白蛋白活化肾小管上皮细胞对肾小管周微血管的影响及机制研究

目的 探讨人类血清白蛋白（HSA）超负荷损伤肾小管上皮细胞后对肾间质微血管损伤的影响及可能机制。 方法 激光共聚焦显微镜观察肾小管上皮细胞（HKC）吞饮罗丹明标记白蛋白（TRITC-BSA）以及吞饮受体cubilin siRNA对其的抑制效应。氢化乙锭标记的荧光探针检测白蛋白刺激HKC产生O2－以及白蛋白吞饮受体cubilin siRNA和线粒体呼吸链复合物I抑制剂鱼藤酮对HKC产生O2－的抑制作用。培养上清H2O2采用化学比色方法检测。倒置显微镜下观察HSA激活的HKC以及cubilin siRNA或鱼藤酮预处理的HKC与脐静脉内皮细胞（HUVEC）共培养后血管内皮管样结构形成情况。四甲基偶氮唑盐（MTT）比色法测定内皮细胞活力。用流式细胞仪以AnnexinV FITC/PI双染法检测内皮细胞凋亡率。 结果 （1）cubilin siRNA可显著抑制HKC吞饮白蛋白(P ＜ 0.05)。（2）HSA刺激HKC产生 ROS呈剂量及时间依赖性(P ＜ 0.05)；cubilin siRNA及鱼藤酮均可抑制白蛋白超负荷诱导HKC产生大量ROS (P ＜ 0.05)。（3）HKC与HUVEC共培养体系中，HSA活化的HKC抑制内皮细胞管样结构的形成，内皮细胞增殖显著减少(P ＜ 0.05)，细胞凋亡率显著增高(P ＜ 0.05)；而cubilin siRNA和鱼藤酮干预组内皮细胞管样结构数及内皮细胞活力显著增加(P ＜ 0.05)，细胞凋亡率显著降低(P ＜ 0.05)。 结论 白蛋白可通过吞饮受体cubilin激活肾小管上皮细胞线粒体呼吸链复合物I产生大量ROS，后者可能是慢性肾病时大量蛋白尿活化肾小管上皮细胞进而损伤肾小管周微血管网的重要介质。     

阿托伐他汀调控PERK/eIF2α/CHOP通路减轻造影剂诱导的大鼠肾小管上皮细胞凋亡

目的研究造影剂肾病大鼠肾脏中葡萄糖调节蛋白78(GRP78)、内质网调节激酶(PERK)、真核起始因子2α(eIF2α)及C/EBP同源蛋白质(CHOP)的表达情况,探讨内质网应激在造影剂肾病发病中的作用及阿托伐他汀的干预作用。 方法60只大鼠随机分为4组：对照组、模型组和高、低剂量阿托伐他汀组(80 mg,40 mg),每组15只。分别于注射造影剂后24、48、72 h留取血清;检测各组大鼠的血清尿素氮(BUN)、血清肌酐(Scr);TUNEL法及Western印迹法测casepase-3的表达检测肾小管上皮细胞凋亡;免疫组化和Western印迹法检测各组大鼠肾组织GRP78、p-eIF2α、p-PERK及CHOP的表达。 结果与对照组相比,模型组大鼠BUN、Scr显著升高,细胞凋亡严重,GRP78、p-eIF2α、p-PERK及CHOP的表达均显著升高(P< 0.05);与模型组相比,高、低剂量阿托伐他汀组,BUN、Scr显著下降,凋亡指数降低,GRP78、p-eIF2α、p-PERK及CHOP的表达显著下调,但仍高于对照组,差异均达到统计学意义(P<0.05);高、低剂量阿托伐他汀组之间上述各指标差异均不显著。 结论PERK/eIF2α/CHOP通路介导的内质网应激可能参与大鼠造影剂肾病的发生发展;阿托伐他汀在造影剂诱导的肾脏损伤中发挥保护作用,这可能与其调节PERK/eIF2α/CHOP通路,从而减轻内质网应激有关。     

