RESUMO
Enzymatic properties of the enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD), which confers mineralocorticoid selectivity, have been explored in the aldosterone-sensitive collecting duct (CCD) and the aldosterone-insensitive Pars Recta (PR) of the rat kidney. After incubation of freshly isolated tubular segments with [3H]corticosterone (3H-B) or [3H]dehydrocorticosterone (3H-A), the rate of transformation of 3H-B into 3H-A (dehydrogenase activity), or the reverse reaction (reductase activity) were measured by HPLC, Vmax for dehydrogenase activity was found to be 8- to 10-fold higher in CCD than PR. The enzyme functions over a very wide range (0.1-5000 nM) of corticosterone concentration. In CCD, enzyme kinetics suggest either the presence of two 11-HSD forms, differing by their affinity for corticosterone, or complex kinetics. Addition of NAD or NADP to permeabilized tubules revealed that dehydrogenase activity is NAD-dependent in CCD and NADP-dependent in PR. Cofactor addition was ineffective in intact tubules. CCD exhibited an exclusive dehydrogenase activity, whereas in PR dehydrogenase and reductase activity were found. No regulation of dehydrogenase activity could be evidenced in adrenalectomized rats receiving or not aldosterone, corticosterone or dexamethasone, for 2 h, 3 days or 4 days. We conclude that 11-HSD in the CCD and PR differs by its Vmax and cofactor dependence. Corticosteroid hormones do not influence 11-HSD activity.
Assuntos
Homeostase , Hidroxiesteroide Desidrogenases/metabolismo , Túbulos Renais Distais/enzimologia , Túbulos Renais Proximais/enzimologia , 11-beta-Hidroxiesteroide Desidrogenases , Adrenalectomia , Aldosterona/farmacologia , Animais , Permeabilidade da Membrana Celular , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Corticosterona/farmacologia , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Cinética , NAD/farmacologia , NADP/farmacologia , Ratos , Ratos Wistar , TrítioRESUMO
N-Acetyl-beta-hexosaminidase, beta-galactosidase and beta-glucuronidase activities were shown to be present in cultured rabbit articular chondrocytes. Secretion of enzyme activity seems to preferentially result in the accumulation of N-acetyl-beta-hexosaminidase. Three days after seeding, the amount of N-acetyl-beta-hexosaminidase activity found in the medium accounts for about 140% of the total N-acetyl-beta-hexosaminidase activity after complete disruption of the cell pellet. Optimal conditions of incubation time, cell numbers, substrate concentration, and pH for glycosidase activities were determined in 0.1% Triton X-100. Intracellular and secreted glycosidases have shown similar elution profiles by chromatofocusing. N-acetyl-beta-hexosaminidase exhibits two major forms which may play a role in the catabolism of glycosaminoglycans.
Assuntos
Cartilagem Articular/enzimologia , Glucosidases/análise , Fosfatase Ácida/metabolismo , Animais , Cartilagem Articular/citologia , Células Cultivadas , Focalização Isoelétrica , Isoenzimas/análise , CoelhosRESUMO
The objective of this study was to evaluate the renal tolerance of a new magnetic resonance contrast agent, AMI 25. This agent has an affinity for the reticuloendothelial system and is used for the detection of focal liver lesions. A combination of renal ischemia and intra-arterial iodinated contrast agent infusion (diatrizoate) leads to a reproducible and reversible model of acute renal failure in the rat. Using this model, AMI 25 was perfused directly into the aorta at the dose of 1 ml/kg--ten times the dose used in humans. AMI 25 induced no change in serum creatinine (45 +/- 7, 40 +/- 6, 40 +/- 9 mumol/L before infusion and at 24 and 48 hours, respectively), in creatinine clearance (2.1 +/- 0.6, 2.1 +/- 0.6, 2.1 +/- 0.6 mL/mn), or in urinary N-acetyl glucosaminidase (NAG) excretion (72 +/- 16, 98 +/- 12, 58 +/- 9.8 mumol hour-1/mmol creatinine). Blinded histologic analysis of 11 kidneys perfused with AMI 25 revealed no abnormalities, whereas diatrizoate induced acute tubular necrosis in four of the seven kidneys examined. In our animal model, AMI 25 has no nephrotoxicity, even at ten times the expected clinical dose for humans.
