脱氢抗坏血酸对高糖诱导系膜细胞产生氧自由基的影响

目的 探讨脱氢抗坏血酸(DHA)对高糖诱导系膜细胞产生氧自由基(ROS)的影响。 方法 (1)原代培养大鼠系膜细胞；(2)以Fe3+还原法检测细胞内抗坏血酸(AA)和DHA浓度，观察系膜细胞摄取AA和DHA的情况及葡萄糖、葡萄糖转运蛋白(GLUT)抑制剂细胞松弛素B对其的影响；(3)采用激光扫描共聚焦显微镜检测细胞内ROS，观察高糖诱导系膜细胞ROS产生的情况及不同浓度DHA对其的影响；(4)采用凝胶电泳迁移率法(EMSA)检测活性蛋白1(AP-1)和DNA的结合活性，观察DHA对高糖诱导的系膜细胞内AP-1 活性的影响。结果 (1)AA不能由细胞外进入系膜细胞，而DHA可以进入，并且随着细胞外葡萄糖浓度的增加，其进入速度减慢；细胞松弛素B则完全抑制了DHA进入到系膜细胞。(2)高糖快速诱导系膜细胞ROS产生增多；DHA抑制了高糖的这种作用，并且该抑制作用在≤4 mmol/L的浓度范围内呈浓度依赖性。(3)DHA抑制了高糖诱导的系膜细胞内AP-1 的激活。 结论 (1)系膜细胞是依赖DHA利用Vit C的细胞型；DHA进入该细胞依赖GLUT介导，高糖可抑制其进入细胞。(2)DHA可有效抑制高糖诱导的系膜细胞ROS产生增多，并在一定范围内呈浓度依赖性。(3)DHA在抑制ROS产生的同时，也显著抑制了高糖诱导的AP-1 的激活。     

白蛋白诱导的肾小管细胞凋亡与丝裂素激活蛋白激酶信号通路

目的 探讨白蛋白诱导肾小管上皮细胞凋亡以及诱导凋亡的信号传导机制｡ 方法 将培养的大鼠肾小管细胞NRK-52E分别与不同浓度(10､ 20､ 30 mg/ml)的去脂无内毒素牛血清白蛋白(BSA)共同孵育6､ 12､ 18和24 h｡透射电镜､共聚焦激光显微镜和流式细胞仪检测细胞凋亡｡BSA 20 mg/ml刺激NRK-52E细胞15､ 30､ 60和120 min后, Westen印迹测定p38､氨基末端激酶(JNK)和细胞外信号调节激酶(ERK)活性｡将SB202190(20 μmol/L, p38抑制剂)､SP600125(10 μmol/L, JNK抑制剂)和PD98059(20 μmol/L, ERK抑制剂)分别与白蛋白和NRK-52E细胞共同孵育24 h后检测细胞凋亡｡结果 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡｡白蛋白与NRK-52E细胞共孵育后,p38和JNK活性明显升高,ERK活性显著降低｡SB202190和SP600125可分别抑制白蛋白诱导NRK-52E细胞凋亡,而PD98059促进白蛋白诱导的NRK-52E细胞凋亡｡结论 白蛋白以时间和剂量依赖方式诱导肾小管细胞凋亡,而p38和JNK激活与ERK抑制介导了白蛋白诱导的肾小管细胞凋亡｡     

人尿酸转运蛋白在肾小管上皮细胞的定位表达研究

目的 明确人尿酸转运蛋白(hUAT)在人肾小管上皮细胞(HKC)内的定位表达情况&#65377; 方法 利用DNA重组技术构建hUAT&#65377;绿色荧光蛋白的融合基因分别导入人HKC及非洲爪蟾卵细胞&#65377;构建hUAT的谷胱甘肽转移酶(GST)融合表达载体并制备抗hUAT的多克隆抗体&#65377;利用免疫荧光&#65380;Western印迹及激光共聚焦显微镜等技术观察hUAT在人HKC的定位表达&#65377;结果 成功制备了兔抗hUAT-GST多克隆抗体&#65377;利用该抗体及构建的pEGFP-hUAT荧光表达载体进行Western印迹和免疫荧光检测,结果表明hUAT是一种膜蛋白,并且表达于人HKC胞膜上,Northern blot结果也表明人HKC在高尿酸环境中的hUAT表达水平明显上调(P < 0.05)&#65377;结论 hUAT并非典型的膜转运蛋白,可能是以二聚体的形式才能够表达在细胞膜上,它在细胞内的定位表达可能需要特殊的转录调控机制&#65377;     

