高糖对人肾小球系膜细胞SREBP-1、FAS表达的影响

目的探讨高糖环境中人肾小球系膜细胞（human mesangial cells,HMC）SREBP-1、FAS表达。方法体外培养HMC细胞,随机分为正常糖组、高糖组,免疫细胞化学、Western Blot和RT-PCR方法检测固醇调节元件结合蛋白1（sterol regulatory element binding protein-1,SREBP-1）和脂肪酸合酶（fatty acid synthase,FAS）表达。采用脂质体转染技术将特异性SREBP-1质粒引入细胞内并进行表达,进一步采用RT-PCR方法检测脂肪酸合酶FAS的表达。结果与正常糖组比较,高糖培养的人肾小球系膜细胞固醇调节元件结合蛋白1前体和成熟体以及FAS mRNA表达均升高,差异有统计学意义。质粒转染后HMC细胞经免疫组化和Western blot检测证实特异性质粒能够在细胞内高表达SREBP-1蛋白,进一步对FAS的检测证实了FAS mRNA表达升高。结论高糖可诱导人肾小球系膜细胞固醇调节元件结合蛋白1和FAS表达增强且SREBP-1和FAS之间存在有直接关系。     

X盒子结合蛋白1对肾小管上皮细胞脂质代谢的影响研究

目的明确拼接型和非拼接型X盒子结合蛋白1对肾小管上皮细胞脂质代谢的影响。方法体外培养人肾小管上皮细胞,采用脂质体2000转染拼接型和非拼接型XBP-1质粒,培养48h后,免疫细胞化学和Western blot检测XBP-1表达,反转录PCR检测脂肪酸合成酶和乙酰辅酶A羧化酶mRNA表达,免疫细胞化学检测脂肪分化相关蛋白表达,油红O检测细胞内脂滴。结果 HKC细胞转染XBP-1拼接型和非拼接型质粒48h后,免疫细胞化学和Western blot证实XBP-1蛋白均明显升高。而拼接型XBP-1质粒转染上调了FASN和ACC mRNA表达,细胞内ADRP表达升高,细胞内脂滴增多。非拼接型XBP-1质粒对FASN、ACC mRNA,ADRP和细胞内脂滴未产生影响。结论拼接型XBP-1上调可导致肾小管上皮细胞脂质代谢关键酶升高和细胞内脂质沉积,可能是多种肾脏脂质沉积疾病的调控因子之一。     

Wnt/β-catenin信号途径在高糖诱导肾小管上皮细胞转分化中的作用

目的探讨Wnt/β-catenin信号途径在高糖诱导肾小管上皮细胞转分化中的作用。方法体外培养人近端肾小管上皮细胞（HKC）,分为正常糖组、甘露醇对照组及高糖组。采用免疫细胞化学观察β-连环蛋白（β-catenin）表达情况;Westernblot检测Wnt4、β-catenin、E-钙粘蛋白（E-cadherin）和α-平滑肌肌动蛋白（α-SMA）表达水平;逆转录-聚合酶链反应检测Wnt4和β-cateninmRNA表达水平。结果高糖组较正常糖及渗透浓度对照组Wnt4蛋白及mRNA、α-SMA蛋白表达增高,E-cadherin表达降低,β-catenin总蛋白及mRNA水平无明显变化,细胞浆及核内蛋白表达增强。高糖刺激肾小管上皮细胞Wnt4及核β-catenin蛋白表达呈时间依赖性,于高糖刺激后12h增强,24h达到高峰。结论Wnt/β-catenin信号通路可能参与了高糖介导的肾小管上皮细胞转分化过程。     

