Evidence for low-density lipoprotein–induced expression of connective tissue growth factor in mesangial cells  Mimi Sohn, Yan Tan, Richard L. Klein, Ayad A. Jaffa  Kidney International  Volume 67, Issue 4, Pages 1286-1296 (April 2005) DOI: 10.1111/j.1523-1755.2005.00206.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Low-density lipoprotein (LDL) stimulates the regulation of collagen I protein in mesangial cells. Quiescent mesangial cells were stimulated with 50 μg/mL of LDL for 24 hours. Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies against collagen I (1:1000) and actin (1:1000). Bar graph represents intensities of collagen I bands relative to actin and expressed as percent above control. Blots shown are representative of 22 experiments. *P < 0.0005 vs. control. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Low-density lipoprotein (LDL) stimulates the regulation of connective tissue growth factor (CTGF) in mesangial cells. Quiescent mesangial cells were stimulated with 50 μg/mL of LDL for 24 hours. Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with an antibody against CTGF (1:1000) and actin (1:1000). Bar graph represents intensities of CTGF bands relative to actin expressed as percentage above control. Blots shown are representative of 23 experiments. *P < 0.0001 vs. control. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Induction of transforming growth factor-β1 (TGF-β1) protein secretion by low-density lipoprotein (LDL) in mesangial cells. Quiescent mesangial cells were stimulated by LDL (50 μg/mL) for 24 hours. Conditioned media were collected and activated to determine the total level of TGF-β1 protein, using colorimetric enzyme-linked immunosorbent assay (ELISA) kit assay. Values are means ± SE of TGF-β1 levels expressed in pg/mL (N = 26). *P < 0.0001 vs. control. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Induction of connective tissue growth factor (CTGF) and collagen I by transforming growth factor-β (TGF-β). Mesangial cells were treated with various concentration of TGF-β 1 (0, 0.2, 0.5, 1, 5, and 10 ng/mL) for 24 hours. (A) Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with an antibody against CTGF (1:1000) and actin (1:1000). Bar graph represents intensities of CTGF bands relative to actin expressed as percentage above control. Blots shown are representative of five separate experiments. *P < 0.05 vs. control. (B) Cell proteins were separate by SDS-PAGE (7.5%) and immunoblotted with an antibody against collagen I (1:1000) and actin (1:1000). Bar graph represents intensities of collagen I bands relative to actin expressed as percentage above control. Blots shown are representative of three separate experiments. *P < 0.05 vs. control. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Low-density lipoprotein (LDL) induces connective tissue growth factor (CTGF) and collagen I expression via autocrine activation of transforming growth factor-β (TGF-β). Mesangial cells were stimulated with 50 μg/mL of LDL or 5 ng/mL of TGF-β1 for 24 hours in presence or absence of 5 μg/mL of neutralizing anti-TGF-β antibody (TGF-β NA). (A) Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with an antibody against CTGF (1:1000) and actin (1:1000). Bar graph represents intensities of CTGF bands relative to actin expressed as percentage above control. Blots shown are representative of nine experiments. *P < 0.01 vs. control; †P < 0.05 vs. LDL; ††P < 0.05 vs. TGF-β1. (B) Equal amounts of proteins were resolved on SDS-PAGE (7.5%) and immunoblotted with an antibody against collagen I (1:1000) and actin (1:1000). Bar graph represent intensities of collagen I bands relative to actin expressed as percentage above control. Blots shown are representative of eight experiments. *P < 0.05 vs. control; †P < 0.05 vs. LDL; ††P < 0.01 vs. TGF-β1. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Phosphorylation of mitogen-activated protein kinase (MAPK) pathway by low-density lipoprotein (LDL). Quiescent mesangial cells were stimulated with 50 μg/mL of LDL for 5 minutes. Cell proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with a polyclonal antibody against phospho-p44/42 (1:4000) and total-p44/42 (1:4000) (A), phospho-p38 (1:1000) and total-p38 (1:4000) (B), and phospho-c-Jun NH2-terminal kinase (JNK) (1:1000) and total-JNK (1:4000) (C). Blots shown are representative of 12 experiments. Bar graph represents intensities of phospho-MAPK relative to total-MAPK expressed as percent phosphorylation above control. Bars represent means ± SE of 12 experiments. *P < 0.0001 vs. control. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Role of c-Jun NH2-terminal kinase (JNK) in low-density lipoprotein (LDL)-induced transforming growth factor-β (TGF-β) production. Quiescent mesangial cells were stimulated with LDL (50 μg/mL) for 24 hours in presence or absence of p44/42mapk inhibitor, PD98059 (40 μmol/L) (A), p38mapk inhibitor, SB203580 (10 μmol/L) (B), and JNK inhibitor, SP600125 (30 μmol/L) (C). Conditioned media were collected and activated to determine the total level of TGF-β1 protein using colorimetric enzyme-linked immunosorbent assay (ELISA) kit assay. Values are means ± SE of TGF-β1 levels expressed in pg/mL. *P < 0.05 vs. control; #P < 0.0001 vs. LDL (N = 4 to 10 experiments). Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Effect of mitogen-activated protein kinase (MAPK) inhibitors on low-density lipoprotein (LDL)-induced regulation of connective tissue growth factor (CTGF). (A) Mesangial cells were stimulated with LDL (50 μg/mL) for 24 hours in presence or absence of p44/42mapk inhibitor, PD98059 (40 μmol/L) for 45minutes. Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with an antibody against CTGF (1:1000) and actin (1:1000). Bar graph represents intensities of CTGF bands relative to actin expressed as percent above control. Blots shown are representative of eight experiments. *P < 0.05 vs. control; #P < 0.005 vs. LDL. (B) Quiescent mesangial cells were stimulated by LDL (50 μg/mL) for 24 hours in presence or absence of p38mapk inhibitor, SB203580 (10 μmol/L) for 45minutes. Blots shown are representative of four experiments. *P < 0.05 vs. control. (C) Quiescent mesangial cells were stimulated by LDL (50 μg/mL) for 24 hours in presence or absence of c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125 (30 μmol/L) for 45minutes. Blots shown are representative of eight experiments. *P < 0.05 vs. control; #P < 0.001 vs. LDL. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 9 Role of mitogen-activated protein kinase (MAPK) pathway in low-density lipoprotein (LDL)-induced collagen I expression. (A) Quiescent mesangial cells were stimulated with LDL (50 μg/mL) for 24 hours in presence or absence of p44/42mapk inhibitor, PD98059 (40 μmol/L) for 45minutes. Equal amounts of proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and immunoblotted with an antibody against collagen I (1:1000) and actin (1:1000). Bar graph represents intensities of collagen I bands relative to actin expressed as percentage above control. Blots shown are representative of seven experiments. *P < 0.05 vs. control. (B) Quiescent mesangial cells were stimulated by LDL (50 μg/mL) for 24 hours in presence or absence of p38mapk inhibitor, SB203580 (10 μmol/L) for 45minutes. Blots shown are representative of four experiments. *P < 0.05 vs. control. (C) Quiescent mesangial cells were stimulated by LDL (50 μg/mL) for 24 hours in presence or absence of c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125 (30 μmol/L) for 45minutes. Blots shown are representative of eight experiments. *P < 0.05 vs. control; #P < 0.0001 vs. LDL. Kidney International 2005 67, 1286-1296DOI: (10.1111/j.1523-1755.2005.00206.x) Copyright © 2005 International Society of Nephrology Terms and Conditions