Taurine secretion in primary monolayer cultures of flounder renal epithelium: stimulation by low osmolality.

Transepithelial taurine fluxes determined in short-circuited monolayer cultures of flounder renal proximal cells in Ussing chambers revealed net taurine secretion. Both unidirectional secretory and reabsorptive taurine fluxes exhibited saturation kinetics contributed by two distinct saturable transepithelial taurine transport systems operating at different taurine concentration ranges. The taurine secretory system operating below 0. 5 mM had lower affinity but higher capacity than the reabsorptive system, whereas the one operating at high concentrations (0.5-3.0 mM) had higher affinity but the same capacity as the corresponding reabsorptive system. Exposure (2 h) of the cultures to hyposmotic medium in the presence of taurine increased taurine secretory flux twofold with no effect on the reabsorptive flux. The hyposmolality-induced increase in taurine secretion was associated with a decreased peritubular taurine efflux and a concurrent increased luminal taurine efflux; the latter occurred via a pathway that was not affected by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid but inhibited by probenecid. The culture response in hyposmotic medium mimics the in vivo response of the intact marine fish kidney to dilution.

Basolateral taurine transport system in reptilian renal cells.

The characteristics of taurine transport across renal basolateral membranes were examined, using basolateral membrane vesicles (BLMV) isolated from garter snake (Thamnophis spp.) kidneys. BLMV fraction exhibited high enrichment for the basolateral marker enzyme, Na(+)-K(+)-adenosinetriphosphatase (23-fold), with approximately 10% contamination by brush-border membranes. Taurine uptake into BLMV was specifically stimulated by inwardly directed Na+ gradient in the presence of Cl-. Equilibrium NaCl condition and replacement of NaCl gradient by KCl, choline chloride, NaSCN, sodium gluconate, or mannitol inhibited taurine uptake. Unlike brush-border membrane vesicles (BBMV), taurine uptake into BLMV was not stimulated by a Cl- gradient. In further contrast to BBMV, BLMV taurine uptake was smaller in magnitude and not electrogenic. The stoichiometric relationship between Na+ and BLMV taurine uptake, determined by activation method, indicated a 1 Na+:1 taurine interaction, in contrast to the 3 Na+:1 taurine stoichiometry for BBMV taurine transport. Bromcresol green inhibited BBMV taurine transport but had no effect on BLMV taurine uptake. Efflux of taurine from BLMV was faster than that from BBMV. Unlike BBMV, the BLMV efflux was stimulated by external taurine. The observed characteristics of taurine transport on both membranes would integratively result in net transepithelial reabsorption of taurine across renal cells of the garter snake, a species that demonstrates both net reabsorption and secretion of taurine in vivo.

Sodium-taurine cotransport in reptilian renal brush-border membrane vesicles.

The coupled transport of Na+ with taurine into snake renal brush-border membrane vesicles (BBMV) was studied using 5-s uptake conditions. Taurine transport into snake renal BBMV involved two parallel processes, one saturable (Na(+)-dependent) and one (Na(+)-independent) that behaved like passive diffusion. Below 1 mM taurine concentration, the Na(+)-dependent system accounted for 60% of total taurine uptake. Over both low (0.001-0.80 mM) and high (0.8-5.0 mM) taurine concentration ranges, the Na(+)-dependent taurine uptake within each range showed Michaelis-Menten kinetics, suggesting the presence of two independent saturable Na(+)-dependent transport systems for taurine. The high-affinity, low-capacity system saturated above 100 microM with a Km of 71.4 +/- 45.7 microM and a maximum velocity (Vmax) of 21.9 +/- 3.77 pmol (mg protein)-1 (5 s)-1. The low-affinity, high-capacity system saturated above 1 mM, with a Km of 1.11 +/- 0.63 mM and a Vmax of 252 +/- 47 pmol (mg protein)-1 (5 s)-1. The stoichiometric relationship between external Na+ concentration and taurine uptake (at 10 microM) by the high-affinity BBMV transport system was examined by the activation method under short-circuited conditions. The 5-s rate of taurine transport was a sigmoid function of increasing extravesicular Na+ concentration. Kinetic analysis of the interaction of Na+ with the high-affinity taurine transport system suggested that 3 Na+ ions (3.2 +/- 0.7) may be involved with 1 taurine molecule in the transport event.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of renal transport of taurine in reptilian brush-border membranes.

