The long-term objective of this proposal is to provide new and meaningful structural-functional correlative data as it relates to solute transport along the mammalian renal tubule. The experiments described focus our attention primarily on the collecting duct and place a major emphasis on the role of angiotensin II (All) in the regulation of intercalated cell structure and function and specifically in the control of transepithelial H+ and HCO3- transport. The proposal includes four sections with clearly delineated specific aims and hypotheses to be tested.
In Specific Aim I we will examine the effect of All on the ultrastructure of intercalated cells to identify specific intercalated cell subpopulations that respond to All, and determine the effect of All on the subcellular distribution of H+ATPase by immunogold cytochemistry. Hypothesis to be tested: Specific intercalated cell subtypes are responsible for the effect of All on H+-ATPase activity and acid-base transport in the collecting duct.
In Specific Aim II the effects of All on H+-ATPase activity and the signalling transduction pathways involved in mediating these effects will be studied in microdissected collecting duct segments. Hypothesis to be tested: The inhibitory effect of All on H+-ATPase activity in the collecting duct is mediated via specific AT1 receptors and activation of distinct signalling systems which will be identified.
In Specific Aim III we will determine the expression and cellular location of All receptor isoform mRNA in the collecting duct by quantitative RT-PCR and in situ hybridization and determine the effect of acid-base perturbations on All receptor mRNA expression. Hypothesis to be tested: All receptor mRNA expression varies between different populations of intercalated cells and is influenced by acid-base perturbations.
In Specific Aim I V we will determine the effect of All on proton and bicarbonate transport in the isolated perfused rabbit cortical collecting duct and identify the subtype(s) of intercalated cells that responds to All. Hypothesis to be tested: All stimulates HCO3 secretion by type B intercalated cells and inhibits H+ secretion by type A intercalated cells in the cortical collecting duct. The experiments described in this proposal are intended to continue our long-term commitment to delineate the structural and functional characteristics of the renal tubule and specifically to establish the role of intercalated cells in the """"""""fine-tuning"""""""" of acid-base regulation by the kidney. A better understanding of the mechanisms that control H+ and HCO3, transport in this region of the renal tubule will enable physicians to manage complicated clinical problems of acid-base balance that are often life-threatening with greater expertise and success.
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