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）：尽管尚不清楚高脑结晶质血症（HHCY），血浆同型半胱氨酸（HCY）水平升高会导致高血压，但最近的临床数据表明收缩压高血压与HHCY之间存在关联。血管紧张素II（ANG II）和HCY都降低了血液流动。然而，HCY在ANG II中介导的血流降低和血管密度的降低尚不清楚。有趣的是，该提案的初步研究表明，ANG II增加了小鼠血浆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。基于我们的初步研究，在该提案中，我们假设HHCY通过降低血管密度，降低血管的流动，从而降低血管，从而增加氧化，促炎，促纤维化，促纤维化和抗血管生成因子中的ANG II介导的肾血管介导的肾血管结构重塑。通过HCY清除机制进行FA处理可降低血浆HCY水平，从而减轻肾脏重塑。该假设将通过遵循其具体目的来检验：（1）确定HCY是否通过诱导氧化应激（NOX2，GP47PHOX，NOX4和MTROS）来确定ANG II高血压是否有助于加剧肾脏炎症，从而促进MCP-1 ，MIP-2，ICAM-1，VCAM-1，如果FA可以改善这些变化； （2）确定HCY是否通过诱导胶原蛋白IV，MMP -2，-9，-13来激发ANG II高血压和肾血管纤维化； TIMP -1，-2，-3，-4;如果FA减轻这种肾脏纤维化； （3）确定HCY是否会通过不平衡血管生成（VEGF）和抗血管生成因子（Angiostatin和therostatin）以及FA增加血管密度和血液流量来促进ANG II高血压并降低肾血管血流。本研究将使用WT（C57BL/6J）小鼠，HHCY（CBS）的遗传小鼠模型和血管紧张素受体I（AT1R - / - ）缺乏的小鼠。在WT和CBS小鼠中，将通过Ang II（1000 ng/kg/min 4周）通过Alzet Mini Pump来​​产生高血压。控制动物只会接收车辆。在ANG II输注2周后，将引入一组单独的动物（0.015g/L，在饮用水中），并将继续进行直至实验结束（总共2周）。将使用适当的FA控件。 AT1R - / - 小鼠将接受或不带有HCY（1.8 g/L 4周）的治疗，以确定HHCY的效果是否取决于AT1R。卧床血压将通过DSI radiotelemetry（型号TA11PA-C10）进行测量。血浆HCY将通过HPLC测量。血管密度将通过体内软组织硫酸钡对比度X射线血管造影和Moorflpi全填充激光灌注成像仪进行测量。肾功能将通过测量肾小球滤过率（GFR）来确定。组织学肾脏切片将用于检测ROS，胶原蛋白，肾小球延伸和足细胞损伤。等离子体MCP-1，MIP-2，VCAM-1和ICAM-1将由ELISA测量。组织胶原蛋白，MMP，TIMP，NOX2，P47PHOX，NOX4，ICAM-1，VCAM-1蛋白表达将通过蛋白质印迹和免疫染色来测量。 MMP的活性将通过凝胶Zymography，TIMPS通过反向Zymography和Q-PCR的mRNA丰度来测量。除了确认初步研究外，还将扩展该研究的范围，以了解FA治疗在ANG II诱导的高血压中对调节促炎，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的作用的理解。此外，研究结果将提供HHCY缺失的信息，作为加剧高血压的肾血管纤维化的潜在危险因素，并将导致发展或修改当前的肾血管疾病治疗策略。