We examined characteristics of taurine transport across renal brush-border membranes (BBM) of the garter snake (Thamnophis sirtalis), a species that demonstrates both net reabsorption and secretion of taurine in vivo. Transport was examined by a rapid filtration technique at 25 degrees C. Inwardly directed Na+ gradient specifically stimulated taurine uptake. Under initial taurine equilibrium condition, a small overshoot of taurine uptake driven by an inwardly directed NaCl gradient could be observed. No stimulation of taurine uptake was observed under Na+ equilibrium or K+, Li+, or choline gradients conditions. Reptilian renal BBM taurine transport also displayed specific Cl- requirement: replacement of NaCl by NaSCN or Na(+)-gluconate gradients inhibited taurine uptake. The uptake was stimulated under Cl- gradient compared with Cl- equilibrium conditions. Taurine transport was not stimulated by H+ gradient in either direction, although it was inhibited by acidic pH (less than 7.0). Amiloride and furosemide had no effects. The transport was electrogenic, stimulated by an inside negative membrane potential, and inhibited by other beta-amino acids. Overall, the reptilian BBM transport system for taurine resembles those observed in both mammalian and fish renal BBM.

Renal effects of atrial natriuretic peptide in a marine elasmobranch.

The effects of atrial natriuretic peptide on the renal function of the spiny dogfish (Squalus acanthias) in seawater were evaluated. A synthetic mammalian peptide, atriopeptin II (2 micrograms/kg), was injected intravascularly into unanesthetized, unrestrained fish prepared for renal clearance studies. The aortic pressure, glomerular filtration rate (GFR), urine flow rate (V), and urinary excretion of sodium, potassium, and total osmolytes were continuously monitored. Atriopeptin II significantly decreased mean aortic pressure (-12%), GFR (-40%), V (-66%), and the absolute excretion rates of sodium (-47%), potassium (-43%), and total osmolytes (-44%). However, the renal effects of atriopeptin II were temporally dissociated from the vasodepressor effect. Mean aortic pressure decreased quickly and returned to control values approximately 2 h after injection, whereas GFR did not decrease significantly until the third hour after injection. The decreases in renal water and solute excretion in response to atriopeptin could be accounted for by the decrease in GFR, since there were no significant changes in fractional water or solute excretion. Similar decreases in GFR were observed during constant infusions of a lower physiological dose of the peptide (80 pg.kg-1.min-1). The observed antidiuretic and antinatriuretic effect of synthetic atriopeptin in the dogfish contrasts with its putative role as a hormone mediating hypervolemic regulation.

Enzymatic and transport characteristics of isolated snake renal brush-border membranes.

Brush-border membranes (BBM) of proximal tubules were isolated from the kidney of the garter snake (Thamnophis sirtalis) by a procedure involving hypotonic lysis, Ca precipitation, and differential centrifugation. The isolated membranes were enriched 15-fold in brush-border enzyme activities (alkaline phosphatase, gamma-glutamyl transpeptidase) compared with whole kidney homogenates and were substantially free of other contaminating membranes. The yield of the BBM preparation was 40%. The BBM vesicular transport of several organic solutes was characterized by a rapid filtration technique at 25 degrees C. D-glucose, p-aminohippurate (PAH), and urate entered the same osmotically active space (2-3 microliter/mg protein) and binding was minimal (less than 20% for PAH). An uptake overshoot for 3-O-methyl-D-glucose (20 microM) by reptilian BBM was observed only in the presence of an inwardly directed NaCl gradient and was abolished by 0.1 mM phlorizin. Reptilian BBM exhibited Na-gradient-stimulated uptake of PAH (90 microM) with an overshoot that was inhibited by other organic acids and by 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid (SITS). In contrast, urate uptake (30 microM) appeared to be Na independent and not appreciably affected by other organic anions or SITS. The presence of specific transport systems for organic solutes in the isolated membrane preparation distinctly characterizes the BBM of reptilian kidney.

Plasma levels and renal handling of endogenous amino acids in snakes: a comparative study.

Plasma levels of 22 endogenous amino acids were measured by ion-exchange chromatography in four species of snakes: Thamnophis sirtalis, T. radix, Aipysurus laevis, and Python molurus. Despite considerable interspecific variation in the amino acid composition, all species showed relatively high plasma concentrations of histidine, a feature apparently unique to reptiles. The renal handling of these amino acids was studied by renal clearance methods. As in other vertebrates, net tubular absorption of filtered amino acids predominated. However, net tubular secretion of taurine, cysteic acid and/or phosphoserine and beta-alanine was observed, with taurine being the predominant amino acid secreted. The percentage reabsorption of the total amino acids filtered by the snake kidneys ranged from 79 to 95%. Evidence for the postrenal absorption of amino acids in these reptiles is presented. In species that normally undergo hibernation (Thamnophis spp.), the ability of the kidney to reabsorb amino acids was depressed by cold acclimation. Cold acclimation significantly decreased plasma levels of all amino acids except taurine, whose concentration increased. The increase in plasma taurine level may have resulted from cellular osmoregulation. Under these conditions, renal excretion of taurine increased concomitantly with the increase in plasma taurine concentration.

