Gremlin1在高糖环境足细胞损伤中的作用及机制研究
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摘要
目的：糖尿病肾病（diabetic nephropathy，DN）是糖尿病最常见的微血管并发症之一，是目前世界范围内导致终末期肾病（end stage renaldisease, ESRD）的第一位病因，增加了各国卫生保健事业的支出，严重影响患者的生存质量。糖尿病肾病临床主要表现为浮肿、蛋白尿和进行性肾功能减退；肾脏病理主要表现为结节状或弥漫性系膜细胞增殖、系膜细胞外基质积聚、系膜区增宽、基底膜（GBM）增厚，足细胞足突融合，肾小球硬化及小管间质纤维化。近年来的研究显示，肾小球滤过屏障结构和功能的改变，尤其是作为肾小球滤过屏障重要组成成分的足细胞损伤，在糖尿病肾病的进展中发挥了重要作用，与长期的、进行性加重的蛋白尿密切相关。
足细胞是一种高度特异性的终末分化的脏层上皮细胞，附着于肾小球基底膜外侧，与肾小囊壁层上皮细胞辗转延续，功能互补，参与构成肾小球滤过屏障的最外层，对蛋白质的滤过及GBM成分的更新起着举足轻重的作用。糖尿病肾病早期主要表现为足细胞的损伤，这一病损可早于系膜细胞和小管细胞数年，与临床早期持续蛋白尿相关。由于高糖环境导致足细胞损伤，使蛋白漏出增加，而蛋白尿又进一步加重足细胞的损伤，研究显示足细胞在糖尿病肾病的进展中具有重要作用，因此人们把糖尿病肾病也称之为糖尿病足细胞病。在DN的发生发展中，足细胞的病理改变主要包括足细胞数目减少，凋亡增加，流动性增强，从GBM上脱落导致GBM裸露，形成局灶粘连和肾小球硬化。但是，高糖导致足细胞损伤的具体机制尚不完全清楚，有研究显示，gremlin生发基因在糖尿病肾病时重现，调控的损伤修复反应参与了足细胞的损伤过程。
Gremlin是进化上高度保守的、分子量为23-28KD、由184个氨基酸残基构成的分泌型糖蛋白，属于骨形态蛋白(bone morphogenetic protein，BMP)拮抗剂家族成员,在胚胎发育、生长和分化过程中起有重要的作用。有磷酸化和糖基化修饰，这些修饰位点介导蛋白质与蛋白质之间的相互作用。在胚胎发育时期gremlin通过调节由SHH/FGF-4（sonichedgehog/fibroblast growth factor-4）构成的间质极化区（ZPA:zone ofpolarizing activity）和外胚层嵴顶点(apical ectodermal ridge)之间的反馈环路而实现对肢体发育和肺、肾分支形态的调控，随着器官的发育成熟而寂静停止；而这一反馈调节的结果是通过gremlin1对相关组织的凋亡进行时空调控实现的。在成年正常肾组织几乎无gremlin1表达，DN时表达明显增强，并与TGF-β1共定位。Gremlin1可以介导高糖培养近端小管上皮细胞表型转变，分泌大量胶原和纤连蛋白，促进间质纤维化；可以使高糖培养的肾小球系膜细胞增殖，产生大量系膜外基质（ECM），促进系膜区扩张，造成肾小球硬化。关于gremlin是否可以引起足细胞的损伤，文献未见报道。
在早期糖尿病肾病中，足细胞损伤是引起肾小球性蛋白尿的重要原因，而gremlin1在早期DN主要表达在足细胞。足细胞标志蛋白的表达下降和足细胞的凋亡是反应足细胞损伤的主要指标。本研究拟通过高糖环境体外培养条件性永生化足细胞，探讨gremlin1在糖尿病肾小球足细胞损伤中的作用及机制，为进一步阐明糖尿病肾病的发病机制及其防治提供理论依据。
方法：
1Gremlin1在高糖环境足细胞中的表达与分布
条件性永生化小鼠足细胞在33℃，γ-IFN存在的许可条件下培养传代后，在37℃，无γ-IFN的非许可条件下培养10-14天，促进足细胞分化成熟。应用免疫荧光化学检测F-actin以观察分化与未分化足细胞形态；应用免疫荧光化学检测synaptopodin观察足细胞成熟情况，未分化成熟的足细胞不表达synaptopodin，合格的细胞被用于实验。足细胞随机分为3组：正常对照组（Normal Control, NC）、高糖组（High Glucose, HG）和甘露醇对照组（Manitol+Glucose Control，MG）。NC组细胞给予D-glucose5.5mmol/L，HG组给予D-glucose30mmol/L，为了控制高渗透压对细胞实验结果的影响，MG组应用24.5mol/L甘露醇+D-glucose5.5mmol/L作为对照。各组细胞同步培养48小时后收集，应用western blot和real-time PCR方法检测gremlin1蛋白和mRNA在高糖环境足细胞中的表达；应用激光共聚焦显微镜观察gremlin1在足细胞中的分布情况及定位特点。
2重组gremlin1对高糖环境足细胞标志蛋白及凋亡的影响
应用重组的小鼠gremlin1细胞因子与高糖共同刺激足细胞，模仿生物体内高糖环境下gremlin1过表达的情形。①首先探讨gremlin1对高糖环境足细胞发生作用的最佳浓度和时间，即量效和时效关系实验。用高糖分别混合0、0.1、0.25、0.5、0.75、1μg/ml的gremlin1培养分化成熟的足细胞48小时，应用western blot和real-timePCR方法，找出gremlin1对足细胞nephrin和synaptopodin负性调控最明显的浓度作为下一步应用gremlin1的浓度依据。其次应用高糖混合最佳作用浓度的gremlin1因子分别培养成熟足细胞0、6、12、24、48、72小时，找出gremlin1对高糖环境足细胞nephrin和synaptopodin负性调控作用的最佳时间。②在最佳gremlin1作用浓度(0.75μg/ml)和时间(48h)条件下，分别设置正常对照（NC）、高糖对照(HG)和高糖+gremlin1(HG+grem1)对照，应用激光共聚焦显微镜观察gremlin1对足细胞nephrin和synaptopodin表达和定位的影响，直观分析gremlin1对足细胞的损伤作用。③设置对照如上，应用TUNEL检测gremlin1对足细胞凋亡的影响。计数TUNEL阳性细胞数。④应用western、real-time PCR方法检测Bcl-2、Bax、Cleaved Casepase-3等凋亡相关蛋白表达，以弥补TUNEL方法缺陷，与TUNEL一起共同评价gremlin1对高糖足细胞凋亡的影响。
3Gremlin1-siRNA对高糖环境足细胞标志蛋白及凋亡的影响
分化成熟的足细胞分为高糖组(HG)、高糖+gremlin1-siRNA组(HG+grem.siRNA)和高糖+对照siRNA组(HG+con.siRNA)。①优化转染条件，找出化学合成的gremlin1-siRNA沉默gremlin1基因表达的最佳剂量。②应用激光共聚焦显微镜观察gremlin1-siRNA对高糖环境足细胞nephrin和synaptopodin表达及定位的影响，评价gremlin-siRNA对足细胞的影响。③应用western blot和real time PCR的方法观察gremlin1-siRNA对高糖环境足细胞nephrin和synaptopodin表达的影响。④应用TUNEL方法观察gremlin1-siRNA对高糖环境足细胞凋亡的影响，并计数TUNEL阳性细胞。⑤检测gremlin1-siRNA对高糖环境足细胞Bcl-2、Bax和Cleaved Caspase-3等凋亡相关蛋白表达的影响，进一步印证gremlin1-siRNA对足细胞凋亡的影响。
4介导gremlin1对高糖环境足细胞损伤的信号机制研究
①由于在DN肾组织上发现TGF-β1和gremlin1表达共定位，因此我们研究高糖环境时二者之间是否存在特定关系。将分化成熟的高糖培养的足细胞分为两组，一组高糖培养基中加入重组的gremlin1细胞因子（0.75μg/ml）共同孵育48小时，检测TGF-β1的蛋白和mRNA表达情况（HG+grem1组）；另一组采用高糖培养基中加入重组的TGF-β1细胞因子（5ng/ml）共同孵育48小时，检测gremlin1的蛋白和mRNA表达情况（HG+TGF-β1组）；另外设置正常对照组（NC）和高糖对照组(HG)分别检测gremlin1和TGF-β1的表达。②与gremlin1相关的TGF-β信号分子的检测：用高糖与重组gremlin1（0.75μg/ml）共同孵育足细胞0、15、30、60、120、240分钟后，检测smad2/3、p-smad2/3、p38、p-p38、JNK1/2、p-JNK1/2等经典与非经典TGF-β信号分子，找出与gremlin1具有时效关系的信号途径进行阻断实验研究，以期发现其中的调控机制。③上述实验结果发现TGF-β/smad2/3信号途径与gremlin1作用相关。应用TGF-β1受体阻断剂SB431542阻断TGF-β1信号，观察TGF-β1受体阻断后对gremlin1抑制的nephrin和synaptopodin蛋白表达的影响。④应用smad2/3-siRNA转染高糖和gremlin1共同培养的小鼠足细胞，48小时后收获细胞，验证smad2/3敲减效果，并检测smad2/3敲减后对gremlin1抑制的nephrin和synaptopodin蛋白表达的影响。
结果：
1Gremlin1在高糖环境足细胞中的表达与分布
①足细胞F-actin免疫荧光染色结果显示，在33℃，γ-IFN存在的许可条件下培养，足细胞呈梭形或三角形，细胞突起较少。在37℃，无-IFN的非许可条件下培养10-14天后，足细胞胞体上伸出大量足突，胞浆向四周展开，呈树枝状。Synaptopodin免疫荧光染色结果显示，未分化足细胞胞浆中synaptopodin的表达几乎没有，而分化10天后的足细胞中synaptopodin的表达明显增强，提示足细胞已分化成熟。②Western blot结果：在正常对照及甘露醇对照组，gremlin1蛋白表达很少，而HG组gremlin1表达明显上调(p<0.01)。③Real-time PCR检测结果显示，与NC和MG对照组相比，HG组足细胞gremlin mRNA的表达水平在培养48h时明显升高（p<0.01）。④激光共聚焦显微镜观察结果显示：gremlin1在高糖环境足细胞中的分布主要位于胞浆中，少量沿细胞膜分布。胞浆中的gremlin1蛋白主要呈斑点状或颗粒状分布；包膜上的gremlin1蛋白呈细颗粒状或线样分布。
2重组gremlin1对高糖环境足细胞标志蛋白及凋亡的影响
①Gremlin1量效关系实验结果显示：在高糖混合0.75μg/ml gremlin细胞因子时，nephrin和synaptopodin的蛋白和mRNA表达降低显著（p<0.01）。②Gremlin1时效关系实验结果显示：在高糖混合0.75μg/mlgremlin作用48h时，nephrin和synaptopodin的蛋白和mRNA的表达降低显著（p<0.01）。③Gremlin1对足细胞nephrin和synaptopodin影响的共聚焦结果显示：gremlin1可以加重高糖引起的足细胞损伤，二者表达量均较高糖组进一步下降。Nephrin分布由细胞膜为主转变为以胞浆为主，synaptopodin由斑点状沿actin细丝分布转变为团块状或核周、核内分布。这些分布结构的改变与细胞功能的变化密切相关，提示足细胞受损。④TUNEL结果显示，与HG组相比，HG+grem1组细胞凋亡发生率明显升高（p<0.05）。⑤凋亡相关蛋白的检测结果显示：gremlin1使Bcl-2蛋白及mRNA表达较HG组进一步下降，而Bax和Cleaved Caspase-3的表达较HG组进一步升高，提示gremlin1进一步加重高糖引起的足细胞凋亡。3Gremlin1-siRNA对高糖环境足细胞标志蛋白及凋亡的影响
①Gremlin1-siRNA验证实验结果：gremlin1-siRNA能够显著敲减高糖环境足细胞gremlin1蛋白和mRNA的表达，差异显著，（p<0.01）。证实在6孔板中以80pmol/孔转染足细胞敲减效果最好。②激光共聚焦显微镜结果显示：gremlin1-siRNA能够改善或部分恢复高糖环境足细胞nephrin和synaptopodin的表达和分布。Nephrin由高糖时胞浆为主部分恢复转至胞膜，synaptopodin由高糖时的粗颗粒状、团块状恢复至沿actin细丝排列的斑点状。③Western blot和real time PCR实验结果显示gremlin1-siRNA可以上调高糖环境足细胞nephrin和synaptopodin的蛋白及mRNA的表达。
④TUNEL结果：Gremlin1-siRNA可以改善高糖诱导的足细胞凋亡，TUNEL细胞阳性率下降显著（p<0.05）。⑤Gremlin1-siRNA对高糖环境足细胞凋亡相关蛋白的影响结果显示：gremlin-siRNA可以恢复Bcl-2蛋白和mRNA的表达，并下调Bax、Cleaved Casepase-3蛋白及mRNA的表达，较高糖组差异显著（p<0.01）。
4介导gremlin1对高糖环境足细胞损伤的信号机制研究
①Gremlin1与TGF-β1关系研究结果显示：gremlin1与TGF-β1在高糖足细胞中可以互相诱导生成，相互促进，与高糖组比较差异显著（p<0.05）。②与gremlin1相关的TGF-β信号分子研究结果显示：经典的TGF-β1途径信号分子smad2/3蛋白在高糖和gremlin1共培养时存在时间依赖性的磷酸化激活（p<0.05），而非经典途径的p38、JNK1/2信号系统在gremlin1刺激后无明显变化。说明gremlin1发挥作用可能需要经典的TGF-β1/smad2/3信号途径。③TGF-β1受体阻断实验结果显示：SB431542（5μmol/L）可以阻断gremlin1对高糖环境足细胞nephrin和synaptopodin蛋白表达的抑制（p<0.01），说明gremlin1需要内源性TGF-β1参与其活性。④Smad2/3-siRNA阻断实验结果显示：应用smad2/3-siRNA阻断TGF-β1信号可以阻断gremlin对高糖环境足细胞nephrin和synaptopodin蛋白表达的抑制（p<0.01），再次印证体外gremlin1发挥作用需要内源性TGF-β1的存在。
结论：
1在高糖培养的小鼠足细胞中，gremlin1表达上调。Gremlin1蛋白主要分布在足细胞的胞浆中，少部分分布在胞膜上。胞浆部分呈颗粒状分布，包膜部分呈细颗粒状或线样排列。
2Gremlin1加重高糖所致的nephrin和synaptopodin表达下降及分布异常，促进足细胞损伤。Gremlin1加重高糖所致的足细胞凋亡。
3Gremlin1-siRNA治疗可以改善高糖所致的nephrin和synaptopodin表达下降和分布异常，降低高糖所致足细胞凋亡，部分恢复足细胞损伤。
4在高糖环境足细胞中gremlin1与TGF-β1相互诱导生成，协同发挥作用。Gremlin1在高糖环境下可能通过经典的TGF-β1/smad2/3信号途径发挥足细胞损伤作用，阻断TGF-β1/smad2/3信号可以抑制gremlin1对高糖环境足细胞的损伤作用。
综上，本研究提示gremlin1在糖尿病足细胞的损伤和凋亡中发挥了作用，TGF-β1/smad2/3信号途径可能参与了这一过程。
Objective: Diabetic nephropathy (diabetic nephropathy, DN) is one ofthe most common microvascular complications of diabetes, and also the firstcause of end-stage renal disease (ESRD) in the world. It is becaming the hugeburden of health care in developing and developped countries. The patientswith DN mainly show edema, proteinuria, and progressive renal functiondecline. Renal pathology is mainly characterized by excessive amassing ofextracelluar matrix (ECM) with thickening of glomerular and tubularbasement membranes and increased amount of mesangial matrix, whichultimately progress to glomerulosclersis and tubulo-interstitial fibrosis. Recentresearches have shown that the disturbance of the glomerular filtration barrier,especially the podocyte injury (which is an important component ofglomerular filtration barrier), play crucial role in the progression of diabeticnephropathy.
Podocytes are terminally differentiated visceral epithelial cell. Podocytesand their foot processes are unique in that they comprise the outer layer of thekidney ultrafiltration filter and form the glomerula slit diaphragm, which is acomplex cellular structure that prevents the development of the proteinuria inan actin cytoskeleton-dependent manner. In order to highlight the importanceof podocyte in diabetic nephropathy, DN is also called diabetic podocytopathy.In the onset and development in this setting, the pathological changes ofpodocyte mainly include the number decrease, apoptosis, foot effacement,detached from the GBM and GBM bare. However, the detailed mechanism ofpodocyte injury is remaining unclear. The developmental gene gremlinre-emergence in diabetic nephropathy is of great interesting.
