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. ChIP-Seq quantification of binding sites for RNA polymerase II was combined with RNA-Seq quantification of transcript abundances to identify genes whose transcription is regulated by vasopressin. (View curated dataset at https://helixweb.nih.gov/ESBL/Database/Vasopressin/). The analysis revealed only 35 vasopressin-regulated genes (of 3659) including Aqp2. Increases in RNA polymerase II binding and mRNA abundances for Aqp2 far outstripped corresponding measurements for all other genes, consistent with the conclusion that vasopressin-mediated transcriptional regulation is highly selective for Aqp2. Despite the overall selectivity of the net transcriptional response, vasopressin treatment was associated with increased RNA polymerase II binding to the promoter proximal region of a majority of expressed genes, suggesting a nearly global positive regulation of transcriptional initiation with transcriptional pausing. Thus, the overall net selectivity appears to be a result of selective control of transcriptional elongation.

Proteomic profiling of nuclear fractions from native renal inner medullary collecting duct cells.

The control of renal water excretion occurs in part by regulation of transcription in response to vasopressin in cells of the collecting duct. A systems biology-based approach to understanding transcriptional control in renal collecting duct cells depends on knowledge of what transcription factors and other regulatory proteins are present in the cells' nuclei. The goal of this article is to report comprehensive proteomic profiling of cellular fractions enriched in nuclear proteins from native inner medullary collecting duct (IMCD) cells of the rat. Multidimensional separation procedures and state-of-the art protein mass spectrometry produced 18 GB of spectral data that allowed the high-stringency identification of 5,048 proteins in nuclear pellet (NP) and nuclear extract (NE) fractions of biochemically isolated rat IMCD cells (URL: https://helixweb.nih.gov/ESBL/Database/IMCD_Nucleus/). The analysis identified 369 transcription factor proteins out of the 1,371 transcription factors coded by the rat genome. The analysis added 1,511 proteins to the recognized proteome of rat IMCD cells, now amounting to 8,290 unique proteins. Analysis of samples treated with the vasopressin analog dDAVP (1 nM for 30 min) or its vehicle revealed 99 proteins in the NP fraction and 88 proteins in the NE fraction with significant changes in spectral counts (Fisher exact test, P < 0.005). Among those altered by vasopressin were seven distinct histone proteins, all of which showed decreased abundance in the NP fraction, consistent with a possible effect of vasopressin to induce chromatin remodeling. The results provide a data resource for future studies of vasopressin-mediated transcriptional regulation in the renal collecting duct.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. II. Bioinformatic analysis of vasopressin signaling.

Vasopressin controls osmotic water transport in the renal collecting duct through regulation of aquaporin-2 (AQP2). We carried out bioinformatic analysis of quantitative proteomic data from the accompanying article to investigate the mechanisms involved. The experiments used stable isotope labeling by amino acids in cell culture in cultured mpkCCD cells to quantify each protein species in each of five differential-centrifugation (DC) fractions with or without the vasopressin analog 1-desamino-8-d-arginine-vasopressin (dDAVP). The mass spectrometry data and parallel Western blot experiments confirmed that dDAVP addition is associated with an increase in AQP2 abundance in the 17,000-g pellet and a corresponding decrease in the 200,000-g pellet. Remarkably, all subunits of the cytoplasmic ribosome also increased in the 17,000-g pellet in response to dDAVP (P < 10(-34)), with a concomitant decrease in the 200,000-g pellet. Eukaryotic translation initiation complex 3 (eIF3) subunits underwent parallel changes (P < 10(-6)). These findings are consistent with translocation of assembled ribosomes and eIF3 complexes into the rough endoplasmic reticulum in response to dDAVP. Conversely, there was a systematic decrease in small GTPase abundances in the 17,000-g fraction. In contrast, most proteins, including protein kinases, showed no systematic redistribution among DC fractions. Of the 521 protein kinases coded by the mouse genome, 246 were identified, but many fewer were found to colocalize with AQP2 among DC fractions. Bayes' rule was used to integrate the new colocalization data with prior data to identify protein kinases most likely to phosphorylate aquaporin-2 at Ser(256) (Camk2b > Camk2d > Prkaca) and Ser(261) (Mapk1 = Mapk3 > Mapk14).

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). Desmopressin altered the translation rate rather than the stability of most responding proteins, but it significantly increased both the translation rate and the half-life of AQP2. In addition, proteins associated with vasopressin action, including Mal2, Akap12, gelsolin, myosin light chain kinase, annexin-2, and Hsp70, manifested altered translation rates. Interestingly, desmopressin increased the translation of seven glutathione S-transferase proteins and enhanced protein S-glutathionylation, uncovering a previously unexplored vasopressin-induced post-translational modification. Additional bioinformatic analysis of the mpkCCD proteome indicated a correlation between protein function and protein half-life. In particular, processes that are rapidly regulated, such as transcription, endocytosis, cell cycle regulation, and ubiquitylation are associated with proteins with especially short half-lives. These data extend our understanding of the mechanisms underlying vasopressin signaling and provide a broad resource for additional investigation of collecting duct function (http://helixweb.nih.gov/ESBL/Database/ProteinHalfLives/index.html).

