In vivo accumulation of sodium pump inhibitor by normal and uremic erythrocytes.

A uremic toxin fraction (fraction 2-3-1) in the middle molecular mass range (300-2000 Da) containing a sodium potassium inhibitor pump was studied. As fraction 2-3-1 was known to be present in minute quantities in urine and plasma, erythrocyte lysates were used as an alternative source. Results show that fraction 2-3-1 is more abundant in erythrocytes than in normal urine. In addition, the fraction is present in a greater quantity in uremic erythrocytes than in normal blood cells. It is concluded that fraction 2-3-1 is concentrated at the erythrocyte level in uremic patients.

2,3 diphosphoglycerate-haemoglobin binding in uraemic patients treated with erythropoietin. A 31P-nuclear magnetic resonance study.

Using 31P-nuclear magnetic resonance (NMR) measurements of relaxation rate for 2,3 diphosphoglycerate (DPG) phosphorus atoms, we showed previously that in uraemic red blood cells the DPG-haemoglobin binding is stronger, thus stabilizing the deoxyhaemoglobin form and hence facilitating oxygen release. Here we verified if these modifications of spatial environment of DPG remain in uraemic patients treated by human recombinant erythropoietin (rHuEpo). Simultaneously we measured the intraerythrocytic ATP concentration (ATPi) and pH (pHi) of patients. Our results show a slight decrease on pHi and ATPi values during rHuEpo treatment. For the DPG relaxation rates, we observed a very weak but statistically significant increase 6 months after the beginning of treatment, but we cannot attribute a physiopathological significance to these results because of the lack of accuracy of the NMR determination of relaxation rate in red blood cells. Therefore, the DPG-haemoglobin binding is always stronger than in normal subjects.

Sodium pump activity in uremic erythrocytes: a microcalorimetric study.

Erythrocyte thermogenesis was studied by flow microcalorimetry in 25 healthy subjects and 27 uremic patients. The heat production (HP) from cells in plasma, decrease in HP induced by ouabain (a specific sodium pump inhibitor) and index of rate response to ouabain action were measured. HP was higher in uremic patients than controls. Sodium pump inhibition with ouabain induced the same decrease in HP in the two groups. The index of rate response to ouabain action was lower in uremic patients than in controls. The difference in total HP may be due to a different age distribution of erythrocytes. Mean sodium pump activity was identical in the two groups, but some patients had lower activity than controls. Ouabain seems to act more slowly in many patients than in controls, perhaps because of hindered binding of the inhibitor.

The restoration of ATP synthesis may explain the protective effect of calcium antagonists against cyclosporine A nephrotoxicity.

Cyclosporine A at pharmacological doses decreases the rate and yield of ATP synthesis in rat mitochondria. This action seems to be due to the mitochondrial calcium storage induced by the drug. If such an effect occurs in vivo, the ATP deficit will affect calcium extrusion pumps, so triggering vasoconstriction which is the major side effect of Cyclosporine A. Calcium antagonists (Nifedipine and Verapamil) at least partially correct this effect on ATP synthesis: this finding may be related with the beneficial clinical effect conferred on Cyclosporine A toxicity by calcium antagonists. This effect of calcium antagonists may be due to an interaction with Cyclosporine A at the level of mitochondrial calcium efflux.

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.

Recent developments in short-chain fatty acid metabolism.

Bacterial translocation across the bowel wall has recently been proposed as a major problem in the stressed patient. Consequently, there has been considerable interest in fostering bowel wall integrity as a barrier to bacteria and endotoxin. One postulated means to promote this barrier function has been through the provision of the preferred fuels of the bowel wall. Among these are the short-chain fatty acids (SCFAs) which are found to a limited extent in the diet but are primarily produced through the fermentation of non-digestible carbohydrates by the bacterial flora of the colon. In both animal and human studies, SCFAs have been shown to stimulate intestinal mucosal growth. Because non-digestible carbohydrates, after colonic fermentation, are precursors to SCFAs, similar effects to those found with direct provision of SCFAs may be anticipated. Pectin, beta-glucan, and lactulose are among the many available nonabsorbable carbohydrates that could serve as a source of these trophic stimulants to colonic mucosa. Short-chain fatty acids and their precursors deserve extensive and clinical evaluation to define their ultimate role in human disease.

Kinetic modeling of intracellular pH and comparison with 31P NMR experimental values in dialysed uremic patients.

Changes in intra-erythrocytic pH values over time, during and after bicarbonate hemodialysis, were studied with 31P Nuclear Magnetic Resonance. Simultaneously, pH values of whole blood were obtained by a gazometric method. A two-compartment model appeared to be the simplest kinetic model to explain the shifts in proton concentrations in extra- and intra-cellular media. Non-linear regression was used to determine exchange constant values. There was a very good correlation between the experimental and calculated proton concentrations. This model can describe all patients but individual experimental constants must be determined. Under these conditions a single blood pH determination before dialysis will permit determination of the initial intra-erythrocytic pH and monitoring of intra-erythrocytic pH during hemodialysis.

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.

Adaptation of uraemic patients to anaemia: A31 P nuclear magnetic resonance study.

