Expression of CD2AP on podocytes with different pathological types of nephrotic syndrome

目的 观察不同病理类型的原发性肾病综合征(nephrotic syndrome,NS)患者肾小球足细胞中CD2相关蛋白(CD2AP)的表达,探讨其与足细胞损伤的关系.方法 选取原发性NS患者54例,10例同期肾肿瘤切除患者正常肾组织作为对照.肾活检后常规染色观察肾脏组织病理改变,肾组织行免疫荧光法CD2AP和肾小球上皮细胞蛋白-1(GLEPP1)双重标记,对肾小球CD2AP的表达进行定位;分别用real time PCR和免疫组化SP法检测组织中CD2AP的表达,采用real time PCR检测nephrin的表达,透射电镜观察足细胞的结构变化,并定量测量足突密度.结果 (1)NS患者肾小球中CD2AP的表达及nephrin的表达下调,足细胞足突不同程度融合,足突密度降低.(2)病理表现为微小病变性肾病(minimal change disease,MCD)、局灶性节段性肾小球硬化(focal segmental glomerulosclerosis,FSGS)和膜性肾病(membranous nephropathy,MN)的NS患者CD2AP表达及nephrin表达较对照组明显降低,且CD2AP与nephrin表达呈正相关,病理表现为MCD和FSGS的NS患者CD2AP表达与足突密度呈正相关.结论 本研究首次发现原发性NS患者肾小球足细胞中CD2AP的表达降低,且在MCD和FSGS中与足细胞病变程度相关,提示CD2AP低表达在足细胞病变为主的肾小球疾病中发挥重要作用.CD2AP有利于诊断足细胞病变的早期检测,对CD2AP表达减低进行早期干预可能有助于延缓疾病进展.     

CD2相关蛋白在肾病综合征中的表达及意义

目的观察不同病理类型的原发性肾病综合征(nephrotic syndrome,NS)患者肾小球足细胞中CD2相关蛋白(CD2AP)的表达,探讨其与足细胞损伤的关系。方法选取原发性NS患者54例,10例同期肾肿瘤切除患者正常肾组织作为对照。肾活检后常规染色观察肾脏组织病理改变,肾组织行免疫荧光法CD2AP和肾小球上皮细胞蛋白-1(GLEPP1)双重标记,对肾小球CD2AP的表达进行定位;分别用real time PCR和免疫组化SP法检测组织中CD2AP的表达,采用real time PCR检测nephrin的表达,透射电镜观察足细胞的结构变化,并定量测量足突密度。结果 (1)NS患者肾小球中CD2AP的表达及nephrin的表达下调,足细胞足突不同程度融合,足突密度降低。(2)病理表现为微小病变性肾病(minimal change disease,MCD)、局灶性节段性肾小球硬化(focal segmental glomerulosclerosis,FSGS)和膜性肾病(membranous nephropathy,MN)的NS患者CD2AP表达及nephrin表达较对照组明显降低,且CD2AP与nephrin表达呈正相关,病理表现为MCD和FSGS的NS患者CD2AP表达与足突密度呈正相关。结论本研究首次发现原发性NS患者肾小球足细胞中CD2AP的表达降低,且在MCD和FSGS中与足细胞病变程度相关,提示CD2AP低表达在足细胞病变为主的肾小球疾病中发挥重要作用。CD2AP有利于诊断足细胞病变的早期检测,对CD2AP表达减低进行早期干预可能有助于延缓疾病进展。     

