高糖通过转化生长因子β信号诱导肾小球内皮细胞向肌成纤维细胞转分化

目的 探讨高糖(HG)能否通过转化生长因子β(TGF-β)途径诱导大鼠肾小球内皮细胞向肌成纤维细胞转分化(EndMT).方法 体外培养大鼠肾小球内皮细胞(GEnC),分为正常对照组(NG,5.5 mmol/L)、高糖组(HG,15、30 mmol/L)、TGF-β抑制剂组(HG+LY36,30 mmol/L葡萄糖+10 μmol/L LY364947)以及高渗对照组(M,5.5 mmol/L葡萄糖+25.5 mmol/L甘露醇)和溶剂对照组(D,5.5 mmol/L葡萄糖+1 ml/LDMSO).采用Western印迹法检测各组细胞内皮细胞标志物claudin5和肌成纤维细胞标志物α-SMA表达变化;实时定量PCR法检测细胞TGF-β1和TGF-β2 mRNA表达改变;免疫荧光法观察细胞形态学变化以及血管内皮细胞标志物VE-cadherin和肌成纤维细胞标志物α-SMA的表达.结果 与NG组比较,HG组claudin5蛋白的表达量随葡萄糖浓度增加而降低(P＜0.05),α-SMA蛋白表达量随葡萄糖浓度增加而升高(P＜0.05),TGF-β1和TGF-β2 mRNA表达均升高(P＜0.05).与HG组比较,TGF-β抑制剂组claudin5蛋白表达量升高(P＜0.05),α-SMA蛋白表达降低(P＜0.05).高渗对照组和溶剂对照组改变差异无统计学意义.激光共聚焦免疫荧光结果显示,高糖处理可引起细胞形态由卵圆形向梭形改变,VE-cadherin表达减少,α-SMA表达增加;TGF-β抑制剂组细胞形态无明显改变.与HG组比较,TGF-β抑制剂组VE-cadherin表达增加,α-SMA表达降低(P＜0.05).结论 高糖诱导大鼠肾小球内皮细胞TGF-β表达增加及内皮细胞-肌成纤维细胞转分化.抑制TGF-β可抑制高糖引起的转分化,提示TGF-β参与了高糖引起的肾小球内皮细胞转分化过程.     

虫草素通过下调TGF-β抑制高糖诱导的大鼠肾小管上皮细胞转分化

目的：探讨虫草素对高糖诱导的大鼠肾小管上皮细胞转分化的影响。方法：体外培养大鼠近端肾小管上皮细胞株（NRK52E细胞株）,分为正常对照组（葡萄糖5.5 mmol/L,NG组）、高糖组（葡萄糖30 mmol/L,HG组）、高糖＋虫草素组（葡萄糖30 mmol/L＋虫草素10μg/ml,HG＋C组）。分别于刺激12 h,24 h,48 h后收集细胞。应用定量RT-PCR测定NRK52E TGF-β,E-cadherin,α-SMA mRNA的表达;Western印迹方法检测TGF-β、E-cadherin、α-SMA蛋白的表达。结果：高糖刺激后NRK52E细胞的TGF-β和α-SMA mRNA及蛋白表达明显高于正常糖组（P〈0.01）,而虫草素组TGF-β和α-SMA mRNA及蛋白表达显著低于高糖组（P〈0.05）;高糖诱导的NRK52E细胞E-cadherin mRNA及蛋白水平明显降低（P〈0.01）;而虫草素组NRK52E细胞E-cadherin mRNA及蛋白水平显著高于高糖组（P〈0.05）。结论：虫草素可以明显抑制高糖诱导的大鼠肾小管上皮细胞转分化,其机制可能是通过下调TGF-β实现。     

