RESUMEN
Static preservation is currently the most widely used organ preservation strategy; however, decreased donor organ quality is impacting outcome negatively. M101 is an O2 carrier with high-oxygen affinity and the capacity to function at low temperatures. We tested the benefits of M101 both in vitro, on cold preserved LLC-PK1, as well as in vivo, in a large white pig kidney autotransplantation model. In vitro, M101 supplementation reduced cold storage-induced cell death. In vivo, early follow-up demonstrated superiority of M101-supplemented solutions, lowering the peak of serum creatinine and increasing the speed of function recovery. On the longer term, supplementation with M101 reduced kidney inflammation levels and maintained structural integrity, particularly with University of Wisconsin (UW). At the end of the 3-month follow-up, M101 supplementation proved beneficial in terms of survival and function, as well as slowing the advance of interstitial fibrosis. We show that addition of M101 to classic organ preservation protocols with UW and Histidine-Tryptophane-Ketoglutarate, the two most widely used solutions worldwide in kidney preservation, provides significant benefits to grafts, both on early function recovery and outcome. Simple supplementation of the solution with M101 is easily translatable to the clinic and shows promises in terms of outcome.
Asunto(s)
Fibrosis/prevención & control , Riñón/fisiopatología , Modelos Animales , Soluciones Preservantes de Órganos , Preservación de Órganos/métodos , Oxígeno/administración & dosificación , Animales , Microscopía Electrónica de Transmisión , PorcinosRESUMEN
Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.
Asunto(s)
Membrana Celular/enzimología , Serina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , 2,4-Dinitrofenol/farmacología , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Transporte Biológico/efectos de los fármacos , Colchicina/farmacología , Activación Enzimática , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Fosforilación/efectos de los fármacos , Cianuro de Potasio/farmacología , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Roedores , Rubidio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.
Asunto(s)
Forbol 12,13-Dibutirato/metabolismo , Proteína Quinasa C/metabolismo , Serina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Ácido Araquidónico/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Mutagénesis , Ouabaína/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación , ATPasa Intercambiadora de Sodio-Potasio/genética , Temperatura , TransfecciónRESUMEN
The kidney medulla is exposed to very high interstitial osmolarity leading to the activation of mitogen-activated protein kinases (MAPK). However, the respective roles of increased intracellular osmolality and of cell shrinkage in MAPK activation are not known. Similarly, the participation of MAPK in the regulatory volume increase (RVI) following cell shrinkage remains to be investigated. In the rat medullary thick ascending limb of Henle (MTAL), extracellular hypertonicity produced by addition of NaCl or sucrose increased the phosphorylation level of extracellular signal-regulated kinase (ERK) and p38 kinase and to a lesser extent c-Jun NH(2)-terminal kinase with sucrose only. Both hypertonic solutions decreased the MTAL cellular volume in a dose- and time-dependent manner. In contrast, hypertonic urea had no effect. The extent of MAPK activation was correlated with the extent of MTAL cellular volume decrease. Increasing intracellular osmolality without modifying cellular volume did not activate MAPK, whereas cell shrinkage without variation in osmolality activated both ERK and p38. In the presence of 600 mosmol/liter NaCl, the maximal cell shrinkage was observed after 10 min at 37 degrees C and the MTAL cellular volume was reduced to 70% of its initial value. Then, RVI occurred and the cellular volume progressively recovered to reach about 90% of its initial value after 30 min. SB203580, a specific inhibitor of p38, almost completely inhibited the cellular volume recovery, whereas inhibition of ERK did not alter RVI. In conclusion, in rat MTAL: 1) cell shrinkage, but not intracellular hyperosmolality, triggers the activation of both ERK and p38 kinase in response to extracellular hypertonicity; and 2) RVI is dependent on p38 kinase activation.
Asunto(s)
Médula Renal/enzimología , Asa de la Nefrona/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Tamaño de la Célula/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Soluciones Hipertónicas/farmacología , Técnicas In Vitro , Soluciones Isotónicas/farmacología , Médula Renal/citología , Médula Renal/efectos de los fármacos , Asa de la Nefrona/citología , Asa de la Nefrona/efectos de los fármacos , Masculino , Concentración Osmolar , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología , Urea/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.
