Localization of the ammonium transporter proteins RhBG and RhCG in mouse kidney

2003 ◽  
Vol 284 (2) ◽  
pp. F323-F337 ◽  
Author(s):  
Jill W. Verlander ◽  
R. Tyler Miller ◽  
Amy E. Frank ◽  
Ines E. Royaux ◽  
Young-Hee Kim ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Immunoblot Analysis ◽  
Mouse Kidney ◽  
Ammonium Transporter ◽  
Ammonium Transport ◽  
Apical Region ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Medullary Collecting Duct

Ammonia is both produced and transported by renal epithelial cells, and it regulates renal ion transport. Recent studies have identified a family of putative ammonium transporters; mRNA for two members of this family, Rh B-glycoprotein (RhBG) and Rh C-glycoprotein (RhCG), is expressed in the kidney. The purpose of this study was to determine the cellular location of RhBG and RhCG protein in the mouse kidney. We generated RhBG- and RhCG-specific anti-peptide antibodies. Immunoblot analysis confirmed that both proteins were expressed in the mouse kidney. RhBG localization with immunohistochemistry revealed discrete basolateral labeling in the connecting segment (CNT) and in the majority of initial collecting tubule (ICT) and cortical collecting duct (CCD) cells. In the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD) only a subpopulation of cells exhibited basolateral immunoreactivity. Colocalization of RhBG with carbonic anhydrase II, the thiazide-sensitive transporter, and the anion exchangers AE1 and pendrin demonstrated RhBG immunoreactivity in all CNT cells and all CCD and ICT principal cells. In the ICT and CCD, basolateral RhBG immunoreactivity is also present in A-type intercalated cells but not in pendrin-positive CCD intercalated cells. In the OMCD and IMCD, only intercalated cells exhibit RhBG immunoreactivity. Immunoreactivity for a second putative ammonium transporter, RhCG, was present in the apical region of cells with almost the same distribution as RhBG. However, RhCG immunoreactivity was present in all CCD cells, and it was present in outer stripe OMCD principal cells, in addition to OMCD and IMCD intercalated cells. Thus the majority of RhBG and RhCG protein expression is present in the same epithelial cell types in the CNT and collecting duct but with opposite polarity. These findings suggest that RhBG and RhCG may play important and cell-specific roles in ammonium transport and signaling in these regions of the kidney.

Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch

2002 ◽  
Vol 283 (4) ◽  
pp. R983-R992 ◽  
Author(s):  
Peter M. Piermarini ◽  
Jill W. Verlander ◽  
Ines E. Royaux ◽  
David H. Evans
Keyword(s):  
Collecting Duct ◽  
Apical Region ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Gill Epithelium ◽  
Dasyatis Sabina ◽  
Proton Atpase ◽  
Atlantic Stingray ◽  
Freshwater Stingray ◽  
Gill Lamellae

Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl−/HCO[Formula: see text]exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na+-K+-ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl−/HCO[Formula: see text] exchange in gills of Atlantic stingrays from both freshwater and marine environments.

Immunolocalization of the Na+/Ca2+ exchanger in rabbit kidney

1993 ◽  
Vol 265 (2) ◽  
pp. F327-F332 ◽  
Author(s):  
R. F. Reilly ◽  
C. A. Shugrue ◽  
D. Lattanzi ◽  
D. Biemesderfer
Keyword(s):  
Collecting Duct ◽  
Membrane Vesicle ◽  
Immunoblot Analysis ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Intracellular Loop ◽  
Nephron Segments ◽  
Vesicle Preparation

We recently isolated a cDNA encoding a Na+/Ca2+ exchanger from rabbit kidney that was highly similar to the canine cardiac sarcolemmal Na+/Ca2+ exchanger. In the present study, we used two different antibodies to the exchanger to identify the protein and establish its cellular and subcellular localization in the kidney. The first antibody was prepared against a fusion protein consisting of 190 amino acids of the large, presumably intracellular loop of the rabbit renal exchanger fused to the maltose-binding protein. The second was a monoclonal antibody generated against the isolated purified canine cardiac sarcolemmal exchanger. To identify the Na+/Ca2+ exchanger protein, we performed immunoblot analysis against a membrane vesicle preparation from rabbit kidney cortex. Both antibodies immunoblotted proteins of 120 and 70 kDa that are known to be associated with the exchanger. Indirect immunofluorescence revealed that both antisera labeled the basolateral surface of the majority of cells in the connecting tubule (CNT). Since the phase-dense (intercalated) cells in the CNT were not stained, this suggested that the labeled cells were CNT cells. No labeling was detected in other nephron segments with the exception of occasional faint staining of the majority cell population of the cortical collecting duct. The fact that we did not detect labeling in other nephron segments is consistent with either 1) the absence of expression of the Na+/Ca2+ exchanger in these segments, 2) the expression of the exchanger in levels below the threshold of detection of the two antibodies used in this study, or 3) the exchanger in these segments is represented by a different isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Axial heterogeneity of rabbit cortical collecting duct

