Role of P311 in interleukin-1α-induced epithelial to myofibroblast transition in kidney tubular epithelial cells

Abstract

Tubular epithelial-myofibroblast transition (TEMT) is an important process in renal tubulointerstitial fibrosis. Interleukin-1α (IL-1α) and transforming growth factor-β1 (TGF-β1) have been demonstrated to be key inducers of TEMT. In mouse embryonic fibroblast cells (NIH3T3), P311 protein induces phenotypic changes that are consistent to myofibroblast transformation. In the present study, we investigated the role of P311 gene and protein as well as potential mechanisms underlying TEMT in normal rat kidney tubular epithelial cells (NRK52E). Morphological and molecular changes were determined in NRK52E cells that were treated with IL-1α and/or P311 antibodies. The results showed that the NRK52E cells triggered by IL-1α became fibroblast-like cells, exhibiting hypertrophy of elongated and fusiform-shaped cells. IL-1α induced a time-dependent increase in P311 gene expression in NRK52E cells, with a peak time at 4 days. The expression levels of P311 gene were positively correlated with α-SMA and TGF-β1 gene expression levels. Anti-P311 antibody inhibited P311 and α-SMA expression in the presence of IL-1α. In contrast, anti-P311 antibody increased the expression of TGF-β1 gene in cells cultured with IL-1α. Therefore, P311 gene, together with α-SMA and TGF-β1 genes, was induced in the process of TEMT. P311 protein triggered by interleukin-1α may promote TEMT through a TGF-β1-independent pathway.     

Effect and mechanism of fluvastatin on the expression of fibronectin in human peritoneal mesothelial cells induced by high?glucose peritoneal dialysate

Objective To explore the effect and mechanism of fluvastatin on the expression of fibronectin(FN) in human peritoneal mesothelial cells (HPMCs) induced by high?glucose peritoneal dialysate (HGPDS). Methods Cultured HPMCs were randomly divided into control, HGPDS, HGPDS plus GSK650394 10-5 mol/L (the competitive inhibitor of SGK1), different concentrations of fluvastatin, fluvastatin 10-6 mol/L and GSK650394 10-5 mol/L alone. The morphology change of HPMC was observed by light microscopy. The cellular viability was detected by MTT colorimetry. The mRNA and protein expressions of serum and glucocorticoid?inducible kinase 1 (SGK1) and FN were detected by RT?PCR, Western blotting or ELISA. Results After incubation with HGPDS, the cell morphology changed from typical cobblestone?like appearance to fibroblast?like appearance, and the cell viability was inhibited significantly (P<0.05). Fluvastatin 10?6mol/L and GSK650394 could improved the cell morphology and the cell viability injured by HGPDS (P<0.05). Compared with the normal control group, the mRNA and protein expressions of SGK1 and FN increased significantly in HPMC treated with HGPDS(P<0.05). GSK650394 significantly decreased the high expression of SGK1 and FN (P<0.05), also the fluvastatin had same effects as GSK650394 in dose?dependent manner (P<0.05). Conclusions High?glucose peritoneal dialysate can increase FN expression in human peritoneal mesothelial cells, which can be attenuated by fluvastatin. The protective role of fluvastatin in HPMC may be partially achieved through the signal pathway of SGK1.     

Effect of triptolide on expression of thrombospondin-1 and transforming growth factor-β1 in renal tubular cells

Abstract

The objective of our study is to investigate the effect of triptolide on expression of thrombospondin-1 and transforming growth factor β1 in renal tubular cells. Human renal tubular epithelial cells were stimulated by different concentrations of triptolide (0.1, 1, and 10?μg/L) in the presence of angiotensin-II (10^?7?mol/L). Real Time PCR was used to detect the mRNA expression of thrombospondin-1 and transforming growth factor β1. Western blot analysis was used to detect the protein expression. ELISA was used to detect the level of total and active transforming growth factor β1. The mRNA expression of thrombospondin-1 (3.66?±?0.48 vs. 1.33?±?0.26, p?<?0.05) and transforming growth factor β1 (3.58?±?0.59 vs. 1.26?±?0.28, p?<?0.05) were up-regulated obviously when stimulated by angiotensin-II. And the protein expression of thrombospondin-1 (0.5126?±?0.0936 vs. 0.1025?±?0.0761, p?<?0.01) and transforming growth factor β1 (0.5948?±?0.0736 vs. 0.1318?±?0.0614, p?<?0.01) were also up-regulated simultaneously when stimulated by angiotensin-II. The expression of thrombospondin-1 and transforming growth factor β1 induced by angiotensin-II were down-regulated markedly with 1?μg/L and 10?μg/L of triptolide in mRNA and protein levels (p?<?0.05, p?< 0.01). And triptolide (1 and 10?μg/L) could reduce the expression of total and active transforming growth factor β1 (p?<?0.05, p?<?0.01). In conclusion, triptolide can inhibit the expression of thrombospondin-1 and transforming growth factor β1 in mRNA and protein levels and down-regulate the levels of total and active transforming growth factor β1.     

