Role of Intracrine Angiotensin II in Kidney Cells

描述（由申请人提供）：血管紧张素II （Ang II）通过调节近端小管钠重吸收，在维持机体钠和液体平衡和正常血压中起重要作用。Ang II通过激活近端小管细胞刷状边界和基底外膜上的细胞表面受体，对钠转运和细胞生长产生强大的影响。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吸收；b)直接向细胞内微量注射Ang II增加细胞内钙含量；c) Ang II在离体细胞核中诱导RNA转录和表达。我们的研究结果表明，内化的Ang II可能作为细胞内激素在这些细胞中发挥重要的生理和病理作用。在这个项目中，我们提出验证细胞外的Ang II通过AT1A受体介导的内化被近端小管细胞吸收，内化的Ang II结合细胞质和核受体诱导细胞内反应的一般假设。为了验证这一假设，我们将进行体外和体内研究，使用互补的生化，形态学，细胞和分子生物学方法。在Aim I中，我们将研究近端小管细胞是否在体内和体外摄取细胞外的Ang II，并阐明Ang II受体和内吞机制调节Ang II运输的机制。在Aim II中，我们将使用共聚焦显微镜，最先进的电子显微镜放射自显像和免疫组织化学来追踪内化的Ang II到内体室的细胞内运输途径及其在体外和体内的转位到细胞核的途径。在Aim III中，我们将研究微注射Ang II是否通过激活细胞质AT1受体而增加细胞内钙以及所涉及的细胞机制。最后，在Aim IV中，我们将研究内化的Ang II是否与细胞内的Ang II受体结合，激活转录因子NFkappaB及其向细胞核的易位，以及内化的Ang II是否刺激核受体增加Na+/H+交换剂NHE3和促炎细胞因子的转录和表达。这些研究将为内化Ang II在肾脏生理和Ang II引起的高血压肾损伤中的重要作用提供新的见解。

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.