Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis

Abstract

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Figure 1. Deletion of CTGF ameliorated proteinuria and histological changes in anti-GBM nephritis.

(a) An experimental protocol for the study on the anti-GBM nephritis in tamoxifen-inducible systemic CTGF cKO (Rosa-CTGF cKO) mice. Three-week old male ROSA26-CreER^T2; Ctgf^fl/fl mice or control [Cre (−); Ctgf^fl/fl] were intraperitoneally injected with 4-OHT, and anti-GBM nephritis was induced at 8 weeks of age (GBM). Vehicle treatment was also carried out in some animals (veh.). Mice were killed at 4 weeks after induction of anti-GBM nephritis. (b) Double immunofluorescence staining for CTGF (green) and podocin (red) in the glomeruli of control or Rosa-CTGF cKO mice at 4 weeks after induction of anti-GBM nephritis. Bar represents 50 μm. (c) Changes in proteinuria during the course of anti-GBM nephritis. Closed squares, vehicle-treated control mice; open square, vehicle-treated Rosa-CTGF cKO mice; closed circles, control mice with GBM nephritis; open circles, Rosa-CTGF cKO mice with anti-GBM nephritis. (d) Representative photomicrographs of the kidneys at 4 weeks after induction of anti-GBM nephritis (PAS staining). Left upper panel, vehicle-treated control mice; right upper panel, vehicle-treated Rosa-CTGF cKO mice; left lower panel, control mice with anti-GBM nephritis; right lower panel show, Rosa-CTGF cKO mice with anti-GBM nephritis. Bar represents 50 μm. (e) The percentage of crescent formation (left panel) and the mesangial area (right panel) in anti-GBM nephritis. (f) Electron microscopic analysis of glomeruli. Bar represents 2,000 nm. (g) Immunofluorescence studies for rabbit IgG, mouse IgG and mouse C3 deposition in the kidney at 28 days after induction of anti-GBM nephritis in control and in Rosa-CTGF cKO mice. Both Rosa-CTGF cKO and control mice exhibited liner IgG and C3 deposition along the GBM. Bar represents 50 μm. Values are expressed as means ± s.e. ^*P < 0.05, ^**P < 0.01 vs control mice with GBM nephritis. Vehicle-treated control mice (n = 7), vehicle-treated Rosa-CTGF cKO mice (n = 7), control mice with GBM nephritis (n = 7), Rosa-CTGF mice with GBM nephritis (n = 9). Veh, vehicle; GBM, anti-GBM nephritis.

Figure 2. Systemic deletion of CTGF reduced mRNA expression of fibrotic makers in anti-GBM nephritis.

(a) Serum creatinine and BUN levels. No significant difference between control and Rosa-CTGF cKO mice with nephritis. (b) Expression of Ctgf and Tgfb1 mRNA in the glomeruli of the kidney at 4 weeks after induction of anti-GBM nephritis. Glomerular Ctgf expression decreased by 80% in Rosa-CTGF cKO mice with nephritis compared with control mice with nephritis. Tgfb1 expression was also decreased in Rosa-CTGF cKO with nephritis. (c) Expression of Acta2, Fn1 and Itgav mRNA in the glomeruli. Gapdh mRNA expression was used as internal control. (d) Glomerular CTGF protein at 4 weeks after induction of anti-GBM nephritis by Western blotting (n = 4, each). β-actin was used as internal control. Full-length blots are presented in Supplementary Figure S10. Values are expressed as means ± s.e. ^*P < 0.05, ^**P < 0.01. Vehicle-treated control mice (n = 7), vehicle-treated Rosa-CTGF cKO mice (n = 7), control mice with anti-GBM nephritis (n = 7), Rosa-CTGF cKO mice with anti-GBM nephritis (n = 9). Veh, vehicle; GBM, anti-GBM nephritis.

Figure 3. Podocyte-specific inhibition of CTGF did not ameliorate glomerular changes.

