RESUMEN
The mechanisms of proton secretion by the proximal brush-border membrane (BBM) were compared in carnivorous (dog), omnivorous (human, pig, rat), and herbivorous (rabbit, hamster) species. The activity of the proton pump (V-type bafilomycin-sensitive H(+)-adenosinetriphosphatase) and of the Na+/H+ exchanger (amiloride-sensitive quenching of acridine orange fluorescence), the two major proton secretion mechanisms, was measured. The enzymatic activity of the H(+)-adenosinetriphosphatase activity was measured in intact (endosomes) and solubilized (0.1% deoxycholate or Triton X-100) BBM vesicles isolated by conventional Mg2+ precipitation techniques. In all species, but not in humans, the fraction of the ATP turnover energizing the proton pump (bafilomycin-sensitive respiration) was also measured in isolated proximal tubules. Significant differences in acid transport mechanisms were noted between species, with the proton pump predominating in the BBM of carnivorous species and the Na+/H+ exchanger predominating in the BBM of herbivorous species. The fraction of respiration suppressible by bafilomycin in proximal tubules was also different in all the species considered. This may indicate a different organization of proximal H+ transport related to the species-specific menace to acid-base balance.
Asunto(s)
Riñón/metabolismo , Protones , Animales , Cricetinae , Perros , Túbulos Renales/metabolismo , Microvellosidades/metabolismo , Consumo de Oxígeno , Bombas de Protones/metabolismo , ATPasas de Translocación de Protón/metabolismo , Conejos , Ratas , Ratas Wistar , Intercambiadores de Sodio-Hidrógeno/metabolismo , Especificidad de la Especie , PorcinosRESUMEN
The cellular energy required for the activity of the Na(+)-K(+)-ATPase and of the H(+)-ATPase was estimated in intact proximal tubules in suspension. Both the fall in oxygen consumption (directly measured) and NADH oxidation (as estimated from exogenous substrate metabolism) were measured before and following application of ouabain (1 mM) to inhibit the sodium pump, following bafilomycin (0.1 mM) to inhibit the proton pump or following a combination of these inhibitors. The data demonstrate that the sodium pump utilizes 43% and the proton pump 19% of the phosphorylating NADH turnover of canine proximal tubules studied in vitro. However, a significant and stoichiometric stimulation of one pump was observed upon inhibition of the other. The NADH turnover related to the sodium pump increased from 308 to 402 (delta = 94) mumol.g-1 wet weight.h-1 following bafilomycin application and that of the proton pump from 136 to 230 (delta = 94) following ouabain application. This stimulation was largely abolished by inhibition of the Na+/H+ exchange occurring in either direction by amiloride or methylisobutylamiloride. It is concluded that a cross-talk occurs between the basolateral sodium pumps and the proton pumps located on the brush border membrane and/or on endosomes in proximal tubules. This cross-talk appears to be mediated by Na+/H+ exchange suggesting that both the proton pump and the Na+/H+ exchanger may contribute in a cooperative fashion to the proximal secretion of protons.
Asunto(s)
Túbulos Renales Proximales/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Amilorida/farmacología , Animales , Ácidos Cumáricos/farmacología , Perros , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Microvellosidades/efectos de los fármacos , Microvellosidades/enzimología , Microvellosidades/metabolismo , NAD/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Ácido Valproico/farmacologíaRESUMEN
The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.
