The metabolome, the small molecule chemical entities involved in metabolism, has traditionally been studied with the aim of identifying biomarkers in the diagnosis and prediction of disease. However, the value of metabolomics has been redefined from a simple biomarker identification tool to a technology for the discovery of active drivers of biological processes. In this review, we describe the molecular mechanisms by which the active cell metabolome affects cellular physiology through modulation of other ‘omic’ levels, including the genome, epi-genome, transcriptome and proteome. This concept of activity screening guided by metabolomics to identify biologically active metabolites, or “activity metabolomics”, is having broad impact on biology.

Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown. We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli. Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations. Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

Vasopressin's action in renal cells to regulate water transport depends on protein phosphorylation. Here we used mass spectrometry-based quantitative phosphoproteomics to identify signaling pathways involved in the short-term V2-receptor-mediated response in cultured collecting duct cells (mpkCCD) from mouse. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) with two treatment groups (0.1 nM dDAVP or vehicle for 30 min), we carried out quantification of 2884 phosphopeptides. The majority (82%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called "basophilic" motifs consistent with activation of kinases of the AGC family. Increases in phosphorylation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of socalled "proline-directed" motifs, consistent with down-regulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphorylation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/ Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Both ERK and JNK were able to phosphorylate residue S261of aquaporin-2 in vitro, a site showing a decrease in phosphorylation in response to dDAVP in vivo. The data support roles for multiple vasopressin V2-receptor-dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function.aquaporin-2 | MAP kinase | mass spectrometry | protein kinase A | SILAC R egulation of osmotic water transport across the renal collecting duct epithelium is responsible for precise control of renal water excretion and regulation of the osmolality of body fluids. This precise control is achieved largely by actions of the peptide hormone vasopressin to regulate the water channel aquaporin-2 (AQP2) in collecting duct cells. Vasopressin binds to a G protein-coupled receptor (the type 2 vasopressin receptor or V2R) in the basolateral plasma membrane of collecting duct (CD) principal cells, triggering a complex signaling response that involves an increase in intracellular cAMP and spike-like, aperiodic increases in intracellular calcium (1, 2).The advent of methodologies for large-scale, mass spectrometry (MS)-based phosphoproteomics offers the opportunity for comprehensive discovery of signaling pathways in mammalian cells. In particular, the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) methodology is a metabolic-labeling approach that allows high-precision quantification of individual peptides in cultured cells using tandem MS (3), leading to powerful quantitative approaches to discovery of signaling pathways (4-6...

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We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).

