1. Volume 92, Issue 4, Pages 850-863 (October 2017)
Connective tissue growth factor regulates fibrosis-associated renal lymphangiogenesis 
Hiroshi Kinashi, Lucas L. Falke, Tri Q. Nguyen, Niels Bovenschen, Jan Aten, Andrew Leask, Yasuhiko Ito, Roel Goldschmeding 
Kidney International 
Volume 92, Issue 4, Pages (October 2017)
DOI: /j.kint
Copyright © 2017 International Society of Nephrology Terms and Conditions

2. Figure 1
Connective tissue growth factor (CTGF), vascular endothelial growth factor C (VEGF-C), and D2-40 expression were increased in the human renal cortex undergoing fibrosis due to ureteral obstruction. A human obstructed kidney (OBK) and unobstructed contralateral kidney (CLK) were derived from the same patient undergoing bilateral nephrectomy for bilateral renal pelvis tumors of which only 1 caused ureteral obstruction. Images depict representative renal cortical areas in consecutive sections stained with Sirius red and Masson’s trichrome, and for CTGF, VEGF-C, and D2-40. The increase of D2-40–positive lymphatic vessels in OBK undergoing tubulointerstitial fibrosis was associated with an increased expression of CTGF and VEGF-C. Arrows indicate the same renal tubules expressing both CTGF and VEGF-C. Bar = 100 μm. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

3. Figure 2
Expression of connective tissue growth factor (CTGF) was dramatically decreased in CTGF knockout (CTGF–/–) mouse kidneys. (a,b) Western blot analysis showed that CTGF protein levels were significantly decreased in both contralateral (CLK) and obstructed (OBK) kidneys of CTGF–/– mice compared with wild-type (WT) mice. Actin was shown as a loading control. (c) Quantitative real-time polymerase chain reaction analysis showed that CTGF mRNA expression was significantly decreased in both CLK and OBK of CTGF–/– mice compared with WT mice. Data are expressed as mean ± SEM (N = 5 for WT mice and N = 9 for CTGF–/– mice). TATA-box binding protein (TBP) was used as an internal control. *P < 0.05, **P < 0.01, and ***P < To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

4. Figure 3
Tubulointerstitial fibrosis was decreased in connective tissue growth factor knockout (CTGF–/–) obstructed kidneys (OBK). (a) Representative micrographs of mouse renal cortex stained with periodic acid-Schiff (PAS), Masson’s trichrome, Sirius red, and α-smooth muscle actin (α-SMA). Bar = 50 μm. (b–d) Quantification of Masson’s trichrome staining, Sirius red staining, and immunohistochemistry of α-SMA showed that an increased expression of tubulointerstitial fibrosis and α-SMA in OBKs was significantly decreased in CTGF–/– mice compared with wild-type (WT) mice. (e,f) Quantitative real-time polymerase chain reaction analysis showed that an increased expression of collagen type I alpha 2 (Col1α2) and transforming growth factor β (TGF-β) mRNA in OBK was significantly decreased in CTGF–/– mice compared with WT mice. Data are expressed as mean ± SEM (N = 5 for WT mice and N = 9 for CTGF–/– mice). *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. denotes not significant. CLK, contralateral kidneys; TBP, TATA-box binding protein. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

5. Figure 3
Tubulointerstitial fibrosis was decreased in connective tissue growth factor knockout (CTGF–/–) obstructed kidneys (OBK). (a) Representative micrographs of mouse renal cortex stained with periodic acid-Schiff (PAS), Masson’s trichrome, Sirius red, and α-smooth muscle actin (α-SMA). Bar = 50 μm. (b–d) Quantification of Masson’s trichrome staining, Sirius red staining, and immunohistochemistry of α-SMA showed that an increased expression of tubulointerstitial fibrosis and α-SMA in OBKs was significantly decreased in CTGF–/– mice compared with wild-type (WT) mice. (e,f) Quantitative real-time polymerase chain reaction analysis showed that an increased expression of collagen type I alpha 2 (Col1α2) and transforming growth factor β (TGF-β) mRNA in OBK was significantly decreased in CTGF–/– mice compared with WT mice. Data are expressed as mean ± SEM (N = 5 for WT mice and N = 9 for CTGF–/– mice). *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. denotes not significant. CLK, contralateral kidneys; TBP, TATA-box binding protein. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

