Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase.

The morphologically heterogeneous cell populations in the collecting ducts of the rat kidney were studied using immunocytochemical detection of Na+-K+-ATPase and the anion channel (band 3) glycoprotein. Both enzymes were localized to the basal aspect of separate and morphologically distinct subpopulations of cells in various segments of the collecting duct. Na+-K+-ATPase appeared to be present exclusively in principal cells as identified by their morphology, whereas band 3 antibodies reacted only with intercalated cells. However, 5-20% of cells with the morphological characteristics of intercalated cells failed to react with either antisera in various segments of collecting ducts. As band 3 glycoprotein serves in exchanging intracellular bicarbonate for chloride, it is highly likely that the cells positive for this antigen secrete protons. The method introduced here appears thus useful for distinguishing between principal and intercalated cells by differences in their enzyme content and further for revealing two subpopulations of intercalated cells. This method promises to provide a useful approach for studying the principal and intercalated cell populations in various metabolic states.

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Expression and Immunolocalization of AQP6 in Intercalated Cells of the Rat Kidney Collecting Duct.

Archives of Histology and Cytology ◽

10.1679/aohc.64.329 ◽

2001 ◽

Vol 64 (3) ◽

pp. 329-338 ◽

Cited By ~ 24

Author(s):

Kazufumi OHSHIRO ◽

Eishin YAOITA ◽

Yutaka YOSHIDA ◽

Hidehiko FUJINAKA ◽

Asako MATSUKI ◽

...

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

Intercalated Cells ◽

Kidney Collecting Duct

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Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel

AJP Cell Physiology ◽

10.1152/ajpcell.1994.266.1.c189 ◽

1994 ◽

Vol 266 (1) ◽

pp. C189-C197 ◽

Cited By ~ 45

Author(s):

T. Ma ◽

H. Hasegawa ◽

W. R. Skach ◽

A. Frigeri ◽

A. S. Verkman

Keyword(s):

Base Pair ◽

Collecting Duct ◽

Rat Kidney ◽

Water Channel ◽

Cloning And Expression ◽

Base Pairs ◽

Kidney Medulla ◽

Coding Sequence ◽

Kidney Collecting Duct ◽

Protein Size

The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

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Activation of acid-secreting intercalated cells in rabbit collecting duct with ammonium chloride loading

AJP Renal Physiology ◽

10.1152/ajprenal.1994.266.4.f633 ◽

1994 ◽

Vol 266 (4) ◽

pp. F633-F645 ◽

Cited By ~ 22

Author(s):

J. W. Verlander ◽

K. M. Madsen ◽

J. K. Cannon ◽

C. C. Tisher

Keyword(s):

Plasma Membrane ◽

Morphometric Analysis ◽

Collecting Duct ◽

Band 3 ◽

Apical Plasma Membrane ◽

Multivesicular Bodies ◽

Intercalated Cells ◽

Cytoplasmic Vesicles ◽

Basolateral Plasma Membrane ◽

Acid Loading

In normal rabbit, immunolabeling of intercalated cells in the outer medullary collecting duct (OMCD) demonstrates band 3-like protein in the basolateral plasma membrane (15) and H(+)-adenosinetriphosphatase (H(+)-ATPase) in the apical plasma membrane and cytoplasmic vesicles (30). However, in type A intercalated cells in the cortical collecting duct (CCD), band 3-like protein is located primarily in multivesicular bodies and cytoplasmic vesicles (15), whereas H(+)-ATPase is present in cytoplasmic vesicles only in most intercalated cells (30). In this study, we observed the effect of chronic acid loading on immunolocalization of these transporters in the collecting duct. Adult New Zealand White rabbits received either normal tap water (controls) or 75 mM NH4Cl for 12 days plus eight daily gavages of 2-6 meq NH4Cl/kg body wt. At time of death, mean urine pH of acid-loaded animals was 5.96 (SD = 0.69), vs. 8.47 (SD = 0.07) in controls. Kidneys were fixed by in vivo perfusion and processed for light and electron microscopic immunoperoxidase localization of band 3-like protein and immunogold localization of H(+)-ATPase. In controls, band 3-like protein was largely confined to multivesicular bodies in the majority of positive-staining intercalated cells in the CCD and to the basolateral plasma membrane of intercalated cells in the OMCD. In acid-loaded rabbits, band 3 protein-positive intercalated cells in the inner CCD and the in the outer stripe of the OMCD (OMCDo) were strikingly stellate in form. Basolateral plasma membrane label was intensified, while the number of labeled multivesicular bodies was diminished. Morphometric analysis demonstrated an increase in the amount of basolateral plasma membrane in these intercalated cells. In control rabbits, H(+)-ATPase immunoreactivity in intercalated cells in the CCD was located predominantly over cytoplasmic vesicles. A minority of intercalated cells exhibited basolateral plasma membrane label, and only an occasional cell displayed apical plasma membrane label. In acid-loaded rabbits, H(+)-ATPase immunoreactivity was enhanced along the apical plasma membrane of intercalated cells in the inner CCD, and morphometric analysis demonstrated increased apical plasma membrane in band 3-positive intercalated cells in this segment. These results suggest that rabbits respond to acid loading via enhancement of both electrogenic proton secretion and Cl-/HCO3- exchange in intercalated cells in the inner CCD and the OMCDo.

