The pattern of expression and function of urea transport proteins is essential to the formation of concentrated urine and thus the conservation of water by the mammalian kidney. During the past decade, we and others have made tremendous progress in understanding the regulation of urea transport proteins. Major advances include the cloning of the two urea transporter genes and of several eDNA isoforms, creation of polyclonal antibodies, and physiologic studies of urea transporter function, abundance, and phosphorylation in inner medullary collecting duct (IMCD). However, very little progress has been made in understanding the cell biology of urea transporters, primarily because an appropriate cell culture system does not exist. We have preliminary data showing that we have created Madin-Darby Canine Kidney (MDCK) cell lines that stably expresses individual UT-A urea transporters. The overall goal of this project is to develop and characterize polarized epithelial cell lines that stably express individual urea transporter proteins. By comparing the functional properties of each cell line, we hope to gain insight into the question of why nature created so many different UT-A isoforms.
Specific Aim 1. Determine the transport properties of MDCK cells that stably express UT-A1, UT-A2, and UT-A4 and are grown in culture as a high resistance epithelium. Create MDCK cell lines that stably express UT-A3 and determine their transport properties. Properties to be measured: 1) UT-A protein synthesis; 2) urea concentration dependence; 3) inhibitor Ki values for phloretin, thionicotinamide, and dimethylurea; 4) surface expression (apical vs. basolateral); and 5) phosphorylation status of UT-A protein.
Specific Aim 2. Determine the role of protein kinase A (PKA) pathways in regulating UT-A transport and phosphorylation. Interventions to be studied: 1) concentration dependence and time course of flux and phosphorylation activation by vasopressin, dDAVP, forskolin, and cyclic AMP analogs; 2) effects of PKA inhibitors (H-89, PKA peptide inhibitor); and 3) effects of lithium.
Specific Aim 3. Determine the role of protein kinase C (PKC) pathways in regulating UT-A transport and phosphorylation. Interventions to be studied: 1) concentration dependence and time course of flux and phosphorylation activation by angiotensin II, hyperosmolality, and phorbol esters; 2) dependence upon intracellular calcium; and 3) effects of PKC inhibitors (general and isoform-family-specific PKC irLhibitors).
Specific Aim 4. Identify the site(s) within UT-A proteins that are phosphorylated in response to PKA or PKC. Interventions to be studied: urea flux and UT-A phosphorylation in MDCK cells expressing UT-A proteins in which one or more PKA or PKC consensus sites have been mutated.
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