6. Figure 4
Expression of lymphatic vessels and vascular endothelial growth factor C (VEGF-C) was decreased in connective tissue growth factor knockout (CTGF–/–) obstructed kidneys (OBKs). (a) Representative micrographs of immunohistochemistry of lymphatic endothelial hyaluronan receptor 1 (LYVE-1) and VEGF-C in a mouse renal cortex. Arrows indicate LYVE-1 positive lymphatic vessels. Insets indicate magnification of the black boxed area. Bar = 100 μm. (b,c) Quantification of immunohistochemistry showed that an increased number of LYVE-1–positive lymphatic vessels and an increased expression of VEGF-C in OBKs were significantly decreased in CTGF–/– mice compared with wild-type (WT) mice. (d,e) Quantitative real-time polymerase chain reaction analysis showed that LYVE-1 and VEGF-C mRNA expression were significantly decreased in CTGF–/– OBK compared with WT OBK. Data are expressed as mean ± SEM (N = 5 for WT mice and N = 9 for CTGF–/– mice). *P < 0.05, ***P < 0.001, and n.s. denotes not significant. CLK, contralateral kidneys; TBP, TATA-box binding protein. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

7. Figure 4
Expression of lymphatic vessels and vascular endothelial growth factor C (VEGF-C) was decreased in connective tissue growth factor knockout (CTGF–/–) obstructed kidneys (OBKs). (a) Representative micrographs of immunohistochemistry of lymphatic endothelial hyaluronan receptor 1 (LYVE-1) and VEGF-C in a mouse renal cortex. Arrows indicate LYVE-1 positive lymphatic vessels. Insets indicate magnification of the black boxed area. Bar = 100 μm. (b,c) Quantification of immunohistochemistry showed that an increased number of LYVE-1–positive lymphatic vessels and an increased expression of VEGF-C in OBKs were significantly decreased in CTGF–/– mice compared with wild-type (WT) mice. (d,e) Quantitative real-time polymerase chain reaction analysis showed that LYVE-1 and VEGF-C mRNA expression were significantly decreased in CTGF–/– OBK compared with WT OBK. Data are expressed as mean ± SEM (N = 5 for WT mice and N = 9 for CTGF–/– mice). *P < 0.05, ***P < 0.001, and n.s. denotes not significant. CLK, contralateral kidneys; TBP, TATA-box binding protein. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

8. Figure 5
Lymphatic endothelial hyaluronan receptor 1 (LYVE-1) mRNA expression was decreased in renal cortices of connective tissue growth factor knockout (CTGF–/–) mice after ischemia-reperfusion injury (IRI). (a,b) Quantitative real-time polymerase chain reaction analysis showed a tendency that an increased expression of LYVE-1 and VEGF-C mRNA was decreased in CTGF–/– kidneys compared with wild-type (WT) kidneys 3 days after IRI. (c,d) An increased expression of LYVE-1 mRNA was significantly decreased in CTGF–/– kidneys compared with WT kidneys 6 weeks after IRI. VEGF-C mRNA showed similar levels in all kidney groups 6 weeks after IRI. (e) Representative micrographs of immunohistochemistry of LYVE-1 in mouse renal cortices 6 weeks after IRI. Arrows indicate LYVE-1–positive lymphatic vessels. Bar = 50 μm. Data are expressed as mean ± SEM. Sample numbers (WT sham, CTGF–/– sham, WT IRI, and CTGF–/– IRI) = 4, 5, 5, and 6 in 3 days and 6, 5, 6, and 13 in 6 weeks, respectively. *P < 0.05 and n.s. denotes not significant. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

