Renin-stimulated TGF-β1 expression is regulated by a mitogen-activated protein kinase in mesangial cells  Y. Huang, N.A. Noble, J. Zhang, C. Xu, W.A. Border  Kidney International  Volume 72, Issue 1, Pages 45-52 (July 2007) DOI: 10.1038/sj.ki.5002243 Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 1 Time course of intracellular MAP kinases ERK1/2 phosphorylation in mesangial cells treated with or without renin. (a) Quiescent rat mesangial cells were incubated in the absence (left blot) or presence of 10-8 M Renin (right blot) for the indicated times, and Western blotting was performed. (b) Densitometric analysis of Western blots as shown in (a). The extent of ERK1/2 phosphorylation was determined by normalizing phospho-ERK1/2 levels to the total ERK1/2. Values are expressed relative to the no-additive, zero-time control, which was set at unity. *P<0.05 compared to the untreated control. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 2 Effect of renin dose on ERK1/2 phosphorylation in mesangial cells. (a) Quiescent rat mesangial cells were incubated for 2.5 min in the presence of various concentrations of renin, and Western blotting was performed. (b) Graph shows the relative levels of intracellular phosphorylated ERK1/2. Values are expressed relative to the no-additive, control, which was set at unity. *P<0.05 compared to the control. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 3 Angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor antagonist does not affect renin-induced ERK1/2 phosphorylation in mesangial cells. (a) Quiescent mesangial cells were incubated with the angiotensin-converting enzyme inhibitor enalaprilate (the active form of enalapril) (Ena) at 10-5 M, or the angiotensin II type 1 receptor antagonist losartan (Los) at 10-5 M or vehicle control for 30 min before stimulation with 10-8 M renin for 15 min, and Western blotting was performed. (b) Graph shows the relative levels of intracellular phosphorylated ERK1/2. Values are expressed relative to the no-additive, control (Con), which was set at unity. *P<0.05 compared to the control. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 4 Effect of renin on the proliferation of mitogen-activated rat mesangial cells. (a) Quiescent rat mesangial cells were incubated with 10% FBS RPMI 1640 and increasing concentrations (0–10-8 M) of renin in serum-free RPMI 1640 for 24 h (n=8). (b) Quiescent rat mesangial cells were treated with or without 10-7 M of enalaprilate (Ena), or 10-7 M of losartant (Los) or 50 μM of U0126, in addition to 10-9 M of renin for 24 h (n=8). The administration of 10% FBS was used as the positive control. The proliferative response was evaluated by MTT assay. Results were expressed as the mean optical density of MTT absorbance in control and treated wells. *P<0.05 compared to the untreated control. #P<0.05 compared to the renin-alone-treated cells. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 5 Effect of Stealth™ siRNA of renin receptor on renin-induced cellular ERK1/2 phosphorylation in mesangial cells. (a) After pre-transfection with renin receptor Stealth™ siRNA or Lipofectamine™2000 for 48 h, mesangial cell expressions of renin receptor mRNA and pERK1/2 and total ERK1/2 were evaluated by Northern blotting and Western blotting, respectively followed by 15 min incubation with 10-8 M renin. (b) Renin receptor mRNA levels were standardized for densitometric analysis to GAPDH mRNA levels. (c) Graph shows the relative levels of intracellular phosphorylated ERK1/2. Densitometric values are expressed relative to the Lipofectamine™2000 (Lipo)-transfected, no renin added control, which was set at unity. *P<0.05 vs control, #P<0.05 vs Stealth™ siRNA-transfected cells plus renin. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 6 Effect of ERK inhibitor U0126 on cellular baseline or renin-induced cellular ERK1/2 phosphorylation in mesangial cells. Quiescent rat mesangial cells were incubated with various concentrations of U0126 (a) in the absence of renin or (b) in the presence of 10-8 M renin for 10 min, and Western blotting was performed. Graphs show the relative levels of intracellular phosphorylated ERK1/2 (below). Values are expressed relative to the no-additive control, which was set at unity. *P<0.05 compared to the untreated control. #P<0.05 vs renin-alone-treated cells. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 7 Renin-induced TGF-β1 mRNA expression is ERK dependent. Quiescent rat mesangial cells were co-incubated with 10-8 M renin and the ERK inhibitor U0126 (50 μM) for 2 h. TGF-β1 mRNA levels were determined by Northern blot analysis and standardized for densitometric analysis to GAPDH mRNA levels. Densitometric values are expressed relative to the no-additive control, which was set at unity. *P<0.05 compared to the untreated control. #P<0.05 vs renin-alone-treated cells. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 8 Renin-induced PAI-1 mRNA expression is ERK dependent. Quiescent rat mesangial cells were co-incubated with 10-8 M renin and the ERK inhibitor U0126 (50 μM) for 2 h. PAI-1mRNA levels were determined by Northern blot analysis and standardized for densitometric analysis to GAPDH mRNA levels. Densitometric values are expressed relative to the no-additive control, which was set at unity. *P<0.05 compared to the untreated control. #P<0.05 vs renin-alone-treated cells. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions

Figure 9 ERK inhibitor U0126 blocks renin-mediated TGF-β 1action on PAI-1 mRNA expression. Quiescent rat mesangial cells were co-incubated with 10-8 M renin, the neutralizing antibody, 1D11, and/or the ERK inhibitor U0126 (50 μM) for 2 h. PAI-1mRNA levels were determined by Northern blot and standardized for densitometric analysis to GAPDH mRNA levels. Densitometric values are expressed relative to the U0126 alone treated control, which was set at unity. *P<0.05 compared to the U0126-alone-treated control. #P<0.05 vs renin-alone-treated cells. Kidney International 2007 72, 45-52DOI: (10.1038/sj.ki.5002243) Copyright © 2007 International Society of Nephrology Terms and Conditions