We used a systems biology-based approach to investigate the basis of cell-specific expression of the water channel aquaporin-2 (AQP2) in the renal collecting duct. Computational analysis of the 5-flanking region of the AQP2 gene (Genomatix) revealed 2 conserved clusters of putative transcriptional regulator (TR) binding elements (BEs) centered at ؊513 bp (corresponding to the SF1, NFAT, and FKHD TR families) and ؊224 bp (corresponding to the AP2, SRF, CREB, GATA, and HOX TR families). Three other conserved motifs corresponded to the ETS, EBOX, and RXR TR families. To identify TRs that potentially bind to these BEs, we carried out mRNA profiling (Affymetrix) in mouse mpkCCDc14 collecting duct cells, revealing expression of 25 TRs that are also expressed in native inner medullary collecting duct. One showed a significant positive correlation with AQP2 mRNA abundance among mpkCCD subclones (Ets1), and 2 showed a significant negative correlation (Elf1 and an orphan nuclear receptor Nr1h2). Transcriptomic profiling in native proximal tubules (PT), medullary thick ascending limbs (MTAL), and IMCDs from kidney identified 14 TRs (including Ets1 and HoxD3) expressed in the IMCD but not PT or MTAL (candidate AQP2 enhancer roles), and 5 TRs (including HoxA5, HoxA9 and HoxA10) expressed in PT and MTAL but not in IMCD (candidate AQP2 repressor roles). In luciferase reporter assays, overexpression of 3 ETS family TRs transactivated the mouse proximal AQP2 promoter. The results implicate ETS family TRs in cell-specific expression of AQP2 and point to HOX, RXR, CREB and GATA family TRs as playing likely additional roles.aquaporin 2 ͉ kidney ͉ microarrays ͉ transcription ͉ vasopressin R enal water excretion is tightly regulated chiefly through effects of vasopressin on the molecular water channel, aquaporin-2 (AQP2) (1). AQP2 gene expression in the kidney is restricted to collecting duct principal cells and connecting tubule cells (2, 3). Aside from control of trafficking of AQP2-containing vesicles (1), AQP2 is regulated through changes in the total abundance of the AQP2 protein in collecting duct cells. Vasopressin increases the renal abundance of the AQP2 protein (4) via changes in AQP2 mRNA levels (5), in part by transcriptional regulation. Studies in transgenic mice in which 14-15 kb of the 5Ј-flanking region of the AQP2 gene was coupled to reporters established that cell-specific expression of the AQP2 gene in the collecting duct is dependent on cis-elements in this region (6, 7). Altered AQP2 protein abundance in the renal collecting duct is largely responsible for water balance abnormalities associated with diverse clinical states including lithium-induced diabetes insipidus, congestive heart failure, and the syndrome of inappropriate antidiuresis (1). Understanding the roles of AQP2 in these clinical states hinges largely on understanding the mechanism of cell-specific expression of the AQP2 gene.Sequencing of the 5Ј-flanking region of the AQP2 gene revealed several putative cis-binding element (BE) motifs including a c...

Primary cilia are sensory, antennae-like organelles present on the surface of many cell types. They have been involved in a variety of diseases collectively termed ciliopathies. As cilia are essential regulators of cell signaling, the composition of the ciliary membrane needs to be strictly regulated. To understand regulatory processes at the ciliary membrane, we report the targeting of a genetically engineered enzyme specifically to the ciliary membrane to allow biotinylation and identification of the membrane-associated proteome. Bioinformatic analysis of the comprehensive dataset reveals high-stoichiometric presence of actin-binding proteins inside the cilium. Immunofluorescence stainings and complementary interaction proteomic analyses confirm these findings. Depolymerization of branched F-actin causes further enrichment of the actin-binding and actin-related proteins in cilia, including Myosin 5a (Myo5a). Interestingly, Myo5a knockout decreases ciliation while enhanced levels of Myo5a are observed in cilia upon induction of ciliary disassembly. In summary, we present a novel approach to investigate dynamics of the ciliary membrane proteome in mammalian cells and identify actin-binding proteins as mechanosensitive components of cilia that might have important functions in cilia membrane dynamics.

Quantitative mass spectrometry was used to identify hormonedependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCl across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin). mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by "proline-directed" motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins. Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the Na + :K + :2Cl − cotransporter NKCC2, at Ser552 of the Na + :H + exchanger NHE3, and at Ser552 of β-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved.protein phosphatase | glucose transporters | mass spectrometry | ion transporters | protein kinase T he thick ascending limb (TAL) of Henle's loop is a nephron segment that plays a critical role in the control of mammalian water excretion. Active NaCl transport by the medullary TAL (mTAL) drives the countercurrent multiplication process that concentrates the urine (1). Hormones that increase the concentration of the intracellular second messenger, cAMP, have been shown to enhance the rate of NaCl transport in mTAL cells (2). These hormones include parathyroid hormone (PTH), calcitonin, glucagon, and vasopressin (2). Among these, only vasopressin plays a selective role in regulation of water balance. The molecular targets for cAMP-mediated regulation in the mTAL include the apical Na + : K + :2Cl − cotransporter NKCC2 (gene symbol: Slc12a1) and the apical Na + :H + exchanger NHE3 (Slc9a3) (3).The signaling network that accounts for cAMP-dependent regulation of these transporters is largely unk...