ROLES OF THE PROXIMAL TUBLE RENIN ANGIOTENSIN SYSTEM

各种生理学研究提供了令人信服的证据 血管紧张素II(Ang II)在肾脏近端有重要作用 曲折的小管。生化和分子生物学技术已经 允许鉴定肾素、血管紧张素原(Ang-n)，以及 肾脏内的血管紧张素转换酶(ACE)蛋白和mRNA。 多条证据表明，肾内的一个主要部位 肾素血管紧张素系统(RAS)是近端小管(PT)。在 PT、Ang-n和ACE是合成的，而肾素更有可能是合成的 重新吸收。因为在PT内也有血管紧张素受体 内源性局部产生Ang II，体内有完整的PT。 此外，有说服力的数据支持一个完整的RAS的概念 近端小管内在各种肾小管中起着重要作用 功能包括钠和水的重吸收，蛋白质加工， 或许，还有细胞肥大。 本项目旨在研究细胞的调节和功能。 近曲小管肾素血管紧张素系统利用近曲小管细胞的实验研究 文化。许多信息支持这样一种概念，即近端 肾小管RAS具有自分泌、旁分泌和内分泌功能。 现有的已建立的类似PT的细胞系，如OK细胞，并不是真正的 类似于体内的PT细胞；原代培养的PT细胞已经证明 难以生长和传代，而且去分化速度快。 因此，开发新的方法来研究原代培养和 同时建立转化的永生化的PT细胞系或 从转基因动物克隆的细胞系将对 对PT RAS的进一步研究。因此，我们的目标是：1.演绎函数 永生化PT细胞系的建立及其对PTRAs的影响 细胞和亚细胞内RAS组分的调控和定位 将这些细胞与原代培养的细胞进行比较；2.研究自分泌和 PTRAS的旁分泌作用机制，假设局部PT 血管紧张素原为管状血管紧张素提供局部底物 PT ACE兼有多种功能；3.考察其影响 (A)与肾素分泌细胞共培养对PT细胞肾素的影响 细胞，(B)PT细胞暴露于已添加肾素的培养液中 以及(C)将肾素导入永生化的PT细胞。因此， 本申请书旨在研究 在细胞和亚细胞水平上的PT-RAS。我们预计这些数据 获得的结果将是新颖的，并对PT的方式产生影响 肾素-血管紧张素转换酶参与肾脏和心血管的动态平衡。

A variety of physiological studies provides compelling evidence that angiotensin II (Ang II) has important actions in the renal proximal convoluted tubule. Biochemical and molecular biologic techniques have permitted the identification of renin, angiotensinogen (ang-n), and angiotensin converting enzyme (ACE) proteins and mRNAs within the kidney. Several lines of evidence suggest that a major site of the intrarenal renin angiotensin system (RAS) is the proximal tubule (PT). Within the PT, ang-n and ACE mRNAs are synthesized, whereas renin is more likely reabsorbed. As there are also angiotensin receptors within PT as well as endogenous local Ang II production, there is in vivo a complete PT. Furthermore, persuasive data support the concept that a complete RAS within the proximal tubule plays important roles in a variety of tubular functions including sodium and water reabsorption, protein processing, and, perhaps, cellular hypertrophy. The present project is designed to study regulation and function of the proximal tubule (PT) renin angiotensin system (RAS) utilizing PT cells in culture. Much information lends support to the concept that the proximal tubular RAS subserves autocrine, paracrine, and intracrine functions. Available established PT-like cell lines such as OK cells are not truly analogous to PT cells in vivo; primary PT cells in culture have proven difficult to grow and passage, and also de-dedifferentiate rapidly. Thus, developing new ways to study PT cells in primary culture and concomitantly establishing transformed, immortalized PT cell lines or cell lines cloned from transgenic animals would be of great benefit to further study of the PT RAS. Thus, our aims are: 1. To deduce functions of the PT RAS by developing immortalized PT cell lines and studying cellular and subcellular regulation and localization of RAS components in these cells compared to primary cultures; 2. To study the autocrine and paracrine mechanisms of action of the PT RAS, hypothesizing that local PT angiotensinogen provides-local substrate for tubular angiotensins and that PT ACE subserves multiple functions; 3. To investigate the effects of renin on PT cells by (a) co-culture of PT cells with renin-producing cells, (b) exposure of PT cells to media into which renin has been added and (c) by transfecting renin into immortalized PT cells. Thus, the present application seeks to examine the regulation and functions of the PT RAS on a cellular and subcellular level. We anticipate that the data obtained will be novel and have implications for the ways in which the PT RAS is involved in both renal and cardiovascular homeostasis.