AE4 is a DIDS-sensitive Cl−/HCO 3 − exchanger in the basolateral membrane of the renal CCD and the SMG duct

2002 ◽  
Vol 283 (4) ◽  
pp. C1206-C1218 ◽  
Author(s):  
Shigeru B. H. Ko ◽  
Xiang Luo ◽  
Henrik Hager ◽  
Alexandra Rojek ◽  
Joo Young Choi ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Specific Expression ◽  
Hek 293 ◽  
Acid Base Status ◽  
Species Specific

The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The β-intercalated cells secrete most of the HCO[Formula: see text], which is mediated by a luminal, DIDS-insensitive, Cl−/HCO[Formula: see text] exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180–8189, 2001). By contrast, it was proposed (Royaux IE et al. Proc Natl Acad Sci USA 98: 4221–4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK1 cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pHi) revealed that AE4 indeed functions as a Cl−/HCO[Formula: see text] exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in α-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl−/HCO[Formula: see text] exchanger in the basolateral membrane of α-intercalated cells and may participate in HCO[Formula: see text] absorption. In the rabbit AE4 may contribute to HCO[Formula: see text] secretion.

Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status

1994 ◽  
Vol 266 (4) ◽  
pp. F528-F535 ◽  
Author(s):  
C. Emmons ◽  
J. B. Stokes
Keyword(s):  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Acid Base Status

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

Intracellular band 3 immunostaining in type A intercalated cells of rabbit kidney

1992 ◽  
Vol 262 (6) ◽  
pp. F1015-F1022
Author(s):  
K. M. Madsen ◽  
J. Kim ◽  
C. C. Tisher
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Type A ◽  
Band 3 ◽  
Rabbit Kidney ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Electron Microscopic

Intercalated cells (ICs) in the collecting duct and the connecting tubule (CNT) are involved in H+ secretion and HCO3- reabsorption. H+ secretion is mediated by an H(+)-adenosinetriphosphatase in the apical plasma membrane, whereas a band 3-like Cl(-)-HCO3- exchanger in the basolateral membrane is responsible for HCO3- reabsorption. Recent studies have reported that a band 3-like protein is also present in mitochondria in rabbit ICs. The purpose of this study was to establish the subcellular location of the band 3-like Cl(-)-HCO3- exchanger in rabbit ICs by electron microscopic immunocytochemistry using a monoclonal antibody, IVF12, against erythrocyte band 3 protein. Rabbit kidneys were preserved by in vivo perfusion with a paraformaldehyde-lysine-periodate solution and processed for immunocytochemistry using a horseradish peroxidase preembedding technique. Band 3 immunostaining was observed on the basolateral plasma membrane of ICs in the outer medullary collecting duct and type A cells in the cortical collecting duct (CCD) and CNT. In addition, distinct staining for band 3 was present in numerous small vesicles and in multivesicular bodies in type A ICs in the CCD and CNT. However, there was no evidence of band 3 immunostaining of mitochondria or of the apical plasma membrane in any cells of the collecting duct. These observations suggest that basolateral Cl(-)-HCO3- exchangers in type A ICs in the rabbit kidney are stored in intracellular vesicles and possibly degraded in the vascular-lysosomal system when these cells are in a resting state. The previously reported band 3 immunolabeling of mitochondria could not be confirmed.

Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance

1998 ◽  
Vol 274 (3) ◽  
pp. F596-F601 ◽  
Author(s):  
Géza Fejes-Tóth ◽  
Erzsébet Rusvai ◽  
Emily S. Cleaveland ◽  
Anikó Náray-Fejes-Tóth
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Cell Types ◽  
Alkaline Media ◽  
Mrna Levels ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base Balance ◽  
Acid Base ◽  
Base Balance

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3 (base) loading of rabbits for ∼18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for β-actin levels. We found that CCD cells express high levels of AE2 mRNA (∼500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 ± 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, α-intercalated cells, and β-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+ concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of β-intercalated cells.

