Endothelial Cell Mineralocorticoid Receptor and Tubulointerstitial Fibrosis

内皮细胞盐皮质激素受体与肾小管间质纤维化

• 批准号: 9457155
• 负责人: Adam T Whaley Connell
• 金额: --
• 依托单位: HARRY S. TRUMAN MEMORIAL VA HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9457155
• 关键词: Address; Adoption; Aldosterone; Atomic Force Microscopy; Attenuated; Automobile Driving; Bioavailable; Blood Vessels; Carbohydrates; Cells; Chemotaxis; Chronic Kidney Failure; Complications of Diabetes Mellitus; Consumption; Data; Development; Diet; Disease Progression; Doppler Ultrasound; Electron Microscopy; End stage renal failure; Endocrine system; Endothelial Cells; Enzymes; Epidemic; Extracellular Matrix; Fatty acid glycerol esters; Fibrosis; Flow Cytometry; Future; Histology; Hormones; Hypertension; Image; Immune system; Immunologic Markers; Individual; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Insulin Resistance; Interruption; Intervention; Kidney; Kidney Diseases; Knockout Mice; Lead; Link; Magnetic Resonance Imaging; Measurement; Measures; Mediating; Mineralocorticoid Receptor; Mineralocorticoids; Modeling; Molecular; Mus; Myocardial; NOS3 gene; Nitric Oxide; Nitric Oxide Donors; Obesity; Overnutrition; Oxidative Stress; Pathway interactions; Play; Population; Prevalence; Receptor Activation; Regulation; Renin-Angiotensin-Aldosterone System; Reporting; Resistance; Risk; Role; Sodium Chloride; Sucrose; System; Testing; Therapeutic; Tissues; Ultrasonography; Veterans; Work; adaptive immune response; arteriole; blood pressure regulation; chemokine receptor; crosslink; cytokine; design; disorder prevention; extracellular; high risk; immune activation; in vivo; inhibitor/antagonist; innovation; interstitial; intravital microscopy; light microscopy; macrophage; migration; monocyte; nutrition; obesity development; prevent; receptor expression; resistance mechanism; response; sugar; transglutaminase 2; western diet

Project Summary/Abstract Consumption of a western diet (WD) is a major contributor to the epidemic of obesity and associated insulin resistance, hypertension and kidney disease that afflict our Veteran population. Chronic kidney disease is increasing despite widespread use of renin-angiotensin-aldosterone system (RAAS) inhibition and aggressive blood pressure control. Higher circulating aldosterone levels and excess mineralocorticoid receptor (MR) activation, prevalent in obese, insulin resistant individuals, may be driving the increased risk for progression of kidney disease, often in the face of adequate blood pressure control. Tubulointerstitial fibrosis plays a major role in the development and progression of kidney disease. What little is known about MR activation in tubulointerstitial fibrosis has been extrapolated from vascular studies where the development of endothelial cell stiffness can be directly related to reductions in bioavailable nitric oxide (NO). Lowered NO levels activate transglutaminase 2 (TG2) known to promote the extracellular matrix remodeling, synthesis of fibrotic products and macrophage infiltration that lead to fibrosis. My pilot data indicate that consumption of a WD promotes MR activation resulting in lower endothelial nitric oxide synthase (eNOS) activity, cell stiffness, macrophage infiltration, a shift to an inflammatory M1/M2 polarization and vascular fibrosis. Preliminary data link activation of the endothelial cell MR and TG2 activation but the role of endothelial MR activation and its regulation of eNOS and TG2 in tubulointerstitial fibrosis remain to be determined. In this context, the Objectives have been designed to investigate the mechanisms by which over-nutrition promotes tubulointerstitial fibrosis through the endothelial aldosterone/MR system. We posit that the endothelial MR regulates bioavailable NO and TG2, leading to stiffness and immune system activation. We will use a high fat/sugar/salt diet (WD) known to activate the aldosterone/MR system and an endothelial-cell specific MR knockout mouse (ECMR-/-) model to more specifically implicate the endothelial MR in tubulointerstitial fibrosis. Fibrosis will be determined by light and electron microscopy, expression of fibrotic markers and magnetization transfer MRI. To address Objective 1, I will measure endothelial cell stiffness using atomic force microscopy, vascular reactivity using intravital microscopy, and resistance using ultrasound imaging. To address Objective 2, I will focus on monocyte chemotaxis and Mφ polarization through FACS and flow cytometry analysis of immune markers and M2 polarization along with other measures of inflammation, such as cytokine expression. Objective 3 has been designed to investigate the importance of MR-, NO-, and TG2-dependent actions on kidney fibrosis. I will employ a NO-donor and/or a TG2 inhibitor and measure oxidative, inflammatory, extracellular cross-linking enzymes and fibrotic pathways to gauge molecular responses to the interventions. These results will be correlated to the degree of tubulointerstitial fibrosis and endothelial cell stiffness (Objective 1) and monocyte chemotaxis and Mφ polarization (Objective 2). In summary, results from these studies will define a heretofore unknown link between activation of the endothelial cell MR and the contribution of this activation to the inflammatory response and endothelial cell stiffness and tubulointerstitial fibrosis seen in obesity (Objectives 1 and 2). Detailed exploration of the mechanisms of ECMR regulation of NO and TG2 (Objective 3) will allow identification of newer targets for the development of innovative therapeutic strategies to prevent/interrupt progression of kidney disease.