CD2相关蛋白在肾脏细胞中的分布及其在足细胞中的作用

目的 观察CD2相关蛋白(cD2AP)在肾脏不同细胞系中的表达分布,及其与足细胞裂孔隔膜分子nephrin和细胞骨架蛋白F-actin之间的联系.方法 以DMEM培养基培养人肾小球系膜细胞(HMC)和人肾小管上皮细胞系(HK-2),RPMI 1640培养基培养条件永生化小鼠足细胞系.以RT-PCR及Western blotting方法检测足细胞内CD2AP和nephrin的表达.间接免疫荧光结合激光共焦方法观察CD2AP在HMC、HK-2、未分化及已分化足细胞中的表达情况,及CD2AP与nephtin在足细胞中的共存.直接免疫荧光结合激光共焦方法观察F-actin在足细胞的表达及其与CD2AP的共存.结果 CD2AP均匀分布于HK-2及未分化足细胞的核周及胞浆,而不表达于HMC细胞.在足细胞分化过程中,CD2AP的分布发生了变化,出现向周边聚集的现象.CD2AP与足细胞裂孔隔膜分子nephrin及细胞骨架蛋白F-actin在足细胞中存在共定位关系.结论 CD2AP表达于上皮来源的肾脏固有细胞.CD2AP在足细胞中的分布特点,提示CD2AP可能参与足细胞的分化过程,并与裂孔隔膜分子功能及细胞骨架凋节有关.     

CD2AP、F-actin在肾病大鼠中的表达及意义

目的：观察CD2-associated protein（CD2AP）、肌动蛋白微丝（F-actin）在氨基核苷肾病大鼠肾小球中的表达变化及其意义。 方法： 通过建立大鼠氨基核苷肾病模型，采用免疫组织化学、蛋白质印迹技术观察CD2AP在不同时点肾病大鼠肾小球中的表达和分布变化，采用荧光技术检测肾小球F-actin含量的变化。 结果： ①肾小球足细胞CD2AP的表达在肾病模型建立的早期即有下调；在肾病大鼠蛋白尿的高峰期，CD2AP的表达明显下降（P<0.01）；在肾病大鼠的疾病恢复期，CD2AP的表达逐步恢复正常。②肾小球足细胞CD2AP表达量的变化与肾病大鼠24 h尿蛋白的变化呈负相关（r=-0.865，P<0.01）。③在肾病大鼠蛋白尿的高峰期，肾小球F-actin含量明显下降（P<0.05）。④肾小球CD2AP蛋白表达量和肾小球F-actin荧光定量变化呈正相关（r=0.873，P<0.01）。 结论： ①CD2AP、F-actin的表达变化在蛋白尿的发生机制中有着重要作用。②CD2AP可能是判断肾小球足细胞损伤的一个早期重要指标。     

CD2AP在肾脏病学的研究进展

CD2AP是新发现的一种肾小球足细胞裂孔膜蛋白。CD2AP / 小鼠表现为先天性肾病综合症,出生后6~7周将死于肾功能衰竭;CD2AP / 小鼠肾脏病理表现类似于人类FSGS。CD2AP基因突变可能与人类一些肾脏疾病的发病有关。此外,CD2AP还可能通过与其他裂孔膜蛋白相互作用,在足细胞信号转导和功能调节过程中发挥重要作用。     

miR-155 在TGF-Ｂ1 诱导足细胞损伤中的表达及其与synaptopodin、 CD2AP 表达的相关性

目的:探讨TGF-β1诱导的足细胞损伤中miR-155的表达变化及其与足细胞损伤的关系。方法:体外培养小鼠肾小球足细胞,用TGF-β1刺激足细胞构建足细胞损伤模型,用CCK8法检测各浓度梯度(0、4、8、12 ng/ml)、时间梯度(0、24、48、72 h)的细胞增殖活性。实时荧光定量PCR技术检测足细胞损伤模型中synaptpotodin、CD2AP mRNA及miR-155的表达。Western blot法检测足细胞损伤模型中synaptpotodin、CD2AP蛋白的表达。结果:一定浓度的TGF-β1可抑制足细胞增殖,降低细胞存活率(P 0. 05)。TGF-β1诱导的足细胞损伤模型中miR-155表达水平上调(P 0. 05),TGF-β1诱导足细胞损伤后,可引起synaptpotodin、CD2AP mRNA和蛋白表达水平下调,且与miR-155表达水平呈负相关(P 0. 05)。结论:miR-155表达上调与TGF-β1诱导的足细胞损伤有关,可能作为足细胞损伤诊断和治疗的靶点。     