白介素-1β对高糖刺激的人肾小管上皮细胞转分化的影响

目的探讨炎症细胞因子白介素-1β（interleukin-1βIL-1β）对高糖刺激的人肾小管上皮细胞转分化的影响。-方法体外培养人肾近曲小管上皮细胞株（HKCs）,随机分为正常对照组（5.5 mmol/L normal glucose）;高糖组（30 mmol/L high glucose）;高糖＋IL-1β（5ng/ml）组。分别于处理后24h、48h、72h收集细胞,采用免疫细胞化学染色和Western蛋白印迹法检测细胞角蛋白-18（cytokeratin-18 CK-18）、α-平滑肌肌动蛋白（α-smooth muscle actinα-SMA）水平。结果高糖能够诱导肾小管上皮细胞α-SMA蛋白的合成增加,而肾小管上皮细胞的标志物CK-18的表达逐渐减少;IL-1β与高糖同时刺激可使肾小管上皮细胞α-SMA蛋白表达进一步增多,而其自身标志物CK-18的表达则明显下降。结论炎症因子IL-1β能增强高糖对肾小管上皮细胞转分化的作用。     

高脂喂养大鼠肾小管上皮细胞SREBP-1、TGF-β1、α-SMA的表达和细胞外基质沉积

目的:探讨高脂喂养对大鼠肾脏小管上皮细胞SREBP-1、TGF-β1、α-SMA表达和细胞外基质(ECM)的影响。方法:高脂饲料喂养大鼠12周后,油红O检测肾脏脂质沉积,Masson染色检测肾小管间质细胞外基质沉积,免疫组化、Western blot和原位杂交检测SREBP-1、TGF-β1、α-SMA和FN的表达。结果:高脂喂养后大鼠体重明显增加,血糖、甘油三酯和胰岛素均升高,油红O检测显示大鼠肾小管上皮细胞内出现明显脂滴。SREBP-1蛋白和mRNA在肾小管上皮细胞内表达,高脂组高于正常对照组,分别是正常组的1.88倍和1.85倍;TGF-β1和α-SMA也定位于肾小管上皮细胞胞浆并出现上调。Masson染色显示高脂喂养大鼠肾间质ECM沉积增多,纤维粘连蛋白FN检测也显示模型组表达强于对照组。结论:高脂饮食喂养可能通过上调肾脏小管上皮细胞SREBP-1表达使细胞内脂滴沉积,并进一步诱导TGF-β1、α-SMA合成而导致细胞外基质堆积。     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

p38 MAPK介导高糖诱导的肾小管上皮细胞向间充质细胞转变

本文旨在观察p38MAPK与高糖诱导的肾小管上皮细胞向间充质细胞转变之间的关系。将雄性Sprague—Dawley（SD）大鼠随机分为对照组、糖尿病组、胰岛素治疗组,用免疫组织化学、Western blot检测p38MAPK和磷酸化p38MAPK（P—p38MAPK）蛋白表达。采用机械分离和酶消化获取SD大鼠肾小管节段,进行肾小管上皮细胞培养,将肾小管上皮细胞分为对照组、高渗组（20mmol／L D—mannitol）、高糖组（20mmol／L D—glucose）和SB202190（p38MAPK特异性抑制剂）＋高糖组,处理72h后收集细胞,用免疫细胞化学检测α-平滑肌肌动蛋白（α—smooth muscleactin,α-SMA）、p-p38MAPK和Snaill蛋白表达,Western blot检测p38MAPK、p-p38MAPK、Snaill、转化生长因子β1（transforming growth factor—β1,TGF-β1）、α-SMA和E-cadherin的表达,RT-PCR检测α-SMA和E-cadherin mRNA的表达。体内和体外结果均显示,高糖状态激活了p38MAPK,这种活化作用在体内可因胰岛素控制血糖而被消除,在体外可被p38MAPK特异性抑制剂SB202190显著抑制;高糖组α-SMA蛋白和mRNA在原代培养肾小管上皮细胞的表达较对照组分别增加12倍和8倍（P〈0．01）,SB202190处理组其表达则较高糖组分别减少67％和50％（P〈0．01）。SB202190不影响TGF—β1蛋白表达,但下调Snaill蛋白表达,并部分恢复高糖组E—cadherin蛋白和mRNA的表达。上述结果提示,p38MAPK可能通过转录因子Snaill介导高糖诱导的肾小管上皮细胞向间充质细胞转变。     

