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

OBJECTIVETo study the effects and possible underlying mechanism of Qufeng Tongluo Prescription (, QFTL) on the regulation of mesangial cells (MCs) proliferation and apoptosis.

METHODSThe MCs used in this experiment have undergone five passages induced by lipopolysaccharide (LPS). Changes in the proliferation, apoptosis, cell cycle regulatory proteins and mRNA expression levels of the MCs after administration of Benazepril or QFTL were measured by methyl thiazolyl tetrazolium (MTT) reduction assay, flow cytometry, Western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively.

RESULTSThe addition of Benazepril or QFTL serum inhibited LPS-induced MC proliferation after treatment for 24, 48 and 72 h, respectively (P<0.05 or P<0.01). Moreover, the inhibitory effect is more significant in the QFTL group at 48 h (P<0.05). Compared with the control group, LPS-induced cell proliferation decreased the number of cells in G1 phase versus cells in S and G2/M phases, while the addition of QFTL and Benazepril serum increased the ratio of cells at G1 phase (P<0.05 or P<0.01) to cells at S phase (P<0.01), implicating the cell cycle inhibition effect exerted by QFTL. LPS decreased the level of MC apoptosis, compared with the control group (P<0.05), while QFTL and Benazepril serum increased the level of MC apoptosis (P<0.01). Moreover, the difference between the QFTL group and the Benazepril group was statistically significant (P<0.01). Compared with the control group, the protein and mRNA expression levels of cylinD1, cyclin dependent kinase 2 (CDK2) and p21 were significantly increased (P<0.05 or P<0.01), p27 was decreased but with no statistical significance (P>0.05); After being treated with QFTL and Benazepril serum, the protein and mRNA expression levels of cylinD1, CDK2, p21 were decreased and p27 increased significantly (P<0.05 or P<0.01); Compared with the Benazepril group, QFTL show better effects on protein and mRNA expression levels of cylinD1, CDK2 (P<0.05 or P<0.01) and p21 protein expression (P<0.05).

CONCLUSIONQFTL inhibits MCs proliferation, promotes MCs apoptosis through an underlying mechanism of down-regulating the protein and mRNA expression levels of cylinD1, CDK2, p21 and up-regulation of the expression level of p27.

Animals ; Apoptosis ; drug effects ; Base Sequence ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; DNA Primers ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Glomerular Mesangium ; cytology ; drug effects ; metabolism ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

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 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

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

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

AIMTo study the mechanisms of anti-diabetic nephropathy of rhein on cultured human mesangial cells (HMCs).

METHODSTo mimic the hyperglycemic (HG) environment of diabetic nephropathy, 30 mmol x L(-1) glucose were added to 10% FBS RPMI 1640. The HMCs were treated with rhein for 8, 24, 48 or 72 h, at these time, the bioactivity, total activity of transforming growth factor-beta1 (TGFbeta1), activity of p38MAPK (p38 mitogen-activated protein kinases, by using immunoprecipitate and Western blot), MMP-2 (matrix metalloproteinase-2), and MMP-9 (matrix metalloproteinase-9, by using gelatinase zymography) and the proliferation of HMCs in high glucose media were measured. Meanwhile the levels of secretion of FN in cultured HMCs were measured.

RESULTSThe results showed that rhein markedly inhibit the proliferation of HMCs, significantly reduce the bioactivity of TGFbeta1 and FN secretion in HMCs, and decrease the increased activity of p38MAPK, but showed no action on the activities of MMP-2 and MMP-9.

CONCLUSIONRhein reduced the secretion of FN and inhibited the proliferation of HMCs may through inhibiting the bioactivities of TGFbeta1 and p38MAPK.

Animals ; Anthraquinones ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Epithelial Cells ; cytology ; metabolism ; Fibronectins ; secretion ; Glomerular Mesangium ; cytology ; metabolism ; Glucose ; antagonists & inhibitors ; pharmacology ; Humans ; Lung ; cytology ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mink ; Transforming Growth Factor beta ; metabolism ; Transforming Growth Factor beta1 ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the role of c-Jun N-terminal kinase (JNK)-c-Jun/activator protein-1 (AP-1) signal pathway in expression of monocyte chemoattractant protein-1 (MCP-1) in experimental rat glomerulonephritis.

METHODSNephrotoxic sera nephritis (NTN) was induced by injection of anti-GBM antibody into the tail veins of rats. Electrophoretic mobility shift assay (EMSA) and non-radioactive kinase assay were used to detect the activity of AP-1 and JNK in kidneys and angiotensin II-stimulated human mesangial cells. Ribonuclear protection assay was used to detect MCP-1 expression in cultured human mesangial cells.

RESULTSSignificant up-regulation of JNK and AP-1 was observed in NTN rats (3.82 +/- 0.58) folds and (5.36 +/- 0.61) folds, as compared with the controls. Supershift assay demonstrated that c-Jun and c-Fos were the predominant subunits involved. Activation of JNK and AP-1 significantly correlated with MCP-1 expression in NTN rats. Angiotensin II enhanced the expression of MCP-1 and activation of JNK and AP-1 in cultured human mesangial cells in a dose-dependent manner, with maximal stimulation seen at 100 nmol/L (20.99 +/- 4.71) folds, (6.91 +/- 1.65) folds and (7.82 +/- 1.32) folds respectively. Significant down-regulation of AP-1 activation and MCP-1 expression were observed in angiotensin II-induced human mesangial cells pretreated with JNK specific inhibitor SP600125.

CONCLUSIONSAngiotensin II and MCP-1 may play an important role in glomerulosclerosis via the JNK-c-Jun/AP-1 signal pathway.

Angiotensin II ; pharmacology ; Animals ; Cells, Cultured ; Chemokine CCL2 ; metabolism ; Glomerular Mesangium ; cytology ; metabolism ; Glomerulonephritis ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Proto-Oncogene Proteins c-fos ; metabolism ; Proto-Oncogene Proteins c-jun ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; metabolism

Angiotensin II ; pharmacology ; Blotting, Western ; Cells, Cultured ; Glomerular Mesangium ; cytology ; drug effects ; metabolism ; Humans ; I-kappa B Kinase ; NF-kappa B ; analysis ; Peptide Fragments ; pharmacology ; Protein-Serine-Threonine Kinases ; analysis