TMPRSS4 promotes cancer stem cell traits by regulating CLDN1 in hepatocellular carcinoma.

Encouraging advances in the treatment of hepatocellular carcinoma(HCC) have been achieved; however, a considerable part of patients still relapse or metastasize after therapy, and the underlying mechanisms have not been clarified yet. Here, we found that CLDN1 was markedly up-regulated in HCC tissues, and correlated with poor prognosis. Overexpression of CLDN1 dramatically promoted the capability of tumorsphere formation and cancer stem cell (CSC) traits. Furthermore, we found that TMPRSS4 was up-regulated in HCC tissues and there was a positive correlation between TMPRSS4 and CLDN1. In addition, the expression of CLDN1 was regulated by TMPRSS4. Moreover, TMPRSS4 mediated CSC properties and up-regulated CLDN1 by activating ERK1/2 signaling pathway. Taken together, our results revealed that CLDN1 contributed to CSC features of HCC, which was altered by TMPRSS4 expression via ERK1/2 signaling pathway, providing promising targets for novel specific therapies.

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.

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells.

AIMS: Monocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1. MAIN METHODS: The effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and 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). KEY FINDINGS: DN+IS, DH, and DH+IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH+IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells. SIGNIFICANCE: Indoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.

Indoxyl sulfate induces endothelial cell senescence by increasing reactive oxygen species production and p53 activity.

BACKGROUND/AIM: We have reported that indoxyl sulfate (IS), a uremic toxin, accelerates proximal tubular cell senescence. Asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, has been reported to induce endothelial cell senescence. This study aimed to determine whether IS induces endothelial cell senescence in comparison with ADMA, and to investigate its molecular mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with IS (250 µM) and/or ADMA (10 µM). These concentrations were comparable with their mean serum levels in hemodialysis patients. Cell senescence was evaluated by measuring senescence-associated beta-galactosidase (SA-ß-gal) activity. N-acetylcysteine, an antioxidant, and pifithrin alpha p-nitro, a p53 inhibitor, were used to determine the role of reactive oxygen species (ROS) and p53 in the induction of cell senescence. RESULTS: Both IS and ADMA significantly increased SA-ß-gal activity in HUVECs. Further, some additional increase in SA-ß-gal activity was observed when IS and ADMA were co-incubated. Preincubation of N-acetylcysteine or pifithrin alpha p-nitro significantly inhibited SA-ß-gal activity induced by IS and ADMA in HUVECs. Thus, both IS and ADMA induced endothelial senescence through ROS and p53. CONCLUSION: IS induces endothelial cell senescence by increasing ROS production and p53 activity, like ADMA.

AST-120 ameliorates epithelial-to-mesenchymal transition and interstitial fibrosis in the kidneys of chronic kidney disease rats.

OBJECTIVE: Indoxyl sulfate (IS), a uremic toxin, is a risk factor for progression of chronic kidney disease (CKD). AST-120 reduces serum IS and delays the progression of CKD. This study aimed to examine whether AST-120 inhibits epithelial-to-mesenchymal transition (EMT) in the kidneys of CKD rats. METHODS: CKD rats were produced by 5/6 nephrectomy and were divided into 2 groups: (1) CKD rats and (2) AST-120-treated CKD rats at a dosage of 4 g/kg body weight/day. After 10 weeks, their kidneys were excised for histological and immunohistochemical analysis. EMT was evaluated by immunohistochemistry of zonula occludens (ZO-1), an epithelial marker, and alpha-smooth muscle actin (α-SMA), a mesenchymal marker. Interstitial fibrosis was evaluated by Masson's trichrome staining. RESULTS: CKD rats showed reduced expression of ZO-1 and enhanced expression of α-SMA as compared with normal rats. Administration of AST-120 to CKD rats increased expression of ZO-1 and decreased expression of α-SMA as compared with CKD rats. Further, CKD rats showed enhanced extent of interstitial fibrosis as compared with normal rats, and administration of AST-120 to CKD rats ameliorated interstitial fibrosis. CKD rats showed increased serum level of IS as compared with normal rats, whereas administration of AST-120 to CKD rats decreased both serum and urine levels of IS. CONCLUSION: We conclude that AST-120 ameliorated EMT and interstitial fibrosis in the kidneys of CKD rats, probably by alleviating IS overload on the kidneys.

NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells.

We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated ß-galactosidase activity and expression of p53, transforming growth factor (TGF)-ß1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, ß-galactosidase, TGF-ß1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.

Indoxyl sulfate induces epithelial-to-mesenchymal transition in rat kidneys and human proximal tubular cells.

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin that accelerates the progression of chronic kidney disease (CKD). This study aimed to determine if IS induces epithelial-to-mesenchymal transition (EMT) in the kidneys of hypertensive rats and human proximal tubular cells (HK-2). METHODS: EMT was evaluated by immunohistochemistry, reverse transcription-polymerase chain reaction and immunoblotting of the epithelial markers E-cadherin and zonula occludens-1 (ZO-1), and the mesenchymal marker α-smooth muscle actin (α-SMA). Rat groups consisted of (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). HK-2 cells were incubated with or without IS. RESULTS: In kidneys, DH rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN rats. DN+IS and DH+IS rats showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA compared with DN and DH rats, respectively. DH+IS and DH rats showed increased Masson's trichrome-positive fibrosis areas compared with DH and DN, respectively. IS-treated HK-2 cells showed reduced expression of E-cadherin and ZO-1, and enhanced expression of α-SMA. CONCLUSION: IS induces EMT in the kidneys of hypertensive rats and in human proximal tubular cells.

Indoxyl sulfate downregulates renal expression of Klotho through production of ROS and activation of nuclear factor-ĸB.

BACKGROUND/AIM: Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease (CKD). The present study aimed to examine whether renal expression of Klotho is regulated by indoxyl sulfate, a uremic toxin, using rat kidneys and human proximal tubular cells (HK-2). METHODS: The effect of indoxyl sulfate on renal expression of Klotho was examined using (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). The effects of indoxyl sulfate, inhibitors of nuclear factor-κB (NF-κB) and an antioxidant on the expression of Klotho in HK-2 cells were examined. RESULTS: DH+IS and DN+IS rats showed decreased expression of Klotho mRNA in the kidneys as compared with DH and DN rats, respectively. Indoxyl sulfate suppressed the expression of Klotho mRNA and protein in HK-2 cells, whereas an antioxidant, N-acetylcysteine, and NF-κB inhibitors, pyrrolidine dithiocarbamate and isohelenin, alleviated these effects. CONCLUSIONS: Indoxyl sulfate downregulates Klotho expression in kidneys through production of reactive oxygen species and activation of NF-κB in proximal tubular cells. Indoxyl sulfate may be involved in reduced renal expression of Klotho in CKD.

Senescence and dysfunction of proximal tubular cells are associated with activated p53 expression by indoxyl sulfate.

Various uremic toxins accumulate in patients with chronic renal failure (CRF) and one of them is indoxyl sulfate, which accelerates the progression of CRF through unknown mechanisms. The present study investigates how indoxyl sulfate promotes CRF using the proximal tubular cell line HK-2 and CRF rats. Indoxyl sulfate inhibited serum-induced cell proliferation and promoted the activation of senescence-associated ß-galactosidase, a marker of cellular senescence, and the expression of α-smooth muscle actin (α-SMA), a marker of fibrosis, through inducing p53 expression and phosphorylation. Pifithrin-α, p-nitro, a p53 inhibitor, blocked these effects. Indoxyl sulfate evoked reactive oxygen species (ROS), and the antioxidant N-acetylcysteine inhibited indoxyl sulfate-induced p53 expression and phosphorylation, as well as indoxyl sulfate-induced α-SMA expression. We previously demonstrated that although cellular senescence and fibrosis are detectable in the kidneys of CRF rats, the oral adsorbent AST-120 repressed these effects. Here, we found that ß-galactosidase, p53 and α-SMA were expressed and colocalized in the renal tubules of CRF rats, whereas AST-120 decreased the expression of these genes. Taken together, these findings indicate that indoxyl sulfate induces the expression and phosphorylation of p53 though ROS production, thus inhibiting cell proliferation and promoting cellular senescence and renal fibrosis.