Gremlin, an antagonist of bone morphogenetic protein (BMP), is a23-28KD highly conservative secreted glycoprotein, which is present as both soluble and cell-associated forms and is found within the Golgi apparatus andendoplasmic reticulum. It consists of184amino acid residues and regulatesembryonic development, growth and differentiation. There are potentialnuclear localization sites near its carboxyl-terminus, N-linked glycosylationsites, and a number of phosphorylation sites, which mediate the interactionsbetween proteins. During embryonic development, gremlin regulates thedevelopment of limb bud、lung and kidney branching morphogenesis viaSHH/FGF-4(sonic hedgehog/fibroblast growth factor4) feedback loopbetween stromal polarization area (ZPA: zone of polarizing activity) and theapical ectodermal ridge (AER). This process is achieved through the control ofapoptosis. In adult, normal kidney tissues has hardly expression of gremlin,wheras in conditions of diabetic nephropathy the expression of gremlinre-emerges and co-localizes with TGF-beta1. Gremlin results in epithelialmesenchymal transition of proximal tubular epithelial cell and mesangial cellscultured in high ambient glucose, drive them to secrete large amounts ofcollagen and fibronectin and promotes renal fibrosis. Whether gremlin causespodocyte injury, there are limited reports.
The podocyte injury results in proteinuria in the early stage of diabeticnephropathy. The marker proteins’ expression and apoptosis of podocyte aremajor indicators of podocyte injury. The present study aims to investigate therole and mechanisms of gremlin in hyperglycemia-induced podocyte injuryand to explore the probably involved signaling pathway to provide theoreticalbasis for the prevention and management of DN.
Methods:
1The expression and distribution of gremlin in podocyte treated with highglucose.
The conditionally immortalized mouse podocytes were cultured at33℃with γ–IFN (the permissive conditions) to induce proliferation, and thencultured at37℃withoutγ–IFN (the unpermissive conditions) for10-14daysto induce podocytes differentiation and mature. F-actin and synaptopodin weredetected by immunofluorescence to validate the morphology and differentiation of podocytes. Qualified podocytes were used in experiments.Differentiated podocytes were randomly divided into3groups: normal control(NC) group, high glucose group (HG) and normal glucose+mannitol group(MG) group. NC group was treated with D-glucose5.5mmol/L, HG groupwas treated with D-glucose30mmol/L and MG group was treated with5.5mmol/L D-glucose+24.5mol/L mannitol in order to control the influence ofhigh osmotic pressure to the experimental results. After synchronous culturedfor48hours three group cells were harvested to abstract the total protein andmRNA. The expression of gremlin was detected by western blot and real-timePCR. The distribution and localizing characteristics of gremlin in podocyteswere obseved by the Laser confocal microscope.
2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose ambient.
Recombinant mice gremlin and high glucose co-stimulated the podocytesto mimic the conditions of diabetic nephropathy in vivo.①Firstly, to explorethe time and dose relationship between gremlin and high glucose podocytes,the differentiated podocytes were cultivated with high glucose and gremlin (0,0.1,0.25,0.5,0.75,1μg/ml) for48hours respectively. Using western blotand real-time PCR method found out the maximal negative regulatoryconcentration of gremlin on nephrin and synaptopodin, then this optimalconcentration was used in the experiments below. Secondly, podocytes werecultivated with the optimal concentration of gremlin contaminating highglucose for0,6,12,24,48and72hours to find out the maximal negativeregulatory time of gremlin on nephrin and synaptopodin.②Normal control(NC) group, high glucose control (HG) group and high glucose+gremlin (HG+grem1) group were set. According to the results of experiment①, thepodocytes were incubated with0.75ug/ml gremlin for48h, then theexpression and distribution of nephrin and synaptopodin were visualized bylaser confocal microscope to analyze the podocyte injury.③Setting controlsas above, the apoptosis podocytes were detected by TUNEL and TUNELpositive cells were counted.④Bcl-2, Bax and Cleaved Casepase–3, which compensate for the false-positive of TUNEL, were putative indicators ofapoptosis. They were detected by western blot and real-time PCR method tofurther assess the podocyte injury.
3The effect of gremlin-siRNA on the markers and apoptosis of podocyte inhigh glucose ambient.
Differentiated podocytes were divided into three groups: high glucosegroup (HG), high glucose+gremlin-siRNA group (HG+grem.siRNA) andhigh glucose+control siRNA group (HG+con.siRNA).①Optimizing thetransfection conditions: The optimal dose of gremlin–siRNA was confirmedby abviously knockdown the expression of gremlin gene.②The effect ofgremlin-siRNA on the location and distribution of nephrin and synaptopodinin high glucose ambient podocyte was observed by laser confocal microscope.③The effect of gremlin-siRNA on the expression of nephrin andsynaptopodin in high glucose ambient podocyte was assessed by western blotand real time PCR.④The apoptosis effect of gremlin-siRNA on podocyteswere detected by TUNEL. TUNEL positive cells were counted.⑤Bcl-2,Bax and Cleaved Casepase-3were detected by western blot and real-timePCR to further confirm the apoptosis effect of gremlin-siRNA on podocyte.
4The probably signal mechanism involved in gremlin-mediated podocyteinjury in high glucose ambient.
①Due to TGF–β1colocalization with gremlin in renal tissue of DN,there maybe exists a certain relationship between TGF–β1and gremlin inhigh glucose environment. Differentiated mature podocytes were divided intotwo groups: one group was treated with high glucose+gremlin (0.75μg/ml)for48h, the protein and mRNA expression of TGF-β1was detected. Theother group was treated with high glucose+TGF-β1(5ng/ml) for48h, theprotein and mRNA expression of gremlin was detected. Additionally，normalcontrol (NC) and high glucose control (HG) were set.②The cannoniacl andnoncannonical TGF–β1signaling molecules detection in high glucosepodocyte exposed to gremlin: Podocytes were incubated with high glucoseand recombinant mice gremlin (0.75μg/ml) for0,15,30,60,120and240 minutes, the cannonical (smad2/p-smad2, smad3/p-smad3) and noncannonical(p38/p-p38, JNK1/2/p-jnk1/2) signling molecules were detected to find outthe signal pathway mediating the role of gremlin on hyperglycemic podocyte.③The above experimental results implicated that the TGF–β/smad2/3signaling pathways involved in the pathogenesis of gremlin. SB431542, aTGF–β receptor blocker, blocked the suprression of gremlin on the expressionof nephrin and synaptopodin protein in hyperglycemic podocytes.④Podocytes treated with high glucose and gremlin was transfected withsmad2/3–siRNA and harvested after48hours to verified the knocking downeffect. Smad2/3knocking down inhibited the supression of gremlin on theexpression of nephrin and synaptopodin protein.
Results:
1The expression and distribution of gremlin1in high glucose ambientpodocytes.
①F-actin immunofluorescence assay showed that there were twodifferent phenotypes in cultured podocytes. At33℃with γ–IFN (thepermissive conditions), cells proliferated and maintained cobblestone-likemorphology. When cultured at37℃without γ–IFN (nonpermissivecondition), cells stretched out differently shaped long processes from cellbodies，arised many spindle-like projections from their primary processes andstopped proliferation. Synaptopodin is one marker indicating maturedifferentiated podocytes. Immunofluorescence staining showed that there wassparsely expression of synaptopodin in undifferentiated podocytes, while itsexpression in differentiated podocytes was enhanced obviously.②The resultsof Western blot showed that in NC group and MG group, there was hardlyexpression of gremlin protein, while in HG group its expression was obviouslyenhanced (p<0.01).③The results of Real-time PCR displayed that comparedwith NC group and MG group, the expression level of gremlin mRNA in HGgroup was increased significantly when the podocyte was cultured for48hours (p<0.01).④Confocal staining demonstrated that the distribution ofgremlin1in podocytes was located predominantly in the cytoplasm with a punctate pattern., and sparsely on the cell membrane with a thin linear or fineparticles pattern.
