Renal responses during a dry saturation dive to 450 msw.

Four subjects were compressed to a simulated depth of 450 msw (46 bar) for 37 days in the main research chamber of the German underwater simulator diving facility at the GKSS Research Center, Geesthacht. The ambient gas was trimix. Urine was collected at 0700, 1300, and 1900 h each day for analysis of Na+, K+, volume, osmolality, and creatinine. Urine, antidiuretic hormone (ADH), and aldosterone were analyzed separately. Daily fluid, Na+, and K+ intake were analyzed throughout the dive. The aim of the investigation was to confirm the existence of a diuresis and natriuresis which had been observed in earlier saturation dives to 31 atm abs using He-O2. A significant diuresis was observed during compression despite a decrease in fluid intake. After compression the diuresis decreased somewhat but remained significantly above precompression control levels during the entire hyperbaric exposure. No significant change in fluid intake was observed. Daily Na+ and K+ excretion increased significantly during compression, which was accompanied by a significant increase in nocturnal excretion of Na+ and K+. Daily intake of Na+ and K+ decreased throughout the dive. Daily urine ADH decreased immediately upon compression and was associated with a parallel decrease in urine osmolality. In contrast, urinary aldosterone excretion exhibited no change during the dive despite the increase in Na+ and K+ excretion and decrease in Na+ intake.

Urinary vasopressin and aldosterone and plasma volume during a saturation dive to 450 m.

Urinary vasopressin (VP), aldosterone (ALDO), osmotic substances, sodium excretion, and plasma volume were assessed in 4 healthy male divers during 2 predive control days, 2 compression days, 6 days at 46 atm abs, and 26 days of decompression with stops at 37 and 27 atm abs. At pressure the ambient gas was trimix (0.5 atm abs O2:5% N2:remainder He). All urine was collected throughout the dive. Samples were divided into daytime (0700-1900) and nighttime (1900-0700). Indocyanine green dye dilution was used to determine plasma volume at predive 1, 46, and 24 atm abs. In agreement with previous dives at 31 atm abs, there was a decrease in VP excretion during compression lasting until return to 1 atm abs (P less than 0.05). Also similar to the shallower dives at 31 atm abs, the normal diurnal pattern of VP excretion, daytime higher than nighttime (P less than 0.05), disappeared at pressure. Urine osmolality showed alterations compatible with responses to VP. In contrast to previous studies at 31 atm abs, but in agreement with a previous study at 49.5 atm abs, there was no sustained increase in urinary ALDO excretion and only a transient natriuresis during the compression phase, followed by a reduced sodium excretion. In confirmation of earlier conclusions from indirect evidence, direct measurements of plasma volume indicated a reduction of about 20% (P less than 0.05) at 46 atm abs which remained reduced after decompression to 24 atm abs.

Effects of methylguanidine on sodium and organic ion transport.

We studied the acute effect of methylguanidine (MG), a suspected uremic toxin that accumulates in renal failure, on p-aminohippurate (PAH) and tetraethylammonium (TEA) uptake in rabbit kidney slices, on Na+,K+ ATPase activity in the microsomal fraction of rabbit kidneys, and on transepithelial active Na transport across toad skin. MG at concentrations ranging from 0.05 (similar to that reported in uremic patients) to 1.0 mM does not affect the organic anion (PAH) uptake, although it exhibits a concentration-dependent inhibition of organic cation (TEA) uptake. MG at concentrations from 0.05 to 5 mM had no effect on kidney Na+,K+ ATPase activity or on active transepithelial Na transport across toad skins when applied to the outside bathing solution; however, MG (greater than 1 mM) stimulated Na transport when applied to the inside bathing solution. These results are not consistent with the hypothesis that MG is a potential uremic toxin that causes the natriuresis and other toxic effects. However, long-term toxic effects of MG on the kidney were not assessed in the present study.

Interaction of heptanol and pressure on sodium and chloride transport by toad skin.

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.

Absence of diuresis during a 7-day saturation dive at 2.5 ATA N2-O2.