Assuntos
Meios de Contraste/toxicidade , Ferro/toxicidade , Rim/efeitos dos fármacos , Óxidos/toxicidade , Acetilglucosaminidase/urina , Animais , Creatinina/metabolismo , Dextranos , Diatrizoato/toxicidade , Óxido Ferroso-Férrico , Rim/patologia , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Masculino , Ratos , Ratos EndogâmicosRESUMO
RATIONALE AND OBJECTIVES: Although gadolinium chelates are mainly eliminated by the kidney, there is limited information about their effects. The renal tolerance of these compounds on renal function in an in vivo rat model are evaluated. METHODS: A combination of renal ischemia and intrarenal iodinated contrast agent infusion (diatrizoate) led to a reproducible and reversible model of acute renal failure (n = 5). Using this model, the renal tolerance of gadolinium DOTA (Gd-DOTA) (n = 10) and gadolinium DTPA (Gd-DTPA) (n = 10) were evaluated. The effects of the association of Gd-DOTA with diatrizoate (n = 5) on renal function also were assessed. RESULTS: Gadolinium DOTA induced no change in serum creatinine and creatinine clearance. Gadolinium DTPA induced a significant increase in serum creatinine (50 to 83 +/- 5 and 70 +/- 6 mumol/L) before and at 24 and 48 hours, respectively (P < .05), and a decrease in creatinine clearance from 1.6 +/- 0.1 to 0.8 +/- 0.1; 1.2 +/- 0.1 mL/mL before and at 24 and 48 hours, respectively (P < .05). In this model, Gd-DOTA did not modify the renal tolerance of diatrizoate as assessed with serum creatinine and creatinine clearance. CONCLUSIONS: Gadolinium DOTA is not nephrotoxic and can be infused in association with iodinated contrast media. In this model, Gd-DTPA induced reversible renal failure.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Meios de Contraste/efeitos adversos , Compostos Heterocíclicos/efeitos adversos , Rim/efeitos dos fármacos , Compostos Organometálicos/efeitos adversos , Ácido Pentético/análogos & derivados , Animais , Diatrizoato/efeitos adversos , Gadolínio DTPA , Imageamento por Ressonância Magnética , Masculino , Ácido Pentético/efeitos adversos , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE AND OBJECTIVES: A possible involvement of endothelium derived relaxing nitric oxide (NO) in the pathogenesis of iodinated contrast media (CM)-induced nephrotoxicity was investigated in the rat. METHODS: Male rats (6 to 12 per group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery and a low-osmolar contrast medium (CM), ioxaglate, was injected (1 mL/min; 3 minutes) via an aortic puncture in the single remaining kidney. Contrast medium was injected with or without the NO-synthase inhibitor L-NAME (100 mg/kg intravenously [i.v.] 5 minutes before CM). One group received L-Arginine, the physiological precursor of NO (100 mg/kg i.v.), 5 minutes before L-NAME. Phenylephrine (300 micrograms/kg; 30 min) was used as a vasoconstrictive NO-independent control. The effects of iohexol, another low-osmolar CM, on creatinine clearance (CrCl) were also studied with and without pretreatment with L-NAME. A control group was subjected to a 3-minute renal ischemia only. Creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined before, and 24 and 48 hours after CM administration. Blinded histologic analysis was carried out after completion of the study. RESULTS: When administered alone, neither L-NAME nor L-arginine modified CrCl. Ioxaglate mildly but significantly decreased CrCl at 24 hours (-26.5% of preinjection value). This was similar to the effect observed in the control group subjected to ischemia only. When associated with L-NAME, ioxaglate markedly decreased CrCl (-58 + 11% at 24 hours, P < .05 vs. ioxaglate alone). A similar interaction was noted in the case of iohexol. L-NAME also markedly increased ioxaglate-induced urinary NAG excretion. Phenylephrine had a similar impact on renal function. L-arginine pretreatment reduced the increase in serum creatinine induced by L-NAME+ioxaglate (68 + 17 mumol/L vs. 175 + 59 mumol/L for L-NAME+ioxaglate; P < .05) and urinary NAG excretion. Ioxaglate alone induced only tubular epithelial vacuolization. When associated with L-NAME, this CM induced tubular and vascular lesions, as well as necrosis in the outer medulla. Such histologic effects were clearly inhibited by L-arginine. CONCLUSION: These data indicate that L-NAME, a specific inhibitor of NO-synthase, and phenylephrine, accentuate the nephrotoxicity of CM in the rat. This is consistent with results from the literature showing that CM-toxicity is enhanced by renal ischemia.