2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose environment
①The dose-effect relationship experiment showed that the protein andmRNA expression of nephrin and synaptopodin decreased significantly at agremlin concentration of0.75μg/ml (p <0.01).②The time-effect relationshipexperiment disclosed that the protein and mRNA expression of nephrin andsynaptopodin decreased significantly when cells were treated with HG+0.75μg/ml gremlin for48h (p <0.01).③The laser confocal image showedthat gremlin aggravated the hyperglycemia-induced podocyte injury. Thedistribution of nephrin differed markedly between podocytes cultured in thepresence of gremlin and control cells. Control cells expressed nephrin in cellprojectons or exhibited a filamentous nephrin appearance. By contrast, cellscultured with gremlin displayed a cytoplastic and non-filamentous distribution.In gremlin-cultured podocytes, synaptopodin distributed mainly at peri-orintra-nuclear regions. In normal control cells, synaptopodin localized mainlyin the foot process or cytoplasm in a punctuated pattern along actin filaments.High-glucose induced synaptopodin shifting to the peri-nucleus anddepolymerization of actin mirofilaments. Gremlin aggravated such injury andinduced a marked decreasement in podocyte size, and rearrangement ofsynaptopodin from a punctuated pattern to a granular mass pattern.④Theresults of TUNEL showed that compared with the HG group, the cellapoptosis rate of HG+gremlin group was increased significantly (p <0.05).
⑤The experimental results of apoptosis related proteins displayed thatcompared with the HG group, gremlin decreased the levels of Bcl-2proteinand mRNA further, while increased those of Bax and Cleaved Caspase-3.Collectively, these results disclosed that gremlin aggravatedhyperglycemia-induced podocytes apoptosis.
3The effect of gremlin1-siRNA on the markers and apoptosis of podocyte inhigh glucose environment ①The verification experiment results of gremlin1-siRNA showed that at80pmol/well gremlin-siRNA knocked down the protein and mRNAexpression of gremlin significantly in podocytes cultured in6well plate (p <0.01).②The confocal images displayed that gremlin-siRNA rescued theexpression and distribution of nephrin and synaptopodin in hyperglycemicpodocyte. Gremlin-siRNA attenuated the distribution of nephrin from thecytoplasm and non-filamental to the cell membrane and filament pattern, andthat of synaptopodin from massive granular to punctate pattern.③Westernimmunoblot and real-time PCR analysis indicated that gremlin1siRNArescued the expression of nephrin and synaptopodin at both the mRNA andprotein levels.④Gremlin1-siRNA decreased the percentage ofTUNEL-positive cells. There were7.021%apoptosis cells in HG group and4.986%apoptosis cells in HG+grem.siRNA group (p <0.05).⑤GremlinsiRNA increased the expression of Bcl-2and decreased that of Bax andCleaved Caspase-3in HG ambient (p <0.01). Gremlin1-siRNA attenuated HGinduced apoptosis effectively.
4The signal mechanism mediating the pathogenesis of gremlin inhyperglycemic podocytes
①Podocytes that were cultured in gremlin1(0.75μg/ml) and highglucose (30mmol/L) expressed TGF-β1more abundantly than high glucoseand normal controls at both the protein (p<0.05) and mRNA (p<0.05) levels.Conversely, podocytes that were cultured in TGF-β1(5ng/ml) concomitantlywith high glucose (30mmol/L) expressed gremlin1more abundantly than highglucose and normal controls at both the protein (p<0.05) and mRNA (p<0.05)levels.②The protein of TGF-β signaling molecules (smad2/p-smad2,smad3/p-smad3, p38/p-p38and JNK1/2/p-JNK1/2) was analyzed by westernblot. In gremlin-activated podocytes, smad2and smad3were phosphorylatedin a time-dependent manner. By contrast, neither JNK1/2nor p38MAPK wereactivated. Gremlin activated canonical, but not non-canonical signalingpathway in podocytes (p<0.05, versus0min controls).③TGF-β receptorinhibitors (SB431542) blocked the ability of gremlin1to suppress nephrin and synaptopodin expression. Inhibition of the TGF-β receptor with SB431542(5μM/L) blocked the effect of gremlin1on high glucose cultured podocytes andrescued the expression of nephrin and synaptopodin (p<0.05). Gremlin injuredpodocytes required endogenous TGF-β1.④Smad2/3-siRNA treatmentinhibited the ability of gremlin to down-regulate the expression of nephrin andsynaptopodin in high-glucose ambient podocytes. Podocytes were transfectedwith smad2/3siRNA (60pmol/well) for6hour before treatment with gremlinand high glucose. Control cells were transfected with scrambled siRNAsequences. Smad2/3siRNA knockdown rescued the expression of bothnephrin and synaptopodin and attenuated injury of podocytes cultured in highglucose ambient (p <0.01).
Conclusions:
1Gremlin is clearly elevated in high glucose cultured mouse podocytes.The distribution of gremlin1in podocytes locates predominantly in thecytoplasm in a punctate pattern and sparsely on the cell membrane in a thinlinear or fine particle pattern.
2Gremlin aggravates hyperglycemia induced podocyte injury, supressesthe expression of nephrin and synaptopodin and deteriorates the apoptosis ofpodocytes.
3Treatment high glucose ambient podocyte with gremlin-siRNArescues the expression of nephrin and synaptopodin and declines apoptosisrate of podocyte.
4Gremlin and TGF-β1induce each other in high glucose conditions.Canonical smad2/3pathway, but not non-canonical pathway of TGF-βsignaling is activated by gremlin, which exerts its effect on the podocytethrough endogenous TGF-β signaling.
In summary, gremlin plays a crucial role in the pathogenesis of podocyteinjury in DN, perhaps by disrupting the homeostatic balance between TGF-βand BMP. There also appears to be a feedback loop between gremlin andTGF-β1whereby they are capable of inducing each other and accelerating thepathogenesis of the disease process. Gremlin activates a canonical smad2/3 pathway, but does not activate a non-canonical pathway of TGF-β signaling.New therapies targeting gremlin may protect the kidney from the developmentand progression of DN.
足细胞是一种高度特异性的终末分化的脏层上皮细胞，附着于肾小球基底膜外侧，与肾小囊壁层上皮细胞辗转延续，功能互补，参与构成肾小球滤过屏障的最外层，对蛋白质的滤过及GBM成分的更新起着举足轻重的作用。糖尿病肾病早期主要表现为足细胞的损伤，这一病损可早于系膜细胞和小管细胞数年，与临床早期持续蛋白尿相关。由于高糖环境导致足细胞损伤，使蛋白漏出增加，而蛋白尿又进一步加重足细胞的损伤，研究显示足细胞在糖尿病肾病的进展中具有重要作用，因此人们把糖尿病肾病也称之为糖尿病足细胞病。在DN的发生发展中，足细胞的病理改变主要包括足细胞数目减少，凋亡增加，流动性增强，从GBM上脱落导致GBM裸露，形成局灶粘连和肾小球硬化。但是，高糖导致足细胞损伤的具体机制尚不完全清楚，有研究显示，gremlin生发基因在糖尿病肾病时重现，调控的损伤修复反应参与了足细胞的损伤过程。
Gremlin是进化上高度保守的、分子量为23-28KD、由184个氨基酸残基构成的分泌型糖蛋白，属于骨形态蛋白(bone morphogenetic protein，BMP)拮抗剂家族成员,在胚胎发育、生长和分化过程中起有重要的作用。有磷酸化和糖基化修饰，这些修饰位点介导蛋白质与蛋白质之间的相互作用。在胚胎发育时期gremlin通过调节由SHH/FGF-4（sonichedgehog/fibroblast growth factor-4）构成的间质极化区（ZPA:zone ofpolarizing activity）和外胚层嵴顶点(apical ectodermal ridge)之间的反馈环路而实现对肢体发育和肺、肾分支形态的调控，随着器官的发育成熟而寂静停止；而这一反馈调节的结果是通过gremlin1对相关组织的凋亡进行时空调控实现的。在成年正常肾组织几乎无gremlin1表达，DN时表达明显增强，并与TGF-β1共定位。Gremlin1可以介导高糖培养近端小管上皮细胞表型转变，分泌大量胶原和纤连蛋白，促进间质纤维化；可以使高糖培养的肾小球系膜细胞增殖，产生大量系膜外基质（ECM），促进系膜区扩张，造成肾小球硬化。关于gremlin是否可以引起足细胞的损伤，文献未见报道。
在早期糖尿病肾病中，足细胞损伤是引起肾小球性蛋白尿的重要原因，而gremlin1在早期DN主要表达在足细胞。足细胞标志蛋白的表达下降和足细胞的凋亡是反应足细胞损伤的主要指标。本研究拟通过高糖环境体外培养条件性永生化足细胞，探讨gremlin1在糖尿病肾小球足细胞损伤中的作用及机制，为进一步阐明糖尿病肾病的发病机制及其防治提供理论依据。
方法：
1Gremlin1在高糖环境足细胞中的表达与分布
条件性永生化小鼠足细胞在33℃，γ-IFN存在的许可条件下培养传代后，在37℃，无γ-IFN的非许可条件下培养10-14天，促进足细胞分化成熟。应用免疫荧光化学检测F-actin以观察分化与未分化足细胞形态；应用免疫荧光化学检测synaptopodin观察足细胞成熟情况，未分化成熟的足细胞不表达synaptopodin，合格的细胞被用于实验。足细胞随机分为3组：正常对照组（Normal Control, NC）、高糖组（High Glucose, HG）和甘露醇对照组（Manitol+Glucose Control，MG）。NC组细胞给予D-glucose5.5mmol/L，HG组给予D-glucose30mmol/L，为了控制高渗透压对细胞实验结果的影响，MG组应用24.5mol/L甘露醇+D-glucose5.5mmol/L作为对照。各组细胞同步培养48小时后收集，应用western blot和real-time PCR方法检测gremlin1蛋白和mRNA在高糖环境足细胞中的表达；应用激光共聚焦显微镜观察gremlin1在足细胞中的分布情况及定位特点。
2重组gremlin1对高糖环境足细胞标志蛋白及凋亡的影响
应用重组的小鼠gremlin1细胞因子与高糖共同刺激足细胞，模仿生物体内高糖环境下gremlin1过表达的情形。①首先探讨gremlin1对高糖环境足细胞发生作用的最佳浓度和时间，即量效和时效关系实验。用高糖分别混合0、0.1、0.25、0.5、0.75、1μg/ml的gremlin1培养分化成熟的足细胞48小时，应用western blot和real-timePCR方法，找出gremlin1对足细胞nephrin和synaptopodin负性调控最明显的浓度作为下一步应用gremlin1的浓度依据。其次应用高糖混合最佳作用浓度的gremlin1因子分别培养成熟足细胞0、6、12、24、48、72小时，找出gremlin1对高糖环境足细胞nephrin和synaptopodin负性调控作用的最佳时间。②在最佳gremlin1作用浓度(0.75μg/ml)和时间(48h)条件下，分别设置正常对照（NC）、高糖对照(HG)和高糖+gremlin1(HG+grem1)对照，应用激光共聚焦显微镜观察gremlin1对足细胞nephrin和synaptopodin表达和定位的影响，直观分析gremlin1对足细胞的损伤作用。③设置对照如上，应用TUNEL检测gremlin1对足细胞凋亡的影响。计数TUNEL阳性细胞数。④应用western、real-time PCR方法检测Bcl-2、Bax、Cleaved Casepase-3等凋亡相关蛋白表达，以弥补TUNEL方法缺陷，与TUNEL一起共同评价gremlin1对高糖足细胞凋亡的影响。
3Gremlin1-siRNA对高糖环境足细胞标志蛋白及凋亡的影响
