The collecting duct is the final site in the kidney for regulation of urine volume and composition and is the primary target for aldosterone and vasopressin, two of the most important hormones known to control whole animal salt and water homeostasis. Under normal conditions the cortical collecting tubule (CCT) actively reabsorbs NaC1 and secretes K+, H+ and HCO3-. Cortical collecting tubules of the rabbit are composed of principal and intercalated cells present in a ratio of ca. 2:1. A large body of evidence suggests that the principal cell is primarily involved in NaC1 reabsorption and K+ secretion. The function of the intercalated cell has been inferred almost entirely from indirect studies, but this cell is thought to be responsible for H+ and HCO3- secretion. The proposed study will determine the transport functions of identified cell types in isolated perfused rabbit CCT. Specifically, solute entry and exit steps will be characterized in principal and intercalated cells by studying the mechanisms of ouabain-induced cell swelling using quantitative differential interference contrast microscopy. Techniques to impale identified cell types with ion and voltage-sensitive microelectrodes will be developed to characterize membrane conductive properties and electro-chemical gradients for transported ions. Electrophysiological studies are important for corroborating and complementing results of the ouabain experiments. Mechanisms of volume regulatory decrease behavior in principal and intercalated cells will be studied using quantitative microscopy and microelectrode techniques. These studies are important not only for determining the transport properties of principal and intercalated cells, but for characterizing intrinsic cellular homeostatic regulatory mechanisms. The technical and experimental approach described in this proposal will characterize normal CCT cell function and will provide a crucially important foundation for future attempts to elucidate the role played by hormones, acute and chronic physiological disturbances and disease states in altering principal and intercalated cell physiology.
