OBJECTIVES: To investigate the possible role of mononuclear cells and their products in the pathogenesis of IgA nephropathy, in vitro expression of ICAM-1 on cultured mouse mesangial cell (MC) was examined after stimulation with mononuclear cell culture supernatant from patients with IgA nephropathy. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated and cultured from 18 patients with primary IgA nephropathy, 8 normal controls and 5 patients with non-IgA nephropathy (FSGS 1, MGN 3, MPGN 1). ICAM-1 expression on cultured mouse MC by TNF-alpha, IL-1 beta and culture supernants of PBMC were analyzed using a cell ELISA method. The concentration of IL-1 beta and TNF-alpha in culture supernatants was measured by using a commercially available radioimmunoassay kit. RESULTS: Addition of human recombinant TNF-alpha induced an increased ICAM-1 expression in a dose-dependent manner. The expression of ICAM-1 was further increased after co-stimulation with TNF-alpha and IL-1 beta. Addition of PBMC culture supernatants into mouse MC induced significantly higher expression of ICAM-1 by supernatants from the patients with IgA nephropathy compared with that from normal controls. The concentration of TNF-alpha and IL-1 beta in supernatants from the patients with IgA nephropathy was significantly higher than that from those with non-IgA nephropathy. CONCLUSION: TNF-alpha and IL-1 released from mononuclear cells induced the up-regulation of ICAM-1 expression and this may be related to the immune pathogenesis of IgA nephropathy.
Animal ; Cells, Cultured ; Glomerular Mesangium/immunology ; Glomerular Mesangium/cytology ; Glomerulonephritis, IGA/immunology* ; Glomerulonephritis, IGA/etiology ; Human ; Intercellular Adhesion Molecule-1/metabolism* ; Interleukin-1/secretion ; Interleukin-1/pharmacology ; Leukocytes, Mononuclear/immunology ; Mice ; Tumor Necrosis Factor/secretion ; Tumor Necrosis Factor/pharmacology

BACKGROUND: Transforming growth factor-beta (TGF-beta) stimulates renal fibrosis in various renal diseases including IgA nephropathy. METHODS: We examined whether immunoglobulin A (IgA) stimulated TGF-beta1 synthesis in human mesangial cells (MCs), and whether this effect was mediated through the protein kinase C (PKC) pathway. We measured the intraglomerular TGF-beta1 mRNA expression by using competitive RT-PCR, and this was compared with various parameters in IgA nephropathy patients. RESULTS: The IgA aggregate increased the TGF-beta1 mRNA expression in MCs, while this expression was not affected by the culture media or IgG aggregate. Phorbol 12-myristate 13-acetate and calphostin C did not influence the TGF-beta1 mRNA expression that was increased by the IgA aggregate. Intraglomerular TGF-beta1 mRNA expression was significantly correlated with creatinine clearance (r=-0.764, p=0.027), daily proteinuria (r=0.781, p=0.022), serum creatinine (r=0.884, p=0.004), and tubulointerstitial changes (r=0.809, p=0.015). Glomerular TGF-beta1 mRNA expression was associated with an increased tendency for glomerulosclerosis (r=0.646, p=0.084). After 4 years, patients with a high expression of intraglomerular TGF-beta1 mRNA showed a tendency for an decrease of their renal function. CONCLUSION: The IgA aggregate increased TGF-beta1 mRNA expression in MCs, and this was independent of the PKC pathway. The evaluation of intraglomerular TGF-beta1 mRNA expression could be useful in predicting the progression of IgA nephropathy.
Cells, Cultured ; Female ; Glomerular Mesangium/*cytology/metabolism ; Glomerulonephritis, IGA/metabolism/pathology ; Humans ; Immunoglobulin A/*pharmacology ; Male ; Proteins/*metabolism ; RNA, Messenger/*metabolism ; Research Support, Non-U.S. Gov't

BACKGROUNDThe renoprotective mechanisms of adenosine monophosphate (AMP)-activated protein kinase (AMPK) agonist - metformin have not been stated clearly. We hypothesized that metformin may ameliorate inflammation via AMPK interaction with critical inflammatory cytokines. The aim of this study was to observe the effects of metformin on expression of nuclear factor-κB (NF-κB), monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-β1) induced by high glucose (HG) in cultured rat glomerular mesangial cells (MCs).

