Multiphoton imaging of the juxtaglomerular apparatus

描述（由申请人提供）：肾小球旁器（JGA）是肾素-血管紧张素系统的主要结构组成部分，是肾脏盐、水保护和血压维持的重要调控位点之一。肾小球滤液形成和肾血流动力学调节的过程，包括来自黄斑致密（MD）的小管肾小球反馈（TGF）机制和肾素释放，涉及多种不同类型JGA细胞的复杂相互作用。JGA的两大功能，即MD对TGF和肾素释放的控制，其机制似乎很复杂，在一些领域尚未得到解决。最近，我们建立了一个新的实验模型，利用多光子成像（petii - peterdi等）。活体肾小球旁器官的双光子激发荧光成像。[J] .肾生理学报，2003,19(1):1 - 2,2002)。与早期的方法相比，这种方法有几个优点，因为在单个孤立的JGA中，可以实时检查复杂的机制，具有高时间和空间分辨率。这可以在单个细胞水平上进行，并且不受系统影响。在初步实验中，我们进行了一些新的观察，包括小管NaCI/渗透压依赖的MD细胞肿胀，肾小球收缩，并在传入小动脉的末端，肾小球内部分确定了括约肌。此外，令我们惊讶的是，这些反应被MD顶端Na:H交换器NHE2异构体的抑制所阻断，而不是速尿敏感的Na:2CI:K共转运体。此外，在NHE2敲除小鼠中，肾素的表达也大大增加。这项新技术将在我们的研究中用于功能和形态学分析JGA中各种细胞类型的离子转运体，以及构成TGF和肾素释放机制的细胞内和细胞间信号机制。由于TGF和jga相关的肾素-血管紧张素系统是肾脏血流动力学的主要调节因子，参与控制体液电解质平衡和血压调节，这些研究应能提供重要的临床信息。

DESCRIPTION (provided by applicant): The juxtaglomerular apparatus (JGA) represents a major structural component of the renin-angiotensin system, and is one of the most important regulatory sites of renal salt & water conservation and blood pressure maintenance. The process of glomerular filtrate formation and regulation of renal hemodynamics, including the tubuloglomerular feedback (TGF) mechanism from the macula densa (MD) and renin release, involves the complex interaction of a number of different cell types of the JGA. The mechanism of these two major JGA functions, the MD control of TGF and renin release, appear to be complex and are unresolved in several areas. Very recently, we established a new experimental model that utilizes multiphoton imaging (Peti-Peterdi et al. Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus. Am J Physiol Renal Physiol 283: F197-F201, 2002) to visualize morphological changes of the living JGA in response to variations in tubular fluid composition. This approach has several advantages over earlier methods, since complex mechanisms, in a single isolated JGA, can be examined in real-time, with high temporal and spatial resolution. This can be performed on the individual cell level, and is free of systemic influences. In preliminary experiments we made several novel observations, including tubular NaCI/osmolality dependent MD cell swelling, glomerular shrinkage and identified a sphincter in the terminal, intraglomerular part of the afferent arteriole. Also, to our great surprise, these responses were blocked by the inhibition of the MD apical Na:H exchanger NHE2 isoform, but not the furosemide sensitive Na:2CI:K cotransporter. In addition, renin expression is greatly increased in NHE2 knockout mice. This new technology will be used in our studies to functionally and morphologically analyze ionic transporters, and intra-, and intercellular signaling mechanisms in various cell types in the JGA that constitute the TGF and renin release mechanisms. These studies should yield clinically important information since TGF and the JGA-associated renin-angiotensin system are major regulators of renal hemodynamics and participate in control of body fluid and electrolyte balance and blood pressure regulation.