Immunohistochemical localization of H-K-ATPase α2c-subunit in rabbit kidney

2001 ◽  
Vol 281 (2) ◽  
pp. F357-F365 ◽  
Author(s):  
Jill W. Verlander ◽  
Robin M. Moudy ◽  
W. Grady Campbell ◽  
Brian D. Cain ◽  
Charles S. Wingo
Keyword(s):  
Collecting Duct ◽  
Type A ◽  
Immunohistochemical Localization ◽  
Macula Densa ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Type B ◽  
Principal Cells ◽  
Medullary Collecting Duct

The rabbit kidney possesses mRNA for the H-K-ATPase α1-subunit (HKα1) and two splice variants of the H-K-ATPase α2-subunit (HKα2). The purpose of this study was to determine the specific distribution of one of these, the H-K-ATPase α2c-subunit isoform (HKα2c), in rabbit kidney by immunohistochemistry. Chicken polyclonal antibodies against a peptide based on the NH2 terminus of HKα2c were used to detect HKα2cimmunoreactivity in tissue sections. Immunohistochemical localization of HKα2c revealed intense apical immunoreactivity in a subpopulation of cells in the connecting segment, cortical collecting duct, and outer medullary collecting duct in both the outer and inner stripe. An additional population of cells exhibited a thin apical band of immunolabel. Immunohistochemical colocalization of HKα2c with carbonic anhydrase II, the Cl−/HCO[Formula: see text] exchanger AE1, and HKα1 indicated that both type A and type B intercalated cells possessed intense apical HKα2c immunoreactivity, whereas principal cells and connecting segment cells had only a thin apical band of HKα2c. Labeled cells were evident through the middle third of the inner medullary collecting duct in the majority of animals. Immunolabel was also present in papillary surface epithelial cells, cells in the cortical thick ascending limb of Henle's loop (cTAL), and the macula densa. Thus in the rabbit kidney, apical HKα2c is present and may contribute to acid secretion or potassium uptake throughout the connecting segment and collecting duct in both type A and type B intercalated cells, principal cells, and connecting segment cells, as well as in cells in papillary surface epithelium, cTAL, and macula densa.

Immunocytochemical localization of band 3 protein in the rat collecting duct

1988 ◽  
Vol 255 (1) ◽  
pp. F115-F125 ◽  
Author(s):  
J. W. Verlander ◽  
K. M. Madsen ◽  
P. S. Low ◽  
D. P. Allen ◽  
C. C. Tisher
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Type A ◽  
Band 3 ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Band 3 Protein ◽  
Type B ◽  
Gold Particles

Band 3 protein is the major anion transport protein of the erythrocyte cell membrane where it catalyzes the exchange of HCO3- for Cl-. There is evidence that band 3 protein is present in the collecting duct of both the rat and rabbit kidney. We used colloidal-gold immunocytochemistry to determine the ultrastructural location of band 3 protein in the rat cortical (CCD) and outer medullary collecting ducts (OMCD). Kidneys of normal Sprague-Dawley rats were fixed by intravascular perfusion with 1% glutaraldehyde and embedded in Lowicryl K4M. Two polyclonal antibodies raised in rabbits were used as the primary antibody in separate experiments, one against the 43-kDa fragment of the cytoplasmic domain of human erythrocyte band 3 protein and the other against rat erythrocyte band 3 protein. This was followed by exposure to gold-conjugated goat anti-rabbit immunoglobulin G. Transmission electron microscopy revealed gold particles along the basal and lateral plasma membranes of all intercalated cells of the OMCD. In the CCD, the basal and lateral plasma membranes of the type A intercalated cells only were labeled with gold particles. The type B intercalated cells and principal cells were devoid of gold particles, as were all cells of the proximal tubule, the distal convoluted tubule, and the thick ascending limb of the loop of Henle. We conclude that a Cl(-)-HCO3- transporter is present in the basal and lateral plasma membranes of the intercalated cells in the OMCD and the type A intercalated cells in the CCD. These findings provide further evidence that these intercalated cells are involved in H+ secretion in the OMCD and CCD of the rat. We have no evidence for the presence of band 3 protein in the type B intercalated cells of the CCD, which supports the hypothesis that type B cells are functionally and structurally distinct from type A cells.

