Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia

2013 ◽  
Vol 216 (15) ◽  
pp. 2917-2930 ◽  
Author(s):  
A. K. Sinha ◽  
H. J. Liew ◽  
C. M. Nawata ◽  
R. Blust ◽  
C. M. Wood ◽  
...  
Keyword(s):  
Ammonia Excretion ◽  
Rh Glycoproteins ◽  
Freshwater Teleosts

Ammonia excretion by the skin of zebrafish (Danio rerio) larvae

2008 ◽  
Vol 295 (6) ◽  
pp. C1625-C1632 ◽  
Author(s):  
Tin-Han Shih ◽  
Jiun-Lin Horng ◽  
Pung-Pung Hwang ◽  
Li-Yih Lin
Keyword(s):  
Direct Evidence ◽  
Ammonia Excretion ◽  
Bafilomycin A1 ◽  
Selective Electrode ◽  
Zebrafish Larvae ◽  
Trapping Mechanism ◽  
Morpholino Oligonucleotides ◽  
Electrode Technique ◽  
External Ph ◽  
Freshwater Teleosts

The mechanism of ammonia excretion in freshwater teleosts is not well understood. In this study, scanning ion-selective electrode technique was applied to measure H+ and NH4+ fluxes in specific cells on the skin of zebrafish larvae. NH4+ extrusion was relatively high in H+ pump-rich cells, which were identified as the H+-secreting ionocyte in zebrafish. Minor NH4+ extrusion was also detected in keratinocytes and other types of ionocytes in larval skin. NH4+ extrusion from the skin was tightly linked to acid secretion. Increases in the external pH and buffer concentration (5 mM MOPS) diminished H+ and NH4+ gradients at the larval surface. Moreover, coupled decreases in NH4+ and H+ extrusion were found in larvae treated with an H+-pump inhibitor (bafilomycin A1) or H+-pump gene ( atp6v1a) knockdown. Knockdown of Rhcg1 with morpholino-oligonucleotides also decreased NH4+ excretion. This study demonstrates ammonia excretion in epithelial cells of larval skin through an acid-trapping mechanism, and it provides direct evidence for the involvement of the H+ pump and an Rh glycoprotein (Rhcg1) in ammonia excretion.

scholarly journals Marine, freshwater and aerially acclimated mangrove rivulus (Kryptolebias marmoratus) use different strategies for cutaneous ammonia excretion

2013 ◽  
Vol 304 (8) ◽  
pp. R599-R612 ◽  
Author(s):  
Christopher A. Cooper ◽  
Jonathan M. Wilson ◽  
Patricia A. Wright
Keyword(s):  
Immunofluorescence Microscopy ◽  
Ammonia Excretion ◽  
Teleost Fishes ◽  
Ammonia Gas ◽  
Ammonia Transport ◽  
Paracellular Pathways ◽  
Kryptolebias Marmoratus ◽  
Rh Glycoproteins ◽  
Mangrove Rivulus ◽  
Ammonia Flux

Rhesus (Rh) glycoproteins are ammonia gas (NH3) channels known to be involved in ammonia transport in animals. Because of the different osmoregulatory and ionoregulatory challenges faced by teleost fishes in marine and freshwater (FW) environments, we hypothesized that ammonia excretion strategies would differ between environments. Also, we hypothesized that cutaneous NH3 volatilization in air-acclimated fish is facilitated by base secretion. To test these hypotheses, we used the skin of the euryhaline amphibious mangrove rivulus ( Kryptolebias marmoratus). The skin excretes ammonia and expresses Rh glycoproteins. Serosal-to-mucosal cutaneous ammonia flux was saturable (0–16 mmol/l ammonia, Km of 6.42 mmol/l). In FW, ammonia excretion increased in response to low mucosal pH but decreased with pharmacological inhibition of Na+/H+ exchangers (NHE) and H+ ATPase. Conversely, in brackish water (BW), lowering the mucosal pH significantly decreased ammonia excretion. Inhibitors of NHE also decreased ammonia excretion in BW fish. Immunofluorescence microscopy demonstrated that both the Rh isoform, Rhcg1, and NHE3 proteins colocalized in Na+/K+ ATPase expressing mitochondrion-rich cells in the gills, kidney, and skin. We propose that the mechanisms of cutaneous ammonia excretion in FW K. marmoratus are consistent with the model for branchial ammonia excretion in FW teleost fish. NH4+ excretion appeared to play a stronger role in BW. NH4+ excretion in BW may be facilitated by apical NHE and/or diffuse through paracellular pathways. In aerially acclimated fish, inhibition of NHE and H+ ATPase, but not the Cl−/HCO3− exchanger, significantly affected cutaneous surface pH, suggesting that direct base excretion is not critical for NH3 volatilization. Overall, K. marmoratus use different strategies for excreting ammonia in three different environments, FW, BW, and air, and Rh glycoproteins and NHE are integral to all.