糖基化终产物对肾间质成纤维细胞DNA损伤的影响

目的 探讨糖基化终产物(AGE)对肾间质成纤维细胞DNA损伤的影响及抗氧化剂的干预作用&#65377; 方法 不同浓度AGE修饰的牛血清白蛋白(AGE-BSA)作用于NRK-49F细胞24 h,普通培养基和牛血清白蛋白 (BSA)作为对照&#65377;N-乙酰半胱氨酸(NAC)预处理细胞以观察抗氧化剂的干预作用&#65377;AlamarBlue还原法测定细胞增殖活力,单细胞凝胶电泳(彗星实验)测定细胞DNA损伤&#65377; 结果 与对照组比较,AGE作用后的细胞增殖活力下降;DNA迁移距离(彗星尾长)增加,差异有统计学意义,且2者呈剂量依赖关系&#65377;各浓度BSA作用后对细胞增殖活力无明显影响;彗星尾长无明显变化&#65377;与相同浓度BSA相比, 400&#65380;800 mg/L AGE分别使AlamarBlue还原率下降14%和15%(P < 0.05);200&#65380;400&#65380;800 mg/L AGE组彗星尾长分别为BSA组的1.45&#65380;2.12&#65380;2.71倍(P < 0.05)&#65377;与未用NAC预处理组相比,NAC预处理可使AlamarBlue还原率上升[(45.15±0.93)% 比(38.40±0.81)%,P < 0.05];彗星尾长变短[(10.02±4.54) μm比(13.48±5.32) μm,P < 0.05]&#65377; 结论 AGE可导致肾间质成纤维细胞DNA损伤,使用抗氧化剂可有效减轻AGE导致的DNA损伤&#65377;细胞内氧化应激增强可能是AGE引起肾间质成纤维细胞DNA损伤的作用机制之一。     

Wilms瘤1基因在肾小管上皮细胞转分化中的表达及作用

目的 探讨Wilms 肿瘤1基因(WT1)在肾小管上皮细胞(TEC)向肌成纤维细胞转分化中的可能作用&#65377;方法 将体外原代培养TEC分别置含IL-1α(10 ng/ml)&#65380; IL-1α+抗WT1中和抗体(10 μg/ml)的DMEM/F12培养基中培养, 观察TEC的形态变化及免疫学特征&#65377;采用RT-PCR检测各组TEC中WT1及α-SMA的表达&#65377;结果 经IL-1α掺入培养5 d后,体外培养的TEC形态特征趋于成纤维细胞化,细胞拉长&#65380;梭形变,失去原有的呈铺路石样的生长方式&#65377;电镜下细胞极性丧失,表面微绒毛消失&#65377;正常情况下,成年TEC不表达WT1&#65377;经IL-1α掺入培养1 d后,TEC重新表达WT1,同时伴有α-SMA的表达;3 d后WT1表达消失,呈一过性特征,而α-SMA的表达则随时间的延长而逐渐增强&#65377;在培养中掺入抗WT1抗体以中和WTl基因产物后,尽管仍给予IL-1α刺激,TEC大都保持原有特征不变,α-SMA及WT1 mRNA仅呈微弱表达&#65377;结论 高浓度IL-1α可导致TEC向肌成纤维细胞的转分化&#65377;在TEC的转分化过程中,WT1基因的重新&#65380;一过性表达可能起着重要作用&#65377;成年TEC重新获得WT1表达,可能是其发生转分化的内在启动机制&#65377;体外中和WT1的基因产物可明显抑制TEC的转分化进程,并可能籍此影响肾脏的纤维化发生&#65377;     

肝细胞生长因子负性调控细胞转分化在肾间质纤维化中的作用

目的 研究肝细胞生长因子(HGF)对单侧输尿管梗阻(UUO)大鼠肾间质纤维化的保护作用及其可能机制&#65377;方法 大鼠随机分为UUO组&#65380;HGF治疗组和假手术组&#65377;用实时荧光定量RT-PCR&#65380;Western杂交和免疫组化检测术后大鼠肾组织结缔组织生长因子(CTGF)和骨形成蛋白7(BMP7)表达量&#65377;免疫组化检测大鼠肾组织TGF-β1&#65380;FN及α-SMA表达&#65377;结果 与假手术组相比,UUO组及HGF治疗组CTGF mRNA&#65380;TGF-β1&#65380;α-SMA&#65380;FN&#65380;CTGF蛋白表达均增高,且UUO组明显高于治疗组;UUO组及HGF治疗组BMP7 mRNA和蛋白表达均减少,且UUO组显著低于治疗组&#65377;结论 HGF能减轻肾间质纤维化,负性调控肾小管上皮细胞-肌成纤维细胞转分化,调节CTGF及BMP7表达可能是其作用途径&#65377;     

