Homocysteine and Angiotensin II in Renovascular Remodeling

同型半胱氨酸和血管紧张素 II 在肾血管重塑中的作用

• 批准号: 8441628
• 负责人: Utpal Sen
• 金额: $ 35.7万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8441628
• 关键词: 3-Mercaptopyruvate sulfurtransferase; Angiogenic Factor; Angiography; Angiostatins; Angiotensin II; Angiotensin Receptor; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Attenuated; Barium; Barium Sulfate; Basement membrane; Blood Pressure; Blood Vessels; Blood flow; C10; CCL2 gene; Clinical Data; Collagen; Collagen Type IV; Control Animal; Cystathionine; Disease; Endostatins; Enzyme-Linked Immunosorbent Assay; Enzymes; Equilibrium; Extracellular Matrix; Extracellular Matrix Proteins; Fibrosis; Folic Acid; Functional disorder; Gel; Gelatinase A; Genetic; Glomerular Capillary; Glomerular Filtration Rate; Goals; Health; High Pressure Liquid Chromatography; Histology; Homocysteine; Homocystine; Hyperhomocysteinemia; Hypertension; Individual; Inflammation; Inflammatory; Infusion procedures; Injury; Intercellular adhesion molecule 1; Kidney; Lasers; Lead; Link; Lyase; MTHFR gene; Matrix Metalloproteinases; Measures; Mediating; Messenger RNA; Metabolism; Methylenetetrahydrofolate reductase (NADPH); Mitochondria; Modeling; Morbidity - disease rate; Mus; NADP; Outcomes Research; Oxidases; Oxidative Stress; Pathogenesis; Patients; Perfusion; Plasma; Pump; Reactive Oxygen Species; Renal Blood Flow; Renal function; Renovascular Hypertension; Research; Reverse Transcriptase Polymerase Chain Reaction; Risk; Risk Factors; Roentgen Rays; Role; Sclerosis; Stress; Testing; Therapeutic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinases; Tissues; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factors; Western Blotting; base; bevacizumab; density; drinking water; glomerulosclerosis; in vivo; inhibitor/antagonist; kidney vascular structure; mortality; mouse model; oxidation; podocyte; protein expression; research study; soft tissue; systolic hypertension

DESCRIPTION (provided by applicant): Although it is not clear whether hyperhomocysteinemia (HHcy), an elevated plasma homocysteine (Hcy) level, causes hypertension, recent clinical data suggest an association between systolic hypertension and HHcy. Both angiotensin II (Ang II) and Hcy decrease blood flow; however, the role of Hcy in Ang II mediated decrease in blood flow and vascular density is unclear. Interestingly, preliminary studies of this proposal suggest that Ang II increases plasma Hcy level in mice. Treatment of Ang II animals with folic acid (FA) decreases plasma Hcy and mitigates hypertension. Based on our preliminary studies, in this proposal, we hypothesize that HHcy decreases blood flow in part by decreasing vascular density, increasing oxidative, pro-inflammatory, pro-fibrotic and anti-angiogenic factors in Ang II mediated renovascular remodeling. FA treatment through Hcy clearance mechanism reduces plasma Hcy level that mitigates renal remodeling. The hypothesis will be tested by following their specific aims: (1) To determine whether the Hcy contributes to Ang II hypertension, in part, by inducing oxidative stress (Nox2, gp47phox, Nox4 and mtROS), exacerbating renal inflammation by inducing MCP-1, MIP-2, ICAM-1, VCAM-1, and if FA ameliorates these changes; (2) To determine whether the Hcy instigates Ang II hypertension and renovascular fibrosis, in part, by inducing collagen IV, MMP-2, -9, -13; TIMP-1,-2, -3, -4; and if FA mitigates this renovascular fibrosis; and (3) To determine whether the Hcy promotes Ang II hypertension and decreases renovascular blood flow, in part, by unbalancing of angiogenic (VEGF) and anti-angiogenic factors (angiostatin and endostatin), and if FA increases vascular density and blood flow. WT (C57BL/6J) mouse, genetic mouse model of HHcy (CBS) and mouse deficient with angiotensin receptor I (AT1R-/-) will be used in this study. In WT and CBS mice, hypertension will be created by infusing Ang II (1000 ng/kg/min for 4 weeks) through alzet mini pump. Control animals will receive only vehicle. In a separate group of animals FA (0.015g/L, in drinking water) will be introduced after 2 weeks of Ang II infusion and will be continued until the end of experiments (total 2 weeks). Appropriate FA controls will be used. AT1R-/- mice will be treated with or without Hcy (1.8 g/L for 4 weeks) to determine whether the effect of HHcy is AT1R dependent. Ambulatory blood pressure will be measured by DSI radiotelemetry (model TA11PA- C10). Plasma Hcy will be measured by HPLC. Vascular density will be measured by in vivo soft-tissue Barium sulfate-contrast X-ray angiography and renal cortical blood flow by moorFLPI full-filled laser perfusion imager. Renal function will be determined by measuring glomerular filtration rate (GFR). Histological kidney sections will be used to detect ROS, collagen, mesangial widening and podocyte injury. Plasma MCP-1, MIP-2, VCAM- 1 and ICAM-1 will be measured by ELISA. Tissue collagen, MMPs, TIMPs, Nox2, p47phox, Nox4, ICAM-1, VCAM-1 protein expressions will be measured by Western blot and immunostaining. MMPs activities will be measured by in gel zymography, TIMPs activity by reverse zymography, and mRNA abundance by Q-PCR. In addition to confirming the preliminary studies, the scope of the research will be extended to understand the implications of FA treatment in Ang II-induced hypertension to modulate pro-inflammatory, pro-fibrotic and anti- angiogenic factors. The results of this study will increase our understanding of role of Hcy in Ang II hypertension and renovascular remodeling. Additionally, the research outcome will provide the missing information of HHcy as a potential risk factor of renovascular fibrosis, which exacerbates hypertension, and will lead to develop or modify current therapeutic strategies of renovascular disease.

