Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-β1 (TGF-β1) expression.Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-β1 in proximal tubular cells

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

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

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

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

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