An intronic locus determines SHROOM3-expression and potentiates renal allograft fibrosis

内含子位点决定 SHROOM3 表达并增强肾同种异体移植纤维化

• 批准号: 9749960
• 负责人: Barbara T. Murphy
• 金额: $ 38.14万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9749960
• 关键词: Agonist; Alleles; Allografting; Binding; Biopsy; Candidate Disease Gene; Cells; Chronic; Chronic Kidney Failure; Chronic rejection of renal transplant; Collagen Type I; Collection; Complex; Consensus; Data; Deterioration; Development; Dominant-Negative Mutation; Donor person; Drug Targeting; End stage renal failure; Enhancers; Epithelial Cells; Etiology; European; F-actin-binding proteins; Fibrosis; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genes; Genetic Transcription; Genomics; Genotype; HK2 gene; Human; In Vitro; Kidney; Kidney Transplantation; Link; Maps; Mediating; Messenger RNA; Microarray Analysis; Modeling; Morphology; Mus; Observational Study; Obstruction; Outcome; Pathogenesis; Pathway interactions; Phosphorylation; Prevention; Proteins; Protocols documentation; Quantitative Trait Loci; Regulation; Renal function; Reporting; Research; Role; Sampling; Serum; Signal Transduction; Single Nucleotide Polymorphism; Specific qualifier value; TCF7L2 gene; Testing; Tissues; Transcriptional Regulation; Transforming Growth Factor beta; Transgenic Model; Transplant Recipients; Transplantation; Untranslated RNA; Variant; allograft rejection; base; beta catenin; cell type; cohort; diabetic; effective therapy; in vivo; in vivo Model; indexing; inhibitor/antagonist; innovation; interstitial; kidney allograft; kidney fibrosis; knock-down; mouse model; mutant; nephrogenesis; novel; overexpression; population based; post-transplant; prevent; promoter; prospective; protein expression; public health relevance; risk variant; second transplant; small hairpin RNA; therapeutic target

 DESCRIPTION (provided by applicant): Tubulo-interstitial fibrosis (TIF) contributes to the progression of chronic kidney disease (CKD) from varied etiologies, including chronic allograft nephropathy (CAN), to end stage renal failure. However, sensitive predictor of kidney fibrosis and effective antifibrotic therapy for CKD do not exist. Therefore, it is critical for us to identiy markers of and drug targets for TIF. Preliminary data demonstrates that SHROOM3 expression in 3 months renal allografts biopsies was associated with indices of poor allograft function (higher CADI score and decline in eGFR) at 12 months post transplantation, but not at 3 months. Furthermore, theSHROOM3rs17319721 SNP (i.e. A/G or A/A) is associated with a higher SHROOM3 expression at 3 months and a greater propensity to developaΔ CADI (chronic allograft index score) ≥2at 12 months, compared to the G/G allele. In the presence of the risk allele regulation of expression appears to be mediated via a Wnt/βcatenin/ TCF7L2 dependent pathway. Furthermore, expression of SHROOM3 enhanced the profibrotic effects of TGFβ. Based on these preliminary data, the central hypothesize of this proposal is that the SHROOM3 snprs17319721 functions as a cisacting expression quantitative trait locus of SHROOM3 to facilitate TGFβ1 signaling contributes to renal fibrosis leading to progression of CAN and CKD. To test this hypothesis, the following specific aims are proposed: Specific aim 1: To validate that SHROOM3 genotype correlates with expression in a second transplant cohort (1a); To determine the correlation of SHROOM3 genotype and expression with long-term graft outcomes (1b); and To determine SHROOM3 protein expression and localization in the allograft (1c).Specific aim 2: (2a)To determine the transcriptional regulation of SHROOM3 expression by the rs1731972 SNP; (2b) To map the molecular interaction between the TCF7L2/βcatenin complex and the consensus binding sequence encompassing rs17319721; (2c) To investigate the mechanism of SHROOM3facilitated TGFβ signaling in kidney fibrosis. Specific aim 3: To determine the role of SHROOM3 on kidney fibrosis in CAN and CKD murine models in vivo. An inducible and tissues pecific SHROOM3 knockdown model (3a) and an overexpression transgenic model (3b) will be used to examine SHROOM3's function in these models of renal fibrosis.Innovation:SHROOM3is a novel candidate gene for which both a mutant allele and expression predict the decline of renal function and the progression of TIF in renal allografts. This is the first description of a CKD associated SNP that functions as an eQTL. Furthermore, a mechanism for transcriptional regulation via the in tronic SNP is proposed. The current research plan will build upon these initial findings to further delineate transcriptional regulation of SHROOM3, the mechanism by which it contributes the development of fibrosis in vitro and its significance in vivo. The proposed research is of Significance because it will determine the role ofShroom3 in the development and progression of CAN and CKD and its potential as a therapeutic target for the prevention of fibrosis.

 描述(申请人提供)：肾小管间质纤维化(TIF)导致慢性肾脏疾病(CKD)从各种病因，包括慢性移植物肾病(CAN)，到终末期肾功能衰竭。然而，肾纤维化的敏感预测指标和CKD的有效抗纤维化治疗尚不存在。因此，确定TIF的标记物和药物靶点是至关重要的。初步数据显示，移植后3个月肾活检组织中SHROOM3的表达与移植后12个月移植肾功能不良指标(CADI评分较高和EGFR下降)相关，但与移植后3个月无关。此外，与G/G等位基因相比，SHROOM3rs17319721SNP(即A/G或A/A)与SHROOM3在3个月时的高表达以及在12个月时发生慢性移植物指数评分(ΔCADI)≥2的倾向更大相关。在风险等位基因存在的情况下，表达的调节似乎是通过Wnt/β连环蛋白/TCF7L2依赖的途径来介导的。此外，SHROOM3的表达增强了转化生长因子β的促纤维化作用。基于这些初步数据，本研究的中心假设是SHROOM3SNPRs17319721作为SHROOM3顺式表达的数量性状基因座，促进转化生长因子β-1信号转导促进肾纤维化，从而导致CAN和慢性肾脏病的进展。为了验证这一假设，提出了以下特定目标：特定目的1：验证SHROOM3基因与第二个移植队列(1a);中表达的相关性，确定SHROOM3基因和表达与长期移植结果(1b);的相关性，并确定SHROOM3蛋白在同种异体移植肾中的表达和定位(1c)。特定目标2：(2a)确定rs1731972 SNP;(2b)对SHROOM3表达的转录调控，绘制TCF7L2/β连环蛋白复合体与包含rs17319721;(2c)的共识结合序列之间的分子相互作用图，以探讨SHROOM3促进转化生长因子信号β在肾脏纤维化中的机制。具体目的3：确定SHROOM3在体内对CAN和CKD模型小鼠肾脏纤维化的作用。我们将使用一个可诱导的和组织特异性的SHOM3基因敲除模型(3a)和一个过表达的转基因模型(3b)来研究SHROOM3在这些肾纤维化模型中的S功能。创新：SHROOM3是一个新的候选基因，突变的等位基因和表达都可以预测肾功能的下降和移植肾TIF的进展。 这是第一次描述与CKD相关的SNP作为eQTL的功能。此外，还提出了一种通过内含子SNP进行转录调控的机制。目前的研究计划将在这些初步发现的基础上，进一步描述SHROOM3的转录调控，它在体外促进纤维化发展的机制及其在体内的意义。这项拟议的研究具有重要意义，因为它将确定Shroom3在CAN和CKD的发生和发展中的作用，以及它作为预防纤维化的治疗靶点的潜力。