Asunto(s)
Asa de la Nefrona/metabolismo , Canales de Potasio/metabolismo , Animales , Bario/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Electrofisiología , Membranas Intracelulares/metabolismo , Corteza Renal , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , ConejosRESUMEN
Intracellular potassium activity (alpha Ki) was measured in control conditions in mid-cortical rabbit proximal convoluted tubule using two methods: (i) by determination of the K+ equilibrium potential (EK) using Ba(2+)-induced variations in the basolateral membrane potential (VBL) during transepithelial current injections and (ii) with double-barrel K-selective microelectrodes. Using the first method, the mean VBL was -48.5 +/- 3.2 mV (n = 16) and the mean EK was -78.4 +/- 4.1 mV corresponding to alpha Ki of 68.7 mM. With K-selective microelectrodes, VBL was -36.6 +/- 1.1 mV (n = 19), EK was -64.0 +/- 1.1 mV and alpha Ki averaged 40.6 +/- 1.7 mM. While these last EK and VBL values are significantly lower than the corresponding values obtained with the first method (P less than 0.001 and P less than 0.01, respectively), the electrochemical driving force for K transport across the basolateral membrane (microK = VBL-EK) is not significantly different for both techniques (30.1 +/- 3.3 mV for the first technique and 27.6 +/- 1.8 mV for ion-selective electrodes). This suggests an adequate functioning of the selective barrel but an underestimation of VBL by the reference barrel of the double-barrel microelectrode. Such double-barrel microelectrodes were used to measure temporal changes in alpha Ki and microK in different experimental conditions where Na reabsorption rate (JNa) was reduced. alpha Ki was shown to increase by 12.2 +/- 2.7 (n = 5) and 14.1 +/- 4.4 mM (n = 5), respectively, when JNa was reduced by omitting in the luminal perfusate: (i) 5.5 mM glucose and 6 mM alanine and (ii) glucose, alanine, other Na-cotransported solutes and 110 mM Na. In terms of the electrochemical driving force for K exit across the basolateral membrane, microK, a decrease of 5.4 +/- 2.0 mV (P less than 0.05, n = 5) was measured when glucose and alanine were omitted in the luminal perfusate while microK remained unchanged when JNa was more severely reduced (mean change = -1.7 +/- 2.1 mV, NS, n = 5). In the latter case, this means that the electrochemical driving force for K efflux across the basolateral membrane has not changed while both the active influx through the Na-K pump and the passive efflux in steady state are certainly reduced. If the main pathway for K transport is through the basolateral K conductance, this implies that this conductance must have decreased in the same proportion as that of the reduction in the Na-K pump activity.
Asunto(s)
Túbulos Renales Proximales/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Alanina/farmacología , Animales , Transporte Biológico Activo , Epitelio/metabolismo , Femenino , Glucosa/farmacología , Técnicas In Vitro , Líquido Intracelular/metabolismo , Meglumina/farmacología , Potenciales de la Membrana , Perfusión , ConejosRESUMEN
We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (Vbl) in rabbit proximal convoluted tubule (PCT, S1 segment). The present study is aimed at understanding the origin of the membrane repolarization following the initial effect of addition of luminal cotransported solutes. Luminal addition of 10-15 mM L-alanine produced a rapid and highly significant depolarization of Vbl (20.3 +/- 1.1 mV, n = 15) which was transient and associated with an increase in the fractional K+ conductance of the basolateral membrane (tK) from 8 to 29% (P less than 0.01, n = 6). Despite the significant increase in tK, the repolarization was only slightly reduced by the presence of basolateral Ba2+ (2 mM, n = 6) or quinine (0.5 mM, n = 5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4'isothiocyamostilbene-2,2'-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pHi) determined with the fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF), averaged 7.39 +/- 0.02 in control solution (n = 9) and increased to 7.50 +/- 0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16 +/- 0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K+ conductance but also through a cellular acidification reducing the basolateral HCO3- exit through the electrogenic Na-3(HCO3) cotransport mechanism.
Asunto(s)
Túbulos Renales Proximales/metabolismo , Sodio/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Bario/farmacología , Membrana Basal/metabolismo , Bicarbonatos/farmacología , Transporte Biológico Activo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Perfusión , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Quinina/farmacología , ConejosRESUMEN
Studies were performed in anesthetized normal dogs to evaluate the effects of maleate on renal metabolism. Intravenous administration of maleate (50 mg/kg) markedly increased urinary excretion of glutamine, glutamate, alpha-ketoglutarate, alanine, lactate, pyruvate, and citrate. Despite a fourfold rise in renal cortical concentration of alpha-ketoglutarate, glutamine utilization expressed per 100 ml glomerular filtration rate almost doubled following maleate administration, whereas total ammonia production increased threefold, most of this ammonia being diverted into the renal vein. The renal production of alpha-ketoglutarate rose in a spectacular fashion and was almost equal to the renal utilization of glutamine, indicating a metabolic block at the alpha-ketoglutarate dehydrogenase step. Maleate reduced renal alanine production but did not change lactate utilization. These findings suggest that 1) in the intact dog the mitochondrial entry of glutamine is not regulated only by alpha-ketoglutarate; 2) the deamination of glutamate into alpha-ketoglutarate is accelerated by maleate, probably through an impaired mitochondrial NADH production; 3) the resulting decrement in intramitochondrial glutamate concentration deinhibits the phosphate-dependent glutaminase.
