Multiphoton Imaging of the Juxtaglomerular Apparatus

说明(申请人提供)：肾素-血管紧张素系统(RAS)是维持体液和电解质平衡和维持血压的最重要的调节机制之一。肾小球旁结构(JGA)是RAS的关键解剖部位，肾素是RAS的限速步骤，肾素及其前体在这里以高度调控的方式合成和释放。最近RAS研究的一个令人兴奋的主题是发现了(PRO)肾素受体[(P)RR]，挑战了现有的简单的RAS模型(血管紧张素原、肾素、转换酶、血管紧张素(Ang)肽和受体)。(Pro)受体与肾素的结合不仅导致酶的非蛋白水解性激活和Angi的产生，而且还触发Angii非依赖性的细胞内信号转导。自从血浆前肾素升高是公认的心血管疾病和糖尿病微血管并发症的预测因子以来，人们很早就怀疑前肾素的功能作用。原肾素及其受体已成为RAS的新成分，成为疾病治疗的热点和靶点。在过去的赠款周期中，我们成功地研究了JGA的重要功能，肾小管球反馈(TGF)和肾素释放机制及其重要组成部分，包括ATP和连接蛋白介导的钙波。此外，我们还开创了一种独特的多光子成像方法，可以直接和定量地显示完整的肾脏，监测体内肾功能的基本参数，包括(PRO)肾素含量、释放和组织活动。我们的总体假设是，(P)RR定位于致密斑细胞的基底膜，它的下游信号是MD和JGA功能的重要和新的调节器。更准确地说，我们假设(P)RR构成了一个短环正反馈，刺激相邻JG细胞的肾素释放，其中包括激活MD MAP激酶和经典的PGE2合成和释放机制。我们还假设，另一种新的但抑制JGA的机制，连接蛋白和ATP介导的血管钙信号有助于平衡肾素的合成和释放。目标1将利用分子技术测试致密斑细胞中(P)RR的表达、调节和信号转导。目的2通过对体内完整肾脏或体外新鲜解剖、微灌流的JGA标本以及一些转基因方法和动物模型的多光子荧光成像，确定致密斑区(P)RR在JGA功能中的作用。目的3将利用多光子成像技术确定Cx45/ATP介导的肾素释放抑制的机制和重要性。这些原创性和新颖性的研究有望提供临床上重要的信息，可用于开发新药和治疗方法，以更好地治疗心血管和肾脏疾病。 公共卫生相关性 高血压是心血管疾病的主要危险因素，影响大约25%的人口。肾脏是维持正常血压和高血压发病的中心，因为它在调节盐分和水分平衡方面起着主导作用。在这项提案中，我们将使用最先进的成像技术直接可视化肾脏中的新机制，这将使我们能够发现肾脏如何实时运行以控制血量和血压。

DESCRIPTION (provided by applicant): The renin-angiotensin system (RAS) is one of the most important regulatory mechanisms of body fluid and electrolyte homeostasis and blood pressure maintenance. The juxtaglomerular apparatus (JGA) is a key anatomical site of RAS where renin, the rate-limiting step of RAS, and its precursor prorenin are synthesized and released in a highly regulated fashion. One of the recent exciting topics in RAS research is the discovery of the (pro)renin receptor [(P)RR] challenging the existing simplistic RAS model (angiotensinogen, renin, converting enzyme, angiotensin (Ang) peptides and receptors). (Pro)renin binding by the receptor not only causes non- proteolytic activation of the enzyme and generation of AngI, but also triggers AngII-independent intracellular signaling. A functional role for prorenin was suspected long ago since elevated plasma prorenin is a well-recognized predictor of microvascular complications in cardiovascular disease and diabetes. Prorenin and its receptor have become new elements of RAS, hot players and therapeutic targets in disease. During the past grant cycle we successfully characterized important JGA functions, the tubuloglomerular feedback (TGF) and renin release mechanisms and their important constituents including the ATP and connexin-mediated calcium wave. Also, we pioneered a unique multi-photon imaging approach to directly and quantitatively visualize the intact kidney, monitor the basic parameters of kidney function in vivo including (pro)renin content, release and tissue activity. Our overall hypothesis is that the (P)RR, localized in the basolateral membrane of macula densa (MD) cells, and its downstream signaling is an important and novel modulator of MD and JGA functions. More precisely, we hypothesize that the (P)RR constitutes a short-loop positive feedback stimulating renin release from adjacent JG cells which includes activation of MD MAP kinases and the classic PGE2 synthetic and release machinery. We also hypothesize that another novel, but inhibitory JGA mechanism, a connexin and ATP-mediated vascular calcium signal helps to balance renin synthesis and release. Aim 1 will test for the expression, regulation, and signaling of the (P)RR in macula densa cells using molecular techniques. Aim 2 will establish the role of macula densa (P)RR in JGA function using multi-photon fluorescence imaging of the intact kidney in vivo or freshly dissected, microperfused JGA preparations in vitro and a number of transgenic approaches and animal models. Aim 3 will identify the mechanism and importance of Cx45/ATP-mediated inhibition of renin release using multi-photon imaging. These original and novel studies are expected to provide clinically important information that can be used to develop new drugs and therapeutic approaches for the better treatment of cardiovascular and kidney diseases. PUBLIC HEALTH RELEVANCE Hypertension is a major risk factor for cardiovascular diseases, affecting about 25% of the population. The kidney is central to maintenance of normal blood pressure and to the pathogenesis of hypertension because of its dominant role in the regulation of salt and water balance. In this proposal we will directly visualize novel mechanisms in the kidney using a state-of- the-art imaging technology which will allow us to discover how the kidney operates in real time to control blood volume and pressure.