The chief goal of the Epithelial Systems Biology Laboratory headed by Mark Knepper, MD, PhD, 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 more than a decade ago, it is now clear that vasopressin regulates aquaporin-2 by two basic processes: 1) regulation of trafficking of aquaporin-2-containing membrane vesicles to and from the apical plasma membrane of collecting duct cells (time frame, minutes); and 2) 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 protein mass spectrometry, deep sequencing of DNA, mathematical modeling and physiological methods.
There are two major areas of focus currently: a) elucidation of the signaling network for vasopressin responses in the renal collecting duct; and b) understanding how vasopressin regulates the abundance of the aquaporin-2 water channel and other proteins in the renal collecting duct.
--- a --- In the first area, we have already published a series of papers using LC-MS/MS 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 completed work to characterize the phosphoproteomic response of collecting duct cells to the vasopressin antagonist satavaptan. We have also completed work on new methodology for profiling individual protein kinases with regard to the target sequence preferences 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 Bayes' rule 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 (CRISPR) followed by assessment of the deletion on the vasopressin-dependent phosphoproteome using protein mass spectrometry.
--- 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 RNA polymerase II-binding along the genome using ChIP-seq 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 dynamic SILAC 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 global profiling of vasopressin-induced nuclear translocation in collecting duct cells, identifying a relatively small number of transcription factors 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 ChIP-seq 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.
| Briggs, Josephine; Palevsky, Paul; Knepper, Mark (2018) JASN this Month: Something Old, Something New. J Am Soc Nephrol 29:1345-1346 |
| Chen, Lihe; Lee, Jae Wook; Chou, Chung-Lin et al. (2018) Reply to Edemir: Physiological regulation and single-cell RNA sequencing. Proc Natl Acad Sci U S A 115:E351-E352 |
| Hyndman, Kelly A; Yang, Chin-Rang; Jung, Hyun Jun et al. (2018) Proteomic determination of the lysine acetylome and phosphoproteome in the rat native inner medullary collecting duct. Physiol Genomics 50:669-679 |
| Jung, Hyun Jun; Raghuram, Viswanathan; Lee, Jae Wook et al. (2018) Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/EBP? in Vasopressin-Sensitive Collecting Duct Cells. J Am Soc Nephrol 29:1490-1500 |
| Lee, Jae Wook; Alsady, Mohammad; Chou, Chung-Lin et al. (2018) Single-tubule RNA-Seq uncovers signaling mechanisms that defend against hyponatremia in SIADH. Kidney Int 93:128-146 |
| Gilmer, Gabrielle G; Deshpande, Venkatesh; Chou, Chung-Ling et al. (2018) Flow Resistance along the Rat Renal Tubule. Am J Physiol Renal Physiol : |
| Rinschen, Markus M; Limbutara, Kavee; Knepper, Mark A et al. (2018) From Molecules to Mechanisms: Functional Proteomics and Its Application to Renal Tubule Physiology. Physiol Rev 98:2571-2606 |
| Chou, Chung-Lin; Hwang, Gloria; Hageman, Daniel J et al. (2018) Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct. Am J Physiol Cell Physiol 314:C99-C117 |
| Guillén-Gómez, Elena; Bardají-de-Quixano, Beatriz; Ferrer, Sílvia et al. (2018) Urinary Proteome Analysis Identified Neprilysin and VCAM as Proteins Involved in Diabetic Nephropathy. J Diabetes Res 2018:6165303 |
| Saethang, Thammakorn; Hodge, Kenneth; Kimkong, Ingorn et al. (2018) AbDesigner3D: a structure-guided tool for peptide-based antibody production. Bioinformatics 34:2158-2160 |
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