scholarly journals LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

2008 ◽  
Vol 295 (6) ◽  
pp. F1799-F1806 ◽  
Author(s):  
Aaron N. Sachs ◽  
Trairak Pisitkun ◽  
Jason D. Hoffert ◽  
Ming-Jiun Yu ◽  
Mark A. Knepper
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Differential Centrifugation ◽  
Proteomic Profiling ◽  
Inner Medullary Collecting Duct ◽  
Ms Analysis ◽  
Alternative Approach ◽  
Baseline Knowledge ◽  
Medullary Collecting Duct

We carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000, 4,000, 17,000, and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000- g fraction to the 200,000- g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000- g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000- and 200,000- g fractions. LC-MS/MS analysis showed that markers of “intracellular vesicles,” chiefly endosomal markers, were present in both the 17,000- and the 200,000- g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000- and 17,000- g fractions. Proteins associated with several multiprotein complexes (e.g., actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000- g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000- and 200,000- g fractions are highly heterogeneous and cannot be equated with “plasma membrane” and “intracellular vesicle” fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.

scholarly journals Syntaxin specificity of aquaporins in the inner medullary collecting duct

2009 ◽  
Vol 297 (2) ◽  
pp. F292-F300 ◽  
Author(s):  
Abinash C. Mistry ◽  
Rickta Mallick ◽  
Janet D. Klein ◽  
Thomas Weimbs ◽  
Jeff M. Sands ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Snare Complex ◽  
Apical Plasma Membrane ◽  
Transport Activity ◽  
Inner Medullary Collecting Duct ◽  
Syntaxin 4 ◽  
Medullary Collecting Duct ◽  
Syntaxin 3

Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (t-SNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-Madin-Darby canine kidney cells subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pull-down studies, snapin detected AQP2, syntaxin-3, syntaxin-4, and SNAP23 from the inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were coinjected with cRNAs of SNARE components (snapin+syntaxin-3+SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin+syntaxin-4+SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP-syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex, leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion.

scholarly journals Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct

2018 ◽  
Vol 314 (1) ◽  
pp. C99-C117 ◽  
Author(s):  
Chung-Lin Chou ◽  
Gloria Hwang ◽  
Daniel J. Hageman ◽  
Lichy Han ◽  
Prashasti Agrawal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinases ◽  
Membrane Trafficking ◽  
Collecting Duct ◽  
Water Channel ◽  
Rab Proteins ◽  
Interacting Proteins ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. Fifty-three proteins that were present in both the UT-A1 and the AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in posttranslational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d, and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.

The vasopressin-regulated urea transporter in renal inner medullary collecting duct

1990 ◽  
Vol 259 (3) ◽  
pp. F393-F401 ◽  
Author(s):  
M. A. Knepper ◽  
R. A. Star
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Toad Urinary Bladder ◽  
Urea Transport ◽  
Urea Transporter ◽  
Transport Pathway ◽  
Inner Medullary Collecting Duct ◽  
Urea Permeability ◽  
Medullary Collecting Duct

The terminal part of the inner medullary collecting duct (terminal IMCD) is unique among collecting duct segments in part because its permeability to urea is regulated by vasopressin. The urea permeability can rise to extremely high levels (greater than 100 x 10(-5) cm/s) in response to vasopressin. Recent studies in isolated perfused IMCD segments have established that the rapid movement of urea across the tubule epithelium occurs via a specialized urea transporter, presumably an intrinsic membrane protein, present in both the apical and basolateral membranes. This urea transporter has properties similar to those of the urea transporters in mammalian erythrocytes and in toad urinary bladder, namely, inhibition by phloretin, inhibition by urea analogues, saturation kinetics in equilibrium-exchange experiments, and regulation by vasopressin. The urea transport pathway is distinct from and independent of the vasopressin-regulated water channel. The increase in transepithelial urea transport in response to vasopressin is mediated by adenosine 3',5'-cyclic monophosphate and is associated with an increase in the urea permeability of the apical membrane. However, little is known about the physical events associated with the activation or insertion of urea transporters in the apical membrane. Because of the importance of this transporter to the urinary concentrating mechanism, efforts toward understanding its molecular structure and the molecular basis of its regulation appear to be justified.

