scholarly journals Immunoelectron Microscopic Localization of the Electrogenic Na/HCO3 Cotransporter in Rat and Ambystoma Kidney

2000 ◽  
Vol 11 (12) ◽  
pp. 2179-2189
Author(s):  
ARVID B. MAUNSBACH ◽  
HENRIK VORUM ◽  
TAE-HWAN KWON ◽  
SØREN NIELSEN ◽  
BRIAN SIMONSEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Rat Kidney Cortex ◽  
C Terminus ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Abstract. Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney

1998 ◽  
Vol 275 (6) ◽  
pp. F885-F893 ◽  
Author(s):  
Søren Nielsen ◽  
Arvid B. Maunsbach ◽  
Carolyn A. Ecelbarger ◽  
Mark A. Knepper
Keyword(s):  
Plasma Membrane ◽  
Rat Kidney ◽  
Ultrastructural Localization ◽  
Macula Densa ◽  
Tubuloglomerular Feedback ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Rat Kidney Cortex ◽  
Intracellular Vesicles

A bumetanide-sensitive Na-K-2Cl cotransporter, BSC-1, is believed to mediate the apical component of transcellular NaCl absorption in the thick ascending limb (TAL) of Henle’s loop. To study its ultrastructural localization in kidney, we used an affinity-purified, peptide-derived polyclonal antibody against rat BSC-1. Immunoblots from rat kidney cortex and outer medulla revealed a solitary 161-kDa band in membrane fractions. Immunocytochemistry of 1-μm cryosections demonstrated strong BSC-1 labeling of the apical and subapical regions of medullary and cortical TAL cells. Notably, macula densa cells also exhibited distinct labeling. Distal convoluted tubules and other renal tubule segments were unlabeled. Immunoelectron microscopy demonstrated that BSC-1 labeling was associated with the apical plasma membrane and with subapical intracellular vesicles in medullary and cortical TAL and in macula densa cells. Smooth-surfaced TAL cells, in particular, had extensive BSC-1 labeling of intracellular vesicles. These results support the view that BSC-1 provides the apical pathway for NaCl transport across the TAL and that an extensive intracellular reservoir of BSC-1 is present in a subpopulation of TAL cells. Furthermore, the BSC-1 localization in the apical plasma membrane of macula densa cells is consistent with its proposed role in tubuloglomerular feedback.

Identification of an apical \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger in rat kidney proximal tubule

2003 ◽  
Vol 285 (3) ◽  
pp. C608-C617 ◽  
Author(s):  
Snezana Petrovic ◽  
Liyun Ma ◽  
Zhaohui Wang ◽  
Manoocher Soleimani
Keyword(s):  
Proximal Tubule ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Immunocytochemical Staining ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Kidney Proximal Tubule ◽  
Anion Transporter

SLC26A6 (or putative anion transporter 1, PAT1) is located on the apical membrane of mouse kidney proximal tubule and mediates [Formula: see text] exchange in in vitro expression systems. We hypothesized that PAT1 along with a [Formula: see text] exchange is present in apical membranes of rat kidney proximal tubules. Northern hybridizations indicated the exclusive expression of SLC26A6 (PAT1 or CFEX) in rat kidney cortex, and immunocytochemical staining localized SLC26A6 on the apical membrane of proximal tubules, with complete prevention of the labeling with the preadsorbed serum. To examine the functional presence of apical [Formula: see text] exchanger, proximal tubules were isolated, microperfused, loaded with the pH-sensitive dye BCPCF-AM, and examined by digital ratiometric imaging. The pH of the perfusate and bath was kept at 7.4. Buffering capacity was measured, and transport rates were calculated as equivalent base flux. The results showed that in the presence of basolateral DIDS (to inhibit [Formula: see text] cotransporter 1) and apical EIPA (to inhibit Na+/H+ exchanger 3), the magnitude of cell acidification in response to addition of luminal Cl– was ∼5.0-fold higher in the presence than in the absence of [Formula: see text]. The Cl–-dependent base transport was inhibited by ∼61% in the presence of 0.5 mM luminal DIDS. The presence of physiological concentrations of oxalate in the lumen (200 μM) did not affect the [Formula: see text] exchange activity. These results are consistent with the presence of SLC26A6 (PAT1) and [Formula: see text] exchanger activity in the apical membrane of rat kidney proximal tubule. We propose that SLC26A6 is likely responsible for the apical [Formula: see text] (and Cl–/OH–) exchanger activities in kidney proximal tubule.