两种病理类型肾病患者尿IgG对人肾小管上皮细胞表达巨噬细胞移动抑制因子的影响

目的 分离纯化表现为肾病综合征(NS)的微小病变型(MCD)及膜性肾病(MN)患者尿IgG，比较它们对人近端小管上皮细胞(HK-2)表达巨噬细胞移动抑制因子(MIF)的影响。方法 采用硫酸铵沉淀&#65380;蛋白G亲和层析纯化尿中IgG，并经SDS-PAGE、Western印迹分析鉴定&#65377;用不同浓度(0&#65380;0.5&#65380;1.0&#65380;2.5&#65380;5.0&#65380;10.0 mg/ml)的上述两种患者的尿IgG分别刺激HK-2细胞6 h，应用RT-PCR检测细胞表达MIF mRNA的变化;应用Western印迹检测细胞中MIF的蛋白水平&#65377; 结果 纯化的尿IgG经SDS-PAGE分析显示其分解为4个片段,以兔抗人IgG抗体进行免疫印迹鉴定，证实这些蛋白条带均为IgG成分&#65377; 两种不同病理类型NS患者的尿IgG均可上调HK-2细胞MIF 的基因及蛋白表达，并呈剂量依赖性&#65377;MN患者的尿IgG 0.1 mg/ml即可明显上调HK-2细胞MIF mRNA和蛋白表达(P < 0.01);而MCD患者的尿IgG需达到2.5 mg/ml才具有显著上调效应&#65377; 结论 呈NS的MCD和MN患者尿IgG可上调HK-2细胞表达MIF&#65377;MN患者尿IgG的作用强于MCD患者,提示这两种不同病理类型患者尿IgG可能存在结构或功能上的差异&#65377;     

通过基因敲低探讨多个足细胞分子作用及分子间反应

目的 研究足细胞裂孔隔膜(SD)复合体分子nephrin&#65380;podocin和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应&#65377;方法 针对nephrin&#65380; podocin&#65380;CD2AP和α-actinin-4 mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以 “敲低”其表达&#65377;免疫荧光染色观察其分布方式&#65377;半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达&#65377; 结果 (1) podocin敲低组(siPod966和siPod54):未检测到podocin及nephrin mRNA,其蛋白分别下降了92%&#65380;79%及82%&#65380;67%&#65377;而CD2AP mRNA和蛋白分别增加了62%&#65380;42%及71%&#65380;46%&#65377;α-actinin-4无变化&#65377;(2)nephrin敲低组(siNep492):未检测到nephrin mRNA和蛋白&#65377;而CD2AP mRNA和蛋白分别增加了35%&#65380;48%&#65377;Podocin和α-actinin-4无变化&#65377;(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2AP mRNA,其蛋白分别下降了92%和83%&#65377;Nephrin mRNA和蛋白分别下降了60%&#65380;48%及76%&#65380;72%;而podocin mRNA和蛋白分别增加了38%&#65380;22%及56%&#65380;44%&#65377;Α-actinin-4无变化&#65377;(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin 的mRNA分别下降了69%&#65380;58%及64%&#65380;49%;蛋白分别下降了81%和55%以及71%&#65380;64%&#65377;而podocin以及CD2AP mRNA分别增加了50%&#65380;34%及45%&#65380;28%;蛋白分别增加了64%&#65380;46%及65%&#65380;42%&#65377;(5)敲低nephrin&#65380;podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主&#65377;α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中&#65377;结论 (1)在SD复合体分子中,nephrin可能具有相对独立的作用&#65377;(2) α-actinin-4对nephrin&#65380;podocin和CD2AP有直接或间接的作用&#65377;(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”&#65380;也可能是“双向的”&#65377;(4)nephrin&#65380;podocin&#65380;CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应&#65377;     