scholarly journals Proteomic determination of the lysine acetylome and phosphoproteome in the rat native inner medullary collecting duct

2018 ◽  
Vol 50 (9) ◽  
pp. 669-679 ◽  
Author(s):  
Kelly A. Hyndman ◽  
Chin-Rang Yang ◽  
Hyun Jun Jung ◽  
Ezigbobiara N. Umejiego ◽  
Chung-Ling Chou ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Collecting Duct ◽  
Water Channel ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Water Reabsorption ◽  
Inner Medullary Collecting Duct ◽  
Phosphorylated Proteins ◽  
V2 Receptor ◽  
Medullary Collecting Duct

Phosphorylation and lysine (K)-acetylation are dynamic posttranslational modifications of proteins. Previous proteomic studies have identified over 170,000 phosphorylation sites and 15,000 K-acetylation sites in mammals. We recently reported that the inner medullary collecting duct (IMCD), which functions in the regulation of water-reabsorption, via the actions of vasopressin, expresses many of the enzymes that can modulated K-acetylation. The purpose of this study was to determine the K-acetylated or phosphorylated proteins expressed in IMCD cells. Second we questioned whether vasopressin V2 receptor activation significantly affects the IMCD acetylome or phosphoproteome? K-acetylated or serine-, threonine-, or tyrosine-phosphorylated peptides were identified from native rat IMCDs by proteomic analysis with four different enzymes (trypsin, chymotrypsin, ASP-N, or Glu-C) to generate a high-resolution proteome. K-acetylation was identified in 431 unique proteins, and 64% of the K-acetylated sites were novel. The acetylated proteins were expressed in all compartments of the cell and were enriched in pathways including glycolysis and vasopressin-regulated water reabsorption. In the vasopressin-regulated water reabsorption pathway, eight proteins were acetylated, including the novel identification of the basolateral water channel, AQP3, acetylated at K282; 215 proteins were phosphorylated in this IMCD cohort, including AQP2 peptides that were phosphorylated at four serines: 256, 261, 264, and 269. Acute dDAVP did not significantly affect the IMCD acetylome; however, it did significantly affect previously known vasopressin-regulated phosphorylation sites. In conclusion, presence of K-acetylated proteins involved in metabolism, ion, and water transport in the IMCD points to multiple roles of K-acetylation beyond its canonical role in transcriptional regulation.

Atrial natriuretic peptide and nitric oxide signaling antagonizes vasopressin-mediated water permeability in inner medullary collecting duct cells

2009 ◽  
Vol 297 (3) ◽  
pp. F693-F703 ◽  
Author(s):  
Jens Klokkers ◽  
Patrik Langehanenberg ◽  
Björn Kemper ◽  
Sebastian Kosmeier ◽  
Gert von Bally ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Water Permeability ◽  
Collecting Duct ◽  
Cell Swelling ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Atrial Natriuretic

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.

Effect of insulin on water and urea transport in the inner medullary collecting duct

1994 ◽  
Vol 266 (3) ◽  
pp. F394-F399 ◽  
Author(s):  
A. J. Magaldi ◽  
K. R. Cesar ◽  
Y. Yano
Keyword(s):  
Water Transport ◽  
Collecting Duct ◽  
Water Channel ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Urea Transport ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Dose Dependent ◽  
Rule Out