Asunto(s)
Insulina/farmacología , Túbulos Renales Proximales/enzimología , Fosfotirosina/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sustitución de Aminoácidos , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Genisteína/farmacología , Antagonistas de Insulina/farmacología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Zarigüeyas , Ouabaína/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , Transfección , Tirosina/genética , Tirosina/metabolismo , Vanadatos/farmacologíaRESUMEN
1. The aim of this study was to investigate the mechanism of control of Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10-3 M dibutyryl-cAMP (db-cAMP) or 8-bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10-5 M forskolin) on Na+,K+-ATPase activity and membrane trafficking. 2. PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+-ATPase by about 40%. Transport activity stimulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive 86Rb+ uptake in a time- and dose-dependent fashion; (2) this effect was abolished by addition of H-89 or Rp-cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activity of Na+,K+-ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP-PKA pathway was implicated in membrane redistribution control. 3. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase activity by db-cAMP was associated with an increase (+40%) in Na+, K+-ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30%) in pump units located in the subcellular fraction corresponding to early endosomes. 4. In conclusion, PKA stimulates Na+,K+-ATPase activity, at least in part, by increasing the number of Na+-K+ pumps in the plasma membrane in proximal convoluted tubule cells.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endosomas/enzimología , Túbulos Renales Proximales/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Biotinilación , Bucladesina/farmacología , Fraccionamiento Celular/métodos , Membrana Celular/enzimología , Endosomas/ultraestructura , Técnicas In Vitro , Masculino , Nefronas/enzimología , Ouabaína/farmacología , Ratas , Ratas Wistar , Rubidio/metabolismoRESUMEN
1. In the rat kidney proximal convoluted tubule, epidermal growth factor and insulin have been reported to stimulate Na+ reabsorption. Because most of the effects of these growth factors are mediated by a process of tyrosine phosphorylation and Na+,K(+)-ATPase drives Na+ reabsorption, the influence of tyrosine kinases and tyrosine phosphatases on Na+,K(+)-ATPase activity located in the proximal convoluted tubule was evaluated. 2. Activation of receptor tyrosine kinases by epidermal growth factor and insulin stimulated ouabain-sensitive 86Rb+ uptake. The effects of epidermal growth factor and insulin were prevented by genistein, a tyrosine kinase inhibitor, but were unaffected by GF109203X, a protein kinase C inhibitor. 3. Inhibition of tyrosine phosphatases by orthovanadate (10(-7) and 10(-6)M) mimicked the effects of activation of receptor tyrosine kinases: stimulation of the ouabain-sensitive 86Rb+ uptake and of the hydrolytic activity of Na+,K(+)-ATPase under rate-limiting Na+ concentration, and absence of modification of the maximal activity (Vmax) of the enzyme. The effects of orthovanadate and insulin on the ouabain-sensitive 86Rb+ uptake were not additive. 4. The present results show that both activation of receptor tyrosine kinases and inhibition of tyrosine phosphatases stimulate the Na+,K(+)-ATPase activity through a common mechanism. Thus, a tyrosine phosphorylation process directly controls the Na+,K(+)-ATPase activity and contributes to the physiological control of water and solute reabsorption in the proximal convoluted tubule.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Nefronas/metabolismo , Proteínas Tirosina Fosfatasas/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Masculino , Nefronas/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas WistarRESUMEN
We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Corteza Renal/metabolismo , Rubidio/metabolismo , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Bucladesina/farmacología , Colforsina/farmacología , Técnicas In Vitro , Transporte Iónico , Corteza Renal/enzimología , Túbulos Renales/enzimología , Túbulos Renales/metabolismo , Masculino , Ouabaína/farmacología , Fosforilación , Ratas , Ratas WistarRESUMEN
1. The collecting duct is involved in the whole antinatriuretic effect of insulin, as indicated in vitro by the stimulatory effect of the hormone on ouabain-sensitive 86Rb+ uptake. Since Na+,K(+)-ATPase drives Na+ reabsorption, the contribution of the Na+ pump to the effect of insulin was investigated in rat isolated cortical and outer medullary collecting duct. 2. Insulin enhanced ouabain-sensitive 86Rb+ uptake in the absence, as well as in the presence, of either 5 x 10(-4) M amiloride or 10(-3) M hydrochlorothiazide (HCT). Maximal ouabain-sensitive 86Rb+ uptake, measured in Na(+)-loaded tubules, was also enhanced by insulin. The insulin effect persisted both in the absence of external Na+, when the Na+,K(+)-ATPase operates in a Rb(+)-Rb+ exchange mode, and in tubules depolarized by a high external concentration (20 mM) of Rb+ or by addition of 3 mM Ba2+. 3. Insulin treatment did not alter the intracellular Na and K concentrations, the specific binding of [3H]ouabain measured in intact tubules, or the hydrolytic activity of Na+,K(+)-ATPase measured after permeabilization of the tubule cells. 4. In conclusion, in the rat collecting duct, insulin increased Na+,K(+)-ATPase-mediated cation transport independently of Na+ availability, membrane potential and recruitment of pump units. The effect of insulin was lost after cell permeabilization, suggesting the presence of a cytosolic factor which controls the turnover of Na+,K(+)-ATPase.