scholarly journals Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis

2014 ◽  
Vol 306 (4) ◽  
pp. F389-F400 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Jill W. Verlander ◽  
Mary E. Handlogten ◽  
Ki-Hwan Han ◽  
I. David Weiner
Keyword(s):  
Metabolic Acidosis ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Severe Metabolic Acidosis ◽  
Urine Ph ◽  
Renal Response ◽  
Rh Glycoproteins ◽  
Acid Loading ◽  
Specific Deletion

The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3− were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1–5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosph enolpyruvate carboxykinase (PEPCK) and Na+/H+ exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis.

Mechanisms of ion transport inPotamotrygon, a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro

2002 ◽  
Vol 205 (19) ◽  
pp. 3039-3054 ◽  
Author(s):  
Chris M. Wood ◽  
Aline Y. O. Matsuo ◽  
R. J. Gonzalez ◽  
Rod W. Wilson ◽  
Marjorie L. Patrick ◽  
...  
Keyword(s):  
Ammonia Excretion ◽  
Hard Water ◽  
Blood Chemistry ◽  
The Body ◽  
Acute Exposure ◽  
Transport Systems ◽  
Glucose Levels ◽  
Rio Negro ◽  
The Family ◽  
Freshwater Teleosts

SUMMARYStingrays of the family Potamotrygonidae are the only stenohaline freshwater elasmobranchs. Potomotrygon sp. collected from the ion-poor blackwaters ([Na+], [Cl-] and[Ca2+]=10-30 μmol l-1, pH 6.1) of the Rio Negro,Amazonas, Brazil, were ammoniotelic (91% ammonia-N, 9% urea-N excretion) and exhibited blood chemistry (Na+, Cl-, urea, ammonia and glucose levels and osmolality) typical of freshwater teleosts. Unidirectional Na+ and Cl- influx rates, measured with radiotracers,displayed saturation kinetics. The relationships for Cl- and Na+ had similar Km values (300-500 μmol l-1), but Jmax values for Cl-(approximately 950 μmol kg-1 h-1) were almost twice those for Na+ (approximately 500 μmol kg-1h-1). Cl- efflux rates varied with external concentration, but Na+ efflux rates did not. There were no differences in the kinetic variables (Km, Jmax) for influx between animals acclimated to their native ion-poor blackwater or to ion-rich hard water, but efflux rates for both Na+ and Cl- were lower in the former, yielding much lower balance points (external Na+ or Cl- levels at which influx and efflux were equal). Na+, Cl- and Ca2+ uptake were all strongly inhibited by acute exposure to pH 4.0, but efflux rates and Ca2+ binding to the body surface did not change. Na+ influx was inhibited by amiloride (10-4 mol l-1) and by two of its analogs, phenamil (4×10-5mol l-1) and HMA (4×10-5 mol l-1), with the latter being slightly more potent, while Cl- fluxes were unaffected. Cl- fluxes were insensitive to DIDS(2×10-5 mol l-1 or 10-4 mol l-1) and SITS (10-4 mol l-1), but both influx and efflux rates were strongly inhibited by DPC (10-4 mol l-1) and thiocyanate (10-4 mol l-1). Ammonia excretion was unresponsive to large changes in water Na+concentration, but was elevated by 70% during acute exposure to pH 4.0 and transiently inhibited by approximately 50% by amiloride and its analogues. The strategy of adaptation to ion-poor blackwater appears similar to that of some Rio Negro teleosts (Cichlidae) in which low-affinity transport systems are relatively sensitive to inhibition by low pH but are complemented by low diffusive loss rates. Ionic transport systems in these freshwater elasmobranchs, although superficially similar to those in some freshwater teleosts, may bear more resemblance to their presumed evolutionary precursors in marine elasmobranchs.

