Maimaiti Yisireyili

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

BackgroundIndoxyl 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.MethodsEffects 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.ResultsIndoxyl 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.ConclusionsIndoxyl 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.

Indoxyl sulfate promotes cardiac fibrosis with enhanced oxidative stress in hypertensive rats.

AIMS Cardiovascular disease (CVD) is common in chronic kidney disease (CKD) patients. Indoxyl sulfate (IS) is a nephrovascular uremic toxin that induces oxidative stress in kidney and vascular system. The present study aimed to examine the effect of IS on fibrosis and oxidative stress in rat heart. MAIN METHODS The effects of IS on heart were examined by Massons trichrome (MT) staining and immunohistochemistry using: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive IS-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive IS-administered rats (DH+IS). KEY FINDINGS DH+IS rats showed significantly increased heart weight and left ventricle weight compared with DN. DH and DH+IS rats showed significantly increased diameter of cardiomyocytes, increased MT-positive fibrotic area, increased staining for transforming growth factor (TGF)-β1, α-smooth muscle actin (SMA), type 1 collagen, NADPH oxidase Nox 4, malondialdehyde (MDA), and 8-hydroxydeoxyguanosine (8-OHdG) and decreased staining for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the heart compared with DN. More notably, DH+IS rats showed significantly increased diameter of cardiomyocytes, increased fibrotic area, increased staining for TGF-β1, SMA, type 1 collagen, Nox4, 8-OHdG and MDA, and decreased staining for Nrf2 and HO-1 in the heart compared with DH. SIGNIFICANCE IS aggravates cardiac fibrosis and cardiomyocyte hypertrophy with enhanced oxidative stress and reduced anti-oxidative defense in hypertensive rats.

Stat3 contributes to indoxyl sulfate-induced inflammatory and fibrotic gene expression and cellular senescence.

Background/Aim: Increased phosphorylation (activation) of signal transducer and activator of transcription 3 (Stat3) on tyrosine 705 leads to renal fibrosis. Indoxyl sulfate, a uremic toxin, induces renal fibrosis through expression of transforming growth factor-β1 (TGF-β1) in proximal tubular cells. The present study aimed to determine whether Stat3 is involved in indoxyl sulfate-induced dysfunction of proximal tubular cells. Methods: Localization of phosphorylated Stat3 in the kidneys of normal, subtotally nephrectomized, and AST-120-treated subtotally nephrectomized rats was examined by immunohistochemistry. The effect of indoxyl sulfate on phosphorylation of Stat3 and the role of Stat3 on indoxyl sulfate-induced cellular effects were examined using human proximal tubular cells (HK-2 cells). Results: Subtotally nephrectomized rats showed increased immunostaining of phosphorylated Stat3 in the renal tubules compared with normal rats. Administration of AST-120, which reduces serum level of indoxyl sulfate, to subtotally nephrectomized rats reduced the immunostaining of phosphorylated Stat3 in the renal tubules. Indoxyl sulfate induced phosphorylation of Stat3 in HK-2 cells. Stat3 small interfering RNA suppressed indoxyl sulfate-induced expression of an inflammation marker gene (monocyte chemotactic protein-1), fibrosis marker genes (TGF-β1, α-smooth muscle actin) and a subunit of nuclear factor-ĸB (p65), and attenuated a cellular senescence marker, senescence-associated β-galactosidase activity. Conclusions: Stat3 is involved in indoxyl sulfate-induced inflammatory and fibrotic gene expression and cellular senescence 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. Conclusion 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.

Indoxyl sulfate enhances p53-TGF-β1-Smad3 pathway in proximal tubular cells.

Background/Aim: Indoxyl sulfate-induced activation of nuclear factor (NF)-ĸB promotes transforming growth factor (TGF)-β1 in human proximal tubular cells (HK-2 cells). The present study aimed to elucidate the cross talk among indoxyl sulfate, p53 and TGF-β1-Smad3 signaling in proximal tubular cells. Methods: The effects of indoxyl sulfate on the expression of TGF-β1, Smad3, and α-smooth muscle actin (α-SMA) were determined using HK-2 cells. As for in vivo experiments the following animals were used: Dahl salt-resistant normotensive rats (DN) and indoxyl sulfate-administered Dahl salt-resistant normotensive rats (DN+IS). Results: Both indoxyl sulfate and nutlin-3, a specific p53 inducer, stimulated TGF-β1 expression, which was suppressed by pifithrin-α, p-nitro, a p53 inhibitor. Further, indoxyl sulfate stimulated TGF-β1-induced expression of α-SMA by enhancing Smad3 expression and TGF-β1-induced Smad3 phosphorylation. Indoxyl sulfate induced phosphorylation of extracellular signal-regulated kinase (ERK). U0126, an inhibitor of ERK pathway, prevented indoxyl sulfate-induced upregulation of Smad3 expression. Immunohistochemistry demonstrated that TGF-β1 and Smad3 were localized in renal tubular cells, and that indoxyl sulfate increased the TGF-β1 and Smad3-positive area in the kidney. Conclusion: Indoxyl sulfate stimulates p53-induced TGF-β1 expression and TGF-β1-induced α-SMA expression in proximal tubular cells. Indoxyl sulfate-induced Smad3 accelerates TGF-β1-induced α-SMA expression through ERK activation. Thus, indoxyl sulfate enhances p53-TGF-β1-Smad3 pathway in proximal tubular cells.

