低分子量肝素对肾小球系膜细胞及细胞外基质作用的研究

目的：探讨低分子量肝素（LMWH）对体外,体内肾小球系膜细胞及细胞外基质增生的影响。方法：体外试验在培养人肾小球系膜细胞中进行;以氮蓝四唑盐方法测定LMWH对系膜细胞增殖的影响;间接竞争抑制酶联免疫吸附法观察LMWH对细胞外基质成分纤维连接蛋白（FN）的影响。体内试验在大鼠抗Thy1.1系膜增生性肾小球肾炎模型（MnsPGN)中进行,用定量病理分析观察LMWH对系膜细胞增殖和系膜基质增殖的影响。结果：体外试验显示,LMWH明显抑制血清刺激的系膜细胞增殖（与对照组比较,P＜0．05）,呈量依赖关系;并减少系膜细胞培养上清及细胞层的FN含量（与对照组比较,P＜0．05）,体内试验中,MsPGN大鼠LMWH治疗组第5及第14d的系膜细胞增殖,系膜基质蓄积均明显减轻（与对照组比较P＜0．05）,结论LMWH能够抑制肾小球系膜细胞增殖及细胞外基质增生。     

通脉口服液对肾小球系膜细胞增殖及白细胞介素β1、白细胞介素10表达的影响

目的：探讨中药复方“通脉口服液”对大鼠肾小球系膜细胞（MsC）增殖及其产生白细胞介素融（IL-β1）和白细胞介素10（IL-10）的影响。方法：应用细胞培养技术进行肾小球系膜细胞的传代培养，采用血清药理学方法，制备通脉口服液含药血清，采用四甲基偶氮唑盐（MTT）法测定通脉口服液含药血清对过度增殖状态下肾小球系膜细胞增殖的影响，酶联免疫吸附法（ELISA）及逆转录聚合酶链反应（RT—PEn）法测定系膜细胞IL—β1 mRNA和IL-10mRNA表达及其蛋白水平。结果：通脉口服液含药血清在5％～20％浓度范围明显抑制脂多糖（LPS）刺激的系膜细胞增殖；在观察的2个时间点（6h、24h）上，LPS均可刺激系膜细胞IL-β1 mRNA的表达及提高IL-β1 分泌水平，在6h时间点上LPS可刺激系膜细胞IL-10mRNA的表达及提高IL-10分泌水平；而通脉口服液在10％浓度上能够明显抑制LPS诱导的系膜细胞IL-β1 分泌及其mRNA的表达，在6h时间点上促进LPS诱导的系膜细胞IL-10分泌及其mRNA的表达。结论：中药复方“通脉口服液”可抑制肾小球系膜细胞的增殖及LPS诱导的系膜细胞IL-β1 的产生，促进LPS诱导的系膜细胞IL-10的产生；肾小球系膜细胞是通脉口服液防治慢性肾炎，延缓肾小球硬化的重要靶细胞之-。     

百令胶囊对大鼠肾小球系膜细胞增殖、Ⅳ型胶原及TGF-β1 mRNA表达的影响

目的：观察百令胶囊对高糖刺激后系膜细胞增殖、Ⅳ型胶原（typeⅣCollagen,ⅣC）、转化生长因子-β1（TGF-β1）mRNA表达的影响。方法：培养正常SD大鼠系膜细胞,高浓度葡萄糖刺激系膜细胞,加入百令胶囊、苯那普利含药血清后,用四甲基偶氮唑盐（MTT）光吸收法观察肾小球系膜细胞增殖;酶联免疫吸附法（ELISA）测量培养细胞上清液中ⅣC的含量;实时荧光逆转录聚合酶链反应（real-time RT-PCR）测定TGF-β1mRNA的表达。结果：百令胶囊、苯那普利含药血清在24 h可抑制高糖对大鼠肾小球系膜细胞的促增殖作用,72 h最为明显（P〈0.01）;高糖刺激后ⅣC、TGF-β1mRNA在肾小球系膜细胞内表达增多（P〈0.01）,百令胶囊、苯那普利含药血清能明显抑制肾小球系膜细胞增殖（P〈0.01）、ⅣC分泌及TGF-β1mRNA的高表达（P〈0.05）。百令胶囊与苯那普利组之间差异无统计学意义（P〉0.05）。结论：百令胶囊可降低高糖致系膜细胞增殖,抑制高糖所致ⅣC分泌,抑制高糖引起的TGF-β1mRNA过度表达,这可能是其防治糖尿病肾病的作用机制。     

