Role of Intracrine Angiotensin II in Kidney Cells

描述（由申请人提供）：血管紧张素II（Ang II）通过调节近端肾小管钠重吸收，在维持体内钠和液体平衡以及正常血压方面发挥重要作用。 血管紧张素II通过激活近曲小管细胞刷状缘和基底膜上的细胞表面受体，对钠转运和细胞生长产生强有力的影响。 然而，我们有新的证据表明：a）细胞外的Ang II在体内和体外被近端小管细胞摄取; B）直接向细胞内显微注射Ang II增加细胞内钙;和c）Ang II诱导分离的细胞核中的RNA转录和表达。 我们的研究结果表明，内化的血管紧张素Ⅱ可能作为一种细胞内激素发挥重要的生理和病理作用，在这些细胞。 在这个项目中，我们建议测试的一般假设，即细胞外的血管紧张素II采取了近端小管细胞通过AT 1A受体介导的内化，和内化的血管紧张素II结合细胞质和核受体，诱导细胞内的反应。 为了验证这一假设，我们将使用互补的生物化学、形态学、细胞和分子生物学方法进行体外和体内研究。 在目的一，我们将研究是否近端肾小管细胞采取细胞外血管紧张素II在体外和体内，并阐明机制，血管紧张素II受体和内吞机制调节血管紧张素II的运输。 在目的II中，我们将使用共聚焦显微镜，国家的最先进的EM放射自显影和免疫组织化学追踪细胞内运输途径的内化血管紧张素II的内体室和translocaUon的核在体外和体内。 在目的III中，我们将研究微注射血管紧张素II是否通过激活细胞质AT 1受体增加细胞内钙离子和相关的细胞机制。 最后，在目的IV中，我们将研究内化的Ang II是否与细胞内Ang II受体结合以激活转录因子NF κ B及其向细胞核的易位，以及内化的Ang II是否刺激核受体以增加Na+/H+交换器NHE 3和促炎细胞因子的转录和表达。 这些研究将提供新的见解的重要作用，内化的血管紧张素II在肾脏生理和血管紧张素II诱导的高血压肾损伤。

DESCRIPTION (provided by applicant): Angiotensin II (Ang II) plays an essential role in maintaining body sodium and fluid balance and normal blood pressure by regulating proximal tubular sodium reabsorption. Ang II exerts powerful effects on sodium transport and cell growth by activating cell surface receptors on brush borders and basolateral membranes of proximal tubule cells. However, we have new evidence that a) extracellular Ang II is taken up by proximal tubule cells in vivo and in vitro; b) microinjection of Ang II directly into the cells increases intracellular calcium; and c) Ang II induces RNA transcription and expression in isolated nuclei. Our results suggest that internalized Ang II may act as an intracellular hormone to play important physiological and pathological roles in these cells. In this project, we propose to test the general hypothesis that extracellular Ang II is taken up by proximal tubule cells through AT1A receptor-mediated internalization, and that internalized Ang II binds to cytoplasmic and nuclear receptors to induce intracellular responses. To test this hypothesis, we will conduct both in vitro and in vivo studies, using complementary biochemical, morphological, cellular and molecular biology approaches. In Aim I, we will study whether proximal tubular cells take up extracellular Ang II in vitro and in vivo and elucidate the mechanisms by which Ang II receptors and the endocytotic machinery regulate Ang II trafficking. In Aim II, we will use confocal microscopy, state-of-the-art EM autoradiography and immunohistochemistry to trace intracellular trafficking pathways of internalized Ang II to the endosomal compartments and its translocaUon to the nucleus in vitro and in vivo. In Aim III we will study whether microinjection of Ang II increases intracellular calcium through activation of cytoplasmic AT1 receptors and the cellular mechanisms involved. Finally, in Aim IV we will investigate whether internalized Ang II binds to intracellular Ang II receptors to activate transcription factor NFkappaB and its translocation to the nucleus, and whether internalized Ang II stimulates nuclear receptors to increase transcription and expression of the Na+/H+ exchanger NHE3 and pro-inflammatory cytokines. These studies will provide new insights into the important role of internalized Ang II in renal physiology and Ang II-induced hypertensive renal injury.