Asunto(s)
Insulina/farmacología , Túbulos Renales Colectores/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/fisiología , Hidrólisis , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Nefronas/efectos de los fármacos , Nefronas/enzimología , Ouabaína/metabolismo , Ouabaína/farmacología , Potasio/metabolismo , Ratas , Ratas Wistar , Radioisótopos de Rubidio/metabolismo , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , TritioRESUMEN
Previous studies have demonstrated the presence of two populations of Na,K-ATPase with distinct kinetic, pharmacological and immunological characteristics along the rabbit nephron, indicating that the proximal segments of the nephron express exclusively the alpha 1 isoform of the catalytic subunit, whereas the collecting duct expresses an alpha 3-like isoform. Because pharmacological studies have shown the existence of two populations of Na,K-ATPase with different sensitivities to ouabain in the rat cortical collecting duct, which may result from the presence in the same nephron segment of the two isoforms demonstrated in the different segments of the rabbit nephron, the present study was undertaken to characterize the properties of Na,K-ATPase along the rat nephron. Results indicate that each segment of the rat nephron contains two subpopulations of Na,K-ATPase: a component highly sensitive to ouabain (IC50 approximately 5.10(-6) M) which is recognized by an anti-alpha 3 antibody and another moiety of lower affinity for ouabain (IC50 approximately 5.10(-4) M) which is recognized by an anti-alpha 1 antibody. Whether these two subpopulations correspond to different isoforms of the alpha subunit of Na,K-ATPase (alpha 1 and alpha 3-like) remains to be determined.
Asunto(s)
Isoenzimas/análisis , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Túbulos Renales/enzimología , Túbulos Renales Colectores/enzimología , Túbulos Renales Proximales/enzimología , Masculino , Datos de Secuencia Molecular , Nefronas/enzimología , Ouabaína/farmacología , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
In rat proximal convoluted tubule (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) was previously reported to inhibit Na(+)-K(+)-ATPase, a paradoxical finding in view of the known stimulatory effect of PKC on Na+ reabsorption. Because this inhibition occurs via phospholipase A2 activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on PKC action on Na(+)-K(+)-ATPase. Results confirmed that PDBu inhibited PCT Na(+)-K(+)-ATPase activity under usual conditions. In contrast, when oxygen supply was increased, PDBu had no effect on Na(+)-K(+)-ATPase hydrolytic activity, but it dose-dependently stimulated ouabain-sensitive 86Rb+ uptake. This latter effect, which was abolished by PKC inhibitors, resulted from an increment of the Na+ sensitivity of Na(+)-K(+)-ATPase. Thus, in oxygenated rat PCTs, activation of PKC primarily stimulated Na(+)-K(+)-ATPase. This likely contributes to increase solute reabsorption. Inhibition of Na(+)-K(+)-ATPase was observed only under hypoxic conditions. It may represent an adaptation to protect PCTs against deleterious effects of hypoxia.
