 0.029, rs = 0.536), cortical accumulation of collagen type I (both days 28 and 42: P = 0.003, rs = 0.720), and cortical α-SMA (day 28: P = 0.011, rs = 0.627; day 42: P = 0.024, rs = 0.558).

      Effects of CCN3 Overexpression in Healthy Rats

      In control experiments, CCN3 systemically was overexpressed in healthy rats (Figure 2) and the kidneys were removed 5 or 7 days later. CCN3-transfected healthy rats as compared with LacZ-transfected rats showed normal kidney morphology by light microscopy in PAS-stained sections. Immunohistology excluded significant differences in glomerular endothelial cell areas (ie, JG-12–positive glomerular area), alterations in glomerular cell proliferation (glomerular mitotic figures, PCNA-positive cells, proliferating PCNA and α-SMA double-positive cells, and PCNA and JG12 double-positive cells), and podocyte activation (ie, de novo expression of desmin) (data not shown). In addition, there were no changes in glomerular ED-1–positive monocyte/macrophage counts (data not shown).
      Similar to nephritic rats (see earlier), overexpression of CCN3 resulted in a down-regulation of endogenous glomerular CCN3 mRNA expression (Figure 7A). With respect to the PDGF system, CCN3 up-regulated glomerular PDGF-C and PDGFR-α and down-regulated PDGF-B, PDGF-D, and PDGFR-β mRNA expression (Figure 7A).
      Figure thumbnail gr7
      Figure 7CCN3 overexpression in healthy rats. A: In healthy rats overexpressed CCN3 predominantly acts as a regulator of gene transcription resulting in an glomerular mRNA overexpression of PDGF-C and PDGFR-α as well as down-regulation of CCN3, PDGF-B, PDGF-D, and PDGFR-β mRNA expression. B: Glomerular collagen type IV protein expression was increased in CCN3-overexpressing rats. C: No changes were observed in glomerular collagen type I and fibronectin mRNA and protein expression. Data are means ± SD relative to the mean of control transfected rats. *P < 0.05 versus LacZ control rats.
      Findings on glomerular matrix accumulation were inconsistent with CCN3 rats versus LacZ rats, showing a minor and transient increase in glomerular accumulation of type IV collagen protein on day 5 (Figure 7B). In contrast, fibronectin and type I collagen mRNA decreased in CCN3-transfected rats but the respective proteins in glomeruli showed no significant changes (Figure 7C).

      CCN3 Overexpression Down-Regulates CCN2 Expression and Smad Phosphorylation

      In renal fibrosis the antifibrotic effect of CCN3 has been reported to involve inhibition of CCN2 expression
      • Riser B.L.
      • Najmabadi F.
      • Perbal B.
      • Peterson D.R.
      • Rambow J.A.
      • Riser M.L.
      • Sukowski E.
      • Yeger H.
      • Riser S.C.
      CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.
      • Riser B.L.
      • Najmabadi F.
      • Perbal B.
      • Rambow J.A.
      • Riser M.L.
      • Sukowski E.
      • Yeger H.
      • Riser S.C.
      • Peterson D.R.
      CCN3/CCN2 regulation and the fibrosis of diabetic renal disease.
      and of the BMP-induced pSmad1/5/8 signaling pathway.
      • Minamizato T.
      • Sakamoto K.
      • Liu T.
      • Kokubo H.
      • Katsube K.
      • Perbal B.
      • Nakamura S.
      • Yamaguchi A.
      CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways.
      • Rydziel S.
      • Stadmeyer L.
      • Zanotti S.
      • Durant D.
      • Smerdel-Ramoya A.
      • Canalis E.
      Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia.
      In our healthy rats CCN2 mRNA and protein expression were indeed suppressed after overexpression of CCN3 compared with control transfected rats (Figure 8). However, in nephritic animals overexpression of CCN3 did not alter the expression of CCN2 (Figure 8). Similarly, in vitro the stimulation of glomerular endothelial and mesangial cells with recombinant CCN3 protein did not result in altered CCN2 expression (see Supplemental Figure S2 at http://ajp.amjpathol.org). The phosphorylation of Smad1/5/8 and Smad2 was slightly reduced by overexpression of CCN3 in nephritic rats but not in healthy rats (Figure 8).
      Figure thumbnail gr8
      Figure 8CCN3 overexpression down-regulates CCN2 expression in healthy but not nephritic rats. A: In healthy but not in nephritic rats (day 5 after disease induction) CCN3 overexpression leads to down-regulation of glomerular CCN2 mRNA expression. B: CCN2 glomerular protein levels were decreased in CCN3-overexpressing compared with control transfected healthy but not in nephritic rats as shown by Western blot analysis of glomerular lysates (day 5 after disease induction). The phosphorylation of Smad1/5/8 as well as Smad2 is not changed after CCN3 overexpression in healthy rats, whereas a slight decrease could be observed in nephritic rats overexpressing CCN3 compared with control rats (day 5 after disease induction). β-actin or Smad2 were used as loading controls. C: Mean values of both animals are shown in the morphometric analysis.

