Molecular mechanisms of Target organ damage in youth with primary hypertension [smallRNA-Seq 2]

High blood pressure or Primary Hypertension is associated with target organ damage (TOD) in both adults and youth, where TOD predicts the adverse cardiovscular (CV) events. The mechanisms involved in blood pressure mediated TOD are limitedly understood in onset of TOD. To understand this, first using RNA sequencing, we identified candidate differentially expressed genes in circulating PBMCs of study participants with and without hypertension and elevated left ventrivlar mass index . Next, through miRNA and whole genome bisulphite sequencing of circulating PBMCs we determined the epigenetic regulators of TOD. Also, using LCMS, serum proteomics, the key circulatory protein involved in advancing CV- TOD events was identified. In conclusion, multiomics approach was utilized to define the circulatory regulators involved in BP mediated CV-TOD. Overall design: The design and methods of the SHIP AHOY (Study of High Blood Pressure in Pediatrics, Adult Hypertension Onset in Youth) project have been discussed elsewhere (Mendizabal B, et al., 2018, Hypertension . 2018 Sep;72(3):625-631.). Briefly, in the current cross-sectional study, youth aged 11 to less than 19 years with BP levels (N=397) across the BP distribution, were enrolled at five clinical sites (Cincinnati, Houston, Philadelphia, Rochester, and Seattle) from 2015 to 2017 from pediatric specialty clinics (four nephrology clinics, and one joint nephrology and cardiology clinic). Efforts were made to achieve body mass index (BMI) balance across BP groups by periodically checking differences in BMI percentile among groups and actively recruiting normotensive obese controls. Out of 397 subjects, 132 adolescents of both sexes and all races with normal (Cut off: SBP <80th%), and high- systolic blood pressure (Cut off: SBP >90th%), and then stratified by with or without TOD, like Low LVMI (Left ventricular mass index) (Cut off: <38.6 gm/m2.7), and High LVMI (Cut off: >38.6 gm/m2.7), were selected in the present study cohort. Urine and Blood samples (for PBNCs and serum) were collected to investigate genetic, epigenetic, and proteomics changes that influence the development of hypertensive TOD in youth. We excluded youth with symptomatic severe hypertension, on antihypertensive or lipid-lowering medication in the past 6 months, with diabetes mellitus (type 1 or 2), kidney disease, or other chronic medical conditions. All study participants and their parents provided written informed consent and assent according to local investigational review board requirements.

高血压（Primary Hypertension，原发性高血压）与成人及青少年的靶器官损害（Target Organ Damage，TOD）密切相关，而靶器官损害可预测不良心血管（Cardiovascular，CV）事件的发生。目前对于血压介导靶器官损害的发病机制，学界的认知仍较为有限。
为阐明该机制，本研究首先通过RNA测序（RNA sequencing），在伴或不伴高血压及左心室质量指数（Left Ventricular Mass Index，LVMI）升高的研究受试者的循环外周血单个核细胞（Peripheral Blood Mononuclear Cells，PBMCs）中筛选出差异表达的候选基因。随后，通过对循环PBMCs进行miRNA测序及全基因组亚硫酸氢盐测序，明确了靶器官损害的表观遗传调控因子。此外，借助液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry，LCMS）技术开展血清蛋白质组学分析，鉴定出了参与进展性心血管靶器官损害事件的关键循环蛋白。综上，本研究采用多组学方法，明确了血压介导心血管靶器官损害过程中的循环调控因子。
总体研究设计：SHIP AHOY（Study of High Blood Pressure in Pediatrics, Adult Hypertension Onset in Youth，儿科高血压研究、青少年成人高血压发病研究）项目的设计与方法已在既往研究中详述（Mendizabal B等，2018，Hypertension. 2018 Sep;72(3):625-631.）。简言之，本项横断面研究于2015年至2017年间，从5个临床中心（辛辛那提、休斯顿、费城、罗切斯特及西雅图）的儿科专科门诊（4家肾病专科门诊及1家肾病与心血管联合专科门诊）招募了397名年龄介于11岁至不足19岁的青少年，覆盖全血压分布区间。研究通过定期监测各组间体质量指数（Body Mass Index，BMI）百分位数差异，并主动招募血压正常的肥胖对照人群，力求实现各血压组间的BMI均衡。在397名受试者中，本研究队列最终纳入132名不同性别、不同种族的青少年，其收缩压分别符合正常截断值（Cutoff：收缩压<80th百分位）及高收缩压截断值（Cutoff：收缩压>90th百分位），并进一步按是否存在靶器官损害分层，即低LVMI组（截断值：<38.6 gm/m².7）与高LVMI组（截断值：>38.6 gm/m².7）。研究收集了尿液及血液样本（用于分离PBMCs及血清），以探究影响青少年高血压性靶器官损害发生发展的遗传、表观遗传及蛋白质组学变化。本研究排除了近6个月内接受降压或调脂药物治疗、患有症状性重度高血压、糖尿病（1型或2型）、肾脏疾病或其他慢性疾病的青少年。所有研究受试者及其监护人均已按照当地伦理审查委员会的要求签署了书面知情同意书，受试者本人亦签署了受试同意书。