It is well established that ESRD patients can tolerate a greater degree of anaemia than anaemic subjects of other aetiologies. It is widely accepted that this is partly due to a decrease in the oxygen affinity of haemoglobin (Hb) associated with a greater concentration of 2,3-diphosphoglycerate (2,3-DPG) observed in uraemic patients. In order to verify whether this is a concentration effect or whether the binding of 2,3-DPG in erythrocytes is structurally affected, we have studied 2,3-DPG haemoglobin binding by measuring spin-spin relaxation times (T2) of 31P in living erythrocytes (RBC) by NMR. In uraemic RBC, 2,3-DPG relaxation is faster. This difference is not due to the reduced intracellular pH of uraemic RBC: we have verified that increased presence of paramagnetic substances can also be discarded since intra-RBC proton T2 is longer. Our results are compatible with stronger binding of 2,3-DPG to haemoglobin in uraemic erythrocytes, stabilising the deoxyhaemoglobin form and therefore facilitating oxygen release.

Intraerythrocytic pH variations during hemodialysis: a 31P NMR study.

Before hemodialysis, patients have an intraerythrocytic pH (pHi) and an extracellular pH, measured in whole blood (pHo), which are lower than those of healthy controls. During bicarbonate hemodialysis, pHi values continuously increase, approaching a normal value at the end of the session. Concomitantly, pHo values follow similar variations. During acetate hemodialysis, pHi values exhibit a steep initial decrease, reaching a minimum after about 15 minutes. Concurrently, however, pHo values decrease only slightly. This phenomenon seems to originate in the intraerythrocytic medium and might be due to a shift in intracellular CO2/bicarbonate equilibrium. This drop in pHi exhibits interpatient variability, suggesting that the magnitude of pH decrease would be correlated with the degree of the problems observed in some patients undergoing acetate hemodialysis.

Sequential sodium therapy allows correction of sodium-volume balance and reduces morbidity.

We investigated whether individually adjusting Na+ dialysis levels (Na+Di) combined with Na+ and UFR (ultrafiltration rate) programming, and a sodium/volume model (sequential sodium therapy, SST) can improve the end stage renal failure (ESRD) patient's homeostatic equilibrium intra- and interdialytically. One hundred and fifty patients were included in the study over a one year period. The results show that the patients are divided into two groups: 50 patients respond according to the sodium/volume model developed by F. Gotch [1983]. In this group it is possible to predict pre- and post dialysis plasma Na+ concentration (Na+o, Na+t) as a function of Na+Di and it becomes possible to choose Na+Di to allow Na+o and Na+t to virtually coincide, eliminating severe shifts in plasma tonicity. In the second group two subgroups can be distinguished: excess Na+ or excess H2O post dialysis, without possible correction at a single sodium level. SST corrects sodium/volume balance in this group by using sequential intermittent hypo or hypertonic dialysate, combined with fluid removal adapted to each episode. In both groups there was a significant improvement in the clinical condition of the patients who previously were less equilibrated. It is possible to conclude that SST improves tolerance intradialytically and achieves better equilibrium interdialytically. Implementation of SST requires precise control of the concentrate and the water, and equipment adapted for accurate, programmable sequential control of Na+Di and ultrafiltration rate.

Action on mitochondrial calcium metabolism of an ionophorous compound isolated from uremic plasma or normal urine.

An ionophorous compound that is one of the uremic middle molecules is able to inhibit the mitochondrial storage of calcium. Its active concentration is equivalent to that found in uremic plasma. This result can explain the diminution of phosphate calcium granules observed in mitochondria from uremic children. Moreover, this phenomenon may be involved in the calcium pool decrease observed in chronic renal insufficiency.

Dialysate sodium control during modeling in hemodialysis.

Dialysate sodium (Na+) modeling in hemodialysis requires precise individual adjustment and control of Na+ dialysate concentration. In practice, variations of Na+ concentration can be important and can affect the accuracy of Na+ modeling. Variations relate mainly to the low accuracy of dialysate concentrate (+/- 2.5% for Na+ is tolerated by the European Pharmacopeias), purity of hemodialysis water (2.17 mEq/L is the limit fixed by the French Pharmacopeia), and precision of the proportioning delivery systems for hemodialysis bath preparation. To minimize these difficulties, this study focused on the following points: (a) Na+ concentration is maintained constant (133 mEq/L) in the dialysate manufacturing unit. In 1982, 268 dialysate preparations (177,000 L) were made, and the mean value for Na+ concentration was 133.1 +/- 0.3 mEq/L with a probability of 99.9%. (b) The purity of the water, especially for Na+, Ca2+, and K+, is controlled for two times a day. (c) The accuracy of the proportioning delivery system is controlled by two conductivity monitors, and the variation of Na+ concentration around 133 mEq/L is smaller than +/- 0.5%. As the composition of the basic dialyzing fluid remains constant before adaptation, conductivity values reflect exactly Na+ variation, and are not affected by variations of other elements (K+, Ca2+, Mg2+) that may occur when modification of the dialysate is used for Na+ modeling. (d) Na+ dialysate concentration is adapted for each patient's need by a bedside monitor with sterile Na+ solutions (5 mol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition in vitro of the polymerization of tubulin by uremic middle molecules: corrective effect of isaxonine.

The polymerization of tubulin leads to the formation of microtubules which are one of the components of the axons of nerve cells. This reaction is the limiting factor in the growth of axons. Uremic middle molecules inhibit in vitro the polymerization of tubulin in a dose dependent way. It is possible that a similar phenomenon could occur in vivo in uremic patients, and this might be involved in the development of neuropathy. In addition, isaxonine phosphate counteracts the inhibitory effect of uremic middle molecules on the polymerization of tubulin.