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.

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.

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.