Indoxyl sulfate-induced activation of (pro)renin receptor promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs.IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells

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

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 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

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

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