SNAP-23 in rat kidney: colocalization with aquaporin-2 in collecting duct vesicles

1998 ◽  
Vol 275 (5) ◽  
pp. F752-F760 ◽  
Author(s):  
Takeaki Inoue ◽  
Søren Nielsen ◽  
Beatrice Mandon ◽  
James Terris ◽  
Bellamkonda K. Kishore ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Thick Ascending Limb ◽  
Principal Cells ◽  
Aquaporin 2 ◽  
Collecting Ducts ◽  
Medullary Collecting Duct ◽  
Intracellular Vesicles ◽  
Regulated Trafficking

Vesicle targeting proteins (“SNAREs”) have been proposed to direct vasopressin-induced trafficking of aquaporin-2 water channels in kidney collecting ducts. A newly identified SNARE protein, SNAP-23, is proposed to mediate vesicle targeting to the plasma membrane in diverse tissues. The current studies were done to determine whether SNAP-23 is expressed in collecting ducts with an intracellular distribution compatible with a role in aquaporin-2 trafficking. RT-PCR demonstrated SNAP-23 mRNA in microdissected collecting ducts and other tubular segments including the proximal tubule and thick ascending limb. Immunoblotting using a polyclonal antibody raised against a COOH-terminal peptide revealed a solitary band at an apparent molecular mass of 30 kDa in renal medullary membrane fractions and inner medullary collecting duct suspensions. Differential centrifugation revealed that SNAP-23 is present in membrane fractions including the low-density fraction enriched in intracellular vesicles. Immunocytochemistry revealed SNAP-23 labeling at both the apex and the cytoplasm of collecting duct principal cells. Immunoblotting of intracellular vesicles immunoisolated using an aquaporin-2 antibody revealed the presence of both SNAP-23 and synaptobrevin-2 (VAMP-2) in aquaporin-2-bearing vesicles. We conclude that SNAP-23 is strongly expressed in collecting duct principal cells, consistent with a role in vasopressin-regulated trafficking of aquaporin-2. However, localization of SNAP-23 in both intracytoplasmic vesicles and plasma membranes suggests a function different from that originally proposed for SNAP-25 in synaptic vesicle targeting.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

Immunolocalization of electroneutral Na-HCO3 − cotransporter in rat kidney

2000 ◽  
Vol 279 (5) ◽  
pp. F901-F909 ◽  
Author(s):  
Henrik Vorum ◽  
Tae-Hwan Kwon ◽  
Christiaan Fulton ◽  
Brian Simonsen ◽  
Inyeong Choi ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Electron Microscopic Level ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Electron Microscopic ◽  
Outer Medulla ◽  
Outer Stripe ◽  
Medullary Collecting Duct

An electroneutral Na-HCO3 − cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ∼180 kDa, in rat kidney membranes. Peptide- N-glycosidase F deglycosylation reduced the band to ∼140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBCN1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3 −-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3 − transport in these cells.

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

2004 ◽  
Vol 286 (5) ◽  
pp. F903-F912 ◽  
Author(s):  
Jeppe Praetorius ◽  
Young-Hee Kim ◽  
Elena V. Bouzinova ◽  
Sebastian Frische ◽  
Aleksandra Rojek ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Thick Ascending Limb ◽  
Medullary Thick Ascending Limb ◽  
Collecting Ducts ◽  
Basolateral Plasma Membrane ◽  
Medullary Collecting Duct ◽  
Plasma Membrane Domain

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na+ dependently import [Formula: see text] across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral [Formula: see text] cotransport. The mRNA encoding the electroneutral [Formula: see text] cotransporter NBCn1 was detected as the only [Formula: see text] cotransporter in the rat inner medulla (IM) among the five characterized Na+-dependent [Formula: see text] transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henle's loop. Immunoblotting confirmed the presence of NBCn1 as an ∼180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na+- and [Formula: see text]-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO3-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH4Cl loading. In conclusion, NBCn1 is a basolateral [Formula: see text] cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney

