Net Excretion Research Articles

Blood Kinetics of Lipophilic and Proteinophilic Pollutants during Two Types of Long-Term Fast in King Penguins.

In vertebrates, fasting is an intricate physiological process associated with strong metabolic changes, yet its effect on pollutant residue variation is poorly understood. Here, we quantified long-term changes in plasma concentrations of 20 organochlorine and 16 perfluoroalkyl pollutants in king penguins Aptenodytes patagonicus during the breeding and molting fasts, which are marked by low and high levels of protein catabolism, respectively, and by strong lipid use. The profile of measured pollutants in plasma was dominated by perfluorooctanesulfonic acid (PFOS, initial relative contribution of 60%). Initial total pollutant concentrations were similar in molting (3.3-5.7 ng g-1 ww) and breeding penguins (range of 4.2-7.3 ng g-1 wet weight, ww). Long-term fasting (25 days) for molting and breeding led, respectively, to a 1.8- and 2.2-fold increase in total plasma pollutant concentrations, although the rate and direction of change were compound-specific. Hexachlorbenzene (HCB) and PFOS concentrations increased in plasma (net mobilization) during both types of fasting, likely due to lipid use. Plasma perfluoroundecanoate (PFUnDA) and perfluorotridecanoate (PFTrDA) concentrations increased in breeders (net mobilization) but decreased in molting individuals (net excretion), suggesting a significant incorporation of these pollutants into feathers. This study is a key contribution to our understanding of pollutant variation in blood during long-term fasting in wildlife.

Role of the kidneys in acid-base regulation and ammonia excretion in freshwater and seawater fish: implications for nephrocalcinosis.

Maintaining normal pH levels in the body fluids is essential for homeostasis and represents one of the most tightly regulated physiological processes among vertebrates. Fish are generally ammoniotelic and inhabit diverse aquatic environments that present many respiratory, acidifying, alkalinizing, ionic and osmotic stressors to which they are able to adapt. They have evolved flexible strategies for the regulation of acid-base equivalents (H+, NH4 +, OH- and HCO3 -), ammonia and phosphate to cope with these stressors. The gills are the main regulatory organ, while the kidneys play an important, often overlooked accessory role in acid-base regulation. Here we outline the kidneys role in regulation of acid-base equivalents and two of the key 'urinary buffers', ammonia and phosphate, by integrating known aspects of renal physiology with recent advances in the molecular and cellular physiology of membrane transport systems in the teleost kidneys. The renal transporters (NHE3, NBC1, AE1, SLC26A6) and enzymes (V-type H+ATPase, CAc, CA IV, ammoniagenic enzymes) involved in H+ secretion, bicarbonate reabsorption, and the net excretion of acidic and basic equivalents, ammonia, and inorganic phosphate are addressed. The role of sodium-phosphate cotransporter (Slc34a2b) and rhesus (Rh) glycoproteins (ammonia channels) in conjunction with apical V-type H+ ATPase and NHE3 exchangers in these processes are also explored. Nephrocalcinosis is an inflammation-like disorder due to the precipitation of calcareous material in the kidneys, and is listed as one of the most prevalent pathologies in land-based production of salmonids in recirculating aquaculture systems. The causative links underlying the pathogenesis and etiology of nephrocalcinosis in teleosts is speculative at best, but acid-base perturbation is probably a central pathophysiological cause. Relevant risk factors associated with nephrocalcinosis are hypercapnia and hyperoxia in the culture water. These raise internal CO2 levels in the fish, triggering complex branchial and renal acid-base compensations which may promote formation of kidney stones. However, increased salt loads through the rearing water and the feed may increase the prevalence of nephrocalcinosis. An increased understanding of the kidneys role in acid-base and ion regulation and how this relates to renal diseases such as nephrocalcinosis will have applied relevance for the biologist and aquaculturist alike.

Uptake of Sulfate from Ambient Water by Freshwater Animals.