项目总结/摘要 西方饮食（WD）的消费是肥胖和相关胰岛素流行的主要原因 抵抗力，高血压和肾脏疾病折磨着我们的退伍军人。慢性肾脏疾病是 尽管广泛使用了肾素-血管紧张素-醛固酮系统（RAAS）抑制剂， 积极控制血压。较高的循环醛固酮水平和过量的盐皮质激素 胰岛素受体（MR）激活，在肥胖、胰岛素抵抗个体中普遍存在，可能导致以下风险增加： 肾脏疾病的进展，往往是在充分控制血压的情况下。肾小管间质纤维化 在肾脏疾病的发展和进展中起主要作用。关于MR所知甚少 已经从血管研究中推断出肾小管间质纤维化的激活， 内皮细胞硬度可与生物可利用的一氧化氮（NO）的减少直接相关。降低NO 水平激活转氨酶2（TG 2），已知促进细胞外基质重塑，合成 导致纤维化的纤维化产物和巨噬细胞浸润。我的试验数据表明， WD促进MR活化，导致较低的内皮一氧化氮合酶（eNOS）活性，细胞硬度， 巨噬细胞浸润、向炎性M1/M2极化的转变和血管纤维化。初步数据 连接激活内皮细胞MR和TG 2激活，但内皮细胞MR激活的作用及其 eNOS和TG 2在肾小管间质纤维化中的调节仍有待确定。 在这方面，制定这些目标的目的是调查营养过剩的机制， 通过内皮醛固酮/MR系统促进肾小管间质纤维化。我们认为， 内皮MR调节生物可利用的NO和TG 2，导致僵硬和免疫系统激活。我们将 使用高脂肪/糖/盐饮食（WD），已知可激活醛固酮/MR系统和内皮细胞 特异性MR敲除小鼠（ECMR-/-）模型，以更特异性地涉及内皮MR， 肾小管间质纤维化纤维化将通过光学和电子显微镜来确定，纤维化的表达将通过免疫组织化学来确定。 标记和磁化转移MRI。为了解决目标1，我将测量内皮细胞硬度， 使用原子力显微镜，血管反应性使用活体显微镜，和阻力使用超声波 显像为了解决目标2，我将通过流式细胞仪重点研究单核细胞趋化性和Mφ极化， 免疫标记物和M2极化的流式细胞术分析沿着其他炎症测量， 例如细胞因子表达。目的3探讨MR-、NO-、 TG 2依赖性对肾纤维化的作用。我将使用NO供体和/或TG 2抑制剂并测量 氧化、炎症、细胞外交联酶和纤维化途径来测量分子 对干预措施的回应。这些结果将与肾小管间质纤维化的程度相关， 内皮细胞硬度（目标1）和单核细胞趋化性和Mφ极化（目标2）。 总之，这些研究的结果将确定一个迄今为止未知的联系之间的激活， 内皮细胞MR以及这种激活对炎症反应和内皮细胞MR的贡献。 在肥胖症中观察到的僵硬和肾小管间质纤维化（目的1和2）。详细探索 NO和TG 2的ECMR调节机制（目标3）将允许识别新的靶点， 开发创新的治疗策略，以预防/中断肾脏疾病的进展。