The ability to concentrate urine, and to vary urine osmolality, is critical for maintaining fluid balance. A urea transporter is critically important to the theories proposed to explain the physiologic processes occurring when urine is concentrated. Only 12 years ago, evidence for such a transporter was largely speculative, but investigations in this laboratory provided experimental evidence for facilitated urea transport in kidney. Subsequently, 2 cDNA isoforms for facilitated urea transporters were cloned from kidney medulla (UT-Al, UT-A2) and one from erythrocytes (UT-B). During the previous grant period, the applicants cloned 2 additional kidney cDNA isoforms (UT-A3, UT-A4) and the UT-A gene. Vasopressin stimulates urea permeability in perfused terminal inner medullary collecting ducts (IMCDs) within 5 minutes, and the application provides preliminary data showing that vasopressin increases the phosphorylation of UT-A 1 in rat IMCDs within 2-5 minutes. There has been progress, through those studies, in understanding the long-term regulation of urea transport. In other experiments, five animal models associated with impaired urine concentrating ability were studied. The surprising finding was that facilitated urea transport and UT-A l protein abundance are increased during in vivo conditions associated with an impaired urine concentrating ability and a reduced plasma vasopressin level. The current application proposes to: 1) identify the mechanism that regulates urea transport rapidly to test the hypothesis that rapid regulation occurs by changes in phosphorylation of specific UT-A proteins; and 2) evaluate the mechanism of long-term regulation of urea transport in a rat model of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) to test the hypothesis that down-regulation of UT-A proteins is involved in the mechanisms responsible for vasopressin-escape.
Specific Aim 1 will test whether urea transporter phosphorylation is the mechanism by which vasopressin rapidly increases urea permeability in rat IMCDs.
Specific Aim 2 will identify the site(s) within UT-A proteins that are phosphorylated in response to vasopressin.
Specific Aim 3 will test whether phosphorylation is the mechanism by which other agonists (hyperosmolality, angiotensin II) rapidly stimulate urea permeability in rat IMCDs.
Specific Aim 4 will test whether UT-A expression or regulation is altered in rats with a model of SIADH or during vasopressin-escape.
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