To better understand how the sulfate (SO42−) anion may contribute to the adverse effects associated with elevated ionic strength or salinity in freshwaters, we measured the uptake and efflux of SO42− in four freshwater species: the fathead minnow (Pimephales promelas, Teleostei: Cyprinidae), paper pondshell (Utterbackia imbecillis, Bivalvia: Unionidae), red swamp crayfish (Procambarus clarkii, Crustacea: Cambaridae), and two-lined mayfly (Hexagenia bilineata, Insecta: Ephemeridae). Using δ(34S/32S) stable isotope ratios and the concentrations of S and SO42−, we measured the SO42− influx rate (Jin), net flux (Jnet), and efflux rate (Jout) during a 24 h exposure period. For all four species, the means of Jin for SO42− were positive, and Jin was significantly greater than 0 at both target SO42− concentrations in the fish and mollusk and at the lower SO42− concentration in the crayfish. The means of Jout and Jnet were much more variable than those for Jin, but several species by target SO42− concentration combinations for Jout and Jnet, were negative, which suggests the net excretion of SO42− by the animals. The results of our experiments suggest a greater regulation of SO42− in freshwater animals than has been previously reported.

Deciphering genetic signatures by whole exome sequencing in a case of co-prevalence of severe renal hypouricemia and diabetes with impaired insulin secretion

BackgroundRenal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic β-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism.Case presentationWe describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8–7.0 mg/dl), 41.6 μmol/l (226–416 μmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 μg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic β-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation.ConclusionWe found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic β-cell functions that deserve further scrutiny.

Clinical study on the treatment of small calculi in the middle and lower segment of ureter by extracorporeal physical vibrating lithotomy

Objective To compare the clinical effects of active lithotripsy(extracorporeal physical vibration lithotripsy combined with drugs) and passive lithotripsy(drug therapy alone) in the treatment of small stones in the middle and lower ureter. Methods All the included subjects were patients with primary small ureteral calculi in the middle and lower segment(<6 mm) admitted to our hospital from January 2018 to September 2018 outpatient department. Patients in the two groups were randomly divided into the active stone drainage group (in vitro physical vibration stone drainage combined with drug therapy) and the passive stone drainage group(purely drug therapy). Urinary CT was reexamined 1, 2 and 4 weeks after treatment. Comparison indexes: the rate of ureteral calculi excretion and net excretion at 1, 2 and 4 weeks after treatment, and the incidence of complications in the two groups were compared.Logistic regression analysis was performed on related parameters. Results A total of 138 patients were included in the study, among which 76 were males and 62 were females. There was no statistical difference in gender composition, age, stone size, CT value and stone distribution between the two groups(P>0.05). The stone removal rate of active rock discharging group on 1, 2 and 4 weeks was 52.6%, 63.2% and 78.9% respectively, while the corresponding passive stone discharging group was 33.9%, 40.3% and 61.3% respectively(P 0.05). Conclusions Physical vibration stone drainage is a safe and effective auxiliary treatment of stone drainage, compared with conservative treatment, can improve the ureteral stone drainage rate. Key words: Ureteral Calculi; Lithotripsy; Drug Therapy

Nitrogen supply in cattle coupled with appropriate supply of utilisable crude protein at the duodenum, a precursor to metabolisable protein

ABSTRACTThe overall objective of this study was to calculate the amount of nitrogen (N) that cattle feed must contain in order to utilise the potential supply of utilisable crude protein at the duodenum provided by their energy intake without incurring a negative N balance, that is, without having to break down body protein. For this purpose, the literature was screened for measurements of net degradation and renal excretion of urea as well as N balances (N intake, faecal N and urinary N) in ruminants (cattle, sheep and goats) fed diets with varying N concentrations. Irreversible loss of N from the body urea pool increased with increasing N intake, but net degradation of urea as a proportion of irreversible loss decreased concurrently. Faecal N appeared not to be influenced by N intake and exceeded 11 g/kg dry matter intake (DMI) only in 7% of the data sets available. Urinary non-urea-N rarely exceeded 4 g/kg DMI and appeared independent of N intake. Urinary urea-N showed a clear dependence of N intake, and it is concluded that 1 g N/kg DMI is sufficient for compensating inevitable N losses in the form of urinary urea. In conclusion, ruminant rations should contain the following N concentrations (per kg DM) to account for obligatory losses: 11 g for compensating losses as faecal N, 4 g for compensating losses as urinary non-urea-N and 1 g for compensating inevitable losses as urinary urea-N. The derived recommendations should be helpful for limiting N excretion where this is desirable for ecological reasons.

