Endothelial Cell Mineralocorticoid Receptor and Tubulointerstitial Fibrosis

项目摘要/摘要 吃西式饮食(Wd)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。慢性肾脏病是 尽管广泛使用肾素-血管紧张素-醛固酮系统(RAAS)抑制和 积极控制血压。循环中较高的醛固酮水平与过量的盐皮质激素 受体(MR)激活，在肥胖和胰岛素抵抗的个体中普遍存在，可能是导致高血压风险增加的原因。 肾脏疾病的进展，通常是在血压得到适当控制的情况下。肾小管间质纤维化 在肾脏疾病的发生和发展中起着重要作用。人们对他知之甚少。 肾小管间质纤维化的活化是从血管研究推断出来的 内皮细胞硬度与生物可利用的一氧化氮(NO)的减少直接相关。已降下编号 水平激活转谷氨酰胺酶2(TG2)，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。我的试点数据显示， WD促进MR激活，导致内皮型一氧化氮合酶(ENOS)活性降低，细胞刚性降低， 巨噬细胞浸润，转变为炎症性M1/M2极化和血管纤维化。初步数据 内皮细胞MR和TG2激活的环节激活，但内皮细胞MR的激活及其作用 ENOS和TG2在肾小管间质纤维化中的调控作用尚不清楚。 在这种背景下，这些目标旨在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。我们假设 内皮MR调节生物可利用的NO和TG2，导致僵硬和免疫系统激活。我们会 使用已知的高脂肪/糖/盐饮食(WD)来激活醛固酮/MR系统和内皮细胞 特异性MR基因敲除小鼠(eCMR-/-)模型更特异地牵涉内皮细胞MR 肾小管间质纤维化。纤维化程度将通过光镜和电子显微镜确定，纤维化的表达 标记物和磁化转移磁共振。为了达到目标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，阻力使用超声波 成像。为了解决目标2，我将集中在单核细胞趋化和Mφ极化通过流式细胞术和 流式细胞术分析免疫标志物和M2极化以及其他炎症指标， 如细胞因子的表达。目标3旨在调查MR-、NO-和 TG2在肾脏纤维化中的依赖作用。我将使用非供体和/或TG2抑制剂并采取措施 氧化、炎症、细胞外交联酶和纤维化途径以测量分子 对干预措施的反应。这些结果将与肾小管间质纤维化的程度和 内皮细胞僵硬(目标1)和单核细胞趋化和Mφ极化(目标2)。 综上所述，这些研究的结果将定义一种迄今未知的联系 内皮细胞MR及其在炎症反应和内皮细胞中的作用 肥胖患者的僵硬和肾小管间质纤维化(目标1和2)。详细探讨了 ECMR调节NO和TG2的机制(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。

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.