Renal secretion of amino acids in ophidian reptiles.

Net renal tubular secretion of endogenous beta-amino acids (taurine, beta-alanine, beta-aminoisobutyric acid) and their endogenous analogue (L-cysteic acid) was revealed in the olive sea snake, Aipysurus laevis, and in the garter snake, Thamnophis sirtalis, by renal clearance methods. The net secretory rates ranged from 16 to 795 nmol X kg-1 X h-1, with taurine being secreted at the highest rates. These rates of secretion are comparable to those observed in marine fish, the only other group of vertebrates exhibiting renal tubular secretion of L-amino acids under physiological conditions. However, only some snakes (8-58%) demonstrated net tubular secretion; the others (0-58%) showed net tubular reabsorption of these amino acids. Net tubular secretion was consistently observed whenever the filtered load of the amino acid was low, and net reabsorption was apparent whenever the filtered load was high; the two variables being significantly correlated. An analysis of the net amino acid transport rates, both secretory and reabsorptive, as a function of the filtered load suggests that in snakes the beta-amino acids are reabsorbed and secreted at discrete tubular sites, with the secretory sites located beyond the major reabsorptive sites. Taurine, beta-alanine, and L-cysteic acid appear to share a common transport system in the snake renal tubule cells.

Renal function in sea snakes. I. Glomerular filtration rate and water handling.

Renal clearance studies were performed on anesthetized sea snakes (Aipysurus laevis) under various salt and volume loads. The median values for the control glomerular filtration rate (GFR) and urine flow rate (V) were 0.78 and 0.11 ml X kg-1 X h-1, respectively. Acute salt loading with 1 mol/l NaCl significantly increased V to 0.68 ml X kg-1 X h-1, whereas acute water loading significantly decreased GFR to 0.17 ml X kg-1 X h-1. Acute loading with either isosmotic NaCl or seawater did not significantly alter either V or GFR. Chronic loading with water or seawater by intraperitoneal injection significantly increased both GFR and V. No consistent relationship between plasma osmolality and GFR was observed. Instead a threshold-type relationship existed between GFR and plasma K concentration. Within each treatment group, high plasma K concentrations were correlated with low GFRs. There was an abrupt increase in GFR at low plasma K concentrations, the inflection point ranging from 2.5 to 4 mmol/l K depending on the experimental treatment. A curvilinear relationship existed between V and GFR; as GFR increased, V approached a maximal limiting value. At low GFRs (less than 0.2 ml X kg-1 X h-1), V often exceeded GFR, indicating net fluid secretion that was correlated with net Na movement. In addition, there was an inverse relationship between urine-to-plasma osmolality ratio and V. At low values of V, urine osmolalities modestly exceeded those of the plasma. Overall, renal function of A. laevis appears to be adapted to conserve water.

Renal function in sea snakes. II. Sodium, potassium, and magnesium excretion.

Renal clearance studies performed on the sea snake Aipysurus laevis demonstrated net tubular reabsorption of Na and both net tubular secretion and reabsorption of Mg and K. Fractional excretion of Na (FENa) was 0.05 under control conditions and increased markedly with hyperosmotic salt loading. Within each group, however, higher glomerular filtration rates were accompanied by disproportionately greater Na reabsorption. FEMg was 0.66 under control conditions and increased with Mg loading. The rate of Mg secretion was positively correlated with the urine flow rate. However, Mg loading caused a flow-independent increase in Mg secretion that was correlated with increasing plasma Mg concentration. FEK was 0.86 under control conditions and increased with K loading as a result of enhanced K secretion by the renal tubules. Like renal Mg secretion, renal K secretion showed a flow-dependent component as well as a flow-independent component related to plasma K concentration.

Comparative aspects of glomerular filtration in vertebrates.