单核细胞化学吸引蛋白质1 小分子干扰RNA人肾小管上皮细胞株的建立

目的 建立单核细胞化学吸引蛋白质1（MCP-1） 小分子干扰RNA（siRNA）人肾小管上皮细胞株，并检测MCP-1 siRNA对人肾小管上皮细胞（HKC）MCP-1基因的抑制效应。 方法 针对人MCP-1 mRNA 67、116、142位点设计并合成3对MCP-1 siRNA序列。构建MCP-1特异性siRNA真核表达载体，以脂质体法瞬时转染至HKC。转染24 h后分别应用实时定量RT-PCR及Western印迹技术检测HKC内MCP-1 mRNA、蛋白表达，筛选出抑制效率最高的MCP-1 siRNA。以筛选出的MCP-1 siRNA序列构建慢病毒穿梭质粒。使用慢病毒穿梭质粒进行慢病毒颗粒的包装和生产，得到病毒液并确定其滴度。以病毒液感染HKC，筛选MCP-1 siRNA人肾小管上皮细胞株，并应用实时定量RT-PCR及Western印迹技术检测HKC内MCP-1 mRNA、蛋白表达。 结果 成功建立MCP-1特异性siRNA人肾小管上皮细胞株。MCP-1 siRNA稳定转染能下调人肾小管上皮细胞MCP-1 mRNA (68.49±6.38)%、蛋白(72.97±6.13)%，与对照组比较差异有统计学意义(P < 0.01)。 结论 MCP-1特异性siRNA能高效抑制HKC内MCP-1基因表达。MCP-1 siRNA人肾小管上皮细胞株的建立为MCP-1基因功能及肾间质纤维化防治研究提供实验材料和依据。     

内质网应激相关凋亡途径参与单侧输尿管梗阻大鼠肾间质纤维化

目的 探讨内质网应激（ERS）相关凋亡途径在单侧输尿管梗阻（UUO）大鼠肾间质纤维化发生、发展中的作用。 方法 健康雄性Wistar大鼠25只,按随机数字表法分为UUO模型组（n=18）和假手术组（n=7）,UUO模型组行左侧输尿管结扎术,假手术组仅分离输尿管不结扎,分别于术后3 d、7 d、14 d处死各组大鼠,行HE和Masson染色,观察肾脏病理变化;比色法测定肾组织羟脯氨酸（HYP）含量;免疫组化法检测α平滑肌肌动蛋白（α-SMA）;原位末端标记法（TUNEL）与DNA电泳观察肾小管间质细胞凋亡情况;RT-PCR法检测梗阻侧肾组织ERS相关分子葡萄糖调节蛋白78（GRP78）mRNA表达变化;Western印迹法分析凋亡相关蛋白半胱氨酸天门冬氨酸蛋白酶3（caspase-3）和GRP78的蛋白表达变化。 结果 与假手术组比较,UUO模型组肾脏病理改变加重,肾间质纤维化程度随梗阻时间延长逐渐加重,肾组织HYP含量显著升高（P < 0.05）,肾组织α-SMA也在肾小管间质细胞广泛表达,TUNEL染色及DNA琼脂糖凝胶电泳提示大量的肾小管间质细胞凋亡。UUO模型组GRP78 mRNA表达于术后3 d即发生显著上调,而蛋白表达在术后7 d开始出现显著变化,与假手术组差异均有统计学意义（均P < 0.01）;在此后观察期间内GRP78 mRNA和蛋白均持续高水平表达。模型组大鼠肾组织caspase-3的蛋白表达在UUO术后3 d即有显著上调（P < 0.05）,且随着梗阻时间延长进行性升高,于术后7 d、14 d增多更为显著,与假手术组差异均有统计学意义（P < 0.05）。相关分析显示GRP78蛋白表达与肾组织HYP含量和caspase-3蛋白表达均呈正相关（r = 0.657,P < 0.01;r = 0.714,P < 0.01）。 结论 UUO早期即可诱导ERS标志蛋白表达变化,触发ERS。长期ERS可诱导肾小管间质细胞凋亡;caspase-3介导的ERS相关凋亡途径可能参与了肾间质纤维化过程。     