利拉鲁肽对糖尿病大鼠阴茎海绵体eNOS表达的影响

目的:研究胰高血糖素样肽-1(GLP-1)类似物利拉鲁肽对糖尿病(DM)大鼠阴茎海绵体内皮型一氧化氮合酶(e NOS)表达的影响,探讨利拉鲁肽对DM勃起功能障碍(DED)大鼠勃起功能的作用。方法:取6周龄雄性SD大鼠,分为正常对照组(NC,n=10)与实验组(n=20),实验组构建DM大鼠模型,随机将实验组分为DM组(n=8)与GLP-1组(n=8)。干预12周后,检测各组空腹血糖(FPG)、空腹胰岛素(FINS)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、睾酮、白介素-6,计算胰岛素敏感指标Homa-IR与Homa-β,比较各组大鼠勃起功能;Western印迹检测各组大鼠阴茎海绵体组织Akt/p-Akt、e NOS/p-e NOS的表达。结果:DM组大鼠勃起次数(0.90±1.14)及勃起率(37.5%)较GLP-1组(2.90±1.53,25.0%)、NC组(4.20±1.05,100%)均明显减少(P0.05);GLP-1组大鼠勃起率及勃起次数亦少于NC组大鼠(P0.05)。免疫荧光染色提示e NOS主要表达在海绵体血管、血窦内皮细胞的细胞质中,DM组、GLP-1组e NOS蛋白表达水平显著低于正常对照组(P0.05),且GLP-1组明显高于DM组(P0.05)。DM、GLP-1组大鼠阴茎组织e NOS/p-e NOS表达水平较NC组明显下降(P0.01或0.05)。与DM组相比,GLP-1组大鼠阴茎组织p-e NOS表达水平明显升高(P0.05)。3组大鼠阴茎组织Akt比较无显著差异(P0.05)。DM、GLP-1组大鼠阴茎组织p-Akt表达水平较NC组明显下降(P0.01或0.05)。与DM组相比,GLP-1组大鼠阴茎组织p-e NOS表达水平明显升高(P0.05)。结论:GLP-1可能通过调节Akt/e NOS信号通路,保护阴茎海绵体组织内皮细胞功能,改善DED大鼠的勃起功能,提示GLP-1的使用可能是将来治疗和预防DED的重要方法之一。     

高糖通过PI3K-AKT-mTOR信号通路增加足细胞自噬

目的 研究高糖引起足细胞自噬变化及其相关的信号机制.方法 培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成;Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达;通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果 高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论 高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.     

FGF2/FGFR1/ERK信号通路在高糖诱导人肾小管上皮细胞转分化中的机制及肾元颗粒的干预作用

目的:探讨FGF2/FGFR1/ERK在高糖诱导人肾小管上皮细胞转分化中的信号转导机制及肾元颗粒的干预作用。方法:以高糖(30 mmol/L)诱导HK-2细胞上皮-间充质细胞转分化建立模型,分为正常组、模型组、高渗对照组、肾元颗粒组、厄贝沙坦组及ERK阻断剂组。采用CCK-8法确定大鼠血清加入浓度,Western blot及RT-PCR检测FGF2、CollgenⅣ蛋白及mRNA表达水平,ELISA法分析各组细胞ERK及磷酸化ERK(p-ERK)水平。结果:与正常组比较,模型组FGF2、CollgenⅣ蛋白及mRNA表达显著增高(P0.05),p-ERK水平显著增高(P0.05)。与模型组比较,肾元颗粒组FGF2、CollgenⅣ蛋白及mRNA表达显著降低(P0.05),p-ERK水平显著降低(P0.05)。结论:肾元颗粒含药血清能通过下调高糖诱导的HK-2细胞FGF2的表达,抑制FGF2/FGFR信号转导途径从而改善肾小管上皮细胞转分化,这可能是其防治糖尿病肾病的机制之一。     

