Multiphoton Imaging of the Juxtaglomerular Apparatus

描述（由申请人提供）：肾素-血管紧张素系统（RAS）是体液和电解质稳态以及血压维持的最重要调节机制之一。肾小球体（JGA）是RAS的关键解剖部位，其中RAS的限速步骤肾素及其前体原肾素以高度调节的方式合成和释放。RAS研究中最近令人兴奋的主题之一是（原）肾素受体[（P）RR]的发现挑战了现有的简单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）肾素与受体的结合不仅引起酶的非蛋白水解活化和AngI的产生，而且触发AngII非依赖性细胞内信号传导。很久以前就怀疑前肾素的功能作用，因为血浆前肾素升高是心血管疾病和糖尿病微血管并发症的公认预测因子。肾素原及其受体已成为RAS的新元件、疾病的热点参与者和治疗靶点。在过去的资助周期中，我们成功地表征了重要的JGA功能，肾小管肾小球反馈（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，我们开创了一种独特的多光子成像方法，可以直接定量地可视化完整的肾脏，监测体内肾功能的基本参数，包括（原）肾素含量，释放和组织活性。我们的总体假设是，定位于致密斑（MD）细胞基底外侧膜的（P）RR及其下游信号传导是MD和JGA功能的重要且新颖的调节剂。更准确地说，我们假设（P）RR构成了一个短环正反馈刺激从相邻的JG细胞，其中包括激活MD MAP激酶和经典的PGE 2合成和释放机制的肾素释放。我们还假设，另一种新的，但抑制JGA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。目的1利用分子生物学技术检测（P）RR在致密斑细胞中的表达、调节和信号传导。目的2将建立致密斑（P）RR在JGA功能中的作用，使用多光子荧光成像的完整肾脏在体内或新鲜解剖，微灌注JGA制剂在体外和一些转基因方法和动物模型。目的3：利用多光子成像技术研究Cx45/ATP介导的肾素释放抑制作用的机制和意义。这些原创和新颖的研究有望提供临床上重要的信息，可用于开发新的药物和治疗方法，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，影响约25%的人口。肾是维持正常血压和高血压发病机制的中心，因为它在调节盐和水平衡中起主导作用。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在真实的时间内控制血容量和血压。

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.