RESUMO
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.
Assuntos
Inibidores Enzimáticos/análise , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Uremia/metabolismo , Trifosfato de Adenosina/metabolismo , Hemofiltração , Humanos , Cinética , Ouabaína , Potássio/metabolismo , Valores de ReferênciaRESUMO
Adenosine is involved in a large number of physiological processes including immune response and vasomotor function. But its precise involvement in renal physiology is poorly understood. We have investigated the putative relationships between cyclosporine A (CsA) and adenosine (ADO) metabolism in kidney transplant recipients (KTR). We first compared ADO plasma levels in three groups of patients and in 10 controls: the first group (N = 14) was composed of CsA-treated KTR; the second group (N = 5) was KTR not treated with CsA, and the third (N = 6) was chronic kidney failure patients. We also measured ADO plasma level in two KTR treated with FK506, a CsA analog. ADO plasma levels in CsA-treated KTR were significantly higher (mean 0.76 microM +/- 0.27) than in the control group (mean 0.31 +/- 0.13; Mean-Whitney test, S = 8.5; P = 2.1 x 10(-4)) and than in the chronic kidney failure group (0.37 +/- 0.16, Mann-Whitney, S = 5.5; P = 1.6 x 10(-3)). In CsA-treated KTR, CsA and ADO plasma levels were significantly correlated (Spearman's, r = 0.8, P = 1.9 x 10(-3)). No significant differences in ADO plasma levels were found between patients with chronic kidney failure and controls (P < 0.05). ADO plasma levels in KTR not treated with CsA were in the same range as those in controls. Finally, the ADO plasma level was increased in the two FK506-treated patients. We also investigated the action of CsA on ADO plasma degradation and uptake by erythrocytes in vitro. No interaction between adenosine deaminase and CsA was found because CsA, in the presence of adenosine deaminase, did not modify the plasma half-life of ADO. Conversely, in the presence of CsA (500 and 1000 ng/ml), the uptake of ADO by erythrocytes was significantly decreased in adenosine deaminase-free samples (analysis of variance, P = 1.8.10(-3) and 1.2 x 10(-4), respectively). We conclude that ADO plasma levels are significantly elevated and correlate with CsA blood level in CsA-treated KTR, and that these high levels are due to CsA inhibition of ADO uptake by red cells. Since ADO and metabolites have well known immunosuppressive and vascular effects, ADO is likely to participate in the immune defect and in the vasoconstriction induced by CsA.
Assuntos
Adenosina/sangue , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Transplante de Rim , Adolescente , Adulto , Feminino , Humanos , Rim/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Vasoconstrição/efeitos dos fármacosRESUMO
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.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Celulose/análogos & derivados , Mitocôndrias Hepáticas/efeitos dos fármacos , Diálise Renal/efeitos adversos , Animais , Cálcio/metabolismo , Celulose/toxicidade , Masculino , Mitocôndrias Hepáticas/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
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.
Assuntos
Materiais Biocompatíveis/farmacologia , Membranas Artificiais , Mitocôndrias Hepáticas/efeitos dos fármacos , Diálise Renal/instrumentação , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Animais , Masculino , Mitocôndrias Hepáticas/metabolismo , Ratos , Ratos Wistar , Soluções/farmacologiaRESUMO
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.
Assuntos
Anemia/sangue , Hemoglobinas/metabolismo , Falência Renal Crônica/sangue , Oxigênio/metabolismo , Ureia/farmacologia , 2,3-Difosfoglicerato , Ácidos Difosfoglicéricos/metabolismo , HumanosRESUMO
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.
Assuntos
Trifosfato de Adenosina/biossíntese , Bloqueadores dos Canais de Cálcio/farmacologia , Ciclosporina/toxicidade , Animais , Cálcio/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
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.
Assuntos
Ácido Ascórbico/análogos & derivados , Toxinas Biológicas/química , Uremia/sangue , Ácido Ascórbico/metabolismo , Cromatografia por Troca Iônica , Humanos , Espectroscopia de Ressonância Magnética , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Toxinas Biológicas/isolamento & purificação , Humanos , Toxinas Biológicas/sangueRESUMO
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.
Assuntos
Eritrócitos/metabolismo , Sódio/sangue , Uremia/metabolismo , Cátions , Membrana Eritrocítica/metabolismo , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Ouabaína/farmacologia , Fosfatos/sangue , Potássio/sangue , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
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.
Assuntos
Ácido Ascórbico/análogos & derivados , Uremia/metabolismo , Ácido Ascórbico/análise , Cromatografia Líquida de Alta Pressão , Transferência de Energia , Humanos , Espectroscopia de Ressonância Magnética , Microtúbulos/metabolismo , Estrutura Molecular , Espectrofotometria Ultravioleta , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/fisiologiaRESUMO
Undecalcified sections of doubly tetracycline-labeled transiliac bone biopsy specimens obtained from ten hemodialyzed patients before and 10 to 16 months after parathyroidectomy (PTX) were analyzed. Before parathyroidectomy (total PTX with autotransplant in six patients and subtotal PTX in four patients), all the patients demonstrated histological evidence of hyperparathyroidism with increased resorption parameters. A high bone formation rate (BFR) was noted in all patients but one who had both an increase in the osteoid seam thickness and a low calcification rate characteristic of osteomalacia. A significant correlation was found between immunoreactive parathyroid hormone (iPTH) levels and BFR at the tissue and at the basic multicellular unit (BMU) levels. Parathyroidectomy was associated with a dramatic drop in resorption surfaces and osteoclast number as well as in bone formation rate at the tissue, BMU, and cell-levels. After PTX, the bone formation rate at the tissue level was low or in the lower range of normal values in six patients. The thickness index of osteoid seams was significantly reduced and no evidence of osteomalacia was present even in the six patients showing bone aluminum deposits after PTX. One of the three patients, who had an iPTH level within the normal range after PTX, showed an osteoid excess associated with a low bone formation rate. These date demonstrate that increased PTH secretion is an important factor of bone formation in dialyzed patients and that excessive reduction of the PTH secretion leads to an inactive bone.