      Discussion

      In a recent work, we identified CCN3 as a potent endogenous growth inhibitor of mesangial cells in vitro.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.
      To further elucidate the role of CCN3 in glomerular disease, we systemically overexpressed CCN3 in healthy and nephritic rats. By using this approach, we identified novel pro-angiogenic and antimesangioproliferative effects of CCN3 in experimental glomerulonephritis.
      Our first major finding was an increased glomerular endothelial area on day 5 after induction of nephritis in rats receiving the CCN3 plasmid. A role of CCN3 in angiogenesis already has been postulated in vitro because in human umbilical vein endothelial cells (HUVEC), CCN3 increased endothelial cell adhesion, migration, and cell survival.
      • Lin C.G.
      • Leu S.J.
      • Chen N.
      • Tebeau C.M.
      • Lin S.X.
      • Yeung C.Y.
      • Lau L.F.
      CCN3 (NOV) is a novel angiogenic regulator of the CCN protein family.
      However, in that study CCN3 failed to increase proliferation of HUVEC. In contrast, in the present study CCN3 induced a trend toward an increased number of proliferating glomerular endothelial cells in vivo and markedly augmented proliferation of a glomerular endothelial cell line in vitro. Thus, potentially CCN3 exerts different effects in different endothelial cell types. Indeed, there is ample evidence that glomerular endothelial cells are highly specialized and different in their biology from, for example, HUVEC.
      • Fogo A.B.
      • Kon V.
      The glomerulus–a view from the inside–the endothelial cell.
      In addition, in our study a mitogenic effect of CCN3 on endothelial cells was observed only in full growth medium, suggesting that CCN3 requires other angiogenic factors to exert proliferative effects in glomerular endothelial cells, a similar finding to another pro-angiogenic factor, PDGF-CC.
      • Boor P.
      • van Roeyen C.R.
      • Kunter U.
      • Villa L.
      • Bucher E.
      • Hohenstein B.
      • Hugo C.P.
      • Eriksson U.
      • Satchell S.C.
      • Mathieson P.W.
      • Eitner F.
      • Floege J.
      • Ostendorf T.
      PDGF-C mediates glomerular capillary repair.
      The pro-angiogenic CCN3 effect in our model appeared to derive from release of angiogenic factors from glomerular endothelial cells in an autocrine fashion as suggested by our in vitro experiments and by the observation that CCN3 in vivo resulted in an increased glomerular mRNA expression of two pro-angiogenic factors, VEGF and PDGF-C, both of which were shown to induce glomerular endothelial cell proliferation in experimental nephritis in vivo.
      • Boor P.
      • van Roeyen C.R.
      • Kunter U.
      • Villa L.
      • Bucher E.
      • Hohenstein B.
      • Hugo C.P.
      • Eriksson U.
      • Satchell S.C.
      • Mathieson P.W.
      • Eitner F.
      • Floege J.
      • Ostendorf T.
      PDGF-C mediates glomerular capillary repair.
      • Masuda Y.
      • Shimizu A.
      • Mori T.
      • Ishiwata T.
      • Kitamura H.
      • Ohashi R.
      • Ishizaki M.
      • Asano G.
      • Sugisaki Y.
      • Yamanaka N.
      Vascular endothelial growth factor enhances glomerular capillary repair and accelerates resolution of experimentally induced glomerulonephritis.
      However, the CCN3-induced proliferation of glomerular endothelial cells in vitro could not be inhibited by VEGF-specific aptamer.
      In addition, the Smad signaling pathway was identified as a downstream signaling pathway of CCN3 overexpression. In agreement with previous studies showing inhibition of Smad1/5/8 phosphorylation in stromal cells and osteoblasts,