Three male divers were studied for 2 days during each of the predive and postdive 1 ATA air control periods and for 7 days at 2.5 ATA (2.3 ATA N2 and 0.2 ATA O2). The chamber temperature was always maintained at a comfort level. Average urine flow remained at 1500 ml.day-1 during both predive and 2.5 ATA periods; urine osmolality also remained constant at around 700 mOSM/kg. On the other hand, daily excretion of Na increased significantly from 139 mEq during the predive period to 178 mEq at 2.5 ATA (P less than 0.05) but returned to the predive level during the postdive period. In contrast, daily K excretion decreased progressively with a significant decrease during the postdive period (P less than 0.05). Plasma osmolality, Na, and K remained unchanged, whereas a 6% reduction of total protein concentration at 2.5 ATA (P less than 0.05) was observed. A quantitatively similar decrease (8%) was observed for hematocrit during the 2.5 ATA period, which did not recover at postdive. These changes were accompanied by a significant increase in urinary excretion of antidiuretic hormone (P less than 0.05) and by decreases in both plasma renin and aldosterone (P less than 0.05) level and urinary excretion of aldosterone (P less than 0.05). Plasma atrial natriuretic factor remained unchanged throughout the entire dive period.

Hyperbaric oxygen effect on active Na+ transport across isolated toad skin.

The effect of hyperbaric oxygen (HBO) on Na+ transport across the isolated toad (Bufo marinus) skin was studied by measuring the transepithelial short-circuit current (ISC) and resistance (R) at 5, 8, and 10 ATA PO2 and 15 ATA normoxia during steady state conditions. The imposition of 5, 8, and 10 ATA PO2 for 2 h resulted in 45, 52, and 85% decrease in ISC, respectively. This decrease in ISC was always accompanied by an increase in R. When amiloride (10(-4) M) was added to the bathing medium, ISC decreased to zero within 15 min regardless of the PO2 level, indicating that the HBO-induced decrease in ISC is caused by an inhibition of amiloride-sensitive Na+ transport. Addition of both superoxide dismutase (SOD) and catalase to the medium bathing both sides of the skin markedly attenuated the HBO effect on ISC and R. Applying HBO to the serosal or mucosal surface independently produced similar effects on ISC. However, the presence of antioxidant enzymes (SOD and catalase) with 10 ATA PO2 prevented the toxic HBO effect only from the serosal side; no protection by these antioxidant enzymes was observed from the mucosal side. These findings are consistent with a view that free radicals are involved in the HBO-induced inhibition of ISC. However, further studies involving the site(s) of radical generation as well as site(s) of toxic action are needed to understand the cellular and molecular mechanism of HBO toxicity.

Size selected mRNA induces expression of P-aminohippurate transport in Xenopus oocytes.

Xenopus oocytes were injected with size-fractionated mRNA isolated from the renal cortex of rabbit kidney and after 4 days incubation, PAH uptake in oocytes injected with mRNA (0.7-1.3 kb) was 8 to 45 fold that of the water injected controls. The oocyte to medium ratio of accumulated PAH was 1.95. The Km and Vmax for transport were 333 microM and 66.6 nmoles.oocyte-1.min-1, respectively. This Km is similar to that reported for PAH transport in intact kidneys and slices. The uptake of PAH was unaffected by the absence of Na+ or the presence of probenecid. Expression of the transport represents the first step in an effort to clone and identify the gene for PAH transport.

Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells.

The organic anion p-aminohippurate (PAH) is specifically secreted by the renal proximal tubule. The possibility was examined that the probenecid sensitive PAH transport system (which is involved in this secretory process in renal proximal tubule cells in vivo) is retained in primary cultures of rabbit kidney proximal tubule cells. Significant 3H-PAH uptake into primary cultures of proximal tubule cells was observed. After 10 min, 150 pmole PAH/mg protein had accumulated intracellularly. Given an intracellular fluid volume of 10 microliter/mg protein, the intracellular PAH concentration was estimated to be 15 microM. The initial rate of PAH uptake (when 50 microM PAH was in the uptake buffer) was inhibited 50% by 2 mM probenecid. Intact monolayers also exhibited Na+-dependent alpha methyl-D-glucoside uptake (an apical marker). Basolateral membranes were purified from primary rabbit kidney proximal tubule cell cultures. Probenecid sensitive PAH uptake into the membrane vesicles derived from the primary cultures was observed. The rate of PAH uptake was equivalent to that obtained with vesicles obtained from the rabbit renal cortex. No significant Na+-dependent D-glucose uptake into the vesicles was observed, indicating that primarily basolateral membrane vesicles had indeed been obtained.

