MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups: normal glucose (NG, 5.5 mmol/L glucose) group, hypertonic (MA, 5.5 mmol/L glucose+ 19.5 mmol/L mannitol) group and high-glucose (HG, 25.0 mmol/L glucose) group. MiR-148b expression was detected by real time PCR. Then miR-148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKα1, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), fibronectin (FN) and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down-regulated AMPKα1 mRNA and protein expressions, and up-regulated CHOP, GRP78, collagen Ⅳ and FN expressions (all P＜0.05). HG-induced mesangial cells with miR-148b inhibitor had up-regulated AMPKα1 mRNA and protein expressions, and down-regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P＜0.05). Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.     

Extracellular matrix protein: gene expression and synthesis in cultured rat mesangial cells.

Production of extracellular matrix (ECM) by mesangial cells (MCs) contributes to progressive glomerulosclerosis. Our previous immunocytochemical studies on ECM production by MCs [1] were extended by examining the gene expression and protein synthesis using Northern blotting and ELISA. ELISA demonstrated that fetal calf serum (FCS) stimulated the synthesis of collagen I (CI), III (CIII) and IV (CIV), and laminin (LM). At and after confluence, the amounts of CIII, CIV and LM per DNA amount began to decrease, whereas that of CI did not. Northern blotting indicated that the mRNA expressions for CIV and LM were transiently increased by FCS. The mRNA expressions for CI and CIII decreased until confluence and then returned to the initial levels. These results suggest that FCS and cellular confluence affected the synthesis of ECM at both the level of gene expression and protein synthesis. The mRNA expressions for CIV and LM paralleled the protein synthesis and appeared tightly regulated. CI and CIII synthesis and mRNA expression were not coordinate and were not regulated in the same manner as the basement membrane matrix components.     

HGF protects rat mesangial cells from high-glucose-mediated oxidative stress

BACKGROUND: Oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy. Recent observations suggested that hepatocyte growth factor (HGF) was an antioxidant growth factor; thus, its renoprotective effects in diabetic nephropathy might be related to antioxidant mechanism. The aim of the present study was to evaluate whether HGF could prevent rat mesangial cells (RMC) from high-glucose-mediated oxidative stress and explore its relevant mechanism. METHODS: RMC were cultured in 5.6 mM (NG) or 30 mM (HG) glucose in the absence or presence of HGF (20 ng/ml) and c-met inhibitor SU11274 (5 microM) for 24 h. RESULTS: c-met expression in HG was markedly increased. Enhanced oxidative stress was observed in HG as evidenced by elevated reactive oxygen species and malondialdehyde levels and decreased glutathione level, which was markedly attenuated by HGF. HGF also inhibited HG-induced p22(phox) and aldose reductase upregulation and prevented HG-reduced glutamate-cysteine ligase catalytic subunit (GCLC) expression through inhibiting USF binding to negative regulatory region of GCLC promoter. Reduced glucose-6-phosphate dehydrogenase activity and expression in RMC by HG was rescued by HGF. CONCLUSION: HGF could function as an antioxidant factor and protect against HG-mediated oxidative stress by enhancing ROS scavenging and suppressing ROS production.     

Impairment of glutathione biosynthetic pathway in uraemia and dialysis.

BACKGROUND: Glutathione (GSH), the predominant intracellular antioxidant, reportedly has been shown to be decreased in chronic renal failure patients, which renders these patients more susceptible to oxidative damage by free radicals. To our knowledge, the ability of erythrocytes to normalize the GSH level by de novo synthesis in uraemic and dialysis patients has not been studied previously. The main goal of the present study was to measure the activities of the enzymes that are responsible for de novo GSH generation, namely gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSH-S), in erythrocytes from uraemic and dialysis patients. METHODS: Erythrocyte total GSH level and gamma-GCS and GSH-S activities as well as plasma malondialdehyde (MDA) levels were measured in 19 non-dialysis patients (ND), 34 haemodialysis patients (HD), 22 continuous ambulatory peritoneal dialysis patients (CAPD) and 21 normal healthy controls. The effect of a single haemodialysis session was determined in 16 HD patients. RESULTS: Significant decreases in GSH levels and gamma-GCS activity but not GSH-S were observed in ND, HD and CAPD patients compared with controls. However, GSH levels as well as gamma-GCS and GSH-S activities were not different among the ND, HD and CAPD patients. The decrease in GSH was strongly and positively correlated with the decrease in gamma-GCS in ND, HD and CAPD patients (r = 0.717, P<0.001; r = 0.854, P<0.001; and r = 0.603, P<0.01, respectively). In addition, plasma MDA was negatively correlated with gamma-GCS in ND, HD and CAPD patients (r = 0.721, P<0.001; r = 0.560, P<0.01; and r = 0.585, P<0.01, respectively). A single dialysis session had no effect on GSH level or on gamma-GCS and GSH-S activities. Only a significant reduction in MDA was observed at the end of dialysis. CONCLUSIONS: The activity of the rate-limiting enzyme in GSH biosynthesis, gamma-GCS, was significantly decreased in uraemic and dialysis patients, which explains, at least in part, frequent reports of reduced GSH levels in these patients. The decrease in gamma-GCS activity may have been secondary to inhibitory effects from uraemic factors that are not removed by standard dialysis. However, this assumption does not exclude the possibility of down-regulation of gamma-GCS protein expression and further studies in this context are recommended.     