The effect of insulin on water and urea transport was examined in normal isolated rat inner medullary collecting duct (IMCD). Hydraulic conductivity (Lp, x 10(-6) cm.atm-1.s-1), diffusional water permeability (Pdw, x 10(-5) cm/s) and [14C]urea permeability (x 10(-5) cm/s) were studied at 37 degrees C and pH 7.4. Insulin (6 x 10(-8) M; 200 microU/ml) added to the bath fluid enhanced Lp from 0.40 +/- 0.10 to 1.21 +/- 1.40 (P < 0.01) and Pdw from 42.40 +/- 3.40 to 58.50 +/- 5.00 (P < 0.02) and also stimulated Lp in a dose-dependent manner. In the presence of antidiuretic hormone (ADH)-stimulated Pdw (10 microU/ml), insulin increased Pdw even more. Prostaglandin E2 (10(-5) M) added to the bath reversibly increased insulin-induced Lp. Forskolin (10(-4) M) blocked the action of insulin. Colchicine (10(-4) M) and V1-receptor antagonist (10(-4) M) inhibited the development but not the maintenance of insulin-stimulated Pdw. Vanadate (2.5 x 10(-6) M) enhanced Pdw. Polymyxin B (10(-5) M) inhibited the insulin-stimulated Pdw, whereas in a glucose-free medium insulin did not enhance Pdw. Urea transport was not affected by insulin. These data suggest that insulin may enhance water transport, probably by stimulating glucose transporters, which would serve as a water channel. We cannot rule out the possibility that insulin may be eliciting existing ADH-like mechanisms of water transport, beyond the microtubule step, to establish water transport.

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

2005 ◽  
Vol 289 (1) ◽  
pp. F194-F199 ◽  
Author(s):  
Ryan G. Morris ◽  
Shinichi Uchida ◽  
Heddwen Brooks ◽  
Mark A. Knepper ◽  
Chung-Lin Chou
Keyword(s):  
Expression Profile ◽  
Knockout Mice ◽  
Collecting Duct ◽  
Water Channel ◽  
Major Protein ◽  
Altered Expression ◽  
Aquaporin 1 ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Concentrating Defect

Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 −/−) mice. Here, we have investigated the long-term consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (−/−) mice vs. heterozygotes [AQP1 (+/−)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (−/−) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits β-ENaC and γ-ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, β-ENaC, γ-ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (−/−) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (−/−) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (−/−) mice showed decreased expression of UT-A1 and increased expression of β-ENaC and γ-ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 −/− mice and this manifestation is related to the associated concentrating defect.

Vasopressin depolymerizes apical F-actin in rat inner medullary collecting duct

1993 ◽  
Vol 265 (3) ◽  
pp. C757-C762 ◽  
Author(s):  
H. Simon ◽  
Y. Gao ◽  
N. Franki ◽  
R. M. Hays
Keyword(s):  
Collecting Duct ◽  
Water Channel ◽  
Granular Cell ◽  
Immunogold Labeling ◽  
Target Cells ◽  
Apical Region ◽  
Rhodamine Phalloidin ◽  
Consistent Finding ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

In amphibian bladder, arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the granular cell, promoting fusion of water channel-carrying vesicles with the apical membrane. We now report the effect of AVP on F-actin in the mid- and terminal segments of rat inner medullary collecting duct (IMCD2 and IMCD3). In IMCD3, 5 min of stimulation by 2.5-250 nM AVP significantly depolymerized F-actin by 13-24% in whole cell assays employing the rhodamine-phalloidin binding technique. The IMCD2 was more sensitive, responding to subnanomolar (0.25 nM) AVP with 6 +/- 2% depolymerization. Depolymerization occurred as early as 2 min after 2.5 and 25 nM but not 250 nM AVP. 8-Bromoadenosine 3',5'-cyclic monophosphate depolymerized F-actin in IMCD3 at both 2 and 5 min. Immunogold labeling of the apical actin pool in IMCD3 principal cells was reduced by 26 +/- 5% (P < 0.05) by 2.5 nM AVP; the lateral and basal pools showed no significant changes. Capillary endothelial, thin limb of Henle, and intercalated cells showed no changes in immunogold labeling after AVP. Thus reorganization of the apical actin network by AVP is a consistent finding in both mammalian and amphibian target cells.

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