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Quantitative LC-MS/MS Analysis of Apical Membrane Proteome of mpkCCD Cells

ABSTRACT: This is a 2-plex SILAC-based quantitative proteomic study. It quantifies abundance changes of the apical membrane proteins of the kidney collecting duct model cells (mpkCCD) in response to vasopressin. Cells were labeled with normal or heavy lysine and arginine and then exposed to vehicle or the vasopressin analogy dDAVP (1 nM, 1 hour). Apical membrane proteins of vehicle and dDAVP-treated cells were enriched via surface biotinylation and affinity purification prior to LC-MS/MS analysis. Database search and protein quantification were performed using the SEQUEST algorithm included in the Proteome Discover Version 1.3 (Thermo Scientific). The database used for the spectral searching was prepared from the mouse RefSeq database plus common contaminants.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Ming-Jiun Yu

PROVIDER: PXD000399 | Pride | 2013-10-02

REPOSITORIES: Pride

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Quantitative apical membrane proteomics reveals vasopressin-induced actin dynamics in collecting duct cells.

Loo Chin-San CS Chen Cheng-Wei CW Wang Po-Jen PJ Chen Pei-Yu PY Lin Shu-Yu SY Khoo Kay-Hooi KH Fenton Robert A RA Knepper Mark A MA Yu Ming-Jiun MJ

Proceedings of the National Academy of Sciences of the United States of America 20131001 42

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vas ...[more]

PMID: 24085853

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Expression data from mouse collecting duct cell, mpkCCD, in response to a peptide hormone vasopressin analog, dDAVP

Project description:Vasopressin is the major hormone that regulates renal water excretion. It does so by binding to a receptor in renal collecting duct cells, triggering signaling pathways that ultimately regulate the abundance, location, and activity of the water channel protein aquaporin 2. We took an advantage of quantitative large scale proteomic technologies and oligonucleotide microarrays to quantify steady state changes in protein and transcript abundances in response to vasopressin in a collecting duct cell line, mpkCCD clone 11 (Yu et al. PNAS 2009, 106:2441-2446). This cell line originally developed by Alan Vandewalle’s group recapitulates vasopressin-mediated AQP2 expression and phosphorylation as seen in native colleting duct cells. The mpkCCD cells were grown on membrane supports to permit polarization. Once transepithelial resistance reached 5kohm per centimeter square and higher, the cells were exposed to the vasopressin V2 receptor analog, dDAVP, at a physiological concentration, 0.1nM, for 5 days. Control experiments were done with cells exposed to vehicle alone. Total RNA was harvested and processed for transcript expression analysis using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Each experimental treatment, vehicle and dDAVP, was repeated 3 times.

2010-12-09 | E-GEOD-19938 | biostudies-arrayexpress

Time-course transcriptome profiling in vasopressin-treated mouse kidney collecting duct mpkCCD

Project description:Purpose: The goal of this study is to identify vasopressin-regulated genes in mouse kidney collecting duct cell line mpkCCD. To explore dynamic regulation of vasoressin-mediated transcriptional regulation, transcriptome of vasopressin-treated cells at four different time points (3h, 6h, 12h, and 24h) were profiled and analyzed. Methods: Total RNAs were isolated from vasopressin-treated mpkCCD cells at different time points (3h, 6h, 12h, and 24h). Three replicates for vehicle- or vasopressin-treated group were generated at each tested time point. cDNA libraries were prepared using a Nextera DNA library preparation protocol. The sequence reads from Illumina HiSeq3000 platform were qualified and quantified at the transcript level using Salmon (0.14.1). Differential expression analysis were performed using edgeR. Results: mRNA profiles of mouse kidney collecting duct mpkCCD cells treated with vasopressin analog (dDAVP) for four different time potins (3h, 6h, 12h, and 24h) were generated using an optimized RNA-Seq workflow. Transcript level quantification using a pseudo-alignment quantification method (Salmon) was performed to calculate transcript abundance in each sample. Then differential expression analysis identified the differentially expressed genes including Aqp2 gene at each time point comparison (dDAVP vs vehicle, FDR < 0.05). In addition, several new vasopressin-responsive genes that have not been elucidated before were identified. Conclusions: This study revealed dynamic changes of vasopressin-responsive gene expression within 24 hours in mouse kidney collecting duct cells. The results from time-course transcriptome profiling identified the known vasopressin-responsive genes and several novel gene that are regulated temporally.