二甲基乙二酰基甘氨酸对缺氧复氧诱导的肾小管上皮细胞损伤的保护作用及其机制

目的 通过脯氨酸羟化酶抑制剂二甲基乙二酰基甘氨酸（DMOG）稳定缺氧诱导因子1α（HIF-1α）表达,探讨其对缺氧复氧诱导的肾小管上皮细胞（HKC）损伤的保护作用及其机制。 方法 制作无糖缺氧复氧细胞损伤模型,用不同浓度的DMOG预处理,锥虫蓝染色和乳酸脱氢酶（LDH）活性方法检测细胞活力及损伤;Annexin V和PI染色流式细胞仪技术检测细胞凋亡;实时荧光定量PCR方法检测红细胞生成素（EPO）、热休克蛋白70（HSP70）和血红素氧合酶1（HO-1） mRNA的表达;Western印迹法检测HIF-1α、活性caspase-3和Bcl-2蛋白表达。 结果 正常情况下HKC细胞内几乎无HIF-1α蛋白表达,DMOG刺激6 h后HIF-1α蛋白及其靶基因EPO、HSP70和HO-1 mRNA表达均显著上调（均P ＜ 0.01）,且呈浓度依赖性。500 μmol/L或1 mmol/L DMOG预处理可明显改善缺氧复氧诱导的细胞损伤,表现为细胞存活率升高（95.6%±1.8%、96.1%±1.0%比 83.3%±3.1%）;培养上清液中LDH 活性下降;细胞凋亡减少（8.6%±2.7%、6.1%±2.3%比19.2%±4.0%）（均P ＜ 0.05）。另外,细胞内活性caspase-3蛋白表达显著下调,而Bcl-2蛋白表达则显著上调（均P ＜ 0.05）。 结论 DMOG预处理可稳定肾小管上皮细胞内HIF-1α表达,对缺氧复氧诱导的肾小管上皮细胞损伤具有一定保护作用。其机制可能与促进EPO、HSP70和HO-1表达,抑制caspase-3活化,上调Bcl-2表达有关。     

小檗碱预先给药对缺氧/复氧诱导人肾小管上皮细胞凋亡的影响

目的 探讨小檗碱预先给药对缺氧/复氧诱导人肾小管上皮细胞凋亡的影响.方法 培养人肾小管上皮细胞(HK-2),以1×106个/ml密度接种于培养皿(2 ml/皿)和96孔培养板(200μl/孔),采用随机数字表法,将其分为4组(n=30):正常对照组(C组)、小檗碱组(B组)、缺氧/复氧组(H/R组)和缺氧/复氧+小檗碱组(H/R+B组).B组和H/R+B组加入10 μmol/L小檗碱孵育2h,随后H/R组和H/R+B组进行缺氧24h复氧3h.测定细胞活力、细胞凋亡率、丙二醛(MDA)、超氧化物歧化酶(SOD)、caspase-3、活化caspase-3、细胞色素c、葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)表达,并计算Bax表达与Bcl-2表达的比值(Bax/Bcl-2).结果 与C组比较,H/R组和H/R+B组细胞活力、SOD活性和caspase-3表达降低,细胞凋亡率、MDA浓度和Bax/Bcl-2升高,活化caspase-3、细胞色素c、GRP78和CHOP表达上调(P＜0.05),B组上述指标差异无统计学意义(P＞0.05);与H/R组比较,H/R+B组细胞活力、SOD活性和caspase-3表达升高,细胞凋亡率、MDA浓度和Bax /Bcl-2降低,活化caspase-3、细胞色素c、GRP78和CHOP表达下调(P＜0.05).结论 小檗碱预先给药可抑制缺氧/复氧诱导人肾小管上皮细胞凋亡,其机制与抑制线粒体应激通路和内质网应激通路有关.     

