Targeting T lymphocyte Keap1 for acute kidney injury

靶向 T 淋巴细胞 Keap1 治疗急性肾损伤

• 批准号: 9333374
• 负责人: HAMID RABB
• 金额: $ 44.66万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-16 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333374
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Adoptive Transfer; Antioxidants; Apoptosis; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CRISPR/Cas technology; Cell Therapy; Cells; Chalcones; Cisplatin; Clinical; Cues; Data; Disease; Environment; FOXP3 gene; Frequencies; Future; Health Care Costs; Homeostasis; Human; Immune; Inflammation; Inflammatory; Inflammatory Response; Injury; Interferon Type II; Interleukin-2; Intervention; Ischemia; Kidney; Kidney Diseases; Knockout Mice; Link; Mediating; MicroRNAs; Modeling; Morbidity - disease rate; Mus; Nephrotoxic; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmacology; Phenotype; Population; Predisposition; Process; Production; Recovery; Regulatory T-Lymphocyte; Renal function; Reperfusion Therapy; Role; Small Interfering RNA; Specificity; Sulforaphane; T cell response; T-Cell Immunologic Specificity; T-Lymphocyte; TNF gene; Technology; Testing; Therapeutic; Therapeutic Studies; Thymus Gland; Wild Type Mouse; clinical translation; cytokine; design; experimental study; improved; in vitro Assay; in vivo; inhibitor/antagonist; insight; mortality; mouse model; nephrotoxicity; novel; novel therapeutics; overexpression; prevent; protective effect; repaired; response; targeted treatment; transcription factor

PROJECT SUMMARY /ABSTRACT Acute kidney Injury (AKI)-associated morbidity and mortality is a major clinical problem that involves multiple overlapping pathophysiological mechanisms. Our lab and others have established the modulatory roles of T lymphocytes as well as the protective role of transcription factor Nrf2 in ischemia reperfusion (IR) and cisplatin induced AKI. We recently demonstrated that T cell deletion of Keap1 which augments specific Nrf2 activity provides significant protection from IR induced AKI in knockout mice, and that adoptive transfer of T cells with augmented Nrf2 activity improves kidney function and survival following AKI in wild type mice. These findings were accompanied by a significant increase in regulatory T (Treg) cell frequency and numbers, and reduced proinflammatory cytokine production by T cells in the kidneys of mice with Keap1 deficient T cells. These novel observations reveal an unexpected relationship between Keap1 and T cell homeostasis and function in AKI, however the underlying mechanisms are unknown. The overarching aim of this proposed study is to test the hypothesis that Keap1 regulates the expansion of Treg cell population that subsequently suppresses harmful inflammatory responses during AKI. In order to test our hypotheses, we will determine whether Treg cells with Keap1 deletion have enhanced proliferation or decreased apoptosis as compared to Treg cells from wild type (WT) mice. We will also evaluate if Keap1 deletion increases the suppressive function of Treg cells, enhances TCR sensitivity in T cells and regulates response to IL-2 under steady state and in two different models (IR and cisplatin) of AKI in mice. To further establish the specificity of Keap1 deletion and to investigate any Nrf2 independent effects of Keap1 in T cells we will generate mice lacking Keap1 specifically in Treg, CD4 and CD8 T cells and determine whether Treg specific Keap1 deletion is sufficient to prevent ischemic and nephrotoxic AKI. We will further generate mice with T cell specific deletion of either Nrf2 alone or Keap1 and Nrf2 together (double KO) to delineate Nrf2 dependent and independent effects. In the final aim we will transition from mechanistic studies to experiments that will set the stage for clinical translation. We will explore therapeutic approaches including pharmacologic Nrf2 activators to activate Nrf2 and Keap1 specific CRISPR/Cas9 technology, siRNAs and miRNAs to delete Keap1 in mouse T cells ex vivo. We will then examine the effect of these Nrf2 activation/Keap1 deletion strategies on phenotypic and functional T cell responses as well as AKI outcome in IR and cisplatin AKI models. We will test the most promising Nrf2 activation/Keap1 deletion strategy in human primary T lymphocytes and examine its effects on Nrf2 regulated antioxidant response, proinflammatory cytokine production, and T cell dynamics. Successful completion of these studies should result in major advances regarding our understanding on the role of Keap1/Nrf2 in T cell homeostasis and function, and also set the stage for future clinical interventions targeting Keap1-Nrf2 pathway for AKI and other inflammatory diseases.

项目摘要/摘要 急性肾损伤(AKI)相关的发病率和死亡率是一个主要的临床问题，涉及 重叠的病理生理机制。我们的实验室和其他实验室已经确定了T的调节作用 淋巴细胞及转录因子Nrf2在缺血再灌注和顺铂中的保护作用 诱导AKI。我们最近证明了T细胞Keap1的缺失可以增强Nrf2的特异性活性 在基因敲除小鼠中对IR诱导的AKI提供显著的保护，T细胞的过继转移 增强的Nrf2活性改善了野生型小鼠AKI后的肾功能和存活率。这些发现 伴随着调节性T(Treg)细胞频率和数量的显著增加，以及 Keap1缺陷小鼠肾脏T细胞产生促炎细胞因子的研究这些小说 观察揭示了AKI中Keap1和T细胞稳态与功能之间意想不到的关系， 然而，潜在的机制尚不清楚。这项拟议的研究的总体目标是测试 Keap1调节Treg细胞群体扩张的假说 抑制AKI期间的有害炎症反应。为了检验我们的假设，我们将确定 Keap1缺失的Treg细胞是否促进了增殖或减少了凋亡 野生型(WT)小鼠Treg细胞。我们还将评估Keap1缺失是否会增加抑制功能 增强T细胞对TCR的敏感性，并调节对IL-2的反应 不同模型(IR和顺铂)的AKI小鼠模型。为了进一步确定Keap1缺失的特异性，并 研究Keap1在T细胞中对Nrf2的非依赖性作用，我们将建立缺乏Keap1的小鼠 并确定Treg特异性Keap1的缺失是否足以防止 缺血和肾毒性AKI。我们将进一步产生T细胞特异性缺失的Nrf2单独或 Keap1和Nrf2一起(双KO)描述Nrf2依赖和独立的效应。在最终目标中 我们将从机械研究过渡到为临床奠定基础的实验 翻译。我们将探索包括药物NRF2激活剂在内的治疗方法来激活NRF2 和Keap1特异的CRISPR/Cas9技术，siRNAs和miRNAs在体外删除小鼠T细胞中的Keap1。 然后我们将研究这些Nrf2激活/Keap1缺失策略对表型和功能的影响 IR和顺铂AKI模型中T细胞应答和AKI结局。我们将测试最有希望的NRF2 人原代T淋巴细胞激活/Keap1缺失策略及其对Nrf2调控的影响 抗氧化反应、致炎细胞因子的产生和T细胞动力学。成功完成 这些研究应该会在理解Keap1/Nrf2在T细胞中的作用方面取得重大进展 并为将来针对Keap1-Nrf2通路的临床干预奠定了基础 用于AKI和其他炎症性疾病。