Altered antioxidant capacity in human renal cell carcinoma: role of glutathione associated enzymes

PURPOSE: We aimed to discern the role of glutathione (GSH) associated enzymes in maintaining high GSH levels in renal cell carcinoma (RCC) of the clear cell type and analyze RCC enzyme antioxidant capacity. Since changes in cellular redox balance in RCC might also be related to alterations of glutathione S-transferase (GST) phenotype, GST class alpha and pi expression was also explored. METHODS AND MATERIALS: Human kidney specimens of tumor and distant nontumor regions were obtained from 15 patients with RCC at the time of surgery. The activities of GSH-replenishing enzymes, gamma-glutamylcysteine synthetase (gamma-GCS), gamma-glutamyl transferase (gamma-GT), and glutathione reductase (GR), as well as the activities of antioxidant enzymes glutathione peroxidase (GPX) and catalase (CAT) were determined spectrophotometrically. GST alpha and pi class expression was determined by immunoblot. RESULTS: In the course of renal cancerization, significant changes appear in the activities of GSH-replenishing and antioxidant enzymes. The activity of the key enzyme of GSH synthesis, gamma-GCS, is up-regulated (P < 0.001), while the activities of gamma-GT and GR are down-regulated in renal tumors compared to nontumor tissue (P < 0.001 and P < 0.05, respectively). Activities of GPX and CAT were also down-regulated (P < 0.001 and P < 0.05, respectively) in RCC. Changes in enzyme antioxidant capacity in RCC were associated with decreased GST class alpha (P < 0.001) and unchanged GST pi expression at the protein level. CONCLUSIONS: Changes in redox status in RCC as a consequence of decreased enzyme antioxidant capacity, together with altered GST alpha expression, may be important factors in development and tumor growth. The up-regulation of gamma-GCS and high levels of GSH in RCC may be an attempt to limit injury caused by oxidative stress.     

过氧化物酶体增殖物激活受体α激动剂非诺贝特抑制高糖条件下的肾脏系膜细胞增殖

目的探讨非诺贝特（Fenofibrate）对高糖条件下大鼠肾脏系膜细胞（mesangial cells,MCs）HBZY-1增殖的影响及其作用机制。方法将对数生长期大鼠肾脏系膜细胞分成正常糖组（NG组）、高糖组（HG组）和非诺贝特组（FN组）。NG组细胞体系常规培养,HG组细胞体系加入40mmol／L葡萄糖,FN组细胞培养体系中加入40mmol／L葡萄糖和非诺贝特100μmol／L。各组细胞培养48h后,利用MTT方法检测细胞增殖;realtime-PCR（实时定量PCR）检测各组细胞PPAR-α、JAK2和STAT3基因表达;Westernblotting检测细胞中过氧化物酶体增殖物激活受体Q激动剂（peroxisome proliferators activedreceptor-α,PPAR-α）、p-PPAR-a、JAK2、STAT3、p-JAK2和p-STAT3蛋白的变化。结果与NG组比较,HG组系膜细胞增殖率明显增高（P〈0．05）,JAK2与STAT3mRNA表达水平无统计学差异（P〉0．05）,PPAR-α、JAK2和STAT3蛋白无明显变化（P〉0．05）,但p-PPAR-α、p-JAK2、p-STAT3蛋白明显增加（P〈0．05）;与HG组比较,FN组系膜细胞增值率和JAK2、p-sTAT3蛋白表达均明显下降（P〈0．05）,PPAR-α、JAK2与STAT3mRNA和JAK2与STAT3mRNA表达均无统计学差异,但p-PPAR-α蛋白表达进一步增加（P〈0．05）。结论PPAR-α激动剂非诺贝特能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制JAK2／STAT3信号激活。     