Proton-facilitated ammonia excretion by ionocytes of medaka (Oryzias latipes) acclimated to seawater

2013 ◽  
Vol 305 (3) ◽  
pp. R242-R251 ◽  
Author(s):  
Sian-Tai Liu ◽  
Lin Tsung ◽  
Jiun-Lin Horng ◽  
Li-Yih Lin
Keyword(s):  
Boundary Layer ◽  
Ammonia Excretion ◽  
Oryzias Latipes ◽  
Yolk Sac ◽  
Pcr Analysis ◽  
Selective Electrode ◽  
High Ammonia ◽  
Rh Glycoproteins ◽  
Electrode Technique

The proton-facilitated ammonia excretion is critical for a fish's ability to excrete ammonia in freshwater. However, it remains unclear whether that mechanism is also critical for ammonia excretion in seawater (SW). Using a scanning ion-selective electrode technique (SIET) to measure H+ gradients, an acidic boundary layer was detected at the yolk-sac surface of SW-acclimated medaka ( Oryzias latipes) larvae. The H+ gradient detected at the surface of ionocytes was higher than that of keratinocytes in the yolk sac. Treatment with Tricine buffer or EIPA (a NHE inhibitor) reduced the H+ gradient and ammonia excretion of larvae. In situ hybridization and immunochemistry showed that slc9a2 (NHE2) and slc9a3 (NHE3) were expressed in the same SW-type ionocytes. A real-time PCR analysis showed that transfer to SW downregulated branchial mRNA expressions of slc9a3 and Rhesus glycoproteins ( rhcg1, rhcg2, and rhbg) but upregulated that of slc9a2. However, slc9a3, rhcg1, rhcg2, and rhbg expressions were induced by high ammonia in SW. This study suggests that SW-type ionocytes play a role in acid and ammonia excretion and that the Na+/H+ exchanger and Rh glycoproteins are involved in the proton-facilitated ammonia excretion mechanism.

scholarly journals Collecting duct-specific Rh C glycoprotein deletion alters basal and acidosis-stimulated renal ammonia excretion

2009 ◽  
Vol 296 (6) ◽  
pp. F1364-F1375 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Jill W. Verlander ◽  
Jesse M. Bishop ◽  
Peter Igarashi ◽  
Mary E. Handlogten ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Collecting Duct ◽  
Ammonia Excretion ◽  
Pcr Analysis ◽  
Lipid Phase ◽  
Urine Ph ◽  
Phase Diffusion ◽  
Ammonia Transport ◽  
Lipid Diffusion ◽  
Rh Glycoproteins

NH3movement across plasma membranes has traditionally been ascribed to passive, lipid-phase diffusion. However, ammonia-specific transporters, Mep/Amt proteins, are present in primitive organisms and mammals express orthologs of Mep/Amt proteins, the Rh glycoproteins. These findings suggest that the mechanisms of NH3movement in mammalian tissues should be reexamined. Rh C glycoprotein (Rhcg) is expressed in the collecting duct, where NH3secretion is necessary for both basal and acidosis-stimulated ammonia transport. To determine whether the collecting duct secretes NH3via Rhcg or via lipid-phase diffusion, we generated mice with collecting duct-specific Rhcg deletion (CD-KO). CD-KO mice had loxP sites flanking exons 5 and 9 of the Rhcg gene (Rhcgfl/fl) and expressed Cre-recombinase under control of the Ksp-cadherin promoter (Ksp-Cre). Control (C) mice were Rhcgfl/flbut Ksp-Cre negative. We confirmed kidney-specific genomic recombination using PCR analysis and collecting duct-specific Rhcg deletion using immunohistochemistry. Under basal conditions, urinary ammonia excretion was less in KO vs. C mice; urine pH was unchanged. After acid-loading for 7 days, CD-KO mice developed more severe metabolic acidosis than did C mice. Urinary ammonia excretion did not increase significantly on the first day of acidosis in CD-KO mice, despite an intact ability to increase urine acidification, whereas it increased significantly in C mice. On subsequent days, urinary ammonia excretion slowly increased in CD-KO mice, but was always significantly less than in C mice. We conclude that collecting duct Rhcg expression contributes to both basal and acidosis-stimulated renal ammonia excretion, indicating that collecting duct ammonia secretion is, at least in part, mediated by Rhcg and not solely by lipid diffusion.

