Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression

2007 ◽  
Vol 293 (4) ◽  
pp. R1743-R1753 ◽  
Author(s):  
Tsutomu Nakada ◽  
Kazuyuki Hoshijima ◽  
Masahiro Esaki ◽  
Saori Nagayoshi ◽  
Koichi Kawakami ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Distal Tubule ◽  
Yolk Sac ◽  
Renal Tubules ◽  
Larval Stages ◽  
Ammonia Transport ◽  
Green Fluorescent

Members of the Rh glycoprotein family have been shown to be involved in ammonia transport in a variety of species. Here we show that zebrafish Rhcg1, a member of the Rh glycoprotein family, is highly expressed in the yolk sac, gill, and renal tubules. Molecular cloning and characterization indicate that zebrafish Rhcg1 shares 82% sequence identity with the pufferfish ortholog fRhcg1. RT-PCR, combined with in situ hybridization, revealed that Rhcg1 is first expressed in vacuolar-type H+-ATPase/mitochondrion-rich cells (vH-MRC) on the yolk sac of larvae at 3 days postfertilization (dpf) and later in vH-MRC-like cells in the gill at 4–5 dpf. Ammonia excretion from zebrafish larvae increased in parallel with the expression of Rhcg1. At larval stages, Rhcg1 mRNA was detected only on the yolk sac and gill; however, the kidney, as well as the gill, becomes a major site of Rhcg1 expression in adults. Using a zebrafish Tol2 transgenic line whose vH-MRC are labeled with green fluorescent protein (GFP) and an antibody against zebrafish Rhcg1, we demonstrate that Rhcg1 is located in the apical regions of 1) vH-MRC on the yolk sac and vH-MRC-like cells (cell population with the expression of Rhcg1 and GFP) in the gill and 2) cells in the renal distal tubule and intercalated cell-like cells in the collecting duct of the kidney. Remarkably, expression of Rhcg1 mRNA at the larval stage was changed by environmental ionic strength. These results suggest that roles of zebrafish Rhcg1 are not solely ammonia secretion to eliminate nitrogen from the gill.

Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway

2005 ◽  
Vol 288 (6) ◽  
pp. C1390-C1401 ◽  
Author(s):  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Malay K. Raychowdhury ◽  
Vladimir Marshansky ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Time Lag ◽  
Renal Medulla ◽  
Degradation Pathway ◽  
Urinary Concentration ◽  
Long Time ◽  
Green Fluorescent ◽  
Golgi Network

Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) “recycling” pathway. To examine this pathway, we expressed V2R-green fluorescent protein (GFP) in LLC-PK1a cells. V2R-GFP showed characteristics similar to those of wild-type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but it colocalized with the lysosomal marker Lysotracker after VP-induced internalization. Western blot analysis of V2R-GFP showed a broad 57- to 68-kDa band and a doublet at 46 and 52 kDa before VP treatment. After 4-h VP exposure, the 57- to 68-kDa band lost 50% of its intensity, whereas the lower 46-kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect, whereas lactacystin, a proteasome inhibitor, had no effect. Incubating cells at 20°C to block trafficking from the trans-Golgi network reduced V2R membrane fluorescence, and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.

scholarly journals More actors in ammonia absorption by the thick ascending limb

2012 ◽  
Vol 302 (3) ◽  
pp. F293-F297 ◽  
Author(s):  
Pascal Houillier ◽  
Soline Bourgeois
Keyword(s):  
Current Knowledge ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Hydrogen Ion ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Transport Mechanisms ◽  
Sodium Hydrogen ◽  
Ammonia Transport ◽  
Ammonia Absorption

This review will briefly summarize current knowledge on the basolateral ammonia transport mechanisms in the thick ascending limb (TAL) of the loop of Henle. This segment transports ammonia against a concentration gradient and is responsible for the accumulation of ammonia in the medullary interstitium, which, in turn, favors ammonia secretion across the collecting duct. Experimental data indicate that the sodium/hydrogen ion exchanger isoform 4 (NHE4; Scl9a4) is a sodium/ammonia exchanger and plays a major role in this process. Disruption of murine NHE4 leads to metabolic acidosis with inappropriate urinary ammonia excretion and decreases the ability of the TAL to absorb ammonia and to build the corticopapillary ammonia gradient. However, NHE4 does not account for the entirety of ammonia absorption by the TAL, indicating that, at least, one more transporter is involved.

Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

2004 ◽  
Vol 287 (5) ◽  
pp. C1366-C1374 ◽  
Author(s):  
Julie A. Nicoletta ◽  
Jonathan J. Ross ◽  
Guangmu Li ◽  
Qingzhang Cheng ◽  
Jonathon Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Snare Complex ◽  
Glutathione S Transferase ◽  
Syntaxin 1A ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Reduced Rate ◽  
Green Fluorescent

Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.

pCO2 in regulating ammonia excretion by renal tubules of dogs

1961 ◽  
Vol 200 (4) ◽  
pp. 881-884 ◽  
Author(s):  
Robert Cade ◽  
Robert J. Shalhoub ◽  
Klaus Hierholzer
Keyword(s):  
Intracellular Ph ◽  
Diffusion Theory ◽  
Ammonia Excretion ◽  
Respiratory Acidosis ◽  
Passive Diffusion ◽  
Urine Flow ◽  
Potassium Depletion ◽  
Renal Tubules ◽  
Urine Ph ◽  
Ammonia Transport

The importance of urine pH, urine flow, availability of substrate and activity of cellular enzymes on the production and excretion of ammonia have been investigated in several species. Little information concerning the effect of intracellular pH on ammonia excretion is available. To investigate this problem we have altered intracellular pH by inducing respiratory acidosis and alkalosis in dogs. An elevated pCO2 uniformly depresses excretion of ammonia while a fall in pCO2 is associated with increased ammonia excretion at any given urine pH. When animals are depleted of potassium, the degree of depression by 10% CO2 is far greater than in the same animals studied before potassium depletion. The data are interpreted in the context of the passive diffusion theory of ammonia transport.

Expression of the unconventional myosin Myo1c alters sodium transport in M1 collecting duct cells

2005 ◽  
Vol 289 (1) ◽  
pp. C120-C129 ◽  
Author(s):  
Mark C. Wagner ◽  
Bonnie L. Blazer-Yost ◽  
Judy Boyd-White ◽  
Anjaiah Srirangam ◽  
Janice Pennington ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Collecting Duct ◽  
Short Circuit ◽  
Direct Interaction ◽  
Short Circuit Current ◽  
Duct Cell ◽  
Unconventional Myosin ◽  
Myosin I ◽  
Green Fluorescent

Epithelial cells rely on proper targeting of cellular components to perform their physiological function. This dynamic process utilizes the cytoskeleton and involves movement of vesicles to and from the plasma membrane, thus traversing the actin cortical cytoskeleton. Studies support both direct interaction of actin with channels and an indirect mechanism whereby actin may serve as a track in the final delivery of the channel to the plasma membrane. Actin-dependent processes are often mediated via a member of the myosin family of proteins. Myosin I family members have been implicated in multiple cellular events occurring at the plasma membrane. In these studies, we investigated the function of the unconventional myosin I Myo1c in the M1 mouse collecting duct cell line. Myo1c was observed to be concentrated at or near the plasma membrane, often in discrete membrane domains. To address the possible role of Myo1c in channel regulation, we expressed a truncated Myo1c, lacking ATP and actin domains, in M1 cells and compared electrophysiological responses to control M1 cells, M1 cells expressing the empty vector, and M1 cells expressing the full-length Myo1c construct. Interestingly, cells expressing the Myo1c constructs had modulated antidiuretic hormone (ADH)-stimulated short-circuit current and showed little inhibition of short-circuit current with amiloride addition. Evaluation of enhanced green fluorescent protein-Myo1c constructs supports the importance of the IQ region in targeting the Myo1c to its respective cellular domain. These data are consistent with Myo1c participating in the regulation of the Na+ channel after ADH stimulation.

Decreased amiloride-sensitive Na+ absorption in collecting duct principal cells isolated from BPK ARPKD mice

2004 ◽  
Vol 286 (2) ◽  
pp. F244-F254 ◽  
Author(s):  
Elias I. Veizis ◽  
Cathleen R. Carlin ◽  
Calvin U. Cotton
Keyword(s):  
Fluorescent Protein ◽  
Kidney Diseases ◽  
Collecting Duct ◽  
Short Circuit ◽  
Expression Patterns ◽  
Renal Cysts ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Principal Cells ◽  
Green Fluorescent

The main feature of polycystic kidney diseases (PKD) is formation and progressive enlargement of renal cysts. Alterations in epithelial cell proliferation, extracellular matrix, and ion transport are thought to contribute to cyst enlargement and loss of renal function. Abnormal Cl- secretion is implicated in cyst enlargement in autosomal dominant PKD (ADPKD), but little is known about transport abnormalities in autosomal recessive PKD (ARPKD). We developed a method to isolate collecting duct (CD) principal cells (site of the lesion in ARPKD) from normal and ARPKD mice. A transgenic mouse (Hoxb7/GFP) in which enhanced green fluorescent protein (GFP) is expressed in CDs was bred with an ARPKD mouse (BPK), and GFP-positive cells from normal and cystic mice were selected by fluorescence-activated cell sorting. GFP-positive CD cells (>95 ± 3%) obtained from either normal or cystic mice formed high-resistance, polarized epithelial monolayers. Expression patterns for marker proteins and the presence of a central cilium confirmed that the monolayers are composed of principal cells. Under basal conditions, the Cl- secretory responses elicited by elevation of cAMP or calcium were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current was significantly reduced in monolayers of cells isolated from cystic mice (12.9 ± 1.6 μA/cm2; n = 10) compared with monolayers of cells isolated from normal mice (27.3 ± 3.4 μA/cm2; n = 12). The results of these studies suggest that epithelial sodium channel-mediated sodium absorption is decreased in principal cells of ARPKD CD cysts and that the reduction in sodium absorption may contribute to the accumulation of luminal fluid.

scholarly journals Kidney intercalated cells are phagocytic and acidify internalized uropathogenic Escherichia coli

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vijay Saxena ◽  
Hongyu Gao ◽  
Samuel Arregui ◽  
Amy Zollman ◽  
Malgorzata Maria Kamocka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Transcriptional Response ◽  
Vacuolar Atpase ◽  
Renal Tubules ◽  
Intercalated Cells ◽  
Intercalated Cell ◽  
Biological Pathway Analysis ◽  
Green Fluorescent

AbstractKidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenicEscherichia coliin two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressingEscherichia colior pHrodo GreenE. coliBioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney’s defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages.