1995 ◽  
Vol 269 (4) ◽  
pp. F461-F468 ◽  
Author(s):  
F. C. Brosius ◽  
K. Nguyen ◽  
A. K. Stuart-Tilley ◽  
C. Haller ◽  
J. P. Briggs ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Chloride Concentration ◽  
Transepithelial Transport ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Base Exchange ◽  
Medullary Thick Ascending Limb ◽  
Nephron Segment ◽  
Medullary Collecting Duct

Chloride/base exchange activity has been detected in every mammalian nephron segment in which it has been sought. However, in contrast to the Cl-/HCO3- exchanger AE1 in type A intercalated cells, localization of AE2 within the kidney has not been reported. We therefore studied AE2 expression in rat kidney. AE2 mRNA was present in cortex, outer medulla, and inner medulla. Semiquantitative polymerase chain reaction of cDNA from microdissected tubules revealed AE2 cDNA levels as follows [copies of cDNA derived per mm tubule (+/- SE)]: proximal convoluted tubule, 688 +/- 161; proximal straight tubule, 652 +/- 189; medullary thick ascending limb, 1,378 +/- 226; cortical thick ascending limb, 741 +/- 24; cortical collecting duct, 909 +/- 71; and outer medullary collecting duct, 579 +/- 132. AE2 cDNA was also amplified in thin limbs and in inner medullary collecting duct. AE2 polypeptide was detected in all kidney regions. AE2 mRNA and protein were also detected in several renal cell lines. The data are compatible with the postulated roles of AE2 in maintenance of intracellular pH and chloride concentration and with its possible participation in transepithelial transport.

Lectin-gold cytochemistry reveals intercalated cell heterogeneity along rat kidney collecting ducts

1985 ◽  
Vol 248 (3) ◽  
pp. C348-C356 ◽  
Author(s):  
D. Brown ◽  
J. Roth ◽  
L. Orci
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Intercalated Cells ◽  
Double Labeling ◽  
Thin Sections ◽  
Intercalated Cell ◽  
Collecting Ducts

The lectin-gold technique was used to detect Helix pomatia and Dolichos biflorus lectin binding sites directly on semithin and thin sections of rat kidney collecting ducts. Intercalated cell apical plasma membranes and the membranes of apical cytoplasmic vesicles were heavily labeled in the cortex and outer stripe of the outer medulla but were negative or very weakly labeled in the inner stripe and inner medulla. In contrast, clear cell apical membranes were labeled along the entire length of the collecting duct. Double labeling of semithin cryostat sections with a specific antibody and lectin-gold complexes was used to demonstrate that the intercalated cells in all regions studied contained carbonic anhydrase, even though the lectin binding differed. These results indicate that, in terms of their glycocalyx composition, intercalated cells represent a heterogeneous population in different regions of the collecting duct.

Aquaporin-2 abundance in the renal collecting duct: new insights from cultured cell models

2009 ◽  
Vol 297 (1) ◽  
pp. F10-F18 ◽  
Author(s):  
Udo Hasler ◽  
Valérie Leroy ◽  
Pierre-Yves Martin ◽  
Eric Féraille
Keyword(s):  
Gene Transcription ◽  
Collecting Duct ◽  
Mrna Translation ◽  
Osmotic Gradient ◽  
Thick Ascending Limb ◽  
Whole Cell ◽  
Cell Abundance ◽  
Principal Cells ◽  
Aquaporin 2 ◽  
Aqp2 Gene