Seadragon VI: a 7-day dry saturation dive at 31 ATA. VII. Erythrocyte functions.

Organic phosphates and the Donnan ratio (cell to plasma Cl ratio) were measured in man during a dry saturation dive to 31 ATA for 7 d. 2,3-DPG increased significantly with a concomitant decrease in ATP and the molar ratios of ATP and ADP on Day 5 at 31 ATA (P less than 0.05). Inorganic phosphate did not change during the dive. The Donnan ratio was relatively high at 31 ATA and the value was significantly different on Day 3 at 31 ATA (0.731 at 1 ATA vs. 0.895 at 31 ATA, P less than 0.05). This suggests a rise in intracellular pH, resulting from a change in the net charge of hemoglobin. It is speculated that an increase in oxygen affinity of hemoglobin occurs during the early phase at 31 ATA and is followed by an increase in 2,3-DPG in response to the resulting tissue hypoxia.

Seadragon VI: a 7-day dry saturation dive at 31 ATA. VIII. Plasma enzyme profiles.

This report summarizes serum profiles of liver enzymes of divers during 2 dry saturation dives to 31 ATA. In both dives, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), and alkaline phosphatase were significantly elevated at 31 ATA when compared to predive control levels. SGPT and SGOT levels returned to control levels during the postdive period. These data provide strong evidence for compromised liver function at high pressure and are consistent with similar observations in other saturation dives. The reason for the parenchymal dysfunction at high pressure remains unknown.

Kidney immunopathology and pathophysiology in rats immunized with proximal tubule cell brush border or basolateral membrane vesicles.

Proximal tubule pathology in Heymann nephritis has been attributed to anti-brush border antibodies, but antibodies with other specificities might also be important. To determine whether injury to the basolateral membranes of proximal tubules could occur independently of brush border injury, LEW rats were immunized either with partially purified basolateral or brush border membrane vesicles. Both immunogens produced glomerular immunopathology and pathophysiology identical in magnitude and time course to that seen in Heymann nephritis. Antibodies eluted from the kidneys of rats immunized with either antigen preparation stained the brush border in vitro. However, circulating anti-brush border antibodies were in significant titers only in rats immunized with brush border vesicles, whereas antibodies that stained the cytoplasm of both proximal and distal tubules predominated in rats immunized with basolateral membranes. With the onset of proteinuria, rats immunized with brush border membranes developed the proximal tubule pathology of Heymann nephritis. In rats immunized with basolateral membranes, the brush border and apical aspect of proximal tubule cells remained essentially normal. However, defects of basolateral membrane transport function were present, indicating that those defects need not necessarily be secondary to brush border damage. The dissociation of brush border damage from glomerular injury suggests that different antibody populations may account for each. Furthermore, anti-brush border antibodies may not account for all aspects of proximal tubule pathology in Heymann nephritis.

Effect of ethanol on organic ion transport in rabbit kidney.

The effect of ethanol on the transport of organic ions in rabbit kidney cortical slices was studied. Ethanol at a concentration of 4 to 10% (v/v) reversibly inhibited the slice uptake of the organic anion, p-aminohippurate (PAH), in a dose-dependent manner, but had no significant effect on that of the organic cation, tetraethylammonium (TEA). Overall, the inhibitory effect on PAH uptake increased with the length of the hydrocarbon chain, with an I50 of 7.7, 0.9, and 0.05% for ethanol, butanol, and heptanol, respectively. The efflux of PAH was significantly decreased in the presence of 8% ethanol. Kinetic analysis indicated that ethanol decreased Vmax without a significant change in Km. Lowering the Na concentration in the incubation medium from 130 to 20 mM resulted in a disappearance of the above described inhibitory effect of ethanol on PAH uptake. Although Na-K-ATPase activity of renal cortical microsomes was significantly inhibited by ethanol (6-10%), butanol (1%), and heptanol (0.1%), there was no clear correlation between the effects of alcohols on PAH transport and/or Na-K-ATPase. Nevertheless, the results suggest that ethanol inhibits reversibly the Na-dependent transport of PAH from the medium into the cell across the basolateral membrane through a mechanism yet to be elucidated. The high degree of correlation between I50 and the partition coefficients of the alcohols suggested that their interaction with membrane lipids is important for the inhibition of PAH uptake, and also that PAH (but not TEA) transport is affected by alteration of the lipid environment of the membrane.

