褪黑素对高糖条件下培养的大鼠系膜细胞增殖、凋亡和细胞周期的影响

糖尿病肾病（DN）晚期肾脏细胞凋亡与肾功能恶化程度成正比，抑制凋亡则可延缓肾小球硬化。褪黑素（MT）是松果体分泌的神经内分泌激素，肾脏是其靶器官之一。研究显示MT对DN肾脏有明显的保护作用，但MT对DN晚期肾小球系膜细胞（MC）凋亡的影响尚未检索到有关报道。本试验选用大鼠MC为研究对象，来观察MT对高糖诱导的MC凋亡的影响。     

Cl-/base exchange in rat mesangial cells: regulation of intracellular pH

The present study was designed to determine whether rat glomerular mesangial cells possess Cl- -dependent intracellular pH (pHi) regulatory processes. Rat glomerular mesangial cells were grown to confluence on glass coverslips. Intracellular pH (pHi) was measured with BCECF. Steady state pHi in HCO3- containing solutions was 7.08 +/- 0.03 (N = 13). When extracellular Cl- was acutely removed, pHi increased at a rate of 0.57 +/- 0.03 pH/min units (N = 8), P less than 0.001. DIDS (0.5 mM) significantly decreased the rate of increase in pHi to 0.34 +/- 0.04 pH/min, P less than 0.01. Na+ removal and amiloride (1 mM) did not alter the increase in pHi induced by Cl- removal. Steady state pHi in the absence of Cl- was significantly increased above control, 7.39 +/- 0.02 (N = 7), P less than 0.001. Following the acute alkalinization of pHi by CO2 removal, pHi recovered at a rate of 0.07 +/- 0.01 pH/min (N = 9). In the absence of Cl-, the pHi recovery rate was significantly decreased to 0.01 +/- 0.008 pH/min (N = 5), P less than 0.01. DIDS (0.5 mM) significantly decreased the rate of pHi recovery to 0.02 +/- 0.01 pH/min (N = 5), P less than 0.01. Na+ removal and amiloride (1 mM) had no effect on the rate of pHi recovery following acute alkaline loading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.

In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.     

褪黑素抑制高糖诱导的大鼠肾小球系膜细胞凋亡

目的探讨褪黑素（MT）对于高糖诱导的肾小球系膜细胞（MC）凋亡中的作用及机制。方法光学显微镜观察大鼠MC凋亡的形态学变化;流式AnnexinV／PI双染法检测不同浓度MT对大鼠MC细胞凋亡率的影响;免疫细胞化学检测细胞色素C的表达改变。结果MT对30mmol／L葡萄糖诱导的MC高凋亡率有明显的抑制作用,呈剂量依赖性关系;并能明显抑制细胞色素C的表达。结论MT可抑制高糖诱导的大鼠MC高凋亡率,这种作用部分通过调控细胞色素C来实现的。     

Effect of elevated glucose on endothelin-induced store-operated and non-store-operated calcium influx in renal mesangial cells

Early diabetic nephropathy exhibits renal glomerular hyperfiltration and an increase in renal plasma flow. The hyperfiltration is a dysfunctional state that may arise from a hyperglycemic-induced hypocontractility of glomerular mesangial cells that may be associated with depressed Ca(2+) signaling events. The present study was designed to determine the effects of acute (minutes) and chronic (days) elevated glucose levels on endothelin-induced calcium signaling with a particular emphasis on the potential influence on stores and store-operated Ca(2+) influx (SOCI; also called capacitative calcium entry) in glomerular mesangial cells. Primary cultures of rat mesangial cells were grown in either high (30 mM) or normal (5 mM) glucose-containing media and tested in the presence of either high (30 mM) or normal (5 mM) glucose levels. Intracellular calcium levels were monitored with the calcium-sensitive fluorophore fura-2 before and after treatment with either endothelin-1 (10 nM), to induce typical Ca(2+) signals, or the endoplasmic reticulum (ER) Ca-ATPase inhibitor thapsagargin (1 microM), to unload ER Ca(2+) stores. Both acute and chronic exposure to high glucose levels depressed the endothelin-induced calcium signal. However, neither release of Ca(2+) from stores nor SOCI were depressed by high glucose levels. In contrast, an endothelin-induced calcium entry pathway (likely receptor-operated calcium influx), separate from SOCI, was markedly depressed in the presence of both acute and chronic high glucose levels. The depressant effect of high glucose was rapidly (minutes) reversible upon returning to normal glucose levels. It is concluded that high glucose levels depress endothelin-induced calcium signaling in rat mesangial cells by inhibiting non-SOCI Ca(2+) entry pathways, namely the receptor-operated Ca(2+) influx pathway. The glucose-induced alterations in the receptor-operated calcium influx pathway may, in part, contribute to the depressed contractile state of glomerular cells during periods of hyperglycemia.     