糖肾汤对糖基化蛋白诱导培养下系膜细胞MMP-2表达的影响

目的：研究糖基化蛋白诱导培养下，糖肾汤对大鼠肾小球系膜细胞表达基质金属蛋白酶2（MMP-2）活性及mRNA的影响。方法：采用血清药理学及体外大鼠肾小球系膜细胞培养方法，用糖基化牛血清白蛋白（AGE-BSA）刺激肾小球系膜细胞，以明胶酶谱法检测培养上清MMP-2的活性，并提取细胞RNA，采用RT-PCR法检测系膜细胞MMP-2mRNA表达。结果：糖基化蛋白可抑制MMP-2的活性及mRNA表达，糖肾汤能提高MMP-2活性及mRNA表达。结论：糖肾汤对糖尿病肾病金属蛋白酶系统紊乱具有一定的调节作用。     

OX-LDL诱导活化的巨噬细胞对肾小球系膜细胞增殖的影响

目的:采用细胞共培养方法,研究氧化低密度脂蛋白(OX-LDL)诱导活化的巨噬细胞对肾小球系膜细胞增殖的影响.方法:用OX-LDL刺激大鼠腹腔巨噬细胞,ELISA测定上清液中IL-1浓度鉴定巨噬细胞活化程度;用普通培养皿和细胞共培养皿分别将活化巨噬细胞上清液和活化巨噬细胞与大鼠肾小球系膜细胞共培养;用MTT法和流式细胞检测细胞周期测定细胞增殖.结果:OX-LDL刺激后大鼠腹腔巨噬细胞上清液中IL-1浓度明显升高;无论是活化的巨噬细胞还是其上清液均能明显促进系膜细胞增殖,但以共培养组最为显著.结论:在细胞共培养状态下由于脂质诱导活化的巨噬细胞及其上清液可能通过促进系膜细胞增殖导致肾脏损伤.     

游离脂肪酸对大鼠肾小球系膜细胞增殖和细胞周期的影响

目的：探讨游离脂肪酸（FFAs）对体外培养的大鼠肾小球系膜细胞增殖和生长周期的影响。方法：用不同浓度的游离脂肪酸处理大鼠HBZY-1细胞株（即大鼠肾小球系膜细胞）24h～72h。采用噻唑蓝比色（MTT）法检测内皮细胞增殖情况,流式细胞术（FCM）分析法测定细胞周期变化。结果：游离脂肪酸可抑制HBZY-1细胞的生长增殖（与对照组比较,P〈0.01）,且这种抑制作用具有剂量和时间依赖性;游离脂肪酸作用于HBZY-1细胞24h、48h、72h,细胞周期发生明显改变,G1期细胞数增多,S期细胞数减少（与对照组比较,P〈0.01）。结论：游离脂肪酸可通过停滞细胞生长于G1期,抑制大鼠肾小球系膜细胞的生长增殖。     

白细胞介素13抑制肾小球系膜细胞增殖及白细胞介素1β的分泌

目的：探讨白细胞介素13（IL-13）对体外培养的大鼠系膜细胞的增殖及其产生白细胞介素1β（IL-1β）的影响。方法：用四甲基偶氮唑（MTT）法测定系膜细胞增殖,用酶联免疫吸附（ELISA）法测定培养系膜细胞的上清液IL-1β蛋白水平。结果：IL-13在1.0、10、100ng/ml浓度范围呈剂量依赖性地抑制系膜细胞的增殖;含5?S的RPMI1640培养状态下的系膜细胞几乎检测不出IL-1β分泌,加入LPS可诱导系膜细胞分泌IL-1β,IL-13在抑制系膜细胞的增殖的相应浓度范围可显地抑制诱导的系膜细胞IL-1β。结论：IL-13对体外培养的系膜细胞增殖具有抑制作用 ,IL-13可能对肾小球系膜细胞炎症具有拮抗作用。     