Glomerular ultrafiltration of the plasma is a fundamental component of vertebrate renal function. The importance of the glomerulus is reflected by its near-universal presence and great elaboration among the vertebrates. Although the general structural features and functional properties of the glomerulus appear to be largely similar among diverse groups, there exists considerable variation in the magnitude of the rate of filtration. The kidney is the primary vertebrate organ responsible for water and metabolic waste excretion, and glomerular filtration plays an important role in these functions. Therefore, the magnitude of the GFR appears to be influenced primarily by the rates of water influx and metabolism. Major phylogenetic differences in morphological, physiological and metabolic design have a decisive impact on the magnitude of the GFR. The endothermic classes, with more numerous glomeruli, high metabolic rates, and high ultrafiltration pressures, have proportionately higher rates of glomerular filtration than the ectothermic groups. As a group, the reptiles, with presumably the lowest rates of water influx, exhibit the lowest GFRs. Within each class, there are trends toward species with greater access to free water having higher GFRs (e.g. fresh water vs. marine; mesic vs. xeric. The clearest examples exist for the teleosts, with marine forms having lower GFRs than their fresh water relatives. The coupling of the GFR to environmental influences is also demonstrated by the response of the animal to environmentally imposed perturbations, such as dehydration. In terrestrial animals during dehydration, reductions in the rate of glomerular filtration occur reducing the rate of urinary water loss. And increases in GFR appears to be important in the rapid elimination of water loads in nonmammalian vertebrates. This short-term modulation of the GFR occurs by either changing glomerular plasma flow or glomerular capillary hydrostatic pressure, or both. In addition, shifts in the filtering populations of glomeruli can take place, as has been demonstrated in birds. Although the mediators of these effects have not been unequivocally identified, several hormones, including antidiuretic hormone, angiotensin, and catecholamines, have been implicated.

Relation of ammonia excretion adaptation to glutaminase activity in acidotic, subtotalnephrectomized rats.

The response of renal ammonia excretion to acidosis was examined in adult rats with reduced renal mass (SNX). Three days after surgical ablation of 70% of renal mass, the activity of renal phosphate-dependent glutaminase (PDG) in SNX rats was 7.7 +/- 1.5 mu moles of ammonia/100 mg of protein min or approximately 50% of the activity in normal rats (14.5 +/- 2.6 mu moles of ammonia/100 mg of protein min), but enhanced ammonia excretion per unit weight was observed in SNX rats (7.2 +/- 0.7 in control vs. 14.6 +/- 3.2 mumoles/g of kidney.hr in SNX rats). The cause (s) of the reduction in the specific activity of PDG (as well as the increase in ammonia excretion) is unknown. The PDG decrease was not due to apparent tissue damage and appeared to be a specific change as the activity of renal succinate dehydrogenase, another mitochondrial inner-membrane enzyme, did not decrease (from the control level) in SNX rats. Ammonia excretion showed no significant response to an acute acid load (ammonium chloride, 5 mmoles/kg of body wt) in SNX rats. Ammonia excretion, however, did adapt to repeated acid-loading (10 mmoles of ammonium chloride per kg of body wt per day for 3 days); ammonia excretion increased more than two-fold by third day of treatment. This adaptive response was associated with a two-fold rise in renal PDG. Administration of actinomycin D, at a dose which produced no gross toxic signs (100 microgram/kg/day i.p.) inhibited virtually all the increase in both ammonia excretion and PDG activity. The correlation of ammonia excretion and PDG adaptations in acidotic SNX rats was similar to that previously observed in infant rats.

Regulation of sheep erythrocyte volume in anisotonic media.

Sheep erythrocytes of high and low potassium types were incubated in non-haemolytic hypotonic and hypertonic media for 4-5 h at 30 degrees. After initial swelling or shrinking, they readjusted their volume toward their initial isotonic volume. The volume regulation was associated with specific changes in cation fluxes. In the swollen cells, efflux of both sodium and potassium was increased and influx of both cations was slightly decreased; the converse was true for the shrunken cells. All four fluxes were changed in a direction that led to return to normal volume. The difference in the response of the two types of sheep erythrocytes to changes of extracellular fluid osmolality resided in the different activity of their cation transport systems. It is concluded that sheep erythrocytes possess some means of regulating their volume in vitro which is linked to cation permeability. The exact nature of the physical mechanisms by which they accomplish this remains to be elucidated.

Renal glutaminase adaptation and ammonia excretion in infant rats.

The purpose of this investigation was to determine the role of enzyme adaptation in the response of ammonia excretion to acidosis in developing rats. The response of renal ammonia excretion was low in infant rats (7-12 days old) following administration of a single dose of acidifying salt (5 mmol NH4CL/kg). However, repeated administration (2 times daily) of the salt increased ammonia excretion two- to threefold within 2 days. This adaptive response was associated with a concomitant rise in renal phosphate-dependent glutaminase (PDG) activity; PDG activity increased from approximately 36% adult level in untreated infants to 79% adult level in infants given NH4Cl for 2 days. Ammonia excretion and PDG activity decreased in parallel following cessation of NH4Cl treatment. Administration of the antibiotic, actinomycin D (100 mug/kg, ip, 2 times daily for 2 days) completely inhibited the response of PDG to repeated NH4Cl administration. In contrast to the situation previously observed in adult rats, actinomycin D treatment prevented the acid-induced rise in renal ammonia excretion. These results suggest that the level of renal PDG plays a more direct role in the adaptation of ammonia excretion to acidosis in infant rats than in adults.