Regulation of Renal TNF Production and Function

描述（由申请人提供）：髓质厚升肢（mTAL）对盐和水稳态的调节至关重要，并受到代谢花生四烯酸的不同单加氧酶的调节。在血管紧张素II （Ang II）刺激和钙敏感受体（CaR）激活后，肿瘤坏死因子- α (TNF)的产生增加，这些激动剂以TNF-和环氧化酶-2 （COX-2）依赖的方式抑制金属tal中的离子运输。因此，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机制的贡献将分别在tal小管和细胞中确定。细胞将从野生型同窝对照（TNF+/+）和TNF缺陷小鼠中分离。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angii依赖性高血压模型中使用无线电遥测法确定。Ang II以依赖tnf的方式调节COX-2表达和活性的能力也将在体内确定。最近，我们的实验室首次证明了CaR激活会增加任何细胞类型中活化T细胞核因子（NFAT）的活性。因此，mTAL中存在的NFAT异构体已被确定，并将与CaR激活后mTAL细胞中TNF和PGE2的产生有关。几种细胞和分子方法，包括电迁移转移试验、激光扫描细胞术、显性阴性NFAT构建体转染和NFAT活性报告构建体的使用，将用于鉴定促进car介导的TNF产生的亚型。TNF对心血管功能的生物学特征表明，这种细胞因子可能以环境依赖的方式表现出促高血压或抗高血压的活性。例如，在tal细胞中，TNF及其与COX-2和PGE2表达的关系可能作为抗压机制的一部分。因此，响应细胞外Ca2+的这一机制的激活可能是由TAL基底外侧膜上的CaR激活启动的，这通过tnf依赖的机制增加了COX-2衍生的PGE2合成。我们的研究结果表明，由于PGE2对TAL中盐和水运输的抑制作用，导致CaR激活后血压降低的机制。

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.