Three pathways to mature macrophages in the early mouse yolk sac

Blood ◽  
2005 ◽  
Vol 106 (9) ◽  
pp. 3004-3011 ◽  
Author(s):  
Julien Y. Bertrand ◽  
Abdelali Jalil ◽  
Michèle Klaine ◽  
Steffen Jung ◽  
Ana Cumano ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yolk Sac ◽  
Third Wave ◽  
Transient Wave ◽  
Differentiation Potential ◽  
Somite Stage ◽  
Early Mouse ◽  
Green Fluorescent ◽  
Second Wave

Abstract The existence of macrophages (Mφ) of yolk-sac (YS) origin has been reported in all vertebrate models. However, the nature of their precursors and pathways of differentiation have not been elucidated. Phenotypic and differentiation potential analyses of YS at 7.5 to 10 postcoital days (dpc), performed in CX3CR1GFP embryos, allowed us to discern 3 independent Mφ populations. A first transient wave consisted of mature, maternal-derived Mφpresent as early as 7.5 to 8 dpc. A second wave of committed Mφ precursors arose at 8 dpc (2-4 somite stage) and was followed by a third wave of erythromyeloid precursors (4-6 somite stage). Both types of precursors displayed similar phenotypes and gave rise to CX3CR1/green fluorescent protein (GFP)–positive Mφ, but differed by their differentiation potential, at the clonal level. The combined data of phenotypic, gene-expression, and in situ analyses allowed us to conclude that the previously named “primitive Mφ” corresponded to a mixture of the first transient wave and committed Mφ precursors. Both YS-derived precursors followed a developmental pathway common to adult Mφ and could be qualified as definitive.

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans

2004 ◽  
Vol 385 (6) ◽  
pp. 565-568 ◽  
Author(s):  
M. Suzuki ◽  
N. Sagoh ◽  
H. Iwasaki ◽  
H. Inoue ◽  
K. Takahashi
Keyword(s):  
Functional Analysis ◽  
Caenorhabditis Elegans ◽  
Genetic Background ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Larval Stages ◽  
Thrombospondin 1 ◽  
Cub Domain ◽  
Epidermal Growth ◽  
Green Fluorescent

AbstractFunctional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family inCaenorhabditis elegansand the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacinlike metalloprotease with an epidermal growth factor (EGF) like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoterdriven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wildtype rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.

scholarly journals Aktivitas Promoter â-aktin Ikan Medaka Jepang (Oryzias latipes) pada Ikan Mas (Cyprinus carpio)

2012 ◽  
Vol 11 (2) ◽  
pp. 70
Author(s):  
Alimuddin ' ◽  
Lola Irma Purwanti ◽  
MH. Fariduddin Ath-thar ◽  
Chairul Muluk ◽  
Odang Carman ◽  
...  
Keyword(s):  
Common Carp ◽  
Cyprinus Carpio ◽  
Fluorescent Protein ◽  
Oryzias Latipes ◽  
Green Fluorescent Protein Gene ◽  
Hatching Rate ◽  
Cell Stage ◽  
Larval Stages ◽  
Renilla Reniformis ◽  
Green Fluorescent

This study was conducted to examine activity of medaka (Oryzias latipes) â-actin promoter (mBP) in common carp(Cyprinus carpio) as the first step towards development of common carp transgenic in country. Gene constructpmBP-hrGFP that consists of mBA promoter and humanized Renilla reniformis green fluorescent protein gene(hrGFP) was injected into cytoplasm of one cell stage of common carp by using microinjector. PmBP-hrGFPconcentration used for microinjection was 50 μg/mL aquabides. Parameters observed were survival rate of embryo(SRe), hatching rate (HR) and expression of hrGFP gene. SRe was calculated before eggs hacthed, while hatchingrate (HR) was after all of eggs hatched. The activity of mBA promoter was analyzed by observation of hrGFP genetransient expression using a fluorescence microscope. The results of experiment showed that SRe (87,5%) andHR (79.2%) of control was respectevily higher than that of injected treatment (75.0% & 61.7%). Expression of hrGFPwas observed firstly at blastula (12 hours after fertilization) to 1-day-old larval stages (24 hours after hatching)with higher gene expression at blastula to late gastrula stages. Percentage of micronjected larvae expressinghrGFP at 6 hours after hatching reached 71.6 ± 6.7%. Conclusion was that mBA promoter could drove hrGFPexpression in common carp, hence it can be used to produce common carp transgenic by changing hrGFP withgenes correlated with important traits in aquaculture.

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