磷脂酶D对高糖培养的肾小球系膜细胞骨架的影响

目的 研究高糖环境下肾小球系膜细胞（GMC）内磷脂酶D（PLD）的活性改变及其对细胞骨架的影响。方法 对高糖（30mmol/L)刺激48h的大鼠GMC，用酶联比色法测定磷脂酰胆碱专一性磷脂酶D（PC-PLD）活性，底物磷酸化法检测蛋白激酶C（PKC）的活性。用免疫荧光标记和共聚焦显微镜显示并测量F-actin的表达。结果 高糖刺激48h后，GMC内PC-PLD和PKC活性明显增高，而F-actin荧光表达减少，排列紊乱，给予PC-PLD抑制剂后，高糖培养的GMCPKC活性明显下降。F-actin的荧光表达和排列都得到显著改善。结论 高糖时PLD活性增高可以通过PKC途径影响GMC骨架的组装状态。改变系膜细胞收缩功能。     

Antioxidants modulate adenosine metabolism in rat mesangial cells cultured under high glucose conditions

Glomerular mesangial cells play a major role in glomerular hemodynamics, considered also as antigen-presenting cells participating in immune response. Mesangial dysfunction and proliferation are typical lesions of diabetic glomerulopathy. Adenosine, a local hormone, produced by mesangial cells is a metabolic regulator of renal blood flow, capable of decreasing glomerular filtration rate (GFR), exerting immunosuppressive, antiproliferative and anti-inflammatory properties. Since it was well established that antioxidants confer protection against increased oxidative stress that occurs in diabetes, the effect of captopril, reduced glutathione and melatonin on adenosine metabolism was investigated. Glomerular mesangial cells obtained from collagenase treated glomeruli, isolated from renal cortex of Sprague-Dowley rats, were grown under high glucose conditions (30 mmol/L) as a model of diabetic microenvironment. The activity of adenosine metabolizing enzymes: 5'-nucleotidease (5'-NU) responsible for its production and adenosine deaminase (ADA) responsible for its degradation were investigated. Hyperglycemic conditions led to decreased adenosine production via 5'-NU and decreased removal via ADA. Captopril, given in therapeutic concentration induced enzyme activities in normoglycemic conditions and restored hyperglycemia-induced decrease. In order to investigate if the presence of SH groups may be responsible for this improvement, the cells were exposed to reduced glutathione, and it exerted almost equal effect, given in physiological and higher concentrations. Melatonin increased 5'-NU activity only in physiological glucose conditions. Presented results confirm potential renoprotective effect of SH-group containing antioxidant supplementation during diabetes in restoring adenosine metabolism.     

蛋白激酶C激活在高糖诱导肾系膜细胞中的作用

目的：探讨高糖对系膜细胞蛋白激酶C（PKC）活性的影响及PKC在系膜细胞增殖、细胞外基质积聚中的作用。方法：采用大鼠系膜细胞进行体外培养,高糖作为激动剂,佛波酯（PMA）作为PKC抑制剂,甘露醇作为渗透压对照,用液闪仪测定PKC活性,^3H-TdR渗入法检测细胞增殖,ELISA法测定培养上清中纤维连接蛋白（FN）含量。结果：高糖可增加系膜细胞颗粒部分PKC活性、抑制细胞增殖、促进FN分泌,且与渗透压无关。抑制PKC后,可阻止高糖诱导的FN分泌。结论：高糖可激活系膜细胞PKC,促进细胞外基质积聚和糖尿病肾症的发生。     