The renal cortico-papillary osmotic gradient is generated by sodium reabsorption in the thick ascending limb. The antidiuretic hormone arginine vasopressin (AVP) increases collecting duct water permeability by enhancing aquaporin-2 (AQP2) water channel insertion in the apical membrane of principal cells, allowing water to passively flow along the osmotic gradient from the tubule lumen to the interstitium. In addition to short-term AQP2 redistribution between intracellular compartments and the cell surface, AQP2 whole cell abundance is tightly regulated. AVP is a major transcriptional activator of the AQP2 gene, and stimulation of insulin- and calcium-sensing receptors respectively potentiate and reduce its action. Extracellular tonicity is another key factor that determines the levels of AQP2 abundance. Its effect is dependent on activation of the tonicity-responsive enhancer binding protein that reinforces AVP-induced AQP2 transcriptional activation. Conversely, activation of the NF-κB transcriptional factor by proinflammatory factors reduces AQP2 gene transcription. Aldosterone additionally regulates AQP2 whole cell abundance by simultaneously reducing AQP2 gene transcription and stimulating AQP2 mRNA translation. These examples illustrate how cross talk between various stimuli regulates AQP2 abundance in collecting duct principal cells and consequently contributes to maintenance of body water homeostasis.

Lack of effect of atrial natriuretic peptide and urodilatin on vasopressin-stimulated cyclic AMP production in microdissected rat inner medullary collecting ducts

1994 ◽  
Vol 12 (2) ◽  
pp. 149-154
Author(s):  
W J Burgess ◽  
M N Perrott ◽  
R J Balment
Keyword(s):  
Cyclic Amp ◽  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Absolute Magnitude ◽  
Camp Production ◽  
Collecting Ducts ◽  
Medullary Collecting Duct ◽  
Atrial Natriuretic

ABSTRACT It is unclear whether the diuretic effects of atrial natriuretic peptide (ANP) result, in part, from an inhibition of the renal actions of vasopressin. Moreover, accruing evidence suggests that the kidneys themselves may produce an ANP-like peptide, urodilatin, which shares many of the renal actions of ANP. The mechanism underlying the diuretic action of urodilatin has not yet been examined. Accordingly, we have investigated the potential modulatory actions of both ANP and urodilatin on vasopressin-stimulated cyclic AMP (cAMP) production in microdissected inner medullary collecting duct (IMCD) segments of rat kidney. ANP and urodilatin alone (at 10−8 or 10−6 m) had no demonstrable effect on cAMP accumulation in IMCD segments. Moreover, neither ANP nor urodilatin (each at 10−6 m) significantly altered either the profile or the absolute magnitude of the cAMP response stimulated by vasopressin. These findings indicate that neither ANP nor urodilatin interacts with the vasopressin-sensitive adenylate cyclase site in the rat IMCD to contribute to its diuretic actions.

Localization of ral, a small Mr GTP-binding protein, to collecting duct cells in bovine and rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F1063-F1070
Author(s):  
A. Gupta ◽  
B. Bastani ◽  
P. Chardin ◽  
K. A. Hruska
Keyword(s):  
Gene Product ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Gtp Binding Protein ◽  
Bovine Kidney ◽  
Gtp Binding ◽  
Collecting Ducts

Plasma membranes from bovine kidney cortex were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting with [alpha-32P]GTP and [35S]GTP gamma S demonstrated specific binding to three and six distinct protein bands, respectively, in the 20,000- to 29,000-Mr range. This indicated the presence of small Mr GTP binding proteins (smg) in bovine kidney cortex. Only one smg with 28,000 Mr was labeled with hydrolysis-resistant GTP photoaffinity probe p3-(4-azidoanilido)-p1-5GTP (AAGTP). The major smg in platelet membranes that binds GTP on nitrocellulose blots has been identified as ral-Mr 29,000. With the use of an antiserum against the ral A gene product, one of the smg with Mr of 29,000 present in bovine renal cortical plasma membranes was identified as ral. Ral was absent from glomerular homogenate, suggesting that it is localized to the tubular segments of the nephron. Ral was detected only in the particulate fraction and not the cytosol. Further subcellular localization of ral was investigated by immunohistochemical staining. Anti-ral antibody immunostained the apical and basolateral membranes of cells in the cortical and medullary collecting ducts in a speckled pattern in the bovine kidney. In the rat kidney, however, uniform linear staining of cortical and medullary collecting ducts predominantly localized to the apical membrane was observed. To date, no function has been assigned to ral. Localization of the ral gene product to the collecting duct suggests a specific functional role for this GTP-binding protein.