黄连素通过PI3K/Akt/mTOR信号通路抑制高糖条件下的肾脏系膜细胞增殖

目的：探讨黄连素（berberine,BBR）对高糖条件下的大鼠肾脏系膜细胞 HBZY-1 cells （MCS）增殖的影响及其作用机制。方法将对数生长期细胞分成正常糖处理组（NG 组）,高糖组（HG组）和黄连素组（BBR组）。NG组细胞体系常规培养培养,HG 组细胞体系加入40 mmol/L 葡萄糖,BBR组细胞培养体系中加入40 mmol/L葡萄糖＋黄连素30μmol/L。各组细胞培养48 h 后,利用MTT检测各组HBZY-1细胞增殖;RT-PCR检测各组细胞p85,Akt和mTOR基因表达;West-ern blotting检测细胞中 p85、p-p85、Akt、p-Akt,mTOR 和 Collagen-Ⅳ蛋白的变化。结果 NG 组与HG组增值率分别为（25%±3%）和（75%±5%）,与 NG组增值率比较,HG组肾脏系膜细胞增殖率明显增高（P〈0.05）,p85与 Akt mRNA表达水平和蛋白水平均无明显变化（P＆gt;0.05）,但 p-p85、p-Akt,Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显增加（P〈0.05）;BBR 组增值率为（42%±5%）,与 HG 组比较,BBR 组肾脏系膜细胞增值率明显降低（P〈0.05）,但 p-p85、p-Akt、Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显下降（P〈0.05）,而 p85和Akt mRNA和蛋白表达水平均无统计学差异（P〈0.05）。结论黄连素能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制PI3K/Akt/mTOR信号通路激活。     

Role of TGF-beta/GLUT1 axis in susceptibility vs resistance to diabetic glomerulopathy in the Milan rat model.

BACKGROUND: GLUT1 upregulation and increased glucose transport activity may contribute to extracellullar matrix (ECM) accumulation characterizing diabetic nephropathy (DN). Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease, due to a haemodynamic protection. On the contrary, those of the Milan normotensive strain (MNS) develop spontaneous glomerulosclerosis, and when rendered diabetic, show typical morphological and haemodynamic changes. METHODS: To assess whether susceptibility to diabetic glomerulopathy in MNS rats is associated with higher glucose transporter 1 (GLUT1) expression (and glucose transport activity) vs MHS rats, diabetic and nondiabetic MNS and MHS rats were followed for 6 months and mesangial cells derived from these animals were exposed to high glucose (HG) vs normal glucose (NG) conditions. RESULTS: Glomerular expression of GLUT1 protein and ECM and transforming growth factor-beta (TGF-beta) mRNA was significantly upregulated in diabetic vs nondiabetic MNS, but not MHS rats. Upon exposure to HG and/or TGF-beta, mesangial cells from 1- and 8-month-old MNS rats showed higher glucose transport activity and GLUT1 membrane expression than those from age-matched MHS rats. Likewise, ECM and TGF-beta production increased more markedly in response to HG and/or TGF-beta in MNS vs MHS mesangial cells. CONCLUSIONS: These data indicate that susceptibility to diabetic glomerulopathy in MNS rats is associated with increased GLUT1-dependent glucose transport activity in response to hyperglycaemia and/or TGF-beta, which may amplify ECM overproduction. Conversely, the haemodynamic protection from glomerulosclerosis in MHS rats is associated with lack of upregulation of TGF-beta/GLUT1 axis, thus supporting the concept that this axis may represent the link between haemodynamic and metabolic mechanisms of injury.     

