--- The chief goal is to understand how the hormone vasopressin regulates water excretion by the kidney. Vasopressin's action is mediated through regulation of the molecular water channel aquaporin-2. Based on our studies a decade ago, it is now clear that vasopressin regulates aquaporin-2 in a time frame of seconds to minutes by altering the distribution of the water channel aquaporin-2 between the plasma membrane and the cytoplasm via vesicular trafficking. Trafficking of aquaporin-2 to the plasma membrane renders the cells permeable to water. We are presently using a systems approach to address the mechanisms involved. For this approach, we are integrating protein mass spectrometry, DNA microarrays, deep sequencing, mathematical modeling and physiological methods. --- There are two major areas of focus currently: 1) elucidation of the signaling network for vasopressin responses in the renal collecting duct;and 2) understanding how vasopressin regulates the abundance of the aquaporin-2 water channel and other proteins in the renal collecting duct. --- In the first area, we have already published a series of papers showing phosphoproteomic responses to vasopressin in the inner medullary collecting duct of rat, in the thick ascending limb of rat, and in cultured cortical collecting duct cells (see reference list). We have completed a dynamic study of the phosphoproteomic response to vasopressin using iTRAQ to track changes in thousands of individual phosphorylation sites over a 15 minute time period after vasopressin exposure. We have also completed work on new methodology for profiling individual protein kinases with regard to the sequence preferences in the target substrate proteins. The current thrust in the first area of focus is to map individual protein kinases to regulated phosphorylation targets in the collecting duct. --- In the second area of focus, we have published an article describing studies in which we have carried out global profiling of vasopressin-induced changes in both protein abundance and transcript abundance in the same collecting duct cells (Khositseth et al. see references). In general, there was a low correlation between these two measures for most proteins. In particular, a large numbers of proteins exhibited changes in protein abundance in response to vasopressin without corresponding changes in transcript abundances. The implication is that there is regulation of translation or protein stability for a large number of proteins. To follow this up, we have carried out global profiling of protein half lives using dynamic SILAC revealing that only a small fraction of post-translationally regulated proteins are regulated by altering half life including aquaporin-2 (Sandoval et al). We are now using SILAC to determine translation rates for all proteins across the proteome. An additional focus is on transcriptional regulation. We have carried out global profiling of vasopressin-induced nuclear translocation, identifying a relatively small number of transcription factors that move into the nucleus in response to vasopressin. Also, we are finalized a study to identify nuclear proteins that become phosphorylated in response to vasopressin to identify additional candidate proteins that may play roles in vasopressin-regulated transcription in collecting duct cells (Bolger et al.).

Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
2013
Total Cost
$1,933,408
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Zhao, Yue; Yang, Chin-Rang; Raghuram, Viswanathan et al. (2016) BIG: A large-scale data integration tool for renal physiology. Am J Physiol Renal Physiol :ajprenal.00249.2016
Huebner, Alyssa R; Cheng, Lei; Somparn, Poorichaya et al. (2016) Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation. Mol Cell Proteomics 15:1556-71
Medvar, Barbara; Raghuram, Viswanathan; Pisitkun, Trairak et al. (2016) Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation. Physiol Genomics 48:502-12
Pickering, Christina M; Grady, Cameron; Medvar, Barbara et al. (2016) Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells. Physiol Genomics 48:154-66
Cheng, Lei; Pisitkun, Trairak; Knepper, Mark A et al. (2016) Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics. Methods Mol Biol 1355:53-70
Knepper, Mark A; Kwon, Tae-Hwan; Nielsen, Soren (2015) Molecular Physiology of Water Balance. N Engl J Med 373:196
Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad et al. (2015) Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions. Am J Physiol Cell Physiol 309:C785-98
Knepper, Mark A (2015) Systems biology of diuretic resistance. J Clin Invest 125:1793-5
Yang, Chin-Rang; Raghuram, Viswanathan; Emamian, Milad et al. (2015) Deep proteomic profiling of vasopressin-sensitive collecting duct cells. II. Bioinformatic analysis of vasopressin signaling. Am J Physiol Cell Physiol 309:C799-812
Lee, Jae Wook; Chou, Chung-Lin; Knepper, Mark A (2015) Deep Sequencing in Microdissected Renal Tubules Identifies Nephron Segment-Specific Transcriptomes. J Am Soc Nephrol 26:2669-77

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