      • Minamizato T.
      • Sakamoto K.
      • Liu T.
      • Kokubo H.
      • Katsube K.
      • Perbal B.
      • Nakamura S.
      • Yamaguchi A.
      CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways.
      • Rydziel S.
      • Stadmeyer L.
      • Zanotti S.
      • Durant D.
      • Smerdel-Ramoya A.
      • Canalis E.
      Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia.
      we also noted a mild reduction of Smad1/5/8 phosphorylation on CCN3 overexpression in nephritic rats. In contrast, the inhibition of Smad2 phosphorylation by CCN3 overexpression was not detected in stromal cells.
      • Rydziel S.
      • Stadmeyer L.
      • Zanotti S.
      • Durant D.
      • Smerdel-Ramoya A.
      • Canalis E.
      Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia.
      Additional effects of CCN3 in vascular disease have been described. CCN3 null-mice showed increased vascular smooth muscle cell proliferation and reduced endothelialization, resulting in an enhanced neointimal thickening after femoral artery injury.
      • Shimoyama T.
      • Hiraoka S.
      • Takemoto M.
      • Koshizaka M.
      • Tokuyama H.
      • Tokuyama T.
      • Watanabe A.
      • Fujimoto M.
      • Kawamura H.
      • Sato S.
      • Tsurutani Y.
      • Saito Y.
      • Perbal B.
      • Koseki H.
      • Yokote K.
      CCN3 inhibits neointimal hyperplasia through modulation of smooth muscle cell growth and migration.
      In HUVEC, CCN3 has anti-inflammatory effects by inhibition of NF-κB activation and reduction of cytokine-mediated vascular adhesion molecule-1 expression.
      • Lin Z.
      • Natesan V.
      • Shi H.
      • Hamik A.
      • Kawanami D.
      • Hao C.
      • Mahabaleshwar G.H.
      • Wang W.
      • Jin Z.G.
      • Atkins G.B.
      • Firth S.M.
      • Rittie L.
      • Perbal B.
      • Jain M.K.
      A novel role of CCN3 in regulating endothelial inflammation.
      CCN1 (Cyr61), another member of the CCN family, also exerts pro-angiogenic activities such as neovascularization in corneal implants, promotion of cell survival, and induction of cell proliferation and tubule formation in HUVEC.
      • Leu S.J.
      • Lam S.C.
      • Lau L.F.
      Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells.
      • Grote K.
      • Salguero G.
      • Ballmaier M.
      • Dangers M.
      • Drexler H.
      • Schieffer B.
      The angiogenic factor CCN1 promotes adhesion and migration of circulating CD34+ progenitor cells: potential role in angiogenesis and endothelial regeneration.
      CCN1-knockout mice are embryonically lethal owing to vascular defects of failure in chorioallantoic fusion or placental vascular insufficiency and compromised vessel integrity.
      • Mo F.E.
      • Muntean A.G.
      • Chen C.C.
      • Stolz D.B.
      • Watkins S.C.
      • Lau L.F.
      CYR61 (CCN1) is essential for placental development and vascular integrity.
      Similar to CCN3, during anti-Thy1.1, nephritis CCN1 is overexpressed by podocytes during the mesangioproliferative phase and has been suggested to inhibit PDGF-B–induced mesangial cell migration.
      • Sawai K.
      • Mori K.
      • Mukoyama M.
      • Sugawara A.
      • Suganami T.
      • Koshikawa M.
      • Yahata K.
      • Makino H.
      • Nagae T.
      • Fujinaga Y.
      • Yokoi H.
      • Yoshioka T.
      • Yoshimoto A.
      • Tanaka I.
      • Nakao K.
      Angiogenic protein Cyr61 is expressed by podocytes in anti-Thy-1 glomerulonephritis.