H(+)V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways

2000 ◽  
Vol 203 (1) ◽  
pp. 137-145 ◽  
Author(s):  
D. Brown ◽  
S. Breton
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Vas Deferens ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
A Cells ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Many vertebrate transporting epithelia contain characteristic ‘mitochondria-rich’ cells that express high levels of a vacuolar proton-pumping ATPase (H(+)V-ATPase) on their plasma membrane and on intracellular vesicles. In the kidney cortex, A-cells and B-cells are involved in proton secretion and bicarbonate secretion, respectively, in the distal nephron and collecting duct. A-cells have an H(+)V-ATPase on their apical plasma membrane and on intracellular vesicles, whereas the cellular location of the H(+)V-ATPase can be apical, basolateral, bipolar or diffuse in B-cells. The rat epididymis and vas deferens also contain a distinct population of H(+)V-ATPase-rich epithelial cells. These cells are involved in generating a low luminal pH, which is involved in sperm maturation and in maintaining sperm in an immotile state during their passage through the epididymis and vas deferens. In both kidney and reproductive tract, H(+)V-ATPase-rich cells have a high rate of apical membrane recycling. H(+)V-ATPase molecules are transported between the cell surface and the cytoplasm in vesicles that have a well-defined ‘coat’ structure formed of the peripheral V(1) subunits of the H(+)V-ATPase. In addition, we propose that B-type intercalated cells have a transcytotic pathway that enables them to shuttle H(+)V-ATPase molecules from apical to basolateral plasma membrane domains. This hypothesis is supported by data showing that A-cells and B-cells have different intracellular trafficking pathways for LGP120, a lysosomal glycoprotein. LGP120 was found both on the basolateral plasma membrane and in lysosomes in B-cells, whereas no LGP120 was detectable in the plasma membrane of A-cells. We propose that the ‘polarity reversal’ of the H(+)V-ATPase in B-intercalated cells is mediated by a physiologically regulated transcytotic pathway that may be similar to that existing in some other cell types.

scholarly journals THE POLARITY OF THE PROXIMAL TUBULE CELL IN RAT KIDNEY

1972 ◽  
Vol 54 (2) ◽  
pp. 232-245 ◽  
Author(s):  
Hans-G Heidrich ◽  
Rolf Kinne ◽  
Eva Kinne-Saffran ◽  
Kurt Hannig
Keyword(s):  
Proximal Tubule ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Rat Kidney ◽  
High Specific Activity ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Surface Charges ◽  
Tubule Cell ◽  
Rat Kidney Cortex

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Divalent metal transporter 1 in the kidney proximal tubule is expressed in late endosomes/lysosomal membranes: implications for renal handling of protein-metal complexes

2006 ◽  
Vol 290 (6) ◽  
pp. F1525-F1533 ◽  
Author(s):  
Marouan Abouhamed ◽  
Jakub Gburek ◽  
Wei Liu ◽  
Blazej Torchalski ◽  
Andreas Wilhelm ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Rt Pcr ◽  
Divalent Metal Transporter 1 ◽  
Rat Kidney Cortex ◽  
Divalent Metal Transporter ◽  
Alexa Fluor ◽  
Metal Transporter ◽  
Late Endosomes

The H+-coupled polyligand transport protein divalent metal transporter 1 (DMT1) plays a key role in mammalian iron homeostasis. It has a widespread pattern of expression including tissues associated with iron acquisition and storage. Interestingly, it is also highly expressed in the kidney, yet its function in this tissue is unknown. The aim of this study was to determine the cellular location of DMT1 in proximal tubule cells as a first step to determining the role of this protein in the kidney. To do this we performed RT-PCR and immunostaining experiments using rat kidney and the S1 proximal tubule-derived WKPT-0293 Cl.2 cell line. RT-PCR revealed that mRNAs encoding all four DMT1 splice variants were present in RNA extracted from rat kidney cortex or WKPT-0293 Cl.2 cells. Immunostaining of rat kidney cortex or WKPT-0293 Cl.2 cells showed that DMT1 protein was expressed intracellularly and was not present in the plasma membrane. Expression of DMT1 partially colocalized with the late endosomal/lysosomal proteins LAMP1 and cathepsin-L. Using immunogold labeling, DMT1 was shown to be expressed in the membranes of late endosomes/lysosomes. Uptake of Alexa Fluor 546-transferrin was only observed following application to the apical membrane of WKPT-0293 Cl.2 cells. Within these cells, Alexa Fluor 546-transferrin colocalized with DMT1. In conclusion, renal proximal tubular cells express DMT1 in the membranes of organelles, including late endosomes/lysosomes, associated with processing of apically sequestered transferrin. These findings have implications for renal iron handling and possibly for the handling of nephrotoxic metals that are also DMT1 ligands, including Cd2+.