Ionic basis of short-circuit current in toad skin at high hydrostatic pressure.

The toad skin and urinary bladder are widely used for the study of water and Na+ transport under high pressure. These tissues can be mounted in Ussing type chambers and ion transport can be measured by evaluating electrical properties of the preparation, e.g., short-circuit current (Isc). The tacit assumption in these experiments is that the preparation behaves in the same manner at high pressure as at 1 ATA; namely, that net Na+ flux is equivalent to Isc. The purpose of the experiments described here was to test that assumption. Toad skins were mounted in an Ussing chamber and Isc was measured as an index of active net Na+ transport under hydrostatic pressures up to 100 ATA. The chamber was modified so that isotopic Na+ flux from the mucosal to serosal compartments could be measured in conjunction with Isc, without decompression. A linear regression of JNa+ms on Isc was computed and found to be described by the equation, JNa+ms = 3.83 + 0.83 Isc; n = 18; r = 0.92. The slope of the line was not significantly different from unity. No correlation was made for JNa+sm because of the difficulty in measuring JNa+sm and JNa+ms in the same skin simultaneously. Independent measurement of JNa+sm demonstrated that this flux accounted for less than 2% of JNa+net. In a second set of experiments, the influence of amiloride on Isc with and without pressure was tested. 10(-4) M amiloride abolished Isc under both circumstances. It is concluded that Isc can be wholly accounted for by net Na+ flux under pressures up to 100 ATA.

Lack of interaction between atrial natriuretic factor and renal organic anion transport system.

The aim of the present investigation was to test the hypothesis that atrial natriuretic factor (ANF) is secreted into the proximal tubule lumen by the organic anion transport mechanism. The rationale for this hypothesis was the reported probenecid attenuation of the natriuretic effect of ANF. Probenecid is widely regarded as an inhibitor of organic acid transport in the proximal tubule. ANF was prepared in varying degrees of purity ranging from a relatively crude extract to a highly purified form. A commercially available form was also used. All forms were bioassayed using the anesthetized rat and a diuresis and natriuresis was observed in each case which was comparable to literature reports. Interaction of ANF with the organic acid transport system was evaluated using the renal cortical slice technique. Over a wide range of concentrations, there was no effect of ANF on cortical slice accumulation of either p-aminohippurate (PAH), the classical substrate of the organic anion transport system or tetraethylammonium (TEA), a typical organic cation. It is concluded that although ANF may indeed exert its effect at the luminal membranes of the nephron, access to the lumen is not mediated by the organic cation or anion transport system in the proximal tubule.

In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses.

The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation.

Modification of pulmonary oxygen toxicity by bleomycin treatment.

The purpose of this study was to determine whether pretreatment of rabbits with bleomycin would modify their response to 100% O2 and, if so, to identify the mechanism of this action. A single intratracheal injection of bleomycin (5 U/kg) resulted in a transient decrease of the arterial Po2, its mean value (+/- SE) 7 days postinjection being 59 +/- 3 Torr. All animals were either killed or exposed to 100% O2 35 days postinjection. At this time, arterial Po2 had returned to its control level. On the other hand, lung hydroxyproline content had doubled and static compliance and the total lung capacity had decreased by 22 and 31%, respectively, indicating the existence of significant lung fibrosis. Furthermore, activities of catalase and superoxide dismutase in lung homogenates were higher than control and were further augmented by exposure to 100% O2 for 64 h. These biochemical changes may account, at least in part, for the mitigation of the toxic effects of hyperoxia, as shown by the delayed appearance of arterial hypoxemia, and the 50% increase in survival time when bleomycin injected rabbits were exposed to 100% O2 35 days postinjection.

Renal transport of gossypol in the rabbit.

