--- 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.).

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Bradford, Davis; Raghuram, Viswanathan; Wilson, Justin L L et al. (2014) Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256. Am J Physiol Cell Physiol 307:C123-39
Knepper, Mark A; Raghuram, Viswanathan; Bradford, Davis et al. (2014) Letter to the editor: "Systems biology versus reductionism in cell physiology". Am J Physiol Cell Physiol 307:C308-9
Miranda, Carlos A; Lee, Jae Wook; Chou, Chung-Lin et al. (2014) Tolvaptan as a tool in renal physiology. Am J Physiol Renal Physiol 306:F359-66
Saeed, Fahad; Hoffert, Jason D; Pisitkun, Trairak et al. (2014) Exploiting Thread-Level and Instruction-Level Parallelism to Cluster Mass Spectrometry Data using Multicore Architectures. Netw Model Anal Health Inform Bioinform 3:54
Trepiccione, Francesco; Pisitkun, Trairak; Hoffert, Jason D et al. (2014) Early targets of lithium in rat kidney inner medullary collecting duct include p38 and ERK1/2. Kidney Int 86:757-67
Sanghi, Akshay; Zaringhalam, Matthew; Corcoran, Callan C et al. (2014) A knowledge base of vasopressin actions in the kidney. Am J Physiol Renal Physiol 307:F747-55
Xie, Luke; Subashi, Ergys; Qi, Yi et al. (2014) Four-dimensional MRI of renal function in the developing mouse. NMR Biomed 27:1094-102
Knepper, Mark A (2014) Proteomic pearl diving versus systems biology in cell physiology. Focus on "proteomic mapping of proteins released during necrosis and apoptosis from cultured neonatal cardiac myocytes". Am J Physiol Cell Physiol 306:C634-5
Hoffert, Jason D; Pisitkun, Trairak; Saeed, Fahad et al. (2014) Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct. Am J Physiol Renal Physiol 306:410-21
Pisitkun, Trairak; Dummer, Patrick; Somparn, Poorichaya et al. (2014) Integrated Design of Antibodies for Systems Biology Using Ab Designer. J Proteomics Bioinform 7:088-94

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