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

项目总结/摘要 透析的年死亡率超过20%，肾移植是这些患者中的首选治疗方法。 然而，对于终末期肾病，虽然短期移植物存活率正在改善， 抗体介导的排斥反应（ABMR）是其主要原因。内皮细胞是主要的 在ABMR中损伤的靶点和包括反映内皮活化的标志物（内皮- 相关转录物或ENDATs）现在被纳入其诊断标准。因为晚期诊断通常 意味着对治疗的反应不佳，迫切需要确定内皮细胞 损伤发生在ABMR，以改善诊断和治疗策略。Krüppel样因子（KLF 4），一种锌指 在ABMR患者的肾活检组织中，一种转录因子的表达最高 与其他类型的移植物功能障碍相比。近年来，KLF 4介导抗肿瘤的关键作用被广泛研究， 已经在心血管系统中证实了内皮细胞中的炎性基因表达。在 此外，KLF 4已显示出抑制细胞因子诱导的信号转导和转录激活因子 （STAT 3）信号传导，其抗炎作用可能通过其介导的潜在机制。但 KLF 4在肾内皮细胞对损伤的反应中的作用还不太清楚。根据这些发现， 根据初步数据，我们的中心假设是，KLF 4的表达是防止抗体介导的细胞凋亡所必需的。 通过抑制STAT 3信号传导来抑制内皮损伤，并将通过以下具体目的进行研究： 假设KLF 4的内皮特异性表达是阻止抗体介导的内皮细胞凋亡所必需的， 2）确定KLF 4-STAT 3信号传导对预防内皮损伤至关重要的机制 在ABMR。在第一个目标（子目标1A）下，将使用以下模型确定KLF 4的保护作用： 在稳定敲低（KLF 4-shRNA）和过表达（KLF 4-ORF）的人中，抗体诱导的损伤 内皮细胞系和适当的对照。在子目标1B中，将诱导抗体介导的内皮损伤， 在内皮特异性Klf 4缺失的小鼠和对照中，使用Cre重组酶系统测定 Klf 4的缺失是否加速了内皮损伤。对于目标1，将确定内皮损伤的程度 通过测量参与粘连和炎症的ENDAT的表达，以及STAT 3的激活， 通过RT-PCR和蛋白质印迹分析信号传导。在目标2（子目标2A）下，KLF 4抑制 将通过免疫共沉淀阐明STAT 3信号传导以防止抗体介导的内皮损伤 问题研究在子目的2B中，将使用流式细胞术从外周血中分离循环内皮细胞 来自ABMR患者的样本，以评估KLF 4和STAT 3靶点（IL-10）的表达模式是否与ABMR患者的免疫反应有关。 6，ICAM-1）与同时活检的内皮损伤程度相关。这一提议将填补目前的这一空白 通过利用细胞培养和小鼠模型的综合框架， 其中KLF 4-STAT 3信号转导的调节对于预防ABMR中的内皮损伤至关重要。

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.