TSP-1 promotes glomerular mesangial cell proliferation and extracellular matrix secretion in Thy-1 nephritis rats

The proliferation of glomerular mesangial cells (GMC) and secretion of the extracellular matrix (ECM) in rat with Thy-1 nephritis (Thy-1N) resembling human mesangioproliferative glomerulonephritis have been explored for many years; however, the molecular mechanisms of GMC proliferation and ECM production remain unclear. Our previous studies have demonstrated that the thrombospondin-1 (TSP-1) gene was involved in mediating rat GMC proliferation and ECM synthesis induced by sublytic C5b-9 in vitro. In the present study, the roles of the TSP-1 gene in GMC proliferation, ECM production, and urinary protein secretion in Thy-1N rats were determined by using TSP-1 small hairpin RNA, and the results revealed that silencing of the TSP-1 gene in rat renal tissues could diminish GMC proliferation (P < 0.01) and ECM secretion (P < 0.01) as well as urinary protein secretion (P < 0.05) in Thy-1N rats. Together, the current findings suggested that TSP-1 gene expression was required for GMC proliferation and ECM production in Thy-1N rats.     

地塞米松对大鼠肾小球系膜细胞增殖和合成胶原物质的影响

本文采用大鼠肾小球系膜细胞体外培养技术，用＾３Ｈ－ＴｄＲ掺入和羟脯氨酸测定方法，观察了地塞米松对大鼠肾小球系膜细胞增殖及合成细胞外基质－胶原的影响。结果显示：地塞米松可抑制大鼠肾小球系膜细胞的增殖。     

雷帕霉素对高糖诱导的大鼠肾系膜细胞增殖、凋亡和细胞周期的影响

目的:评价雷帕霉素对高糖诱导的肾系膜细胞增殖、凋亡和细胞周期的影响,探讨其在糖尿病肾病防治中的意义。方法:体外培养的大鼠肾小球系膜细胞株HBZY-1 分为:正常对照组、高糖组、高糖加不同浓度的雷帕霉素组,应用CCK-8 法观察细胞增殖的变化;流式细胞术检测各组细胞的细胞周期和凋亡情况;Real-time PCR 法检测各组细胞中血管紧张素 (ANG )、转化生长因子β1(TGF-β1)和血管内皮生长因子(VEGF)的表达水平。结果:高糖诱导下HBZY-1 的增殖水平明显上升,凋亡水平下降,ANG ,TGF-β1 和VEGF 的表达水平上升,而雷帕霉素具有明显抑制作用,且有剂量依赖性,并下调ANG ,TGF-β1 和VEGF 的表达;对于细胞周期,高糖组的S 期细胞明显高于正常组(P<0.05);雷帕霉素干预后,S 期细胞比例减少(P<0.05)。结论:雷帕霉素能够抑制高糖状态下HBZY-1 的增殖,促进其凋亡及导致G1/ S 期阻滞,同时下调ANG ,TGF-1β和VEGF 的表达。     

Effects of kynurenine metabolites on mesangial cell proliferation and gene expression

In the present study, we examined the effects of kynurenine metabolites on cultured mesangial cells (MCs) and demonstrated for the first time that they affect MC proliferation and gene expression. Anthranilic acid and 3-hydroxy-dl-kynurenine suppressed MC proliferation by 32% and 43%, respectively at 10^− 6 M compared to the control. In contrast, quinolinic acid and l-kynurenine promoted MC proliferation by 49% and 35% at 10^− 8 M respectively, although promoting activities declined at higher concentrations. In addition, quinolinic acid upregulated the gene expression of platelet-derived growth factor-B, collagen type-Iα1, and collagen type-IVα1. However, the gene expression of hepatocyte growth factor (HGF) was downregulated. We further examined the gene expressions in the glomeruli of high serum IgA (HIGA) mice with IgA nephropathy using microarray technology and found that the gene expression of insulin-like growth factor-1 was higher, but that of HGF was lower at 40 weeks of age compared to 8 weeks of age. In Balb/c mice, the gene expression of three kynurenine pathway enzymes (kynurenine aminotransferase I, kynurenine aminotransferase II, and quinolinate phospho-ribosyltransferase) increased 2- to 3.5-fold, whereas those in HIGA mice did not change significantly. These results suggest that abnormalities in the kynurenine pathway are associated with the dysfunction of MCs and progression of chronic kidney diseases.     

