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 action for arginine vasopressin (AVP). To examine the mode of action of AVP in this segment, we measured the change in transepithelial resistance of cultured rat IMCD cells grown to confluence on collagen-coated Millicell culture plate inserts in response to AVP. Resistance was measured by use of an EVOM voltage-ohm meter. AVP at 10(-11)-10(-8) M caused a fall in resistance of 6.9 +/- 1.3 to 14.0 +/- 1.4 omega.cm2 (P less than 0.05 to less than 0.01 vs. no AVP), which was reversed by removal of AVP or addition of 10(-6) M amiloride. Pretreating the apical surface of IMCD cells with trypsin had no effect on resistance but totally prevented the antidiuretic hormone-induced fall in resistance. Pretreating the apical surface with trypsin and amiloride did not prevent the fall in resistance to AVP. Addition of 10(-9) M AVP or 10(-6) M forskolin increased 2-min adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by 55 or 96%, respectively. Stimulation of endogenous cAMP accumulation by forskolin or the addition of exogenous 8-bromo-cAMP caused no change in resistance. To examine the relationship between intracellular calcium [( Ca2+]i) and AVP action, the response of [Ca2+]i to AVP was measured by use of fura-2. AVP induced no change in [Ca2+]i in IMCD cells in suspension, on glass cover slips, or on permeable supports. Ionomycin (25 nM) increased [Ca2+]i in IMCD cells and lowered resistance across monolayers, but the fall in resistance was not blocked by amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:AVP reduces transepithelial resistance across IMCD cell monolayers. 169 8

A homogeneous population of single cells from the thick ascending limb of Henle's loop (TALH) has been isolated from the rabbit kidney medulla. A total medullary cell suspension was prepared by a series of collagenase, hyaluronidase, and trypsin digestions and separated on a Ficoll gradient (2.6-30.7% wt/wt). Morphologically, the cells isolated from the TALH were homogeneous and showed polarity within their plasma membrane structure, with a few blunt microvilli on their apical surface and deep infoldings of the basal-lateral membrane. Biochemically, the TALH cells were highly enriched in calcitonin-sensitive adenylate cyclase and Na, K-ATPase. Alkaline phosphatase and arginine vasopressin-sensitive adenylate cyclase, highly concentrated in proximal tubule and collecting duct, were present only in low concentrations in the TALH cells. Additionally, furosemide, a diuretic inhibiting sodium chloride transport in the TALH in vivo, inhibited oxygen consumption of the TALH cells in a dose-dependent manner. The TALH cells were viable, as judged by morphological appearance, trypan blue exclusion, the response of oxygen consumption to 2,4-dinitrophenol, succinate and ouabain, and the cellular Na, K and ATP levels.
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PMID:Separation of renal medullary cells: isolation of cells from the thick ascending limb of Henle's loop. 625 27

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4-5 days after seeding. As expected for the involvement of epithelial Na+ channels, alpha-, beta-, and gamma-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10-100 nM) or aldosterone (Aldo, 10(-6)-10(-7) M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl- secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.
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PMID:Regulation of sodium transport in M-1 cells. 984 18

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.
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PMID:Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells. 1056 30

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.
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PMID:Calpain-mediated AQP2 proteolysis in inner medullary collecting duct. 1264 65

Vasopressin acts on renal collecting duct cells to stimulate translocation of aquaporin-2 (AQP2)-containing membrane vesicles from throughout the cytoplasm to the apical region. The vesicles fuse with the plasma membrane to increase water permeability. To identify the intracellular membrane compartments that contain AQP2, we carried out LC-MS/MS-based proteomic analysis of immunoisolated AQP2-containing intracellular vesicles from rat inner medullary collecting duct. Immunogold electron microscopy and immunoblotting confirmed heavy AQP2 labeling of immunoisolated vesicles. Vesicle proteins were separated by SDS-PAGE followed by in-gel trypsin digestion in consecutive gel slices and identification by LC-MS/MS. Identification of Rab GTPases 4, 5, 18, and 21 (associated with early endosomes); Rab7 (late endosomes); and Rab11 and Rab25 (recycling endosomes) indicate that a substantial fraction of intracellular AQP2 is present in endosomal compartments. In addition, several endosome-associated SNARE proteins were identified including syntaxin-7, syntaxin-12, syntaxin-13, Vti1a, vesicle-associated membrane protein 2, and vesicle-associated membrane protein 3. Rab3 was not found, however, either by mass spectrometry or immunoblotting, suggesting a relative lack of AQP2 in secretory vesicles. Additionally, we identified markers of the trans-Golgi network, components of the exocyst complex, and several motor proteins including myosin 1C, non-muscle myosins IIA and IIB, myosin VI, and myosin IXB. Beyond this, identification of multiple endoplasmic reticulum-resident proteins and ribosomal proteins indicated that a substantial fraction of intracellular AQP2 is present in rough endoplasmic reticulum. These results show that AQP2-containing vesicles are heterogeneous and that intracellular AQP2 resides chiefly in endosomes, trans-Golgi network, and rough endoplasmic reticulum.
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PMID:Large scale protein identification in intracellular aquaporin-2 vesicles from renal inner medullary collecting duct. 1590 45

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

In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.
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PMID:The collecting duct is the major source of prorenin in diabetes. 1841 93

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface beta and gammaENaC subunits were assayed by immunoblot. Surface alphaENaC was assessed by membrane fractionation. Most of the gammaENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the first transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this finding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two- to fivefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute significantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.
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PMID:Surface expression of epithelial Na channel protein in rat kidney. 1850 17

Proteases are involved in the processing and activation of the epithelial sodium channel (ENaC). The aim of the present study was to investigate whether the prototypical serine protease trypsin can activate ENaC in microdissected, split-open mouse renal distal tubules. Whole-cell patch-clamp recordings from principal cells of connecting tubules (CNT) or cortical collecting ducts (CCD) demonstrated that addition of trypsin (20 microg/ml) to the bath solution increased the ENaC-mediated amiloride-sensitive whole cell current (DeltaIAmi) in the majority of cells. In contrast, trypsin applied in the presence of an excess of soybean trypsin inhibitor had no stimulatory effect. The DeltaIAmi response to trypsin was variable, ranging from no apparent effect to a twofold increase in DeltaI(Ami) with an average stimulatory effect of 31 or 37% in mice on low-Na+ or standard Na+ diet, respectively. In cultured M-1 mouse collecting duct cells, a robust stimulatory effect of trypsin on DeltaIAmi was only observed in cells pretreated with protease inhibitors. This suggests that endogenous proteases contribute to ENaC activation in renal tubular cells and that the degree of ENaC prestimulation by endogenous proteases determines the magnitude of the stimulatory response to exogenous trypsin. In conclusion, we provide electrophysiological evidence that trypsin can stimulate ENaC activity in native renal mouse tubules. Thus, in the kidney, ENaC stimulation by extracellular proteases may be a relevant regulatory mechanism in vivo.
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PMID:Trypsin can activate the epithelial sodium channel (ENaC) in microdissected mouse distal nephron. 1865 83


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