Electron-microscopic immunogold localization of a collecting-duct antigen (PCD2) in intercalated and principal cells of rabbit kidney

1987 ◽  
Vol 249 (3) ◽  
Author(s):  
S. Bachmann ◽  
P. Gilbert ◽  
W.W. Minuth
Keyword(s):  
Collecting Duct ◽  
Rabbit Kidney ◽  
Immunogold Localization ◽  
Electron Microscopic ◽  
Principal Cells

Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct

1998 ◽  
Vol 274 (2) ◽  
pp. F384-F394 ◽  
Author(s):  
David Marples ◽  
Trina A. Schroer ◽  
Nikolai Ahrens ◽  
Ann Taylor ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Motor Protein ◽  
Cytoplasmic Dynein ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Principal Cells ◽  
Intracellular Vesicles ◽  
Dynactin Complex ◽  
Intermediate Chains ◽  
Regulated Trafficking

We investigated whether the motor protein cytoplasmic dynein and dynactin, a protein complex thought to link dynein with vesicles, are present in rat renal collecting ducts and associated with aquaporin-2 (AQP2)-bearing vesicles. Immunoblotting demonstrated cytoplasmic dynein heavy and intermediate chains in kidney, with relative expression levels of inner medulla > outer medulla > cortex. In addition to being present in cytoplasmic fractions, dynein was abundant in membrane fractions enriched for intracellular vesicles. Dynactin was also abundant in membrane fractions enriched for intracellular vesicles. Furthermore, both dynactin and dynein were present in vesicles specifically immunoisolated using anti-AQP2 antibodies. Immunocytochemistry revealed labeling for dynein in the collecting duct principal cells with a pattern consistent with labeling of intracellular vesicles. Moreover, quantitative double immunogold labeling confirmed colocalization of AQP2 and dynein in the same vesicles at the electron microscopic level. Thus the microtubule-associated motor protein dynein and the associated dynactin complex are present in rat renal collecting duct principal cells and are associated with intracellular vesicles, including those bearing AQP2, consistent with the view that dynein and dynactin are involved in vasopressin-regulated trafficking of AQP2-bearing vesicles.

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.

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.

scholarly journals Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct

2016 ◽  
Vol 310 (4) ◽  
pp. F284-F293 ◽  
Author(s):  
Alexis A. Gonzalez ◽  
Flavia Cifuentes-Araneda ◽  
Cristobal Ibaceta-Gonzalez ◽  
Alex Gonzalez-Vergara ◽  
Leonardo Zamora ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Critical Role ◽  
Combined Treatment ◽  
Receptor Activation ◽  
Ang Ii ◽  
Principal Cells ◽  
Protein Levels ◽  
Renin Synthesis ◽  
Renin Mrna ◽  
Creb Pathway

Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10−6 M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD.

ANP-stimulated cGMP egression in renal principal cells: abrogation of polarity by SV40 large T

1996 ◽  
Vol 270 (4) ◽  
pp. C1051-C1060 ◽  
Author(s):  
V. Millul ◽  
D. Prie ◽  
M. Geniteau-Legendre ◽  
M. C. Verpont ◽  
B. Baudouin ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Collecting Duct ◽  
Physiological Role ◽  
Specific Effect ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Temperature Sensitive ◽  
Cyclic Monophosphate ◽  
First Order ◽  
Principal Cells

Egression of atrial natriuretic peptide (ANP)-stimulated guanosine 3', 5'-cyclic monophosphate (cGMP) was compared with that of isoproterenol-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) in a rabbit collecting duct cell line transformed with a temperature-sensitive strain of simian virus 40 (SV40). At 39.5 degrees C (inactivated large T), cells exhibit major features of principal cells, whereas at 33 degrees C (functional large T) they lose most of their specific properties. When cells were grown on plastic at 39.5 degrees C, both cyclic nucleotides were predominantly released extracellularly via probenecid-sensitive carriers. Probenecid (3mM) reduced the ratios of extracellular cGMP and cAMP by 84 and 70%, respectively. The amount of extracellular cGMP or cAMP ws linearly correlated with the time integral of the intracellular cyclic nucleotide, suggesting first-order kinetics. The apparent first-order rate constant (k) was sixfold greater for cGMP (0.139 +/- 0.037 min-1, n = 3 experiments) than for cAMP (0.022 +/- 0.003(-1), n = 3 experiments). 3-Isobutyl-1-methylxanthine markedly inhibited extrusion of cGMP (k = 0.022 +/- 0.003 min-1), whereas that of cAMP was unchanged. When cells were grown on filters at 39.5 degrees C, both nucleotides were predominantly released in the apical medium but with a greater polarity for cGMP (83 +/- 4%, n = 6 experiments) than for cAMP (60 +/- 6%, n = 3 experiments) and a prevailing apical localization of the probenecid-sensitive carrier. Activation of SV40 large T at 33 degrees C did not alter cyclic nucleotide transport characteristics but abolished the polarity of probenecid-sensitive cyclic nucleotide extrusion. These results suggest a physiological role for luminal cGMP in the rabbit collecting duct and a specific effect of large T on the probenecid-sensitive carrier polarity.