激活素A在高糖诱导的人肾小管上皮细胞中的表达变化

目的 通过体外高糖刺激的人近端肾小管上皮细胞株(HK-2),探讨激活素A(ACT A)的表达变化及卵泡抑素(FS)的干预作用.方法 HK-2细胞生长于DMEM培养基,待细胞生长至亚融合状态时,更换为无血清DMEM培养基使细胞生长同步化24h,然后将细胞分为以下5组:正常对照组(NG,5.5 mmol/L葡萄糖)、甘露醇对照组(MG,5.5 mmol/L葡萄糖+25 mmol/L甘露醇)、高糖组(HG,25 mmol/L葡萄糖)、HG+FS100组(25 mmol/L葡萄糖+100 μg/L FS)、HG+FS500组(25 mmol/L葡萄糖+500 μg/L FS).12、24、48 h后收集各组细胞及细胞上清液.Western印迹法检测各组细胞ACT A和p-Smad2/3的表达;ELISA检测各组细胞上清液转化生长因子β(TGF-β)和纤连蛋白(FN)的含量.结果 NG组细胞ACT A有微量表达,而HG组表达显著增加(P＜0.05),呈时间依赖性.与HG组相比,FS干预组ACT A表达显著减少(P＜0.05),呈剂量依赖性.与NG组相比,p-Smad2/3在HG组培养24 h后表达显著增加(P＜0.05);FS可抑制p-Smad2/3的高表达(P＜0.05).ELISA检测结果显示,与NG组相比,HG组培养12 h后TGF-β表达即显著增加(P＜0.05),呈时间依赖性,FS干预对高糖诱导的TGF-3高表达没有影响.与NG组相比,HG组培养24h后FN表达显著增高(P＜0.01),FS对此有明显抑制作用,呈剂量依赖性(P＜0.01).结论 高糖能刺激HK-2细胞ACT A表达增加,从而促进肾小管上皮细胞FN的合成;FS可通过阻断ACT A的活化,减轻肾小管上皮细胞FN的合成.     

血管紧张素1-7对糖尿病大鼠肾小管间质纤维化的影响

目的 研究血管紧张素1-7(Ang1-7)对糖尿病大鼠肾小管间质纤维化的影响及其可能机制.方法 32只雄性Wistar大鼠被随机分为4组:健康对照组(NC组)、模型组(DM组)、替米沙坦组(TM组)、治疗组(T组).建模成功后第9周末检测各组大鼠24 h尿蛋白量、尿NAG/Cr、血糖、血胰岛素、三酰甘油(TG)、总胆固醇(TC)、BUN、Scr、血K+及血Na+ ;PAS染色观察肾脏病理改变 ;实时定量PCR法检测各组大鼠肾脏组织中转化生长因子β1(TGF-β1)、过氧化物酶体增殖物激活受体(PPAR)γ、α平滑肌肌动蛋白(α-SMA)mRNA水平 ;Western印迹法检测PPARγ、α-SMA、TGF-β1蛋白表达.结果 (1)第9周末,DM组大鼠血压、尿蛋白量、肾质量/体质量较NC组显著升高(P＜0.05),TM组及T组较DM组显著降低(P＜0.05),且T组变化更明显.(2)DM组第9周末肾间质损伤指数显著高于NC组(P＜0.05),TM组及T组则低于DM组(P＜0.05).(3)实时定量PCR结果显示,DM组TGF-β1、α-SMAmRNA水平显著升高(P＜0.05),PPARγ mRNA水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA mRNA水平均显著下降(P＜0.05),PPARγ mRNA水平显著上升(P＜0.05),且T组变化更明显.(4)Western印迹结果显示,DM组TGF-β1、α-SMA蛋白水平显著升高(P＜0.05),PPARγ蛋白水平显著下降(P＜0.05),TM组及T组较DM组TGF-β1、α-SMA蛋白水平均显著下降(P＜0.05),PPARγ蛋白水平显著上升(P＜0.05),且T组变化更明显.结论 Ang1-7在体内可通过上调PPARγ表达,抑制α-SMA表达,对糖尿病大鼠肾小管间质纤维化可能具有抑制作用.     

