Systems-level analysis reveals selective regulation of Aqp2 gene expression by vasopressin.

Vasopressin-mediated regulation of renal water excretion is defective in a variety of water balance disorders in humans. It occurs in part through long-term mechanisms that regulate the abundance of the aquaporin-2 water channel in renal collecting duct cells. Here, we use deep DNA sequencing in mouse collecting duct cells to ask whether vasopressin signaling selectively increases Aqp2 gene transcription or whether it triggers a broadly targeted transcriptional network.

Autophagic degradation of aquaporin-2 is an early event in hypokalemia-induced nephrogenic diabetes insipidus.

Hypokalemia (low serum potassium level) is a common electrolyte imbalance that can cause a defect in urinary concentrating ability, i.e., nephrogenic diabetes insipidus (NDI), but the molecular mechanism is unknown.

Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics.

Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g.

A knowledge base of vasopressin actions in the kidney.

Biological information is growing at a rapid pace, making it difficult for individual investigators to be familiar with all information that is relevant to their own research. Computers are beginning to be used to extract and curate biological information; however, the complexity of human language used in research papers continues to be a critical barrier to full automation of knowledge extraction. Here, we report a manually curated knowledge base of vasopressin actions in renal epithelial cells that is designed to be readable either by humans or by computer programs using natural language processing algorithms.

Exploiting Thread-Level and Instruction-Level Parallelism to Cluster Mass Spectrometry Data using Multicore Architectures.

Modern mass spectrometers can produce large numbers of peptide spectra from complex biological samples in a short time. A substantial amount of redundancy is observed in these data sets from peptides that may get selected multiple times in Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) experiments. A large number of spectra do not get mapped to specific peptide sequences due to low signal-to-noise (S/N) ratio of the spectra from these machines.

Early targets of lithium in rat kidney inner medullary collecting duct include p38 and ERK1/2.

Almost half of patients receiving lithium salts have nephrogenic diabetes insipidus. Chronic lithium exposure induces AQP2 downregulation and changes in the cellular composition of the collecting duct. In order to understand these pathophysiological events, we determined the earliest lithium targets in rat inner medullary collecting duct (IMCD) by examining changes in the IMCD phosphoproteome after acute lithium administration.

Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256.

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known.

PhosSA: Fast and accurate phosphorylation site assignment algorithm for mass spectrometry data.

Phosphorylation site assignment of high throughput tandem mass spectrometry (LC-MS/MS) data is one of the most common and critical aspects of phosphoproteomics. Correctly assigning phosphorylated residues helps us understand their biological significance. The design of common search algorithms (such as Sequest, Mascot etc.

Endogenous carbamylation of renal medullary proteins.

Protein carbamylation is a post-translational modification that can occur in the presence of urea. In solution, urea is in equilibrium with ammonium cyanate, and carbamylation occurs when cyanate ions react with the amino groups of lysines, arginines, protein N-termini, as well as sulfhydryl groups of cysteines. The concentration of urea is elevated in the renal inner medulla compared with other tissues.

CAMS-RS: Clustering Algorithm for Large-Scale Mass Spectrometry Data Using Restricted Search Space and Intelligent Random Sampling.

High-throughput mass spectrometers can produce massive amounts of redundant data at an astonishing rate with many of them having poor signal-to-noise (S/N) ratio. These low S/N ratio spectra may not get interpreted using conventional spectra-to-database matching techniques. In this paper, we present an efficient algorithm, CAMS-RS (Clustering Algorithm for Mass Spectra using Restricted Space and Sampling) for clustering of raw mass spectrometry data.

Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct.

Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit β-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.

Proteome-wide measurement of protein half-lives and translation rates in vasopressin-sensitive collecting duct cells.

Vasopressin regulates water excretion, in part, by controlling the abundances of the water channel aquaporin-2 (AQP2) protein and regulatory proteins in the renal collecting duct. To determine whether vasopressin-induced alterations in protein abundance result from modulation of protein production, protein degradation, or both, we used protein mass spectrometry with dynamic stable isotope labeling in cell culture to achieve a proteome-wide determination of protein half-lives and relative translation rates in mpkCCD cells. Measurements were made at steady state in the absence or presence of the vasopressin analog, desmopressin (dDAVP).

An Efficient Dynamic Programming Algorithm for Phosphorylation Site Assignment of Large-Scale Mass Spectrometry Data.