罗格列酮对高糖刺激的大鼠系膜细胞β1整合素和cyclinD1表达的影响

目的观察过氧化物酶体增殖物激活受体(PPAR)γ激动剂噻唑烷二酮类化合物(罗格列酮)对高糖刺激大鼠肾小球系膜细胞(GMC)β1整合素和细胞周期正调控蛋白cyclinD1表达的影响,为早期糖尿病肾病(DN)防治寻求新的理论依据.方法体外培养大鼠HBZY-1 GMC,分为6组正常糖组、高糖组、甘露醇组、高糖分别加1、5、10 μmol/L罗格列酮组.干预作用24 h后采用间接免疫荧光染色及流式细胞仪检测β1整合素表达,细胞免疫组化染色检测cyclinD1表达,四唑盐比色(MTT法)测定细胞增殖.结果高糖刺激使GMC的β1整合素、cyclinD1表达增加,细胞增殖能力增强,且不依赖于渗透浓度.罗格列酮可抑制高糖刺激GMC的β1整合素、cyclinD1表达,使细胞增殖能力降低,且呈剂量依赖性.相关分析表明β1整合素与cyclinD1、β1整合素与细胞增殖、cyclinD1与细胞增殖间均呈正相关.结论黏附分子参与了DN发病机制,PPAR-γ可能通过参与调节黏附机制及其介导的细胞周期调控而影响DN早期GMC增殖和系膜扩张.     

Growth inhibition of rat mesangial cells by high glucose: effect of vitamin E

Background. We aimed to examine both whether vitamin E prevented and whether it reversed the growth inhibitory effect of high glucose. Methods. For the preventive experiment, rat mesangial cells (RMC) were grown in control glucose medium with the addition of 100 μM of vitamin E. High glucose (27.5 mM) was added to the medium concurrent with the vitamin E addition. The 3-(4,5-di-methylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay was used to measure RMC proliferation. Our data confirmed the growth inhibitory effect of high glucose and showed that the growth inhibition was prevented by vitamin E. To examine whether vitamin E reversed the growth inhibitory effects of high glucose, RMC were grown in control and high glucose medium. Contrary to previous prevention type studies, vitamin E was not added to the medium until growth inhibition of the RMC by the high glucose was established. Results. Our data show that it took 5 days of vitamin E administration to reverse the growth inhibitory effect of high glucose. Conclusion. This is the first time that vitamin E has been shown to reverse this high-glucose-induced inhibition of RMC, suggesting that vitamin E reverses a potentially important pathogenetic process. Received: January 6, 1999 / Accepted: May 20, 1999     

Retinoic acid regulation of mesangial cell apoptosis

Retinoic acid (RA) is recently used for the treatment of experimental glomerular diseases. However, mechanisms underlying its therapeutic effects are largely unknown. We recently reported that RA has the potential for protecting certain cells from particular injury. A typical example is its effect on oxidant-induced apoptosis of mesangial cells. Mesangial cells exposed to hydrogen peroxide undergo apoptosis through activation of the c-Jun N-terminal kinase activator protein 1 pathway. RA dramatically inhibits this process via suppression of c-fos/c-jun expression and inhibition of the c-Jun N-terminal kinase activation. The anti-apoptotic effect of RA is mediated by both nuclear receptor dependent and nuclear receptor independent mechanisms and is, at least in part, mediated by induction of mitogen-activated protein kinase phosphatase 1. In this review, we briefly summarize the current knowledge on molecular mechanisms involved in the anti-apoptotic effects of RA.     

高糖对人类肾小球系膜细胞的影响

目的观察高糖状态下，人类肾小球系膜细胞（ＧＭＣ）增殖，细胞内游离钙，纤维连接蛋白（Ｆｎ）及白介素－６（ＩＬ－６）的变化。方法采用体外ＧＭＣ培养，３Ｈ－胸腺嘧啶（３Ｈ－ＴｄＲ）掺入和细胞计数，Ｆｕｒａ－２双波长荧光法和ＥＬＩＳＡ法。结果高糖对ＧＭＣ增殖有抑制作用，对Ｆｎ及ＩＬ－６的生成有促进作用，而对细胞内游离钙水平无显著影响。结论高血糖可能是糖尿病肾病的启动因素     

Effect of mesangial cell lysis and proliferation on glomerular hemodynamics in the rat.

