Gene expression profiling of hypertensive and hypotensive mice

Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive "blood pressure high" (BPH), normotensive "blood pressure normal" (BPN), and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific gene signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. In order to distinguish causal genes from responsive genes in the kidney gene signature we integrated phenotype associated genes into Genetic Bayesian networks, identifying several novel candidate genes causal for hypertension. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension. Animals: BPH/2J, BPL/1J and BPN/3J (142 ± 5 mmHg, 69 ± 1.7 mmHg, 94 ± 6 mmHg, SBP respectively) male mice (Jackson Laboratory) were maintained on a 12:12-h light-dark cycle and fed with standard chow ad libitum in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were in conformance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Tissue collection: Mice (12 weeks old) were euthanized with CO2. Liver, heart, and kidney were flash frozen in liquid nitrogen. Aorta from the aortic root to the left renal artery was rinsed with PBS, immersed in 300ul of RNeasy Lysis buffer (Qiagen), and flash frozen in liquid nitrogen. RNA preparation: Tissues (~100mg, 5 mice per strain) were collected independently and homogenized in 2ml of Trizol (Invitrogen). After extraction with 0.4 ml of chloroform, RNA was extracted with SV Total RNA extraction kit (Promega) followed by DNase I treatment and purification using the RNeasy Kit (Qiagen). RNA was assayed for quality (Agilent Bioanalyzer) and yield (Ribogreen). Kidney, heart and liver RNA was amplified and labeled using a custom automated version of the RT/IVT protocol and reagents provided by Affymetrix. Aorta RNA was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol (NuGEN). Hybridization, labeling and scanning were according to Affymetrix. All samples were processed independently. Microarray analysis: Merck/Affymetrix mouse 1.0 custom arrays monitoring 38384 individual transcripts (25846 Entrez genes) were used. Raw intensity was normalized using the RMA algorithm.高血压是一种可引发严重心血管与肾脏并发症的疾病，全球罹患人数近十亿。目前可供药物干预的经过验证基因靶点寥寥无几，因此亟需识别调控血压且包含新型治疗靶点的全新生物学通路。遗传型高血压的"血压偏高（BPH）"、正常血压的"血压正常（BPN）"以及低血压的"血压偏低（BPL）"近交系小鼠品系，是研究可代表上述生物学通路的基因表达模式差异的理想模型体系。我们对BPH、BPN及BPL小鼠的肝脏、心脏、肾脏与主动脉进行了基因表达谱分析，并明确了观测到的器官特异性基因特征中富集的生物学过程。最终，我们鉴定出多条与血压表型相关的生物学通路，这些通路可作为高血压候选致病基因的来源。为在肾脏基因特征中区分致病基因与应答基因，我们将表型相关基因整合至遗传贝叶斯网络（Genetic Bayesian networks）中，鉴定出数个全新的高血压候选致病基因。整合基因表达谱与遗传网络的数据，是鉴定高血压药物治疗新型潜在靶点的有效策略。 实验动物：选取来自杰克逊实验室（Jackson Laboratory）的BPH/2J、BPL/1J及BPN/3J品系雄性小鼠，其收缩压（SBP）分别为142±5 mmHg、69±1.7 mmHg与94±6 mmHg。所有小鼠在12:12小时明暗循环环境中饲养，自由摄食标准啮齿类饲料，饲养设施通过国际实验动物评估与认证委员会（Association for Assessment and Accreditation of Laboratory Animal Care, AAALAC）认证。所有实验操作均符合美国国家研究委员会《实验动物护理与使用指南》的要求。 组织采集：选取12周龄的小鼠，以二氧化碳实施安乐死。迅速将肝脏、心脏与肾脏置于液氮中快速速冻。从主动脉根部至左肾动脉的主动脉段用磷酸盐缓冲液（Phosphate Buffered Saline, PBS）冲洗后，浸入300 μl RNeasy裂解缓冲液（Qiagen）中，随后置于液氮中快速速冻。 RNA制备：每个品系取5只小鼠的约100 mg组织，独立进行样本采集与处理。将组织在2 ml Trizol试剂（Invitrogen）中匀浆，经0.4 ml氯仿萃取后，使用SV总RNA提取试剂盒（Promega）提取RNA，随后进行脱氧核糖核酸酶I（DNase I）处理，并通过RNeasy试剂盒（Qiagen）纯化RNA。采用安捷伦生物分析仪（Agilent Bioanalyzer）检测RNA质量，以Ribogreen法测定RNA产量。肾脏、心脏与肝脏的RNA采用定制自动化版本的反转录/体外转录（Reverse Transcription/In Vitro Transcription, RT/IVT）方案及Affymetrix提供的试剂进行扩增与标记。主动脉RNA则采用定制自动化版本的NuGEN Ovation WB方案（NuGEN）进行扩增与标记。杂交、标记与扫描步骤均遵循Affymetrix的标准流程。所有样本均独立完成处理。 微阵列分析：使用默克/Affymetrix小鼠1.0定制芯片，该芯片可检测38384个独立转录本（对应25846个Entrez基因）。原始信号强度采用稳健多阵列平均算法（Robust Multi-array Average, RMA）进行归一化处理。