Volume 62, Issue 2, Pages 412-421 (August 2002) Inhibition of IGF-I–induced Erk 1 and 2 activation and mitogenesis in mesangial cells by bradykinin  Celine Alric, Christiane Pecher, Eric Cellier, Joost P. Schanstra, Bruno Poirier, Jacques Chevalier, Jean-Loup Bascands, Jean-Pierre Girolami  Kidney International  Volume 62, Issue 2, Pages 412-421 (August 2002) DOI: 10.1046/j.1523-1755.2002.00475.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Dose- (A) and time-dependent (B) effect of bradykinin (BK) and pharmacological profile (C) of BK activation of Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated: (A) for 5 min in the presence of increasing concentrations of BK (0 to 1000 nmol/L); (B) in the presence of 100 nmol/L BK for different times (from 1 to 10 min); (C) in the presence of 100 nmol/L BK with either 1 μmol/L of the B2 antagonist HOE 140 (B2A) or 1 μmol/L of the B1 antagonist des-Arg9-Leu8-BK (B1A). Erk 1 and 2 phosphorylation (P-Erk) was measured by Western blotting with an antibody against the phosphorylated forms. Analyses were performed on an equal amount of protein (30 μg) as measured with an antibody against total-Erk (phosphorylated and non-phosphorylated forms). Erk 1 (□) and Erk 2 (▪) phosphorylation (P-Erk 1 and 2) was expressed by the fold increase of the ratio P-Erk versus total-Erk and is shown as mean ± SE of the scanning densitometry of five experiments. *P < 0.05; **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lane 13). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Effects of pertussis toxin (PTX) and phospholipase C inhibitor (U73122) on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated in the presence or absence of 100 nmol/L BK for 5 min and in the presence of PTX (lanes 3 and 4) or of 10 nmol/L U73122 (lanes 7 and 8). Western blotting was performed as described in the legend of Figure 1. Symbols are: (□) Erk 1; (▪) Erk 2. N = 5; *P < 0.05, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 2 and 6). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Effects of protein kinase C stimulation (A) and inhibition (B and C) on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated: (A) in the presence or absence of 100 nmol/L BK for 5 min either in the presence of PMA for 5 min (lanes 1 and 2); (B) in the presence of GF109203, a PKC inhibitor (lanes 6 and 7); and (C) after pretreatment with PMA for 18 hours (lanes 10 and 11). Western blotting was performed as described in the legend of Figure 1. Symbols are: (□) Erk 1; (▪) Erk 2. N = 5, *P < 0.05, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 5 and 9). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Effect of tyrosine kinase and src kinase inhibition on BK-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated in the presence or absence of 100 nmol/L BK for 5 min and PPI, an inhibitor of the src family protein (lanes 3 and 4) or PD090059, an Erk 1 and 2 kinase (MEK) inhibitor (lanes 7 and 8). Western blotting was performed as described in the legend of Figure 1. Symbols are: (□) Erk 1; (▪) Erk 2. N = 5, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 100 nmol/L BK (lanes 2 and 6). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 Effects of BK on IGF-I-stimulated Erk 1 and 2 phosphorylation. Serum-depleted rat MCs were incubated for 5 min: (A) in the presence of increasing concentrations of IGF-I from 0.1 to 10 nmol/L (lanes 2 to 4); (B) in the presence of 10 nmol/L IGF-I and increasing concentrations of BK from 0.1 to 10 nmol/L (lanes 6 to 8); (C) in the presence of 10 nmol/L IGF-I and increasing concentrations of BK from 0.1 to 10 nmol/L and 1 μmol/L of the B2 antagonist HOE 140 (lanes 9 to 12). Western blotting was performed as described in the legend of Figure 1. Symbols are: (□) Erk 1; (▪) Erk 2. N = 5, *P < 0.05, **P < 0.01 P < 0.01 when compared to the level of P-Erk in the absence of BK (lane1). °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 10 nmol/L IGF-I (lane 5). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lanes 7 and 8). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 Effects of orthovanadate, okadoic acid and BAPTA/AM on BK-induced reduction of Erk 1 and 2 phosphorylation stimulated with IGF-I. Serum-depleted rat MCs were incubated in the presence or absence of IGF-I for 5 min (lanes 2 to 8 and 10 to 13), 100 nmol/L BK (lanes 3, 5, 7,8, 11 and 13), okadoic acid, an inhibitor of serine phosphatase (lanes 4 and 5), orthovanadate, an inhibitor of tyrosine phosphatase (lanes 6 to 8), and BAPTA/AM, a chelator of intracellular calcium (lanes 11 and 13). Western blotting was performed as described in the legend of Figure 1. N = 5, **P < 0.01 when compared to the level of P-Erk in the absence of BK. °°P < 0.01 when compared to maximal P-Erk level detected in MCs stimulated with 10 nmol/L IGF-I (lanes 2 and 4). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lanes 3 and 11). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 7 Effects BK on MC proliferation. MCs were plated at a density of 40,000 cells per well and were stimulated with increasing concentrations of BK for 48 hours in two different conditions. (A) MCs cultured in RPMI medium containing 0.5% FCS. (B) MCs cultured in RPMI medium containing 10 nmol/L IGF-I. Cell proliferation is shown as percent of control values obtained in the absence of BK and is expressed as mean ± SE of five experiments. *P < 0.01, **P < 0.01 when compared to proliferation in the absence of BK. Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 8 Time-dependent effect of bradykinin (BK), IGF-I and insulin on Erk 1 and 2 phosphorylation in isolated glomeruli. Isolated glomeruli were incubated in the presence of: (A) 100 nmol/L BK; (B) 65 nmol/L IGF-I; (C) 1 μmol/L insulin for increasing times from 0.5 to 20 min; (D) in the presence of 65 nmol/L IGF-I, 100 nmol/L BK and orthovanadate (O-vanadate). Western blotting was performed as described in the legend of Figure 1. Symbols are: (□) Erk 1; (▪) Erk 2. N = 5, *P < 0.05; **P < 0.01 when compared to the level of P-Erk in the absence of growth factor. °°P < 0.01 when compared to maximal P-Erk level detected in glomeruli stimulated with 10 nmol/L IGF-I (lane 21). ++P < 0.01 when compared to the level of P-Erk in the presence of IGF-I and BK (lane 22). Kidney International 2002 62, 412-421DOI: (10.1046/j.1523-1755.2002.00475.x) Copyright © 2002 International Society of Nephrology Terms and Conditions