Phosphorylation site assignment of large-scale data from high throughput tandem mass spectrometry (LC-MS/MS) data is an important aspect of phosphoproteomics. Correct assignment of phosphorylated residue(s) is important for functional interpretation of the data within a biological context. Common search algorithms (Sequest etc.

An Efficient Algorithm for Clustering of Large-Scale Mass Spectrometry Data.

High-throughput spectrometers are capable of producing data sets containing thousands of spectra for a single biological sample. These data sets contain a substantial amount of redundancy from peptides that may get selected multiple times in a LC-MS/MS experiment. In this paper, we present an efficient algorithm, CAMS (Clustering Algorithm for Mass Spectra) for clustering mass spectrometry data which increases both the sensitivity and confidence of spectral assignment.

Assessment of the effect of 24-hour aldosterone administration on protein abundance in fluorescence-sorted mouse distal renal tubules by mass spectrometry.

Background/aims: Aldosterone exerts multiple long-term effects on the distal renal tubules. The aim of this study was to establish a method for identifying proteins in these tubules that change in abundance by only 24-hour aldosterone administration.

Methods: Mice endogenously expressing green fluorescent protein (eGFP) in the connecting tubule and cortical collecting ducts were treated with a subcutaneous injection of 2.

Vasopressin inhibits apoptosis in renal collecting duct cells.

The peptide hormone arginine vasopressin (AVP) plays a critical role in regulating salt and water transport in the mammalian kidney. Recent studies have also demonstrated that AVP can promote cell survival in neuronal cells through V1 receptors. The current study addresses whether AVP can inhibit apoptosis in kidney collecting duct cells via V2 receptors and also explores the downstream signaling pathways regulating this phenomenon.

Aquaporin-2 regulation in health and disease.

Aquaporin-2 (AQP2), the vasopressin-regulated water channel of the renal collecting duct, is dysregulated in numerous disorders of water balance in people and animals, including those associated with polyuria (urinary tract obstruction, hypokalemia, inflammation, and lithium toxicity) and with dilutional hyponatremia (syndrome of inappropriate antidiuresis, congestive heart failure, cirrhosis). Normal regulation of AQP2 by vasopressin involves 2 independent regulatory mechanisms: (1) short-term regulation of AQP2 trafficking to and from the apical plasma membrane, and (2) long-term regulation of the total abundance of the AQP2 protein in the cells. Most disorders of water balance are the result of dysregulation of processes that regulate the total abundance of AQP2 in collecting duct cells.

CPhos: a program to calculate and visualize evolutionarily conserved functional phosphorylation sites.

Profiling using high-throughput MS has discovered an overwhelming number of novel protein phosphorylation sites ("phosphosites"). However, the functional relevance of these sites is not always clear. In light of recent studies on the evolutionary mechanism of phosphorylation, we have developed CPhos, a Java program that can assess the conservation of phosphosites among species using an information theory-based approach.

Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells.

Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions.

Identifying protein kinase target preferences using mass spectrometry.

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences.

Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells.

Gene expression databases for kidney epithelial cells.

The 21st century has seen an explosion of new high-throughput data from transcriptomic and proteomic studies. These data are highly relevant to the design and interpretation of modern physiological studies but are not always readily accessible to potential users in user-friendly, searchable formats. Data from our own studies involving transcriptomic and proteomic profiling of renal tubule epithelia have been made available on a variety of online databases.

Dynamics of the G protein-coupled vasopressin V2 receptor signaling network revealed by quantitative phosphoproteomics.

G protein-coupled receptors (GPCRs) regulate diverse physiological processes, and many human diseases are due to defects in GPCR signaling. To identify the dynamic response of a signaling network downstream from a prototypical G(s)-coupled GPCR, the vasopressin V2 receptor, we have carried out multireplicate, quantitative phosphoproteomics with iTRAQ labeling at four time points following vasopressin exposure at a physiological concentration in cells isolated from rat kidney. A total of 12,167 phosphopeptides were identified from 2,783 proteins, with 273 changing significantly in abundance with vasopressin.

NHLBI-AbDesigner: an online tool for design of peptide-directed antibodies.

Investigation of physiological mechanisms at a cellular level often requires production of high-quality antibodies, frequently using synthetic peptides as immunogens. Here we describe a new, web-based software tool called NHLBI-AbDesigner that allows the user to visualize the information needed to choose optimal peptide sequences for peptide-directed antibody production (http://helixweb.nih.