Assuntos
Iohexol/toxicidade , Ácido Ioxáglico/toxicidade , Rim/efeitos dos fármacos , Óxido Nítrico/farmacologia , Acetilglucosaminidase/urina , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Creatinina/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico/antagonistas & inibidores , Fenilefrina/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE AND OBJECTIVES: To compare the histologic effects on rat tubular cells of two nonionic contrast media with equivalent osmolalities and viscosities. METHODS: Histologic, functional (creatinine clearance), and biochemical (proteinuria and enzymuria) profiles of iohexol and iobitridol (both at 350 mg I/mL) were compared in the uninephrectomized rat. A control group (n = 14) received compared isotonic saline solution. Test substances (3 mL) were injected into the kidney at a rate of 1 mL/minute while transitory ischemia was induced by clamping the aorta above the renal artery. RESULTS: In terms of their (moderate) effects on creatinine clearance, proteinuria, and urinary N-acetyl-beta-D-glucosaminidase activity, no statistically significant difference was detected between the two low-osmolar contrast agents either 24 or 48 hours after injection. However, blinded histologic analysis of the kidneys showed significantly greater epithelial cell vacuolization in the proximal convoluted tubules of the outer cortex with iohexol (14 of 14 rats versus 3 of 14 rats for iobitridol; P < .001). The same degree of vacuolization in the inner cortex was observed for all three substances. Iobitridol also induced fewer congestive lesions in the glomerular capillaries than iohexol (4 of 14 versus 10 of 14, respectively; P < .05) and saline (5 of 6; P < .05). It is difficult to explain the lesser degree of cytoplasmic vacuolization using standard physicochemical parameters. CONCLUSION: Although iobitridol and iohexol showed similar functional and biochemical profiles when selectively injected into the single remaining kidney of rats, iobitridol induced significantly less tubular vacuolization and capillary congestion than iohexol.