      However, in that latter study, effects of CCN1 on capillary healing were not investigated.
      The second major finding of this study was a reduction of mesangial cell proliferation and matrix protein accumulation after systemic overexpression of CCN3. This finding substantiates our prior data, which identified CCN3 as an endogenous growth inhibitor of mesangial cells.
      • van Roeyen C.R.
      • Eitner F.
      • Scholl T.
      • Boor P.
      • Kunter U.
      • Planque N.
      • Grone H.J.
      • Bleau A.M.
      • Perbal B.
      • Ostendorf T.
      • Floege J.
      CCN3 is a novel endogenous PDGF-regulated inhibitor of glomerular cell proliferation.
      Until now, CCN3 was identified as a growth inhibitor (eg, in chicken embryonic fibroblasts, glioblastoma, choriocarcinoma, or Ewing sarcoma cell lines).
      • Perbal B.
      NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
      • Gupta N.
      • Wang H.
      • McLeod T.L.
      • Naus C.C.G.
      • Kyurkchiev S.
      • Advani S.
      • Yu J.
      • Perbal B.
      • Weichselbaum R.R.
      Inhibition of glioma cell growth and tumorigenic potential by CCN3 (NOV).
      • Bleau A.M.
      • Planque N.
      • Lazar N.
      • Zambelli D.
      • Ori A.
      • Quan T.
      • Fisher G.
      • Scotlandi K.
      • Perbal B.
      Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation.
      • Perbal B.
      CCN proteins: multifunctional signalling regulators.
      • Gellhaus A.
      • Dong X.
      • Propson S.
      • Maass K.
      • Klein-Hitpass L.
      • Kibschull M.
      • Traub O.
      • Willecke K.
      • Perbal B.
      • Lye S.J.
      • Winterhager E.
      Connexin43 interacts with NOV: a possible mechanism for negative regulation of cell growth in choriocarcinoma cells.
      In our model, two major mechanisms may have accounted for the reduction of mesangial cell proliferation and matrix accumulation. First, we noted that CCN3 overexpression led to a down-regulation of PDGFR-β mRNA expression at day 5 after disease induction, which might have reduced the promitogenic activity of the two PDGFR-β ligands PDGF-B and PDGF-D. Indeed, the inhibition of this pathway by the administration of anti–PDGFR-β IgG or the overexpression of a recombinant protein with the extracellular domain of PDGFR-β also results in diminished mesangial cell proliferation and matrix accumulation.
      • Takahashi T.
      • Abe H.
      • Arai H.
      • Matsubara T.
      • Nagai K.
      • Matsuura M.
      • Iehara N.
      • Yokode M.
      • Nishikawa S.
      • Kita T.
      • Doi T.
      Activation of STAT3/Smad1 is a key signaling pathway for progression to glomerulosclerosis in experimental glomerulonephritis.
      • Nakamura H.
      • Isaka Y.
      • Tsujie M.
      • Akagi Y.
      • Sudo T.
      • Ohno N.
      • Imai E.
      • Hori M.
      Electroporation-mediated PDGF receptor-IgG chimera gene transfer ameliorates experimental glomerulonephritis.
      A second mechanism may involve the increased capillary repair associated with the pro-angiogenic activity of CCN3. Indeed, in the anti-Thy1.1 model, the injection of bone marrow–derived endothelial progenitor cells, or VEGF, reduced glomerular endothelial damage and subsequently also augmented mesangial cell activation.
      • Miyamoto K.
      • Kitamoto Y.
      • Tokunaga H.
      • Takeya M.
      • Ezaki T.
      • Imamura T.
      • Tomita K.
      Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat.
      • Uchimura H.
      • Marumo T.
      • Takase O.
      • Kawachi H.
      • Shimizu F.
      • Hayashi M.
      • Saruta T.