Резекция печени изменяет механизмы выведения аммиака почками крыс с хроническим тетрахлорметановым гепатитом

2018 ◽  
pp. 72-79
Author(s):  
П.Н. Савилов ◽  
Д.В. Молчанов
Keyword(s):  
Body Weight ◽  
Toxic Hepatitis ◽  
Ammonia Excretion ◽  
Left Lobe ◽  
Free Form ◽  
Sham Operation ◽  
White Rats ◽  
Urea Reabsorption ◽  
Ambiguous Effect ◽  
Collecting Tubules

Цель исследования - изучение влияния резекции печени (РП) на аммиакэкскретирующую функцию почек при хроническом тетрахлорметановом гепатите. Методика. Опыты выполнены на 265 беспородных белых крысах (самках) массой 180-220 г. Хронический гепатит воспроизводили подкожным введением 50% раствора тетрахлорметана (CCl) на оливковом масле (0,1 мл/100 г массы тела, через сутки, c двумя двухнедельными перерывами между 6, 7 и 13-14 инъекциями). На 65-е сут. (последние) введения тетрахлорметана, удаляли часть левой доли печени (15-20% массы органа). На 3-и, 7-е и 14-е сут. после РП или лапаротомии («ложнооперированные» животные) в почках, артериальной и венозной крови, моче исследовали содержание аммиака, глутамина и мочевины. Результаты. Прогрессирование эндогенной аммиачной интоксикации после РП на фоне тетрахлорметанового гепатита сопровождается повышенной экскрецией аммиака почками. Однако это не устраняет артериальную гипераммониемию и не предотвращает накопление почками аммиака. Инкреция глютамина из почек в кровоток прекращается. К 14-м сут. послеоперационного периода возрастает потребление глютамина из артериальной крови, что приводит к его накоплению в почках. Стимулируя выведение мочевины из организма с мочой, РП одновременно активирует её образование в почках, с дальнейшим поступлением как в кровоток, так и в мочу. В зависимости от сроков послеоперационного периода это сопровождается изменением скорости реабсорбции мочевины в почках. Заключение. Полученные результаты свидетельствуют, что при РП на фоне тетрахлорметанового гепатита почки не предотвращают прогрессирование эндогенной аммиачной интоксикации, патологическое накопление аммиака и глутамина её клетками, но сохраняют способность принимать участие в регуляции повышенного содержания мочевины в артериальной крови. Mechanical (resection) or toxic (hepatitis) liver damage alone has an ambiguous effect on renal ammonia excretion during development of endogenous ammonia intoxication. The aim. The study investigated the effect of liver resection (LR) on renal ammonia excretion in chronic tetrachlorocarbon (CCl)-induced hepatitis. Methods. Experiments were conducted on 240 mongrel white rats (females) weighing 180-220 g. Chronic hepatitis was induced by subcutaneous injection of 50% solution of carbon tetrachloride (CCl) in olive oil (0.1 ml/100g body weight per day with two two-week breaks between injections 6-7 and 13-14). LR with removal of a part of the left lobe (15-20% of body weight) was performed on the 65th (last) day of CCl injections. The animals were examined on the 3rd, 7th and 14th day after LR or laparotomy (sham operation). Contents of ammonia (AM), glutamine (GN), and urea were measured in the kidney, arterial (AB) and venous ( v.renlis ) blood, and urine. Results. Progression of endogenous ammonia intoxication after LR associated with CCl-induced hepatitis and increased renal excretion of Am involves three mechanisms: 1) excretion of Am that is delivered to kidneys in the free form with AB; 2) stimulation of renal tubule secretion of Am that had formed in kidneys by deamidation of «arterial» Gn; and 3) contrary to rules, partial reabsorption of Am from collecting tubules into the blood. However, this does not eliminate arterial hyperammonemia or prevent accumulation of Am in kidneys. The stimulatory effect of LR in CCl-induced hepatitis on Gn incretion from kidneys to the circulation stops by the 14 day after surgery, and the accompanying increased consumption of Gn from AВ results in Gn accumulation in kidneys. LR stimulates urea excretion with urine and simultaneously activates kidney formation of urea, which further enters the bloodstream and urine. Depending on the postoperative period this is associated with changes in the rate of urea reabsorption in kidneys. Conclusions. In RP associated with CCl-induced hepatitis, kidneys cannot prevent progression of endogenous ammonia intoxication and pathological accumulation of ammonia and glutamine in kidney cells but retain the ability to participate in the regulation of the increased urea level in AB.

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