This project will explore the basic mechanism of volume absorption in the renal proximal tubule, including the driving forces for water flow across the tubular epithelium, the pathway by which water crosses the epithelium, and the role of serosal protein in maintaining the structure of the epithelium during water flow and in providing a force for driving water flow out of the intercellular space. The techniques to be based include the perfusion of isolated tubules, the observation of osmotic behavior of isolated apical-membrane blebs, freeze-fracture and fluorescent-membrane-marker studies of isolated membrane blebs, light and electron microscopic examination of epithelial structure in isolated tubules, and the perfusion of isolated basement membrane from proximal tubules.
The specific aims are: 1) To measure the transepithelial reflection coefficients for the major solutes transported by the proximal tubule; this will be used to determine whether significant flow of water occurs between the cells of this epithelium; 2) To measure the osmotic water permeability of the apical membrane of the proximal tubule using isolated membrane blebs; this permeability is an important parameter for determining if the pathway for water flow can be entirely cellular; 3) To explore the effects of osmotically induced water flow on the ultrastructure of the proximal tubule, and to describe the effects of serosal colloid on flow-induced changes in epithelial structure; this work will help define the role of serosal protein in maintaining the structure of the proximal tubule during volume absorption; 4) To determine the permeability properties of the basement membrane to protein and the ability of the basement membrane to act as a semi-permeable membrane for abstraction of fluid from the intracellular space: and 5) To bring together the data that are collected into computer models that will: a) described the route of water flow across the epithelium of the proximal tubule, b) describe the probable concentration of protein in the intercellular space of the proximal tubule and the role that oncotic forces play in abstracting fluid from the epithelium, and c) assess the role of the intercellular space as a compartment that can aid in coupling the transport of solute and water across this epithelium. The long-range objective of this project is to describe the mechanism of volume absorption in the proximal tubule, with the expectation that this knowledge will help in understanding body fluid homeostasis.