Versatility of NaCl transport mechanisms in the cortical collecting duct

2017 ◽  
Vol 313 (6) ◽  
pp. F1254-F1263 ◽  
Author(s):  
Aurélie Edwards ◽  
Gilles Crambert
Keyword(s):  
Collecting Duct ◽  
Type A ◽  
Transport Systems ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Type B ◽  
Cell Type ◽  
Acid Base ◽  
Nacl Transport ◽  
Principal Cells

The cortical collecting duct (CCD) forms part of the aldosterone-sensitive distal nephron and plays an essential role in maintaining the NaCl balance and acid-base status. The CCD epithelium comprises principal cells as well as different types of intercalated cells. Until recently, transcellular Na+ transport was thought to be restricted to principal cells, whereas (acid-secreting) type A and (bicarbonate-secreting) type B intercalated cells were associated with the regulation of acid-base homeostasis. This review describes how this traditional view has been upended by several discoveries in the past decade. A series of studies has shown that type B intercalated cells can mediate electroneutral NaCl reabsorption by a mechanism involving Na+-dependent and Na+-independent Cl−/[Formula: see text] exchange, and that is energetically driven by basolateral vacuolar H+-ATPase pumps. Other research indicates that type A intercalated cells can mediate NaCl secretion, through a bumetanide-sensitive pathway that is energized by apical H+,K+-ATPase type 2 pumps operating as Na+/K+ exchangers. We also review recent findings on the contribution of the paracellular route to NaCl transport in the CCD. Last, we describe cross-talk processes, by which one CCD cell type impacts Na+/Cl− transport in another cell type. The mechanisms that have been identified to date demonstrate clearly the interdependence of NaCl and acid-base transport systems in the CCD. They also highlight the remarkable versatility of this nephron segment.

Intrarenal and Cellular Localization of CLC-K2 Protein in the Mouse Kidney

2001 ◽  
Vol 12 (7) ◽  
pp. 1327-1334 ◽  
Author(s):  
KATSUKI KOBAYASHI ◽  
SHINICHI UCHIDA ◽  
SHUKI MIZUTANI ◽  
SEI SASAKI ◽  
FUMIAKI MARUMO
Keyword(s):  
Cellular Localization ◽  
Collecting Duct ◽  
Type A ◽  
Mouse Kidney ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Basolateral Membranes ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Distal Tubules

Abstract. CLC-K2, a kidney-specific member of the CLC chloride channel family, is thought to play an important role in the transepithelial Cl- transport in the kidney. This consensus was first reached shortly after it was demonstrated that the mutations of the human CLCNKB gene resulted in Bartter's syndrome type III. To clarify the pathogenesis, the exact intrarenal and cellular localization of CLC-K2 by immunohistochemistry of the Clcnk1-/- mouse kidney were investigated by use of an anti-CLC-K antibody that recognized both CLC-K1 and CLC-K2. CLC-K2 is expressed in the thick ascending limb of Henle's loop and distal tubules, where it is localized to the basolateral membranes. The localization of CLC-K2 to these nephron segments strongly implies that CLC-K2 confers the basolateral chloride conductance in the thick ascending limb of Henle's loop and distal tubules, where Cl- is taken up by the bumetanide-sensitive Na-K-2Cl cotransporter or the thiazide-sensitive Na-Cl cotransporter at the apical membranes. CLC-K2 expression was also shown to extend into the connecting tubule in the basolateral membrane. CLC-K2 was found in basolateral membranes of the type A intercalated cells residing along the collecting duct. This localization strongly suggests that CLC-K2 confers the basolateral conductance in the type A intercalated cells where Cl- is taken up by the anion exchanger in exchange for HCO3- at the basolateral membranes. These aspects of CLC-K2 localization suggest that CLC-K2 is important in Cl- transport in the distal nephron segments.