1991 ◽  
Vol 260 (4) ◽  
pp. F498-F505
Author(s):  
C. L. Emmons ◽  
K. Matsuzaki ◽  
J. B. Stokes ◽  
V. L. Schuster
Keyword(s):  
Cross Sections ◽  
Cell Function ◽  
Rate Coefficient ◽  
Collecting Duct ◽  
Lectin Binding ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Inner Cortex

The rabbit cortical collecting duct (CCD) consists of three major cell types: principal cells transport K+, beta-intercalated cells absorb Cl-, and alpha-intercalated cells secrete H+. We used functional and histological methods to assess axial distribution of these cell types along rabbit CCD. In perfused CCDs, lumen-to-bath Rb+ rate coefficient (an index of principal cell K+ transport) was not different in tubules from outer cortex (1 mm from renal surface) compared with those from inner cortex (2 mm from renal surface), suggesting that principal cell function is homogeneous along the CCD. In contrast, Cl- rate coefficient (a measure of beta-intercalated cell function) was twice as high in CCDs from outer compared with inner cortex, suggesting heterogeneity of beta-intercalated cells along the CCD. To further investigate these regional differences, we fixed and embedded kidneys and identified three cell types in CCD cross sections using carbonic anhydrase staining and peanut lectin binding. Comparing tubule cross sections from outer with those from inner cortex, we found no axial difference in the fraction of cells that were either principal cells (64%) or total (lectin binding and nonlectin binding) intercalated cells (36%). However, the lectin-binding intercalated cell subset was significantly increased in outer compared with inner cortex. We conclude that there is not heterogeneity of principal cells along the rabbit CCD; however, beta-cell number and function are increased in outer CCD. Collecting duct heterogeneity begins within the cortical segment.

Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice

1995 ◽  
Vol 269 (6) ◽  
pp. F761-F774 ◽  
Author(s):  
S. Breton ◽  
S. L. Alper ◽  
S. L. Gluck ◽  
W. S. Sly ◽  
J. E. Barker ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunofluorescent Staining ◽  
Specific Marker ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Collecting Tubules ◽  
Deficient Mice

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.

H(+)-K(+)-ATPase activity in rat collecting duct segments

1992 ◽  
Vol 262 (4) ◽  
pp. F692-F695 ◽  
Author(s):  
J. D. Gifford ◽  
L. Rome ◽  
J. H. Galla
Keyword(s):  
Atpase Activity ◽  
Marked Decrease ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Medullary Collecting Duct ◽  
Gastric Proton Pump

Previous studies have suggested the presence of an H(+)-K(+)-ATPase in rat cortical and medullary intercalated cells with similar properties to the gastric proton pump. The purpose of this study was to determine the functional contribution of an H(+)-K(+)-adenosinetriphosphatase(ATPase) to total CO2 (tCO2) transport along the rat collecting duct. After baseline determination of tCO2 transport in isolated perfused collecting duct segments, Sch 28080 (10 microM) was added to either the perfusate or bath. When Sch 28080 was added to the perfusate, there was no effect in the cortical collecting duct (CCD, 20.8 +/- 6.7 vs. 25.3 + 3.0 pmol.mm-1.min-1), but a marked decrease in tCO2 absorption was effected in both the outer medullary (OMCD, 37.6 + 6.2 vs. 10.7 +/- 4.1 pmol.mm-1.min-1) and initial inner medullary collecting duct (IMCD1, 34.4 +/- 8.1 vs. 16.2 +/- 5.6 pmol.mm-1.min-1). In the CCD from rats with acute alkalosis in vivo, Sch 28080 added to the bath inhibited tCO2 secretion in the CCD (-17.1 +/- 4.4 vs 3.5 + 3.3 pmol.mm-1.min-1). These findings suggest that 1) H(+)-K(+)-ATPase is important in tCO2 absorption in the OMCD and IMCD1 and in tCO2 secretion in the CCD, 2) HCO3(-)-absorbing intercalated cells differ functionally in the cortex and medulla, 3) HCO3- secretion is not the reverse process of HCO3- absorption in the CCD, and 4) H(+)-K(+)-ATPase is important in distal acidification under normal and altered acid-base conditions.

Cellular origin and hormonal regulation of K+-ATPase activities sensitive to Sch-28080 in rat collecting duct

2000 ◽  
Vol 279 (6) ◽  
pp. F1053-F1059 ◽  
Author(s):  
Nicolas Laroche-Joubert ◽  
Sophie Marsy ◽  
Alain Doucet
Keyword(s):  
Hormonal Regulation ◽  
Collecting Duct ◽  
Type I ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Type Iii ◽  
Principal Cells ◽  
Cellular Origin ◽  
Hormone Effects ◽  
A Cell

Rat collecting ducts exhibit type I or type III K+-ATPase activities when animals are fed a normal (NK) or a K+-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K+-ATPases. For this purpose, we searched for an effect on K+-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-d-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, α-intercalated cells, and β-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and α-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K+-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K+-ATPase) and by dD-AVP in LK rats (type III K+-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K+-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K+-ATPase activity in α- and β-intercalated CCD cells, whereas in LK rats it stimulates type III K+-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.

