Multiphoton Imaging of the Juxtaglomerular Apparatus

描述（申请人提供）：肾素-血管紧张素系统（RAS）是体液和电解质稳态和血压维持最重要的调节机制之一。肾小球旁器（JGA）是RAS的一个关键解剖部位，在这里，RAS的限速步骤肾素及其前体prorenin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话题是（pro）肾素受体[(P)RR]的发现，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）肾素与受体结合不仅会引起酶的非蛋白水解激活和AngI的生成，还会触发与AngI无关的细胞内信号传导。由于血浆prorentin升高是心血管疾病和糖尿病微血管并发症的一个公认的预测因子，因此早前就怀疑prorentin的功能作用。prorentin及其受体已成为RAS的新元件、热点分子和疾病治疗靶点。在过去的拨款周期中，我们成功地描述了JGA的重要功能、小管肾小球反馈（TGF）和肾素释放机制及其重要成分，包括ATP和连接素介导的钙波。此外，我们还开创了一种独特的多光子成像方法，可以直接定量地观察完整的肾脏，监测体内肾脏功能的基本参数，包括肾素含量、释放和组织活性。我们的总体假设是，定位于黄斑致密（MD）细胞基底外侧膜的(P)RR及其下游信号是MD和JGA功能的重要和新的调节剂。更准确地说，我们假设(P)RR构成了一个短环正反馈，刺激邻近JG细胞释放肾素，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，但抑制性的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将使用分子技术检测(P)RR在黄斑致密细胞中的表达、调控和信号传导。目的2将利用活体或新鲜解剖的完整肾脏的多光子荧光成像、体外微灌注的JGA制剂以及一些转基因方法和动物模型，建立黄斑致密(P)RR在JGA功能中的作用。目的3将利用多光子成像确定Cx45/ atp介导的肾素释放抑制的机制和重要性。这些原创和新颖的研究有望提供临床重要信息，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。

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.