AngII-induced glomerular mesangial cell proliferation inhibited by losartan via changes in intracellular calcium ion concentration

This study investigated the changes in intracellular [Ca²⁺]ⁱ (intracellular calcium ion concentration) and TRPC6 (transient receptor potential channel 6) expression during angiotensin II (AngII)-induced glomerular mesangial cell (GMC) proliferation, as well as the inhibitory effect of losartan. GMC cultures were split into four groups treated for 24 h: Group N (blank control group), Group A (10^?7 mol/L AngII), Group LT (10^?7 mol/L AngII and 10^?5 mol/L losartan), and Group Pred (10^?7 mol/L AngII and 10^?5 mol/L prednisone). GMCs proliferation was measured by the MTT and trypan blue assays. The distribution of TRPC6 was monitored by immunofluorescence, the expression of TRPC6 was detected by RT-PCR and Western blotting, and [Ca²⁺]ⁱ was measured by laser scanning confocal microscopy. The results showed that the maximal proliferation of GMCs was induced by treatment with 10^?7 mol/L AngII for 24 h. In Group A, the distribution of TRPC6 was not uniform in the cell membrane, there was increased accumulation of this protein within the cytoplasm, and the increased expression of TRPC6 and [Ca²⁺]ⁱ was consistent with the proliferation of cells. In Group LT, losartan inhibited the proliferation of GMCs significantly, the levels of TRPC6 and [Ca²⁺]ⁱ were diminished, and the distribution of TRPC6 was improved. Prednisone also significantly inhibited the proliferation of GMCs and had no effects on the expression of TRPC6 and [Ca²⁺]ⁱ in Group Pred. These findings suggested that AngII could enhance the expression of TRPC6, increase [Ca²⁺]^i, and demonstrate a time–dose–response relationship with the proliferation of GMCs, while losartan reversed the effect of AngII on GMC proliferation.     

Thrombospondin induces glomerular mesangial cell adhesion and migration.

BACKGROUND: Extracellular matrix components are known to modulate mesangial cell functions as adhesion, motility and proliferation. Among other extracellular matrix components, mesangial cells have been recently described to secrete thrombospondin (TSP), a high molecular weight glycoprotein, produced by several cell types, and known to play a role in embryogenesis, wound healing, angiogenesis, and tumorigenesis. The aim of this work was the functional and molecular characterization of TSP interactions with mesangial cells. EXPERIMENTAL DESIGN: Adhesion of mesangial cells to TSP-coated plastic, and chemotaxis in the Boyden chamber assay were tested. In order to identify TSP active domains, heparin, known to bind to the amino-terminal region of TSP, four monoclonal antibodies directed against specific domains of the molecule, and TSP fragments obtained by enzymatic digestion were used. RESULTS: We found that TSP induces mesangial cell adhesion and chemotaxis, in a dose dependent manner. Adhesion was inhibited by antiserum against TSP, and by an anti-CD36 monoclonal antibody tested in the presence of heparin, but not by the peptide Gly-Arg-Gly-Asp-Ser. We have also found that only the carboxy-terminal end of TSP retains the adhesive properties of the molecule, while all the fragments tested showed some degree of chemotactic activity. CONCLUSIONS: We conclude that TSP modulates mesangial cell adhesion and motility, thus acting as a potential autocrine and paracrine regulator of mesangial cell functions in normal and pathologic conditions.     

