Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P41181 (collecting duct)
 5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.
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PMID:High-throughput identification of IMCD proteins using LC-MS/MS. 1644 82

Urinary exosomes and microvesicles (EMV) are promising biomarkers for renal diseases. Although the density of EMV is very low in urine, large quantity of urine can be easily obtained. In order to analyze urinary EMV mRNA, a unique filter device to adsorb urinary EMV from 10 mL urine was developed, which is far more convenient than the standard ultracentrifugation protocol. The filter part of the device is detachable and aligned to a 96-well microplate format, therefore multiple samples can be processed simultaneously in a high throughput manner following the isolation step. For EMV mRNA quantification, the EMV on the filter is lysed directly by adding lysis buffer and transferred to an oligo(dT)-immobilized microplate for mRNA isolation followed by cDNA synthesis and real-time PCR. Under the optimized assay condition, our method provided comparable or even superior results to the standard ultracentrifugation method in terms of mRNA assay sensitivity, linearity, intra-assay reproducibility, and ease of use. The assay system was applied to quantification of kidney-specific mRNAs such as NPHN and PDCN (glomerular filtration), SLC12A1 (tubular absorption), UMOD and ALB (tubular secretion), and AQP2 (collecting duct water absorption). 12-hour urine samples were collected from four healthy subjects for two weeks, and day-to-day and individual-to-individual variations were investigated. Kidney-specific genes as well as control genes (GAPDH, ACTB, etc.) were successfully detected and confirmed their stable expressions through the two-week study period. In conclusion, this method is readily available to clinical studies of kidney diseases.
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PMID:Development of glomerulus-, tubule-, and collecting duct-specific mRNA assay in human urinary exosomes and microvesicles. 2527 11

Exosomes are nanometer-scale, cell-derived vesicles that contain various molecules including nucleic acids, proteins, and lipids. These vesicles can release their cargo into adjacent or distant cells and mediate intercellular communication and cellular function. Here we examined the regulation of epithelial sodium channels in mpkCCD cells and distal tubule Xenopus 2F3 cells by exosomes isolated from proximal tubule LLC-PK1 cells. Cultured mpkCCD cells were stained with CTX coupled to a green fluorophore in order to label the cell membranes and freshly isolated exosomes from LLC-PK1 cells were labeled with the red lipophilic dye PKH26 in order to visualize uptake of exosomes into the cells. Single-channel patch clamp recordings showed the open probability of ENaC in Xenopus 2F3 cells and in freshly isolated split-open tubules decreased in response to exogenous application of exosomes derived from LLC-PK1 proximal tubule cells. Active GAPDH was identified within exosomes derived from proximal tubule LLC-PK1 cells. The effect on ENaC activity in Xenopus 2F3 cells was blunted after application of exosomes transfected with the GAPDH inhibitor heptelidic acid. Also, we show GAPDH and ENaC subunits associate in mpkCCD cells. These studies examine a potential role for exosomes in the regulation of ENaC activity and examine a possible mechanism for communication from proximal tubule cells to distal tubule and collecting duct cells.
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PMID:Exosomal GAPDH from Proximal Tubule Cells Regulate ENaC Activity. 2780 15