scholarly journals Identification of a new gene product (diphor-1) regulated by dietary phosphate

1997 ◽  
Vol 273 (5) ◽  
pp. F801-F806 ◽  
Author(s):  
María Custer ◽  
Benjamin Spindler ◽  
François Verrey ◽  
Heini Murer ◽  
Jürg Biber
Keyword(s):  
Cellular Response ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Rat Kidney Cortex ◽  
Reading Frame ◽  
New Gene ◽  
High Degree ◽  
The Impact ◽  
Kidney Proximal Tubules

Chronic restriction of dietary Pi elicits an increased reabsorption of Pi in the kidney proximal tubules, which involves a stimulation of apical Na-Pi cotansport. This adaptation is in part a direct cellular response of which the mechanism(s) are poorly understood. In this study, the impact of dietary Pi restriction on the differential expression of rat kidney cortex mRNAs was visualized to identify gene products regulated by the Pistatus. When kidney cortex mRNAs of rats fed a low- or a high-Pi diet were compared by differential display-polymerase chain reaction (DD-PCR), thirty modulated cDNA bands were observed, of which four were confirmed as being regulated. We focused on one of the upregulated bands, dietary Pi-regulated RNA-1 (diphor-1). A cDNA containing an open reading frame encoding a 52-kDa protein was cloned by library screening. Diphor-1 exhibits a high degree of identity to the Na/H exchanger regulatory factor and to a tyrosine kinase activating protein. Highest expression of diphor-1 mRNA was detected in the kidney (proximal tubules) and in small intestine. Expression experiments showed that diphor-1 specifically increases Na-Picotransport in oocytes of Xenopus laevis coinjected with renal type II Na-Pi cotransporter cRNA. Further characterizations of diphor-1 will show whether diphor-1 is primarily or secondarily involved in the response to dietary Pi.

Epidermal growth factor receptor regulation in rat kidney: two models of renal growth

1989 ◽  
Vol 257 (6) ◽  
pp. F1059-F1064 ◽  
Author(s):  
M. T. Behrens ◽  
A. L. Corbin ◽  
M. K. Hise
Keyword(s):  
Folic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Rat Kidney Cortex ◽  
Epidermal Growth

The binding of 125I-labeled epidermal growth factor (EGF) to plasma membranes prepared from rat kidney cortex was studied following unilateral nephrectomy, a model of proximal tubule cell hypertrophy, and following the administration of folic acid, a model of proximal tubule cell hyperplasia. Binding of 125I-EGF was a linear function of basolateral membrane protein content and time of incubation. Specific binding to luminal brush-border membranes was not evident in these studies. Neither insulin nor insulin-like growth factor I could displace EGF binding, indicating that binding was specific. Scatchard analysis revealed a single binding site. The KD in sham-operated animals 48 h after surgery was 11.2 +/- 1.4 nM, whereas Bmax averaged 95.2 +/- 4.1 fmol/mg protein (n = 3). Similar values were obtained in nephrectomized animals. The Bmax of folic acid-untreated animals averaged 212.5 +/- 6.9 fmol/mg 48 h after administration, whereas that of vehicle-injected controls averaged 85.4 +/- 9.2 (n = 3, P less than 0.001). Differences in binding were not related to changes in affinity, ligand degradation by the preparations, or receptor binding of endogenous EGF. These data indicate that regeneration following folic acid administration is associated with an upregulation of proximal nephron EGF receptors that may play an important role in the mitogenic response.

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