9. Figure 6
Connective tissue growth factor (CTGF)–induced vascular endothelial growth factor C (VEGF-C) production in cultured human renal proximal tubular epithelial cells (HK-2). HK-2 cells were treated with (a,b) recombinant human full-length CTGF (FL-CTGF) or (c) transforming growth factor β1 (TGF-β1). (d–f) HK-2 cells were transfected with CTGF small, interfering RNA (siRNA) and treated with TGF-β1. Nontargeting siRNA was used as a control siRNA. (d) CTGF and (a,c,e) VEGF-C mRNA were determined by quantitative real-time polymerase chain reaction. TATA-box binding protein (TBP) was used as an internal control. (b,f) The VEGF-C protein level in the supernatant was determined by enzyme-linked immunosorbent assay. (a,b) VEGF-C mRNA expression and VEGF-C protein level were increased by FL-CTGF treatment after an 8-hour incubation. (c) VEGF-C mRNA expression was increased by TGF-β1 treatment after an 8-hour incubation. (d) CTGF mRNA expression was significantly increased by TGF-β1 treatment after an 8-hour incubation. CTGF siRNA significantly reduced CTGF mRNA expression both in control and TGF-β1 treatment conditions. (e) TGF-β1–induced VEGF-C mRNA upregulation was significantly suppressed by CTGF siRNA after an 8-hour incubation. (f) VEGF-C protein level upregulated by TGF-β1 treatment was significantly reduced by CTGF siRNA after a 24-hour incubation. Data are expressed as mean ± SEM (N = 3 or 4). *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. denotes not significant.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

10. Figure 7
Connective tissue growth factor (CTGF) directly bound to vascular endothelial growth factor C (VEGF-C). Physical interaction between CTGF and VEGF-C was demonstrated by solid-phase binding assay and surface plasmon resonance analysis. (a) Increasing concentrations of recombinant human full-length CTGF (FL-CTGF) were added to microtiter plates coated with 4 μg/ml recombinant human VEGF-C or 1% bovine serum albumin (BSA). Bound proteins were detected with an alkaline phosphatase-conjugated antibody against CTGF. Data are from 3 independent experiments. **P < 0.01 compared with the value for BSA. (b) Recombinant human VEGF-C was immobilized, and FL-CTGF, NH2- (N-CTGF), and COOH-terminal (C-CTGF) fragments were incubated. Representative curves at 400 nM CTGF are shown (corrected for background binding). (c) FL-CTGF and C-CTGF, but not N-CTGF, dose-dependently interacted with VEGF-C. The response at maximal binding are shown. Data are from triplicates.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

11. Figure 8
Connective tissue growth factor (CTGF) suppressed vascular endothelial growth factor C (VEGF-C)–induced capillary-like tube formation in human dermal lymphatic microvascular endothelial cells (HMVEC-dLy). (a,b) VEGF-C efficiently induced capillary-like tube formation in HMVEC-dLy seeded on Matrigel in serum-free medium for 6 hours. HMVEC-dLy were treated with (c) recombinant human full-length CTGF (FL-CTGF), (d) an NH2-terminal fragment of CTGF (N-CTGF), or (e) a COOH-terminal fragment of CTGF (C-CTGF) in the presence of VEGF-C. Bar = 400 μm. (f) Tube formation was quantified by counting the number of tubes. FL-CTGF suppressed VEGF-C-induced tube formation. N-CTGF and C-CTGF had no significant effect on tube formation induced by VEGF-C. Data are expressed as mean ± SEM (N = 3). **P < 0.01, ***P < 0.001, and n.s. denotes not significant. To optimize viewing of this image, please see the online version of this article at
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions

12. Figure 9
Connective tissue growth factor (CTGF) plays a significant role in renal lymphangiogenesis. Full-length CTGF (FL-CTGF) promotes vascular endothelial growth factor C (VEGF-C) production in renal tubular epithelial cells. On the other hand, FL-CTGF binds to VEGF-C, and suppresses VEGF-C-induced lymphangiogenesis. This direct inhibitory effect of FL-CTGF on VEGF-C could be abrogated by the cleavage of CTGF because the NH2-terminal fragment and the COOH-terminal fragment of CTGF have no influence on VEGF-C function. CTGF knockdown suppresses VEGF-C expression and lymphangiogenesis in a unilateral ureteral obstruction (UUO) and an ischemia-reperfusion injury model.
Kidney International  , DOI: ( /j.kint )
Copyright © 2017 International Society of Nephrology Terms and Conditions