Sodium pump and Na+/H+ antiport restoration in erythrocytes from cancer patients in remission.

We previously showed that in erythrocytes from cancer patients, the sodium pump is decreased and the optimal intracellular pH for Na+/H+ antiport activity is shifted toward an acidic value. We now have studied these sodium transporters in erythrocytes from patients in remission. Moreover, we intended to explain why the transporters were impaired in erythrocytes, which have no apparent bearing on cancer tissues. The sodium pump was studied through a microcalorimetric method, and the Na+/H+ antiport by a titrimetric method. In patients in remission the sodium pump activity returned to normal: 15.10 +/- 6.00 vs. 14.12 +/- 5.28 mW/l cells for remission and control, respectively. The optimal intracellular pH for Na+/H+ antiport activity was identical in remission and control: 6.09 +/- 0.23 vs. 6.10 +/- 0.10. Restoration of sodium pump activity and optimal intracellular pH for Na+/H+ antiport activity in erythrocytes were thus linked to remission. Moreover, we showed that the impairments of the sodium transporters were due to the presence of plasma-borne factors, the existence of which explained why the sodium transporters were impaired in erythrocytes.

A non ouabain-like inhibitor of the sodium pump in uremic plasma ultrafiltrates and urine from healthy subjects.

A non ouabain-like inhibitor of the sodium pump was separated from uremic plasma ultrafiltrates and normal urine. Under the same chromatographic conditions (C18 column and a gradient of acetonitrile as eluant), ouabain was eluted in a fraction different from the inhibitor. Affinity chromatography based on the formation of a complex between Na,K-ATPase and the inhibitor achieved the differentiation ouabain. Without magnesium and sodium phosphate, ouabain could not bind to enzyme whereas the inhibitor did. A study of Na,K-ATPase enzyme kinetics showed the inhibitor was not competitive for K+, which further differentiates it from ouabain. It was uncompetitive for ATP and seemed competitive for Na+. These results indicate that the inhibitor acts inside the cell, unlike ouabain, and thus its action mechanism appears to be original.

Calcium channel blockers correct in vitro mitochondrial toxicity of cellulose acetate.

We studied the action of rinse solutions from cellulose acetate hemodialyzers on isolated mitochondria. We showed that concentrates from the rinses impaired the adenosine 5'-triphosphate (ATP) synthesis as reflected by the decrease in respiration during state 3 and in P/O ratio. This impairment results from a calcium release from mitochondria that is induced by rinse solution concentrates. The release, triggering the mitochondrial calcium carrier, would explain the decrease in ATP synthesis. Moreover, rinse solution concentrates hinder mitochondrial calcium storage. The rise in cytosolic calcium in hemodialyzed patients may be related, at least in part, to these findings, since a lack of ATP impairs the ATP-dependent cellular calcium-extrusion pumps. We also showed that calcium channel blockers, at therapeutically relevant doses, restore ATP synthesis and calcium storage in mitochondria impaired by rinse solution concentrates. Finally, these in vitro results were confirmed by experiments on cells in culture proving that Diltiazem counteracts the cytotoxicity of rinse solution concentrates. These findings are consistent with observations that these drugs suppress the increase in leukocyte cytosolic calcium in dialyzed patients. Moreover, this would help explain the efficiency of calcium channel blockers in cells without L-calcium channels.

In vitro mitochondrial test to assess haemodialyser biocompatibility.

BACKGROUND: This paper describes an in vitro mitochondrial test to assess the biocompatibility of haemodialysers. METHODS: We tested on isolated liver mitochondria the effect of solutions obtained by an aqueous rinse of different haemodialysers (cuprophane, cellulose acetate, Hemophan, polyacrylonitrile, polymethylmethacrylate, polysulphone, polyamide). Moreover, to determine the penetration into the cell and the cytotoxicity of these solutions from haemodialysers, we examined the effect of rinse solutions on HT29-D4 cells. RESULTS: Our results showed that rinse solutions from haemodialysers decrease the mitochondrial ATP synthesis. Cuprophane has the most marked effect, and the synthetic membranes exhibited only mild effects. Rinse solutions penetrated the cell and were cytotoxic by acting on mitochondria in the cell. In this respect, cellulosic membranes were the most toxic. CONCLUSION: Taken together our findings lead to a classification of haemodialyser membranes which is identical to one based on criteria such as activation of complement (cuprophane > other cellulosics > synthetics). Moreover isolated mitochondria make it possible to differentiate among the synthetic membranes. Isolated mitochondria thus appear to be a good in vitro test to assess the biocompatibility of haemodialysers.