Assuntos
Meios de Contraste/farmacologia , Iohexol/farmacologia , Rim/efeitos dos fármacos , Acetilglucosaminidase/metabolismo , Animais , Creatinina/metabolismo , Rim/patologia , Masculino , Proteinúria/urina , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE AND OBJECTIVES: To summarize the chemical synthesis, physicochemical characterization, pharmacokinetic behavior, and biological evaluation of P743, a new macromolecular iodinated contrast medium. METHODS: The synthesis and molecular modeling of the iodinated macromolecule P743 are described. The pharmacokinetic profile was established in rabbits and rats. Acute toxicity in mice, renal tolerance in normal rabbits, and renal tolerance in uninephrectomized, dehydrated rats undergoing selective intrarenal injection was evaluated. In vitro permeability effects on isolated mastocytes and on the coagulation pathways were carried out. Computed tomography vascular imaging was performed after intravenous injection of P743 (300 mg I/kg) in rabbits and compared with the nonspecific nonionic agent iobitridol. RESULTS: P743 is a monodisperse, macromolecular iodinated contrast medium. In both rabbits and rats, P743 showed a pharmacokinetic profile consistent with that of a rapid-clearance blood-pool agent. Its diffusion through the endothelium was found to be low in vitro, thus confirming early confinement of this macromolecule, unlike nonspecific contrast media. In both species, P743 was excreted by glomerular filtration. Acute toxicity disclosed no mortality at the highest volume that could be injected into mice, leading to a median lethal dose greater than 8.9 g I/kg. Renal tolerance was found to be good in both euvolemic rabbits and uninephrectomized, dehydrated rats. No histamine or leukotriene B4 release was found on RBL-2H3 isolated mastocytes. P743 did not interfere with the coagulation pathways. Imaging experiments confirmed that P743 remains in the vascular compartment for a longer time than does iobitridol, thus allowing vascular enhancement that is twice as high as that of iobitridol in the recirculation phase. CONCLUSIONS: The pharmacokinetic and imaging profiles of P743, a new, monodisperse, macromolecular blood-pool iodinated contrast medium, were consistent with those of a rapid-clearance blood-pool agent. Its initial safety profile is satisfactory. Further experimental imaging studies are required to define the clinical interest in such molecules.
Assuntos
Meios de Contraste/análise , Meios de Contraste/farmacologia , Animais , Meios de Contraste/síntese química , Compostos de Iodo , Compostos Orgânicos , Coelhos , RatosRESUMO
The renal hemodynamic and tubular effects of S10036 (fotemustine) were evaluated in seven patients with advanced malignancy. Initial evaluation carried out prior to treatment and repeated 1 day after the first fotemustine infusion and 7 days after the second included clinical, haematological parameters, liver-function tests, and determination of the glomerular filtration rate, renal blood flow and enzymuria. The glomerular filtration rate was 108 +/- 3.7 ml/min before treatment and remained stable after the first (117 +/- 5 ml/min) and second (124 +/- 6 ml/min) fotemustine infusions. Renal blood flow and urinary beta 2-microglobulin and N'-acetylglucosaminidase excretion were also not modified by fotemustine administration. We conclude that fotemustine does not acutely alter renal haemodynamics, nor does it have direct tubular toxicity.
Assuntos
Antineoplásicos/uso terapêutico , Rim/efeitos dos fármacos , Compostos de Nitrosoureia/uso terapêutico , Compostos Organofosforados/uso terapêutico , Acetilglucosaminidase/urina , Antineoplásicos/efeitos adversos , Taxa de Filtração Glomerular/efeitos dos fármacos , Hematócrito , Hemodinâmica/efeitos dos fármacos , Humanos , Rim/fisiopatologia , Túbulos Renais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/fisiopatologia , Compostos de Nitrosoureia/efeitos adversos , Compostos Organofosforados/efeitos adversos , Circulação Renal/efeitos dos fármacos , Microglobulina beta-2/urinaRESUMO
A urinary fraction which inhibits the activity of N-acetyl-beta-D-glucosaminidase (NAG) has been isolated and identified as being urea. Usually present in high concentration, urea appears to be the only urinary component responsible for the frequently observed urinary NAG inhibition. The inhibition of the two urinary NAG isoenzymes A and B is competitive with respective Ki values of about 70 mmol/l and 60 mmol/l. With routine assay conditions, it seems that a dilution of urine prior to enzyme assay is sufficient to abolish the inhibition of the two isoenzymes A and B by endogenous urea.
