Biochemical data of the animals at the end of the study.

<p>Data were cited from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091517#pone.0091517-Bolati1" target="_blank">[11]</a> for animal study 1 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091517#pone.0091517-Adijiang1" target="_blank">[12]</a> for animal study 2.</p><p>Data are expressed as mean±SE.</p><p>Abbreviation: CKD, chronic kidney disease; DN, Dahl normotensive rats; DH, Dahl hypertensive rats; IS, indoxyl sulfate.</p>¶<p>p<0.05 <i>vs</i>. normal, ^ψp<0.05 <i>vs</i>. CKD.</p><p>*p<0.05 <i>vs.</i> DN, ^#p<0.05 <i>vs.</i> DH.</p

Effect of Ang-(1–7) on TGF-β1 expression in IS-treated HK2 cells.

<p>IS enhanced the expression of TGF-β1, whereas Ang-(1–7) inhibited it. 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 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

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

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

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

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 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

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

IS induces prorenin expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs were treated with IS (250 µmol/L). Incubation with IS increased prorenin 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 vs control.</p