Impairment of sodium pump and Na+/H+ antiport in erythrocytes isolated from cancer patients.

We sought to determine whether the impairment of sodium pump activity and Na+/H+ exchange reported in tumorigenic cells was specific to these cells or more general. Sodium pump activity and Na+/H+ exchange were measured in erythrocytes from 49 cancer patients and 51 healthy subjects. Cancer patients with a newly detected cancer or in relapse and without associated pathologies known to modify these sodium transporters were included in this study. Two sodium pump statuses reflecting its physiological modulation were evidenced for healthy subjects (10.3 +/- 0.2 and 19.4 +/- 0.8 mW/liter of cells). In cancer patients, only one basal status lower than those of controls was observed (8.3 +/- 0.5 mW/liter of cells; P < 0.001). Cooperativity of the Na+/H+ antiporter is the same in cancer patients and controls (2.58 +/- 0.27 versus 2.60 +/- 0.15). The intracellular pH (pHi) dependence curve of the antiporter was shifted toward more acidic values, and optimal pH1 was lower in cancer patients than in controls (5.80 +/- 0.03 versus 6.08 +/- 0.02; P < 0.0001). The mean maximal rate and the Km of H+ for the Na+/H+ antiporter were higher: 8.4 +/- 1.2 versus 4.6 +/- 0.4 mmol H+/liter of cells/h (P < 0.01) and 514 +/- 12 versus 322 +/- 16 nM (P < 0.05), respectively. Alterations of these Na+ transporters, therefore, were not restricted to cancerous cells. Among the alterations, the acidic shift in the pHi dependence of Na+/H+ exchange appears associated with cancer because this behavior has never been reported in other pathologies.

Opposite effects of urea on hemoglobin-oxygen affinity in anemia of chronic renal failure.

We studied the action of urea on the spin-spin relaxation rate of 2,3-diphosphoglycerate (2,3-DPG) phosphorus atoms in normal and uremic erythrocytes. At concentrations from 10 to 60 mM, urea increased the relaxation rates of 2,3-DPG P-3 phosphorus atoms. This evidenced a stronger binding of 2,3-DPG to hemoglobin (Hb), suggesting that the deoxyform of Hb was stabilized. This hypothesis was confirmed by measurements of the association constant of oxygen to hemoglobin (K) in normal erythrocytes in presence of urea concentrations in the range of those observed in uremic patients (30 mM). Indeed, the observed decrease in K suggests that the T structure of hemoglobin is stabilized. By contrast, with higher urea concentrations (120 mM), measurements of P50 showed an increase in the hemoglobin affinity for oxygen (decrease in P50). Moreover, the relaxation rates of 2,3-DPG P-3 phosphorus atoms were not modified, which is consistent with the simultaneous increase of K. This may be attributed to the formation of carbamylated hemoglobin in presence of urea. These results suggest two opposite effects of urea on Hb-O2 affinity: the first reinforces 2,3-DPG-Hb binding and leads to a decrease in O2 affinity; the second, mediated by carbamylation of Hb, hinders the binding of 2,3-DPG and increases the O2 affinity. These findings are consistent with the fact that, despite the presence of carbamylated hemoglobin, uremic patients do not present increased Hb-O2 affinity.

Is the beneficial effect of calcium channel blockers against cyclosporine A toxicity related to a restoration of ATP synthesis?

ATP synthesis inhibited by Cyclosporine A is restored by calcium channel blockers: nifedipine, verapamil, bepridil, diltiazem. ATP synthesis was estimated using liver mitochondria by measuring the rate of respiration during state 3 and a measure of the yield of ATP synthesis, the P/O ratio. The study of calcium fluxes through mitochondrial membrane indicates that calcium channel blockers counteract the mitochondrial calcium storage induced by cyclosporine A. If the restoration of ATP synthesis observed in vitro also occurred in vivo, the increase in ATP pool might contribute to a better functioning of the Ca2+ extrusion pumps of the cells, thereby maintaining the cytosolic calcium concentration (Cai), in the normal range. The nephrotoxicity of cyclosporine A appears to be due to a vasoconstrictive effect related to an increased Cai. This result may account for the reduction of clinical cyclosporine A toxicity by calcium channel blockers. Verapamil appears to be the most efficient in restoring ATP synthesis.

