As taken from the abstract: A low-protein diet (LPD) changes fundamental properties of inner medullary collecting duct (IMCD) subsegments. This application's long-term objective is to investigate mechanisms(s) regulating urea and water transporters in the inner medulla using: 1) immunohistochemistry and Western analysis to evaluate changes in immunoreactive protein using a newly developed antibody to the facilitated urea transporter, UT2, and antibodies to aquaporins (AQP); 2) perfused tubules from rat IMCD subsegments to determine which transport processes are expressed; 3) Northern analysis and in situ hybridization to evaluate changes in mRNA; and 4) cultured cells to provide an in vitro model system for testing regulatory mechanisms which are elucidated in the in vivo rat studies. Hypothesis I - There is regulation of urea transporter expression in IMCD subsegments which may contribute to long-term regulation of urine concentrating ability. The rationale is that clinical conditions (e.g. LPD, central diabetes insipidus, and water diuresis) reduce urine and inner medullary osmolality. These changes could increase urea reabsorption to restore interstitial urea. Urea transporters are induced in the initial IMCD by a LPD and Northern analysis and in situ hybridization shows changes in rat UT2 mRNA following a LPD or chronic changes in hydration. These occur in Sprague-Dawley (S-D) and in Brattleboro rats.
Specific Aim #1 : Dr. Sands will test whether the intrarenal and/or subcellular pattern of expression UT2 protein changes in rats fed a LPD.
Specific Aim #2 : He will test whether urea transporter(s) are expressed in IMCD subsegments from Brattleboro rats.
Specific Aim #3 : He will test whether expression of urea transporter(s) changes in IMCD subsegments from S-D rats undergoing a water diuresis. Hypothesis II - Hormonal mechanisms regulate urea transport and may be involved in the adaptive response to a LPD. The rationale is that glucocorticoids and AVP change rat UT2 mRNA abundance.
Specific Aim #4 : He will test whether adrenalectomy changes expression of urea transporter(s).
Specific Aim #5 : He will investigate whether there is evidence for interaction of AVP with glucocorticoid. Hypothesis III - LPD-induced reduction in AVP-stimulated osmotic water permeability in the terminal IMCD is due to a decrease in one or more of the collecting duct AQP proteins.
Specific Aim #6 : He will test whether the apical membrane (AQP2) and/or basolateral membrane (AQP3, AQP4) water channels are reduced in rats fed a LPD. Understanding how a LPD, AVP, and glucocorticoid regulate urea and water transport could lead to improved therapy to slow progression of renal insufficiency, for diseases with abnormal urinary concentration, or even hypertension.
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