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)

scholarly journals Cortical distal nephron Cl− transport in volume homeostasis and blood pressure regulation

2013 ◽  
Vol 305 (4) ◽  
pp. F427-F438 ◽  
Author(s):  
Susan M. Wall ◽  
Alan M. Weinstein
Keyword(s):  
Blood Pressure ◽  
Pressor Response ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Extracellular Volume ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base Balance ◽  
Relative Contribution ◽  
And Function

Renal intercalated cells mediate the secretion or absorption of Cl− and OH−/H+ equivalents in the connecting segment (CNT) and cortical collecting duct (CCD). In so doing, they regulate acid-base balance, vascular volume, and blood pressure. Cl− absorption is either electrogenic and amiloride-sensitive or electroneutral and thiazide-sensitive. However, which Cl− transporter(s) are targeted by these diuretics is debated. While epithelial Na+ channel (ENaC) does not transport Cl−, it modulates Cl− transport probably by generating a lumen-negative voltage, which drives Cl− flux across tight junctions. In addition, recent evidence indicates that ENaC inhibition increases electrogenic Cl− secretion via a type A intercalated cells. During ENaC blockade, Cl− is taken up across the basolateral membrane through the Na+-K+−2Cl− cotransporter (NKCC1) and then secreted across the apical membrane through a conductive pathway (a Cl− channel or an electrogenic exchanger). The mechanism of this apical Cl− secretion is unresolved. In contrast, thiazide diuretics inhibit electroneutral Cl− absorption mediated by a Na+-dependent Cl−/HCO3− exchanger. The relative contribution of the thiazide and the amiloride-sensitive components of Cl− absorption varies between studies and probably depends on the treatment model employed. Cl− absorption increases markedly with angiotensin and aldosterone administration, largely by upregulating the Na+-independent Cl−/HCO3− exchanger pendrin. In the absence of pendrin [ Slc26a4 (−/−) or pendrin null mice], aldosterone-stimulated Cl− absorption is significantly reduced, which attenuates the pressor response to this steroid hormone. Pendrin also modulates aldosterone-induced changes in ENaC abundance and function through a kidney-specific mechanism that does not involve changes in the concentration of a circulating hormone. Instead, pendrin changes ENaC abundance and function, at least in part, by altering luminal HCO3−. This review summarizes mechanisms of Cl− transport in CNT and CCD and how these transporters contribute to the regulation of extracellular volume and blood pressure.

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.

Site and mechanism of action of epidermal growth factor in rabbit cortical collecting duct

1991 ◽  
Vol 260 (2) ◽  
pp. F163-F169 ◽  
Author(s):  
S. Muto ◽  
H. Furuya ◽  
K. Tabei ◽  
Y. Asano
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mechanism Of Action ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Membrane Voltage ◽  
Rabbit Kidney ◽  
Cortical Collecting Duct ◽  
Epidermal Growth

To examine the exact target cell and mechanism of action of epidermal growth factor (EGF) in the isolated cortical collecting duct from rabbit kidney, we compared electrical properties of collecting duct (CD) cells (principal cells) and intercalated (IC) cells in absence and presence of EGF at 10(-8) M. Differentiation of CD and IC cells was based on values of basolateral membrane voltage (Vb) and fractional apical membrane resistance (fRa). In CD cells, upon addition of EGF to bath, lumen-negative transepithelial voltage (VT) was decreased from -8.0 +/- 1.9 to -2.4 +/- 1.3 mV (n = 22, P less than 0.001), but Vb was little changed (from -85.1 +/- 2.8 to -83.1 +/- 2.7 mV, n = 19), indicating that EGF in bath mainly caused changes in apical membrane voltage. In addition, peritubular EGF increased transepithelial resistance (RT) from 132.9 +/- 15.8 to 153.8 +/- 18.4 omega.cm2 (n = 16, P less than 0.001) as well as fRa from 0.31 +/- 0.06 to 0.39 +/- 0.07 (n = 12, P less than 0.01). These actions of EGF were prevented by pretreatment with 50 microM luminal amiloride. Luminal EGF had no effects on VT, Vb, RT, or fRa of CD cells. In IC cells, upon addition of EGF to bath, neither Vb nor fRa was affected. From these results, we conclude that EGF acts on the CD cell at the basolateral border and inhibits mainly the amiloride-sensitive Na+ conductance in the apical membrane.

