Immunohistochemistry of PRR in rat aorta.

<p><b>A</b>. Immonohistochemical localization of PRR in the aortas of normal, CKD and AST-120-treated CKD rats. <b>B</b>. Quantitative data of PRR in the aortas of normal (n = 9), CKD (n = 8) and AST-120-treated CKD rats (n = 8) (mean±SE). ***p<0.001 vs normal, ##p<0.001 vs CKD. <b>C</b>. Immonohistochemical localization of PRR in the aortas of DN, DN+IS, DH and DH+IS rats. <b>D</b>. Quantitative data of PRR-positive area in the aorta of DN, DN+IS, DH and DH+IS rats (mean±SE, n = 8). ***p<0.001vs DN, #p<0.05 vs DH.</p

ROS, OAT3, AhR and NF-κB p65 are involved in IS-induced PRR expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs were pretreated with NAC (2.5 mmol/L) and DPI (10 µmol/L) for 30 min before incubation with IS (250 µmol/L) for 24 h (A,B). HASMCs were transfected with or without OAT3 siRNA (10 nmol/L), AhR siRNA (30 nmol/L) or p65 siRNA (10 nmol/L), and serum starved for 24 h, followed by incubation with IS (250 µmol/L) for 24 h. Cell lysates were immunoblotted using anti-OAT3, anti-AhR, anti-p65 and anti-PRR antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05 vs control, #p<0.05 vs IS-treated group. Ctrl: control.</p

IS induces PRR expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs were treated with IS (250 µmol/L). Incubation with IS increased PRR mRNA and protein expression in HASMCs time- (<b>A</b>, <b>C</b>) and dose- (<b>B</b>, <b>D</b>) dependently. Mean±SE (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control.</p

ROS, OAT3, AhR, and NF-κB p65 are involved in IS-induced prorenin expression in vascular smooth muscle cells.

<p>Serum starved HASMCs were pretreated with NAC (2.5 mmol/L) and DPI (10 µmol/L) for 30 min before incubation with IS (250 µmol/L) for 24 h (<b>A, B</b>). HASMCs were transfected with or without OAT3 siRNA (10 nmol/L), AhR siRNA (30 nmol/L) or p65 siRNA (10 nmol/L), and serum starved for 24 h, followed by incubation with IS (250 µmol/L) for 24 h. Cell lysates were immunoblotted using anti-OAT3, anti-AhR, anti-p65 and anti-prorenin antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05, **p<0.01 vs untreated group, #p<0.01 vs IS-treated group. Ctrl: control.</p

Immunohistochemistry of renin/prorenin in rat aorta.

<p><b>A</b>. Immonohistochemical localization of renin/prorenin in the aortas of normal, CKD and AST-120-treated CKD rats. <b>B</b>. Quantitative data of renin/prorenin-positive area in the aortas of normal (n = 9), CKD (n = 8) and AST-120-treated CKD rats (n = 8) (Mean±SE). ***p<0.001 vs normal, #p<0.05 vs CKD. <b>C</b>. Immonohistochemical localization of renin/prorenin in the aortas of DN, DN+IS, DH and DH+IS rats. <b>D</b>. Quantitative data of renin/prorenin-positive area in the aortas of DN, DN+IS, DH and DH+IS rats (mean±SE, n = 8). ***p<0.001vs DN, ##p<0.01 vs DH.</p

IS-induced PRR activation is involved in cell proliferation and tissue factor expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs (5×10³ cells/well) in a 24-well plate were stimulated with or without IS (250 µmol/L) or prorenin (20 nmol/L) for 24 h (<b>A, B</b>). HASMCs were transfected with siPRR (20 nmol/L) for 48 h, before stimulation with IS or prorenin for 24 h. Thereafter, the cell proliferation reagent MTS (50 µL) was added to each well, and cells were incubated for 4 h. The absorbance was measured at 492 nm using a microplate reader (mean±SE, n = 3). **p<0.01 vs untreated group, #p<0.01 vs IS-treated group. HASMCs were transfected with or without PRR siRNA (20 nmol/L), and then serum starved for 24 h, followed by incubation with IS (250 µmol/L) or prorenin (20 nmol/L) for 24 h. Cell lysates were immunoblotted using anti-PRR and anti-tissue factor antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05, **p<0.01 vs untreated group, #p<0.05 vs IS-treated group. Ctrl: control.</p