细胞因子信号传导抑制蛋白1抑制高糖诱导的肾小球系膜细胞单核细胞趋化蛋白1的表达

目的 探讨细胞因子信号传导抑制蛋白1(SOCS-1)对高糖状态下肾小球系膜细胞单核细胞趋化蛋白1（MCP-1）表达的影响。 方法 体外培养人肾小球系膜细胞,应用脂质体2000分别转染pCR3.1-SOCS-1表达质粒和pCR3.1 空质粒载体,G418筛选阳性克隆。分别采用低糖（5.5 mmol/L）、高糖（30 mmol/L）、低糖+甘露醇（24.5 mmol/L甘露醇）和JAK-STAT信号通路抑制剂AG490 （10 μmol/L）进行刺激。Western印迹检测系膜细胞SOCS-1、信号转导和转录活化因子1、3（STAT1、STAT3）及其磷酸化蛋白（p-STAT1、p-STAT3）的表达。ELISA法和放免法测定细胞上清液中MCP-1、FN和Ⅳ型胶原的含量。RT-PCR法检测SOCS-1和MCP-1 mRNA的表达。 结果 高糖刺激系膜细胞SOCS-1蛋白和mRNA表达呈时间依赖性变化, 4 h表达达到峰值,然后逐渐减低,24 h达基线水平。与低糖组相比,高糖组系膜细胞STAT1和STAT3磷酸化水平显著上调（P < 0.01）; MCP-1 mRNA水平表达显著上调[（0.39±0.05）比（0.16±0.02）,P < 0.01];上清液中MCP-1[（459±67）比（241±19） ng/L]、FN[（5.84±0.61）比（3.41±0.31） mg/L]和Ⅳ型胶原[（16.45±2.30）比（9.56±1.52） μg/L] 含量均显著增加（均P < 0.01）。与空载体对照组相比,SOCS-1过表达组系膜细胞STAT1和STAT3的磷酸化水平显著下降（P < 0.05）;MCP-1 mRNA表达下调[（0.34±0.04）比（0.42±0.05）,P < 0.05]; 上清液中MCP-1[(387±47）比（463±56） ng/L]、 FN[（4.61±0.57）比（5.76±0.74） mg/L]和Ⅳ型胶原[(13.4±2.32)比（17.1±2.57） μg/L] 含量显著减少（均P < 0.05）。与高糖组相比,AG490组系膜细胞MCP-1 mRNA（0.31±0.04）表达显著下调;上清液中MCP-1[（361±53） ng/L]、FN[（5.46±0.71）mg/L]和Ⅳ型胶原[（15.2±1.97） μg/L]含量均减少。 结论 SOCS-1过表达抑制高糖状态下肾小球系膜细胞MCP-1及细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

The effects and underlying mechanism of CD36 in high glucose - induced rat glomerular mesangial cells apoptosis

Objective To investigate the effects and underlying mechanism of the scavenger receptor CD36 in high glucose - induced rat glomerular mesangial cells apoptosis. Methods The mesangial cells of rats were divided into 4 groups: control group (5.6 mmol/L glucose), mannitol group (24.2 mmol/L mannitol+5.6 mmol/L glucose), high glucose group (30 mmol/L glucose), CD36 mono- antibody group (30 mmol/L glucose+CD36 mono-antibody). The intracellular ROS level was detected by confocal microscopy with fluorescent probe CM - H2DCFDA. MDA, GSH - PX, 8 - OHDGA in cell supernatant were detected. Apoptosis was determined by flow cytometry followed by Annexin V-FITC/PI double stains. The expression of CD36, Bax and Bcl-2 were detected by RT-PCR and Western blotting. Results The expression of CD36 was detected in glomerular mesangial cells. The highest level was found in high glucose group in 24 hours. There was no significant difference found between control group and mannitol group with respect to intracellular ROS generation, MDA, 8-OHDG, GSH-PX level, apoptosis rate, expression of CD36, Bax and Bcl-2 (all P＞0.05). There was no significant difference in the expression of CD36 between CD36 mono - antibody group and high glucose group (P＞0.05）. Compared to control group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of CD36 and Bax were significantly increased, the GSH-PX level and the expression of Bcl-2 were significantly lower in high glucose group (all P＜0.05). Compared to the high glucose group, the intracellular ROS generation, MDA and 8-OHDG levels, apoptosis rate, the expression of Bax were suppressed but the GSH-PX level and the expression of Bcl-2 increased in CD36 mono-antibody group (all P＜0.05). The intracellular ROS level was positively correlated with apoptosis rate, protein expression of CD36 and Bax gene, was negatively correlated with Bcl - 2 protein expression. Conclusions CD36 was involved in the high glucose induced apoptosis of mesangial cells which was potentially mediated by an increased level of oxidative stress.     