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. Conclusion 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 Is Involved in Expression of TGF-β1 and α-Smooth Muscle Actin in Proximal Tubular Cells

Activation of (pro)renin receptor (PRR) is involved in the progression of chronic kidney disease. However, the role of indoxyl sulfate, a uremic toxin, in the activation of PRR is not clear. The present study aimed to clarify the role of indoxyl sulfate in activation of PRR, in relation to renal expression of fibrotic genes. Renal expression of PRR and renin/prorenin was up-regulated in chronic kidney disease rats compared with normal rats, whereas AST-120 suppressed these expression by reducing serum levels of indoxyl sulfate. Furthermore, administration of indoxyl sulfate to normotensive and hypertensive rats increased renal expression of PRR and renin/prorenin. Indoxyl sulfate induced expression of PRR and prorenin in cultured human proximal tubular cells (HK-2 cells). Indoxyl sulfate-induced PRR expression was inhibited by small interfering RNAs of signal transducer and activator of transcription 3 (Stat3) and nuclear factor-κB p65 in proximal tubular cells. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed indoxyl sulfate-induced PRR expression in proximal tubular cells. N-acetylcysteine prevented indoxyl sulfate-induced phosphorylation of Stat3 in proximal tubular cells. PRR small interfering RNA inhibited indoxyl sulfate-induced expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Taken together, indoxyl sulfate-induced up-regulation of prorenin expression and activation of PRR through production of reactive oxygen species and activation of Stat3 and nuclear factor-κB play an important role in the expression of TGF-β1 and α-smooth muscle actin in proximal tubular cells. Thus, indoxyl sulfate-induced activation of prorenin/PRR might be involved in renal fibrosis.

Indoxyl Sulfate Induces IL-6 Expression in Vascular Endothelial and Smooth Muscle Cells through OAT3-Mediated Uptake and Activation of AhR/NF-κB Pathway

Background/Aims: Interleukin-6 (IL-6) is one of the inflammation biomarkers with highest predictive value for outcome in chronic kidney disease (CKD) patients. The present study aimed to determine the effects of indoxyl sulfate (IS) on IL-6 expression in vascular cells. Methods: IS was administered to normo- and hypertensive rats. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were incubated with or without IS. Results: Immunohistochemistry revealed that IS-administered rats showed increased expression of IL-6 in the aortic tissues. IS increased IL-6 expression in HUVECs and HASMCs in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3) using small interfering RNA (siRNA) inhibited IS-induced expression of IL-6 in HUVECs and HASMCs. IS induced activation of aryl hydrocarbon receptor (AhR) and nuclear factor-κB (NF-κB) subunit p65 in HUVECs and HASMCs. Both AhR siRNA and p65 siRNA inhibited IS-induced expression of IL-6. AhR siRNA inhibited IS-induced phosphorylation and nuclear translocation of p65 without change in total p65 level. However, p65 siRNA did not inhibit IS-induced nuclear translocation of AhR. Thus, AhR is responsible for IS-induced p65 signaling transduction. Conclusion: IS induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3/AhR/NF-κB pathway.

Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K−Akt/p-HDAC6 Signaling Pathway

Objective—Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. Approach and Results—Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor–induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. Conclusions—This is the first report detailing cross-interaction between toll-like receptor 2–mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

Xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice

Chronic stress is closely linked to the metabolic syndrome, diabetes, hyperuricemia and thromboembolism, but the mechanisms remain elusive. We reported recently that stress targets visceral adipose tissue (VAT), inducing lipolysis, low-grade inflammation with production of inflammatory adipokines, metabolic derangements such as insulin resistance, and prothrombotic state. In the present study, we hypothesized the involvement of VAT xanthine oxidoreductase (XOR), a source of reactive oxygen species (ROS) and uric acid (UA) in the above processes. Restraint stress in mice resulted in upregulation of XOR and xanthine oxidase activity, accumulation of ROS in VAT as well as liver and intestine, increase in serum UA levels, upregulation of NADPH oxidase subunits and downregulation of antioxidant enzymes. Immunohistochemistry and RT-PCR analysis also showed that restraint stress induced VAT monocyte accumulation and proinflammatory adipokine production, resulting in reduced insulin sensitivity and induction of plasminogen activator inhibitor-1 and tissue factor in VAT. Treatment with febuxostat, a potent XO inhibitor, suppressed stress-induced ROS production and VAT inflammation, resulting in improvement of serum UA levels, insulin sensitivity, and prothrombotic tendency. Our results suggest that stress perturbs glucose and UA metabolism, and promotes prothrombotic status, and that XO inhibition by febuxostat might be a potential therapy for stress-related disorders.