肾病综合征患者肾组织podocin的表达

目的 观察不同病理类型&#65380; 不同激素反应性肾病综合征(NS)患者肾小球足细胞中podocin的表达和分布特征&#65377;方法 肾组织作冰冻切片免疫荧光双染色, 以激光共聚焦显微镜采集图像&#65377;与定位标志Ⅳ型胶原α3链比较&#65377; 受检NS患者21例, 其中局灶节段性肾小球硬化(FSGS)12例(难治性7例, 非难治性5例)&#65380; 微小病变(MCD)5例&#65380; 膜性肾病(MN)4例;正常肾组织对照3例&#65377;采用LSM 510图像处理系统进行处理&#65377;荧光强度以吸光度表示&#65377; 结果 (1)podocin在正常肾组织沿肾小球基底膜呈连续&#65380; 线形分布&#65377;部分FSGS患者podocin的分布呈点状&#65380; 短线条状, 少数患者未见podocin沉积&#65377;(2)FSGS患者肾小球中podocin表达量(80.5±33.5)与对照正常肾组织(138.4±38.1)比较,显著减少(P=0.0211), 其中难治性FSGS(67.2±30.5)与正常肾组织的差异有统计学意义(P=0.0131); 非难治性FSGS患者(99.0±31.0)与正常肾组织的差异无统计学意义(P=0.1585)&#65377;(3)MCD(112.1±47.6)&#65380; MN(92.5±34.8)患者podocin的表达量亦降低,与正常肾组织比较,无统计学意义(P=0.4497, P=0.1570)&#65377;(4)不同病理类型各组NS患者肾小球中podocin表达量的差异均无统计学意义(P > 0.05)&#65377;结论 FSGS的NS患者肾小球中podocin的表达减少, 难治性FSGS患者尤为明显, 部分FSGS患者podocin分布发生改变&#65377;podocin的检测可能在FSGS激素疗效评价方面有一定价值&#65377;     

黄芪甲苷对高糖诱导的肾小管上皮细胞损伤的保护作用

目的:旨在探究黄芪甲苷(ASI)对高糖诱导的人肾小管上皮细胞损伤的抑制作用,并阐明其机制。方法:体外培养人肾小管上皮细胞,进行分组:正常对照组:低糖(葡萄糖浓度5.5mmol/L)环境下培养;高糖组:高糖(葡萄糖浓度25mmol/L)环境下培养;ASI各组:分别在高糖培养基内加入不同浓度ASI(包括25、50、100、200μg/ml),在处理后0、1、12、24、48、96h观察,采用TUNEL法和caspase3检测各组细胞凋亡情况,采用ELISA法检测TGF-β1和HGF蛋白含量,采用Western blot检测磷酸化p-38、磷酸化ERK和磷酸化JNK浓度。此外,另在同样高糖培养基内加入p38抑制剂SB202190,1、12、24h观察细胞凋亡情况。最后,取同样高糖环境培养细胞,依次加入HGF50、100、200μg/ml,24h后检测细胞内磷酸化p38蛋白水平。结果:ASI(25～200μg/ml)可抑制高糖诱导的人肾小管上皮细胞凋亡,其抑制作用呈现剂量依赖性与时间依赖性。ASI同样可抑制高糖诱导的TGF-β1蛋白表达和p38MAPK信号通路活性。此外ASI可提高肾小管上皮细胞内HGF浓度;HGF可对p38MAPK信号通路产生抑制作用。结论:ASI抑制高糖诱导的人肾小管上皮细胞凋亡,其机制可能与ASI促进HGF分泌,阻断p38MAPK信号通路,进而抑制细胞凋亡以及TGF-β1的表达有关。ASI可能有助于糖尿病肾病的治疗。     

