Urea's importance to the generation of a concentrated urine has been appreciated since at least 1934. Several studies show that maximal urine concentrating ability is decreased in protein-deprived animals and humans, and is restored by urea infusion. UT-A1/UT-A3, UT-A2, and UT-B knock-out (KO) mice were each shown to have urine concentrating defects, and the defect in the UT-A1/UT-A3 mouse is severe. Thus, any hypothesis regarding the mechanism by which the kidney concentrates urine needs to include some effect derived from urea and urea transporters, especially UT-A1 and/or UT-A3. Due to the nature of the UT-A gene, it is not possible to generate a KO mouse that only expresses UT-A1 using traditional approaches. The first goal of this application is to use a novel approach to create a UT-A1 only mouse. The second goal is to create a UT-A3 only mouse. We will use an innovative approach by starting with the UT-A1/UT-A3 KO mouse and then knock-in either UT-A1 or UT-A3 under the control of an inner medullary collecting duct (IMCD) specific promoter to generate mice expressing only UT-A1 or UT-A3. We have preliminary data showing that we have regenerated the UT-A1/UT-A3 KO mouse, prepared the UT-A1 and UT-A3 constructs for making the transgenic mice, and have mice in which these constructs have been injected. These mice will allow us to determine the effect of each urea transporter independently. The second goal of this proposal is to generate a mouse lacking protein kinase C1 (PKC1) in the IMCD. We have preliminary data suggesting that PKC1 is the specific PKC isozyme involved since: 1) hypertonicity activates PKC1 in rat IMCDs;and 2) PKC1 KO mice have a urine concentrating defect and a reduction in UT-A1 protein abundance. The PKC1 KO mouse is a global KO. Thus, it is possible that the urine concentrating defect in the PKC1 KO mice KO mouse could be due to loss of PKC1 from a site other than the IMCD, such as loss from the brain that results in abnormalities in thirst or vasopressin regulation. To determine whether the concentrating defect is due to loss of PKC1 specifically from the IMCD, we propose to make an IMCD-specific PKC1 dominant-negative mouse. We have obtained a PKC1 dominant-negative construct, which when used with a myocyte-specific promoter, was used to make a mouse lacking PKC1 in myocytes. We propose to use a similar approach to make a mouse with IMCD-specific inhibition of PKC1.
Urea plays a critical role in the urinary concentrating mechanism, and therefore, in the regulation of water balance. Our studies into the regulation of urea transport will advance our understanding of the urinary concentrating mechanism. This could yield new therapies for nephrogenic diabetes insipidus and will become increasingly important with the development of protein kinase C inhibitors for the treatment of diabetes mellitus.
|Klein, Janet D; Wang, Yanhua; Mistry, Abinash et al. (2016) Transgenic Restoration of Urea Transporter A1 Confers Maximal Urinary Concentration in the Absence of Urea Transporter A3. J Am Soc Nephrol 27:1448-55|
|Hoban, Carol A; Black, Lauren N; Ordas, Ronald J et al. (2015) Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct. Am J Physiol Renal Physiol 308:F49-55|
|Weiner, I David; Mitch, William E; Sands, Jeff M (2015) Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion. Clin J Am Soc Nephrol 10:1444-58|
|Sands, Jeff M; Blount, Mitsi A (2014) Genes and proteins of urea transporters. Subcell Biochem 73:45-63|
|Sands, Jeff M; Layton, Harold E (2014) Advances in understanding the urine-concentrating mechanism. Annu Rev Physiol 76:387-409|
|Klein, Janet D (2014) Expression of urea transporters and their regulation. Subcell Biochem 73:79-107|
|Wang, Yanhua; Klein, Janet D; Froehlich, Otto et al. (2013) Role of protein kinase C-Î± in hypertonicity-stimulated urea permeability in mouse inner medullary collecting ducts. Am J Physiol Renal Physiol 304:F233-8|
|Bou Matar, Raed N; Klein, Janet D; Sands, Jeff M (2013) Erlotinib preserves renal function and prevents salt retention in doxorubicin treated nephrotic rats. PLoS One 8:e54738|
|Ilori, Titilayo O; Blount, Mitsi A; Martin, Christopher F et al. (2013) Urine concentration in the diabetic mouse requires both urea and water transporters. Am J Physiol Renal Physiol 304:F103-11|
|Sands, Jeff M (2013) Urea transporter inhibitors: en route to new diuretics. Chem Biol 20:1201-2|
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