CD2相关蛋白启动子区域保守序列的功能分析

目的 在不同物种间通过保守序列分析和荧光素酶功能测定的方法寻找发现CD2相关蛋白(cD2AP)基因启动子重要的调节成分.方法 用BLAST分析软件进行序列比较和同源分析不同种系CD2AP启动子序列,构建人CD2AP启动子不同的缺失变异载体,转染来自不同种类的细胞,测定荧光素酶活性,并用全反式维甲酸处理,观测其对CD2AP启动子活性的影响.结果 在人、牛、猪的CD2AP推断的启动子区域进行同源比较发现推断的sp1(specific protein 1)和下游启动子成分高度进化保守,进行性缺失荧光素酶分析表明在人胚肾细胞株HEK-293、非洲绿猴肾细胞株Vero、仓鼠肾细胞株BHK-21中人CD2AP启动子活性有相似的形式,ATG上游500 bp有基本的启动子活性,再向上100 bp启动子活性增加10倍,两个推断的Sp1位点位于该100 bp区域内,全反式维甲酸可下调CD2AP启动子的活性.结论 我们初步发现推断的Sp1位点和下游启动子成分在CD2AP启动子调控中起重要作用.     

miR-133a-3p靶向调控CD2AP基因减轻RSV感染的人支气管上皮细胞系16-HBE损伤

目的探讨miR-133a-3p对呼吸道合胞病毒(RSV)感染的支气管上皮细胞凋亡、炎性反应的影响及其对CD2相关蛋白(CD2AP)的调控作用。方法收集59例支气管哮喘儿童的支气管黏膜组织标本为AsP组,同时选取59例支气管扩张非哮喘儿童的支气管黏膜组织标本为NC组,用RT-qPCR法检测组织中miR-133a-3p、CD2AP的表达量;RSV感染支气管上皮细胞(16-HBE)建立细胞损伤模型(RSV组),分别将miR-NC、miR-133a-3p mimics、si-NC、si-CD2AP、pcDNA-NC、pcDNA-CD2AP、miR-133a-3p mimics与pcDNA-NC、miR-133a-3p mimics与pcDNA-CD2AP转染至感染RSV的16-HBE细胞;用RT-qPCR法与Western blot法分别检测细胞中miR-133a-3p、CD2AP的表达量;用ELISA法检测TNF-α、IL-6、IL-1α的水平;用流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测miR-133a-3p与CD2AP的靶向关系;Western blot法检测cleaved-caspase-3蛋白表达量。结果与NC组比较,AsP组支气管黏膜组织中miR-133a-3p的表达水平降低(P0.05),CD2AP mRNA的表达水平升高(P0.05);RSV感染的16-HBE细胞中miR-133a-3p的表达水平降低(P0.05),CD2AP的表达水平及TNF-α、IL-6、IL-1α的水平升高(P0.05),凋亡率及cleaved-caspase-3蛋白水平升高(P0.05);RSV感染的16-HBE细胞中转染miR-133a-3p mimics或转染si-CD2AP可明显降低TNF-α、IL-6、IL-1α的水平、凋亡率及cleaved-caspase-3蛋白水平(P0.05);双荧光素酶报告实验证实CD2AP是miR-133a-3p的靶基因;共转染miR-133a-3p mimics与pcDNA-CD2AP可明显逆转miR-133a-3p mimics对16-HBE细胞凋亡及炎性反应。结论 miR-133a-3p过表达可通过负向调控CD2AP的表达而抑制RSV感染的支气管上皮细胞炎性反应及凋亡,从而减轻细胞损伤。     