川芎嗪对高糖培养的大鼠肾小球系膜细胞增殖和氧化应激的影响

目的 探讨川芎嗪对高糖培养的大鼠肾小球系膜细胞增殖和氧化应激的影响.方法 体外培养大鼠肾小球系膜细胞株,分对照组、高糖组和川芎嗪组,采用CCK-8细胞计数法测定系膜细胞增殖,以比色法检测细胞培养液中的超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）、丙二醛（MDA）含量的变化.结果 与对照组比较,高糖组出现系膜细胞增殖增加,上清液中SOD活性、GSH含量下降,MDA含量增加;与高糖组相比,川芎嗪组系膜细胞增殖减少,GSH含量、SOD活性上调,MDA含量下降.结论 川芎嗪能抑制高糖环境下的系膜细胞增殖,并显著减少高糖诱导的氧化应激水平.     

肾必宁冲剂加S-9对肾小球系膜细胞凋亡及其相关因素的影响

目的：探讨肾必宁冲剂对肾小球系膜细胞凋亡及凋亡相关基因ICE和BcI-2表达的影响，旨在阐明其治疗系膜增生性肾病有效的作用机制，为临床利用该药治疗以系膜增生为核心病理环节的肾脏疾病提供实验理论依据。方法：采用肾小球系膜细胞培养方法进行体外 研究，有不同剂量与S－9共同孵育的肾必宁冲剂及含肾必宁大鼠血清刺激4－6代系细胞16h,MTT法测定系膜细胞增殖指数；TUNEL法定量观察凋亡细胞；琼脂糖凝胶电泳法观察细胞核小体DNA的断裂现象，逆转录－聚合酶链反应技术（RT－PCR）检测凋亡相关基因ICE和bcI-2的表达变化。结果：加入肾必宁冲剂（S－9）16h后，系膜细胞出现了典型的凋亡征象，诸如细胞内深棕色颗粒形成、核固缩、碎裂和DNA梯形条带等，凋亡率显高于空白对照组（P＜0．01）；同时肾必宁各组ICE表达亦明显高于空白血清对照组，而抑制凋亡的bcI-2基因各组均表达不明显。结论：肾必宁冲剂可诱导系膜细胞凋亡，促进肾小球增殖性病变消散，并可通过调控ICE和bcI-2基因影响凋亡。     

三七总皂苷对大鼠肾小球系膜细胞增殖及细胞周期的影响

目的：研究三七总皂苷（PNS）对大鼠肾小球系膜细胞（MC）增殖以及细胞周期的影响。方法：用不含或含有高、中、低浓度PNS的RPMI-1640细胞培养液刺激体外培养的正常大鼠MC。于实验终点,运用MTT比色法测定细胞增殖情况以及PNS的细胞毒性作用;运用流式细胞术测定细胞周期。结果：PNS能够显著抑制血清诱导的大鼠MC的增殖;通过减少MC进入S＋G2/M期,从而抑制MC的有丝分裂;且在本实验浓度内（100-400μg/ml）不具有明显的诱导凋亡作用和细胞毒性。结论：PNS能通过抑制MC的有丝分裂而抑制其活化、增殖,在慢性肾脏疾病的治疗中具有一定得应用价值。     

Cerivastatin inhibits fetal calf serunvinduced DNA synthesis in cultured rat mesangial cells

SUMMARY: Inhibition of mevalonate synthesis by several statins has been shown to suppress DNA synthesis in glomerular mesangial cells. In the present study, we investigated the effect of a new statin, cerivastatin, on fetal calf serum (FCS)-induced DNA synthesis of cultured rat mesangial cells. Cultured rat mesangial cells were stimulated by 10% FCS in the presence or absence of cerivastatin and mevalonate. 5-bromo-2-deoxyuridine (BrdU) incorporation was used to assess DNA synthesis. the present study showed that 10% FCS caused marked stimulation of DNA synthesis in the mesangial cells. Cerivastatin inhibited FCS-stimulated BrdU incorporation in a dose-dependent manner. IC₅₀ was approximately 1 umol/L. Exogenous mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate significantly prevented the inhibitory effect of cerivastatin on DNA replication. It appears that cerivastatin, by inhibiting the synthesis of mevalonate, may suppress DNA synthesis in the mesangial cells.     