糖基化终末产物（AGEs）诱导下SOCS基因在肾小管上皮细胞中的表达及意义

目的观察SOCS-1、SOCS-3在肾小管上皮细胞（HKC）中的基础表达及在糖基化终末产物（AGEs）诱导下的表达及意义。方法体外培养HKC细胞,随机分为正常对照组、AGEs组,倒置显微镜观察细胞形态学改变,采用流式细胞术,免疫细胞化学和检测SOCS-1、SOCS-3蛋白表达,RT-PCR法检测HKCSOCS-1、SOCS-3 mRNA表达。结果与正常组比较,AGES诱导的肾小管上皮细胞发生形态学改变;免疫细胞化学、流式细胞学发现SOCS-1、SOCS-3表达以胞浆为主,散在胞核表达;12、24、48hSOCS-1、SOCS-3蛋白表达AGEs组均高于正常对照组,差异有统计学意义,其中SOCS-1以12h表达量最大,SOCS-3以24h表达最高,RT-PCR检测SOCS-3 mRNA表达量,AGEs组高于正常组,差异有统计学意义。结论SOCS-1、SOCS-3在正常肾小管上皮细胞中有基础表达,AGEs可诱导肾小管上皮细胞SOCS-1、SOCS-3表达上调,为我们进一步研究SOCS基因与糖尿病肾病的关系提供了理论依据。     

CTRP3通过调节Sirt1参与高糖条件下肾小管细胞的胆固醇转运

该文探究了C1q/肿瘤坏死因子相关蛋白-3(C1q/TNF-related protein 3, CTRP3)在高糖条件下肾小管细胞胆固醇转运中的作用及机制。体外培养人肾小管上皮细胞HK-2,随机分为正常糖对照组(NG)、正常糖+CTRP3干预组(NG+CT)、高糖培养组(HG)、高糖培养+CTRP3干预组(HG+CT)、高糖培养+CTRP3干预组+siRNA转染组(HG+CT+siRNA)和高糖培养+CTRP3干预组+Sirt1 siRNA转染组(HG+CT+siSirt1)。试剂盒检测细胞内胆固醇含量及胆固醇流出情况; Filipin染色观察细胞内胆固醇蓄积情况;试剂盒检测Sirt1酶活性; Western blot检测CTRP3、Sirt1、ABCA1及LXRα的蛋白表达;实时定量PCR检测CTRP3的mRNA表达。结果显示,重组CTRP3蛋白干预能够抑制高糖条件下HK-2细胞胆固醇蓄积,上调ABCA1及LXRα的表达从而促进胆固醇外流;同时增强Sirt1的蛋白表达及活性;应用Sirt1 siRNA抑制Sirt1的表达后CTRP3的上述调控作用均消失了。以上结果提示, CTRP3对高糖条件下的肾小管细胞的保护作用,可能是部分通过调控Sirt1的表达促进高糖条件下肾小管细胞的胆固醇外流实现的。     

降压通络方对大鼠肾小管上皮细胞凋亡的影响

目的:研究降压通络方对大鼠肾小管上皮细胞凋亡的影响。方法:体外培养大鼠肾小管上皮细胞,经过缺血缺氧,将大鼠肾小管细胞随机分为4组:正常组、模型组、降压通络方组、缬沙坦组。采用CCK-8检测细胞增殖情况,免疫组化法检测大鼠肾小管上皮细胞p-AKT蛋白的表达,蛋白免疫印迹法检测大鼠肾小管上皮细胞凋亡相关基因调控蛋白Bcl-2、Bax的表达,对Bcl-2、Bax蛋白的表达水平进行相关性分析。结果:缺血缺氧呈时间依赖性抑制大鼠肾小管上皮细胞活性;免疫组化结果示:p-AKT在正常组呈高表达,模型组低表达,降压通络方组p-AKT表达明显高于模型组(P0.01),缬沙坦组表达低于模型组(P0.05);蛋白免疫印迹法结果示:模型组Bcl-2表达较正常组明显降低(P0.01),降压通络方组和缬沙坦组Bcl-2的表达均高于模型组(P0.05,P0.01);模型组Bax表达较正常组明显升高(P0.01),降压通络方组和缬沙坦组Bax的表达均较模型组降低(P0.05);Bcl-2蛋白与Bax蛋白表达呈负相关性(r=-0.811,P0.01)。结论:降压通络方可抑制大鼠肾小管上皮细胞凋亡,其作用机制可能与上调p-AKT、Bcl-2蛋白,下调Bax蛋白表达有关。     