2021-07-22 | GSE163566 | GEO

Combined RNA-seq and ChIP-seq Identifies Gene Targets for Transcriptional Regulation in Vasopressin-Sensitive Collecting Duct Cells [RNA-seq]

Project description:We sequenced mRNAs and mapping the binding of RNA polymerase 2 in collecting duct cells treat with Vasopressin or vehicle. Confluent mpkCCD cell monolayers were maintained in serum-free medium for 24 h prior to treatment with or without 0.1 nM dDAVP in serum-free medium for 24 h. followed RNA polymerase 2 Chip seq.

2016-07-01 | E-GEOD-79525 | biostudies-arrayexpress

Transcriptome changes in response to dDAVP stimulation in CRISPR/Cas9 mediated Camk2d knock-out mpkCCD cells

Project description:The Ca2+/CaM-dependent protein kinase 2-delta (CAMK2D) has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water and salt excretion by the kidney. RNA sequancing and quantitative proteomics analyses identified the expression of multiple CAMK2 isoforms, with CAMK2D being the most abundant in collecting duct cells. To investigate the role of CAMK2D in regulating RNA expression in response to vasopressin signaling, the transcriptome of CRISPR/Cas9-mediated Camk2d knock-out mpkCCD cells was profiled using RNA-Seq in the presence of vasopressin analogue dDAVP.

2023-06-01 | GSE228762 | GEO

ATAC-Seq analysis in vasopressin-responsive mouse renal collecting duct mpkCCD cells

Project description:Vasopressin, a peptide hormone, controls renal water excretion, largely through regulation of water channel aquaporin-2 (AQP2) in the renal collecting duct. There are two regulatory mechanisms of AQP2: 1) short-term regulation by membrane trafficking of AQP2; and 2) long-term regulation involving vasopressin-induced changes of protein abundance of AQP2 through regulation of gene transcription and protein half-life. Vasopressin binds a G protein-coupled receptor (V2R) activating several downstream signaling pathways. At downstream of V2R activation, many of transcription factors involve gene transcription process associated with status of chromatin structure. ATAC-Seq (Assay for Transpoase-Accessible Chromatin using Sequencing) is a recent technique to study chromatin accessibility (Buenrostro et al. Nat Methods 2013). We carried out ATAC-Seq following standard an ATAC-Seq protocol in mpkCCD cells treated with vehicle or dDAVP for 30 minutes.

2018-03-26 | GSE108786 | GEO

Transcription profiling of mpkCCDc14 cells cloned into colonies with varying aquaporin 2 (AQP2) expression levels in the presence of vasopressin analogy dDAVP to identify transcripts responsible for cell-specific expression of AQP2

Project description:This series of microarray data contain transcript intensity of mpkCCD cells. Experiment Overall Design: The mpkCCDc14 cells were cloned into colonies with varying aquaporin 2 (AQP2) expression levels in the presence of vasopressin analogy dDAVP. Transcript profiling was done for the original cells and cell clones 2, 3, 9, 10, and 11. By studying transcripts that correlate with AQP2 mRNA levels among cell clones, the objective was to identify transcripts responsible for cell-specific expression of AQP2.

2009-01-31 | E-GEOD-13667 | biostudies-arrayexpress

Vasopressin V2 Receptor, Tolvaptan, and ERK1/2 Phosphorylation in the Renal Collecting Duct II

Project description:Activation of ERK1 and ERK2 is essential in regulation of a wide variety of cellular and physiological processes. In native inner medullary collecting ducts, vasopressin (AVP) working through the V2 subtype vasopressin receptor (V2R)-mediated activation of Gαs, inhibits ERK1 and ERK2 activity. However, it has been reported that V2R can signal independently of Gαs through the activation of β-arrestin, which activates ERK1 and ERK2. Vaptans, V2R antagonists that function as so-called “inverse agonists”, have the potential of promoting cell proliferation via β-arrestin-dependent ERK activation. Here we use the mpkCCD cell line which natively expresses V2R to investigate the effects of AVP, the V2-selective analog dDAVP, and tolvaptan on ERK1 and ERK2 phosphorylation and activation. We demonstrated that ERK1 and ERK2 phosphorylation in mpkCCD cells was significantly reduced by either AVP or dDAVP, in contrast to the increases seen in non-collecting duct cells overexpressing V2R. We also found that tolvaptan has a strong effect to increase ERK1 and ERK2 phosphorylation in the presence of dDAVP and that the tolvaptan effect to increase ERK1 and ERK2 phosphorylation is absent in PKA-null mpkCCD cells. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and, therefore, not mediated by the β-arrestin pathway. Overall, the studies show that AVP decreases and that tolvaptan increases ERK1 and ERK2 activation in cells expressing V2R at endogenous levels, and provide no evidence for a role for β-arrestin in the regulation of ERK1 and ERK2 activity.