Assuntos
Acetilglucosaminidase/antagonistas & inibidores , Hexosaminidases/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Ureia/farmacologia , Acetilglucosaminidase/urina , Ligação Competitiva , Humanos , Técnicas In Vitro , Isoenzimas/urina , Ureia/urinaRESUMO
Secretion of N-acetyl-beta-D-glucosaminidase (NAG) isoenzymes by human blood monocyte-derived macrophages in response to zymosan and human recombinant interferon-gamma was studied. Macrophages were found to release NAG in response to zymosan, but interferon-gamma has no effect on secretion. Isoenzyme separation by isoelectric focusing demonstrates that non stimulated and zymosan or interferon-gamma treated macrophages release predominantly NAG B and, to a lesser extent, NAG A isoenzymes. In all these conditions, the intracellular intermediate form NAG I could not be detected in the media. Thus, activated macrophages may not be the source of NAG intermediate forms I and P in pathological or maternal serum. In contrast, macrophages could contribute to a significant elevation of urinary activity and NAG B excretion in response to inflammatory conditions.
Assuntos
Acetilglucosaminidase/metabolismo , Interferon gama/farmacologia , Isoenzimas/metabolismo , Macrófagos/enzimologia , Zimosan/farmacologia , Acetilglucosaminidase/isolamento & purificação , Humanos , Focalização Isoelétrica , Isoenzimas/isolamento & purificação , Macrófagos/efeitos dos fármacos , Proteínas RecombinantesRESUMO
Monitoring of variations in N-acetyl-beta-D-glucosaminidase (NAG) urinary activity, following renal transplantation, has been proposed for the early diagnosis of rejection episodes. In this study, the measurement of urinary NAG-B activity was conducted as a complement to total NAG (A + B) measurement, which is normally used alone. Selective measurement of NAG-B activity is carried out after fixation of NAG-A on ion exchanger in test tubes. Results of NAG (A + B) activity confirm that the assay of urinary NAG is a useful indicator of rejection, but a positive correlation between NAG-B and NAG (A + B) activities was observed during the various complications which can occur after transplantation. The specific measurement of this isoenzyme does not, therefore, seem to provide additional information in the early monitoring of human renal transplantations. Apart from rejection episodes, other factors are likely to produce marked NAG-B excretion, e.g. gentamicin therapy.
Assuntos
Acetilglucosaminidase/urina , Rejeição de Enxerto , Hexosaminidases/urina , Isoenzimas/urina , Transplante de Rim , Gentamicinas/uso terapêutico , Humanos , Falência Renal Crônica/terapia , Período Pós-Operatório , Diálise RenalRESUMO
N-Acetyl-beta-D-glucosaminidase (NAG) isoenzyme profile in primary cultures of rabbit kidney proximal tubule cells was studied. Confluent cells had high levels of NAG activity, but ion exchange chromatography showed that the NAG isoenzyme profile in cultured cells was different from that of rabbit renal cortex homogenates and freshly isolated cells. Confluent cultured cells contained an atypical acidic isoform, absent in homogenates and freshly isolated cells in which the predominant isoform is NAG-A (a heterodimer alpha beta). The fact that this atypical isoform was able to hydrolyse the synthetic substrate 4-methylumbelliferyl-beta-N-acetylglucosaminide-6-sulphate indicated that it probably was an alpha-subunit homodimer. These results suggest subunit rearrangement within NAG polypeptide chains linked to down-regulation of beta-subunit production in cultured rabbit proximal cells. The change in isoenzyme profile in cultured cells may make it difficult to use primary cultures of rabbit proximal tubule cells to establish correlations between in vitro and in vivo studies using NAG isoenzymes as a nephrotoxicity index, as illustrated by the effects of gentamicin.