Inhibition of geranylgeranylation suppresses the proliferation of rat cultured mesangial cells

SUMMARY: Mesangial hypercellularity is a critical early histopathological finding observed in human and experimental glomerular diseases. the regulation of mesangial cell proliferation may be crucial in the treatment of glomerular injury. Ras proteins are guanosine triphosphatases that regulate proliferation and differentiation by transducing biological information from extracellular signals to the nucleus. the blocking of Ras function by inhibitors of prenyltransferase has been shown to suppress the proliferation in several kinds of cell types. the objective of the present study was to examine the effects of selective inhibitors of farnesylation (FTI-277) and geranylgeranylation (GGTI-286) on proliferation of cultured rat mesangial cells. the incubation of mesangial cells with FTI-277 and GGTI-286 with stimulation by either 10% fetal calf cell (FCS) or platelet-derived growth factor (PDGF) was performed, and bromo-deoxyuridine (BrdU) incorporation was measured. Ras processing and mitogen-active protein (MAP) kinase activation, after the incubation with 10% FCS, were also examined. Geranylgeranylation caused a dose-dependent reduction of FCS or PDGF-stimulated BrdU incorporation. Geranylgeranylation also inhibited the activation of MAP kinase, but not Ras processing, stimulated by 10% FCS. However, FTI-277 had little effect on DNA synthesis and MAP kinase activation induced by FCS or PDGF, despite the inhibition of Ras processing. the present study showed that GGTI-286 inhibited the proliferation of rat cultured mesangial cells. It appears that the antiproliferative effect of the geranylgeranyl transferase I inhibitor may be useful in preventing mesangial cell proliferation.     

Simvastatin modulates angiotensin II signaling pathway by preventing Rac1-mediated upregulation of p27

Recent experimental observations have suggested that statins may exert modulatory effects on a number of pathobiological processes beyond their cholesterol-lowering properties. Some of the pleiotropic effects of statins seem to be mediated by their ability to block the synthesis of isoprenoid intermediates, which serve as important lipid attachments required for the proper function and activation of the small GTP-binding proteins. The current study explored the modulatory effects of simvastatin (SMV) on the angiotensin II (Ang II)-induced Rac1-mediated, upregulation of cyclin-dependent kinase inhibitor p27. Ang II (100 nM) stimulation of rat mesangial cells induced a significant increase in p27 protein expression. Co-treatment of cells with SMV (1 microM) inhibited Ang II-induced upregulation of p27 protein. Addition of mevalonate (200 microM) or geranylgeranyl pyrophosphate (5 microM) reversed the inhibitory effect of SMV on p27 protein expression, suggesting that the effect of SMV is geranylgeranyl dependent. This study also provides evidence for a sequential link between Ang II stimulation and downstream activation of Rac1, intracellular H2O2 production, and Akt kinase leading to upregulation of p27 protein in mesangial cells. It was also shown that SMV, by inhibiting Rac1 activity, reversed Ang II-induced increase in intracellular H2O2 production, Akt activation, and p27 protein expression. The data presented in this study not only elucidate Ang II-mediated signaling cascade in mesangial cells but also demonstrate for the first time the modulatory effects of SMV on Ang II-induced signaling pathway at the cell cycle level.     