Long-term trends in the metabolic profile test results in German Holstein dairy herds in Thuringia, Germany

Systematic metabolic profile tests at regular intervals provide important information to meet the increasing challenges in managing dairy herds. This study aimed at deriving long-term trends from the results of metabolic profile tests in Thuringian German Holstein dairy herds. Blood and urine samples were collected from 710 fresh, high yielding, and dry cows, respectively, to monitor 13 parameters including the serum non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHB), bilirubin, β-carotene and urea concentrations, the serum of aspartate aminotransferase and glutamate dehydrogenase activities, the serum concentrations of the minerals calcium, inorganic phosphate and selenium, the urine potassium and sodium concentrations, and the net acid-base excretion (NABE). A total of 17,070 group samples from 103 herds were collected during 1997-2014. Period prevalence of aberrant groups was calculated for each parameter and year accordingly. Using a linear regression model, a long-term trend of the period prevalence was determined. To account for the 15 aberrancies possible for each stage of lactation, seven significant trends were found for dry, nine for fresh, and ten for lactating cows. A decrease of the period prevalence was detected for increased NEFA, for low serum β-carotene, selenium, inorganic phosphate, and urea concentrations, for low sodium and high potassium urine concentrations, and for increased NABE. In contrast, period prevalence increased significantly for cases with increased serum aspartate aminotransferase and glutamate dehydrogenase activities, increased bilirubin concentration, and low NABE. Metabolic profile tests enable a description of the herd health status using objectively measured data as a basis for the implementation of measures to optimize herd management to improve the herd health status.

A Single Host-Derived Glycan Impacts Key Regulatory Nodes of Symbiont Metabolism in a Coevolved Mutualism.

ABSTRACTMost animal-microbe mutualistic associations are characterized by nutrient exchange between the partners. When the host provides the nutrients, it can gain the capacity to shape its microbial community, control the stability of the interaction, and promote its health and fitness. Using the bioluminescent squid-vibrio model, we demonstrate how a single host-derived glycan, chitin, regulates the metabolism of Vibrio fischeri at key points in the development and maintenance of the symbiosis. We first characterized the pathways for catabolism of chitin sugars by V. fischeri, demonstrating that the Ccr-dependent phosphoenolpyruvate-pyruvate phosphotransferase system (PTS) prioritizes transport of these sugars in V. fischeri by blocking the uptake of non-PTS carbohydrates, such as glycerol. Next, we found that PTS transport of chitin sugars into the bacterium shifted acetate homeostasis toward a net excretion of acetate and was sufficient to override an activation of the acetate switch by AinS-dependent quorum sensing. Finally, we showed that catabolism of chitin sugars decreases the rate of cell-specific oxygen consumption. Collectively, these three metabolic functions define a physiological shift that favors fermentative growth on chitin sugars and may support optimal symbiont luminescence, the functional basis of the squid-vibrio mutualism.

Physiological and molecular responses of the spiny dogfish shark (Squalus acanthias) to high environmental ammonia: scavenging for nitrogen.

In teleosts, a branchial metabolon links ammonia excretion to Na(+) uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs (Rhag, Rhbg and Rhp2) and evaluated gill ammonia handling in Squalus acanthias. Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol(-1) NH4HCO3) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24-48 h. Control ΔP(NH3) (the partial pressure gradient of NH3) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH4(+) electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na(+)/K(+)-ATPase (NKA) increased at 24 h and that of H(+)-ATPase decreased at 48 h, while expression of the potential metabolon components Na(+)/H(+) exchanger2 (NHE2) and carbonic anhydrase IV (CA-IV) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.

Mechanisms of Na+ uptake, ammonia excretion, and their potential linkage in native Rio Negro tetras (Paracheirodon axelrodi, Hemigrammus rhodostomus, and Moenkhausia diktyota).