描述（由申请人提供）：虽然尚不清楚高同型半胱氨酸血症（HHcy）（血浆同型半胱氨酸（Hcy）水平升高）是否会导致高血压，但最近的临床数据表明收缩期高血压与 HHcy 之间存在关联。血管紧张素 II (Ang II) 和 Hcy 都会减少血流量；然而，Hcy 在 Ang II 介导的血流量和血管密度减少中的作用尚不清楚。有趣的是，该提案的初步研究表明，Ang II 会增加小鼠血浆 Hcy 水平。用叶酸 (FA) 治疗 Ang II 动物可降低血浆 Hcy 并减轻高血压。根据我们的初步研究，在本提案中，我们假设 HHcy 部分通过降低血管密度、增加 Ang II 介导的肾血管重塑中的氧化、促炎、促纤维化和抗血管生成因子来减少血流量。通过 Hcy 清除机制进行 FA 治疗可降低血浆 Hcy 水平，从而减轻肾重构。该假设将通过以下具体目标进行检验：（1）确定 Hcy 是否部分通过诱导氧化应激（Nox2、gp47phox、Nox4 和 mtROS）而导致 Ang II 高血压，通过诱导 MCP-1、MIP-2、ICAM-1、VCAM-1 加剧肾脏炎症，以及 FA 是否改善这些变化； (2) 确定Hcy是否部分通过诱导IV型胶原、MMP-2、-9、-13而引发Ang II高血压和肾血管纤维化； TIMP-1、-2、-3、-4；如果 FA 可以减轻这种肾血管纤维化； (3) 确定 Hcy 是否部分通过血管生成 (VEGF) 和抗血管生成因子（血管抑制素和内皮抑素）失衡而促进 Ang II 高血压并减少肾血管血流量，以及 FA 是否会增加血管密度和血流量。本研究将使用 WT (C57BL/6J) 小鼠、HHcy 基因小鼠模型 (CBS) 和血管紧张素受体 I 缺陷型小鼠 (AT1R-/-)。在WT和CBS小鼠中，通过alzet微型泵输注Ang II（1000 ng/kg/min，持续4周）来产生高血压。对照动物将仅接受载体。在另一组动物中，在 Ang II 输注 2 周后将引入 FA（0.015g/L，在饮用水中），并将持续到实验结束（总共 2 周）。将使用适当的 FA 控制。 AT1R-/- 小鼠将接受或不接受 Hcy（1.8 g/L，持续 4 周）治疗，以确定 HHcy 的作用是否依赖于 AT1R。动态血压将通过 DSI 无线电遥测（型号 TA11PA-C10）测量。血浆 Hcy 将通过 HPLC 测量。通过体内软组织硫酸钡对比 X 射线血管造影测量血管密度，通过 moorFLPI 全填充激光灌注成像仪测量肾皮质血流量。通过测量肾小球滤过率（GFR）来确定肾功能。组织学肾脏切片将用于检测 ROS、胶原蛋白、系膜增宽和足细胞损伤。将通过ELISA测量血浆MCP-1、MIP-2、VCAM-1和ICAM-1。通过蛋白质印迹和免疫染色测量组织胶原、MMP、TIMP、Nox2、p47phox、Nox4、ICAM-1、VCAM-1蛋白表达。 MMP 活性将通过凝胶酶谱法测量，TIMP 活性通过反向酶谱法测量，mRNA 丰度通过 Q-PCR 测量。除了证实初步研究外，研究范围还将扩大，以了解 FA 治疗 Ang II 诱导的高血压对调节促炎、促纤维化和抗血管生成因子的影响。这项研究的结果将增加我们对 Hcy 在 Ang II 高血压和肾血管重塑中作用的理解。此外，该研究成果将提供HHcy作为肾血管纤维化的潜在危险因素的缺失信息，从而加剧高血压，并将导致开发或修改当前肾血管疾病的治疗策略。