Inhibitory effect of statins on renal epithelial-to-mesenchymal transition

BACKGROUND/AIM: Recent studies have suggested that statins may play a role in the protection against renal failure which is independent of cholesterol reduction. Activation of RhoGTPases is a key step in renal tubular cells' epithelial-to-mesenchymal transition (EMT) which contributes to renal interstitial fibrosis. We hypothesized that statins could act by inhibiting the synthesis of the isoprenoids, such as geranylgeranyl pyrophosphate, which is essential for membrane attachment and biological activity of RhoGTPases, RhoA and Rac1. METHODS: Human proximal tubular epithelial cells (HK2) were used to examine the inhibitory effect of statins on EMT induced with medium conditioned by activated peripheral blood mononuclear cells. RESULTS: Our study demonstrates that the statins lovastatin, simvastatin, and pravastatin inhibit HK2 cells to undergo EMT. Inhibition of EMT in HK2 cells with these statins resulted in a reduction of RhoA and Rac1 activation in both the cytoplasmic and membrane-bound forms, in preservation of the expression of the epithelial cell markers E-cadherin and cytokeratin-19, and in a decrease in Fn-EDA expression, a marker for the myofibroblast phenotype. The decreased levels of activated RhoA and Rac1 in both the cytoplasmic and membrane fractions of the cells were reversed by geranylgeranyl pyrophosphate and mevalonate, and thus attributable to the inhibition of isoprenylation of RhoGTPases by statins. CONCLUSION: This phenomenon could explain the beneficial effect of statins on EMT and on renal fibrosis prevention.     

Ramipril inhibits in vitro human mesangial cell proliferation and platelet-derived growth factor expression.

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive drugs that have been shown to reduce proteinuria and to slow down the progression of renal function deterioration in different models of chronic glomerular disease. Major pathogenetic features of progressive glomerular injury leading to glomerulosclerosis are mesangial cell proliferation and platelet-derived growth factor (PDGF) expression. The aim of the present study was to evaluate the effect of ramipril, an ACE inhibitor, on these two potential therapeutic targets. Thus, the effect of ramipril on DNA synthesis, cell proliferation and PDGF A and B chain gene expression in fetal calf serum (FCS)-activated cultured human glomerular mesangial cells was investigated. DNA synthesis was evaluated by tritiated thymidine incorporation, cell proliferation by direct cell counting and cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). PDGF A and B chain gene expressions were studied by Northern blot and RT-PCR, respectively. In a dose-dependent manner ramipril inhibited the FCS-induced DNA synthesis and cell proliferation. This effect was not dependent upon a toxic effect as demonstrated by MTT. The antiproliferative effect of ramipril was most likely independent of its ability to inhibit ACE present in the FCS and/or expressed by the cells, since a synthetic peptide that specifically inhibits ACE, at the same molar concentrations, did not inhibit FCS-stimulated DNA synthesis. Moreover, ramipril significantly reduced FCS-induced PDGF A and B chain gene expression. Finally, ramipril completely abolished the PDGF A and B chain gene expression induced by phorbol 12-myristate 13-acetate, a specific protein kinase C activator, suggesting a site of action downstream of this enzyme in the mitogenic signal transduction pathway. Our study would suggest that the modulatory action of ramipril on activated mesangial cell proliferation and PDGF expression is independent of its ability to inhibit ACE and could represent an additional mechanism in the renal protective effects of this drug.     

Prostaglandins and rat glomerular mesangial cell proliferation

Arachidonate metabolites modulate glomerular mesangial cell contractility through specific receptors coupled to phospholipase C or adenylate cyclase. The resulting intracellular signals, including changes of cytosolic Ca2+, pH, and cyclic adenosine 3'5'-monophosphate (cAMP) are known to also regulate the growth of many cell types. Since eicosanoids have been shown to interfere with cell proliferation in culture, we studied DNA synthesis and cell number in rat mesangial cell cultures exposed to a selective phospholipase C activator, prostaglandin F2 alpha (PGF2 alpha), or to the cAMP-stimulating PGI2 analogue, Iloprost. PGF2 alpha dose-dependently enhanced DNA synthesis and cell proliferation in the presence of insulin, with an EC50 of 0.1 microM. This eicosanoid potentiated the effects of platelet-derived growth factor (PDGF) or low concentrations of serum. Maximum stimulatory potency was about one-third that of PDGF. Removal of PGF2 alpha after short-term stimulation (30 min) did not reverse its mitogenic effect. Iloprost had no effect on DNA synthesis of quiescent cells, but potently inhibited growth stimulated by various concentrations of fetal serum. PG released within the glomerular microcirculation may play a regulatory role in both normal and deranged mesangial cell growth.     

