Solute And Water Transport In Renal Epithelia

National Heart, Lung, And Blood Institute

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Abstract

The chief goal of the <b>Epithelial Systems Biology Laboratory</b> headed by Mark Knepper, MD, PhD, is to understand how the hormone <b>vasopressin</b> regulates water excretion by the kidney. Vasopressin's action is mediated through regulation of the molecular water channel <b>aquaporin-2</b>. Based on our studies more than a decade ago, it is now clear that vasopressin regulates aquaporin-2 by two basic processes: <b>1)</b> regulation of trafficking of aquaporin-2-containing membrane vesicles to and from the apical plasma membrane of collecting duct cells (time frame, minutes); and <b>2)</b> regulation of the total abundance of the aquaporin-2 water channel protein (time frame, hours to days). We are presently using a systems approach to address the mechanisms involved. For this approach, we are integrating <b>protein mass spectrometry</b>, <b>deep sequencing of DNA</b>, <b>mathematical modeling</b> and <b>physiological methods</b>.<br> There are <b>two major areas of focus</b> currently: <b>a)</b> elucidation of the signaling network for vasopressin responses in the renal collecting duct; and <b>b)</b> understanding how vasopressin regulates the abundance of the aquaporin-2 water channel and other proteins in the renal collecting duct.<br> <b>---</b> <b>a --- </b>In the first area, we have already published a series of papers using <b>LC-MS/MS</b> 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 <b>iTRAQ</b> to track changes in thousands of individual phosphorylation sites over a 15 minute time period after vasopressin exposure. We have completed work to characterize the phosphoproteomic response of collecting duct cells to the vasopressin antagonist <b>satavaptan</b>. We have also completed work on new methodology for profiling individual <b>protein kinases</b> with regard to the <b>target sequence preferences</b> in 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. This is being pursued computationally using <b>Bayes' rule</b> to integrate multiple relevant data sets and experimentally using broad-spectrum protein kinase inhibitors and phosphoproteomics to identify candidate phosphorylation networks for the vasopressin-response of collecting duct cells. With the resulting prioritized list of candidate protein kinases, we have begun to systematically delete them in cultured mouse collecting duct cells using genome editing techniques (<b>CRISPR</b>) followed by assessment of the deletion on the vasopressin-dependent phosphoproteome using <b>protein mass spectrometry</b>. <br> <b>---</b> <b>b ---</b> In the second area of focus, we have carried out global profiling of vasopressin-induced changes in both protein abundance and transcript abundance in the same collecting duct cells. We have also mapped <b>RNA polymerase II</b>-binding along the genome using <b>ChIP-seq</b> methodology in the presence and absence of vasopressin to identify genes that are likely to undergo changes in transcription. In addition, we have done global profiling of protein half-lives and translation rates throughout the proteome using <b>dynamic SILAC</b> labeling to identify targets of post-transcriptional regulation of protein abundance. Taken together, these approaches identify multiple mechanisms involved in vasopressin-induced protein abundance changes in collecting duct cells, namely regulation of transcription of some genes, regulation of translation of others, and regulation of protein stability for additional gene products. We have carried out <b>global profiling of vasopressin-induced nuclear translocation</b> in collecting duct cells, identifying a relatively small number of <b>transcription factors</b> that move into the nucleus in response to vasopressin. Also, we have published a study that identifies nuclear proteins that become phosphorylated in response to vasopressin to find additional candidate proteins that may play roles in vasopressin-regulated transcription in collecting duct cells. Bayesian analysis of these multiple data sets identify a few transcription factors that have a high probability of involvement in transcriptional regulation of the aquaporin-2 gene. We are presently using <b>ChIP-seq</b> technology for genome-wide mapping of binding sites for each of the highly-ranked transcription factors. Finally, we have assimilated technology for single-cell transcriptomics and are applying it to investigation of the cell types expressed in the renal collecting duct.