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

2001 ◽  
Vol 280 (6) ◽  
pp. F1093-F1106 ◽  
Author(s):  
Henrik Hager ◽  
Tae-Hwan Kwon ◽  
Anna K. Vinnikova ◽  
Shyama Masilamani ◽  
Heddwen L. Brooks ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Subcellular Localization ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Medullary Collecting Duct ◽  
Epithelial Sodium Channel Enac

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) in rat kidney. However, the subcellular localization of ENaC subunits in the principal cells of these cells is undefined. The cellular and subcellular localization of ENaC subunits in rat kidney was therefore examined. Immunocytochemistry demonstrated the presence of all three subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex and outer medulla, confocal microscopy demonstrated a difference in the subcellular localization of subunits. α-ENaC was localized mainly in a zone in the apical domains, whereas β- and γ-ENaC were found throughout the cytoplasm. Immunoelectron microscopy confirmed the presence of ENaC subunits in both the apical plasma membrane and intracellular vesicles. In contrast to the labeling pattern seen in cortex, α-ENaC labeling in IMCD cells was distributed throughout the cytoplasm. In the urothelium covering pelvis, ureters, and bladder, immunoperoxidase and confocal microscopy revealed differences the presence of all ENaC subunits. As seen in CCD, α-ENaC was present in a narrow zone near the apical plasma membrane, whereas β- and γ-ENaC were dispersed throughout the cytoplasm. In conclusion, all three subunits of ENaC are expressed throughout the collecting duct (CD), including the IMCD as well as in the urothelium. The intracellular vesicular pool in CD principal cells suggests ENaC trafficking as a potential mechanism for the regulation of Na+ reabsorption.

AE2 isoforms in rat kidney: immunohistochemical localization and regulation in response to chronic NH4Cl loading

2004 ◽  
Vol 286 (6) ◽  
pp. F1163-F1170 ◽  
Author(s):  
Sebastian Frische ◽  
Alexander S. Zolotarev ◽  
Young-Hee Kim ◽  
Jeppe Praetorius ◽  
Seth Alper ◽  
...  
Keyword(s):  
Splice Variants ◽  
Polyclonal Antibodies ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Immunohistochemical Localization ◽  
Thick Ascending Limb ◽  
Relative Distribution ◽  
Medullary Thick Ascending Limb ◽  
Medullary Collecting Duct ◽  
Antibody Specificities

Three splice variants of anion exchanger (AE)2 (AE2a, b, and c) have been described in the rat, but their relative distribution in rat kidney is not known. The purpose of this study was to describe the segmental and cellular distribution of the AE2 isoforms in the rat kidney and to evaluate whether the expression levels of these AE2 isoforms are regulated independently in response to chronic NH4Cl loading. Two polyclonal antibodies were generated, respectively, recognizing a NH2-terminal peptide unique to AE2a and an amino acid sequence common to AE2a and AE2b. Antibody specificities were tested using cells transfected separately with the AE2a, AE2b, and AE2c isoforms. Immunohistochemistry on sections of paraffin-embedded rat kidneys showed a distribution of AE2a/AE2b labeling in the kidney similar to the distribution of AE2 in the rat kidney reported previously. AE2 is highly expressed in the medullary thick ascending limb, cortical thick ascending limb (cTAL), and macula densa. The pattern of AE2a-specific labeling differed from the pattern of AE2a/AE2b labeling in that relatively more of the total immunolabel was observed in the terminal inner medullary collecting duct. NH4Cl loading (0.033 mmol NH4Cl/g body wt for 7 days) did not change the labeling of AE2 isoforms in the medulla, whereas the labeling in the cortex was intensified and included more distal parts of the cTAL. Immunoblotting confirmed upregulation of AE2a/b expression in the cortex. These results indicate that AE2a and AE2b are differentially expressed and regulated in the rat kidney. The regulation following NH4Cl loading of AE2b in the cTAL suggests a role for AE2 in transepithelial bicarbonate reabsorption in this segment.

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