洛伐他汀对系膜细胞凋亡及Bcl-2和Bax基因表达的影响

目的 观察洛伐他汀对系膜细胞凋亡及Bcl-2和Bax基因表达的影响。并探讨其作用的可能机制。方法 用不同浓度的洛伐他汀处理大鼠系膜细胞，用流式细胞仪观察细胞凋亡指数，吖啶橙荧光活体染色观察细胞凋亡率，电镜观察凋亡细胞形态，免疫印迹法及细胞免疫结合图文分析观察洛伐他汀对系膜细胞Bcl-2和Bax基因表达的影响。结果 洛伐他汀对系膜细胞凋亡有明显诱导作用，呈一定剂量依赖性；并能明显抑制Bcl-2基因表达，促进Bax基因表达，致使两者比例明显下降。结论 洛伐他汀可剂量依赖性地诱导大鼠系膜细胞凋亡，其机制可能通过对凋亡抑制或促进基因的调控，改变Bcl-2/Bax比值而诱导MC的凋亡。     

IL-6、IL-1和PDGF对大鼠肾小球系膜细胞生长的影响

目的：探讨白细胞介素－６（ＩＬ－６）、白细胞介素－１（ＩＬ－１）和血小板源生长因子（ＰＤＧＦ）对大鼠肾小球系膜细胞（ＭｓＣ）增殖的影响。方法：采用改良的ＭＴＴ法和ＢｒｄＵ掺入法检测ＳＤ大鼠体外ＭｓＣ悬液中加入ＩＬ－６、ＩＬ－１及ＰＤＧＦ后１２ｈ及２４ｈ的增殖情况。分７组进行观察。结果：ＩＬ－６组、ＩＬ－１组和ＰＤＧＦ组１２ｈ及２４ｈ的光密度（ＯＤ值）与标记指数（ＬＩ）均为２％ＦＣＳ组也均高,但所有实验组均不能达２０％ＦＣＳ组的细胞增殖水平。联合应用双因子刺激１２ｈ及２４ｈ均未见明显的协同促增殖作用。结论：ＩＬ－６、ＩＬ－１及ＰＤＧＦ均能在一定程度上刺激大鼠肾小球ＭｓＣ增生。联合应用双因子刺激无明显的协同效应。     

Ticlopidine inhibits activation of mitogen-activated protein kinase by platelet-derived growth factor in cultured rat renal mesangial cells

Background We previously found that ticlopidine inhibits the proliferation of cultured rat mesangial cells that is induced by fetal bovine serum. This study was designed to examine the effects of ticlopidine on platelet-derived growth factor (PDGF)-induced DNA synthesis and mitogen-activated protein (MAP) kinase activation in such mesangial cells to clarify the mechanism of the antiproliferative action. Methods Glomerular mesangial cells were isolated from rat kidneys, and cells were incubated with various combinations of ticlopidine, PDGF, epidermal growth factor, phorbol 12-acetate 13-myristate (PMA), cilostazol (a phosphodiesterase inhibitor), and H-89 (a cAMP-dependent protein kinase A [PKA] inhibitor). A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell count involving the trypan blue exclusion test were performed for determination of cell viability. DNA synthesis and MAP kinase activity were assessed by means of a tritiated thymidine ([³H]thymidine) incorporation assay and the Biotrak^TM MAP kinase assay system, respectively. Results Ticlopidine (1 μmol/L) significantly inhibited both PDGF-induced DNA synthesis and MAP kinase activation. Also, 1 μmol/L ticlopidine substantially blocked PMA-induced MAP kinase activation. Pretreatment with H-89 did not abolish the ability of ticlopidine to inhibit PDGF-induced MAP kinase activation, while H-89 pretreatment significantly reserved the inhibitory action of cilostazol on PDGF-induced MAP kinase activation. Conclusion These results suggest that ticlopidine might inhibit PDGF-induced DNA synthesis after MAP kinase activation by intercepting the signal transduction from c-Raf-1 to MAP kinase, independent of the cAMP-PKA pathway.