scholarly journals K + homeostasis is maintained with knockdown of big‐conductance K + channel in principal cells of connecting tubule/collecting duct

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Timo Rieg ◽  
Robert Lukowski ◽  
Jessica Dominguez ◽  
Meaghen Sharik ◽  
Peter Ruth ◽  
...  
Keyword(s):  
Collecting Duct ◽  
K Channel ◽  
Principal Cells ◽  
Connecting Tubule

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.

scholarly journals Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ

2004 ◽  
Vol 286 (4) ◽  
pp. C768-C778 ◽  
Author(s):  
Jaafar El Annan ◽  
Dennis Brown ◽  
Sylvie Breton ◽  
Sylvain Bourgoin ◽  
Dennis A. Ausiello ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blotting ◽  
Collecting Duct ◽  
Small Gtpases ◽  
Renal Physiology ◽  
Principal Cells ◽  
Arf Gtpases ◽  
Kidney Epithelial Cells

ADP-ribosylation factors (Arfs) are small GTPases that regulate vesicular trafficking in exo- and endocytotic pathways. As a first step in understanding the role of Arfs in renal physiology, immunocytochemistry and Western blotting were performed to characterize the expression and targeting of Arf1 and Arf6 in epithelial cells in situ. Arf1 and Arf6 were associated with apical membranes and subapical vesicles in proximal tubules, where they colocalized with megalin. Arf1 was also apically expressed in the distal tubule, connecting segment, and collecting duct (CD). Arf1 was abundant in intercalated cells (IC) and colocalized with V-ATPase in A-IC (apical) and B-IC (apical and/or basolateral). In contrast, Arf6 was associated exclusively with basolateral membranes and vesicles in the CD. In the medulla, basolateral Arf6 was detectable mainly in A-IC. Expression in principal cells became weaker throughout the outer medulla, and Arf6 was not detectable in principal cells in the inner medulla. In some kidney epithelial cells Arf1 but not Arf6 was also targeted to a perinuclear patch, where it colocalized with TGN38, a marker of the trans-Golgi network. Quantitative Western blotting showed that expression of endogenous Arf1 was 26–180 times higher than Arf6. These data indicate that Arf GTPases are expressed and targeted in a cell- and membrane-specific pattern in kidney epithelial cells in situ. The results provide a framework on which to base and interpret future studies on the role of Arf GTPases in the multitude of cellular trafficking events that occur in renal tubular epithelial cells.

Binding and internalization of a fluorescent vasopressin analogue by collecting duct cells

1988 ◽  
Vol 255 (5) ◽  
pp. C622-C632 ◽  
Author(s):  
K. L. Kirk
Keyword(s):  
Arginine Vasopressin ◽  
Collecting Duct ◽  
Computer Assisted ◽  
Principal Cells ◽  
Lysine Vasopressin ◽  
Collecting Tubule ◽  
Basal Pole ◽  
V2 Receptor ◽  
Tubule Cells ◽  
Maximal Binding

The kinetics of binding and internalization of a fluorescent vasopressin analogue [1-desamino-8-rhodamine lysine vasopressin (rhoda LVP)] by principal cells within the microperfused rabbit cortical collecting tubule were quantitatively assessed with computer-assisted video microscopy. At 25 degrees C, binding of rhoda LVP exhibited saturation kinetics with half-maximal binding at 2 nM and maximal binding at concentrations greater than 5 nM. Rhoda LVP binding could be prevented by the simultaneous addition of a 10-fold higher concentration of arginine vasopressin (AVP) or the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (desmopressin). No obvious internalization or rhoda LVP was detected at 25 degrees C, i.e., the rhoda LVP fluorescence remained localized to the basal pole of each principal cell for at least 100 min after rhoda LVP addition and could be largely reversed by the subsequent addition of AVP. Conversely, warming the cells to 38 degrees C after binding was initiated resulted in a rapid (less than 30 min) migration of the fluorescence into the cell interior and a loss of AVP-displaceable binding from the cell surface. These results document the utility of this noninvasive optical strategy for quantitatively monitoring hormone binding to intact collecting tubule cells and demonstrate that rhoda LVP binds reversibly and with high affinity to V2 receptors on principal cells in the collecting tubule. The internalization (and presumed inactivation) of the hormone-receptor complex at 38 degrees C may contribute to the desensitization of collecting tubule cells to vasopressin at physiological temperature.

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