The long-term objective of this proposal is to assess the role of sgk (serum- and glucocorticoid-inducec kinase) in steroid hormone action, with special emphasis on aldosterone regulated sodium transport. The foundation for the proposed studies is the recent identification of sgk as an aldosterone-induced early response gene in the cortical collecting duct (CCD). We also demonstrated that in Xenopus oocytes expressing the epithelial Na channel (ENaC) sgk stimulates amiloride-sensitive Na current. Based on these observations, we hypothesize that sgk plays an important role in mediating the actions of aldosterone on Na transport. As a first step toward elucidating the in vivo role of sgk, in this R21 proposal we will develop mouse models with mineralocorticoid target cell-specific, inducible disruption of the sgk gene, using the Cre recombinase-loxP system. Our overall strategy is to generate double transgenic animals carrying a foxed sgk allele and an inducible Cre recombinase under the control of the endogenous 1 1b-hydroxysteroid dehydrogenase-2 (1 1HSD2) promoter.
In Aim 1 we will generate """"""""knock-in"""""""" mice carrying an inducible Cre recombinase (Cre-ET2) selectively in mineralocorticoid target cells. This will make the selective elimination o the sgk gene in mineralocorticoid target cells possible.
In Aim 2 we will generate mice with """"""""foxed"""""""" sgk gene. Using gene targeting via homologous recombination in embryonic stem cells, we will introduce unidirectional loxP sites (recognition sites for the Cre recombinase) that bracket a region of the mouse sgk gene that is essential for its function.
In Aim 3, we will produce inducible, mineralocorticoid target cell-specific sgl knockout mice by crossing mice from Aim 1 with those from Aim 2, as well as """"""""universal"""""""" sgk knockouts by crossing mice with foxed sgk gene with Cre deleter mice. The inducible, mineralocorticoid target cell-specific disruption of the sgk gene will be verified following administration of tamoxifen. Preliminary characterizatior of sgk knockout mice will include litter size, fertility, growth and urinary Na and K excretion. The ultimate goal of this project is to generate knockout and transgenic animals, which will be suitable tc determine the in vivo function of sgk in a wide variety of tissues. Since our main interest is the mechanism of action of aldosterone action in epithelial cells, our immediate goal is to create mice with inducible disruption of sgk in aldosterone-target cells. Characterization of the renal function of such animals should answer the question whether sgk is necessary and sufficient for aldosterone's action on Na transport. Unraveling the molecular steps leading to hormone-stimulated Na transport in the kidney could lead to the identification o genes that might be mutated in some forms of human hypertension, and may eventually lead to therapeutic measures that interrupt this pathway.
| Naray-Fejes-Toth, Aniko; Helms, My N; Stokes, John B et al. (2004) Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies. Mol Cell Endocrinol 217:197-202 |