分化成熟的足细胞分为高糖组(HG)、高糖+gremlin1-siRNA组(HG+grem.siRNA)和高糖+对照siRNA组(HG+con.siRNA)。①优化转染条件，找出化学合成的gremlin1-siRNA沉默gremlin1基因表达的最佳剂量。②应用激光共聚焦显微镜观察gremlin1-siRNA对高糖环境足细胞nephrin和synaptopodin表达及定位的影响，评价gremlin-siRNA对足细胞的影响。③应用western blot和real time PCR的方法观察gremlin1-siRNA对高糖环境足细胞nephrin和synaptopodin表达的影响。④应用TUNEL方法观察gremlin1-siRNA对高糖环境足细胞凋亡的影响，并计数TUNEL阳性细胞。⑤检测gremlin1-siRNA对高糖环境足细胞Bcl-2、Bax和Cleaved Caspase-3等凋亡相关蛋白表达的影响，进一步印证gremlin1-siRNA对足细胞凋亡的影响。
4介导gremlin1对高糖环境足细胞损伤的信号机制研究
①由于在DN肾组织上发现TGF-β1和gremlin1表达共定位，因此我们研究高糖环境时二者之间是否存在特定关系。将分化成熟的高糖培养的足细胞分为两组，一组高糖培养基中加入重组的gremlin1细胞因子（0.75μg/ml）共同孵育48小时，检测TGF-β1的蛋白和mRNA表达情况（HG+grem1组）；另一组采用高糖培养基中加入重组的TGF-β1细胞因子（5ng/ml）共同孵育48小时，检测gremlin1的蛋白和mRNA表达情况（HG+TGF-β1组）；另外设置正常对照组（NC）和高糖对照组(HG)分别检测gremlin1和TGF-β1的表达。②与gremlin1相关的TGF-β信号分子的检测：用高糖与重组gremlin1（0.75μg/ml）共同孵育足细胞0、15、30、60、120、240分钟后，检测smad2/3、p-smad2/3、p38、p-p38、JNK1/2、p-JNK1/2等经典与非经典TGF-β信号分子，找出与gremlin1具有时效关系的信号途径进行阻断实验研究，以期发现其中的调控机制。③上述实验结果发现TGF-β/smad2/3信号途径与gremlin1作用相关。应用TGF-β1受体阻断剂SB431542阻断TGF-β1信号，观察TGF-β1受体阻断后对gremlin1抑制的nephrin和synaptopodin蛋白表达的影响。④应用smad2/3-siRNA转染高糖和gremlin1共同培养的小鼠足细胞，48小时后收获细胞，验证smad2/3敲减效果，并检测smad2/3敲减后对gremlin1抑制的nephrin和synaptopodin蛋白表达的影响。
结果：
1Gremlin1在高糖环境足细胞中的表达与分布
①足细胞F-actin免疫荧光染色结果显示，在33℃，γ-IFN存在的许可条件下培养，足细胞呈梭形或三角形，细胞突起较少。在37℃，无-IFN的非许可条件下培养10-14天后，足细胞胞体上伸出大量足突，胞浆向四周展开，呈树枝状。Synaptopodin免疫荧光染色结果显示，未分化足细胞胞浆中synaptopodin的表达几乎没有，而分化10天后的足细胞中synaptopodin的表达明显增强，提示足细胞已分化成熟。②Western blot结果：在正常对照及甘露醇对照组，gremlin1蛋白表达很少，而HG组gremlin1表达明显上调(p<0.01)。③Real-time PCR检测结果显示，与NC和MG对照组相比，HG组足细胞gremlin mRNA的表达水平在培养48h时明显升高（p<0.01）。④激光共聚焦显微镜观察结果显示：gremlin1在高糖环境足细胞中的分布主要位于胞浆中，少量沿细胞膜分布。胞浆中的gremlin1蛋白主要呈斑点状或颗粒状分布；包膜上的gremlin1蛋白呈细颗粒状或线样分布。
2重组gremlin1对高糖环境足细胞标志蛋白及凋亡的影响
①Gremlin1量效关系实验结果显示：在高糖混合0.75μg/ml gremlin细胞因子时，nephrin和synaptopodin的蛋白和mRNA表达降低显著（p<0.01）。②Gremlin1时效关系实验结果显示：在高糖混合0.75μg/mlgremlin作用48h时，nephrin和synaptopodin的蛋白和mRNA的表达降低显著（p<0.01）。③Gremlin1对足细胞nephrin和synaptopodin影响的共聚焦结果显示：gremlin1可以加重高糖引起的足细胞损伤，二者表达量均较高糖组进一步下降。Nephrin分布由细胞膜为主转变为以胞浆为主，synaptopodin由斑点状沿actin细丝分布转变为团块状或核周、核内分布。这些分布结构的改变与细胞功能的变化密切相关，提示足细胞受损。④TUNEL结果显示，与HG组相比，HG+grem1组细胞凋亡发生率明显升高（p<0.05）。⑤凋亡相关蛋白的检测结果显示：gremlin1使Bcl-2蛋白及mRNA表达较HG组进一步下降，而Bax和Cleaved Caspase-3的表达较HG组进一步升高，提示gremlin1进一步加重高糖引起的足细胞凋亡。3Gremlin1-siRNA对高糖环境足细胞标志蛋白及凋亡的影响
①Gremlin1-siRNA验证实验结果：gremlin1-siRNA能够显著敲减高糖环境足细胞gremlin1蛋白和mRNA的表达，差异显著，（p<0.01）。证实在6孔板中以80pmol/孔转染足细胞敲减效果最好。②激光共聚焦显微镜结果显示：gremlin1-siRNA能够改善或部分恢复高糖环境足细胞nephrin和synaptopodin的表达和分布。Nephrin由高糖时胞浆为主部分恢复转至胞膜，synaptopodin由高糖时的粗颗粒状、团块状恢复至沿actin细丝排列的斑点状。③Western blot和real time PCR实验结果显示gremlin1-siRNA可以上调高糖环境足细胞nephrin和synaptopodin的蛋白及mRNA的表达。
④TUNEL结果：Gremlin1-siRNA可以改善高糖诱导的足细胞凋亡，TUNEL细胞阳性率下降显著（p<0.05）。⑤Gremlin1-siRNA对高糖环境足细胞凋亡相关蛋白的影响结果显示：gremlin-siRNA可以恢复Bcl-2蛋白和mRNA的表达，并下调Bax、Cleaved Casepase-3蛋白及mRNA的表达，较高糖组差异显著（p<0.01）。
4介导gremlin1对高糖环境足细胞损伤的信号机制研究
①Gremlin1与TGF-β1关系研究结果显示：gremlin1与TGF-β1在高糖足细胞中可以互相诱导生成，相互促进，与高糖组比较差异显著（p<0.05）。②与gremlin1相关的TGF-β信号分子研究结果显示：经典的TGF-β1途径信号分子smad2/3蛋白在高糖和gremlin1共培养时存在时间依赖性的磷酸化激活（p<0.05），而非经典途径的p38、JNK1/2信号系统在gremlin1刺激后无明显变化。说明gremlin1发挥作用可能需要经典的TGF-β1/smad2/3信号途径。③TGF-β1受体阻断实验结果显示：SB431542（5μmol/L）可以阻断gremlin1对高糖环境足细胞nephrin和synaptopodin蛋白表达的抑制（p<0.01），说明gremlin1需要内源性TGF-β1参与其活性。④Smad2/3-siRNA阻断实验结果显示：应用smad2/3-siRNA阻断TGF-β1信号可以阻断gremlin对高糖环境足细胞nephrin和synaptopodin蛋白表达的抑制（p<0.01），再次印证体外gremlin1发挥作用需要内源性TGF-β1的存在。
结论：
1在高糖培养的小鼠足细胞中，gremlin1表达上调。Gremlin1蛋白主要分布在足细胞的胞浆中，少部分分布在胞膜上。胞浆部分呈颗粒状分布，包膜部分呈细颗粒状或线样排列。
2Gremlin1加重高糖所致的nephrin和synaptopodin表达下降及分布异常，促进足细胞损伤。Gremlin1加重高糖所致的足细胞凋亡。
3Gremlin1-siRNA治疗可以改善高糖所致的nephrin和synaptopodin表达下降和分布异常，降低高糖所致足细胞凋亡，部分恢复足细胞损伤。
4在高糖环境足细胞中gremlin1与TGF-β1相互诱导生成，协同发挥作用。Gremlin1在高糖环境下可能通过经典的TGF-β1/smad2/3信号途径发挥足细胞损伤作用，阻断TGF-β1/smad2/3信号可以抑制gremlin1对高糖环境足细胞的损伤作用。
综上，本研究提示gremlin1在糖尿病足细胞的损伤和凋亡中发挥了作用，TGF-β1/smad2/3信号途径可能参与了这一过程。
Objective: Diabetic nephropathy (diabetic nephropathy, DN) is one ofthe most common microvascular complications of diabetes, and also the firstcause of end-stage renal disease (ESRD) in the world. It is becaming the hugeburden of health care in developing and developped countries. The patientswith DN mainly show edema, proteinuria, and progressive renal functiondecline. Renal pathology is mainly characterized by excessive amassing ofextracelluar matrix (ECM) with thickening of glomerular and tubularbasement membranes and increased amount of mesangial matrix, whichultimately progress to glomerulosclersis and tubulo-interstitial fibrosis. Recentresearches have shown that the disturbance of the glomerular filtration barrier,especially the podocyte injury (which is an important component ofglomerular filtration barrier), play crucial role in the progression of diabeticnephropathy.
Podocytes are terminally differentiated visceral epithelial cell. Podocytesand their foot processes are unique in that they comprise the outer layer of thekidney ultrafiltration filter and form the glomerula slit diaphragm, which is acomplex cellular structure that prevents the development of the proteinuria inan actin cytoskeleton-dependent manner. In order to highlight the importanceof podocyte in diabetic nephropathy, DN is also called diabetic podocytopathy.In the onset and development in this setting, the pathological changes ofpodocyte mainly include the number decrease, apoptosis, foot effacement,detached from the GBM and GBM bare. However, the detailed mechanism ofpodocyte injury is remaining unclear. The developmental gene gremlinre-emergence in diabetic nephropathy is of great interesting.
Gremlin, an antagonist of bone morphogenetic protein (BMP), is a23-28KD highly conservative secreted glycoprotein, which is present as both soluble and cell-associated forms and is found within the Golgi apparatus andendoplasmic reticulum. It consists of184amino acid residues and regulatesembryonic development, growth and differentiation. There are potentialnuclear localization sites near its carboxyl-terminus, N-linked glycosylationsites, and a number of phosphorylation sites, which mediate the interactionsbetween proteins. During embryonic development, gremlin regulates thedevelopment of limb bud、lung and kidney branching morphogenesis viaSHH/FGF-4(sonic hedgehog/fibroblast growth factor4) feedback loopbetween stromal polarization area (ZPA: zone of polarizing activity) and theapical ectodermal ridge (AER). This process is achieved through the control ofapoptosis. In adult, normal kidney tissues has hardly expression of gremlin,wheras in conditions of diabetic nephropathy the expression of gremlinre-emerges and co-localizes with TGF-beta1. Gremlin results in epithelialmesenchymal transition of proximal tubular epithelial cell and mesangial cellscultured in high ambient glucose, drive them to secrete large amounts ofcollagen and fibronectin and promotes renal fibrosis. Whether gremlin causespodocyte injury, there are limited reports.
The podocyte injury results in proteinuria in the early stage of diabeticnephropathy. The marker proteins’ expression and apoptosis of podocyte aremajor indicators of podocyte injury. The present study aims to investigate therole and mechanisms of gremlin in hyperglycemia-induced podocyte injuryand to explore the probably involved signaling pathway to provide theoreticalbasis for the prevention and management of DN.
Methods:
1The expression and distribution of gremlin in podocyte treated with highglucose.
The conditionally immortalized mouse podocytes were cultured at33℃with γ–IFN (the permissive conditions) to induce proliferation, and thencultured at37℃withoutγ–IFN (the unpermissive conditions) for10-14daysto induce podocytes differentiation and mature. F-actin and synaptopodin weredetected by immunofluorescence to validate the morphology and differentiation of podocytes. Qualified podocytes were used in experiments.Differentiated podocytes were randomly divided into3groups: normal control(NC) group, high glucose group (HG) and normal glucose+mannitol group(MG) group. NC group was treated with D-glucose5.5mmol/L, HG groupwas treated with D-glucose30mmol/L and MG group was treated with5.5mmol/L D-glucose+24.5mol/L mannitol in order to control the influence ofhigh osmotic pressure to the experimental results. After synchronous culturedfor48hours three group cells were harvested to abstract the total protein andmRNA. The expression of gremlin was detected by western blot and real-timePCR. The distribution and localizing characteristics of gremlin in podocyteswere obseved by the Laser confocal microscope.
