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.

The "coat" of kidney intercalated cell tubulovesicles does not contain clathrin

1986 ◽  
Vol 250 (4) ◽  
pp. C605-C608 ◽  
Author(s):  
D. Brown ◽  
L. Orci
Keyword(s):  
Coat Protein ◽  
Rat Kidney ◽  
Cell Types ◽  
Intercalated Cells ◽  
Coated Pits ◽  
Thin Sections ◽  
Intercalated Cell ◽  
Apical Cytoplasm ◽  
Collecting Ducts ◽  
Lowicryl K4m

Intercalated cells of kidney collecting ducts contain a population of tubulovesicles in their apical cytoplasm, whose limiting membranes are decorated by arrays of dense, club-shaped projections oriented toward the cytoplasm. These tubulovesicles have been implicated in endo-exocytotic events in these cells. To determine a possible relationship between this “coating” material and clathrin, the coat protein associated with endocytotic coated pits and coated vesicles in other cell types, we applied a monospecific, affinity-purified anti-clathrin antibody to thin sections of rat kidney embedded at low temperature in Lowicryl K4M. We found that no specific labeling was associated with the studlike material of intercalated cell tubulovesicles.

Three distinct cell populations in rat kidney collecting duct

1987 ◽  
Vol 253 (2) ◽  
pp. C323-C328 ◽  
Author(s):  
H. Holthofer ◽  
B. A. Schulte ◽  
G. Pasternack ◽  
G. J. Siegel ◽  
S. S. Spicer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Morphological Characteristics ◽  
Anion Channel ◽  
Band 3 ◽  
Cell Populations ◽  
Intercalated Cells ◽  
Distinct Cell ◽  
Collecting Ducts ◽  
Principal And Intercalated Cells

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.

Lectin-gold labeling of glycoconjugates in normal and Brattleboro rat papilla: effect of vasopressin

1988 ◽  
Vol 254 (3) ◽  
pp. C450-C458 ◽  
Author(s):  
P. Weyer ◽  
D. Brown ◽  
L. Orci
Keyword(s):  
Epithelial Cell ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Helix Pomatia ◽  
Major Effect ◽  
Urinary Concentration ◽  
Brattleboro Rats ◽  
Cell Plasma ◽  
Collecting Ducts ◽  
Helix Pomatia Lectin

Some reports suggest that the plasma membrane glycocalyx of collecting duct epithelial cells, as well as interstitial glycoconjugates, may be involved in vasopressin action and urinary concentration. In view of this, we have used the lectin-gold technique to map and quantify Helix pomatia lectin (HPL)-binding sites in the inner medulla of kidneys from normal Long-Evans rats, vasopressin-deficient Brattleboro rats, and Brattleboro rats treated for up to 5 wk with exogenous vasopressin. The results show that the labeling of epithelial cell plasma membranes from collecting ducts and thin limbs of Henle is not different between normal and Brattleboro rats, and the labeling is not modified by chronic vasopressin treatment. In contrast, the heavy interstitial labeling seen in normal rats is virtually absent from Brattleboro rats, but it is progressively restored by chronic vasopressin treatment of Brattleboro rats. These results show that vasopressin does not modify HPL-binding glycoconjugates on epithelial cell plasma membranes, but that vasopressin treatment has a major effect on HPL-binding glycoconjugates in the medullary interstitium.

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.

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.

Postnatal maturation of rabbit renal collecting duct: intercalated cell function

1987 ◽  
Vol 253 (4) ◽  
pp. F622-F635 ◽  
Author(s):  
L. M. Satlin ◽  
G. J. Schwartz
Keyword(s):  
Fluorescent Dyes ◽  
Cell Function ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Mitochondrial Potential ◽  
Postnatal Maturation ◽  
Intercalated Cell ◽  
Cell Ph ◽  
Cytoplasmic Vesicles ◽  
Collecting Ducts