淫羊藿苷对大鼠骨折模型愈合及转化生长因子β1、血管内皮生长因子和骨形态发生蛋白-7表达的影响

目的:探讨淫羊藿苷对大鼠骨折模型的愈合作用及转化生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)和骨形态发生蛋白-7(BMP-7)表达的影响。方法:将45只成年SPF级雄性SD大鼠均分为模型组(model组)、淫羊藿苷低剂量组(ICA-L组)和淫羊藿苷高剂量组(ICA-H组),每组都建立骨折模型;X线片观察各组大鼠骨折线的变化及达到愈合的时间;micro-CT检测各组大鼠的骨密度(BMD)和骨体积分数(BV/TV);ELISA检测各组大鼠血清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、碱性磷酸酶(ALP)、骨钙素、TGF-β1、VEGF和BMP-7的表达;Western blotting检测各组大鼠骨组织中TGF-β1、VEGF和BMP-7蛋白的表达。结果:淫羊藿苷使骨折大鼠在第7周时基本完全愈合,BMD和BV/TV均升高(P0.05);与model组相比,ICA-L和ICA-H组大鼠血清中IL-6、TNF-α的表达水平均下降,IL-10的表达水平升高,ALP活性升高,骨钙素含量升高,差异有统计学意义(P0.05);淫羊藿苷使大鼠血清中TGF-β1、VEGF和BMP-7表达水平上升,骨组织中TGF-β1、VEGF和BMP-7蛋白表达水平上调,差异有统计学意义(P0.05)。结论:淫羊藿苷使大鼠骨折愈合程度加快,可能与TGF-β1、VEGF和BMP-7的表达有关。     

内皮-间充质转分化在腹膜透析相关腹膜纤维化中的研究进展

腹膜透析是终末期肾脏病患者的肾脏替代治疗之一。腹膜透析相关腹膜纤维化是患者退出腹膜透析最重要的原因。既往研究认为腹膜纤维化的主要机制是上皮-间充质转分化(epithelial-mesenchymal transition, EMT)。近年来,研究发现内皮-间充质转分化(endothelial-mesenchymal transition, EndMT)可能是腹膜纤维化的机制之一,高糖、炎症、氧化应激等均可影响腹膜组织中EndMT的发生发展。因此,本文将综述EndMT在腹膜透析相关腹膜纤维化的研究进展。     

糖尿病大鼠阴茎海绵体GSH、GSSG和GSH/GSSG对eNOS表达的影响

目的探究在糖尿病大鼠阴茎海绵体中还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)和GSH/GSSG对内皮型一氧化氮合酶(eNOS)表达的影响。方法 30只SD级8周龄健康雄性大鼠,8只作为正常对照组(C组),剩余22只作为糖尿病实验组(D组);D组大鼠高脂高糖喂养4周后腹腔注射链脲佐菌素(40mg/kg)建DM模型;C组大鼠未作特殊处理。继续喂养8周后,注射阿扑吗啡(apomorphine,APO),观察阴茎勃起情况;所有大鼠测定阴茎海绵体内压/平均颈动脉压(ICP_(max)/MAP)后处死,采集血液、阴茎组织,测定GSH、GSSG、睾酮含量,免疫组化和Western印迹法检测eNOS的表达。结果与C组相比较,D组大鼠阴茎组织中GSH含量、GSH/GSSG比值、eNOS含量明显降低(P0.05),而GSSG的含量明显升高(P0.05)。结论糖尿病大鼠阴茎海绵体中GSH和GSSG、GSH/GSSG影响eNOS的表达,在糖尿病性阴茎勃起功能障碍机制中起重要作用。     

Parathyroid hormone induces endothelial-to-adipocyte transition in endothelial cells by Wnt/β-catenin pathway

