Indoxyl sulfate, a uremic toxin, downregulates renal expression of Nrf2 through activation of NF-κB

BACKGROUND: Indoxyl sulfate, a uremic toxin, is accumulated in the serum of chronic kidney disease (CKD) patients, accelerating the progression of CKD. In CKD rat kidney, the expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its related genes are downregulated. AST-120, an oral sorbent, reduces serum indoxyl sulfate and slows the progression of CKD. The present study aimed to determine whether indoxyl sulfate downregulates Nrf2 expression in human proximal tubular cells and rat kidneys and whether AST-120 upregulates Nrf2 expression in CKD rat kidneys. METHODS: Effects of indoxyl sulfate on expression of Nrf2 were determined using HK-2 cells as human proximal tubular cells and the following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). Further, AST-120 was administered to subtotally nephrectomized CKD rats to determine its effect on the expression of Nrf2. RESULTS: Indoxyl sulfate downregulated Nrf2 expression in HK-2 cells. The indoxyl sulfate-induced downregulation of Nrf2 expression was alleviated by an inhibitor of nuclear factor-κB (NF-κB) (pyrrolidine dithiocarbamate) and small interfering RNA specific to NF-κB p65. DN+IS, DH, and DH+IS rats showed decreased renal expression of Nrf2 and its downstream target genes, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), and increased renal expression of 8-hydroxydeoxyguanosine (8-OHdG), a marker of reactive oxygen species (ROS), compared with DN. Thus, indoxyl sulfate, as well as hypertension, downregulated renal expression of Nrf2 in rats. AST-120 upregulated renal expression of Nrf2, HO-1 and NQO1 and suppressed renal expression of 8-OHdG compared with control CKD rats. CONCLUSIONS: Indoxyl sulfate downregulates renal expression of Nrf2 through activation of NF-κB, followed by downregulation of HO-1 and NQO1 and increased production of ROS. Further, AST-120 upregulates renal expression of Nrf2 in CKD rats by removing serum indoxyl sulfate, followed by upregulation of HO-1 and NQO1 and decreased production of ROS

In vitro activity of AST-120 that suppresses indole signaling in Escherichia coli, which attenuates drug tolerance and virulence.

AST-120 (Kremezin) is used to treat progressive chronic kidney disease (CKD) by adsorbing uremic toxin precursors produced by gut microbiota, such as indole and phenols. In this study, we propose that AST-120 reduces indole level, consequently suppresses indole effects on induction of drug tolerance and virulence in Escherichia coli including enterohaemorrhagic strains. In experiments, AST-120 adsorbed both indole and tryptophan, a precursor of indole production, and led to decreased expression of acrD and mdtEF which encode drug efflux pumps, and elevated glpT, which encodes a transporter for fosfomycin uptake and increases susceptibility to aztreonam, rhodamine 6G, and fosfomycin. AST-120 also decreased the production of EspB, which contributes to pathogenicity of enterohaemorrhagic E. coli (EHEC). Aztreonam, ciprofloxacin, minocycline, trimethoprim, and sulfamethoxazole were also adsorbed by AST-120. However, fosfomycin, in addition to rifampicin, colistin and amikacin were not adsorbed, thus AST-120 can be used together with these drugs for therapy to treat infections. These results suggest another benefit of AST-120, i.e., that it assists antibacterial chemotherapy

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

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Cardiovascular death commonly occurs in patients with chronic kidney disease. Indoxyl sulfate (IS), a uremic toxin, has been demonstrated in vitro as a contributory factor in cardiac fibrosis, a typical pathological finding in uremic cardiomyopathy. This study aimed to determine if cardiac fibrosis is reversible by lowering serum IS levels using an oral charcoal adsorbent, AST-120. Subtotal-nephrectomized (5/6-STNx) Sprague-Dawley rats were randomized to receive either AST-120 (AST-120, n=13) or no treatment (vehicle, n=17) for 12 weeks. Sham operated rats (n=12) were used as controls. Early left ventricular (LV) diastolic dysfunction was demonstrated by an increase in peak velocity of atrial filling [A and A' waves] and a decrease of E/A and E'/A' ratios obtained by echocardiography. This was accompanied by a 4.5-fold increase in serum IS (p<0.001) as well as elevated tail-cuff blood pressure (p<0.001) and heart weight (p<0.001). Increased LV fibrosis (p<0.001), gene expression of pro-fibrotic (TGF-β, CTGF) and hypertrophic (ANP, β-MHC and α-skeletal muscle actin) markers, as well as TGF-β and phosphorylated NF-κB protein expression were observed in STNx + vehicle rats. Treatment with AST-120 reduced serum creatinine (by 54%, p<0.05) and urine total protein (by 27%, p<0.05) vs vehicle whilst having no effect on blood pressure (AST-120=227 ± 11 vs vehicle  =224 ± 8 mmHg, ns) and heart weight. The increase in serum IS was prevented with AST-120 (by 100%, p<0.001) which was accompanied by reduced LV fibrosis (68%, p<0.01) and TGF-β and phosphorylated NF-κB protein expression (back to sham levels, p<0.05) despite no significant change in LV function. In conclusion, STNx resulted in increased cardiac fibrosis and circulating IS levels. Reduction of IS with AST-120 normalizes cardiac fibrosis, in a blood pressure independent manner