祛痰通络汤对糖尿病大鼠肾组织内质网应激相关分子GRP78和CHOP表达的影响

目的:观察祛痰通络汤对糖尿病大鼠肾组织中内质网应激相关分子GRP78和CHOP表达的影响。方法:高糖高脂饲料联合腹腔注射STZ制备糖尿病模型,随机分为4组,分别为正常对照组、模型组、祛痰通络汤组、四苯基丁酸组,治疗8周。观察各组24 h尿蛋白定量、肾脏病理学变化;免疫组化和western-blot检测肾组织内质网应激相关蛋白GRP78,CHOP的表达。结果:祛痰通络汤降低尿蛋白,减轻病理改变;模型组肾组织的GRP78、CHOP表达明显升高(P0.05),祛痰通络汤组GRP78、CHOP表达明显低于模型组(P0.05)。结论:糖尿病大鼠肾脏中GRP78、CHOP表达明显增强,祛痰通络汤降低内质网应激,可能是其肾脏保护作用的重要机制。     

脯氨酸羟化酶2 siRNA对抗霉素诱导肾小管上皮细胞凋亡的影响

目的 探讨是否脯氨酸羟化酶2 (PHD2) siRNA 通过减少缺氧诱导因子(HIF)1的降解而减轻缺氧引起的肾小管上皮细胞凋亡。方法 构建针对人PHD2的表达质粒,将其转染到培养的人肾小管上皮细胞系(HKC)中。用RT-PCR方法观察其对PHD2表达的抑制作用。Western印迹观察HIF-1α的表达。培养的HKC细胞分为对照组、模型组和siRNA组、siRNA组经PHD2 siRNA转染后与模型组同时用抗霉素诱导,用Western印迹方法观察3组细胞HIF-1α的表达,用流式细胞仪观察3组细胞的凋亡情况。 结果 经PHD2 siRNA转染的HKC细胞PHD2 mRNA表达明显下调(P < 0.01),HIF-1α蛋白表达明显上调(P < 0.05)。经抗霉素处理后,模型组和siRNA组HIF-1α蛋白表达均明显高于对照组(P < 0.01),但siRNA组HIF-1α蛋白表达高于模型组(P < 0.05。与模型组相比,siRNA组细胞凋亡明显减轻(P < 0.05)。 结论 通过RNA干扰技术抑制PHD2的表达可以增强HKC细胞HIF-1的蛋白表达,从而增强其在缺氧条件下的抗凋亡能力。     

miR-520激活内质网应激导致滋养细胞凋亡的相关研究

目的探讨miR-520通过激活内质网应激导致滋养细胞凋亡,从而参与复发性流产(RSA)的作用机制。方法利用miR-520慢病毒(实验组)和空载慢病毒(对照组)分别感染人滋养细胞系HTR8细胞,并用嘌呤霉素筛选出稳转株。以未感染细胞为空白对照,利用流式细胞术及荧光显微镜观察确定病毒转染效率、实时荧光定量PCR(qRT-PCR)检测各组细胞miR-520的表达水平;通过CCK-8法测定细胞增殖能力、流式细胞术分析细胞凋亡率、乳酸脱氢酶(LDH)试剂盒检测细胞损伤程度、Mito-Tracker Red CMXRos试剂盒检测线粒体膜电位变化;通过Western blot检测凋亡通路中cleaved Caspase-3、cleaved Caspase-9以及cleaved Caspase-12水平的变化,同时检测内质网应激相关蛋白CHOP、葡萄糖调节蛋白GRP78的水平变化。结果与对照组比较,miR-520在人滋养细胞系HTR8细胞中过表达后,HTR8细胞增殖能力显著下降(P0.05),LDH活性升高(P0.05),HTR8细胞线粒体膜电位降低(P0.05),细胞凋亡数显著增加(P0.05);凋亡相关蛋白cleaved Caspase-3、cleaved Caspase-9以及cleaved Caspase-12水平均有不同程度的增高(P0.05),同时内质网应激相关蛋白CHOP、GRP78表达水平上调(P0.05)。结论 miR-520可能通过激活内质网应激诱导HTR8细胞发生凋亡,从而参与RSA的发生。     