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

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.3.c348 ◽

1985 ◽

Vol 248 (3) ◽

pp. C348-C356 ◽

Cited By ~ 41

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.

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Cell volume regulation of rat kidney collecting duct epithelial cells in hypotonic medium

Doklady Biological Sciences ◽

10.1134/s0012496611010108 ◽

2011 ◽

Vol 436 (1) ◽

pp. 13-15 ◽

Cited By ~ 1

Author(s):

E. I. Solenov ◽

G. S. Baturina ◽

A. V. Ilyaskin ◽

L. Ye. Katkova ◽

L. N. Ivanova

Keyword(s):

Epithelial Cells ◽

Cell Volume ◽

Volume Regulation ◽

Collecting Duct ◽

Rat Kidney ◽

Cell Volume Regulation ◽

Hypotonic Medium ◽

Kidney Collecting Duct

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Differential localization of two glucose transporter isoforms in rat kidney

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.2.c286 ◽

1990 ◽

Vol 259 (2) ◽

pp. C286-C294 ◽

Cited By ~ 78

Author(s):

B. Thorens ◽

H. F. Lodish ◽

D. Brown

Keyword(s):

Proximal Tubule ◽

Glucose Transporter ◽

Collecting Duct ◽

Rat Kidney ◽

Basolateral Membrane ◽

Staining Intensity ◽

Intercalated Cells ◽

Brain Glucose ◽

Nephron Segments ◽

Glucose Transporter Isoforms

The localization of two glucose transporter isoforms was mapped in the rat kidney: the high-Michaelis constant (Km; 15-20 mM) low-affinity "liver" transporter and the low-Km (1-2 mM) high-affinity "erythroid/brain" transporter. Both are basolateral membrane proteins, but the liver transporter was present exclusively in the S1 part of the proximal tubule, whereas the erythroid/brain transporter was expressed at variable levels in different nephron segments. Staining intensity was low in the straight proximal tubule (S3), intermediate in the medullary thin and thick ascending limbs, and highest in connecting segments and collecting ducts. In the collecting duct, the erythroid/brain glucose transporter was expressed at the highest level in intercalated cells; less was present in principal cells. In the papilla, only intercalated cells expressed this transporter isoform. These results suggest specific involvements of each transporter isoform in transepithelial glucose reabsorption by different segments of the proximal tubule. They also indicate that while the liver glucose transporter is present in gluconeogenic cells, there is a good correlation between the level of expression of the erythroid/brain glucose transporter and the glycolytic activity of the different nephron segments.

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Intercalated Cell Subtypes in Connecting Tubule and Cortical Collecting Duct of Rat and Mouse

Journal of the American Society of Nephrology ◽

10.1681/asn.v1011 ◽

1999 ◽

Vol 10 (1) ◽

pp. 1-12 ◽

Cited By ~ 1

Author(s):

JIN KIM ◽

YOUNG-HEE KIM ◽

JUNG-HO CHA ◽

C. CRAIG TISHER ◽

KIRSTEN M. MADSEN

Keyword(s):