2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose ambient.
Recombinant mice gremlin and high glucose co-stimulated the podocytesto mimic the conditions of diabetic nephropathy in vivo.①Firstly, to explorethe time and dose relationship between gremlin and high glucose podocytes,the differentiated podocytes were cultivated with high glucose and gremlin (0,0.1,0.25,0.5,0.75,1μg/ml) for48hours respectively. Using western blotand real-time PCR method found out the maximal negative regulatoryconcentration of gremlin on nephrin and synaptopodin, then this optimalconcentration was used in the experiments below. Secondly, podocytes werecultivated with the optimal concentration of gremlin contaminating highglucose for0,6,12,24,48and72hours to find out the maximal negativeregulatory time of gremlin on nephrin and synaptopodin.②Normal control(NC) group, high glucose control (HG) group and high glucose+gremlin (HG+grem1) group were set. According to the results of experiment①, thepodocytes were incubated with0.75ug/ml gremlin for48h, then theexpression and distribution of nephrin and synaptopodin were visualized bylaser confocal microscope to analyze the podocyte injury.③Setting controlsas above, the apoptosis podocytes were detected by TUNEL and TUNELpositive cells were counted.④Bcl-2, Bax and Cleaved Casepase–3, which compensate for the false-positive of TUNEL, were putative indicators ofapoptosis. They were detected by western blot and real-time PCR method tofurther assess the podocyte injury.
3The effect of gremlin-siRNA on the markers and apoptosis of podocyte inhigh glucose ambient.
Differentiated podocytes were divided into three groups: high glucosegroup (HG), high glucose+gremlin-siRNA group (HG+grem.siRNA) andhigh glucose+control siRNA group (HG+con.siRNA).①Optimizing thetransfection conditions: The optimal dose of gremlin–siRNA was confirmedby abviously knockdown the expression of gremlin gene.②The effect ofgremlin-siRNA on the location and distribution of nephrin and synaptopodinin high glucose ambient podocyte was observed by laser confocal microscope.③The effect of gremlin-siRNA on the expression of nephrin andsynaptopodin in high glucose ambient podocyte was assessed by western blotand real time PCR.④The apoptosis effect of gremlin-siRNA on podocyteswere detected by TUNEL. TUNEL positive cells were counted.⑤Bcl-2,Bax and Cleaved Casepase-3were detected by western blot and real-timePCR to further confirm the apoptosis effect of gremlin-siRNA on podocyte.
4The probably signal mechanism involved in gremlin-mediated podocyteinjury in high glucose ambient.
①Due to TGF–β1colocalization with gremlin in renal tissue of DN,there maybe exists a certain relationship between TGF–β1and gremlin inhigh glucose environment. Differentiated mature podocytes were divided intotwo groups: one group was treated with high glucose+gremlin (0.75μg/ml)for48h, the protein and mRNA expression of TGF-β1was detected. Theother group was treated with high glucose+TGF-β1(5ng/ml) for48h, theprotein and mRNA expression of gremlin was detected. Additionally，normalcontrol (NC) and high glucose control (HG) were set.②The cannoniacl andnoncannonical TGF–β1signaling molecules detection in high glucosepodocyte exposed to gremlin: Podocytes were incubated with high glucoseand recombinant mice gremlin (0.75μg/ml) for0,15,30,60,120and240 minutes, the cannonical (smad2/p-smad2, smad3/p-smad3) and noncannonical(p38/p-p38, JNK1/2/p-jnk1/2) signling molecules were detected to find outthe signal pathway mediating the role of gremlin on hyperglycemic podocyte.③The above experimental results implicated that the TGF–β/smad2/3signaling pathways involved in the pathogenesis of gremlin. SB431542, aTGF–β receptor blocker, blocked the suprression of gremlin on the expressionof nephrin and synaptopodin protein in hyperglycemic podocytes.④Podocytes treated with high glucose and gremlin was transfected withsmad2/3–siRNA and harvested after48hours to verified the knocking downeffect. Smad2/3knocking down inhibited the supression of gremlin on theexpression of nephrin and synaptopodin protein.
Results:
1The expression and distribution of gremlin1in high glucose ambientpodocytes.
①F-actin immunofluorescence assay showed that there were twodifferent phenotypes in cultured podocytes. At33℃with γ–IFN (thepermissive conditions), cells proliferated and maintained cobblestone-likemorphology. When cultured at37℃without γ–IFN (nonpermissivecondition), cells stretched out differently shaped long processes from cellbodies，arised many spindle-like projections from their primary processes andstopped proliferation. Synaptopodin is one marker indicating maturedifferentiated podocytes. Immunofluorescence staining showed that there wassparsely expression of synaptopodin in undifferentiated podocytes, while itsexpression in differentiated podocytes was enhanced obviously.②The resultsof Western blot showed that in NC group and MG group, there was hardlyexpression of gremlin protein, while in HG group its expression was obviouslyenhanced (p<0.01).③The results of Real-time PCR displayed that comparedwith NC group and MG group, the expression level of gremlin mRNA in HGgroup was increased significantly when the podocyte was cultured for48hours (p<0.01).④Confocal staining demonstrated that the distribution ofgremlin1in podocytes was located predominantly in the cytoplasm with a punctate pattern., and sparsely on the cell membrane with a thin linear or fineparticles pattern.
2The effect of recombinant mice gremlin on the markers and apoptosis ofpodocytes in high glucose environment
①The dose-effect relationship experiment showed that the protein andmRNA expression of nephrin and synaptopodin decreased significantly at agremlin concentration of0.75μg/ml (p <0.01).②The time-effect relationshipexperiment disclosed that the protein and mRNA expression of nephrin andsynaptopodin decreased significantly when cells were treated with HG+0.75μg/ml gremlin for48h (p <0.01).③The laser confocal image showedthat gremlin aggravated the hyperglycemia-induced podocyte injury. Thedistribution of nephrin differed markedly between podocytes cultured in thepresence of gremlin and control cells. Control cells expressed nephrin in cellprojectons or exhibited a filamentous nephrin appearance. By contrast, cellscultured with gremlin displayed a cytoplastic and non-filamentous distribution.In gremlin-cultured podocytes, synaptopodin distributed mainly at peri-orintra-nuclear regions. In normal control cells, synaptopodin localized mainlyin the foot process or cytoplasm in a punctuated pattern along actin filaments.High-glucose induced synaptopodin shifting to the peri-nucleus anddepolymerization of actin mirofilaments. Gremlin aggravated such injury andinduced a marked decreasement in podocyte size, and rearrangement ofsynaptopodin from a punctuated pattern to a granular mass pattern.④Theresults of TUNEL showed that compared with the HG group, the cellapoptosis rate of HG+gremlin group was increased significantly (p <0.05).
⑤The experimental results of apoptosis related proteins displayed thatcompared with the HG group, gremlin decreased the levels of Bcl-2proteinand mRNA further, while increased those of Bax and Cleaved Caspase-3.Collectively, these results disclosed that gremlin aggravatedhyperglycemia-induced podocytes apoptosis.
3The effect of gremlin1-siRNA on the markers and apoptosis of podocyte inhigh glucose environment ①The verification experiment results of gremlin1-siRNA showed that at80pmol/well gremlin-siRNA knocked down the protein and mRNAexpression of gremlin significantly in podocytes cultured in6well plate (p <0.01).②The confocal images displayed that gremlin-siRNA rescued theexpression and distribution of nephrin and synaptopodin in hyperglycemicpodocyte. Gremlin-siRNA attenuated the distribution of nephrin from thecytoplasm and non-filamental to the cell membrane and filament pattern, andthat of synaptopodin from massive granular to punctate pattern.③Westernimmunoblot and real-time PCR analysis indicated that gremlin1siRNArescued the expression of nephrin and synaptopodin at both the mRNA andprotein levels.④Gremlin1-siRNA decreased the percentage ofTUNEL-positive cells. There were7.021%apoptosis cells in HG group and4.986%apoptosis cells in HG+grem.siRNA group (p <0.05).⑤GremlinsiRNA increased the expression of Bcl-2and decreased that of Bax andCleaved Caspase-3in HG ambient (p <0.01). Gremlin1-siRNA attenuated HGinduced apoptosis effectively.
4The signal mechanism mediating the pathogenesis of gremlin inhyperglycemic podocytes
①Podocytes that were cultured in gremlin1(0.75μg/ml) and highglucose (30mmol/L) expressed TGF-β1more abundantly than high glucoseand normal controls at both the protein (p<0.05) and mRNA (p<0.05) levels.Conversely, podocytes that were cultured in TGF-β1(5ng/ml) concomitantlywith high glucose (30mmol/L) expressed gremlin1more abundantly than highglucose and normal controls at both the protein (p<0.05) and mRNA (p<0.05)levels.②The protein of TGF-β signaling molecules (smad2/p-smad2,smad3/p-smad3, p38/p-p38and JNK1/2/p-JNK1/2) was analyzed by westernblot. In gremlin-activated podocytes, smad2and smad3were phosphorylatedin a time-dependent manner. By contrast, neither JNK1/2nor p38MAPK wereactivated. Gremlin activated canonical, but not non-canonical signalingpathway in podocytes (p<0.05, versus0min controls).③TGF-β receptorinhibitors (SB431542) blocked the ability of gremlin1to suppress nephrin and synaptopodin expression. Inhibition of the TGF-β receptor with SB431542(5μM/L) blocked the effect of gremlin1on high glucose cultured podocytes andrescued the expression of nephrin and synaptopodin (p<0.05). Gremlin injuredpodocytes required endogenous TGF-β1.④Smad2/3-siRNA treatmentinhibited the ability of gremlin to down-regulate the expression of nephrin andsynaptopodin in high-glucose ambient podocytes. Podocytes were transfectedwith smad2/3siRNA (60pmol/well) for6hour before treatment with gremlinand high glucose. Control cells were transfected with scrambled siRNAsequences. Smad2/3siRNA knockdown rescued the expression of bothnephrin and synaptopodin and attenuated injury of podocytes cultured in highglucose ambient (p <0.01).
Conclusions:
1Gremlin is clearly elevated in high glucose cultured mouse podocytes.The distribution of gremlin1in podocytes locates predominantly in thecytoplasm in a punctate pattern and sparsely on the cell membrane in a thinlinear or fine particle pattern.
2Gremlin aggravates hyperglycemia induced podocyte injury, supressesthe expression of nephrin and synaptopodin and deteriorates the apoptosis ofpodocytes.
3Treatment high glucose ambient podocyte with gremlin-siRNArescues the expression of nephrin and synaptopodin and declines apoptosisrate of podocyte.
4Gremlin and TGF-β1induce each other in high glucose conditions.Canonical smad2/3pathway, but not non-canonical pathway of TGF-βsignaling is activated by gremlin, which exerts its effect on the podocytethrough endogenous TGF-β signaling.
In summary, gremlin plays a crucial role in the pathogenesis of podocyteinjury in DN, perhaps by disrupting the homeostatic balance between TGF-βand BMP. There also appears to be a feedback loop between gremlin andTGF-β1whereby they are capable of inducing each other and accelerating thepathogenesis of the disease process. Gremlin activates a canonical smad2/3 pathway, but does not activate a non-canonical pathway of TGF-β signaling.New therapies targeting gremlin may protect the kidney from the developmentand progression of DN.