高渗和低渗造影剂对人肾小管上皮细胞的毒性作用

目的 比较高渗造影剂（HOCM，泛影葡胺）和低渗造影剂（LOCM，欧乃派克）对人肾小管上皮细胞的毒性，探讨Bax／Bcl-2，半胱氨酸天冬氨酸蛋白酶（caspase）-3在造影剂诱导肾小管上皮细胞凋亡中的作用。方法 以HKC细胞株为研究对象，实验分为7组：HOCM1组（111mgI／ml），HOCM2组（74mgI／m1），LOCMl组（111mgI／m1），LOCM2组（74mgI／ml），甘露醇高渗对照组，甘露醇低渗对照组，培养基对照组。采用四甲基偶氮唑蓝（MTT）试验和乳酸脱氢酶（LDH）检测造影剂对体外培养HKC细胞的毒性作用。采用Hoechst染色、TUNEL染色、DNA琼脂糖凝胶电泳、电镜和流式细胞仪DNA分析方法观察造影剂对HKC细胞凋亡的作用。采用Westem印迹方法检测Bax和Bcl-2蛋白含量的变化。采用荧光比色法检测caspase-3活性。结果 HOCM和LOCM组培养液中LDH水平较对照组显著增高（P〈0．05），细胞活力较对照组显著降低（P〈0．05），且与渗透浓度，作用时间及碘离子浓度有关。HOCM可诱导肾小管上皮细胞凋亡，且明显高于甘露醇高渗对照（P〈0．05）。LOCM在本实验剂量不能诱导肾小管上皮细胞凋亡。HOCM在诱导HKC细胞凋亡时伴随有Bax、Bcl-2表达的上调，caspase-3活性升高。结论 HOCM和LOCM对肾小管上皮细胞均有毒性作用，LOCM的毒性作用明显小于HOCM。HOCM可诱导人肾小管上皮细胞凋亡且与高渗及碘离子浓度有关。Bax／Bcl-2、caspase-3可能参与了造影剂诱导人肾小管上皮细胞凋亡的调控。     

内质网应激反应在严重烧伤大鼠心肌细胞凋亡中的作用

目的内质网应激反应(endoplasmic reticulum stress,ERS)介导的凋亡是真核细胞重要凋亡途径之一,通过观察严重烧伤大鼠心肌ERS不同通路蛋白表达变化,探讨其在心肌细胞凋亡中的可能作用。方法雄性7周龄Wistar大鼠64只,体重200～220 g;随机分为两组,每组32只。实验组大鼠背部制备30%体表面积Ⅲ度烫伤;对照组制备假伤模型。伤后1、4、7、14 d两组各处死8只大鼠取心肌组织,透射电镜观察心肌超微结构变化,TUNEL法检测心肌细胞凋亡,Western blot检测ERS相关蛋白,如葡萄糖调节蛋白78(glucose regulated protein 78,GRP 78)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、半胱氨酸天冬氨酸蛋白酶12(Caspase 12)剪切体表达变化。结果大鼠均存活至实验结束。透射电镜观察示实验组大鼠心肌细胞呈凋亡改变。伤后各时间点实验组心肌细胞凋亡指数均明显高于对照组(P<0.05),伤后1、4、7 d凋亡指数逐渐升高,14 d时下降,各时间点间比较差异均有统计学意义(P<0.05)。实验组心肌细胞GRP78、CHOP及Caspase 12剪切体蛋白表达持续升高,其中各时间点GRP 78及Caspase 12剪切体表达均较对照组显著升高,差异有统计学意义(P<0.05);除伤后1 d外,其余各时间点实验组CHOP蛋白表达均较对照组升高(P<0.05)。结论严重烧伤后大鼠心肌发生ERS,其中CHOP、Caspase 12介导的凋亡通路活化,ERS可能是心肌细胞凋亡的途径之一。     

Lisinopril suppresses the endoplasmic reticulum stress associated tubular cell apoptosis induced by proteinuria in adriamycin nephropathy

