Role of Intracrine Angiotensin II in Kidney Cells

内分泌血管紧张素 II 在肾细胞中的作用

• 批准号: 6860073
• 负责人: Jia L. Zhuo
• 金额: $ 24.76万
• 依托单位: HENRY FORD HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6860073
• 关键词: angiotensin II; angiotensin receptor; autoradiography; calcium ion; cytokine; electron microscopy; endocytosis; extracellular; hormone regulation /control mechanism; immunocytochemistry; intracellular transport; kidney cell; laboratory mouse; laboratory rat; microinjections; northern blottings; nuclear factor kappa beta; polymerase chain reaction; receptor binding; renal tubule; sodium hydrogen exchanger; tissue /cell culture; vesicle /vacuole

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.

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的重吸收来维持身体钠和液体平衡和正常血压中起着至关重要的作用。 ANG II通过激活近端小管细胞的刷子边界和基底外侧膜上激活细胞表面受体对钠转运和细胞生长产生强大影响。 但是，我们有新的证据表明a）细胞外ANG II被体内和体外近端小管细胞吸收； b）ANG II直接对细胞的显微注射会增加细胞内钙； c）ANG II在分离的核中诱导RNA转录和表达。 我们的结果表明，内部化的ANG II可以充当细胞内激素，在这些细胞中起重要的生理和病理作用。 在该项目中，我们建议检验一个普遍的假设，即细胞外ANG II通过AT1A受体介导的内在化来吸收细胞外ANG II，并且内部化ANG II与细胞质和核受体结合以诱导细胞内反应。 为了检验这一假设，我们将使用互补的生化，形态学，细胞和分子生物学方法进行体外和体内研究。 在AIM I中，我们将研究近端管状细胞在体外和体内占据细胞外ANG II，并阐明ANG II受体和内吞机械调节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EM自显影和免疫组织化学来追踪内部化ANG II的细胞内运输途径到内体隔室及其易位到体外和体内的核心。 在AIM III中，我们将研究ANG II的显微注射是否通过激活细胞质AT1受体和所涉及的细胞机制来增加细胞内钙。 最后，在AIM IV中，我们将研究内部化的ANG II与细胞内ANG II受体结合，以激活转录因子NFKappab及其转移到核，以及内部化的ANG II是否刺激核受体增加Na+/H+交换器NHE3的转录和表达。 这些研究将为内部化ANG II在肾脏生理学和ANG II引起的高血压肾脏损伤中的重要作用提供新的见解。