引文
1Gaede P, Lund-Andersen H, Parving HH, et al. Effect of a multifactorialintervention on mortality in type2diabetes. N Engl J Med.2008;358:580-591
2Diez-Sampedro A, Lenz Ol, Fornoni A. Podocytopathy in Diabetes: AMetabolic and Endocrine Disorder. Am J Kidney Dis.58(4):637-646
3Jefferson JA, Alpers CE. Shankland SJ. Podocyte Biology for the BedsideJ.Am J Kidney Dis.2011,58(5):835-845
4Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
5M ichos O, Panm an L, V intersten K, et al. Gremlin mediated BMPantagonism induces the epithelial mesenchym a lfeedback signaling controlling metanephric kidney and limb organogenesis. Development,2004,131:3401-3410
6Lappin DW, McMahon R, Murphy M et al.Gremlin: an example of there-emergence of developmental programmes in diabetic nephropathyNephrol Dial Transplant (2002)17[Suppl9]:65–67
7Mezzano S, Droguett A, Burgos ME et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in glomerular crescents ofpauci-immune glomerulonephritis. Nephrol Dial Transplant (2007)22:1882–1890
8Mundel P, Reiser J, Zú iga Mejía Borja A, et al. Rearrangements of thecytoskeleton and cell contacts induce process formation duringdifferentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res,1997,236(1):248-258
9Weil EJ, Lemley KV, Yee B et al. Podocyte Detachment in Type2Diabetic Nephropathy. Am J Nephrol2011;33(suppl1):21–24
10Patrakka J, Tryggvason K. New insights into the role of podocytes inproteinuria. Nat Rev Nephrol.2009;5(8):463-468
11Johnstone DB, Holzman LB.Clinical impact of research on the podocyteslit diaphragm. Nat Clin Pract Nephr20062(5)271-282
12Jefferson JA, Shankland SJ, Pichler RH. Proteinuria in diabetic kidneydisease: a mechanistic viewpoint. Kidney Int.2008;74(1):22-36
13Smerdel-Ramoya A, Zanotti S, Canalis E et al. NephroblastomaOverexpressed (Nov) Induces Gremlin in ST-2Stromal Cell Lines byPost-Transcriptional Mechanisms J Cell Biochem.2011,112:715–722
14Gazzerro E, Pereira RC, Jorgetti V, et al. Skeletal Overexpression ofGremlin Impairs Bone Formation and Causes Osteopenia. Endocrinology146(2):655–665
15Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
16Bénazet JD, Bischofberger M, Tiecke E, et al. A Self-Regulatory Systemof Interlinked Signaling Feedback Loops Controls Mouse Limb Patterning.SCIENCE,2009,323(20)1050-1053
17Murphy M，McMahon R， Lappin D W.P.et al. Gremlins: Is This WhatRenal Fibrogenesis Has Come To? Exp Nephrol2002;10:241–244
18Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS(Proceedings of the National Academy of Sciences)2006103(40):14842-14847
19McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
20Murphy M, Godson C, Cannon S et al. Suppression subtractivehybridisation identifies high glucose levels as a stimulus for expression ofconnective tissue growth factor and o ther genes in human mesangial cells.J Biol Chem1999;274:5830–5834
21Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
22Murphy M, Macmahon R, Clarkson M, et al. Induction of gremlinexpression in the remnant kidney in vivo and during TGF-inducedepithelial mesenchymal transformation. J Am Soc Nephrol,2000,11,3298A
23Zhang QX, Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plus one,2010,5(7) e11709.
2424Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS(Proceedings of the National Academy of Sciences)2006103(40):14842-14847
25Topol LZ, Marx M, Bogdonova D, et al. Identification of drm, a novelgene whose expression is suppressed in transformed cells and which caninhibit growth of normal but not transformed cells in culture. Mol CellBiol1997;15:4801-4810
26Gazzerro E, Canalis E. Bone morphogenetic proteins and their
antagonists.Rev Endocr Metab Disord2006;7:51–65
1McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
2Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
3Zhang QX., Shi YH., Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709.
4Jefferson JA, Alpers CE,. Shankland SJ. Podocyte Biology for theBedsideJ. Am J Kidney Dis.2011,58(5):835-845
5Li HP, Lemay S, Aoudjit L, et al. Scr-family kinsase fyn phosphorylatesthe cytoplasmic domain of nephrin and modulates its interaction withpodocin. J Am Soc Nephrol2004,15:3006–3015
6Asanuma K， Kim K, Oh J, et al. Synaptopodin regulates theactin-bundling activity of a-actinin in an isoform-specific manner. J. Clin.Invest.115(5),1188–1198(2005)
7Diaz-sampedro A, Lenz O, Fornoni A. Podocytopathy in diabetes: ametabolic and endocrine disorder. Am J Kidney Dis,2011,58(4):637-646.
8Drummond K, Mauer M The early natural history of nephropathy in type1diabetes. II. Early renal structural changes in type1diabetes. Diabetes(2002)51:1580–1587
9Patrakka J, Kestila M, Wartiovarra J, et al. Congenital nephrotic syndrome(NPSH1):features resulting from different mutation in Finnish patients.Kidney Int.2000,58(3):972-980
10Zhu J, Sun N, Aoudjit L, et al. Nephrin mediates actin reorgniazation viaphosphoinositide3-kinase in podocytes.Kidney Int.200873(5):556-566
11Saleem MA, Ni L, Witherden I, et al. Co-Localization of Nephrin, Podocin,and the Actin Cytoskeleton. Evidence for a Role in Podocyte Foot ProcessFormation Am J of Pathology,2002,161(4):1459-1466
12Patrie KM，Drescher AJ，Welihinda A，et al．Interaction of twoactinbinding proteins，synaptopodin and alpha‐actinin‐４，with thetight junction protein MAGI-1．J BiolChem,2002,277(33):30183-30192
13Johnstone DB, Holzman LB.Clinical impact of research on the podocyteslit diaphragm. NATURE Nat Clin Pract Nephr20062(5)271-282
14Asanuma K, Kim K, Oh J, et al. Synaptopodin regulates the actin-bundlingactivity of α-actinin in an isoform-specific manner J. Clin. Invest.115:1188–1198(2005)
15Moeller MJ, Holzman LB. Imaging Podocyte Dynamics. Nephron ExpNephrol2006;103:e69–e74
16Schlondorff J. Nephrin AKTs on actin: The slit diaphragm–actincytoskeleton signaling network expands Kidney International (2008)73524-526
17Zhang C, Xia M, Boini KM et al. Epithelial-to-mesenchymal transition inpodocytes mediated by activation of NADPH oxidase inhyperhomocystein-emia. Pflugers Arch-Eur J Physiol (2011)462:455–467
18Jung DS, Lee SH, Kwak SJ,et al. Apoptosis occurs differentially accordingto glomerular size in diabetic kidney disease.Nephrol Dial Transplant(2011)0:1–9
1Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
2Zhang Y，Zhang Q. Bone morphogenetic protein-7and Gremlin: Newemerging therapeutic targets for diabetic nephropathy..Biochemical andBiophysical Research Communications2009,383:1–3.
3Ueda H，Miyazaki Y，Matsusaka T,et al. Bmp in Podocytes Is Essential forNormal Glomerular Capillary Formation. J Am Soc Nephrol200819:685–694.
4Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)：5251-5259
5LiuYH. New Insights into Epithelial-Mesenchymal Transition in KidneyFibrosis. J Am Soc Nephrol201021:212–222
6Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
7Zhang QX., Shi YH., Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
8Roxburgh SA, Kattla JJ, Curran SP, et al. Allelic depletion of grem1attenuates diabetic kidney disease. Diabetes,200958(7)1641-1650
9Mellitzer G, Hallonet M, Chen L, et al. Spatial and temporal knock down'of gene expression by electroporation of double-stranded RNA andmorpholinos into early postimplantation mouse embroys. MechDev.2002;118(1-2):57-56
10Lee G, Saito I. Role of nucleotide sequences of Loxp spacer region inCre-medizted recombination.Gene,1998.216(1)55-65
11Meister G, Tuschl T (2004) Mechanisms of gene silencing bydouble-stranded RNA. Nature431:343–349
12Khatri N, Rathi M, Baradia D,et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012;29(6):487-527
13Braun JE, Huntzinger E, Izaurralde E. The role of GW182proteins inmiRNA-mediated gene silencing. Adv Exp Med Biol.2013;768:147-63
14Weide T, Huber TB. Signaling at the Slit: Podocytes Chat by SynapticTransmission. J Am Soc Nephrol2009.20:1862–1864
15Asanuma K, Kim K, Oh J, et al. Synaptopodin regulates the actin-bundlingactivity of α-actinin in an isoform-specific manner. J Clin Invest.2005,115:1188–1198
1White KE, Bilous RW, Marshall SM, et al. Podocyte number innormotensive type1diabetic patients with albuminuria. Diabetes,2002,51(10):3083-3089
2Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
3Meng XM, Chung AC, Lan HY. Role of the TGF-β/BMP-7/Smadpathways in renal diseases. Clin Sci (Lond).2013,124(4):243-54
4McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
5Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)：5251-5259
6Zarjou A, Agarwal A. Heme oxygenase-1as a target for TGF-β in kidneydisease. Semin Nephrol.2012May;32(3):277-86.