高糖对人肾小管上皮细胞和系膜细胞表达血清和糖皮质激素诱导蛋白激酶1的影响

目的研究高糖作用下人近端肾小管上皮细胞(HKC)和系膜细胞(HMC)中血清和糖皮质激素诱导蛋白激酶1(SGK1)的表达,并初步探讨SGK1在介导高糖致肾细胞(HKC和HMC)过度合成细胞外基质(ECM)中的作用。方法将HKC和HMC细胞分别分为正常对照组(NG组,5.5mmol/LD-葡萄糖)、高糖组(HG组,25mmol/LD-葡萄糖)和渗透浓度对照组(MG组,19.5mmol/L甘露醇和5.5mmol/LD-葡萄糖)。SGK1mRNA水平及蛋白水平的检测分别采用RT-PCR方法和Western印迹方法。培养液中纤连蛋白(FN)水平检测采用ELISA方法。结果HKC和HMC中均存在SGK1基因和蛋白的表达。HMC中SGK1的表达明显高于HKC(P<0.01)。高糖刺激8h后,两种细胞SGK1表达均明显升高(P<0.01);同时,甘露醇也上调HKC和HMCSGK1的表达(P<0.01),但其作用明显弱于高糖(P<0.05)。FN在高糖环境下表达上调,且高峰出现时间滞后于SGK1。结论高糖能促进近端肾小管上皮细胞和系膜细胞SGK1的表达,并可能通过SGK1介导的信号转导途径在糖尿病肾病ECM积聚中发挥重要作用。     

内皮祖细胞对糖尿病心肌病大鼠氧化应激作用的研究

目的研究内皮祖细胞(Endothelial progenitor cells,EPCs)对链脲佐菌素诱导的糖尿病心肌病大鼠的氧化应激作用。方法通过腹腔注射链脲佐素诱导大鼠糖尿病心脏病模型,治疗组给予EPCs进行治疗。HE染色观察各组心肌组织的形态学改变;葡萄糖氧化酶法进行空腹血糖测定;检测各组大鼠血清中GSH和MDA含量;采用RT-PCR和Western blot方法检测大鼠心肌组织中iNOS和eNOS的表达水平。结果与正常对照组比较,糖尿病心肌病模型组心肌肥大,组织排列紊乱;血清GSH含量显著下降(P<0.01),MDA含量显著升高(P<0.01),心肌组织中iNOS和eNOSmRNA及蛋白的表达均显著升高(P<0.05)。经EPCs治疗后,GSH含量显著升高(P<0.01),MDA、iNOS和eNOS水平显著降低(P<0.05);心肌病理损伤得到修复。结论 EPCs可缓解由链脲佐菌素诱导的糖尿病心肌病,其作用机制可能与减少氧化应激、降低NOS水平有关。     

Stretch-induced overproduction of fibronectin in mesangial cells is mediated by the activation of mitogen-activated protein kinase

An excessive production of extracellular matrix (ECM) proteins in glomerular mesangial cells is considered to be responsible for the development of mesangial expansion seen in diabetic nephropathy. Mechanical stretch due to glomerular hypertension has been proposed as one of the factors leading to an increase in the production of ECM proteins in mesangial cells, but the precise mechanism of stretch-induced overproduction of ECM proteins has not been elucidated. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) may play a key role in the overproduction of fibronectin (FN) in mesangial cells exposed to mechanical stretch. MAPK, also termed extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), was activated by mechanical stretch in time- and intensity-dependent manners. Stretch-induced activation of ERK was inhibited by herbimycin A, a tyrosine kinase inhibitor, but not by GF109203X or calphostin C, the inhibitors of protein kinase C. Mechanical stretch also enhanced DNA-binding activity of AP-1, and this enhancement was inhibited by PD98059, an inhibitor of MAPK or ERK kinase (MEK). Furthermore, mechanical stretch stimulated the expression of FN mRNA followed by a significant increase in its protein accumulation. PD98059 could prevent stretch-induced increase in the expression of FN mRNA and protein. These results indicate that the activation of ERK may mediate the overproduction of ECM proteins in mesangial cells exposed to mechanical stretch, an in vitro model for glomerular hypertension seen in diabetes.     