The present work was carried out to investigate the transport characteristics of gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, into the rabbit renal cortical slice. The uptake of gossypol increased linearly during a 2-hr incubation after which it leveled off with the average slice-to-medium concentration ratio (S/M) slightly above 20. In the presence of metabolic inhibitors, the S/M gossypol leveled off at about 9, suggesting an extensive binding of gossypol to tissue proteins. The uptake of gossypol was significantly inhibited by p-aminohippurate (PAH), probenecid, ouabain, and DIDS, all of which are known inhibitors of renal organic anion transport. However, the gossypol uptake was not affected by tetraethylammonium (TEA), a prototypical organic cation. Kinetic studies indicated that the apparent Km for gossypol transport is 0.28 mM, and also that probenecid inhibits gossypol transport in a competitive manner. It is concluded that gossypol is transported by the renal tubule through the classic organic anion system.

Photoaffinity labeling of organic anion transport system in proximal tubule.

The present investigation compares brush-border (BBM) and basolateral membrane (BLM) vesicles in terms of purity, function, appearance on sodium dodecyl sulfate (SDS) gels, and labeling pattern by use of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid (NAP-taurine), a photoaffinity analogue of p-aminohippurate (PAH). Both BLM and BBM were vesicular by demonstration of PAH uptake into an osmotically active space and had probenecid-inhibitable uptake of PAH. Time courses for uptake were similar. 250 microM NAP-taurine resulted in a 35% inhibition of PAH uptake in BLM but it did not significantly effect PAH uptake into BBM. The latter was affected by 1 mM NAP-taurine. A comparison of Coomassie blue SDS gels of BLM and BBM showed markedly different staining patterns. A major band at approximately 52,000 daltons was more intensely stained in BLM than BBM. Major bands at approximately 40,000 and approximately 80,000 were stained more heavily in BBM than BLM. A minor protein at 26,000 in the BLM did not appear in BBM. An irreversible inhibition of PAH uptake in BLM was observed after photolysis in the presence of NAP-taurine. This was associated with the labeling of four protein bands on SDS polyacrylamide gels. In contrast no labeling was observed in BBM.

Effect of high hydrostatic pressure on sodium transport across toad skin.

The short-circuit current (Isc) was measured as an index of active net sodium transport across the isolated toad skin under various hydrostatic pressures up to 300 ATA. Upon compression, the base-line Isc increased transiently during the first 10 min by 10-15 microA/cm2 (congruent to 20%) and then decreased continuously, leveling off at 40 min under pressure. The latter decrease in base-line Isc (P less than 0.05 at all pressures) was pressure dependent, and its magnitude was 30 microA/cm2 (60% inhibition) at 300 ATA. Similarly, the transepithelial electric potential difference (PD) tended to increase slightly during the early phase of compression and decreased during the steady-state phase of compression. The transepithelial resistance (R), calculated from PD/Isc ratios, generally increased under pressure. The addition of vasotocin to the inside bathing medium resulted in an increase in Isc and PD and a reduction in R at all pressures. The magnitude of peak Isc response to vasotocin was 50-60 microA/cm2 at pressures up to 100 ATA, but decreased to 30 microA/cm2 at 200-300 ATA (0.05 less than P less than 0.10 as compared to the response at 1 ATA). On the other hand the stimulatory effect of 1 mM cyclic adenosine monophosphate (cAMP), added to the inside bathing medium, on Isc was not affected by pressure between 1 and 300 ATA. From these results it is postulated that the inhibition of base-line Na transport under high hydrostatic pressure may be primarily due to a decrease in the outer membrane permeability to Na rather than an inhibition of the Na-K-ATPase.

Effects of gossypol on PAH transport in the rabbit kidney slice.

The present work was carried out to investigate if gossypol, a toxic weak organic acid (pK = 7.2) contained in cottonseed, is secreted by the renal proximal tubule through the organic anion transport system in the rabbit. The slice uptake of p-aminohippurate (PAH), a prototypical organic anion, was significantly inhibited (by 30%) only when the medium concentration of gossypol was raised to 10(-4) M. However, gossypol at the latter concentration also induced a 30% inhibition of the slice uptake of tetraethylammonium (TEA), a prototypical organic cation. Moreover, gossypol at 10(-4) M significantly decreased the slice oxygen consumption (congruent to 30%) and Mg-ATPase (70%) and Na-K-ATPase (90%) activities of renal cortical microsomes, while it significantly decreased the intracellular (K+). These results indicate that gossypol inhibits PAH uptake through nonspecific nephrotoxic effects on cell metabolism and Na-K-ATPase activity rather than through its specific interaction with the organic anion transport system.