To elucidate an involvement of mesangial cells in the regulation of glomerular hemodynamics, renal micropuncture techniques and glomerular morphometry were employed in Munich-Wistar rats with mesangial cell lytic or proliferative lesions induced by administration of an antibody reactive with Thy-1.1-like antigens on the mesangial cell surface. The antibody-induced mesangial cell lysis at day 1 resulted in a significant decrease in glomerular ultrafiltration coefficient, leading to reduction in single nephron glomerular filtration rate (SNGFR) in spite of a significant increase in both glomerular hydrostatic pressure and single nephron plasma flow (SNPF). During the antibody-induced proliferative lesion at day 6, glomerular ultrafiltration coefficient and SNGFR remained reduced; however, SNPF was now decreased. Morphometric analysis showed the enlargement of capillary luminal volume and the development of new open space in the mesangium accessible for blood flow in the mesangial cell-lytic glomeruli at day 1. An increase in mesangial cell volume was found in the proliferative glomeruli at day 6. The total area of peripheral glomerular basement membrane, presumed as the probable filtration area, was unchanged in these glomeruli. These results indicate that mesangial lesions decrease glomerular ultrafiltration coefficient, and suggest that mesangial cells participate in regulation of glomerular filtration rate.     

Neuraminidase treatment of isolated rat adipocytes and differential regulation of basal and insulin-stimulated glucose transport

The role of membrane carbohydrate in the function of insulin receptors and glucose transporters was investigated with neuraminidase to release sialic acid from isolated rat adipocytes. Pretreatment of adipocytes with neuraminidase resulted in an increase in basal glucose transport. At the same time, insulin-stimulated glucose transport was reduced by an average of 30%. The enzyme action on glucose transport was not due to a nonspecific membrane perturbation because neuraminidase caused only a nonsignificant decrease in the uptake of the amino acid analog alpha-(methylamino)isobutyric acid and had no effect on basal or insulin-stimulated protein synthesis. Insulin binding was slightly increased in neuraminidase-treated cells, yet the shapes of the dose-response curves for insulin stimulation of glucose transport were similar (EC50 = 0.087 +/- 0.010 and 0.082 +/- 0.008 nM for control and treated cells, respectively). The neuraminidase-induced increase in basal transport was the result of an increase in the affinity of the glucose-transport system (Km = 7.3 +/- 1.4 and 3.6 +/- 0.7 mM before and after treatment, respectively) with no change in Vmax. Conversely, enzyme treatment decreased the Vmax of insulin-stimulated 3-O-methylglucose transport from 87.8 +/- 13.2 to 41.3 +/- 4.9 pmol.2 x 10(5) cells-1.s-1 while not altering the Km. These changes in glucose-transport activity resulting from enzyme treatment were not due to alterations in glucose-transporter concentration on the plasma membrane as measured by the D-glucose-inhibitable binding of [3H]cytochalasin B. Thus, sialic acid plays multiple roles in the control of glucose-transport activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

氯胺酮对大鼠心肌细胞内游离钙的影响

目的　探讨氯胺酮对大鼠心肌细胞内游离钙的影响及其作用机制。方法　原代培养的SD大鼠心室肌细胞 ,用钙敏荧光探针Fluo 3/AM负载染色后 ,根据实验中加入氯胺酮终浓度不同分为六组 ,采用激光扫描共聚焦显微镜 ,测定心肌细胞内钙荧光强度的基础值及加入氯胺酮后 5min以及后续加入 4 0mM氯化钾 (KCl)或 1 0mM咖啡因后细胞内钙荧光强度值。结果　 (1 )≤ 1 0 0 μM的氯胺酮对静息心肌细胞内钙荧光强度无明显影响 ,而 30 0 μM的氯胺酮降低钙荧光强度 (P <0 0 1 ) ;(2 )KCl和咖啡因使细胞内钙荧光强度较基础值均明显升高 (P <0 0 1 ) ;氯胺酮剂量依赖性地抑制KCl诱发胞内钙荧光强度升高的幅度 ,尤以 1 0 0和 30 0 μM氯胺酮组明显 ;而各浓度的氯胺酮不抑制咖啡因诱发胞内钙荧光强度的升高 (P >0 0 5 )。结论　 1、1 0和 6 0 μM的氯胺酮可能不抑制整体状态下的心肌收缩 ;而 1 0 0和 30 0 μM的氯胺酮对心肌有剂量依赖性的负性肌力效应 ,其机制可能是氯胺酮抑制了胞外Ca2 + 经膜电压依赖性钙通道内流 ,胞内游离钙浓度降低所致 ,但不影响肌浆网释钙功能。     