Mechanisms of Na(+) uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J in), efflux (J out), and net flux rates (J net) of Na(+) and Cl(-), and the net excretion rate of ammonia (J Amm). The fish were collected directly from the Rio Negro, and studied in their native "blackwater" which is acidic (pH 4.5), ion-poor (Na(+), Cl(-) ~20µM), and rich in dissolved organic matter (DOM11.5mg Cl(-1)). J in (Na) , J in (Cl) , and J Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J in (Na) was unaffected by amiloride (10(-4)M, NHE and Na(+) channel blocker), but both J in (Na) and J in (Cl) were virtually eliminated (85-99% blockade) by AgNO3 (10(-7)M). A time course study on cardinal tetras demonstrated that J in (Na) blockade by AgNO3 was very rapid (<5min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10(-4)M) caused a 50% reduction in J in (Na) .. Additionally, J in (Na) was unaffected by phenamil (10(-5)M, Na(+) channel blocker), bumetanide (10(-4)M, NKCC blocker), hydrochlorothiazide (5×10(-3)M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J in (Na) (by acetazolamide and AgNO3 respectively), had any inhibitory effect on J Amm. Therefore, Na(+) uptake in Rio Negro tetras depends on an internal supply of H(+) from CA, but does not fit any of the currently accepted H(+)-dependent models (NHE, Na(+) channel/V-type H(+)-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current "Na(+)/NH4 (+) exchange metabolon" paradigm. Na(+), K(+)-ATPase and V-type H(+)-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH4Cl, 10(-3)M) significantly increased J in (Na) , and NH4 (+) was equally or more effective than K(+) in activating branchial Na(+),(K(+)) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na(+) uptake, but that Na(+) uptake (by an as yet unknown H(+)-dependent apical mechanism) depends on ammonia excretion, driven by active NH4 (+) entry via basolateral Na(+),(K(+))-ATPase.

Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW

Previous studies have shown that the deletion of brnQ from the Corynebacterium glutamicum chromosome results in a significant reduction in L-isoleucine uptake rates, while overexpression of brnFE leads to enhanced L-isoleucine export rates. Given that net excretion rates would be an important factor for high titers of L-isoleucine accumulation, we have tested the notion that decreased L-isoleucine uptake combined with increased L-isoleucine excretion will further improve high-yield strains that are currently used for the industrial-scale production of L-isoleucine. To examine the effect of the two carriers on L-isoleucine accumulation in L-isoleucine producer C. glutamicum YILW, we constructed a brnQ deletion mutant (C. glutamicum YILW∆brnQ) and two brnFE overexpressors (C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW∆brnQpXMJ19brnFE). Compared to the original strain, the efflux rate of the brnQ mutant increased from 19.0 to 23.6 nmol min(-1) mg (dry wt)(-1) and its L-isoleucine titer increased from 154.3 mM (20.2 g l(-1)) to 170.3 mM (22.3 g l(-1)). The efflux rates of C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW∆brnQpXMJ19brnFE were 33.5 and 39.1 nmol min(-1) mg (dry wt)(-1), and their L-isoleucine production titers were 197.2 mM (25.9 g l(-1)) and 221.0 mM (29.0 g l(-1)), respectively. Our results suggest that modifications of the transport system could provide a promising avenue for further increasing L-isoleucine yield in the L-isoleucine producer.

Laboratory diagnostics in the urine of young and pregnant ewes

The purpose of this study was to identify values for net acid base excretion (NABE) which are significant indicators of the acid-base equilibrium in pregnant and young ewes and to show its relationship with other parameters (base, acid, ammonium [NH4], base-acid quotient, sodium [Na], potassium [K], calcium [Ca]) in ovine urine. In contrast to dairy cows, data are rare on these parameters in ewes. A total of 99 animals were used in the study, consisting of 56 young (average of 5.6±1.1 months) and 43 pregnant ewes (average of 35.2±18.8 months). Measurement of fractional NABE in urine samples was carried out according to the method reported by Kutas. The pH value of the urine was measured with a laboratory pH meter. Na, K and total Ca were measured with a flame photometer. For all values except Na significant differences occurred between urine samples of pregnant ewes and young ewes (p<0.001). Base, acid, NH4, NABE, K and Ca values were significantly higher in the urine of the youngs than in pregnant ewes. In young ewes, a strong correlation was found between NABE and base values while a weak correlation could be observed between pH and base values. In pregnant ewes, strong NABE-base, NABE-K, K-acid and K-base correlations were found as well as weak NH4-base, NH4-NABE and NH4-K correlations. There was a strongly positive correlation between NABE and NH4 in pregnant ewes, while a weak negative correlation between those values was observed in young ewes. For the first time, we established values for NABE and certain other parameters in urine of pregnant ewes and young ewes. It was shown that the acid-base balance in pregnant ewes and young ewes can be evaluated by measuring NABE and certain trace elements in urine like in cattle.

