Renal Dopamine and Angiotensin II Receptor Function in Age-Related Hypertension

描述(申请人提供)：肾多巴胺D1和血管紧张素II AT1受体通过调节近端肾小管钠离子转运体维持钠稳态和血压。最近，我们报道了老年(21月龄)Fischer 344 X Brown挪威(FBN)大鼠AT1的过度表达和D1受体功能的降低与氧化应激和高血压的增加有关，提示氧化应激在这一现象中起作用。我们的初步研究表明，这些变化与老年大鼠近端小管AT1受体mRNAs增加、D1受体mRNAs减少以及氧化还原敏感转录因子NFkB和SP3核水平升高有关。在细胞培养中，NFkB表达载体能增加AT1受体的mRNA和蛋白表达，而SP3表达载体则能降低D1受体的mRNA和蛋白表达。基于这些发现，我们建议检验“氧化应激下转录因子NFkB和Sp3的激活增加AT1受体功能(NFkB)和抑制D1受体功能(Sp3)从而导致老年大鼠高血压。补充抗氧化剂可以减少老年大鼠的氧化应激，使NFkB和Sp3活性正常化，恢复AT1和D1受体功能，并降低老年大鼠的血压”。将采取两个具体目标来检验这一假设。目的1研究氧化应激在清醒老年大鼠肾脏血管紧张素转换酶1(AT1)过度表达和D1受体功能减弱导致高血压中的作用。目的2研究NFkB和SP3通过表观遗传过程分别激活AT1和抑制D1受体转录，从而导致氧化应激时AT1和D1受体功能改变和血压升高的机制。氧化应激在不利的AT1和D1受体功能和血压中的作用将通过使用抗氧化剂降低老年大鼠的氧化应激来研究。NFkB和SP3在这一现象中的作用将通过将各自的shRNA慢病毒颗粒输送到大鼠肾脏来耗尽这些蛋白，以及通过使用各自的表达载体在细胞培养中过度表达来研究。这些研究应该能更好地理解氧化应激引起高血压的机制，从而导致对高血压的靶向治疗。

DESCRIPTION (provided by applicant): Renal dopamine D1 and angiotensin II AT1 receptors by regulating proximal tubular sodium transporters maintain sodium homeostasis and blood pressure. Recently we reported that exaggerated AT1 and reduced D1 receptor function in old (21-month) Fischer 344 X Brown Norway (FBN) rats were associated with increased oxidative stress and high blood pressure suggesting role of oxidative stress in this phenomenon. Our preliminary studies show that these changes are associated with increased AT1 and decreased D1 receptor mRNAs as well as higher nuclear levels of redox-sensitive transcription factors NFkB and Sp3 in proximal tubules of old rats. And, in cell culture NFkB expression plasmid increased AT1 receptor mRNA and protein while Sp3 expression plasmid decreased D1 receptor mRNA and proteins. Based upon these findings we propose to test the central hypothesis that "Activation of transcription factors NFkB and Sp3 upon oxidative stress increases AT1 receptor function (NFkB) and suppresses D1 receptor function (Sp3) thus contributing to high blood pressure in old rats. Antioxidant supplementation reduces oxidative stress, normalizes NFkB and Sp3 activity, restores AT1 and D1 receptor function, and lowers blood pressure in old rats". Two specific aims will be undertaken to test the hypothesis. Aim 1 will identify causative role of oxidative stress in exaggerated renal AT1 and diminished D1 receptor function contributing to high blood pressure as measured in conscious old rats. Aim 2 will study the mechanism that NFkB and Sp3 by engaging epigenetic process activates AT1 while represses D1 receptor transcription, respectively, contributing to altered AT1 and D1 receptor function and increase in blood pressure during oxidative stress. The causative role of oxidative stress in adverse AT1 and D1 receptor function and blood pressure will be studied by reducing oxidative stress in old rats with antioxidant. The role of NFkB and Sp3 in this phenomenon will be studied by depleting these proteins with respective shRNA lentiviral particle delivery to rat kidneys and by their over-expression using respective expression vector in cell cultures. These studies should provide a better understanding of the mechanism by which oxidative stress causes hypertension which would subsequently lead to targeted therapy for hypertension.