OBJECTIVETo study the effects and mechanism of lovastatin on cell cycle phase and proliferation of cultured human glomerular mesangial cells in vitro.

METHODSHMC proliferation was determined by 3H-Thymidine incorporation. HMC cell cycle was measured by flow cytometric analysis.

RESULTSLovastatin was found to inhibit HMC proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that lovstatin induced G1/S transition arrest. Concomitant addition of mevalonate or farnesol restored all the inhibitory effect of lovstatin on HMC.

CONCLUSIONLovastatin is a HMC proliferation inhibitor. It provides an experimental evidence for re-evaluate renal protective effect of HRI, which has already been widely used in clinical treatment.

Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flow Cytometry ; Glomerular Mesangium ; cytology ; Humans ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Kinetics ; Lovastatin ; pharmacology

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

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 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

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 study the effects and mechanism of lovastatin on cell proliferation and expression of proinflammatory cytokines in cultured human glomerular mesangial cells.

METHODSThe influence of lovastatin on HMC proliferation was evaluated with 3H-thymidine incorporation. mRNA expression of proinflammatory cytokines (IL-1 beta, IL-6, TNF-alpha, and MCP-1) and activation of NF-kappa B of HMC were measured using Reverse transcription-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assay (EMSA) respectively.

RESULTSLovastatin was found to have inhibitory effects on human mesangial cell (HMC) proliferation and lipopolysaccharide (LPS)-mediated human mesangine cell HMC mRNA expression of proinflammatory cytokines via activation of NF-kappa B. The effect of lovstatin on HMC could be prevented when the mevalonate and farnesol were added to the culture.

CONCLUSIONLovastatin may decrease HMC proliferation and production of proinflammatory cytokines through the inhibition of NF-kappa B activation. This provided experimental evidence for further evaluation of the renal protective effect of HRI, suggesting that it may be a potent agent for prevention of progressive renal diseases aside from its lipid-lowering effect.

Cell Division ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Chemokine CCL2 ; analysis ; Glomerular Mesangium ; cytology ; drug effects ; Humans ; Interleukin-1 ; analysis ; Interleukin-6 ; analysis ; Lovastatin ; pharmacology ; NF-kappa B ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; analysis

BACKGROUNDAdrenomedullin (ADM), a potent hypotensive small peptide, was recently found to inhibit the proliferation of glomerular mesangial cells (MsC) in vitro and to attenuate glomerular lesions in vivo, however the mechanisms remain poorly understood. In this study, we attempted to elucidate them using molecular signal transduction.

METHODSCultured rat MsC were treated with ADM and several inhibitors of signalling molecules. Methyl thiazoleterazolium (MTT) assay and BrdU incorporation method were employed for examining MsC proliferation. Western blot analysis was used for detecting total mitogen activated protein kinases (t-MAPKs) and phosphorylated MAPKs (p-MAPKs) proteins.

RESULTSADM suppressed MsC proliferation in a concentration- and time-dependent fashion. This response was inhibited by ADM receptor antagonist CGRP8-37 and a potent protein kinase-A (PKA) inhibitor, H89. Forskolin, a direct adenylate cyclase activator, also significantly inhibited MsC proliferation. SB203580, a P38MAPK inhibitor, and U0126, a MEK inhibitor, both completely blocked ADM mediated responses in MsC. However, curcumin, a SAPK/JNK inhibitor, and GF109203X, a potent protein kinase-C (PKC) inhibitor, had no effect on MsC growth. Western blot analysis showed that ADM did not change the expression of t-MAPKs but increased p-SAPK/JNK and p-P38MAPK levels and decreased p-ERK level. These responses were inhibited by CGRP8-37. All these kinase phosphorylations, except for the increase in p-SAPK/JNK, could be stimulated using forskolin. In addition, only ADM mediated changes in ERK and P38MAPK phosphorylations were inhibited by H89. GF109203X did not affect ADM induced changes in three p-MAPKs expressions.

CONCLUSIONSADM inhibits MsC proliferation possibly through cAMP-PKA pathway. Both phosphorylations of ERK and P38MAPK pathways were necessary in mediating the antiproliferative response of ADM. It does not preclude the involvement of cAMP independent pathways in the ADM mediated responses.

Adrenomedullin ; Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Glomerular Mesangium ; cytology ; drug effects ; JNK Mitogen-Activated Protein Kinases ; physiology ; Peptides ; pharmacology ; Rats ; Signal Transduction ; physiology ; p38 Mitogen-Activated Protein Kinases ; physiology

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 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

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