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Signaling Pathways in Pathogenesis of Renal Fibrosis

肾纤维化发病机制中的信号通路

基本信息

批准号：
6788757
负责人：
JOSEPH PETER GRANDE
金额：
$ 25.4万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-09-01 至 2006-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6788757
关键词：
biological signal transduction collagen fibrosis gel mobility shift assay gene expression gene targeting genetic promoter element genetic transcription genetically modified animals laboratory rat mitogen activated protein kinase nucleic acid sequence pathologic process transforming growth factors

项目摘要

DESCRIPTION (Applicant's abstract): Diabetic arteriopathy, diabetic nephropathy, and many forms of progressive renal disease are all characterized by excessive deposition of collagen IV. The lack of effective therapies for these forms of chronic tissue injury are in large part due to a lack of understanding of basic mechanisms of fibrogenesis. Although TGF-13 1 has emerged as the dominant determinant of collagen IV expression and is considered an essential fibrogenic cytokine, the intracellular signaling pathways elicited by TGF-B 1 and the mechanism by which TGF-J3 1 stimulates transcription of the collagen IV genes in pathobiologic states is not known. The central hypothesis to be tested in this application is that TGF-B 1 increases transcription of the collagen IV genes by triggering several functionally distinct intracellular signaling pathways, involving the Smad proteins and one or more of the mitogen activated protein kinase cascades. In Specific Aim 1, functional characterization of the collagen IV promoter and flanking regions will be completed. Deletion constructs of chimeric collagen IV promoter-CAT vectors will be used to define sequences which function as orientation-specific activator regions that direct transcription of the a1(IV) and a2(IV) collagen genes. Nuclear proteins that bind these critical regulatory regions will be defined by gel mobility shift assays. In Specific Aim 2, the role of TGF-B 1 elicited signaling pathways on collagen IV transcription, steady state mRNA expression, and protein production will be ascertained. Rat mesangial cells and aortic smooth muscle cells will be transfected with constitutively active or dominant negative Smad constructs and with constructs to constitutively activate the ERK, iNK, and p38 signaling pathways; the role of these interventions on basal and TOP-B 1 stimulated collagen IV expression will be ascertained. Potential sites of interaction between the Smad and MAPK signaling pathways will be defined. Based on these studies, transfection analysis and gel mobility shift assays will be employed to define sequence elements within the collagen N promoter or flanking region that confer a transcriptional response to TGF-beta 1(TGF-B1, Specific Aim 3). Finally, mesangial cells and vascular smooth muscle cells isolated from TGF-B 1 knockout animals will be used to determine whether activation of the Smad and/or MAP kinase signaling cascades can activate transcription of the collagen IV genes in a TGF-B 1-independent manner. Delineation of the collagen IV signaling pathway will reveal basic cellular mechanisms of enhanced fibrogenesis underlying progressive tissue injury, and may provide the rational basis for the design of new pharmaco-therapeutic interventions targeted to specific identified signaling steps. If successful, these interventions may retard progression of tissue injury to end-stage disease.

描述（申请人摘要）：糖尿病动脉病，糖尿病肾病和许多形式的进行性肾病都以 IV型胶原过度沉积缺乏有效的治疗方法，这些形式的慢性组织损伤在很大程度上是由于缺乏了解纤维化的基本机制。虽然TGF-13 1具有作为IV型胶原蛋白表达的主要决定因素，一种重要的纤维化细胞因子，细胞内信号通路引起通过TGF-β 1和TGF-β 3 1刺激转录的机制，病理生物学状态下的IV型胶原基因是未知的。核心假设在本申请中要测试的是，TGF-β 1增加了TGF-B1受体的转录。 IV型胶原基因通过触发几种功能不同的细胞内信号通路，涉及Smad蛋白和一种或多种有丝分裂原激活的蛋白激酶级联。在具体目标1中，功能性胶原蛋白IV启动子和侧翼区的表征将是完成嵌合胶原IV启动子-CAT载体的缺失构建体将用于定义作为定向特异性功能的序列指导a1（IV）和a2（IV）胶原转录的激活区基因.结合这些关键调控区域的核蛋白将被通过凝胶迁移率变动测定来确定。在特异性目标2中，TGF-β 1的作用诱导IV型胶原转录、稳态mRNA 表达和蛋白质生产将被确定。大鼠肾小球系膜细胞和主动脉平滑肌细胞将用组成型活性或显性负性Smad结构和结构组成性激活ERK、iNK和p38信号通路;这些信号通路的作用对基础和TOP-B1刺激的IV型胶原蛋白表达的干预将是确定。Smad和MAPK信号转导之间相互作用的潜在位点路径将被定义。基于这些研究，转染分析和凝胶电泳显示，将采用迁移率变动分析来确定赋予转录应答的胶原蛋白N启动子或侧翼区转化为TGF-β 1（TGF-β 1，特异性目标3）。最后，系膜细胞和血管从TGF-B1敲除动物分离的平滑肌细胞将用于确定Smad和/或MAP激酶信号级联的激活是否可以激活IV型胶原基因的转录，方式IV型胶原蛋白信号通路的描述将揭示基本的进行性组织纤维化增强的细胞机制损伤，并可提供合理的依据，设计新的针对特定已识别信号的药物治疗干预步如果成功，这些干预措施可能会延缓组织的进展，对终末期疾病的伤害。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Is body size a biomarker for optimizing dosing of omega-3 polyunsaturated fatty acids in the treatment of patients with IgA nephropathy?

DOI：
10.2215/cjn.00260106
发表时间：
2006-09-01
期刊：
CLINICAL JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY
影响因子：
9.8
作者：
Donadio, James V.;Bergstralh, Eric J.;Grande, Joseph P.
通讯作者：
Grande, Joseph P.

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease.

环核苷酸磷酸二酯酶（PDE）抑制剂：治疗进行性肾病的新型药物。

DOI：
发表时间：
2007
期刊：
Experimental biology and medicine (Maywood, N.J.)
影响因子：
0
作者：
Cheng,Jingfei;Grande,JosephP
通讯作者：
Grande,JosephP

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells.

Lixazinone 刺激 Madin-Darby 犬肾细胞的有丝分裂。

DOI：
10.1177/153537020623100308
发表时间：
2006
期刊：
Experimental biology and medicine (Maywood, N.J.)
影响因子：
0
作者：
Cheng,Jingfei;Thompson,MichaelA;Walker,HenryJ;Gray,CatherineE;Warner,GinaM;Zhou,Wei;Grande,JosephP
通讯作者：
Grande,JosephP

cADP-ribose/ryanodine channel/Ca2+-release signal transduction pathway in mesangial cells.

系膜细胞中的cADP-核糖/兰尼碱通道/Ca2-释放信号转导途径。