Objective To investigate whether elevated parathyroid hormone (PTH) levels could induce endothelial-to-mesenchymal transition (EndMT) and adipocyte transition in endothelial cells (ECs), and to determine the possible underlying mechanism. Methods (1) A rat model of secondary hyperparathyroidism and chronic kidney disease (CKD) was established. The adiposity in bone marrow was detected by oil red O staining. Immunofluorescence staining was performed to detect the expression and localization of cluster of differentiation 31 (CD31) and fibroblast-specific protein 1 (FSP1). (2) The human umbilical vein ECs were cultured in vitro. Western blotting was performed to detect protein expressions of EndMT-related markers CD31, FSP1 and α-smooth muscle actin (α-SMA) in interference groups with different PTH concentrations (0, 10-11, 10-9, 10-7 mol/L PTH for 48 h) and times (0, 12, 24, 48 h, 10-7 mol/L PTH), as well as the expression of β-catenin in interference groups with different PTH concentrations. The localizations of CD31, FSP1 and β-catenin were observed by cell immunofluorescence. Protein expressions of adipocytes markers peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α) by Western blotting and the degree of adipogenesis by oil red O staining were detected after transformed ECs were cultured in adipogenic culture medium for one week. Small interfering RNA (siRNA) was performed to silence β-catenin expression. ECs were divided into control siRNA group, β-catenin siRNA group, PTH+control siRNA group and PTH+β-catenin siRNA group. Protein expressions of CD31, FSP1 and PPAR-γ by Western blotting and the degree of adipogenesis by oil red O staining were determined. Results (1) In vivo, compared with the control, CKD rats had increased adipocytes in bone marrow (P＜0.05), and the co-expression of CD31 and FSP1 in bone marrow ECs. (2) In vitro, PTH significantly inhibited the expression of endothelial marker CD31 and increased the expressions of mesenchymal markers FSP1 and α-SMA in concentration-and time-dependent manners. These indexes in 10-7 mol/L PTH group and 0 mol/L PTH group, in 48 h group and 0 h group showed statistical differences (all P＜0.05). In PTH group ECs with 10-7 mol/L PTH for 48 h showed FSP1 accumulation in the cytoplasm and reduced expressions of CD31, and ECs had higher expressions of PPAR-γ and C/EBP-α as well as the degree of adipogenesis than those in control group (all P＜0.05). Furthermore, PTH enhanced the nuclea β-catenin protein levels in ECs in concentration-dependent. The expressions of β-catenin in 10-7 mol/L PTH group and 0 mol/L PTH group showed statistical differences (P＜0.05). β-catenin expressed in the cytoplasm in control group, while it enter into the nucleus in PTH group. Compared with those in PTH+control siRNA group, the expressions of CD31 and PPAR-γ as well as the degree of adipogenesis decreased in PTH+β-catenin siRNA group (all P＜0.05), while the expression of FSP1 increased (P＜0.05). Conclusions PTH induces ECs- to-adipocytes transition by the canonical Wnt/β-catenin signaling pathway, which might account for bone loss in CKD. Silenced β-catenin expression can inhibit PTH-induced EndMT and adipogenesis.     

High glucose induces endothelial-to-chondrocyte transition in human aortic endothelial cells

Objective To explore whether high glucose (HG)-induced endothelial-to-mesenchymal transition (EndMT) could be transitioned into mesenchymal stem cells (MSCs) and further differentiated into chondrocytes. Methods Human aortic endothelial cells (HAECs) were divided into three groups: normal glucose (NG,5.5 mmol/L glucose) group, HG (30 mmol/L glucose) group, and mannitol (5.5 mmol/L glucose＋24.5 mmol/L mannitol) group, and were cultured for 48 h. Immunofluorescence staining was performed to detect the co-expression of CD31 (endothelial markers), and fibroblast-specific protein 1 (FSP1, fibroblast markers). The expression of CD31 and FSP1 mRNA and protein was detected by real-time PCR and Western blotting. When endothelial-derived MSCs were grown in MSC medium for one week, the expression of the MSCs markers CD44, CD10 and the chondrocyte marker SOX9 was detected by Western blotting and RT-PCR. Chondrocyte expression was detected by alcian blue staining. Calcium deposit was analyzed by alizarin red staining. Pathological changes were investigated using electron microscopy. Results The expression of FSP1 mRNA and protein was significantly increased, but the expression of CD31 mRNA and protein was decreased (P＜0.01), and the cells undergoing EndMT also significantly expressed CD10, CD44 and SOX9 in the HG group compared with those in normal glucose group (P＜0.01). The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype, wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm. Double staining of the HAECs indicated a co-localization of CD31 and FSP1. After one week culture for chondrocyte medium, the expression of MSCs marker STRO-1 was significantly increased by immunofluorescence staining. Additionally, alcian blue staining in the HG group was positive compared to the NG group. Consistent with the elevation of SOX9 expression, calcium deposit also enhanced in the HG group. Conclusion HG can induce endothelial cells transdifferentiation into chondrocyte-like cells via the EndMT.     