Hyperglycemia inhibits the uptake of dehydroascorbate in tubular epithelial cell

BACKGROUND/AIMS: Oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy. But the reason why renal cells are susceptible to oxidative injury in diabetes is not clear. Vitamin C plays a central role in the antioxidant defense system and exists in two major forms. The charged form, ascorbate, is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate, enters cells via glucose transporter and is then converted back to ascorbate within these cells. Because dehydroascorbate and glucose compete for glucose transporters, hyperglycemia will exclude vitamin C from the cell and resulted in a decreased antioxidant capacity in some cell type that is dehydroascorbate dependent. As such, we hypothesized that some renal cells were dehydroascorbate dependent and the susceptibility of renal cells to glucose-induced injury was mediated by hyperglycemic exclusion of dehydroascorbate uptake through competing for glucose transporter. The aims of the present study were to determine whether tubular epithelial cell was dehydroascorbate dependent and the effect of dehydroascorbate on the production of reactive oxygen species in cells incubated by high glucose. METHODS: Tubular epithelial cell was cultured in RPMI-1640 medium containing 10% newborn calf serum. Intracellular ascorbate and dehydroascorbate contents were measured with vitamin C assay system. The intracellular formation of reactive oxygen species was detected with the fluorescent probe CM-H(2)DCFDA by using confocal microscopy. RESULTS: Ascorbate entry into the cells was not significantly different from background noise. In contrast, we observed a significant increase in the uptake of dehydroascorbate in tubular cell. At a dehydroascorbate concentration of 1 mM, increasing concentrations of glucose competitively inhibited dehydroascorbate entry into the cells such that the accumulation of dehydroascorbate was smaller than half maximal at about 22 mM glucose. Cytochalasin B, a kind of hexose transporter inhibitor, inhibited dehydroascorbate entry into the cells. At a glucose concentration of 25 mM, increasing concentrations of dehydroascorbate reduced reactive oxygen species generation in a dose-dependent manner when dehydroascorbate concentration was smaller than 4 mM. However, the inhibitory effect was not observed at 8 mM of dehydroascorbate. CONCLUSIONS: Tubular epithelial cells are dehydroascorbate dependent. Vitamin C exclusion from tubular epithelial cells through competition of glucose and dehydroascorbate for common transport mechanism in diabetes will deprive the cells of antioxidant ability and could lead to reactive oxygen species accumulation.     

高糖加重造影剂诱导的肾小管上皮细胞凋亡

目的探讨高糖对造影剂诱导的肾小管上皮细胞凋亡的影响及其分子机制。方法将NRK52E细胞分成6组：正常对照组（A组）;造影剂组（B组）;葡萄糖（5mmol／L）＋造影剂组（C组）;葡萄糖（15retool／L）＋造影剂组（D组）;葡萄糖（30mmol／L）＋造影剂组（E组）;8]3203580＋葡萄糖（30mmol／L）＋造影剂组（F组）。F组SB203580（20μmol／L）在造影剂加入30min前加入。结果C组细胞葡萄糖作用6、12、24h及D、E组细胞葡萄糖作用6h,未见造影剂诱导的肾损伤和肾小管上皮细胞凋亡加重。D、E组细胞葡萄糖作用12h,造影剂肾损伤和肾小管上皮细胞凋亡重于B组,葡萄糖作用24h,肾损伤和肾小管上皮细胞凋亡重于葡萄糖作用12h,且E组重于D组（P〈0．05）。高糖显著增加造影剂诱导的肾小管上皮细胞ROS水平及p-p38和caspase3蛋白表达,SB203580抑制1〉p38和caspase3蛋白表达。ROS与p-p38、caspase3及p-p38与caspase3呈显著正相关（r分别为0．70、0．68和0．65,P〈0．05）。结论高糖以时间和剂量依赖方式加重造影剂诱导的肾小管上皮细胞损伤,其分子机制与高糖显著增加细胞内ROS水平及上调p-p38、caspase3表达有关。     

Astragalosides IV inhibits high glucose-induced cell apoptosis through HGF activation in cultured human tubular epithelial cells

Astragaloside IV (ASI) in Radix Astragali is believed to be the active component. The study aims to investigate whether ASI inhibits tubular epithelial cells apoptosis induced by high glucose and its mechanisms. Tubular epithelial cells in this paper were isolated from human kidney. The cells apoptosis was detected by TUNEL and caspase 3 assay. The protein levels of HGF and TGF-β1 were measured by ELISA. The phospho-p38 production, ERK and JNK were determined by Western blot. ASI could inhibit cells apoptosis induced by high glucose (25?mmol/L) in dose-dependent and time-dependent manners. ASI also inhibited high glucose-induced expression of TGF-β1 and activation of p38 MAPK pathway at the protein level. Furthermore, ASI increased HGF production in human tubular epithelial cells. The ASI inhibition of tubular epithelial cells apoptosis and reduction of TGF-β1 expression induced by high glucose may represent a new treatment for diabetic kidney injury. The mechanism underlying this inhibitory effect may be related to the inhibition of p38 MAPK signaling pathway activation and HGF overproduction.     