Intercalated cells play a major role in renal regulation of acid-base balance. We used fluorescent dyes to characterize postnatal maturation of intercalated cells. We stained rabbit collecting ducts with the pH-sensitive dye 6-carboxyfluorescein diacetate and identified individual intercalated cells by their bright green fluorescence. Number of fluorescent cells per millimeter tubule doubled during maturation in midcortex (68 +/- 7 to 121 +/- 9; P less than 0.01) but did not change in outer stripe of outer medulla. Excitation-ratio (490/450 nm) fluorometry of individual cells in nonperfused tubules revealed an increase in pH of cortical intercalated cell from 7.28 +/- 0.03 in newborn to 7.43 +/- 0.03 in adult (P less than 0.005); principal cell pH did not change with age, averaging 7.10 +/- 0.05. The smaller difference in pH between intercalated and principal cells in neonates suggested a paucity of H+ pumps in immature intercalated cells. Indeed, few cortical intercalated cells trapped the weak base acridine orange in cytoplasmic vesicles that contained H+ pumps or demonstrated selective uptake of 3,3'+-dipentyloxacarbocyanine, a fluorescent cation that probes for mitochondrial potential. Intercalated cells in neonatal medullary collecting ducts had a cell pH similar to that measured in the adult, as well as numerous acidic cytoplasmic vesicles and significant mitochondrial potentials. In conclusion, intercalated cells in cortical collecting duct underwent postnatal proliferation and maturation, whereas those cells present in outer medullary collecting duct, where proliferation was virtually complete by 1 wk of age, were nearly differentiated. Signals directing this centrifugal pattern of postnatal renal maturation are presently unknown.

Postnatal maturation of rabbit renal collecting duct. III. Peanut lectin-binding intercalated cells

1992 ◽  
Vol 262 (2) ◽  
pp. F199-F208 ◽  
Author(s):  
L. M. Satlin ◽  
T. Matsumoto ◽  
G. J. Schwartz
Keyword(s):  
Collecting Duct ◽  
Lectin Binding ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Outer Cortex ◽  
Cell Surface Antigens ◽  
Postnatal Maturation ◽  
Intercalated Cell ◽  
Cell Ph ◽  
Alkaline Cell

Measurements of transepithelial HCO3 transport in the rabbit cortical collecting duct (CCD) indicate that net HCO3 secretion becomes apparent only after the first month of life [F. M. Mehrgut, L. M. Satlin, and G. J. Schwartz, Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F801-F808, 1990]. We used fluorescent probes and immunocytochemistry to trace the postnatal functional development of the beta-intercalated cell, the HCO3-secreting cell of the fully differentiated CCD. Throughout maturation, the beta-intercalated cell was empirically identified by its selective uptake of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, an alkaline cell pH, apical binding to peanut agglutinin (PNA) and monoclonal antibody B63, and by its functional capacity for apical Cl-HCO3 exchange as manifested by Cl-dependent extrusion of an intracellular alkali load. Compared with the mature segment, the neonatal mid-CCD exhibited fewer intercalated cells, which were characterized by a less alkaline cell pH, reduced apical Cl-HCO3 exchange activity, and shorter apical binding profiles for PNA. There was evidence for basolateral Cl conductance and similar buffering capacity at all ages. In the neonatal outer cortex there was little or no binding to PNA or to B63. As soon as cell surface antigens characteristic of the fully differentiated beta-cell were detected, functional studies indicated the presence, albeit reduced, of apical Cl-HCO3 exchange. Thus there is postnatal proliferation and maturation of HCO3-secreting intercalated cells in the rabbit kidney; the origin of these cells remains to be determined.

Calcitonin stimulates H+ secretion in rat kidney intercalated cells

1996 ◽  
Vol 271 (6) ◽  
pp. F1217-F1223 ◽  
Author(s):  
E. Siga ◽  
P. Houillier ◽  
B. Mandon ◽  
G. Moine ◽  
C. de Rouffignac
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Intercalated Cells ◽  
Specific Function ◽  
Cortical Collecting Duct ◽  
Intercalated Cell ◽  
Transepithelial Voltage ◽  
Net Fluxes