      • Hishikawa K.
      • Fujita T.
      Intrarenal injection of bone marrow-derived angiogenic cells reduces endothelial injury and mesangial cell activation in experimental glomerulonephritis.
      Systemic overexpression of CCN3 increased glomerular regulated on activation normal T cell expressed and secreted and monocyte chemoattractant protein-1 mRNA expression. The latter is in agreement with data from Le Dreau et al in astrocytes stimulated with CCN3.
      • Le Dreau G.
      • Kular L.
      • Nicot A.B.
      • Calmel C.
      • Melik-Parsadaniantz S.
      • Kitabgi P.
      • Laurent M.
      • Martinerie C.
      NOV/CCN3 upregulates CCL2 and CXCL1 expression in astrocytes through beta1 and beta5 integrins.
      However, systemic overexpression of CCN3 did not alter glomerular monocyte/macrophage counts. We therefore speculate that other anti-inflammatory chemokines may have counteracted monocyte chemoattractant protein-1 and regulated on activation normal T cell expressed and secreted. In addition, the role of CCN3 in macrophage maturation, activation, and polarization is completely unknown.
      In progressive glomerulonephritis, overexpressed CCN3 reduced glomerulosclerosis and cortical accumulation of collagen type I, our third major finding. In addition, a trend toward diminished tubulointerstitial damage could be detected at day 56. These observations are well in line with our previous data, in which an inhibition of the pro-angiogenic action of VEGF164 in this model led to fulminant glomerulosclerosis and renal interstitial damage within 4 weeks,
      • Ostendorf T.
      • Kunter U.
      • Eitner F.
      • Loos A.
      • Regele H.
      • Kerjaschki D.
      • Henninger D.D.
      • Janjic N.
      • Floege J.
      VEGF(165) mediates glomerular endothelial repair.
      and also data in which a reduction of mesangioproliferative changes in the anti-Thy1.1 nephritis model translated into long-term preservation of renal morphology and function.
      • Ostendorf T.
      • Kunter U.
      • Grone H.J.
      • Bahlmann F.
      • Kawachi H.
      • Shimizu F.
      • Koch K.M.
      • Janjic N.
      • Floege J.
      Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis.
      • Ostendorf T.
      • Rong S.
      • Boor P.
      • Wiedemann S.
      • Kunter U.
      • Haubold U.
      • van Roeyen C.R.
      • Eitner F.
      • Kawachi H.
      • Starling G.
      • Alvarez E.
      • Smithson G.
      • Floege J.
      Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis.
      • Boor P.
      • Konieczny A.
      • Villa L.
      • Kunter U.
      • van Roeyen C.R.
      • LaRochelle W.J.
      • Smithson G.
      • Arrol S.
      • Ostendorf T.
      • Floege J.
      PDGF-D inhibition by CR002 ameliorates tubulointerstitial fibrosis following experimental glomerulonephritis.
      Finally, we addressed the question of effects induced by CCN3 overexpression in healthy rats. Until now, transgenic animal models overexpressing CCN3 have not been described. We found that in healthy rats, overexpressed CCN3 predominantly acted as a regulator of gene transcription. The described nuclear localization of CCN3 in several cell lines and the interaction with the rpb7 subunit of RNA polymerase in a yeast two-hybrid system indicated that CCN3 indeed is involved in gene regulation at the transcriptional level.
      • Perbal B.
      NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues.
      • Perbal B.
      Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression.