(a) Experimental protocol for anti-GBM nephritis in podocyte-specific CTGF cKO mice (pod-CTGF cKO). (b) Double immunofluorescence staining for CTGF (green) and podocin (red) in glomeruli of pod-CTGF cKO mice at 4 weeks after induction of anti-GBM nephritis. Pod-CTGF cKO mice with anti-GBM nephritis exhibited a decrease in CTGF expression by podocytes. Lower panel, pod-CTGF cKO mice with anti-GBM nephritis. Bar represents 50 μm. (c) Changes in proteinuria during the course of the anti-GBM nephritis. Closed squares, vehicle-treated control mice; open squares, pod-CTGF cKO mice with vehicle; closed circles, control mice with anti-GBM nephritis; open circles, pod-CTGF cKO mice with anti-GBM nephritis. (d) Representative photomicrographs of the kidneys at 4 weeks after induction of anti-GBM nephritis (PAS staining). Left upper panel, vehicle-treated control mice; right upper panel, vehicle-treated pod-CTGF cKO mice; left lower panel, control mice with anti-GBM nephritis; right lower panel, pod-CTGF cKO mice with anti-GBM nephritis. Bar represents 50 μm. (e) The percentage of crescent formation (left panel) and the mesangial area (right panel) in anti-GBM nephritis in pod-CTGF cKO mice. (f) Electron microscopic analysis of glomeruli in anti-GBM nephritis in pod-CTGF cKO mice. No difference in dense deposit accumulation (white arrows) between control and pod-CTGF cKO mice with nephritis. Bar represents 2,000 nm. (g) Serum creatinine and BUN levels in pod-CTGF cKO mice with anti-GBM nephritis. (h) Expression of Ctgf and Tgfb1 mRNA in glomeruli at 4 weeks after induction of anti-GBM nephritis. (i) Expression of Acta2, Fn1and Itgav mRNA in glomeruli. (j) Representative image of glomerular CTGF protein by Western blotting. Immunoblot of β-actin was used as control. Full-lengh blots are presented in Supplementary Figure S11. Values are expressed as means ± s.e. ^*P < 0.05, ^**P < 0.01. Vehicle-treated control mice (n = 9), vehicle-treated pod-CTGF cKO mice (n = 6), control mice with anti-GBM nephritis (n = 7), pod-CTGF mice with anti-GBM nephritis (n = 7). Veh, vehicle; GBM, anti-GBM nephritis.

Figure 4. Reduction of CTGF in mesangial cell ameliorated glomerulonephritis.

(a) Experimental protocol anti-GBM nephritis in Pdgfra-CTGF cKO mice. Anti-GBM nephritis was induced at 8 weeks of age. Mice were killed at 4 weeks after induction of anti-GBM nephritis. (b) Double immunofluorescence staining for CTGF (green) and podocin (red) in the glomeruli of Pdgfra-CTGF cKO or control mice at 4 weeks after induction of anti-GBM nephritis. Bar represents 50 μm. (c) Changes in proteinuria during the course of the anti-GBM nephritis. Closed squares, vehicle-treated control mice; open squares, vehicle-treated Pdgfra-CTGF cKO mice; closed circles, control mice with nephritis; open circles, Pdgfra-CTGF cKO mice with nephritis. (d) Representative photomicrographs of the kidneys at 4 weeks after induction of anti-GBM nephritis (PAS staining). Left upper panel, vehicle-treated control mice; right upper panel, vehicle-treated Pdgfra-CTGF cKO mice; left lower panel, control mice with anti-GBM nephritis; right lower panel, Pdgfra-CTGF cKO mice with anti-GBM nephritis. Bar represents 50 μm. (e) The percentage of crescent formation (left panel) and the mesangial area (right panel) in anti-GBM nephritis. (f) Electron microscopic analysis of glomeruli in anti-GBM nephritis in Pdgfra-CTGF cKO mice. Dense deposit accumulation along GBM was reduced in Pdgfra-CTGF cKO mice with nephritis (white arrows). Bar represents 2,000 nm. (g) Serum creatinine and BUN levels in anti-GBM nephritis in Pdgfra-CTGF cKO mice. (h) Expression of Ctgf and Tgfb1 mRNA in glomeruli at 4 weeks after induction of anti-GBM nephritis. (i) Expression of Acta2, Fn1 and Itgav mRNA in glomeruli. (j) Representative image of glomerular CTGF protein by Western blotting. Immunoblot of β-actin was used as control. Full-length blots are presented in Supplementary Figure S12. Values were expressed as means ± s.e. ^*P < 0.05, ^**P < 0.01 vs control mice with anti-GBM nephritis. Vehicle-treated control mice (n = 6), vehicle-treated Pdgfra-CTGF cKO mice (n = 6), control mice with anti-GBM nephritis (n = 9), Pdgfra-CTGF cKO mice with anti-GBM nephritis (n = 6). Veh, vehicle; GBM, anti-GBM nephritis.

Figure 5. Role of CTGF on macrophage accumulation in anti-GBM nephritis.