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.

Cellular distribution of the potassium channel KCNQ1 in normal mouse kidney

2007 ◽  
Vol 292 (1) ◽  
pp. F456-F466 ◽  
Author(s):  
Wencui Zheng ◽  
Jill W. Verlander ◽  
I. Jeanette Lynch ◽  
Melanie Cash ◽  
Jiahong Shao ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Mouse Kidney ◽  
Cellular Heterogeneity ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Distal Nephron ◽  
Rt Pcr ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Principal Cells

Mechanisms of K+ secretion and absorption along the collecting duct are not understood fully. Because KCNQ1 participates in K+ secretion within the inner ear and stomach, distribution of KCNQ1 in mouse kidney was studied using Northern and Western analyses, RT-PCR of isolated tubules, and immunohistochemistry. Northern blots demonstrated KCNQ1 transcripts in whole kidney. RT-PCR showed KCNQ1 mRNA in isolated distal convoluted tubule (DCT), connecting segment (CNT), collecting ducts (CD), and glomeruli. Immunoblots of kidney and stomach revealed a ∼75-kDa protein, the expected mobility for KCNQ1. KCNQ1 was detected by immunohistochemistry throughout the distal nephron and CD. Thick ascending limbs exhibited weak basolateral immunolabel. In DCT and CNT cells, immunolabel was intense and basolateral, although KCNQ1 label was stronger in late than in early DCT. Initial collecting tubule and cortical CD KCNQ1 immunolabel was predominantly diffuse, but many cells exhibited discrete apical label. Double-labeling experiments demonstrated that principal cells, type B intercalated cells, and a few type A intercalated cells exhibited distinct apical KCNQ1 immunolabel. In inner medullary CD, principal cells exhibited distinct basolateral KCNQ1 immunolabel, whereas intercalated cells showed diffuse cytoplasmic staining. Thus KCNQ1 protein is widely distributed in mouse distal nephron and CD, with significant axial and cellular heterogeneity in location and intensity. These findings suggest that KCNQ1 has cell-specific roles in renal ion transport and may participate in K+ secretion and/or absorption along the thick ascending limb, DCT, connecting tubule, and CD.

Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: colocalization with H+-ATPase

1999 ◽  
Vol 277 (6) ◽  
pp. F957-F965 ◽  
Author(s):  
Hisato Sakamoto ◽  
Yoshikazu Sado ◽  
Ichiro Naito ◽  
Tae-Hwan Kwon ◽  
Shinichi Inoue ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Collecting Duct ◽  
Type A ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Double Labeling ◽  
Cytoplasmic Staining ◽  
Confocal Images

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H+-ATPase and analysis by confocal images showed that ClC-5 and H+-ATPase colocalized in these ClC-5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H+ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.

scholarly journals Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.

1998 ◽  
Vol 9 (6) ◽  
pp. 946-959 ◽  
Author(s):  
A K Stuart-Tilley ◽  
B E Shmukler ◽  
D Brown ◽  
S L Alper
Keyword(s):  
Epithelial Cells ◽  
Anion Exchanger ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Sodium Dodecyl ◽  
Mouse Kidney ◽  
Pcr Analysis ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Reverse Transcription Pcr

In this study, an epitope-unmasking technique was used to immunolocalize AE2 anion exchanger polypeptide to basolateral plasma membranes of tubular epithelial cells in mouse kidney. Kidney AE2 immunostaining in mouse kidney was less prominent than in rat, consistent with the relative levels of AE2 mRNA and polypeptide in these two species. Glomeruli showed faint but consistent AE2 immunostaining, whereas proximal tubules were generally unstained. Macula densa epithelial cells displayed bright AE2 immunostaining, and cortical thick limbs were stained at a lower intensity. AE2 immunostaining was weak or absent in type B intercalated cells and principal cells of the cortical collecting duct, but increased in intensity in principal cells of the inner stripe of the outer medulla. AE2 staining in medullary thick limbs was also of greater intensity than in cortical thick limbs. AE2 staining was strong and uniform in the epithelial cells of the inner medullary collecting duct, and in epithelial cells of the papillary surface, the ureter, and the urinary bladder. Extratubular and epithelial cells of the inner medulla also showed punctate intracellular AE2 staining in a Golgi-like distribution that, in contrast to cell surface staining, was sodium dodecyl sulfate-sensitive. Golgi localization of AE2 epitope was confirmed by immunoperoxidase electron microscopy. Reverse transcription-PCR analysis of mouse kidney RNA detected AE2a, AE2b, and an AE2c2 transcript, but an AE2c1 transcript was absent. Unlike in rat, the mouse AE2c2 mRNA splice variant encoded a polypeptide with a novel predicted N-terminal amino acid sequence.

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