METHODSMCs were cultured in the medium with normal concentration glucose (group NG, 5.6 mmol/L), high concentration glucose (group HG, 25 mmol/L) and different concentrations of metformin (group M1, M2, M3). After 48-hour exposure, the supernatants and MCs were collected. The expression of NF-κB, MCP-1, ICAM-1, and TGF-β1 mRNA was analyzed by real time polymerase chain reaction. Western blotting was used to detect the expression of AMPK, phospho-Thr-172 AMPK (p-AMPK), NF-κB p65, MCP-1, ICAM-1, and TGF-β1 protein.

RESULTSAfter stimulated by HG, the expression of NF-κB, MCP-1, ICAM-1, TGF-β1 mRNA and protein of MCs in group HG increased significantly compared with group NG (P < 0.05). Both genes and protein expression of NF-κB, MCP-1, ICAM-1, TGF-β1 of MCs induced by high glucose were markedly reduced after metformin treatment in a dose-dependent manner (P < 0.05). The expression of p-AMPK increased with the rising of metformin concentration, presenting the opposite trend, while the level of total-AMPK protein was unchanged with exposure to HG or metformin. Conlusion Metformin can suppress the expression of NF-κB, MCP-1, ICAM-1 and TGF-β1 of glomerular MCs induced by high glucose via AMPK activation, which may partly contribute to its reno-protection.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Cells, Cultured ; Glomerular Mesangium ; cytology ; Glucose ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Metformin ; pharmacology ; NF-kappa B ; metabolism ; Rats

OBJECTIVETo investigate the effect of exendin-4 on the expression of monocyte chemoattractant protein-1 (MCP-1) and fibronectin (FN) in rat glomerular mesangial cells in vitro.

METHODSRat glomerular mesangial cells were divided into 5 groups, namely control group, tumor necrosis factor-α (TNF-α) group (10 ng/ml), TNF-α (10 ng/ml)+E1 (1 nmol/L exendin-4) group, TNF-α (10 ng/ml)+E5 (5 nmol/L exendin-4) group, and TNF-α (10 ng/ml)+E10 (10 nmol/L exendin-4) group. After cultured 24 h or 48 h, RNA were extracted to determine the expression of MCP-1 with real-time PCR, the supernatant were collected to determine the expression of MCP-1 and FN with ELISA.

RESULTSCompared with control group, the cells treated with TNF-α for 24 h showed significantly increase the expression of MCP-1 and FN (P<0.01), exendin-4 significantly reduced the expression of MCP-1 and FN in TNF-α+E5 group and TNF-α+E10 group (P<0.05). After 48h incubation, the expression of MCP-1 and FN increased significantly in TNF-α group (P<0.01), which was lowered by exendin-4 in TNF-α+E1,TNF-α+E5 and TNF-α+E10 groups (P<0.05).

CONCLUSIONExendin-4 has an intrinsic capability to concentration- and time-dependently inhibit TNF-α-induced expression of MCP-1 and FN in rat mesangial cells, suggesting the beneficial effect of exendin-4 in preventing and treating diabetic nephropathy.

Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Diabetic Nephropathies ; metabolism ; Glomerular Mesangium ; cytology ; Mesangial Cells ; drug effects ; metabolism ; Peptides ; pharmacology ; Rats ; Tumor Necrosis Factor-alpha ; pharmacology ; Venoms ; pharmacology

Interleukin (IL)-6 is an autocrine growth factor for mesangial cells. It is not known whether high glucose influences IL-6 production in mesangial cells. Angiotensin II (AGII) is involved in the progression of renal diseases including diabetic nephropathy. Therefore, we evaluated the effects of high glucose in concert with AGII on IL-6 production in human mesangial cells and the modulation by blocking AGII. After 48 hr of culture, IL-6 mRNA expression was analyzed by reverse transcription and polymerase chain reaction (PCR). Quantitative determination of IL-6 concentrations in the culture supernatants of mesangial cells was performed using a sandwich enzyme immunoassay kit. Incubation of mesangial cells with high glucose (450 mg/dL) reduced the ratio of PCR products for IL-6 to beta-actin on densitometric results, while AGII (10(-7)M) increased it. The IL-6 secretion in the supernatant was also increased by AGII and decreased by high glucose. The IL-6 mRNA expression and IL-6 secretion in combination of high glucose and AGII were higher than those in high glucose and similar with those in control media. The addition of losartan (10(-6)M) or captopril (10(-6)M) to high glucose had no additional effects on IL-6 production. These results suggest that whereas AGII increases IL-6 production, high glucose decreases it. The IL-6 production of mesangial cells in diabetic milieu may be complicated and depend on the local effects of high glucose and/or AGII.
Angiotensin II/*pharmacology ; Captopril/pharmacology ; Cells, Cultured ; Gene Expression/drug effects ; Glomerular Mesangium/cytology/*metabolism ; Glucose/*pharmacology ; Humans ; Interleukin-6/*biosynthesis/genetics/secretion ; Losartan/pharmacology

