The Role of Krüppel-Like Factor 4 as a Key Mediator of Renal Endothelial Injury In Antibody Mediated Rejection.

项目摘要/摘要 由于每年透析的死亡率超过20%，肾移植是这些疾病的首选治疗方法。 然而，对于终末期肾病，虽然短期移植物存活率正在改善，但长期移植物存活率 仍然停滞不前，抗体介导的排斥(ABMR)是其主要原因。血管内皮细胞是主要的 ABMR的损伤靶点和包括反映内皮细胞激活的标记物的概念 相关转录本或ENDAT)现在包括在其诊断标准中。因为这通常是一个很晚的诊断 意味着对治疗的次优反应，迫切需要确定内皮细胞 在ABMR中发生损伤，以改进诊断和治疗策略。类KRüppel因子(KLF4)--锌指 基因芯片显示转录因子在ABMR患者肾活检组织中的最高表达 与其他类型的移植物功能障碍相比。近年来，KLF4在介导抗肿瘤中的关键作用。 炎症基因在血管内皮细胞中的表达已在心血管系统中得到证实。在……里面 此外，KLF4还能抑制细胞因子诱导的信号转导和转录激活因子 (STAT3)信号转导，可能是其抗炎作用的一种潜在机制。然而， KLF4在肾内皮细胞损伤反应中的作用尚不清楚。基于这些发现和 初步数据，我们的中心假设是KLF4的表达是防止抗体介导的必需的 通过抑制STAT3信号通路引起的内皮损伤，将通过以下特定目的进行研究：1)测试 需要内皮特异性表达KLF4来阻止抗体介导的内皮细胞的假说 损伤和2)确定KLF4-STAT3信号在预防内皮损伤中的关键机制 在ABMR。在第一个目标(次级目标1A)下，将使用以下模型确定KLF4的保护作用 抗体对稳定敲除(KLF4-shRNA)和过表达(KLF4-ORF)人的损伤作用 血管内皮细胞株和适当的对照。在亚目标1B中，抗体介导的内皮损伤将被诱导 在内皮特异性KLF4缺失的小鼠和对照组中使用Cre-重组酶系统确定 KLF4的丢失是否会加速内皮损伤。对于目标1，将确定内皮损伤的程度 通过检测参与黏附和炎症的ENDAT的表达以及STAT3的激活 RT-PCR和蛋白质印迹法检测信号转导。在目标2(次级目标2A)下，KLF4抑制的机制 通过免疫共沉淀阐明STAT3信号在预防抗体介导的内皮损伤中的作用 学习。在SubAim 2B中，将使用流式细胞术从外周血中分离循环内皮细胞 从ABMR患者的样本中评估KLF4和STAT3靶标(IL-1)的表达模式 6、ICAM-1)与同期活检时内皮细胞损伤程度相关。这项提议将填补目前的空白。 在该领域中，通过利用细胞培养和小鼠模型的综合框架，通过 在ABMR中，KLF4-STAT3信号的哪一种调节对于预防内皮损伤至关重要。

Project Summary/Abstract With annual mortality on dialysis of over 20%, kidney transplantation is the treatment of choice amongst those with end stage renal disease, however, while short term graft survival is improving, long term graft survival remains stagnant with antibody mediated rejection (ABMR) as its leading cause. Endothelial cells are the primary targets of injury in ABMR and the concept of including markers reflecting endothelial activation (endothelial- associated transcripts or ENDATs) is now included in its diagnostic criteria. Being that a late diagnosis often means suboptimal response to therapy, there is an urgent need to identify the mechanism by which endothelial injury occurs in ABMR to improve diagnostic and treatment strategies. Krüppel-like factor (KLF4), a zinc-finger transcription factor exhibited the highest expression by microarray in kidney biopsies from patients with ABMR as compared to other types of graft dysfunction. In recent years, the critical role of KLF4 mediating anti- inflammatory gene expression in endothelial cells has been demonstrated in the cardiovascular system. In addition, KLF4 has been shown to inhibit cytokine induced signal transduction and activator of transcription (STAT3) signaling, a potential mechanism by which its anti-inflammatory effects may be mediated. However, the role of KLF4 in the renal endothelial response to injury is less well delineated. Based on these findings and preliminary data, our central hypothesis is that expression of KLF4 is required to prevent antibody-mediated endothelial injury by inhibition of STAT3 signaling and will be investigated by the following specific aims: 1) Test the hypothesis that endothelial-specific expression of KLF4 is required to prevent antibody mediated endothelial injury and 2) Determine the mechanism by which KLF4-STAT3 signaling is critical to preventing endothelial injury in ABMR. Under the first aim (subaim 1A), the protective role of KLF4 will be determined using a model of antibody induced injury in both stable knockdown (KLF4-shRNA) and overexpression (KLF4-ORF) human endothelial cell lines and appropriate controls. In subaim 1B, antibody mediated endothelial injury will be induced in mice with endothelial-specific Klf4 deletion and controls using the Cre-recombinase system to determine whether the loss of Klf4 accelerates endothelial damage. For aim 1, extent of endothelial injury will be determined by measuring the expression of ENDATs involved in adhesion and inflammation, as well as activation of STAT3 signaling by RT-PCR and western blot. Under the Aim 2 (subaim 2A), the mechanism by which KLF4 inhibits STAT3 signaling to prevent antibody-mediated endothelial injury will be elucidated by co-immunoprecipitation studies. In subaim 2B, flow cytometry will be used to isolate circulating endothelial cells from peripheral blood samples from patients with ABMR to evaluate whether the pattern of expression of KLF4 and STAT3 targets (IL- 6, ICAM-1) correlates with extent of endothelial injury on concurrent biopsy. This proposal will fill this current gap in the field by utilizing an integrative framework of cell culture and murine models to ascertain the mechanism by which regulation of KLF4-STAT3 signaling is critical to the prevention of endothelial injury in ABMR.