Ascorbic acid derivatives in two different fractions of uremic toxins.

The middle-molecular-weight uremic toxins which accumulate in uremic plasma seem to be associated with various uremic disorders such as uremic neuropathy and defects in the sodium pump. By a multi-step chromatographic method, two fractions of these toxins were isolated and studied because one inhibits microtubule formation in vitro (fraction 2-5), and the other impairs the sodium pump in living erythrocytes (fraction 2-3). An additional chromatographic method allows the separation of these fractions and isolation of two components: fractions 2-3-V and 2-5-III. Analyses by UV and 1H NMR spectrometry identified these compounds as two different ascorbic acid derivatives. 2-3-V is not yet totally identified and 2-5-III corresponds to ascorbic acid 2-sulfate. These two metabolites exert no toxic effects but they have the same chromatographic behavior as uremic toxins.

Scaling up in isolation of medium-size uraemic toxins.

Plasmatic accumulation of uraemic toxins in the middle molecular mass range has been reported to be associated with several pathologies observed in uraemic patients. The very low concentration of these toxins in uraemic body fluids makes classical chromatography techniques inadequate in isolating sufficient amounts of these endogenous substances, thus precluding their identification. A scaling up of gel permeation and ion-exchange chromatographies was therefore developed. This considerably increased the amount of uraemic toxins isolated, thus allowing the study of their chemical nature and facilitating understanding of their biological activities.

High-resolution NMR studies of transmembrane cation transport in uremic patients.

Cation transport in erythrocytes of some uremic patients is impaired. Most studies have focused on the defect of the erythrocyte Na+/K+ pump in these diseased states. Herein, this cation transport defect was studied by using nuclear magnetic resonance spectroscopy (NMR) which is a non-invasive method permitting study on living erythrocytes. Firstly, we verified that the Na+ transport defect in uremic erythrocytes was not due to non-specific causes such as membrane alteration or a modification of the intracellular metabolism. The proton relaxation data, determined using a paramagnetic doping method, are consistent with a lack of erythrocytic membrane damage in uremic patients. Also, 31P-NMR results showed that in our experimental conditions, uremic and normal erythrocytes exhibit similar variations of ATP level over time. Lastly, the use of anionic paramagnetic shift reagent in 23Na-NMR revealed a defect in the Na+/K+ pump of erythrocytes from uremic patients with high Nain concentration. This defect seems to be due to a reduced number of pump units and to the presence of an endogenous inhibitor in uremic plasma.

Identification of an ascorbic acid metabolite among "uremic middle molecules".

Among uremic toxins in the middle molecular mass range, 1H, 13C-nuclear magnetic resonance, ultraviolet spectrometry, and chromatographic analyses allow identification of the main component of the so-called "2-5-3 fraction" as ascorbic acid 2-sulfate, a conjugated metabolite of ascorbic acid. We previously (Clin Nephrol 1986;25:212-8) showed an inhibitory effect of the 2-5-3 fraction on microtubule formation. Therefore, we tested the action of ascorbic acid 2-sulfate and its synthetized enantiomers on tubulin polymerization. Because these molecules did not exert any inhibitory effect, we hypothesize that the 2-5-3 fraction is a mixture of compounds in which only a very low quantity of the inhibitory factor is present.

Kinetic modeling of intradialytic and interdialytic pH shifts during and after acetate and bicarbonate hemodialysis.

A kinetic model involving intraerythrocytic and whole blood H+ concentrations during and after bicarbonate and acetate hemodialysis is proposed to account for experimental data. A two-compartment model appeared to be the simplest kinetic model to explain the decrease in proton concentration during bicarbonate hemodialysis and its increase between two dialysis sessions, whether acetate or bicarbonate. This model takes into account the hemoglobin buffer power and the cellular metabolic acidosis. During acetate hemodialysis, one must introduce a new compartment to explain the initial increase in H+ concentration in erythrocytes. This compartment, which generates protons, seems to correspond to the carbonic anhydrase cycle. The various parameters obtained show no significant variations between patients receiving bicarbonate hemodialysis. For acetate hemodialysis, the model describes equally well patients with a great initial increase in H+ concentration and those with a slight initial increase. The variations observed in the parameters are due mainly to the carbonic anhydrase compartment. It is suggested the magnitude of this initial increase and the degree of acetate intolerance are correlated.