白细胞介素10基因转移抑制肾小球系膜细胞诱生炎症细胞因子

目的：通过建立白细胞介素１０（ＩＬ－１０）的重组逆转录病毒载体基因转移系统,观察ＩＬ－１０对脂多糖（ＬＰＳ）诱导的大鼠肾小球系膜细胞（ｇｌｏｍｅｒｕｌａｒｍｅｓｅｎｇｉａｌｃｅｌ,ＧＭＣ）中细胞因子的产生及其基因表达的影响。方法：通过构建的重组逆转录病毒载体ｐＬＸ（ＩＬ－１０）ＳＮ将外源基因ＩＬ－１０转移至大鼠ＧＭＣ：（１）应用聚合酶链反应（ＰＣＲ）,反转录聚合酶链反应（ＲＴ－ＰＣＲ）和ＥＬＩＳＡ检测ＩＬ－１０基因的整合和表达;（２）以ＲＴ－ＰＣＲ观察ＩＬ－１０基因转移对ＬＰＳ诱导的ＧＭＣ肿瘤坏死因子－α（ＴＮＦ－α）的ｍＲＮＡ表达的影响,以ＥＬＩＳＡ测定白细胞介素－１β（ＩＬ－１β）和ＴＮＦ－α的蛋白质表达。结果：外源性ＩＬ－１０基因已整合到靶细胞染色体ＤＮＡ并有效地表达,它能抑制ＬＰＳ诱生ＧＭＣ过度产生ＩＬ－１β,ＴＮＦ－α。结论：外源性ＩＬ－１０基因可以转移到ＧＭＣ并稳定表达,它能抑制ＧＭＣ炎症效应中细胞因子的产生及其基因表达。     

The importance of cellular adhesion for mesangial cell proliferation in serum sickness nephritis of the rat: A co-culture study of glomerular macrophages and mesangial cells

The role of cell-cell contact on activating mesangial cell proliferation by nephritic macrophage was investigated. Nephritic glomerular macrophages were obtained from serum sickness nephritis (SSN) rat kidneys at 14 days after the cessation of sensitization, when proliferating cells were most increased in the glomeruli in the course of the SSN. The effect of the nephritic macrophages on mesangial cell proliferation was greater than that of control by co-culture allowing cellular contact. However, nephritic macrophages did not enhance mesangial cell proliferation by co-culture without direct contact even though the nephritic macrophages were activated with lipopolysaccharides. Conditioned medium from co-culture of the nephritic macrophages and mesangial cells did not enhance mesangial cell proliferation. Anti-intercellular adhesion molecules (ICAM)-1 antibody inhibited mesangial cell proliferation by direct co-culture dose-dependently. From these results, cellular contact was important for stimulation of mesangial cell proliferation by macrophages and ICAM-1 participated in these interactions.     

Basic fibroblast growth factor stimulates glomerular mesangial cell proliferation through a protein kinase C-independent pathway.

Basic Fibroblast Growth Factor (bFGF) is shown to be a potent mitogen for cultured glomerular mesangial cells. bFGF induces an increase in cell number and stimulates DNA synthesis measured by [3H]thymidine incorporation in normal as well as in protein kinase C-depleted cells. The ED50 observed in both cases are nearly identical (approximately 0.04 nM) and maximal responses are obtained at 1 nM. Staurosporine, a potent protein kinase C inhibitor, does not prevent bFGF from inducing mitogenesis. On the contrary, the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the bradykinin derivative Des-Arg9bradykinin that we have previously shown as mitogens for mesangial cells, fail to trigger DNA synthesis or cell proliferation upon staurosporine treatment or in protein kinase C-depleted cells. bFGF is unable to induce the association of the enzyme to membranes, the so-called translocation process, although the growth factor induces a slight production of diacylglycerol. Using a highly resolutive two-dimensional electrophoresis, we show that bFGF, in contrast to TPA, is unable to stimulate the phosphorylation of a Mr 80,000/pI 4.5 protein, a major and specific protein kinase C substrate. By contrast, bFGF stimulates the phosphorylation of a Mr 28,000/pI 5.7-5.9 protein in normal as well as in protein kinase C-depleted cells while TPA induces this protein phosphorylation only in normal cells. Our results suggest that bFGF exerts its proliferative action on mesangial cells through a protein kinase C-independent pathway and that the growth factor does not activate anyway the enzyme in this cell type.     