Evaluation of cellular plasticity in the collecting duct during recovery from lithium-induced nephrogenic diabetes insipidus

2013 ◽  
Vol 305 (6) ◽  
pp. F919-F929 ◽  
Author(s):  
Francesco Trepiccione ◽  
Giovambattista Capasso ◽  
Søren Nielsen ◽  
Birgitte Mønster Christensen
Keyword(s):  
Time Course ◽  
Morphological Changes ◽  
Collecting Duct ◽  
Lithium Treatment ◽  
Transition Element ◽  
Type A ◽  
Functional Markers ◽  
Intercalated Cells ◽  
Anion Exchanger 1 ◽  
Principal Cells

The cellular morphology of the collecting duct is altered by chronic lithium treatment. We have previously shown that lithium increases the fraction of type-A intercalated cells and lowers the fraction of principal cells along the collecting duct. Moreover, type-A intercalated cells acquire a long-row distribution pattern along the tubules. In the present study, we show that these morphological changes reverse progressively after discontinuation of lithium and finally disappear after 19 days from lithium suspension. In this time frame we have identified for the first time, in vivo, a novel cellular type positive for both intercalated and principal cells functional markers, as recognized by colabeling with H+-ATPase/aquaporin-4 (AQP4) and anion exchanger-1 (AE-1)/AQP2 and Foxi1/AQP4. This cell type is mainly present after 6 days of lithium washout, and it disappears in parallel with the long-row pattern of the type-A intercalated cells. It usually localizes either in the middle or at the edge of the long-row pattern. Its ultrastructure resembles the intercalated cells as shown both by differential interference contrast and by electron microscopy. The time course of appearance, the localization along the collecting duct, and the ultrastructure suggest that the cells double labeled for principal and intercalated cells markers could represent a transition element driving the conversion of intercalated cells into principal cells.

Structural-functional relationships along the distal nephron

1986 ◽  
Vol 250 (1) ◽  
pp. F1-F15 ◽  
Author(s):  
K. M. Madsen ◽  
C. C. Tisher
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Structural Heterogeneity ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Functional Relationships

The distal tubule, which includes the thick ascending limb (TAL), the macula densa, and the distal convoluted tubule (DCT), and the collecting duct are structurally heterogeneous, thus reflecting the functional heterogeneity that is also present. As the TAL ascends from medulla to cortex, the surface area of the apical plasma membrane increases while that of the basolateral membrane decreases. The structure of the DCT resembles that of the medullary TAL. An excellent correlation exists between structure, Na-K-ATPase activity, and NaCl reabsorptive capacity in the distal tubule. The collecting duct is subdivided into the initial collecting tubule (ICT), and cortical (CCD), outer medullary (OMCD), and inner medullary (IMCD) collecting ducts. Between the distal tubule and the collecting duct is a transition region termed the connecting segment or connecting tubule (CNT). Considerable structural heterogeneity exists along the collecting duct within the two major cell populations, the intercalated cells and the principal cells. In the CNT, the ICT, and the CCD, potassium loading and mineralocorticoids stimulate Na-K-ATPase activity and cause proliferation of the basolateral membrane of CNT cells and principal cells, thus identifying the cells responsible for mineralocorticoid-stimulated potassium secretion in these regions. Finally, at least two morphologically distinct populations of intercalated cells exist, types A and B. In the rat, type A predominates in the CNT and the OMCD and is believed to be responsible for H+ secretion, at least in the OMCD. Type B predominates in the CCD, where it may be involved in bicarbonate secretion.

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

1999 ◽  
Vol 10 (1) ◽  
pp. 1-12 ◽  
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.

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.

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.

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.

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.

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