OBJECTIVETo explore effects of beraprost sodium (BPS) on the metabolism of extracellular matrix (ECM) in rat mesangial cells cultured in the presence of high glucose and the possible mechanism.

METHODSRat mesangial cells were cultured in the presence of high glucose with or without BPS for 24 or 48 h. The levels of transforming growth factor β1 (TGFβ1), fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) protein in the culture supernatants were measured by enzyme-linked immunosorbent assay, and photoshop-Smad3 was detected by Western blotting.

RESULTSCompared with the cells in normal glucose, the cells cultured in the presence of high glucose for 24 and 48 h showed significantly increased TGFβ 1 and FN protein expression and lowered MMP-2 protein expression (P<0.01). Compared with the cells cultured in high glucose, BPS exposure at the concentration of 1, 2, and 5 µmol/L for 24 and 48 h significantly lowered TGFβ 1 protein expression (P<0.01), and at 2 and 5 µmol/L, BPS significantly decreased FN protein expression and increased MMP-2 protein expression in high glucose-induced cells (P<0.05). High glucose exposure also significantly increased the expression phosphorylated Smad3 (P<0.01), which was lowered by BPS treatment at 2 and 5 µmol/L (P<0.01).

CONCLUSIONBPS can regulate ECM metabolism in rat mesangial cells cultured in high glucose by inhibiting TGFβ 1/Smad3 pathway, suggesting the beneficial effects of BPS in preventing and treating diabetic nephropathy.

Animals ; Cell Line ; Cells, Cultured ; Diabetic Nephropathies ; Enzyme-Linked Immunosorbent Assay ; Epoprostenol ; analogs & derivatives ; pharmacology ; Extracellular Matrix ; metabolism ; Fibronectins ; metabolism ; Glomerular Mesangium ; cytology ; Glucose ; Matrix Metalloproteinase 2 ; metabolism ; Mesangial Cells ; drug effects ; Rats ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo study the inhibitive effect of tea polyphenols (TP) on oxidized low density lipoprotein (OX-LDL) intake in cultured rat mesangial cells.

METHODRat mesangial cells incubated in 80 mg.L-1 OX-LDL were treated with TP at different concentrations. Total cholesterol (TC) content of the mesangial cells and proportion of mesangial cells containing lipid droplets were measured, and ultrastructure observation was performed.

RESULTFoam cells were observed in mesangial cells stimulated by OX-LDL, and both the TC content and the proportion of lipid-droplet-containing cell were higher in these cells than in mesangial cells cultured in lipid free conditions. There were concentration-dependent decreases both in TC content and lipid-droplet-containing cell proportions when treated with TP from 40 ng.L-1 to 40 mg.L-1. The number of lipid droplets was also decreased in TP treated mesangial cells.

CONCLUSIONTP has an inhibitive effect on OX-LDL intake in cultured rat mesangial cells, which is in a concentration-dependent manner from 40 ng.L-1 to 40 mg.L-1.

Animals ; Cells, Cultured ; Cholesterol ; metabolism ; Flavonoids ; isolation & purification ; pharmacology ; Foam Cells ; metabolism ; Glomerular Mesangium ; cytology ; metabolism ; Lipoproteins, LDL ; metabolism ; Male ; Phenols ; isolation & purification ; pharmacology ; Polyphenols ; Rats ; Rats, Wistar ; Tea ; chemistry

OBJECTIVETo investigate the effects of rapamycin and 3-methyladenine on autophagy impairment, oxidative stress and premature senescence induced by high-glucose in primarily cultured rat mesangial cells.