系膜增殖性肾炎中花生四烯酸产物对系膜细胞增殖的作用

在抗Ｔｈｙ１．１抗体引起的大鼠系膜增殖性肾炎中，肾小球的增殖性细胞核抗原（ＰＣＮＡ）阳性细胞增多，＾３Ｈ－胸腺嘧啶核苷（＾３Ｈ－ｔｈｙｍｉｄｉｎｅ，＾３Ｈ－ＴｄＲ）掺入法测定的肾小球增殖活性（ＧＰＡ）升高。对肾炎鼠给予５－脂氧合酶抑制剂ＭＫ８８６或血栓素（ＴＸＡ２）合成酶抑制剂Ｆｕｒｅｇｒｅｌａｔｅ或１２－脂氧合酶抑制剂Ｂａｉｃａｌｅｉｎ进行处理，可分别抑制肾小球合成的白三烯（ＬＴ）Ｂ４及１２－羟     

内皮素-1对人肾小球系膜细胞层黏连蛋白的影响

内皮素-1(endothelin-1,ET-1)是一种强烈的缩血管活性肽,与血管紧张素Ⅱ(Ang Ⅱ)具有很多相似之处,且二者之间具有相瓦促进作用[1].阻断肾素-血管紧张素(RAS)系统已被广泛应用于慢性肾脏病(CKD)的治疗,但是ET系统拮抗剂还未得到重视.本实验通过研究ET-1及其受体拮抗剂(Bosentan)对层黏连蛋白(laminin,LN)代谢的影响,为ET-1受体拮抗剂应用于CKD的治疗提供依据.     

Relationship between glomerular mesangial cell proliferation and amyloid deposition as seen by ultrastructural and morphometric analysis in experimental kala-azar of the hamster

Light and electron microscopic studies combined with a morphometric analysis of the hamster glomerulus in experimental kala-azar showed progressive hyperplasia of mesangial cells beginning on the 10th day and reaching a peak on the 20th day after infection. Afterward, the number of mesangial cells declined and a progressive rise of amyloid deposits over the mesangial matrix was observed. This system for amyloid production is unique if we consider that probably one cell, the mesangial cell, is involved in glomerular amyloid deposition. Our data support a slight modification in the sequence of events of the biphasic theory of amyloid formation. We observed that the number of mesangial cells declines when amyloid deposition increases and that mesangial cell morphology in this stage is not that of an actively secreting cell. It is therefore hypothesized that amyloid precursor material is secreted into the matrix during the proliferative phase. In the second phase, amyloid deposits occur in the extracellular media close to functionally impaired mesangial cells.     

TLR/STAT通路在HMGB1诱导的系膜细胞增殖中的作用

目的:探讨TLR/STAT通路在HMGB1诱导的系膜细胞增殖中的作用.方法:将体外培养的人系膜细胞分为正常对照组和HMGB1刺激组,培养6、12和24小时后收集细胞,免疫细胞化学检测PCNA表达的变化;免疫细胞化学和流式细胞术检测TLR2蛋白表达的变化;PCR技术检测STAT1/STAT3mRNA表达的变化.结果:人重组HMGB1刺激系膜细胞后,能够促使其增殖;与正常对照组相比,HMGB1刺激组中TLR2蛋白表达增强;与正常对照组相比,HMGB1刺激组中STAT1和STAT3mRNA表达增强;TLR2蛋白与STAT1、STAT3mRNA表达均呈显著正相关(r=0.830,P＜0.001;r=0.926,P＜0.001);PCNA阳性表达率与STAT1、STAT3mRNA表达亦呈显著正相关(r=0.817,P＜0.001;r=0.863,P＜0.001).结论:HMGB1可能通过与其受体蛋白TLR2结合,进而激活STAT1/STAT3,促进系膜细胞增生,从而导致肾脏损害.     