Objective To investigate the role of endoplasmic reticulum stress in tubular epithelial cell apoptosis in chronic proteinuria rat model and the effect of lisinopril intervention. Methods Adriamycin nephropathy was induced in male Wistar rats (n=12) by a single injection of adriamycin at 2 mg/kg body weight. Rats were then randomly assigned to model group or treatment group, to which distilled water or lisinopril were administered respectively for 12 weeks. Six normal rats serving as controls were administered distilled water. 24 h urine samples were collected at week 4, 8, 12 and the urine protein was measured. At the end of study, serum was obtained and physiological parameters (serum creatinine, urea, total protein and albumin) were measured. Renal tubular epithelial cell apoptosis was assessed by TUNEL. GRP78, CHOP protein expression in kidney was quantified by immunohistochemistry and Western blotting. Results Compared to control group rats, increased proteinuria was observed in model group rats at week 4, 8, 12 (P＜0.05). Lisinopril treatment attenuated urine protein excretion significantly (P＜0.05). At week 12, hypoalbuminemia was detected in model group rats (P＜0.05), whereas the condition was alleviated by lisinopril (P＜0.05). There were no significant differences of serum creatinine, urea and total protein in each group (P＞0.05). Compared to control group rats, increased TUNEL positive tubular epithelial cells and tubular GRP78 and CHOP expression were also observed in model group rats (P＜0.05); however, these conditions in the kidney were significantly decreased in treatment group (P＜0.05). Conclusions Endoplasmic reticulum stress may be involved in the process of tubular epithelial cell apoptosis induced by proteinuria. Lisinopril may attenuate tubular epithelial cell apoptosis through regulating this signal pathway.     

肾小管上皮细胞内质网应激时NGAL表达改变的调控机制研究

目的:探讨人肾小管上皮细胞(HK-2细胞)发生内质网应激时,NGAL表达增加的上游调控机制。方法:将HK-2细胞分为对照组(正常HK-2细胞),TG(毒胡萝卜素,thapsigargin)组(5μmol/L TG处理8 h),单纯转染组(siRNAATF4试剂转染24 h),转染+TG组(siRNA-ATF4试剂转染24 h后,5μmol/L TG处理8 h),阴性对照组(siRNA-阴性对照物转染24 h),DMSO组(5μmol/L DMSO处理8 h)。采用Western blot检测各组细胞内质网源性转录因子(CHOP)、内质网分子伴侣葡萄糖调节蛋白78(GRP78)、中性粒细胞明胶酶相关性载脂蛋白(NGAL)、激活转录因子4(ATF4)的表达,采用Real-time PCR方法测得ATF4mRNA、NGALmRNA表达量。结果:与对照组相比,TG组细胞NGAL、ATF4、ATF4mRNA、NGALmRNA表达量显著提高(P <0. 05),而转染+TG组、单纯转染组、阴性对照组、DMSO组中ATF4及NGAL差异无统计学意义(P> 0. 05)。与TG组相比,转染+TG组ATF4、NGAL、ATF4mRNA及NGALmRNA表达量呈显著降低趋势(P <0. 05)。在TG组与转染+TG组细胞中,CHOP和GRP78呈过表达状态(P <0. 05),而转染+TG组细胞CHOP和GRP78提升趋势明显低于TG组细胞(P <0. 05)。结论:(1) TG可诱导人肾小管上皮HK-2细胞发生内质网应激反应。(2) HK-2细胞发生内质网应激反应时,抑制ATF4表达会引起NGAL降低,提示ATF4是NGAL表达的上游调控因子。(3) HK-2细胞发生内质网应激反应时,抑制ATF4不能阻止CHOP和GRP78发生过表达,但可降低其升高程度,提示ATF4及NGAL降低可能对内质网应激反应介导HK-2细胞损伤起到一定的缓解作用。     

The protective effect of hydroxytyrosol on contrast - induced nephropathy and endoplasmic reticulum stress