2023-11-17 | GSE243773 | GEO

Glucocorticoid receptor maintains vasopressin responses in kidney collecting duct cells

Project description:Water permeability of the kidney collecting ducts is regulated in part by the amount of the molecular water channel protein aquaporin-2 (AQP2), whose expression, in turn, is regulated by the pituitary peptide hormone vasopressin. We previously showed that stable glucocorticoid receptor knockdown diminished the vasopressin-induced Aqp2 gene expression in the collecting duct cell model mpkCCD. Here, we investigated the pathways regulated by the glucocorticoid receptor by comparing transcriptomes of the mpkCCD cells with or without stable glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown downregulated 5,394 transcripts associated with 55 KEGG pathways including “vasopressin-regulated water reabsorption,” indicative of positive regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the downregulation of the vasopressin V2 receptor transcript upon glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown upregulated 3,785 transcripts associated with 42 KEGG pathways including the “TNF signaling pathway” and “TGFβ signaling pathway,” suggesting the negative regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the upregulation of TNF and TGFβ receptor transcripts upon glucocorticoid receptor knockdown. TNF or TGFβ inhibitor alone, in the absence of vasopressin, did not induce Aqp2 gene transcription. However, TNF or TGFβ blunted the vasopressin-induced Aqp2 gene expression. In particular, TGFβ reduced vasopressin-induced increases in Akt phosphorylation without inducing epithelial-to-mesenchymal transition or interfering with vasopressin-induced apical AQP2 trafficking. In summary, our RNA-seq transcriptomic comparison revealed positive and negative regulatory pathways maintained by the glucocorticoid receptor for the vasopressin-induced Aqp2 gene expression.

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Effect of dDAVP on outer medullary gene expression in Brattleboro rats

Project description:Activation of the renal urine concentrating mechanism by vasopressin requires the coordinated regulation of multiple gene products including ion transporters, and water channels as well as regulatory proteins like protein kinases and phosphatases or enzymes involved in the energy-metabolism of the cells. We used microarray analysis of AVP-regulated gene products in a rat model of central diabetes insipidus (DI) to generate a comprehensive database documenting these changes. For microarray studies young (8 weeks) adult male Brattleboro rats were randomly divided into 2 groups (n = 3 per group) and treated for 3 days with either 1-desamino-8-D-Arg vasopressin (dDAVP; 5ng/h; Sigma Aldrich, Germany) or vehicle via osmotic minipump (ALZET minipump model 2001, Charles River, Sulzfeld, Germany). At the end of the treatment period animals were sacrificed and the kidneys removed. The outer medulla was isolated and used for cDNA generation and subsequent microarray analysis using [Rat230_2] Affymetrix Rat Genome 230 2.0 Array.

2012-03-01 | E-GEOD-34225 | biostudies-arrayexpress

Vasopressin (AVP) and the V2R specific agonist dDAVP 7 day infusion into Aquaporin 1 knockout animals

Project description:Vasopressin, the antidiuretic hormone, acts on the renal collecting duct. In this experiment both vasopressin (AVP) and the V2R specific agonist dDAVP were infused into Aquaporin 1 knockout animals for 7 days. The aim of the experiment was to identify genes increased by vasopressin receptors in the renal medullary collecting ducts, in the absence of an increase in renal medullary osmolarity (the AQP1 knockouts are concentrating mechanism knockouts). All experiments used inner medulla tissue for the RNA isolation. Hybridizations were performed that compared kidney inner medulla total RNA from three control mice against kidney medulla total RNA from 3 mice infused with either arginine vasopressin (AVP) or des-amino-D-arginine vasopressin (dDAVP).

2007-10-06 | E-GEOD-5953 | biostudies-arrayexpress

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