Diffuse mesangial cell proliferation in focal sclerosing glomerulonephritis

Morphometrical and clinical investigations were performed in 34 patients with the so-called hypercellular form of focal glomerulosclerosis (FGS), i.e., a form showing clear diffuse mesangial hypercellularity beside focal sclerosis with the light microscope. This form was compared with focal glomerulosclerosis without remarkable mesangial hypercellularity, with mild mesangioproliferative glomerulonephritis (gn), as well as with normal kidneys.The results were as follows:

Defensin α1对大鼠系膜细胞增殖的影响

目的： 探讨defensin α₁ 对体外培养的大鼠肾小球系膜细胞株1097增殖的影响以及可能的机制。方法： 利用MTT掺入观察不同浓度的基因重组人类defensin α₁在不同作用时间对系膜细胞增殖的影响，选择能够刺激系膜细胞生长的浓度及作用时间给予U0126预孵育，观察能否阻断defensin α₁促系膜细胞增殖作用，使用Western blotting了解defensin α₁对系膜细胞ERK1/2磷酸化及Ⅳ型胶原合成的影响。结果： Defensin α₁在3-20 mg/L 范围内对系膜细胞具有促生长作用，12 h达到峰值（P＜0.01）。当浓度大于20 mg/L时可抑制系膜细胞的生长，浓度在15-25 μmol/L U0126可以阻断defensin α₁对系膜细胞的促生长作用。浓度为3 mg/L defensin α₁刺激5 min后可使ERK1/2磷酸化明显增加，作用12 h后系膜细胞的IV胶原蛋白表达高于对照组并持续到48 h（P＜0.01）。结论： 一定浓度的defensin α₁可以促进系膜细胞的增殖和细胞外基质的合成，这种促增殖作用可能是通过MAPK途径实现的。     

Akt/NF-κB信号途径在免疫复合物诱导肾系膜细胞增殖中的作用

目的 :观察免疫复合物 (IC)能否诱导体外培养的肾小球系膜细胞 (MC)增殖及其Akt/NF κB信号在其中的作用。方法 :实验设对照组、刺激组及Akt1正义 (SODN)、错义 (MSODN)与反义寡核苷酸 (ASODN)组。寡核苷酸组 :以lipofectin分别介导Akt1SODN、MSODN、ASODN转染MC 8h ;对照组和刺激组 :以lipofectin作用MC 8h。然后刺激组和寡核苷酸组均用聚合IgG(aggregatedIgG ,AIgG ,一种标准的IC模型 )刺激 ,对照组用等量的单体IgG刺激。用MTT比色法检测细胞的增殖 ,流式细胞术分析MC的细胞周期 ,RT PCR分析mRNA的表达 ,Westernblot检测蛋白的表达 ,电泳迁移率变动实验 (EMSA)检测NF κB的活性。结果 :IC可诱导MC中NF κB的活化 ,上调细胞周期蛋白cyclinD1的mRNA及蛋白表达 ,促进MC进入S期。Akt1ASODN可抑制Akt1蛋白的表达 ,抑制IC诱导的MC中NF κB活性 ,抑制cyclinD1mRNA及其蛋白的表达 ,进而抑制IC促进MC进入S期和增殖。Akt1SODN、MSODN则无上述抑制作用。结论 :IC可通过Akt/NF κB信号途径 ,介导体外培养的MC增殖。提示Akt/NF κB信号途径 ,可作为干预IC诱导MC过度增生的一个新的治疗靶点。     

Effects of human immunodeficiency virus sera and macrophage supernatants on mesangial cell proliferation and matrix synthesis.