Asunto(s)
Túbulos Renales Proximales/enzimología , Proteína Quinasa C/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Activación Enzimática , Masculino , Ouabaína/farmacología , Oxígeno/farmacología , Forbol 12,13-Dibutirato/farmacología , Ratas , Ratas Wistar , Rubidio/farmacocinética , Sodio/metabolismoRESUMEN
1. Hyperinsulinaemia is considered to be a pathogenic factor for human and experimental hypertension. Thus, the contribution of the known insulin-stimulated tubular sodium reabsorption to this aetiological process has to be discussed. 2. Rats fed a fructose-enriched diet develop hyperinsulinaemia and hypertension, providing a model for studying the regulation of the tubular sodium handling and its possible relationship to hypertension. For this purpose, the sodium transport capacity of isolated nephron segments from control rats and from rats fed a fructose-enriched diet was investigated by measurement of ouabain-sensitive 86Rb uptake and of the hydrolytic activity of Na,K-ATPase. The number and affinity of insulin receptors were estimated from the specific [125I]insulin binding. 3. In rats fed a fructose-enriched diet, mild hypertension developed during the 14-day fructose diet. There were no differences, along the nephron, in basal 86Rb uptakes and ATPase activities between control rats and fructose-induced hypertensive rats. In control rats, insulin stimulated 86Rb uptake in the proximal convoluted tubule and cortical collecting duct, but exhibited an inhibitory action in the medullary thick ascending limb. In contrast, in fructose-induced hypertensive rats, 86Rb influx remained unresponsive to insulin concentrations ranging from 10(-11) to 10(-7) mol/l in the proximal convoluted tubule and cortical collecting duct. In the medullary thick ascending limb, the threshold of inhibition was displaced from 10(-11) mol/l up to 10(-7) mol/l. Insulin binding to the proximal convoluted tubule, medullary thick ascending limb and collecting duct were similar in control rats and in rats fed a fructose-enriched diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Hipertensión/metabolismo , Insulina/metabolismo , Túbulos Renales/metabolismo , Sodio/metabolismo , Animales , Fructosa , Hipertensión/inducido químicamente , Masculino , Unión Proteica , Ratas , Ratas Wistar , Receptor de Insulina/metabolismo , Rubidio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Insulin has been shown to stimulate the rate of ouabain-sensitive 86Rb influx in the isolated rat proximal convoluted tubule (PCT). To study the mechanism of this activation of Na-K-adenosinetriphosphatase (Na-K-ATPase), we determined the actions of insulin on 1) the maximal activity (Vmax) of Na-K-ATPase hydrolytic activity; 2) the maximal rate of ouabain-sensitive 86Rb influx (after intracellular Na loading); 3) the rate of ouabain-sensitive 86Rb influx under conditions where intracellular Na concentration is rate limiting, either in the presence or in the absence of 5 x 10(-4) M amiloride and/or low extracellular Na concentration (3 mM); and 4) the Na sensitivity of the Na-K-ATPase hydrolytic activity. The maximal rates of Na-K-ATPase hydrolytic activity and of ouabain-sensitive 86Rb uptake were unchanged by insulin. In contrast, we confirmed that insulin enhanced 86Rb uptake (in peq.mm-1.min-1) in the absence of inhibitor of the Na/H exchanger [18.2 +/- 1.7 to 24.1 +/- 1.3 (SE), P < 0.03] and, in addition, demonstrated a similar stimulation in the presence of either 5 x 10(-4) M amiloride (7.2 +/- 0.6 to 10.7 +/- 0.9, P < 0.01), 3 mM extracellular Na (4.1 +/- 0.4 to 5.6 +/- 0.2, P < 0.05), and both amiloride and 3 mM extracellular Na (2.1 +/- 0.7 to 4.5 +/- 0.4, P < 0.03). Finally, insulin increased the sensitivity of Na-K-ATPase to Na as the apparent dissociation constant decreased from 46.5 +/- 5.3 to 27.6 +/- 3.0 mM (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Insulina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/farmacología , Amilorida/farmacología , Animales , Espacio Extracelular/metabolismo , Técnicas In Vitro , Túbulos Renales Proximales/metabolismo , Cinética , Masculino , Modelos Biológicos , Concentración Osmolar , Ouabaína/farmacología , Permeabilidad , Ratas , Ratas Wistar , Rubidio/farmacocinética , Sodio/metabolismoRESUMEN
The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.
Asunto(s)
Corteza Renal/enzimología , Túbulos Renales Colectores/enzimología , Síndrome Nefrótico/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Hidrólisis , Masculino , Ouabaína/metabolismo , Ouabaína/farmacología , Unión Proteica , Ratas , Ratas Wistar , Radioisótopos de Rubidio/metabolismoRESUMEN
Infertile eggs, dead embryos and tissues from laying geese (airsacs, peritoneum, oviduct, ovary, ova) were examined for presence of myco-plasmas. Forty-three of 110 eggs and the birds laying mycoplasma-containing eggs proved to be positive for mycoplasmas. One of the strains was used for experimental infection of laying geese. A reduction in egg production, an increased number of infertile eggs, egg transmission of mycoplasmas and loss of body weight of hatched goslings, were observed due to the mycoplasma infection.