蟾蜍干预对大鼠肾病模型CD2AP表达的影响

目的 探讨CD2-associated protein（CD2AP）在蟾蜍干预阿霉素肾病模型肾小球中的表达及意义。方法 通过建立阿霉素（ADR）肾病模型，用蟾蜍等干预后，采用免疫组织化学技术观察各组模型大鼠肾小球中CD2AP的表达变化。结果 蟾蜍能使肾小球足细胞CD2AP的表达上调。结论 蟾蜍对肾病大鼠模型病理损伤的修复可能与CD2AP表达上调有关。     

肾脏足细胞裂孔隔膜的分子组成

足细胞裂孔隔膜结构的改变是导致肾小球疾病产生蛋白尿的主要原因 ,已陆续发现包括nephrin在内的与裂孔隔膜相关的多个蛋白分子 ,如ZO 1,CD2AP ,P cadherin ,podocin ,NEPH1,FAT及Filtrin等 ,它们的存在及相互作用对于保证裂孔隔膜的完整性和滤过功能有着直接或间接的作用 ,这些蛋白分子的发现有利于进一步阐明蛋白尿的发病机制 ,同时也为临床监测蛋白尿进展及制定治疗方案提供新的思路     

CD2AP Localizes to the Slit Diaphragm and Binds to Nephrin via a Novel C-Terminal Domain

CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. Here we demonstrate that CD2AP localizes to the slit diaphragm in podocytes using immunoelectron microscopy and that nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm.     

Nephrin forms a complex with adherens junction proteins and CASK in podocytes and in Madin-Darby canine kidney cells expressing nephrin

Mutations in the NPHS1 gene encoding nephrin lead to congenital nephrotic syndrome of the Finnish type. Nephrin is a key component of the glomerular slit diaphragms between epithelial foot processes, but its role in the pathogenesis of this disease is poorly understood. To further clarify the molecular mechanisms involved we investigated the interactions between nephrin and other components of the foot processes and filtration slits, especially adherens junction proteins, and searched for novel nephrin interacting proteins. Using co-immunoprecipitation and pull-down assays we show here that nephrin forms a multiprotein complex with cadherins and p120 catenin and with three scaffolding proteins, ZO-1, CD2AP, and CASK, in kidney glomeruli and when expressed in Madin-Darby canine kidney cells. CASK was identified as a novel binding partner of nephrin by mass spectrometry and was localized to podocytes in the glomerulus. CASK is a scaffolding protein that participates in maintenance of polarized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton. Our results support a model whereby the glomerular slit diaphragms are composed of cell adhesion molecules of the immunoglobulin and cadherin superfamilies that are connected to each other and to the actin cytoskeleton and signaling networks via the cytoplasmic scaffolding proteins CASK, CD2AP, and ZO-1.     

T-cell activation and immunologic synapse

The focus of my laboratory is in three areas. The first area is our long-standing interest in the mechanics of T-cell activation. In addition to our interest in protein phosphorylation mediated by T-cell receptor engagement, we have been interested in defining the function of the immunologic synapse. The second area of focus is on the pathogenesis of glomerular disease. Here we are interested in the function of the podocyte and trying to understand how genetic deficiency of CD2AP in podocytes leads to kidney failure. The third area is the effect of modifiers of protein kinase cascades. Here we are focused on scaffold proteins, namely KSR and its role in controlling the activation of mitogen-activated protein kinase.     