Effect of partial outflow obstruction on rat detrusor contractility and intracellular free calcium concentration

Partial outlet obstruction induces significant alteration in detrusor contractility. A mild obstruction can result in increased contractile force, whereas severe obstruction results in marked contractile dysfunction. The cellular mechanisms mediating these alterations in the contractile response are presently not known. In the current study, we have investigated the effect of both mild and severe partial outlet obstruction on detrusor contractility and correlated the contractile response to field stimulation, bethanechol, adenosine triphosphate (ATP), and KCl with the level of intracellular free calcium using FURA-2 fluorescence. Our results are as follows. Severe obstruction induced a significantly greater increase in bladder weight than mild obstruction. In general, mild outlet obstruction induced an increase in the contractile responses to field stimulation, bethanechol, ATP, and KCl, whereas severe outlet obstruction induced a decrease to field stimulation and ATP. In general, the stimulated increase in intracellular free calcium paralleled the contractile response. The results indicate that the alterations in the contractile response to field stimulation and pharmacological stimulation induced by partial outlet obstruction may be mediated by altered calcium translocation and intracellular release. © 1994 Wiley-Liss, Inc.     

罗格列酮对高糖环境中大鼠肾小管上皮细胞的影响

罗格列酮（RGZ）是一种噻唑烷二酮类（TZDs）胰岛素增敏剂，是核转录因子过氧化物酶体增殖物激活受体1（PPARγ）的配体。除了具有改善机体胰岛素抵抗等代谢调节作用外，TZDs还具有抗炎、影响细胞增殖和转型、抗脏器纤维化等作用，与肾脏疾病有着密切的联系。但TZDs对肾小管细胞直接作用的报道国内外较少，我们利用体外实验观察RGZ对高糖条件下肾小管细胞的影响。     

内皮素-1对高糖溶液培养下肾小管上皮细胞转分化的影响

目的 探讨内皮素-1对高糖溶液培养肾小管上皮细胞转分化的影响和可能的机制.方法 采用甲基噻唑法检测0.1～1 000 nmol/L内皮素-1对肾小管上皮细胞增殖的影响.采用无小牛血清的低糖DMEM培养基（一种含各种氨基酸和葡萄糖的培养基）同步化肾小管上皮细胞24 h,分为4组,分别采用低糖DMEM、高糖DMEM、高糖DMEM加100 nmol/L内皮素-1、高糖DMEM加100 nmol/L内皮素-1和转化生长因子-β1中和抗体培养肾小管上皮细胞.分别检测肾小管上皮细胞内转化生长因子-β1、α-平滑肌肌动蛋白和E-钙黏蛋白mRNA和蛋白的表达.结果 0.1～1 000nmol/L内皮素-1对肾小管上皮细胞增殖无明显影响,内皮素-1明显上调大鼠近端肾小管上皮细胞α-平滑肌肌动蛋白mRNA和蛋白表达,下调E-钙黏蛋白mRNA和蛋白的表达;内皮素-1明显升高大鼠近端肾小管上皮细胞转化生长因子-β1 mRNA和蛋白的表达;转化生长因子-β1中和抗体干预后,大鼠近端肾小管上皮细胞表达的E-钙黏蛋白和mRNA明显高于内皮素-1组,而α-平滑肌肌动蛋白和mRNA明显低于内皮素-1组.结论 糖尿病肾脏病中,内皮素-1可促进肾小管上皮向肌成纤维细胞及间质细胞转分化,可能通过转化生长因子-β1发挥作用.