黄芪甲苷的抗氧化应激作用对体外高糖刺激足细胞的保护作用

目的:观察黄芪甲苷的抗氧化应激作用对体外高糖刺激下的足细胞的保护作用。方法:将条件性永生的小鼠足细胞随机分为正常对照组(NG)、高糖组(HG)、甘露醇高渗对照组(MA)、DMSO组及HG+不同剂量(5、15、30μg/ml)AS-IV干预组。采用CCK-8法检测各组足细胞活性,氧化应激试剂盒检测细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)水平;同时应用western印迹法检测细胞整合素连接激酶ILK蛋白表达量。结果:(1)足细胞活性:与NG组相比,MA组差异无统计学意义,HG组足细胞活性显著降低(P<0.05);与HG组相比,HG+30μg/mlAS-IV组足细胞活性显著升高(P<0.05),DMSO组、HG+15μg/mlAS-IV组和HG+5μg/mlAS-IV组差异无统计学意义(P<0.05)。(2)氧化应激及整合素连接激酶蛋白表达量:与NG组相比,HG组足细胞MDA含量升高,SOD、CAT和GSH-Px活性下降,ILK蛋白表达量下降(P<0.05)。与HG组相比,HG+30μg/mlAS-IV组MDA含量下降,SOD、CAT和GSH-Px活性升高,ILK蛋白表达量水平升高(P<0.05);HG+15μg/mlAS-IV组及HG+5μg/mlAS-IV组差异无统计学意义(P<0.05)。结论:黄芪甲苷(30μg/ml)的抗氧化应激作用对高糖诱导的足细胞损伤具有一定保护作用,可能与下调整合素连接激酶ILK系统有关。     

Calcium channel blockers inhibit proliferation and matrix production in rat mesangial cells: possible mechanism of suppression of AP-1 and CREB activities

BACKGROUND: Calcium channel blockers (CCBs) are reported to attenuate the loss of renal function in various glomerulonephritides. METHODS: To determine the mechanism of action of these drugs, we investigated the effects of CCBs on cell proliferation and extracellular matrix (ECM) production in cultured rat mesangial cells. RESULTS: While stimulation with 5% fetal calf serum (FCS) increased [(3)H]thymidine and [(3)H]proline incorporation into quiescent mesangial cells, incubation with nifedipine and cilnidipine inhibited the increase in a dose-dependent manner. Northern blot analysis demonstrated that 5% FCS increased the expression of transforming growth factor beta (TGF-beta) and fibronectin (FN) mRNA and that CCBs significantly reduced this induction, indicating that CCBs may reduce ECM production through inhibiting TGF-beta and FN. Since activator protein 1 (AP-1) regulates cell proliferation and TGF-beta expression, we evaluated the AP-1 activity by gel mobility shift analysis. Nuclear extracts of FCS-treated cells showed a strong binding to AP-1-specific oligonucleotides which was suppressed by CCBs, suggesting that these agents may inhibit cell proliferation by suppressing AP-1. CCBs also inhibited the binding activity of cyclic adenosine monophosphate responsive element binding protein which regulates FN gene expression. However, neither CCBs nor FCS affected the NFkappaB activity. CONCLUSION: These results suggest that CCBs may, in part, inhibit the progression of glomerulonephritis through non-hemodynamic actions that include the suppression of mesangial cell proliferation and the production of ECM.     

Developmental damage, increased lipid peroxidation, diminished cyclooxygenase-2 gene expression, and lowered prostaglandin E2 levels in rat embryos exposed to a diabetic environment

Previous experimental studies suggest that diabetic embryopathy is associated with an excess of radical oxygen species (ROS), as well as with a disturbance of prostaglandin (PG) metabolism. We aimed to investigate the relationship between these pathways and used hyperglycemia in vitro (embryo culture for 24-48 h) and maternal diabetes in vivo to affect embryonic development. Subsequently, we assessed lipid peroxidation and gene expression of cyclooxygenase (COX)-1 and -2 and measured the concentration of prostaglandin E2 (PGE2) in embryos and membranes. Both hyperglycemia in vitro and maternal diabetes in vivo caused embryonic dysmorphogenesis and increased embryonic levels of 8-epi-PGF2alpha, an indicator of lipid peroxidation. Addition of N-acetylcysteine (NAC) to the culture medium normalized the morphology and 8-epi-PGF2alpha concentration of the embryos exposed to high glucose. Neither hyperglycemia nor diabetes altered COX-1 expression, but embryonic COX-2 expression was diminished on gestational day 10. The PGE2 concentration of day 10 embryos and membranes was decreased after exposure to high glucose in vitro or diabetes in vivo. In vitro addition of NAC to high glucose cultures largely rectified morphology and restored PGE2 concentration, but without normalizing the COX-2 expression in embryos and membranes. Hyperglycemia/diabetes-induced downregulation of embryonic COX-2 gene expression may be a primary event in diabetic embryopathy, leading to lowered PGE2 levels and dysmorphogenesis. Antioxidant treatment does not prevent the decrease in COX-2 mRNA levels but restores PGE2 concentrations, suggesting that diabetes-induced oxidative stress aggravates the loss of COX-2 activity. This may explain in part the antiteratogenic effect of antioxidant treatment.