Connective tissue growth factor mediates the profibrotic effects of transforming growth factor-β produced by tubular epithelial cells in response to high glucose

Background It was reported that connective tissue growth factor (CTGF) was expressed in the tubular epithelial cells of the diabetic kidney. CTGF has, among other factors, been implicated in mediating the downstream, profibrotic effects of transforming growth factor-β (TGF-β), though is precise role in interstitial fibrogenesis in the diabetic kidney has not yet been clarified.Methods We employed a coculture system involving cultured murine proximal tubular epithelial cells (mProx24) and renal fibroblasts (TFB), as a model of the subepithelial mesenchyme in the kidney in order to examine the profibrotic effects of CTGF derived from mProx24 cells in response to high glucose (30 mM).Results We showed that glucose stimulated CTGF expression in cultured mProx24 in both a dose- and a time-dependent manner, and that this effect was mediated by increased levels of TGF-β. We also found that high glucose significantly stimulated TFB cells to produce profibrotic molecules, such as type I collagen, the EIIIA isoform of fibronectin, and plasminogen activator inhibitor-1. The induction of these molecules was both direct and indirect, the latter induction being mediated by mProx24 cell-derived CTGF, which, in turn, was induced by TGF-β that was produced by the mProx24 cells.Conclusions CTGF plays an important role in mediating renal interstitial fibrogenesis in response to high glucose and, as such, is a reasonable target for anti-fibrotic therapy.     

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

Objective To investigate the role of tet methylcytosine dioxygenase 2 (TET2) in the regulation of transforming growth factor-β1 (TGF-β1) expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group (5.5 mmol/L glucose) and high glucose group (30.0 mmol/L glucose) which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group (30.0 mmol/L glucose+DMEM), DMSO group (30.0 mmol/L glucose+0.1%DMSO) and SC1 group (30.0 mmol/L glucose+3 μmol/L SC1). The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin (α-SMA) was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF-β1 was detected by bisulfite sequencing PCR (BSP). The activity of mesangial cell proliferation was assessed by colorimetry of thiazolyl blue (MTT). Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time-dependent manner (all P＜0.05), but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 (P＜0.01), but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently (P＜0.05), the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased (all P＜0.05). Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.     

β-Adrenoceptors regulate matrix metalloproteinase expression in human urothelial cells under hydrostatic pressure

The bladder wall is constantly subjected to intravesical pressure during the filling and voiding cycles. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) under elevated intravesical pressure contributes to pathological changes in the bladder. To investigate the changes in human urothelial cells (HUCs) under elevated intravesical pressure, this study analyzed the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs exposed to pathological hydrostatic pressure (HP) (70 cm H₂O) for 6 hours. Quantitative polymerase chain reaction, Western blot analysis, and cell fluorescence staining were used to explore the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs after agonist and/or antagonist treatment. The expression levels of β₂- and β₃-adrenoceptor, MMP1, and MMP2 were greatly downregulated, while the expression of TIMP1 was greatly upregulated. Formoterol and BRL 37344, which are agonists of β₂- and β₃-adrenoceptor, respectively, significantly increased MMP1 and MMP2 expression under 70 cm H₂O. As a classic downstream pathway of β₂- and β₃-adrenoceptor, protein kinase A (PKA) signaling inhibited MMP1 and MMP2 expression by regulating cAMP response element binding protein (CREB) activity. MMP1 and MMP2 expression in HUCs under 70 cm H₂O was modified by β₂- and β₃-adrenoceptor via the PKA/CREB pathway. This outcome suggests that MMPs likely participate in the pathological effects of elevated intravesical pressure. The underlying mechanism of β₂- and β₃-adrenoceptor in elevated intravesical pressure was also revealed; this mechanism constitutes a new potential therapeutic target for partial bladder outlet obstruction.     