7Zhang QX, Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
8Gnudi L, Thomas SM, Viberti G. Mechanical forces in diabetic kidneydisease: a trigger for impaired glucose metabolism. J Am SocNephrol.200718(8):2226-32
9Schnaper HW, Hayashida T, Hubchak SC et al, TGF-β signaltransduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol
2003284: F243–F252
10Yang YL, Liu YS, Chuang LY et al. Bone Morphogenetic Protein-2Antagonizes Renal Interstitial Fibrosis by Promoting Catabolism of Type ITransforming Growth Factor-β Receptors. Endocrinology2009150(2):727–740
11Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
1212Mitola S, Moroni E, Ravelli C, et al. Angiopoietin-1mediates theproangiogenic activity of the bone morphogenic protein antagonist Drm.Blood.2008;112(4):1154-1157
13Chiodelli P, Mitola S, Ravelli C et al. Heparan Sulfate ProteoglycansMediate the Angiogenic activity of the Vascular Endothelial GrowthFactor Receptor-2Agonist Gremlin. Arterioscler Thromb Vasc Biol2011;31:e116-e127
14Chen B, Blair DG, Plisov S, et al. Cutting edge: bone morphogeneticprotein antagonists Drm/Gremlin and Dan interact with Slits and act asnegative regulators of monocyte chemotaxis. J Immunol2004;173:5914–5917
1515Khokha MK, Hsu D, Brunet LJ, et al. Gremlin is the BMPantagonist required for maintenance of Shh and Fgf signals during limb patterning. Nature Genetics2003;34:303–307
16Khatri N, Rathi M, Baradia D, et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012,29(6):487-527
1Diaz-sampedro A, Lenz O, Fornoni A. Podocytopathy in diabetes: ametabolic and endocrine disorder. Am J Kidney Dis,2011,58(4):637-646
2Shankland SJ. The podocyte’s response to injury: role in proteinuria andglomerulosclerosis. Kidney Int2006,69:2131–2147
3Yu D, Petermann A, Kunter U, Rong S, Shankland SJ, Floege J. Urinarypodocyte loss is a more specific marker of ongoing glomerular damagethan proteinuria. J Am Soc Nephrol2005,16(6):1733-41
4Drummond K, Mauer M. The early natural history of nephropathy in type1diabetes. II. Early renal structural changes in type1diabetes. Diabetes2002,51:1580–1587
5Jefferson JA, Alpers CE, Shankland SJ. Podocyte biology for the bedside.Am J Kidney Disease.2011,58(5):835-845
6M ichos O, Panm an L, V intersten K, et al. Gremlin mediated BMPantagonism induces the epithelial mesenchym a lfeedback signaling controlling metanephric kidney and limb organogenesis. Development,2004,131:3401-3410
7Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
8Bénazet JD, Bischofberger M, Tiecke E, et al. A Self-Regulatory System ofInterlinked Signaling Feedback Loops Controls Mouse Limb Patterning.SCIENCE,2009,323(20)1050-1053
9Murphy M，McMahon R， Lappin D W.P.et al. Gremlins: Is This WhatRenal Fibrogenesis Has Come To? Exp Nephrol2002,10:241–244
10Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS (Proceedingsof the National Academy of Sciences)2006,103(40):14842-1484
11Gazzerro E, Pereira RC, Jorgetti V, et al. Skeletal Overexpression ofGremlin Impairs Bone Formation and Causes Osteopenia. Endocrinology2005,146(2):655–665
12McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
13Murphy M, Godson C, Cannon S et al. Suppression subtractivehybridisation identifies high glucose levels as a stimulus for expression ofconnective tissue growth factor and o ther genes in human mesangial cells.J Biol Chem1999;274:5830–5834
14Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
15Wang SN, Lapage J, Hirschber R. Loss of Tubular Bone MorphogeneticProtein–7in Diabetic Nephropathy. J Am Soc Nephrol2001,12:2392–2399
16Zhang QX., Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
17Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)5251-5259
18Li G, Li Y, Liu S, et al.Gremlin Aggravates Hyperglycemia-InducedPodocyte Injury by a TGFβ/Smad Dependent Signaling Pathway: gremlinand hyperglycemia-induced podocyte injury. J Cellular Biochem Acceptedmanuscript online:31MAR201311:25PM EST|DOI:10.1002/jcb.24559
19LiuYH. New Insights into Epithelial-Mesenchymal Transition in KidneyFibrosis. J Am Soc Nephrol201021:212–222
20Wallner EI, Wada J, Lin S, et al. Renal gene expression in emnbryonic andnewborn diabetic mice. Exp Nephrol2002;10:130-138
21Valdes F, Alvarez AM, Locascio A,et al: The epithelial mesenchymaltransition confers resistance to the apoptotic effects of transforming growthfactor-beta in fetal rat hepatocytes. Mol Cancer Res2002;1:68–78
22Strutz FM: EMT and proteinuria as progression factors. Kidney Int200975:475–481
23Huang H, Huang H, Li Y, et al. Gremlin induces cell proliferation andextra cellular matrix accumulation in mouse mesangial cells exposed tohigh glucose via the ERK1/2pathway. BMC Nephrol.2013Feb11;14:33.doi:10.1186/1471-2369-14-33
24Montero JA, Ga an Y, Macias D et al. Role of FGFs in the control ofprogrammed cell death during limb development. Development2001128,2075-2084
25Costello1CM, Cahill1E, Martin E et al. Role of Gremlin in the LungDevelopment and Disease. Am J Respir Cell Mol Biol,201042:517–523
26Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
27Shibuya M, Claesson-Welsh L. Signal transduction by VEGF receptors inregulation of angiogenesis and lymphangiogenesis. Exp Cell Res.2006;312(5):549-560
28Mitola S, Moroni E, Ravelli C, et al. Angiopoietin-1mediates theproangiogenic activity of the bone morphogenic protein antagonist Drm.Blood.2008;112(4):1154-1157
29Chiodelli P, Mitola S, Ravelli C et al. Heparan Sulfate ProteoglycansMediate the Angiogenic activity of the Vascular Endothelial GrowthFactor Receptor-2Agonist Gremlin. Arterioscler Thromb Vasc Biol2011;31:e116-e127
30Jara A, Chacon C, Burgos ME et al. Expression of gremlin, bonemorphogenetic protein antagonist, is associated with vascular calcificationin uraemia. Nephrol Dial Transplant (2009)24:121–129
31Burns WC, Twigg SM, Forbes JM et al. Connective tissue growth factorplays an important role in advanced glycation end product-induced tubularepithelial-to-mesenchymal transition: Implications for diabetic renaldisease. J Am Soc Nephrol2006,17:2484–2494
32Turk T, Leeuwis JW, Gray J et al. BMP Signaling and Podocyte MarkersAre Decreased in Human Diabetic Nephropathy in Association With CTGFOverexpression. Journal of Histochemistry&Cytochemistry2009,57(7):623–631
33Nguyen TQ，Roestenberg P, van Nieuwenhoven FA. CTGF InhibitsBMP-7Signaling in Diabetic Nephropathy. Am Soc Nephrol2008,19:2098–2107
34Chen B, Blair DG, Plisov S, et al. Cutting edge: bone morphogeneticprotein antagonists Drm/Gremlin and Dan interact with Slits and act asnegative regulators of monocyte chemotaxis. J Immunol2004,173:5914–5917
35Stabile H, Mitola S, Moroni E, et al. Bone morphogenic protein antagonistDrm/gremlin is a novel proangiogenic factor. Blood2007,109:1834–1840
36Khokha MK, Hsu D, Brunet LJ, et al. Gremlin is the BMP antagonistrequired for maintenance of Shh and Fgf signals during limb patterning.Nature Genetics2003,34:303–307
37Mellitzer G, Hallonet M, Chen L，et al. Spatial and temporal knock down'of gene expression by electroporation of double-stranded RNA andmorpholinos into early postimplantation mouse embroys. Mech Dev.2002,118(1-2):57-63
38Roxburgh SA, Kattla JJ, Curran SP, et al. Allelic depletion of grem1attenuates diabetic kidney disease. Diabetes,2009,58(7)1641-1650
39Khatri N, Rathi M, Baradia D,et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012,29(6):487-527
40Braun JE, Huntzinger E, Izaurralde E. The role of GW182proteins inmiRNA-mediated gene silencing. Adv Exp Med Biol.2013,768:147-163
41Guo W，Chen WB Yu WD,et al. Small interfering RNA-based moleculartherapy of cancers. Chinese journal of cancer.2013,1DOI:10.5732/CJC.012.10280
42Wang SN, Lapage J, Hirschberg R.Loss of Tubular Bone MorphogeneticProtein–7in Diabetic Nephropathy. J Am Soc Nephrol2001,12:2392–2399
2Diez-Sampedro A, Lenz Ol, Fornoni A. Podocytopathy in Diabetes: AMetabolic and Endocrine Disorder. Am J Kidney Dis.58(4):637-646
3Jefferson JA, Alpers CE. Shankland SJ. Podocyte Biology for the BedsideJ.Am J Kidney Dis.2011,58(5):835-845
4Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
5M ichos O, Panm an L, V intersten K, et al. Gremlin mediated BMPantagonism induces the epithelial mesenchym a lfeedback signaling controlling metanephric kidney and limb organogenesis. Development,2004,131:3401-3410
6Lappin DW, McMahon R, Murphy M et al.Gremlin: an example of there-emergence of developmental programmes in diabetic nephropathyNephrol Dial Transplant (2002)17[Suppl9]:65–67
7Mezzano S, Droguett A, Burgos ME et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in glomerular crescents ofpauci-immune glomerulonephritis. Nephrol Dial Transplant (2007)22:1882–1890
8Mundel P, Reiser J, Zú iga Mejía Borja A, et al. Rearrangements of thecytoskeleton and cell contacts induce process formation duringdifferentiation of conditionally immortalized mouse podocyte cell lines.Exp Cell Res,1997,236(1):248-258
9Weil EJ, Lemley KV, Yee B et al. Podocyte Detachment in Type2Diabetic Nephropathy. Am J Nephrol2011;33(suppl1):21–24
10Patrakka J, Tryggvason K. New insights into the role of podocytes inproteinuria. Nat Rev Nephrol.2009;5(8):463-468
11Johnstone DB, Holzman LB.Clinical impact of research on the podocyteslit diaphragm. Nat Clin Pract Nephr20062(5)271-282
12Jefferson JA, Shankland SJ, Pichler RH. Proteinuria in diabetic kidneydisease: a mechanistic viewpoint. Kidney Int.2008;74(1):22-36
13Smerdel-Ramoya A, Zanotti S, Canalis E et al. NephroblastomaOverexpressed (Nov) Induces Gremlin in ST-2Stromal Cell Lines byPost-Transcriptional Mechanisms J Cell Biochem.2011,112:715–722
14Gazzerro E, Pereira RC, Jorgetti V, et al. Skeletal Overexpression ofGremlin Impairs Bone Formation and Causes Osteopenia. Endocrinology146(2):655–665
15Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
16Bénazet JD, Bischofberger M, Tiecke E, et al. A Self-Regulatory Systemof Interlinked Signaling Feedback Loops Controls Mouse Limb Patterning.SCIENCE,2009,323(20)1050-1053
17Murphy M，McMahon R， Lappin D W.P.et al. Gremlins: Is This WhatRenal Fibrogenesis Has Come To? Exp Nephrol2002;10:241–244
18Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS(Proceedings of the National Academy of Sciences)2006103(40):14842-14847
19McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
20Murphy M, Godson C, Cannon S et al. Suppression subtractivehybridisation identifies high glucose levels as a stimulus for expression ofconnective tissue growth factor and o ther genes in human mesangial cells.J Biol Chem1999;274:5830–5834
21Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
22Murphy M, Macmahon R, Clarkson M, et al. Induction of gremlinexpression in the remnant kidney in vivo and during TGF-inducedepithelial mesenchymal transformation. J Am Soc Nephrol,2000,11,3298A
23Zhang QX, Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plus one,2010,5(7) e11709.
2424Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS(Proceedings of the National Academy of Sciences)2006103(40):14842-14847
25Topol LZ, Marx M, Bogdonova D, et al. Identification of drm, a novelgene whose expression is suppressed in transformed cells and which caninhibit growth of normal but not transformed cells in culture. Mol CellBiol1997;15:4801-4810
26Gazzerro E, Canalis E. Bone morphogenetic proteins and their
antagonists.Rev Endocr Metab Disord2006;7:51–65
1McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
2Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
3Zhang QX., Shi YH., Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709.
4Jefferson JA, Alpers CE,. Shankland SJ. Podocyte Biology for theBedsideJ. Am J Kidney Dis.2011,58(5):835-845
5Li HP, Lemay S, Aoudjit L, et al. Scr-family kinsase fyn phosphorylatesthe cytoplasmic domain of nephrin and modulates its interaction withpodocin. J Am Soc Nephrol2004,15:3006–3015
6Asanuma K， Kim K, Oh J, et al. Synaptopodin regulates theactin-bundling activity of a-actinin in an isoform-specific manner. J. Clin.Invest.115(5),1188–1198(2005)
7Diaz-sampedro A, Lenz O, Fornoni A. Podocytopathy in diabetes: ametabolic and endocrine disorder. Am J Kidney Dis,2011,58(4):637-646.
8Drummond K, Mauer M The early natural history of nephropathy in type1diabetes. II. Early renal structural changes in type1diabetes. Diabetes(2002)51:1580–1587
9Patrakka J, Kestila M, Wartiovarra J, et al. Congenital nephrotic syndrome(NPSH1):features resulting from different mutation in Finnish patients.Kidney Int.2000,58(3):972-980
10Zhu J, Sun N, Aoudjit L, et al. Nephrin mediates actin reorgniazation viaphosphoinositide3-kinase in podocytes.Kidney Int.200873(5):556-566
11Saleem MA, Ni L, Witherden I, et al. Co-Localization of Nephrin, Podocin,and the Actin Cytoskeleton. Evidence for a Role in Podocyte Foot ProcessFormation Am J of Pathology,2002,161(4):1459-1466
12Patrie KM，Drescher AJ，Welihinda A，et al．Interaction of twoactinbinding proteins，synaptopodin and alpha‐actinin‐４，with thetight junction protein MAGI-1．J BiolChem,2002,277(33):30183-30192
13Johnstone DB, Holzman LB.Clinical impact of research on the podocyteslit diaphragm. NATURE Nat Clin Pract Nephr20062(5)271-282
14Asanuma K, Kim K, Oh J, et al. Synaptopodin regulates the actin-bundlingactivity of α-actinin in an isoform-specific manner J. Clin. Invest.115:1188–1198(2005)
15Moeller MJ, Holzman LB. Imaging Podocyte Dynamics. Nephron ExpNephrol2006;103:e69–e74
16Schlondorff J. Nephrin AKTs on actin: The slit diaphragm–actincytoskeleton signaling network expands Kidney International (2008)73524-526
17Zhang C, Xia M, Boini KM et al. Epithelial-to-mesenchymal transition inpodocytes mediated by activation of NADPH oxidase inhyperhomocystein-emia. Pflugers Arch-Eur J Physiol (2011)462:455–467
18Jung DS, Lee SH, Kwak SJ,et al. Apoptosis occurs differentially accordingto glomerular size in diabetic kidney disease.Nephrol Dial Transplant(2011)0:1–9
1Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
2Zhang Y，Zhang Q. Bone morphogenetic protein-7and Gremlin: Newemerging therapeutic targets for diabetic nephropathy..Biochemical andBiophysical Research Communications2009,383:1–3.