Co-localization of nephrin,podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation

The discovery of the genes for nephrin and podocin, which are mutated in two types of congenital nephrotic syndrome, was pivotal in establishing the podocyte as the central component of the glomerular filtration barrier. In vivo the proteins have been localized to the podocyte slit diaphragm, and there is recent evidence for interaction between the two via the adapter molecule CD2AP. We describe in a human podocyte cell line, the subcellular distribution of nephrin, podocins, and CD2AP and their functional interaction with the cytoskeleton. In addition to membrane expression, nephrin and podocin were detected intracellularly in a filamentous pattern. Double immunolabeling and depolymerization studies showed that nephrin and podocin partially co-localize with actin, most strikingly seen protruding from the tips of actin filaments, and are dependent on intact actin polymers for their intracellular distribution. Treatment of differentiated podocytes with puromycin aminonucleoside, an agent that causes foot process effacement in vivo, disrupted actin and nephrin simultaneously, with loss of cell surface localization. We demonstrate an intimate relationship between nephrin podocin and filamentous actin, and reason that disruption of nephrin/podocin could be a final common pathway leading to foot process effacement in proteinuric diseases.     

Montelukast improves the changes of cytoskeletal and adaptor proteins of human podocytes by interleukin-13

Objective and design

Interleukin-13 (IL-13) has recently been reported to be a potential cytokine in the pathogenesis of minimal-change nephrotic syndrome (MCNS). However, the mechanistic insights associated with podocyte dysfunction mediated by IL-13-induced changes in various slit diaphragm (SD) and cytoskeletal molecules have not yet been shown in cultured human podocytes in vitro.

Materials

Human conditionally immortalized podocytes were used.

Treatment

Podocytes were incubated with various concentrations of IL-13 during the indicated time periods (6, 12, and 24 h) and montelukast was administered with the dose of 0.1 μg.

Results

Treatment of IL-13 resulted in a progressive decrease in distinct processes or projections of the human podocytes and high dose of IL-13 increased podocyte permeability in vitro at 6 h. IL-13 had a substantial impact on the redistribution and rearrangement of zonula occludens (ZO)-1, synaptopodin, α-actinin, CD2-associated protein (CD2AP) in podocytes and disrupted the cytoskeletal connections in a concentration-dependent manner on confocal microscopy. IL-13 also down-modulated ZO-1, synaptopodin, α-actinin, CD2AP, and p130Cas at protein levels and upregulated β-catenin and B7-1 in podocytes. Furthermore, we demonstrated that down-modulated changes in various SD and cytoskeletal structures of human podocytes induced by IL-13 was significantly restored after treatment with montelukast with upregulation of B7-1.

Conclusion

Our results suggest that targeting IL-13 may be one of the important cytokines in the pathogenesis of MCNS and targeting IL-13 could be one of the potential therapeutic strategies in MCNS.

     

Synaptopodin protects against proteinuria by disrupting Cdc42:IRSp53:Mena signaling complexes in kidney podocytes

The actin-based foot processes of kidney podocytes and the interposed slit diaphragm form the final barrier to proteinuria. Mutations affecting several podocyte proteins cause disruption of the filtration barrier and rearrangement of the highly dynamic podocyte actin cytoskeleton. Proteins regulating the plasticity of the podocyte actin cytoskeleton are therefore of critical importance for sustained kidney barrier function. Synaptopodin is an actin-associated protein essential for the integrity of the podocyte actin cytoskeleton because synaptopodin-deficient mice display impaired recovery from protamine sulfate-induced foot process effacement and lipopolysaccharide-induced nephrotic syndrome. Moreover, bigenic heterozygosity for synaptopodin and CD2AP is sufficient to induce spontaneous proteinuria and focal segmental glomerulosclerosis-like glomerular damage in mice. Mechanistically, synaptopodin induces stress fibers by blocking the proteasomal degradation of RhoA. Here we show that synaptopodin directly binds to IRSp53 and suppresses Cdc42:IRSp53:Mena-initiated filopodia formation by blocking the binding of Cdc42 and Mena to IRSp53. The Mena inhibitor FP(4)-Mito suppresses aberrant filopodia formation in synaptopodin knockdown podocytes, and when delivered into mice protects against lipopolysaccharide-induced proteinuria. The identification of synaptopodin as an inhibitor of Cdc42:IRSp53:Mena signaling defines a novel antiproteinuric signaling pathway and offers new targets for the development of antiproteinuric therapeutic modalities.