Active vitamin D reduces macrophage infiltration by TREM-1 in renal tissue of diabetic nephropathy rats

Objective To investigate the effects of active vitamin D (VD) on the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in renal tissue of diabetic nephropathies (DN) rats and to explore the impact of TREM-1 on adhesion and migration capacity of macrophage. Methods DN rat models were established by streptozotocin. Rats were randomly distributed into four groups: control (NC) group, VD group, DN group and DN+VD group (DN rats with 0.1 μg?kg-1?d-1 calcitriol by gavages). Rats were sacrificed respectively at 8 weeks and 12 weeks after treatment. Pathological changes in kidney tissue were detected and the expressions of CD68 and TREM-1 were acquired by immunohistochemistry stain and Western blotting. In vitro, RAW264.7 cells were divided into NC group, VD group, high glucose (HG) group and HG+VD group. In HG+VD group rats were treated by high glucose with 10-8 mol/L 1,25(OH)2D3. TREM-1 expression was measured by immunohistochemistry stain and Western blotting, and the ability of macrophage in migration and adhesion was evaluated by Transwell migration assay and adhesion assay. TREM-1 siRNA was transferred to silence TREM-1 expression, while plasmid of TREM-1 was transferred for high expression. Their ability of adhesion and migration in macrophage and the effect of 1,25(OH)2D3 were examined. Results (1) Compared with the NC group, the expressions of CD68 and TREM-1 were increased in DN group (P＜0.05), whereas markedly decreased in DN+VD group (P＜0.05). (2) The number of adhesion and migration cells, and the expression of TREM-1 protein in macrophage were obviously increased in HG group as compared with those in NC group (all P＜0.05); whereas above changes were markedly decreased in HG+VD group than those in HG group (P＜0.05). (3) The number of adhesion and migrated macrophage was reduced after TREM-1 siRNA intervention (all P＜0.05). VD could significantly decrease the effect of high glucose on adhesion and migrated macrophages after TREM-1 siRNA (all P＜0.05). (4) Adhesion and migration of macrophage were increased via TREM-1 overexpression (all P＜0.05), but the effects of VD on high glucose-induced adhesion and migration of macrophage were disappeared. Conclusions VD can suppress the adhesion and migration of macrophage via reducing the expression of TREM-1, and inhibit infiltration of macrophage in renal tissue of DN rats.     

Role of Irbesartan on cardiac endothelial - to - mesenchymal transition in diabetic rats

Objective To explore the effect of irbesartan on cardiac endothelial-mesenchymal transition (EndMT) in diabetic rats. Methods The model of diabetic rat was induced by intraperitoneal injection with streptozotocin (STZ, 35 mg/kg) in spontaneous hypertensive rats (SHR). Diabetic rats were divided into diabetic group and the Irbesartan treated group. The pathological changes were investigated by fluorescence microscope and electron microscope. The EndMT was studied in human aortic endothelial cells (HAEC) exposure to high glucose. The concentration of angiotensin II in the supernatant was detected by radioimmunoassay. Immunofluorescence staining was performed to detect the co - localization of CD31 and FSP1. Results The significant myocardial fibrosis was presented in the diabetic group. Endothelial protrusions were prominent feature in myocardial microvascular of diabetic rat compared with the control group rats. Double staining of HAEC showed co-localization of CD31 and FSP1, which was decreased by the treatment of Irbesartan (P＜0.05). When HAEC was exposed to high glucose, it showed some cells acquired spindle-shaped morphology and lost CD31 staining, and FSP1 and α - SMA protein expression levels were markedly upregulated, which attenuated by the treatment of Irbesartan. Conclusion Irbesartan might prevent diabetes from myocardial fibrosis via inhibition of EndMT in diabetic rats.     

