Regulation of Renal TNF Production and Function

描述（由申请人提供）：髓质粗升支（mTAL）对盐和水稳态的调节至关重要，并通过代谢花生四烯酸的不同单加氧酶进行调节。在用血管紧张素II（Ang II）激发的mTAL细胞中以及在钙敏感受体（CaR）活化后，肿瘤坏死因子-α（TNF）的产生增加，并且这些激动剂以TNF和环氧合酶-2（考克斯-2）依赖性方式抑制mTAL中的离子转运。因此，将分别在mTAL小管和细胞中确定TNF对影响顶端K+通道和Na+-K+-2Cl-共转运蛋白活性调节的前列腺素依赖性机制的贡献。将从野生型同窝对照（TNF+/+）和TNF缺陷小鼠中分离细胞。这些小鼠也将用于体内实验，旨在确定TNF对血压调节的贡献，在Ang II依赖性高血压模型中使用无线电遥测法确定。还将在体内测定Ang II以TNF依赖性方式调节考克斯-2表达和活性的能力。我们的实验室最近首次证明了CaR活化可增加任何细胞类型中活化T细胞核因子（NFAT）的活性。因此，已经鉴定了mTAL中存在的NFAT同种型，并且将其与CaR活化后mTAL细胞中TNF和PGE 2的产生相关联。几种细胞和分子的方法，包括电迁移率变动测定，激光扫描细胞术，转染显性负NFAT结构，和使用NFAT活性报告构建体，将被用来确定的异构体，有助于钙调素介导的TNF生产。TNF对心血管功能的生物学特性表明，这种细胞因子可能以环境依赖性方式表现出促高血压或抗高血压活性。例如，在mTAL细胞中，TNF及其与考克斯-2和PGE 2表达的关系可能作为抗升压机制的一部分。因此，响应于细胞外Ca 2+的该机制的激活可通过TAL的基底外侧膜上的CaR的激活来启动，其通过TNF依赖性机制增加考克斯-2衍生的PGE 2合成。我们的研究结果表明，一种机制，负责血压降低响应CaR激活凭借的抑制作用，PGE 2对盐和水的运输在TAL。

DESCRIPTION (provided by applicant): The medullary thick ascending limb (mTAL) is critical to the regulation of salt and water homeostasis and subject to regulation via different monooxygenases that metabolize arachidonic acid. Tumor necrosis factor-alpha (TNF) production is increased in mTAL cells challenged with Angiotensin II (Ang II) and after activation of the calcium-sensing receptor (CaR), and these agonists inhibit ion transport in the mTAL in a TNF- and cyclooxygenase-2 (COX-2)-dependent manner. Thus, the contribution of TNF to prostanoid-dependent mechanisms affecting the regulation of apical K+ channels and Na+-K+-2Cl- cotransporter activity will be determined in mTAL tubules and cells, respectively. Cell will be isolated from wild type littermate control (TNF+/+) and TNF deficient mice. These mice also will be used for in vivo experiments designed to determine the contribution of TNF to the regulation of blood pressure, determined using radiotelemetry, in an Ang II-dependent model of hypertension. The ability of Ang II to regulate COX-2 expression and activity in a TNF-dependent manner also will be determined in vivo. The first demonstration that CaR activation increases nuclear factor of activated T cell (NFAT) activity in any cell type was recently provided by our laboratory. Accordingly, NFAT isoforms present in the mTAL have been identified and will be linked to the production of TNF and PGE2 in mTAL cells after CaR activation. Several cellular and molecular approaches, including electromobility shift assays, laser scanning cytometry, transfection with dominant negative NFAT constructs, and use of NFAT activity reporter constructs, will be used to identify the isoforms that contribute to CaR-mediated TNF production. The biological profile of TNF on cardiovascular function indicates that this cytokine may exhibit either pro- or anti-hypertensive activities in a context-dependent manner. For instance, in mTAL cells, TNF and its relationship to expression of COX-2 and PGE2 may act as part of an antipressor mechanism. Thus, activation of this mechanism in response to extracellular Ca2+ may be initiated by activation of CaR on the basolateral membrane of the TAL, which increases COX-2- derived PGE2 synthesis via a TNF-dependent mechanism. Our findings indicate a mechanism responsible for blood pressure reduction in response to CaR activation by virtue of the inhibitory effect of PGE2 on salt and water transport in the TAL.