Objective To investigate the expression of glucose-regulated protein 78(GRP78) and cysteine aspartic acid protease 12(Caspase - 12) and evaluate the endoplasmic reticulum stress (ERS) in rats with contrast - induced nephropathy (CIN), and observe the protective effects of hydroxytyrosol on CIN rats. Methods Eighty-four Wistar rats, (220±20) g, were randomly divided into control group, CIN group, hydroxytyrosol treated group (group C+H). At 12th, 24th, 48th, 72th day after the rats model were established, BUN and Scr were detected. ELISA were used to detect the expression of methane dicarboxylic aldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). HE staining were used to evaluate the pathological change of kidney. TUNEL were used to detect the apoptosis of tubular cells. Real-time PCR were used to detect the expression of GRP78 mRNA and Caspase-12 mRNA in tubular cells. Immunohistochemistry and Western blotting were used to detect the expression of GRP78 and Caspase - 12 protein in tubular cells. Results BUN, Scr, the mRNA and protein expression of GRP78, Caspase-12 in hydroxytyrosol treated group were higher than that in control group(P＜0.05), but were significantly lower than that in CIN group (P＜0.05). Pathological changes and the apoptosis of tubular cells in CIN group were more serious than that in hydroxytyrosol treated group (P＜0.05). Conclusions Endoplasmic reticulum stress may be associated with contrast-induced nephropathy. Hydroxytyrosol can protect kidney from contrast medium via reducing the endoplasmic reticulum stress.     

Relationship of angiotensin II type 1 receptor autoantibody and renal cell apoptosis induced by caspase-12 in diabetic nephropathy rats

Objective To study the relationship of angiotensin II type 1 receptor (AT1R) autoantibody (AT1-AA) and renal cell apoptosis induced by caspase-12 in diabetic nephropathy (DN) rats. Methods High-sucrose and high-fat diet and intraperitoneal injection of streptozotocin (35 mg/kg) were utilized to establish DN rat model. Serum AT1-AA was detected by enzyme-linked immunosorbent assay (ELISA) and renal cell apoptosis was detected by TUNEL staining. Furthermore, the mRNA levels of the endoplasmic reticulum stress (ERS) chaperone protein glucose regulated protein 78 (GRP78) and ERS-associated apoptosis protein caspase-12 were measured by real-time quantitative PCR. Additionally, the levels of GRP78 and caspase-12 protein were measured by Western blotting. Results The renal cell apoptosis rate in DN group was increased significantly (P＜0.01), and the renal cells apoptosis rate in AT1-AA positive DN group was higher than that in AT1-AA negative DN group [(20.05±1.71)% vs (13.24±4.93)%, P＜0.01]. The mRNA expressions of GRP78 and caspase-12 in DN group, in comparison to NC group, were increased significantly (P＜0.01), as well as the proteins (P＜0.01). And the expression of these mRNA and proteins had significant increment in AT1-AA positive DN rats when compared with AT1-AA negative DN rats (P＜0.05). Conclusions AT1-AA can induce ERS in the renal tissue of DN rats, and promote renal cell apoptosis likely via the modulation of caspase-12 signaling pathway.     

Inhibition effect of small interfering RNA of connective tissue growth factor on the expression of extracellular matrix molecules in cultured human renal proximal tubular cells

Objective: In this study, we investigated the effect of small interfering RNA (siRNA) of connective tissue growth factor (CTGF) by pRetro-Super (PRS) retrovirus vector on the expression of CTGF and related extracellular matrix molecules in human renal proximal tubular cells (HKCs) induced by high glucose, to provide help for renal tubulointerstitial fibrosis therapy. Methods: HKCs were exposed to d-glucose to observe their dose and time effect, while the mannitol as osmotic control. Retrovirus producing CTGF siRNA were constructed from the inverted oligonucleotides and transferred into packaging cell line PT67 with lipofectamine, and the virus supernatant was used to infect HKC. The expression of CTGF, fibronectin (FN) and collagen-type I (col1) were measured by semi-quantitative RT-PCR and Western blot. Results: In response to high glucose, CTGF expression in HKCs was increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Introduction of PRS-CTGF-siRNA resulted in the significant reduction of CTGF, FN, col1 mRNA (p?0.01, respectively) and CTGF, col1 protein (p?0.05, respectively) expression, while PRS void vector group did not have these effects (p?>?0.05). Conclusions: CTGF siRNA therapy can effectively reduce the levels of CTGF, FN and col1 induced by high glucose in cultured HKCs, which suggested that it may be a potential therapeutic strategy to prevent the renal interstitial fibrosis in the future.