scholarly journals Insights into acidosis-induced regulation of SLC26A4 (pendrin) and SLC4A9 (AE4) transporters using three-dimensional morphometric analysis of β-intercalated cells

2014 ◽  
Vol 307 (5) ◽  
pp. F601-F611 ◽  
Author(s):  
Jeffrey M. Purkerson ◽  
Eric V. Heintz ◽  
Aya Nakamori ◽  
George J. Schwartz
Keyword(s):  
Collecting Duct ◽  
Three Dimensional ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Recycling Endosomes ◽  
Coordinate Regulation ◽  
Anion Transporters ◽  
Early Endosomes ◽  
Zona Occludens

The purpose of this study was to examine the three-dimensional (3-D) expression and distribution of anion transporters pendrin (SLC26A4) and anion exchanger (AE)4 (SLC4A9) in β-intercalated cells (β-ICs) of the rabbit cortical collecting duct (CCD) to better characterize the adaptation to acid-base disturbances. Confocal analysis and 3-D reconstruction of β-ICs, using identifiers of the nucleus and zona occludens, permitted the specific orientation of cells from normal, acidotic, and recovering rabbits, so that adaptive changes could be quantified and compared. The pendrin cap likely mediates apical Cl−/HCO3− exchange, but it was also found beneath the zona occludens and in early endosomes, some of which may recycle back to the apical membrane via Rab11a+ vesicles. Acidosis reduced the size of the pendrin cap, observed as a large decrease in cap volume above and below the zona occludens, and the volume of the Rab11a+ apical recycling compartment. Correction of the acidosis over 12–18 h reversed these changes. Consistent with its proposed function in the basolateral exit of Na+ via Na+-HCO3− cotransport, AE4 was expressed as a barrel-like structure in the lateral membrane of β-ICs. Acidosis reduced AE4 expression in β-ICs, but this was rapidly reversed during the recovery from acidosis. The coordinate regulation of pendrin and AE4 during acidosis and recovery is likely to affect the magnitude of acid-base and possibly Na+ transport across the CCD. In conclusion, acidosis induces a downregulation of AE expression in β-ICs and a diminished presence of pendrin in apical recycling endosomes.

scholarly journals Life-threatening metabolic alkalosis in Pendred syndrome

2011 ◽  
Vol 165 (1) ◽  
pp. 167-170 ◽  
Author(s):  
Narayanan Kandasamy ◽  
Laura Fugazzola ◽  
Mark Evans ◽  
Krishna Chatterjee ◽  
Fiona Karet
Keyword(s):  
Metabolic Alkalosis ◽  
Collecting Duct ◽  
Sensorineural Deafness ◽  
Apical Plasma Membrane ◽  
Cortical Collecting Duct ◽  
Loss Of Function ◽  
Pendred Syndrome ◽  
Acid Base ◽  
Life Threatening ◽  
Acid Base Status

IntroductionPendred syndrome, a combination of sensorineural deafness, impaired organification of iodide in the thyroid and goitre, results from biallelic defects in pendrin (encoded by SLC26A4), which transports chloride and iodide in the inner ear and thyroid respectively. Recently, pendrin has also been identified in the kidneys, where it is found in the apical plasma membrane of non-α-type intercalated cells of the cortical collecting duct. Here, it functions as a chloride–bicarbonate exchanger, capable of secreting bicarbonate into the urine. Despite this function, patients with Pendred syndrome have not been reported to develop any significant acid–base disturbances, except a single previous reported case of metabolic alkalosis in the context of Pendred syndrome in a child started on a diuretic.Case reportWe describe a 46-year-old female with sensorineural deafness and hypothyroidism, who presented with severe hypokalaemic metabolic alkalosis during inter-current illnesses on two occasions, and who was found to be homozygous for a loss-of-function mutation (V138F) in SLC26A4. Her acid–base status and electrolytes were unremarkable when she was well.ConclusionThis case illustrates that, although pendrin is not usually required to maintain acid–base homeostasis under ambient condition, loss of renal bicarbonate excretion by pendrin during a metabolic alkalotic challenge may contribute to life-threatening acid–base disturbances in patients with Pendred syndrome.

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