Asunto(s)
Glutamina/metabolismo , Riñón/metabolismo , Maleatos/farmacología , Animales , Arterias , Análisis Químico de la Sangre , Diuresis/efectos de los fármacos , Perros , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Corteza Renal/metabolismo , Hígado/metabolismo , Masculino , Estimulación Química , Orina/análisisRESUMEN
Acute respiratory alkalosis (blood pH, 7.60; arterial PCO2, 15 mmHg (1 mmHg = 133.322 Pa); plasma bicarbonate, 14 mM) was induced in nine anesthetized dogs by increasing their respiratory rate and depth. Renal glutamine extraction and ammonia production expressed per 100 mL of glomerular filtration rate did not change during acute hypocapnia, whereas arterial glutamine concentration decreased significantly from 0.47 to 0.36 mM. Hypocapnia did not change plasma potassium concentration and its urinary excretion. Acute hypocapnia increased lactate extraction and pyruvate production, whereas citrate extraction and glutamate and alanine production did not change. Citraturia remained minimal. Renal cortical glutamine concentration fell from 0.64 to 0.38 mM during hypocapnia while alpha-ketoglutarate, glutamate, malate, oxaloacetate, and citrate did not change. Lactate concentration rose from 1.1 to 2.0 mM. Glutamine concentration in the liver and muscle decreased following acute hypocapnia. Our data are compatible with the hypothesis that an acute respiratory alkalosis might not result in any change in the hydrogen ion concentration and (or) gradient between the mitochondrial matrix and the cytosol. Consequently, renal glutamine extraction and ammonia production are not reduced, renal cortical concentrations of relevant metabolites in the ammoniagenic pathway are not changed, and renal handling of citrate remains unaffected.
Asunto(s)
Alcalosis Respiratoria/metabolismo , Amoníaco/metabolismo , Riñón/metabolismo , Enfermedad Aguda , Animales , Perros , Femenino , Tasa de Filtración Glomerular , Glutamina/metabolismo , Concentración de Iones de Hidrógeno , Ácidos Cetoglutáricos/metabolismo , Corteza Renal/metabolismo , Hígado/metabolismo , Masculino , Músculos/metabolismoRESUMEN
Studies were performed in anesthetized dogs to characterize the effect of a progressive volume expansion on the acetazolamide-induced bicarbonaturia. A closed system with urine reinfusion was used in all these experiments. In normovolemic dogs, 24% of the filtered bicarbonate was excreted into the urine while this value reached 62% when a 10% expansion was superimposed on a continuous infusion of acetazolamide. When a single dose of acetazolamide was given, fractional bicarbonate excretion increased from 21% in normovolemic dogs to 46% during 10% expansion. Without acetazolamide administration, 13% of the filtered bicarbonate was excreted during a 10% expansion. The continuous infusion of acetazolamide in normovolemic dogs increased fractional bicarbonate excretion in a progressive fashion, from 25 to 40%. This study shows that an acute volume expansion potentiates markedly the bicarbonaturic effect of acetazolamide, fractional bicarbonate excretion exceeding by far the simple additive effect of acetazolamide and expansion. We speculate that volume expansion might prevent a compensatory rise in acetazolamide-insensitive bicarbonate reabsorption in sites other than the superficial proximal convoluted tubules.
Asunto(s)
Acetazolamida/farmacología , Bicarbonatos/orina , Líquidos Corporales/fisiología , Acetazolamida/administración & dosificación , Acetazolamida/sangre , Animales , Femenino , Plasma/análisisRESUMEN
Studies were performed to determine whether ammoniagenesis could adapt instantaneously to acidosis in the dog kidney. Following acute respiratory acidosis, renal glutamine extraction rose acutely in dogs with stable renal blood flow but did not change when the renal blood flow fell by more than 25%. Acute hypercapnia immediately increased renal ammonia production in both groups of dogs. The rate of both glutamine extraction and ammonia production in acutely hypercapnic dogs without hemodynamic changes was comparable to the rates observed in dogs with chronic metabolic acidosis. Furthermore, the renal metabolite profile observed in acute hypercapnia was similar to the pattern described in chronic metabolic acidosis, i.e., a marked fall in renal glutamate and alpha-ketoglutarate concentrations and a fivefold increase in malate and oxaloacetate concentrations. In the liver and muscle, acute hypercapnia induced no significant change in glutamine concentration but glutamate and alpha-ketoglutarate concentrations decreased. Our findings demonstrate that the dog kidney can adapt immediately to acidosis but that hemodynamic change may mask this adaptation.