The mineralocorticoid receptor (MR) is a transcription factor expressed in many cells that regulates the expression of genes in involved myriad cell type-specific functions. Inappropriate activation of the MR causes hypertension and pathological tissue remodeling even in conditions of normal aldosterone levels. Activity of the MR is regulated by post-translational modifications including phosphorylation of serines and threonines. Phosphorylation activates or suppresses MR activity depending upon the amino acid involved. The kinase ULK1 was recently reported to phosphorylate serine 843 of the MR, thereby suppressing its activity, and to be expressed with the MR only in intercalated cells of the distal nephron. Our own specific antibodies reveal more extensive co-expression of ULK1 and MR in the kidney. ULK1 expression is widespread in the body and in multiple cell lines. Other kinases also have the potential to phosphorylate the MR at S843, thus inhibit MR activity. The MR has similar affinity for aldo, cortisol or corticosterone. Specificity for aldo is conferred by conversion of cortisol and corticosterone to the inactive cortisone and 11-dehydrocorticosterone by the 11β-hydroxysteroid dehydrogenase 2 (11-HSD2), however this enzyme is not present in most non-epithelial aldo target tissues. Cortisol and corticosterone are also converted to 20β-dihydro-cortisol and -corticosterone by carbonyl reductase 1 (Cbr1). We that found the 20β-dihydro metabolites do not activate the MR, thus providing an alternative mechanism for MR specificity for aldo where 11-HSD2 is not expressed. Hypotheses: 1) Phosphorylation of the MR at serine 843 is an important negative regulator of MR action that occurs in multiple tissues. 2) Reduction of the 20-keto of corticosterone and cortisol by carbonyl reductase 1 regulates ligand selectivity for the MR by converting corticosterone and cortisol into the inactive metabolites 20β-dihydrocorticosterone (20β-DHC) and 20β-dihydrocortisol. Both potentially modulate MR activity. Specific Aim 1: Study the role of phosphorylation of the MR at serine 843 (human) on genomic and non- genomic activity of the MR. Study the effects of over-expression and suppression of several kinases, ULK1, ULK2, TBK1, NEK2 and PAK1, with putative ability to phosphorylate the MR at S843 in cells expressing an MR reporter gene system. Study the distribution and co-localization of the MR with ULK1, ULK2 and other kinases that catalyze S843 phosphorylation in the kidney, heart, vessels and brain. Specific Aim 2: Study the role of the 20-keto reduction of cortisol and corticosterone on MR ligand selectivity. Determine the dynamics of the 20-keto reduction of corticosterone and cortisol by the human and rat Cbr1 in cells. Measure the conversion of corticosterone to 20β-dihydrocorticosterone in various tissues including in micropunches from specific areas of the rat brain where we demonstrate that Cbr1 and MR are co-expressed by immunofluorescent histochemi...