Effects of OAT3 siRNA on Mas receptor and peNOS expression in HK-2 cells.

<p>HK-2 cells were treated with or without OAT3 siRNA (siOAT3, 10 nM) (A). Mas receptor (B) and peNOS (C) protein expression was abolished by knocking down OAT3. Data are means±SE, expressed as relative change in comparison with the basal value (n<i>≥3</i> for every experiment). *p<0.05 <i>vs.</i> control; <b>#</b>p<0.05 <i>vs.</i> IS.</p

Effects of Stat3 siRNA on Mas receptor and peNOS expression in HK-2 cells.

<p>HK-2 cells were treated with or without Stat3 siRNA (siStat3, 10 nM) (A). Knockdown of Stat3 inhibited IS-induced downregulation of Mas receptor (B) and peNOS (C) expression. Data are means±SE, expressed as relative change in comparison with the basal value (n<i>≥3</i> for every experiment). *p<0.05 <i>vs.</i> control; #p<0.05 <i>vs.</i> IS.</p

Immunohistochemical staining of Mas receptor in kidneys of CKD and Dahl rats.

<p>CKD rats showed significantly lower level of Mas receptor expression than normal. By administrating AST-120, Mas receptor expression was restored (A). The expression of Mas receptor was downregulated by IS in normotensive and hypertensive Dahl rats with normal renal function (B). The pictures were taken under ×400 magnification (n = 8 for each group). Data are expressed as mean±SE. ¶p<0.05 <i>vs.</i> normal; ψp<0.05 <i>vs.</i> CKD; *p<0.05 <i>vs.</i> DN; #p<0.05 <i>vs.</i> DH.</p

Time- and dose-dependent effects of IS on Mas receptor protein expression in HK-2 cells.

<p>Mas receptor in the HK-2 was reduced by IS in a time- (A) and dose- (B) dependent manner. Bars represent means±SE, expressed as relative change in comparison with the basal value (n<i>≥3</i> for every experiment).*p<0.05 <i>vs.</i> basal value; **p<0.001 <i>vs.</i> basal value.</p

Schema of mechanism of IS-induced Mas receptor downregulation.

<p>IS accumulates in HK-2 cells via OAT3. In the cells, IS acts as a ligand of AhR. The IS-AhR complex then interacts with Stat3. In turn, Mas receptor is downregulated by IS-AhR-Stat3 or IS-AhR complex. This figure was created using Servier Medical Art (<a href="http://www.servier.com" target="_blank">www.servier.com</a>).</p

Effects of NAC on Mas receptor and peNOS expression in HK-2 cells.

<p>NAC (5 mM) inhibited IS-induced downregulation of Mas receptor (A) and peNOS (B) expression. Data are means±SE, expressed as relative change in comparison with the basal value (n<i>≥3</i> for every experiment). *p<0.05 <i>vs.</i> control; #p<0.05 <i>vs.</i> IS.</p

Effects of AhR siRNA on Mas receptor and peNOS expression in HK-2 cells.

<p>HK-2 cells were treated with or without AhR siRNA (siAhR, 30 nM) (A). Knockdown of AhR inhibited IS-induced downregulation of Mas receptor (B) and peNOS (C) expression. Data are means±SE, expressed as relative change in comparison with the basal value (n<i>≥3</i> for every experiment). *p<0.05 <i>vs.</i> control; <b>#</b>p<0.05 <i>vs.</i> IS.</p

Renal tubular expression of kidney injury molecule-1 (KIM-1) and correlation with serum IS levels.

<p>Increased tubular expression of KIM-1 was observed in MI+Vehicle animals, AST-120 treatment significantly reduced KIM-1 expression to sham levels (A). Serum IS was significantly and positively correlated with KIM-1 expression (B, p&lt;0.01), *p&lt;0.05 vs Sham. ^##p&lt;0.01 vs MI+Vehicle.</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

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