Effect of human neural progenitor cells on injured spinal cord

Human central nervous system ( CNS) has verylimited regenerative potentials. Patients withsevere injuries in the CNS such as spinal cordinjury (SCI) frequently endure lifelong disability. Avariety of methods have been tried to prevent spinalcord from further injury and foster regeneration afterSCI. Despite these efforts, an effective treatment forthis disease is still lacking. Since neural progenitorcells have already committed to become neural cells inthe CNS, they appear to be a good c…     

Effect of propofoi on LPS-induced TLR4 expression in rat alveolar type Ⅱ epithelial cells

Objective To investigate the effect of propofol on LPS-induced TLR4 expression in rat alveolar type Ⅱ epithelial cells. Methods The primarily cultured alveolar type Ⅱ epithelial cells isolated from male rats were randomly assigned to one of 5 groups: group Ⅰ cells were incubated for 3 h without any additive (control) ; group Ⅱ cells were incubated with LPS 1 μg/ml for 3 h (LPS) ; group Ⅲ , Ⅳ , Ⅴ cells were incubated with LPS 1 μg/ml + propofol 25, 50 and 100 μmol/L respectively for 3 b (P1-3). TLR4 mRNA and TLR4 protein expression was detected by real time PCR and Western blot. TNF-α release amount was measured using ELISA. Results LPS significantly' increased TLR4 mRNA and protein expression in alveolar type Ⅱ epithelial cells as well as TNF-α release amount. Propefol at 50 and 100 μmol/L significantly inhibited LPS-imluced increase in TLR4 mRNA and protein expression and TNF-α release amount. Conclusion Propofol can dose-dependently inhibit LPS-induced inflammation in alveolar type Ⅱ epithelial ceils, through down-regnlation of TLR4 gene and protein expression.     

Possible roles of tumor necrosis factor-α and angiotensin II type 1 receptor on high glucose-induced damage in renal proximal tubular cells

Abstract

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22^phox protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30?mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22^phox protein levels compared with those in regular glucose medium (5.6?mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22^phox protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22^phox protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22^phox protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.     

Effect of endothelin-1 on testosterone production by rat Leydig cells in vitro

The effect of endothelin-1 on testosterone production by ratLeydig cells in vitro was studied. Results indicated that 10~(-9)-l0~(-10) mol/L of endothelin-1 significantly ( P<0.05) inhibit the     

Effect of 635 nm irradiation on high glucose-boosted inflammatory responses in LPS-induced MC3T3-E1 cells

Hyperglycemia occurs in patients with poorly controlled diabetes mellitus and contributes to bone resorption and increased susceptibility to bacterial infections. Hyperglycemia can incite low-grade inflammation that can contribute to the resorption of bone, especially the periodontal bone. The increased susceptibility to periodontal infections can contribute to bone resorption through the activation of osteoclasts. In this study, the osteoblastic, clonal cell line, MC3T3-E1, was used in an in vitro model of hyperglycemia and lipopolysaccharide-induced reactive oxygen species generation to determine the potential anti-inflammatory effect of 635 nm light-emitting diode (LED) irradiation or whether 635 nm LED irradiation can be a potential anti-inflammatory treatment. LED irradiation of MC3T3-E1 cells stimulated with lipopolysaccharide in a high glucose-containing medium decreased the level of cyclooxygenase gene and protein expression and reduced the level of prostaglandin E2 expression by decreasing the amount of reactive oxygen species generation. LED irradiation also inhibited the osteoclastogenesis in MC3T3-E1 cells by regulating the receptor activator of nuclear factor kappa-B ligand and osteoprotegerin. These findings reveal the mechanisms which are important in the pathogenesis of diabetic periodontitis and highlight the beneficial effects of 635 nm LED irradiation in reducing the adverse effects of diabetic periodontitis.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.     

Effect of intercellular adhesion molecule-1 and hyaluronic acid on monocyte-renal tubular epithelial cell adhesion induced by tumor necrosis factor α

Objective To investigate the effect of tumor necrosis factor ot (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line (HK2 cells)and monocyte cell line (U937)were used to perform cell co-culture. TNF-α (10 μg/L)was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1 (ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P<0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CDI8 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.