Comparison of the impact of different anionic salts on the acid–base status and calcium metabolism in non-lactating, non-pregnant dairy cows

Eleven mature, non-lactating, non-pregnant, Holstein-Friesian crossbred cows were given, via a ruminal cannula, 2000mEq of one of three chloride salts, four sulfate salts, two combinations of anionic salts (AS), sodium chloride (as neutral salt), or water as control. The salts and controls were assigned in an 11x11 Latin square and the cows were randomly distributed. All of the AS induced a metabolic acidosis that resulted in a small reduction of blood pH, base excess, and bicarbonate (P<0.001), and notable changes in urinary pH, net acid base excretion (P<0.001), and urinary calcium excretion (P<0.001). Only calcium chloride had a significantly greater impact on acid-base status (ABS) than the sulfate salts. The effect of other chloride salts did not differ from calcium sulfate. There was no indication that chloride salts in general have a greater impact than sulfate salts on the ABS.

S2063 Calcium Sensing Receptor (CaSR) Function in Intestine Epithelial Cells of Rodents and Humans

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.

S2064 Intestinal Alkaline Phosphatase Maintains the Normal Homeostasis of Gut Microbiota

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.

Using omeprazole to link the components of the post-prandial alkaline tide in the spiny dogfish,Squalus acanthias

After a meal, dogfish exhibit a metabolic alkalosis in the bloodstream and a marked excretion of basic equivalents across the gills to the external seawater. We used the H(+), K(+)-ATPase pump inhibitor omeprazole to determine whether these post-prandial alkaline tide events were linked to secretion of H(+) (accompanied by Cl(-)) in the stomach. Sharks were fitted with indwelling stomach tubes for pretreatment with omeprazole (five doses of 5 mg omeprazole per kilogram over 48 h) or comparable volumes of vehicle (saline containing 2% DMSO) and for sampling of gastric chyme. Fish were then fed an involuntary meal by means of the stomach tube consisting of minced flatfish muscle (2% of body mass) suspended in saline (4% of body mass total volume). Omeprazole pre-treatment delayed the post-prandial acidification of the gastric chyme, slowed the rise in Cl(-) concentration of the chyme and altered the patterns of other ions, indicating inhibition of H(+) and accompanying Cl(-) secretion. Omeprazole also greatly attenuated the rise in arterial pH and bicarbonate concentrations and reduced the net excretion of basic equivalents to the water by 56% over 48 h. Arterial blood CO(2) pressure (Pa(CO(2))) and plasma ions were not substantially altered. These results indicate that elevated gastric H(+) secretion (as HCl) in the digestive process is the major cause of the systemic metabolic alkalosis and the accompanying rise in base excretion across the gills that constitute the alkaline tide in the dogfish.

The alkaline tide and ammonia excretion after voluntary feeding in freshwater rainbow trout

We investigated the potential acid-base and nitrogenous waste excretion challenges created by voluntary feeding in freshwater rainbow trout, with particular focus on the possible occurrence of an alkaline tide (a metabolic alkalosis created by gastric HCl secretion during digestion). Plasma metabolites (glucose, urea and ammonia) were measured at various time points before and after voluntary feeding to satiation (approximately 5% body mass meal of dry commercial pellets), as was the net flux of ammonia and titratable alkalinity to the water from unfed and fed fish. Arterial blood, sampled by indwelling catheter, was examined for post-prandial effects on pH, plasma bicarbonate and plasma CO2 tension. There was no significant change in plasma glucose or urea concentrations following feeding, whereas plasma ammonia transiently increased, peaking at threefold above resting values at 12 h after the meal and remaining elevated for 24 h. The increased plasma ammonia was correlated with an increase in net ammonia excretion to the water, with fed fish significantly elevating their net ammonia excretion two- to threefold between 12 and 48 h post feeding. These parameters did not change in unfed control fish. Fed fish likewise increased the net titratable base flux to the water by approximately threefold, which resulted in a transition from a small net acid flux seen in unfed fish to a large net base flux in fed fish. Over 48 h, this resulted in a net excretion of 13 867 micromol kg(-1) more base to the external water than in unfed fish. The arterial blood exhibited a corresponding rise in pH (between 6 and 12 h) and plasma bicarbonate (between 3 and 12 h) following feeding; however, no respiratory compensation was observed, as PaCO2 remained constant. Overall, there was evidence of numerous challenges created by feeding in a freshwater teleost fish, including the occurrence of an alkaline tide, and its compensation by excretion of base to the external water. The possible influence of feeding ecology and environmental salinity on these challenges, as well as discrepancies in the literature, are discussed.