Human umbilical cord mesenchymal stem cells co-culture ameliorates the innate immunity of podocytes induced by high glucose

Objective To explore the effects of human umbilical cord mesenchymal stem cells(HUC-MSCs) on the innate immunity of podocytes mediated by Toll-like receptor (TLR) signaling pathway under high glucose (HG) condition. Methods Podocytes were divided into four groups according to the treatment: normal glucose group (NG), mannitol control group (NG+MA), high glucose group (HG) and HUC-MSCs co-culture group (HG+MSC). After 72 hours treatment, the protein levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), heat shock protein 70 (HSP70), high-mobility group box-1 (HMGB1) in culture medium were measured by ELISA. Real-time PCR was used to detect the mRNA expressions of TLR2 and TLR4. Western blotting was used to detect the protein expressions of TLR2, TLR4, myeloid differentiation factor 88 (MyD-88) and phospho-P65 (p-P65). Immunofluorescence staining was used to study the localization of p-P65 in podocytes. Results High glucose induced the inflammation of podocytes by activating the TLR signaling, which increased the secretion of IL-6, TNF-α, HSP70, HMGB1, the mRNA level of TLR2, TLR4 and the protein level of TLR2, TLR4, MyD-88 and p-P65 (all P＜0.05). High glucose also activated NF-κB and induced its nuclear translocation. HUC-MSCs co-culture decreased the inflammation and restrained the TLR signaling. Conclusions HUC-MSCs co-culture decreases the inflammation and innate immunity of podocytes induced by HG.     

Effect of mycophenolate mofetil on CD26 expression in the kidneys of diabetic rats

Objective To investigate the expression of CD26 (dipeptidyl peptidase 4) in the kidney tissues of diabetic rats and the effects of mycophenolate mofetil (MMF) on the renal CD26 expression. Methods Wistar rats were randomly divided into three groups: normal control group (NC group, n=7), diabetic model group (DM group, n=7) and MMF-treated group (MMF group, n=7). Wistar rats were fed with high-sucrose-high-fat diet and injected with streptozotocin into abdominal cavity to induce diabetes. Sixteen weeks later, blood glucose (BG), blood urea nitrogen (BUN), serum creatinine (Scr), renal hypertrophy index (kidney weight/body weight) and 24 hour urinary protein (24Upro) were measured. The number of CD3+/CD4+ T cells in renal tissues were measured through flow cytometry. The expression of CD26 in kidney was examined by using Western blotting and immunohistochemistry. Results Compared with NC group, BG, BUN, Scr, kidney weight/body weight, 24Upro were significantly increased in DM group (P﹤0.05). Except BG and kidney weight/body weight, the above-mentioned parameters were lower in MMF group compared with that in DM group (P﹤0.05). Intrarenal CD3+/CD4+ T cells were significantly up-regulated in DM group compared with that in NC group (P﹤0.01). CD26 in renal tissue was mainly expressed in T lymphocytes of renal interstitium. CD26 expression in DM group was significantly higher than that in NC group, and also higher than that in MMF group (P﹤0.05). In DM group, CD26+ T lymphocytes infiltration of renal interstitium was positively correlated with 24Upro (r2=0.770, P﹤0.05). Conclusions CD26 is related with diabetic nephropathy. MMF maybe inhibit T lymphocytes infiltration to reduce the expression of CD26 in renal interstitium, thus protecting the kidney function.     