Patients with human immunodeficiency virus (HIV) infection are prone to the development of focal segmental glomerulosclerosis, a lesion in which increased mesangial cell proliferation and matrix synthesis may play a role. We undertook the present study to determine whether HIV sera may affect mesangial cell proliferation and matrix synthesis either directly or indirectly via effects on macrophage supernatants. Pooled HIV sera was found to significantly enhance (P < 0.01) mesangial cell proliferation in a concentration-related manner. Mesangial cell proliferation was significantly suppressed by two medications commonly utilized in HIV-infected patients, azidothymidine and trimethoprim/sulfamethoxazole, and was not significantly altered by lipopolysaccharide, suggesting that these medications as well as recurrent infection are unlikely to account for the proliferative effect of HIV sera. Supernatants from HIV sera-treated macrophages were found to significantly enhance (P < 0.01) mesangial cell incorporation of [3H]proline, a marker for synthesis of the matrix component collagen, compared to supernatants from control sera-treated macrophages. These results suggest that HIV sera may directly enhance mesangial cell proliferation and may indirectly increase mesangial cell matrix synthesis by altering macrophage secretory products. These effects may play a role in the development of glomerulosclerosis in patients with HIV infection.     

Modulation of glomerular mesangial cell growth and prostaglandin release by T-lymphocyte products

Inflammation Research -     

Akt／NF-kB信号途径在免疫复合物诱导肾系膜细胞增殖中的作用

目的：观察免疫复合物(IC)能否诱导体外培养的肾小球系膜细胞(MC)增殖及其Akt/NF—kB信号在其中的作用。方法：实验设对照组、刺激组及Akt1正义(SODN)、错义(MSODN)与反义寡核苷酸(ASODN)组。寡核苷酸组：以lipofectin分别介导Akt1 SODN、MSODN、ASODN转染MC8h；对照组和刺激组：以lipofectin作用MC8h。然后刺激组和寡核苷酸组均用聚合IgG(aggregated IgG，AIgG，一种标准的IC模型)刺激，对照组用等量的单体IgG刺激。用MTT比色法检测细胞的增殖，流式细胞术分析MC的细胞周期，RT-PCR分析mRNA的表达，Westem blot检测蛋白的表达，电泳迁移率变动实验(EMSA)检测NF—KB的活性。结果：IC可诱导MC中NF-kB的活化，上调细胞周期蛋白cyclinD1的mRNA及蛋白表达，促进MC进入S期。Akt1 ASODN可抑制Akt1蛋白的表达，抑制IC诱导的MC中NF—kB活性，抑制cyclinD1 mRNA及其蛋白的表达，进而抑制IC促进MC进入S期和增殖。Akt1 SODN、MSODN则无上述抑制作用。结论：IC可通过Akt/NF—kB信号途径，介导体外培养的MC增殖。提示Akt／NF-kB信号途径，可作为干预IC诱导MC过度增生的一个新的治疗靶点。     

Signalling mechanisms in sphingosine 1-phosphate-promoted mesangial cell proliferation

BACKGROUND: The bioactive sphingolipid sphingosine 1-phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet-derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P-induced proliferation of mesangial cells is poorly known. RESULTS: We examined the signalling mechanisms by which S1P and dihydro-S1P (DHS1P), another S1P receptor agonist, induce mesangial cell proliferation. We first observed that exogenous S1P/DHS1P had additive effects on the PDGF-promoted proliferation of mesangial cells. Treatment of mesangial cells with pertussis toxin almost completely inhibited S1P- and DHS1P-induced, and slightly inhibited PDGF-induced cell proliferation. Additionally, the ERK kinase inhibitor PD98059 partially blocked the proliferation of mesangial cells induced by all these ligands. N,N-dimethylsphingosine, a competitive inhibitor of SPHK, reduced PDGF-induced mesangial cell proliferation, whereas over-expression of SPHK promoted it. We also revealed that PDGF induces SPHK mRNA expression and SPHK activity, suggesting that SPHK, which links the PDGF to the S1P signalling cascade, is, at least in part, involved in PDGF-induced mesangial cell proliferation. Moreover, we found that extracellular S1P stimulates two S1P receptors, EDG3 and EDG5, which leads to cell proliferation and survival. CONCLUSIONS: The data show that S1P-induced mesangial cell proliferation is mediated by EDG-dependent and -independent signalling pathways. S1P may cooperate with PDGF to increase the proliferation of mesangial cells during pathophysiological processes.