The kidney responds to respiratory acidosis by altering the fractional clearance of chloride as against sodium in pregnant ewes

Using Stewart’s physiochemical acid-base theory, we hypothesized that the kidney will balance the net excretion of chloride as against sodium in response to acute respiratory acidosis. The experiment was performed in pregnant ewes on days 118 and 125 of gestation. Ewes were placed in carts where the head resides within a sealed plexiglass chamber. Inspired CO2 concentration was manipulated for 1 hour to produce acute respiratory acidosis. When the arterial partial pressure of carbon dioxide (PaCO2) and pH changed from 32 ± 1 mm Hg and 7.52 ± 0.02 at the 0th hour to 74 ± 2 mm Hg and 7.22 ± 0.02 respectively at the 1st hour, the renal fractional clearance of chloride with respect to sodium increased significantly by 71 ± 28%. The clearance returned to baseline at 2nd hour when PaCO2 and pH were 37 ± 0.6 mm Hg and 7.49 ± 0.01 respectively. Thus, we conclude that the renal response to respiratory acidosis is to increase the strong ion difference by differential reabsorption of sodium and chloride. Since sodium reabsorption is strongly related to renal regulation of extracellular fluid volume, net chloride excretion relative to sodium is the primary mechanism for renal regulation of acid-base balance. Supported by NIAAA 10940 (T.A.C.) and the Pediatric Initiatives.

Experimental studies on the impact of an increased dose of anionic salts on the metabolism of dairy cows

Summary This study was initiated to investigate the influence of a daily dose of anionic salts (AS) above the valid upper limit at present on metabolism of dairy cows. Eleven non‐pregnant and non‐lactating Holstein‐Friesian crossbred cows with a permanent rumen cannula were used in a study with a controlled feeding design. The initial daily dose was 2500 meq/day, which resulted in a Dietary Cation Anion Difference (DCAD) of ‐211 meq/kg dry matter. Every seven days, the daily dose was raised by 500 meq. If a cow stopped eating, the application of AS was stopped and these cows were monitored over the next seven days. On day 30 another batch of hay, having the same DCAD but higher concentrations of minerals and energy, was fed. Blood and urine samples were taken to monitor acid‐base balance and calcium concentrations. Acid‐base balance was strongly influenced by AS. Blood pH dropped steadily and reached values around 7.23. Urine pH dropped quickly below 6 and remained at that level regardless of the increased dosage of AS. Net acid base excretion (NABE) fell continuously with the increase of the dosage of AS and reached values below ‐200 mmol/l. Calcium concentrations in the serum were nearly stable, but those in urine increased sharply and remained on an elevated level with increasing doses of AS. A few days before the individual cow's refusal of feed intake, calcium excretion in urine decreased. The majority of cows stopped eating while consuming a diet containing 3500 to 4000 meq AS except two animals who consumed up to 6000 meq/day AS but they received the better hay in the second half of the treatment period. In this time pH in blood increased slowly. NABE remained stable on a low level at ‐100 mmol/l. The results showed that with an increasing amount of AS fed the risk of clinical acidosis increased. The decreasing urine concentrations of calcium indicate a breakdown of the compensation capability of the single cow. Besides the dose of AS fed, the quality of the feed stuff might be another factor concerning the tolerance of cows against AS.

Interaction of Methotrexate with Organic-Anion Transporting Polypeptide 1A2 and Its Genetic Variants

Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 +/- 118 microM; Vmax = 17.5 +/- 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V(max) for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.