Activation of CXCL16 pathway by inflammation accelerates the progression of diabetic nephropathy

Objective To investigate the potential role of CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway in the progression of diabetic nephropathy (DN). Methods 8 - week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK-2 cells were treated with high glucose (HG), and IL-1β + HG to investigate the effect of inflammatory stress on HK-2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXCl16, then cells were divided into HG + IL-1β group, HG + IL-1β + siCXCL16 group and HG + IL-1β + vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N-acetyl-β-D-glucosaminidase (NAG) during 8 weeks. The ultra- microstructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK - 2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXCl16, CXCR6, a disintegrin and metalloproteinase-10 (ADAM10), fibronectin and α smooth muscle actin (α - SMA) in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXCl16, CXCR6, ADAM10, fibronectin and α-SMA in HK-2 cells were detected by real-time PCR and Western blotting, and protein expressions of CXCl16, CXCR6 and ADAM10 in HK - 2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week (all P＜0.05). Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular cells, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α-SMA were increased in DN inflamed group when compared with DN group (all P＜ 0.05). Further, the expressions of CXCL16, CXCR6, ADAM10 were significantly increased in DN inflamed group (all P＜0.05). In vitro, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α-SMA, and lipid accumulation were increased in high glucose plus IL-1β group when compared with high glucose group (all P＜0.05). However, after siRNA of CXCL16 transfection, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α-SMA were down-regulated in HG+IL-1β+siCXCL16 group as compared with high glucose+IL-1β group (all P＜0.05). Furthermore, lipid accumulation was decreased (P＜0.05). Conclusion Inflammation accelerates tubulointerstitial injury in DN partly through the activation of CXCL16 pathway, which may facilitate the lipid accumulation in tubular epithelial cells.     

Effects of 1,25-dihydroxyvitamin D3 on the expression of connective tissue growth factor and heat shock protein 47 in peritoneum fibrosis rats

Objective To investigate the expression of connective tissue growth factor (CTGF) and heat shock protein 47 (HSP47) in peritoneum fibrosis rats, and the mechanism of 1,25-dihydroxyvitamin D3 [1,25-(OH)2-VitD3] in inhibiting the peritoneum fibrosis. Methods Adult male Sprague-Dawley rats were randomly divided into 3 groups: control group (n=8), model group (n=8) and 1,25-dihydroxyvitamin D3 group (VitD3, n=8). The model of peritoneum fibrosis rats were induced by daily intraperitoneally injection of 15% chlorhexidine gluconate (CHX) 0.2 ml/d with 0.1% glucose for 4 weeks. Rats in VitD3 group were also treated with 1,25-(OH)2-VitD3 [i.p. 6 ng?(100 g)-1?d-1]. Peritoneal transport function, renal function, peritoneum thickness and serum level of 25 hydroxyvitamin D3 were detected. In vitro, primary cultured peritoneal mesothelial cells were divided into control group, high glucose group (HG, 2.5%), CTGF siRNA intervention group (CTGF siRNA+HG), VitD3 intervention group (VitD3+HG) and combined intervention group (CTGF siRNA+VitD3+HG). Real-time PCR, Western blotting and immunofluorescence were applied to measure the expression of CTGF and HSP47, also ELISA was used to detect the protein level of FN in peritoneum and peritoneal mesothelial cells. Results Compared with control group, the peritoneal ultrafiltration in peritoneum fibrosis rats were significantly decreased (P＜0.05), the absorbance level of peritoneal fibrosis, peritoneum thickness, the rate of dialysate urea nitrogen and blood urea nitrogen (DUN/BUN) and the expressions of CTGF and HSP47 were increased (all P＜0.05). After application of 1,25-dihydroxyvitamin D3, peritoneal fibrosis lesion was significantly improved, the peritoneum thickness, the expressions of CTGF and HSP47 were decreased (all P＜0.05). In vitro, 2.5% high glucose induced-peritoneal mesothelial cells were respectively treated by CTGF siRNA, 1,25-(OH)2-VitD3 and combined interventions, the expression of FN, CTGF and HSP47 was significantly lower than that in high glucose group (all P＜ 0.05). Conclusions The expression of CTGF and HSP47 is significantly increased in peritoneal fibrosis rats. 1,25-(OH)2-VitD3 may ameliorate the progression of peritoneal fibrosis via reducing the expression of CTGF, decreasing the expression of HSP47 and FN.