SREBP-1基因过表达促进人肾小管上皮细胞甘油三酯形成

目的 SREBP-1重组质粒转染人肾小管上皮细胞(HKC)检测SREBP-1基因表达和细胞内脂滴的关系。方法体外培养人肾小管上皮细胞并随机分为空白对照组、pcDNA3.1空质粒对照组和pcDNA3.1-SC1重组质粒转染组,采用阳离子脂质体法将SREBP-1特异性质粒pcDNA3.1-SC1及pcDNA3.1空质粒转染到细胞内并培养48小时,半定量RT-PCR和Westernblot分析目标基因表达丰度的变化,并采用油红O染色检测细胞内脂滴。结果 pcDNA3.1-SC1重组质粒转染的细胞内SREBP-1mRNA表达呈现明显升高,扩增条带积分光密度值分别是空白对照组和阴性对照组的6.158倍和4.194倍,SREBP-1蛋白也出现明显上调,条带积分光密度值为3.092±0.254。空白对照组和pcDNA3.1阴性对照组细胞内均未见有红染脂滴颗粒,而pcDNA3.1-SC1重组质粒转染组中出现了清晰的红染颗粒。结论 SREBP-1表达可增加人肾小管上皮细胞脂肪合成证实HKC细胞中SREBP-1表达和脂滴形成之间存在有直接关系。     

Phospho-mTOR: A novel target in regulation of renal lipid metabolism abnormality of diabetes

The activation of Akt has been proved to involve in the lipogenesis of diabetic nephropathy. However, it's still not clear whether mTOR, another main gene in PI3K/Akt pathway, is also involved in the renal lipogenesis of diabetes. In the present study, it was revealed that the phosphorylation of mTOR was up-regulated in the renal tubular cells of diabetic rats, followed by the over-expression of SREBP-1, ADRP and lipogenesis. Again, high glucose increased the expression of phospho-mTOR accompanied with SREBP-1 and ADRP up-regulation and lipid accumulation in HKC cells. Rapamycin, known as mTOR inhibitor, was used to inhibit the activation of mTOR, which prevented effectively high glucose-induced SREBP-1 up-regulation and lipogenesis in HKC cells. Furthermore, high glucose-stimulated HKC cells transfected with wild-type mTOR vector showed the enhanced SREBP-1 and lipid droplets, however, TE mTOR vector (kinase dead)-transfected HKC cells presented resistance to high glucose and decreased SREBP-1 expression and lipogenesis. These above data suggested that phospho-mTOR mediated lipid accumulation in renal tubular cells of diabetes and might be the potential targets for treating lipogenesis of diabetic nephropathy.     

肥胖大鼠模型的建立及其脂代谢相关分子机制研究

目的建立饮食诱导的肥胖(diet-induced obesity,DIO)大鼠模型并初步探讨其发病的分子机制。方法用脂肪含量30%的高脂饲料饲喂雄性SD大鼠25周,观察大鼠体重、Lee’s指数、肝组织病理改变,检测大鼠空腹血糖及空腹血清胰岛素水平,并通过real-time PCR,检测成模大鼠肝脏中乙酰辅酶A羧化酶(ACC)、脂肪酸合酶(FAS)、激素敏感酯酶(HSL)以及固醇调节元件结合蛋白-1c(SREBP-1c)的表达变化。结果高脂饲料饲喂6周后,DIO组大鼠体重、Lee’s指数均显著增加;25周后肝脏脂肪异常蓄积,出现中重度脂肪肝,空腹血糖及胰岛素水平显著升高,出现明显的胰岛素抵抗。肝脏中ACC、FAS和HSL表达显著增加,SREBP-1c表达水平达到正常组的2.56倍,两组间差异极其显著。结论成功建立了DIO大鼠模型,通过检测脂代谢相关基因的表达水平,初步阐释了营养性肥胖的发生与脂代谢变化之间的关系,SREBP-1c,ACC,FAS和HSL参与了DIO的形成,从而初步揭示了脂代谢变化与营养性肥胖的发生的关系。     