Effects of mycophenolic Acid on high glucose-induced fibronectin secretion and cellular reactive oxygen species in rat vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) proliferation, migration, and matrix protein accumulation play important roles in the development and progression of vascular disease including diabetic vascular complications and chronic allograft vasculopathy. Mycophenolic acid (MPA) inhibits various mesenchymal cell proliferation and matrix protein accumulation and reactive oxygen species (ROS). In this study, we investigated the effects of MPA on high glucose (HG)-induced fibronectin secretion and the role of ROS in rat VSMCs. Primary cultured rat VSMCs from Sprague-Dawley rats were exposed for 1 hour before stimulation with media containing 5.6 mmol/L glucose (low glucose [LG]), 30 mmol/L mannitol (M), or 30 mmol/L glucose (HG) with or without MPA (0.1-10 micromol/L) or N-acetylcysteine (NAC; 5 mmol/L). Fibronectin secretion was measured by Western blot analysis and dichlorofluorescein (DCF)-sensitive cellular ROS by flow cytometry. HG significantly increased fibronectin secretion by 1.7-fold. The increment of DCF-sensitive cellular ROS was 1.5-fold at 1 hour by HG. MPA at concentrations above 1 micromol/L effectively inhibited HG-induced fibronectin secretion and cellular ROS in a dose-dependent manner. NAC at 5 mmol/L also inhibited HG-induced rat VSMC activation. These results suggested that MPA inhibits HG-induced VSMC activation partially through inhibiting cellular ROS.     

促红细胞生成素抑制过氧化氢诱导的肾小管细胞凋亡

目的探讨促红细胞生成素（erythropoietin,EPO）是否可以抑制过氧化氢（H2O2）诱导的氧化应激和细胞凋亡及其抑制凋亡的相关机制。方法传代培养大鼠近端肾小管上皮细胞（NRK-52E）,分为正常对照组（NC组）、H1组（1mmol／L H2O2）、H2组（5mmol／LH2O2、E组（H2U＋EPO组）。细胞免疫荧光检测NRK-52E细胞是否表达EPO受体（erythropoietin receptor,EPOR）。荧光探针氯甲基二氯二氢荧光素二乙酯（CM—H2DCFDA）标记检测细胞内活性氧（reactive oxygen species,ROS）水平。流式细胞仪AnnexinV-FITC／PI双染法检测细胞凋亡指数。RTPCR检测凋亡相关基因B细胞淋巴瘤／白血病-2（B-cell lymphoma／L eukemia-2,Bcl-2）家族中Bcl-2和BaxmRNA的表达。结果NRK52E细胞表达EPOR。H2O2引起NRK-52E细胞内ROS水平升高,上调BaxmRNA表达、下凋Bcl-2mRNA表达,诱导NRK-52E细胞凋亡。EPO可以抑制H2O2诱导的ROS水平升高、Bcl2mRNA表达下调、BaxmRNA表达上调及NRK-52E细胞凋亡。结论EPO可以缓解H2O2诱导的氧化应激、上调Bcl-2mRNA表达、下调BaxmRNA表达,抑制H2O2诱导的NRK52E细胞凋亡,发挥肾小管细胞保护效应。     

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。     

内质网应激PERK/eIF2α通路在草酸钙诱导肾小管上皮细胞损伤中的作用

目的 探讨内质网应激蛋白激酶R样内质网激酶（PERK）/真核翻译起始因子2α（eIF2α）通路在草酸钙诱导的肾小管上皮细胞损伤中的作用机制。〖HT5”H〗方法〓〖HT5”SS〗将人肾小管上皮细胞（HK-2）分为对照组、草酸钙组和草酸钙+4 苯基丁酸组。草酸钙组和草酸钙+4-苯基丁酸组在培养基中加入终浓度为5 mmol/L的草酸钙诱导细胞损伤模型,草酸钙+4-苯基丁酸组在培养基中另加入终浓度为2 mmol/L的4-苯基丁酸。通过CCK-8法、流式细胞术和DCFH DA荧光探针检测细胞活力、凋亡和细胞内活性氧（ROS）水平,并分析细胞内PERK/eIF2α通路中蛋白以及Caspase12和Caspase3表达水平。结果 草酸钙组的OD值（0.42±0.05）低于对照组（0.71±0.08）,草酸钙+4-苯基丁酸组的OD值（0.65±0.07）高于草酸钙组（均P<0.05）。草酸钙组的细胞凋亡率、ROS水平、PERK和eIF2α蛋白水平、Caspase12、Caspase3 mRNA和蛋白水平高于对照组,差异均有统计学意义（均P<0.05）。草酸钙+4-苯基丁酸组的细胞凋亡率、ROS水平、PERK和eIF2α蛋白水平、Caspase12、Caspase3的mRNA和蛋白水平低于草酸钙组,差异均有统计学意义（P<0.05）。结论 在草酸钙诱导的肾小管上皮细胞损伤中使用4-苯基丁酸抑制内质网应激可抑制PERK/eIF2α通路并缓解细胞凋亡。     

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.