Calcitonin (CT) modulates rat intercalated cell (IC) functions of the rat cortical collecting duct (CCD) [E. Siga, B. Mandon, N. Roinel, and C. de Rouffignac. Am.J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F221-F227, 1993]. To characterize the specific function regulated by CT, rat CCDs were perfused in vitro. Total CO2 net fluxes (JtCO2, pmol.mm-1.min-1) and transepithelial voltage (Vt) were measured. Bath CT induced a significant tCO2 reabsorption. This effect was higher on CCDs harvested from acid-loaded than from control rats. When HCO3- secretion was blocked, CT also raised JtCO2 and Vt. When H+ secretion was blocked, CT was ineffective on JtCO2 and Vt. When HCO3- secretion was increased and H+ secretion was inhibited, CT did not change JtCO2, whereas isoproterenol (ISO) increased tCO2 secretion from -13.5 +/- 2.0 (control) to -19.0 +/- 2.4 (ISO). In rat CCD studied under these same preceding conditions plus luminal amiloride to block the Na(+)-dependent Vt, CT did not alter Vt, whereas ISO increased it by 4.5 +/- 0.7 mV. We conclude from these data that, in the rat CCD, calcitonin stimulates H+ secretion, likely by so-called alpha-intercalated (alpha-IC) cells, whereas ISO stimulates HCO3- secretion, likely by so-called beta-IC cells.

scholarly journals Expression of ammonia transporter family members, Rh B glycoprotein and Rh C glycoprotein, in the developing rat kidney

2010 ◽  
Vol 299 (1) ◽  
pp. F187-F198 ◽  
Author(s):  
Ki-Hwan Han ◽  
Su-Youn Lee ◽  
Wan-Young Kim ◽  
Jung-A Shin ◽  
Jin Kim ◽  
...  
Keyword(s):  
Kidney Development ◽  
Collecting Duct ◽  
Family Members ◽  
Ammonia Excretion ◽  
Intercalated Cells ◽  
Double Labeling ◽  
Ammonia Metabolism ◽  
Intercalated Cell ◽  
Transporter Family ◽  
In Cells

Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This study's purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 ( E16), 18 ( E18), and 20 ( E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity.

scholarly journals Ultrastructural localization of H+ATPase in rabbit cortical collecting duct.

1994 ◽  
Vol 4 (8) ◽  
pp. 1546-1557 ◽  
Author(s):  
J W Verlander ◽  
K M Madsen ◽  
D K Stone ◽  
C C Tisher
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rabbit Kidney ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Thin Sections ◽  
Cytoplasmic Vesicles ◽  
Basolateral Plasma Membrane

In contrast to results obtained in the rat kidney, studies of H+ATPase localization in the rabbit kidney have failed to demonstrate basolateral plasma membrane H+ATPase immunoreactivity in intercalated cells in the cortical collecting duct (CCD). Previous studies have relied on light microscopic immunofluorescence techniques, which have limited resolution. Therefore, the immunogold procedure was used to localize H+ATPase in rabbit collecting ducts at the ultrastructural level. Rabbit kidneys were preserved in vivo with periodate-lysine-paraformaldehyde or glutaraldehyde solutions, and samples of cortex were embedded in Lowicryl K4M. Thin sections were labeled for H+ATPase by the immunogold procedure with a rabbit polyclonal antibody against the 70-kd subunit of bovine brain H+ATPase. Three patterns of localization of H+ATPase were observed. The majority of intercalated cells in the CCD exhibited label over cytoplasmic vesicles only. In these cells, no label was associated with either the apical or basolateral plasma membranes. In a second group of cells, label for H+ATPase was observed along the basolateral plasma membrane and over cytoplasmic vesicles throughout the cell. Rarely, intercalated cells with H+ATPase label along the apical plasma membrane and over the apical cytoplasmic vesicles were observed in the CCD. In the initial collecting tubule and connecting segment, intercalated cells with either pronounced apical or basolateral plasma membrane label prevailed, whereas few cells exhibited label restricted to the cytoplasmic vesicles. In summary, in the rabbit CCD, three patterns of H+ATPase distribution exist in intercalated cells, two of which conform to published models of type A and type B intercalated cells.

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