Regulation of Renal TNF Production and Function

描述(由申请人提供)：髓质粗升肢(MTAL)对盐和水的动态平衡的调节至关重要，并通过代谢花生四烯酸的不同单加氧酶进行调节。血管紧张素II(Ang II)和钙敏感受体(CAR)激活后，mTAL细胞产生的肿瘤坏死因子-α(TNF-α)增加，这些激动剂以依赖于肿瘤坏死因子和环氧合酶-2(COX-2)的方式抑制mTAL中的离子转运。因此，肿瘤坏死因子在前列腺素依赖影响心尖K通道调节和Na-K-2Cl-共转运体活性调节机制中的作用将分别在mTAL小管和细胞中确定。将从野生型产仔对照(TNF/)和肿瘤坏死因子缺陷小鼠分离细胞。这些小鼠还将用于体内实验，旨在确定在血管紧张素Ⅱ依赖的高血压模型中，通过无线电遥测确定肿瘤坏死因子对血压调节的贡献。Ang II以肿瘤坏死因子依赖的方式调节COX-2表达和活性的能力也将在体内确定。本实验室最近首次证明CAR活化可增加任何类型细胞中活化T细胞的核因子(NFAT)活性。因此，mTAL中存在的NFAT亚型已被鉴定，并将与CAR激活后mTAL细胞中产生的肿瘤坏死因子和前列腺素E_2有关。几种细胞和分子方法，包括电迁移率改变分析、激光扫描细胞术、显性负NFAT结构的转基因以及NFAT活性报告结构的使用，将被用来鉴定与CAR介导的肿瘤坏死因子产生有关的异构体。肿瘤坏死因子对心血管功能的生物学作用表明，这种细胞因子可能以一种上下文依赖的方式表现出促高血压或抗高血压活性。例如，在mTAL细胞中，肿瘤坏死因子及其与COX-2和PGE2表达的关系可能是抗升压机制的一部分。因此，细胞外钙激活这一机制可能是通过激活TAL基底膜上的CAR，通过肿瘤坏死因子依赖的机制增加COX-2衍生的PGE2的合成。我们的发现表明，由于PGE2对TAL中盐和水运输的抑制作用，CAR激活导致血压下降的机制之一。

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.