Sterol regulatory element binding protein (SREBP) -1 mediates oxidized low-density lipoprotein (oxLDL) induced macrophage foam cell formation through NLRP3 inflammasome activation

Macrophage foam cell formation (FCF) has long been known to play a critical role during atherosclerotic plaque development. In the presence of atherogenic molecules such as oxidized low-density lipoprotein (oxLDL) macrophages accumulate massive amounts of lipid through uptake. However, in the presence of oxLDL mechanism of dysregulated lipid homeostasis in the macrophages remains largely unknown. Herein we have investigated the role of Sterol regulatory element binding protein (SREBP)-1 in oxLDL-induced inflammation and altered lipid homeostasis in macrophages. The U937 monocytes and monocyte-derived macrophages (MDMs) were stimulated with different doses of oxLDL. MTT assay to study the effect of oxLDL on cell viability, Oil-Red-O (ORO) staining to observe cytosolic lipid accumulation, semi-quantitative PCR and Western blotting to analyze mRNA and protein expressions, respectively, and spectrophotometric assay to measure the lipid synthesizing enzyme's activity were performed. Our results indicate that oxLDL increased proliferation in monocytes and decreased the viability in MDMs in a time- and dose-dependent manner. The oxLDL (100 μg/ml) enhanced lipid accumulation via increased expressions of SREBP-1 and its downstream proteins such as fatty acid synthase (FAS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) at both RNA and protein levels in monocytes as well as in MDMs. Inhibiting SREBP-1 by a synthetic inhibitor prevented excessive lipid accumulation by downregulating the expression of its downstream proteins. Further, oxLDL increased reactive oxygen species (ROS) levels, NLRP3 inflammasome activation and active interleukin 1β (IL-1β) release in both the cell types. The oxLDL-induced NLRP3 could be responsible for SREBP-1 and downstream proteins overexpression as siRNA silencing of NLRP3 decreased SERBP-1 levels. In summary, we have demonstrated that SREBP-1 could be a key player in oxLDL-induced excessive lipid accumulation leading to macrophage FCF via ROS-mediated NLRP3/IL-1β/SREBP-1 pathway.     

1α,25(OH)2D3 alleviates high glucose–induced lipid accumulation in rat renal tubular epithelial cells by inhibiting SREBPs

Lipid accumulation is a vital event in the progression of diabetic nephropathy. 1,25-Dihydroxyvitamin D3 (1α,25(OH)₂D₃) is considered to have a protective effect on diabetic nephropathy. However, it remains unclear whether 1α,25(OH)₂D₃ can inhibit lipid accumulation, and the potential mechanisms responsible for lipid metabolism are incompletely understood. In this study, we evaluated the effects of 1α,25(OH)₂D₃ on lipid metabolism in high glucose–exposed rat renal tubular epithelial NRK-52E cells. Results indicated that high glucose–enhanced lipid accumulation in NRK-52E cells and 1α,25(OH)₂D₃ can remarkably decrease high glucose–induced lipid accumulation. Western blot showed that 1α,25(OH)₂D₃ alleviated high glucose–induced upregulation of sterol regulatory element-binding protein-1c (SREBP-1c) and SREBP2, along with their established target genes fatty acid synthase (FASN) and hydroxymethylglutaryl CoA reductases (HMGCR). Overall, these findings suggest that 1α,25(OH)₂D₃ downregulated the expressions of SREBPs to inhibit high glucose–induced lipid accumulation, which provides new sights into the protective effects of 1α,25(OH)₂D₃ on diabetic nephropathy.