Indoxyl Sulfate Downregulates Expression of Mas Receptor via OAT3/AhR/Stat3 Pathway in Proximal Tubular Cells

Abstract

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.ConclusionMas 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 Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells

“…Furthermore, IS promotes dysregulation of oxidative stress [26], providing another explanation for IS-induced smooth muscle cell proliferation. The activation of Mas receptor induces endothelial nitric oxide synthase [14] and nitric oxide production. Ang-(1-7) suppresses the oxidative stress via activation of Mas receptor in cells [27].…”

“…Immunohistochemistry was performed using the streptavidin-biotin complex method, as described previously [14]. Paraffin-embedded fixed tissue sections (4 μm) were deparaffinized with xylene and dehydrated with ethanol.…”

“…Immunoblotting was performed, as described previously [14]. Cell lysates were fractionated by sodium dodecyl sulfate-polyaclylamide gel electrophoresis on polyacrylamide gels (8-12%), and proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Bedford, Mass., USA).…”

“…Previous study by the authors demonstrated that IS downregulated the Mas receptor expression in renal proximal tubular cells [14]. While ACE2 activity is increased in CKD patients [9], the effect of IS on ACE2/Mas receptor axis in vascular smooth muscle cells (VSMCs) is still unclear.…”

Indoxyl sulphate and kidney disease: Causes, consequences and interventions

“…The Mas receptor, a G‐protein coupled receptor, may also be involved in IS‐mediated regulation of the RAS. Activation of the Mas receptor was able to reduce IS‐induced TGF‐β1 expression, following treatment with Ang‐(1–7) (alternate cleavage products of Ang‐II) . IS was found to inhibit Mas receptor expression through Stat3‐associated dioxin receptor signalling, indicating that Mas receptor agonists may reduce IS‐induced TGF‐β expression .…”