Plasma Membrane ◽

B Cells ◽

Collecting Duct ◽

Type A ◽

Band 3 ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Type B ◽

Intercalated Cell ◽

Basolateral Plasma Membrane

Abstract. At least two populations of intercalated cells, type A and type B, exist in the connecting tubule (CNT), initial collecting tubule (ICT), and cortical collecting duct (CCD). Type A intercalated cells secrete protons via an apical H+ - ATPase and reabsorb bicarbonate by a band 3-like Cl-/HCO3- exchanger, AE1, located in the basolateral plasma membrane. Type B intercalated cells secrete bicarbonate by an apical Cl-/HCO3- exchanger that is distinct from AE1 and remains to be identified. They express H+ -ATPase in the basolateral plasma membrane and in vesicles throughout the cytoplasm. A third type of intercalated cell with apical H+ -ATPase, but no AE1, has been described in the CNT and CCD of both rat and mouse. The prevalence of the third cell type is not known. The aim of this study was to characterize and quantify intercalated cell subtypes, including the newly described third non A-non B cell, in the CNT, ICT, and CCD of the rat and mouse. A triple immunolabeling procedure was developed in which antibodies to H+ -ATPase and band 3 protein were used to identify subpopulations of intercalated cells, and segment-specific antibodies were used to identify distal tubule and collecting duct segments. In both rat and mouse, intercalated cells constituted approximately 40% of the cells in the CNT, ICT, and CCD. Type A, type B, and non A-non B intercalated cells were observed in all of the three segments, with type A cells being the most prevalent in both species. In the mouse, however, non A-non B cells constituted more than half of the intercalated cells in the CNT, 39% in the ICT, and 22% in the CCD, compared with 14, 7, and 5%, respectively, in the rat. In contrast, type B intercalated cells accounted for only 8 to 16% of the intercalated cells in the three segments in the mouse compared with 26 to 39% in the rat. It is concluded that striking differences exist in the prevalence and distribution of the different types of intercalated cells in the CNT, ICT, and CCD of rat and mouse. In the rat, the non A-non B cells are fairly rare, whereas in the mouse, they constitute a major fraction of the intercalated cells, primarily at the expense of the type B intercalated cells.

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Immunolocalization of electroneutral Na-HCO3 − cotransporter in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.5.f901 ◽

2000 ◽

Vol 279 (5) ◽

pp. F901-F909 ◽

Cited By ~ 42

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.

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Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.1994.267.4.f573 ◽

1994 ◽

Vol 267 (4) ◽

pp. F573-F582 ◽

Cited By ~ 13

Author(s):

K. Fushimi ◽

S. Sasaki ◽

T. Yamamoto ◽

M. Hayashi ◽

T. Furukawa ◽

...

Keyword(s):

Water Permeability ◽

Expression Vector ◽

Collecting Duct ◽

Rat Kidney ◽

Functional Characterization ◽

Protein A ◽

Water Channel ◽

Kidney Medulla ◽

Principal Cells ◽

Kidney Collecting Duct

Vasopressin-regulated water permeability of the kidney collecting duct is a key component of the urine concentration machinery. Recently, a cDNA for AQP-CD, the vasopressin-regulated water channel, initially reported as WCH-CD, has been isolated (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). AQP-CD was expressed in oocyte membrane using a Xenopus expression vector, and functional characteristics of AQP-CD were examined. Osmotic water permeability (Pf) of oocytes expressing AQP-CD was 138 +/- 19 microns/s (mean +/- SE), 12 times greater than the control (11 +/- 3 microns/s), 90% inhibited by 0.3 mM HgCl2, and weakly temperature dependent (energy of activation for Pf was 4.0 kcal/mol). Urea influx measured from 15-min [14C]urea uptake by oocytes injected with AQP-CD/expression vector 1 cRNA was 86 +/- 17% of the control. Two-electrode voltage-clamp experiments revealed insignificant ion conductance of AQP-CD. Immunoblots of membranes from rat kidney medulla and oocytes expressing AQP-CD using anti-AQP-CD COOH-terminal antibody showed a 29-kDa protein and 35- to 50-kDa high-molecular-mass forms. Immunohistochemistry showed apical and subapical localization of AQP-CD in the collecting duct principal cells. Our results indicated that AQP-CD is a 29-kDa protein, a selective water channel, distinct from a urea channel, and localized to the membranes of vasopressin-sensitive components in kidney collecting duct principal cells.

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