      In healthy and nephritic rats the systemic overexpression of CCN3 resulted in a down-regulation of endogenous glomerular CCN3 expression. Until now, an autoregulatory feedback loop has not been described for CCN3. In contrast, cross-regulation of CCN3 and CCN2 was observed in CCN2-deficient mice, which overexpress CCN3,
      • Kawaki H.
      • Kubota S.
      • Suzuki A.
      • Lazar N.
      • Yamada T.
      • Matsumura T.
      • Ohgawara T.
      • Maeda T.
      • Perbal B.
      • Lyons K.M.
      • Takigawa M.
      Cooperative regulation of chondrocyte differentiation by CCN2 and CCN3 shown by a comprehensive analysis of the CCN family proteins in cartilage.
      and rat mesangial cells stimulated with transforming growth factor-β, which showed a down-regulation of CCN3 and up-regulation of CCN2.
      • Riser B.L.
      • Najmabadi F.
      • Perbal B.
      • Peterson D.R.
      • Rambow J.A.
      • Riser M.L.
      • Sukowski E.
      • Yeger H.
      • Riser S.C.
      CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.
      This “yin-yang” relation between CCN3 and CCN2 expression described by Riser et al
      • Riser B.L.
      • Najmabadi F.
      • Perbal B.
      • Peterson D.R.
      • Rambow J.A.
      • Riser M.L.
      • Sukowski E.
      • Yeger H.
      • Riser S.C.
      CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease.
      • Riser B.L.
      • Najmabadi F.
      • Perbal B.
      • Rambow J.A.
      • Riser M.L.
      • Sukowski E.
      • Yeger H.
      • Riser S.C.
      • Peterson D.R.
      CCN3/CCN2 regulation and the fibrosis of diabetic renal disease.
      in models of renal fibrosis was confirmed in healthy rats but not in our rats with mesangioproliferative glomerulonephritis. Furthermore, in agreement with results obtained in hepatocytes,
      • Borkham-Kamphorst E.
      • van Roeyen C.R.
      • Van de Leur E.
      • Floege J.
      • Weiskirchen R.
      CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC.
      CCN2 expression was not changed by CCN3 stimulation of glomerular endothelial and mesangial cells in vitro.
      In conclusion, we identified a dual role of CCN3 in experimental glomerulonephritis with both pro-angiogenic and antimesangioproliferative effects, both of which serve to reconstitute normal glomerular architecture. CCN3 therefore represents a potentially novel therapeutic tool to help repair glomerular endothelial damage and mesangioproliferative changes.

      Acknowledgments

      We thank Dr. Peter W. Mathieson and Dr. Simon C. Satchell for kindly providing the glomerular endothelial cells (CiGEnC). We are grateful for expert technical assistance by Lydia Zimmermanns, Andrea Cosler, Gabriele Dietzel, Gertrud Minnartz, Christina Gianusis, Nicole Bataille, and Dagmar Wieland.

      Supplementary data

      • Supplemental Figure S1

        Effect of VEGF inhibition in CCN3-induced glomerular endothelial cell proliferation in vivo. The activity of VEGF was inhibited using VEGF-specific aptamers and the proliferation of CiGEnC was measured in a proliferation assay using BrdU incorporation. The CCN3-induced cell proliferation was not changed by the incubation with VEGF-specific aptamers.

      • Supplemental Figure S2

        CCN3 did not regulate CCN2 expression in glomerular endothelial and mesangial cells in vitro. A: Stimulation of endothelial and mesangial cells with recombinant CCN3 protein did not regulate CCN2 mRNA expression. B: CCN2 protein levels were not changed in endothelial cells stimulated with recombinant CCN3. Mean values are shown in the morphometric analysis.

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