Multiphoton imaging of the juxtaglomerular apparatus

描述（由申请人提供）：肾小球体（JGA）是肾素-血管紧张素系统的主要结构组成部分，是肾脏水盐保存和血压维持的最重要调节部位之一。肾小球滤液形成和肾血流动力学调节的过程，包括致密斑（MD）和肾素释放的肾小管肾小球反馈（TGF）机制，涉及许多不同类型的JGA细胞的复杂相互作用。这两个主要的JGA功能的机制，TGF和肾素释放的MD控制，似乎是复杂的，并在几个领域尚未解决。最近，我们建立了利用多光子成像的新实验模型（Peti-Peterdi等人，Two-photon excitation fluorescence imaging of the living approximataglomerular apparatus. Am J Physiol Renal Physiol 283：F197-F201，2002）来可视化响应于小管流体组成的变化的活JGA的形态变化。这种方法有几个优点，比以前的方法，因为复杂的机制，在一个单一的孤立的JGA，可以在实时检查，具有高的时间和空间分辨率。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，我们进行了几个新的观察，包括管状NaCl/渗透压依赖性MD细胞肿胀，肾小球收缩，并确定了一个括约肌的终端，肾小球内部分的传入小动脉。此外，令我们非常惊讶的是，这些反应被阻止的抑制MD顶端Na：H交换NHE 2亚型，但不是呋塞米敏感的Na：2CI：K协同转运。此外，在NHE 2敲除小鼠中，肾素表达大大增加。这项新技术将用于我们的研究中，功能和形态分析离子转运蛋白，以及在JGA中构成TGF和肾素释放机制的各种细胞类型中的细胞内和细胞间信号传导机制。这些研究应产生临床上重要的信息，因为TGF和JGA相关的肾素-血管紧张素系统是肾血流动力学的主要调节剂，并参与控制体液和电解质平衡和血压调节。

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.