3Ueda H，Miyazaki Y，Matsusaka T,et al. Bmp in Podocytes Is Essential forNormal Glomerular Capillary Formation. J Am Soc Nephrol200819:685–694.
4Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)：5251-5259
5LiuYH. New Insights into Epithelial-Mesenchymal Transition in KidneyFibrosis. J Am Soc Nephrol201021:212–222
6Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
7Zhang QX., Shi YH., Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
8Roxburgh SA, Kattla JJ, Curran SP, et al. Allelic depletion of grem1attenuates diabetic kidney disease. Diabetes,200958(7)1641-1650
9Mellitzer G, Hallonet M, Chen L, et al. Spatial and temporal knock down'of gene expression by electroporation of double-stranded RNA andmorpholinos into early postimplantation mouse embroys. MechDev.2002;118(1-2):57-56
10Lee G, Saito I. Role of nucleotide sequences of Loxp spacer region inCre-medizted recombination.Gene,1998.216(1)55-65
11Meister G, Tuschl T (2004) Mechanisms of gene silencing bydouble-stranded RNA. Nature431:343–349
12Khatri N, Rathi M, Baradia D,et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012;29(6):487-527
13Braun JE, Huntzinger E, Izaurralde E. The role of GW182proteins inmiRNA-mediated gene silencing. Adv Exp Med Biol.2013;768:147-63
14Weide T, Huber TB. Signaling at the Slit: Podocytes Chat by SynapticTransmission. J Am Soc Nephrol2009.20:1862–1864
15Asanuma K, Kim K, Oh J, et al. Synaptopodin regulates the actin-bundlingactivity of α-actinin in an isoform-specific manner. J Clin Invest.2005,115:1188–1198
1White KE, Bilous RW, Marshall SM, et al. Podocyte number innormotensive type1diabetic patients with albuminuria. Diabetes,2002,51(10):3083-3089
2Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
3Meng XM, Chung AC, Lan HY. Role of the TGF-β/BMP-7/Smadpathways in renal diseases. Clin Sci (Lond).2013,124(4):243-54
4McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
5Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)：5251-5259
6Zarjou A, Agarwal A. Heme oxygenase-1as a target for TGF-β in kidneydisease. Semin Nephrol.2012May;32(3):277-86.
7Zhang QX, Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
8Gnudi L, Thomas SM, Viberti G. Mechanical forces in diabetic kidneydisease: a trigger for impaired glucose metabolism. J Am SocNephrol.200718(8):2226-32
9Schnaper HW, Hayashida T, Hubchak SC et al, TGF-β signaltransduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol
2003284: F243–F252
10Yang YL, Liu YS, Chuang LY et al. Bone Morphogenetic Protein-2Antagonizes Renal Interstitial Fibrosis by Promoting Catabolism of Type ITransforming Growth Factor-β Receptors. Endocrinology2009150(2):727–740
11Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
1212Mitola S, Moroni E, Ravelli C, et al. Angiopoietin-1mediates theproangiogenic activity of the bone morphogenic protein antagonist Drm.Blood.2008;112(4):1154-1157
13Chiodelli P, Mitola S, Ravelli C et al. Heparan Sulfate ProteoglycansMediate the Angiogenic activity of the Vascular Endothelial GrowthFactor Receptor-2Agonist Gremlin. Arterioscler Thromb Vasc Biol2011;31:e116-e127
14Chen B, Blair DG, Plisov S, et al. Cutting edge: bone morphogeneticprotein antagonists Drm/Gremlin and Dan interact with Slits and act asnegative regulators of monocyte chemotaxis. J Immunol2004;173:5914–5917
1515Khokha MK, Hsu D, Brunet LJ, et al. Gremlin is the BMPantagonist required for maintenance of Shh and Fgf signals during limb patterning. Nature Genetics2003;34:303–307
16Khatri N, Rathi M, Baradia D, et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012,29(6):487-527
1Diaz-sampedro A, Lenz O, Fornoni A. Podocytopathy in diabetes: ametabolic and endocrine disorder. Am J Kidney Dis,2011,58(4):637-646
2Shankland SJ. The podocyte’s response to injury: role in proteinuria andglomerulosclerosis. Kidney Int2006,69:2131–2147
3Yu D, Petermann A, Kunter U, Rong S, Shankland SJ, Floege J. Urinarypodocyte loss is a more specific marker of ongoing glomerular damagethan proteinuria. J Am Soc Nephrol2005,16(6):1733-41
4Drummond K, Mauer M. The early natural history of nephropathy in type1diabetes. II. Early renal structural changes in type1diabetes. Diabetes2002,51:1580–1587
5Jefferson JA, Alpers CE, Shankland SJ. Podocyte biology for the bedside.Am J Kidney Disease.2011,58(5):835-845
6M ichos O, Panm an L, V intersten K, et al. Gremlin mediated BMPantagonism induces the epithelial mesenchym a lfeedback signaling controlling metanephric kidney and limb organogenesis. Development,2004,131:3401-3410
7Topol LZ, Bardot B，Zhang Qy, et al. Biosynthesis, Post-translationModification, and Functional Characterization of Drm/Gremlin. J BiolChem,2000,275(12):8785-8793
8Bénazet JD, Bischofberger M, Tiecke E, et al. A Self-Regulatory System ofInterlinked Signaling Feedback Loops Controls Mouse Limb Patterning.SCIENCE,2009,323(20)1050-1053
9Murphy M，McMahon R， Lappin D W.P.et al. Gremlins: Is This WhatRenal Fibrogenesis Has Come To? Exp Nephrol2002,10:241–244
10Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogeneticprotein antagonist gremlin1is widely expressed by cancer-associatedstromal cells and can promote tumor cell proliferation. PNAS (Proceedingsof the National Academy of Sciences)2006,103(40):14842-1484
11Gazzerro E, Pereira RC, Jorgetti V, et al. Skeletal Overexpression ofGremlin Impairs Bone Formation and Causes Osteopenia. Endocrinology2005,146(2):655–665
12McMahon RA, Murphy M, Clarkson M et al. IHG-2, a mesangial cell geneinduced by high glucose, is human gremlin. J Biol Chem2000;275:901–9904
13Murphy M, Godson C, Cannon S et al. Suppression subtractivehybridisation identifies high glucose levels as a stimulus for expression ofconnective tissue growth factor and o ther genes in human mesangial cells.J Biol Chem1999;274:5830–5834
14Dolan V, Murphy M, Sadlier D et al. Expression of gremlin, a bonemorphogenetic protein antagonist, in human diabetic nephropathy. Am Jkidney disease.200545(6):1034-1039
15Wang SN, Lapage J, Hirschber R. Loss of Tubular Bone MorphogeneticProtein–7in Diabetic Nephropathy. J Am Soc Nephrol2001,12:2392–2399
16Zhang QX., Shi YH, Wada J, et al. In Vivo Delivery of Gremlin siRNAPlasmid Reveals Therapeutic Potential against Diabetic Nephropathy byRecovering Bone Morphogenetic Protein-7.Plos One,2010,5(7) e11709
17Sethi A, Jain A, Zode GS et al. Role of TGF-β/Smad Signaling in GremlinInduction of Human Trabecular Meshwork Extracellular Matrix Proteins.IOVS,2011, Vol.52(8)5251-5259
18Li G, Li Y, Liu S, et al.Gremlin Aggravates Hyperglycemia-InducedPodocyte Injury by a TGFβ/Smad Dependent Signaling Pathway: gremlinand hyperglycemia-induced podocyte injury. J Cellular Biochem Acceptedmanuscript online:31MAR201311:25PM EST|DOI:10.1002/jcb.24559
19LiuYH. New Insights into Epithelial-Mesenchymal Transition in KidneyFibrosis. J Am Soc Nephrol201021:212–222
20Wallner EI, Wada J, Lin S, et al. Renal gene expression in emnbryonic andnewborn diabetic mice. Exp Nephrol2002;10:130-138
21Valdes F, Alvarez AM, Locascio A,et al: The epithelial mesenchymaltransition confers resistance to the apoptotic effects of transforming growthfactor-beta in fetal rat hepatocytes. Mol Cancer Res2002;1:68–78
22Strutz FM: EMT and proteinuria as progression factors. Kidney Int200975:475–481
23Huang H, Huang H, Li Y, et al. Gremlin induces cell proliferation andextra cellular matrix accumulation in mouse mesangial cells exposed tohigh glucose via the ERK1/2pathway. BMC Nephrol.2013Feb11;14:33.doi:10.1186/1471-2369-14-33
24Montero JA, Ga an Y, Macias D et al. Role of FGFs in the control ofprogrammed cell death during limb development. Development2001128,2075-2084
25Costello1CM, Cahill1E, Martin E et al. Role of Gremlin in the LungDevelopment and Disease. Am J Respir Cell Mol Biol,201042:517–523
26Mitola S, Ravelli C, Moroni E, et al. Gremlin is a novel agonist of themajor pro-angiogenic receptor VEGFR2. Blood.2010;116(18):3677-3680
27Shibuya M, Claesson-Welsh L. Signal transduction by VEGF receptors inregulation of angiogenesis and lymphangiogenesis. Exp Cell Res.2006;312(5):549-560
28Mitola S, Moroni E, Ravelli C, et al. Angiopoietin-1mediates theproangiogenic activity of the bone morphogenic protein antagonist Drm.Blood.2008;112(4):1154-1157
29Chiodelli P, Mitola S, Ravelli C et al. Heparan Sulfate ProteoglycansMediate the Angiogenic activity of the Vascular Endothelial GrowthFactor Receptor-2Agonist Gremlin. Arterioscler Thromb Vasc Biol2011;31:e116-e127
30Jara A, Chacon C, Burgos ME et al. Expression of gremlin, bonemorphogenetic protein antagonist, is associated with vascular calcificationin uraemia. Nephrol Dial Transplant (2009)24:121–129
31Burns WC, Twigg SM, Forbes JM et al. Connective tissue growth factorplays an important role in advanced glycation end product-induced tubularepithelial-to-mesenchymal transition: Implications for diabetic renaldisease. J Am Soc Nephrol2006,17:2484–2494
32Turk T, Leeuwis JW, Gray J et al. BMP Signaling and Podocyte MarkersAre Decreased in Human Diabetic Nephropathy in Association With CTGFOverexpression. Journal of Histochemistry&Cytochemistry2009,57(7):623–631
33Nguyen TQ，Roestenberg P, van Nieuwenhoven FA. CTGF InhibitsBMP-7Signaling in Diabetic Nephropathy. Am Soc Nephrol2008,19:2098–2107
34Chen B, Blair DG, Plisov S, et al. Cutting edge: bone morphogeneticprotein antagonists Drm/Gremlin and Dan interact with Slits and act asnegative regulators of monocyte chemotaxis. J Immunol2004,173:5914–5917
35Stabile H, Mitola S, Moroni E, et al. Bone morphogenic protein antagonistDrm/gremlin is a novel proangiogenic factor. Blood2007,109:1834–1840
36Khokha MK, Hsu D, Brunet LJ, et al. Gremlin is the BMP antagonistrequired for maintenance of Shh and Fgf signals during limb patterning.Nature Genetics2003,34:303–307
37Mellitzer G, Hallonet M, Chen L，et al. Spatial and temporal knock down'of gene expression by electroporation of double-stranded RNA andmorpholinos into early postimplantation mouse embroys. Mech Dev.2002,118(1-2):57-63
38Roxburgh SA, Kattla JJ, Curran SP, et al. Allelic depletion of grem1attenuates diabetic kidney disease. Diabetes,2009,58(7)1641-1650
39Khatri N, Rathi M, Baradia D,et al. In vivo delivery aspects of miRNA,shRNA and siRNA. Crit Rev Ther Drug Carrier Syst.2012,29(6):487-527
40Braun JE, Huntzinger E, Izaurralde E. The role of GW182proteins inmiRNA-mediated gene silencing. Adv Exp Med Biol.2013,768:147-163
41Guo W，Chen WB Yu WD,et al. Small interfering RNA-based moleculartherapy of cancers. Chinese journal of cancer.2013,1DOI:10.5732/CJC.012.10280
42Wang SN, Lapage J, Hirschberg R.Loss of Tubular Bone MorphogeneticProtein–7in Diabetic Nephropathy. J Am Soc Nephrol2001,12:2392–2399