METHODSRat glomerular mesangial cells (GMCs) were isolated and cultured in normal glucose, high glucose, high glucose with 3-methyladenine (3-MA), or high glucose with rapamycin. At 24 h, 72 h and 10 days of culture, the cells were examined for expression levels of autophagy markers LC3 and p62/SQSTM1, malondialdehyde (MDA) and protein carbonyl, β-galactosidase (SA-β-gal) activity and heterochromatin foci (SAHF).

RESULTSCompared with those of normal cell culture, the cells exposed to high glucose for 72 h and 10 days showed down-regulated LC3 expression, up-regulated p62/SQSTM1 expression, elevated MDA and protein carbonyl levels, and increased SAHF formation and percentage of SA-β-gal-positive cells. These changes were reversed in GMCs exposed to high glucose and rapamycin for 72 h and 10 days, but exacerbated in cells incubated with 3-MA.

CONCLUSIONHigh glucose can suppress autophagic function of rat GMCs to result in oxidative damage and cell senescence. Rapamycin can attenuate autophagy impairment, oxidative damage and senescence induced by high glucose, whereas 3-MA can further aggravate high glucose-induced cell injuries in rat GMCs.

Animals ; Autophagy ; drug effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; Glomerular Mesangium ; cytology ; Glucose ; adverse effects ; Male ; Mesangial Cells ; cytology ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sirolimus ; pharmacology

Rapamycin, a macrocyclic lactone, is effective in reducing the incidence of acute rejection after renal transplantation. The inhibitory effects of rapamycin on lymphocyte proliferation and the molecular mechanisms that were involved have been described. However, its effects on glomerular mesangial cells have not been clearly understood, and here, we examined the effect of rapamycin on platelet-derived growth factor (PDGF) - induced extracellular matrix synthesis as well as cell proliferation in mesangial cells. Rat mesangial cells were isolated from the glomeruli of Sprague-Dawley rats and cultured with Dulbecco's modified Eagles medium containing 20% fetal bovine serum. Different concentrations of rapamycin were administered 1 hour before the addition of 10 ng/ml of PDGF into growth arrested and synchronized cells. Cell proliferation was assessed by [3H]thymidine incorporation, total collagen synthesis by [3H]proline incorporation, and fibronectin secretion into the medium by Western blot analysis. In the mesangial cells, PDGF increased cell proliferation by 4.6-fold, total collagen synthesis by 1.8-fold, and fibronectin secretion by 3.2-fold. Rapamycin above 10 nM significantly inhibited PDGF-induced proliferation and collagen synthesis, but the treatment of rapamycin up to 1micrometer did not show any significant effects on PDGF-induced fibronectin secretion. These inhibitory effects of rapamycin on PDGF-induced mesangial cell proliferation and collagen synthesis reflect the potential value of rapamycin in the prevention and treatment of glomerulosclerosis in patients with chronic allograft nephropathy.
Animals ; Cells, Cultured ; Collagen/*antagonists & inhibitors/biosynthesis ; Fibronectins/*biosynthesis ; Glomerular Mesangium/cytology/drug effects/*metabolism ; Immunosuppressive Agents/*pharmacology ; Male ; Platelet-Derived Growth Factor/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Sirolimus/*pharmacology

OBJECTIVETo observe the effect of Kanglaite injection(KLT) on the proliferation and telomerase activity of mesangial cells in rats.

METHODMTT, telomere repeat amplification protocal (TRAP), ELISA, PAGE and silver-stain were applied to detect the growth rate and telomerase activity of MC after stimulation of KLT and IL-1.

RESULTThe growth rate of MC was enhanced by IL-1 stimulation, which was accompanied with a redection of the activity of telomerase. Adversely, the growth rate of MC was reduced by KLT, which was accompanied with an enhancement of activity of telomerase. Moreover, the growth rate of MC and the activity of telomerase were both inhibited by the combinative use of IL-1 and KLT without any influence from the sequence of their administration.

CONCLUSIONKLT could inhibit proliferation and telomerase activity of MC with or without pre-stimulation with IL-1. KLT might be useful to prevent and treat glomerular nephritis related to MC proliferation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coix ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Glomerular Mesangium ; cytology ; enzymology ; Injections ; Plant Oils ; administration & dosage ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Rats ; Seeds ; chemistry ; Telomerase ; metabolism