DESCRIPTION: (Adapted from the application) This proposal will investigate the mechanisms by which production of tumor necrosis factor-a (TNF) is regulated by angiotensin II (Ang II), and determine the effects of TNF-Ang II interactions on ion transport in the medullary thick ascending limb of Henle's loop (mTALH). The demonstration that TNF is rapidly produced by the mTALH after stimulation with Ang II serves as a starting point for determining whether some effects of this peptide are mediated/modulated by TNF. The mechanisms by which the MTALH produces TNF in response to Ang II will be characterized using cellular, molecular, and immunochemical (immunofluorescence and Western blot) protocols. These experiments will reveal the contribution of transcriptional and post-transcriptional mechanisms to TNF production induced by Ang II, and will provide a framework around which strategies for altering these interactions can be devised. The close association of the renin-angiotensin system with metabolites of arachidonic acid, and the putative interactions of these hormonal systems with TNF suggest that important regulatory mechanisms involving these three distinct classes of mediators, cytokines (TNF), eicosanoids, and angiotensins, may have relevance to ion transport mechanisms in the kidney. The contribution of TNF to Ang II-mediated increases in PGE2 production will be determined by neutralization of TNF bioactivity with anti-TNF antisera, and a recombinant TNF receptor fusion protein. Three complementary techniques (digital imaging microscopy, patch-clamp analysis and 86 Rb transport) will be used to assess the effects of AngII on ion transport in the mTALH. The ability of TNF and prostanoids to mediate/modulate these effects also will be determined. Based on these considerations, this proposal is designed to test the hypotheses: 1) Ang II is an endogenous regulator of TNF production in the mTALH; 2)TNF mediates Ang II-induced prostanoid production in the mTALH, a regulatory action that may be linked to induction of cyclooxygenase (COX-2); and 3)TNF, released in response to Ang II, mediates/modulates the effects of the peptide on mTALH ion transport mechanisms.