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. Due to the high urea concentration, we hypothesized that carbamylation can occur endogenously within the rat inner medulla. Using immunoblotting of rat kidney cortical and medullary homogenates with a carbamyl-lysine specific antibody, we showed that carbamylation is present in a large number of inner medullary proteins. Using protein mass spectrometry (LC-MS/MS) of rat renal inner medulla, we identified 456 unique carbamylated sites in 403 proteins, including many that play important physiological roles in the renal medulla [Data can be accessed at https://helixweb.nih.gov/ESBL/Database/Carbamylation/Carbamylation_peptide_sorted.html]. We conclude that protein carbamylation occurs endogenously in the kidney, modifying many physiologically important proteins.

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. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis and caspase cleavage assays demonstrated that 1-desamino-8-d-arginine vasopressin (dDAVP) inhibited apoptosis induced by various agents (staurosporine, actinomycin D, and cycloheximide) in cultured mouse cortical collecting duct cells (mpkCCD). Incubation with dDAVP also inhibited apoptosis induced by the phosphatidylinositol 3-kinase (PI3K) pathway inhibitor LY294002, suggesting that the antiapoptotic effects of dDAVP are largely independent of PI3K signaling. The V2 receptor antagonist SR121463 completely abolished the antiapoptotic effects of dDAVP. In addition, incubation with 8-cpt-cAMP, a cell-permeable analog of cAMP, reproduced the antiapoptotic effects of dDAVP. Both dDAVP and 8-cpt-cAMP increased phosphorylation of proapoptotic Bcl-2 family members Bad and Bok. Bad phosphorylation at Ser-112 and Ser-155 is known to inhibit its proapoptotic activity. Preincubation with H89 blocked dDAVP-induced phosphorylation of both Bad and Bok, suggesting dependence on protein kinase A (PKA). This study provides evidence that AVP can inhibit apoptosis through the V2 receptor and downstream cAMP-mediated pathways in mammalian kidney. The antiapoptotic action of AVP may be relevant to a number of physiological and pathophysiological conditions including osmotic tolerance in the inner medulla, escape from AVP-induced antidiuresis, and polycystic kidney disease.

Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal.

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.

Proneural gene requirement for hair cell differentiation in the zebrafish lateral line.

The lateral line system comprises an array of mechanosensory organs, the neuromasts, distributed over the body surface. Each neuromast consists of a patch of mechanosensory hair cells surrounded by support cells. We show that, in the zebrafish, two proneural genes are essential for differentiation of the hair cells, neuroD (nrd) and atonal homolog 1 (ath1). Gene knockdown experiments demonstrate that loss of function of either gene, but not of the related proneural gene neurogenin1 (ngn1), abrogate the appearance of hair cell markers. This is in contrast to other sensory systems, such as the neurons of the lateral line ganglion, where nrd is regulated by ngn1 and not by ath1. Overexpression of ath1 can induce nrd, and the phenotype produced by loss of ath1 function can be partially rescued by injection of nrd mRNA. This supports the conclusion that the activation of nrd probably requires ath1 in the hair cell lineage, whereas in sensory neurons nrd activation requires ngn1. We propose that the emergence of two atonal homologs, ath1 and ngn1, allowed the cellular segregation of mechanoreception and signal transmission that were originally performed by a single cell type as found in insects.

Control of cell migration in the zebrafish lateral line: implication of the gene "tumour-associated calcium signal transducer," tacstd.

The sensory organs of the zebrafish lateral-line system (neuromasts) originate from migrating primordia that move along precise pathways. The posterior primordium, which deposits the neuromasts on the body and tail of the embryo, migrates along the horizontal myoseptum from the otic region to the tip of the tail. This migration is controlled by the chemokine SDF1, which is expressed along the prospective pathway, and by its receptor CXCR4, which is expressed by the migrating cells. In this report, we describe another zebrafish gene that is heterogeneously expressed in the migrating cells, tacstd. This gene codes for a membrane protein that is homologous to the TACSTD1/2 mammalian proteins. Inactivation of the zebrafish tacstd gene results in a decrease in proneuromast deposition, suggesting that tacstd is required for the deposition process.

Activation of the neuronal c-Abl tyrosine kinase by amyloid-beta-peptide and reactive oxygen species.

The deposition and accumulation of amyloid-beta-peptide (Abeta) in the brain are considered a sine qua non for Alzheimer's disease. The experimental delivery of fibrilized Abeta serves as a cellular model for several facets of the disease including the induction of synaptic dysfunction and apoptosis. c-Abl kinase is involved in the regulation of apoptosis and its pro-apoptotic function is in part mediated by its interaction with p73, a p53 homologue. We found that c-Abl activation is involved in cell signals that regulate neuronal death response to Abeta fibrils. Abeta peptide fibrils induced an increase of the c-Abl activity in rat hippocampal neurons as well as an increase in nuclear p73 protein levels and the p73-c-Abl complex. The neuronal cell death induced by Abeta fibrils was prevented by the inhibition of c-Abl with imatinib mesylate (Gleevec or STI571) and by the inhibition c-Abl expression by RNAi. These results directly point to a therapeutic strategy for the treatment of Alzheimer's disease.