Assuntos
Acetilglucosaminidase/metabolismo , Isoenzimas/metabolismo , Túbulos Renais Proximais/enzimologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/toxicidade , Células Cultivadas , Cromatografia por Troca Iônica , Regulação para Baixo , Gentamicinas/administração & dosagem , Gentamicinas/toxicidade , Hidrólise , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Inibidores da Síntese de Proteínas/administração & dosagem , Inibidores da Síntese de Proteínas/toxicidade , CoelhosAssuntos
Glucosidases/isolamento & purificação , Plantas/enzimologia , Sulfato de Amônio , Catálise , Cromatografia , Cromatografia por Troca Iônica , Diálise , Grão Comestível/enzimologia , Grão Comestível/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Glucosidases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Métodos , Peso Molecular , Relação Estrutura-AtividadeRESUMO
The urinary excretion of N-acetyl-beta-D-glucosaminidase isoenzymes A and B following kidney transplantation was studied in rats. High enzymuria with permanent marked isoenzyme B excretion occurred from the immediate post-operative period to the irreversible rejection episode. Isoenzyme B could represent as much as 10-40% of total N-acetyl-beta-D-glucosaminidase activity and it reflected the intensity of tubular lesions as observed by histological examination of allograft specimens. Thus, N-acetyl-beta-D-glucosaminidase B isoenzyme determination may reinforce the diagnostic value of total (A + B) urinary N-acetyl-beta-D-glucosaminidase activity determination during the various complications which can occur after transplantation.
Assuntos
Acetilglucosaminidase/urina , Hexosaminidases/urina , Isoenzimas/urina , Transplante de Rim , Animais , Cromatografia DEAE-Celulose , Ratos , Ratos EndogâmicosRESUMO
This work describes the purification of a beta-glucosidase (beta-D-glucoside-glucohydrolase EC 3.2.1.21) from the digestive juice of Helix pomatia and the study of the enzyme's active site by using different reversible and irreversible inhibitors. The catalytic constants of arylglycosides and their pH-dependent variations have also been determined. The inhibition studies demonstrate that conduritol epoxides are irreversible inhibitors of beta-glucosidase from the digestive juice of H. pomatia, and that nojirimicin shows tight binding with glucosidase: the formation and dissociation of the enzyme-inhibitor complex (dissociation constant 1.1 mumol/1) required several minutes.
Assuntos
Glucosamina/farmacologia , Glucosidases/metabolismo , Caracois Helix/metabolismo , Inositol/análogos & derivados , beta-Glucosidase/metabolismo , 1-Desoxinojirimicina , Animais , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Concentração de Íons de Hidrogênio , Inositol/farmacologia , Cinética , Especificidade por Substrato , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/isolamento & purificaçãoRESUMO
An alpha-L-rhamnosidase from the seeds of Fagopyrum esculentum (saracen corn) has previously been identified, and the effect of the enzyme on rhamnoisic bonds has been studied with various flavonoid glycosides. This alpha-L-rhamnosidase can be useful in structural studies, and a preliminary report of this study has appeared. The present paper describes the extensive purification of the enzyme and the determination of its properties. The purification involved extraction, ammonium sulfate fractionation and chromatography on Sephadex G 75, DEAE-Sephadex and Ultrogel AcA-44. The alpha-L-rhamnosidase was purified about 9600 fold and the final enzyme preparation was practically pure according to the criteria of disc electrophoresis. The molecular weight of this alpha-L-rhamnosidase, calculated from data obtained by disc gel electrophoresis and gel filtration, was about 70 000. Isoelectric focusing established the isoelectric point to be 3.7. The behaviour of the enzyme on a concanavalin-A-Sepharose column suggests the presence of residues resembling alpha-D-mannose or alpha-D-glucose in the protein. The various kinetic parameters, Kcat, Km and the Kcat/Km ratio have been determined at pH 5 on the following substrates: p-nitrophenyl-alpha-L-rhamnoside and rutinose (6-O-alpha-L-rhamnosyl-D-glucopyranose). All kinetics exhibit a Michaelian behaviour and the Km for the former substrate was 0.33 mM and for the latter, 2.2 mM. The Kcat/Km ratio corroborates the greater specificity of the enzyme for p-nitrophenyl-alpha-L-rhamnoside. L-Rhamnose, L-lyxose, 6-deoxy-D-glucose and methyl-alpha-D-mannoside were shown to behave strictly as competitive inhibitors of alpha-L-rhamnosidase activity; it seems that the methyl group of L-rhamnose is important for substrate binding to the enzyme.