(a–c) Immunohistochemical studies for MAC-2 to analyze the number of MAC-2-positive cells per a glomerulus of Rosa-CTGF cKO mice (a), pod-CTGF cKO mice (b) and Pdgfra-CTGF cKO mice (c) at 4 weeks after induction of anti-GBM nephritis. Bar represents 50 μm. (d–f) Expression of Adgre1 (F4/80) mRNA in the glomeruli of Rosa-CTGF cKO mice (d), pod-CTGF cKO mice (e) and Pdgfra-CTGF cKO mice (f) at 4 weeks after induction of anti-GBM nephritis. (g–i) Ccl2 mRNA expression in glomeruli of Rosa-CTGF cKO mice (g), pod-CTGF cKO mice (h) and Pdgfra-CTGF cKO mice (i) at 4 weeks after induction of anti-GBM nephritis. Veh., vehicle; GBM, anti-GBM nephritis; Rosa cKO, Rosa-CTGF cKO mice; pod cKO, pod-CTGF cKO mice; Pdgfra cKO, Pdgfra-CTGF cKO mice. Values were expressed as the means ± s.e. ^*P < 0.05, ^**P < 0.01.

Figure 6. Relevance of CTGF to macrophage phenotypes in anti-GBM nephritis.

(a–c) Expression of Itgax (CD11c, M1 marker) mRNA in the glomeruli of Rosa-CTGF cKO mice (a), pod-CTGF cKO mice (b) and Pdgfra-CTGF cKO mice (c) at 4 weeks after induction of anti-GBM nephritis. (d–f) Mrc1 (CD206, M2 marker) mRNA expression in the glomeruli of Rosa-CTGF cKO mice (d), pod-CTGF cKO mice (e) and Pdgfra-CTGF cKO mice (f). (g–i) Ratio of mRNA expression of M1/M2 macrophage markers in the glomeruli of Rosa-CTGF cKO mice (g), pod-CTGF cKO mice (h) and Pdgfra-CTGF cKO mice (i).Veh, vehicle; GBM, anti-GBM nephritis; Rosa cKO, Rosa-CTGF cKO mice; pod cKO, pod-CTGF cKO mice; Pdgfra cKO, Pdgfra-CTGF cKO mice. Values were expressed as the means ± s.e. ^*P < 0.05, ^**P < 0.01.

Figure 7. The effects of CTGF on macrophage subtypes.

(a,b) Expression of Itgax (a) and Mrc1 (b) by 10 ng/ml TNF-α in RAW264.7 cells at 3 h after stimulation. (c) Ratio of mRNA expression of M1/M2 macrophage markers in RAW264.7 cells after stimulation with TNF-α. (d,e) Expression of Itgax (d) and Mrc1 (e) by 1000 ng/ml CTGF in RAW264.7 cells. (f) Ratio of mRNA expression of M1/M2 macrophage markers in RAW264.7 cells after stimulation with 1000 ng/ml CTGF. (g) Induction of Tnfa expression by treatment with 1000 ng/ml CTGF in RAW264.7 cells. hCTGF, recombinant human CTGF. n = 6, each. Values were expressed as the means ± s.e. ^*P < 0.05, ^**P < 0.01.

Figure 8. The effects of CTGF on chemotasis in cultured mesangial cells and adhesion of cultured macrophage to activated mesangial cells.

(a) Induction of Ccl2 expression by treatment with 500 ng/ml CTGF and/or 1 ng/ml TGF-β1 in cultured mesangial cells. (b) Reduction of Ccl2 expression by CTGF siRNA in cultured mesangial cells. (c) Amelioration of siRNA-mediated Ccl2 reduction by treatment of exogenous CTGF (100 ng/ml). hCTGF, recombinant human CTGF; siCTGF, siRNA against CTGF. (d) Immunoblt of CTGF by CTGF siRNA in cultured mesangial cells by Western blotting. Full-length blots are presented in Supplementary Figure S13. (e–g) Macrophage adhesion to cultured mesangial cells on culture plates. (e) Experimental protocol of adhesion of fluorescein-dye-labeled RAW264.7 cells to 10 ng/ml TNF-α-stimulated mesangial cells. (f) Inhibition of CTGF by siRNA reduced macrophage adhesion, and this reduction was ameliorated by exogenous human CTGF (100 ng/ml). Bar represents 100 μm. (g) Quantitative analyses of adhesion of RAW264.7 cells to cultured mesangial cells. (h) Ctgf mRNA expression in cultured mesangial cells with the stimulation of 10 ng/ml TNF-α. (i–l) Expression of Icam1 (i), Vcam1 (j), Fn1 (k) and Itgav (l) mRNA in mesangial cells after stimulation of 10 ng/ml TNF-α. n = 6, each. ^*P < 0.05, ^**P < 0.01.