Cortical Brush-border Membrane Vesicles Research Articles

Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

Procainamide transport in rabbit renal cortical brush border membrane vesicles.

Previous studies in canine and rat renal cortical brush border membrane vesicles (BBMV) and some results in isolated perfused rabbit proximal tubules indicate that organic cations may be transported across the apical cell membrane by an organic cation/proton exchange process. To determine more directly whether organic cations are transported across the apical cell membrane of rabbit proximal tubules, [3H]procainamide uptake in BBMV was studied. Procainamide uptake was linearly related to the inverse of the media osmolarity, indicating uptake into an intravesicular space. A proton gradient directed from vesicle interior outwardly stimulated and an opposite gradient inhibited procainamide uptake. pH-stimulated uptake was inhibited by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) and was also reduced by an inwardly directed sodium gradient. pH-stimulated procainamide uptake was inhibited by other organic cations including the quarternary ammonium ion tetramethylammonium, indicating that the effect of proton gradients was not due to changes in nonionic diffusion. pH-stimulated procainamide uptake at 10 s was saturable with an apparent Km of 5.4 X 10(-4) M and Vmax of 4.7 X 10(-10) mol X mg protein-1. Uptake of [3H]procainamide was enhanced when BBMV were preloaded with nonradioactive procainamide but this was prevented by FCCP and valinomycin. Finally, an outwardly directed potassium gradient in the presence of valinomycin failed to significantly stimulate procainamide uptake. These results are consistent with a mechanism of secretion that involves electroneutral exchange of procainamide for protons across the apical cell membrane of rabbit proximal tubules.

Nephron adaptation to renal injury or ablation.

In early stages of permanent renal injury or extensive ablation, structural and functional adaptations associated with hypertrophy partially compensate for nephron losses. Glomerulotubular balance is maintained in these conditioned nephrons by intrinsic tubule and peritubular capillary adaptations that parallel single nephron glomerular filtration rate (SNGFR). Studies of Na+-H+ exchange in renal cortical brush border membrane vesicles indicate that tubule functional adaptation is not tied to loss of renal mass per se but rather to factors such as dietary protein content that set the level of SNGFR. Likewise, the structural heterogeneity that follows chronic renal injury or extreme ablation of renal mass is less a consequence of nephron injury than of adaptation linked to dietary protein intake. Indeed, since dietary protein restriction blunts the need for compensatory glomerular hyperfiltration, there is neither a stimulus for nephron hypertrophy nor for enhanced tubule ion and fluid transport. In rats with remnant kidneys, experimentally induced diabetes mellitus, or severe hypertension, increases in glomerular pressures and flows precede proteinuria, glomerular sclerosis, and azotemia. Protein restriction prevents these hemodynamic adaptations as well as the late complications. Similar conclusions appear to be applicable to a wide spectrum of clinical circumstances characterized by reduced nephron number.

Radiation inactivation studies on the rabbit kidney sodium-dependent glucose transporter.

Rabbit kidney cortical brush-border membrane vesicles were irradiated in the frozen state with increasing doses of high energy electrons from a Van de Graaff generator. Sodium-dependent D-glucose and L-alanine transport showed a simple exponential loss of activity with increasing radiation dosage. Target size calculation based on these data gives estimates of 1.0 X 10(6) daltons for the glucose transporter and 1.2 X 10(6) daltons for the alanine transporter. A highly purified glucose transport protein extracted from rabbit kidney cortex was similarly irradiated both before and after reconstitution into liposomes. The target size of this purified glucose transporter was 343,000 daltons, based on inactivation of transport. The intensity of the major 165,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation was decreased by radiation. The decrease in staining intensity was dose-dependent, yielding a target size of 298,000 daltons, in situ. We propose that the purified glucose transporter reconstituted into liposomes is a tetramer comprised of 85,000-dalton subunits.

Cation dependence of renal outer cortical brush border membrane L-glutamate transport.

The cation dependence of L-glutamate transport in renal outer cortical brush border membrane vesicles was studied. Both cis sodium and trans potassium were required for optimal L-glutamate flux. Relative to simultaneous sodium (out greater than in) and potassium (in greater than out) gradient conditions, flux was reduced 50-fold by potassium removal and more than 100-fold by sodium removal. The effect of potassium removal in this preparation was markedly larger than that observed in other renal brush border membrane preparations. No other monovalent cation tested was effective in replacing sodium. However, Rb+ and Cs+ and to a lesser degree NH+4 were found to be effective potassium substitutes. Kinetic analysis of the Na/K-dependent component of L-glutamate flux indicated a single transport system of Km = 13 microM and Vmax = 1.3 nmol X min-1 X mg protein-1. Studies of the dependence of L-glutamate flux on potassium and sodium concentrations yielded Hill coefficients of 0.9 and 1.9, respectively, consistent with involvement of one potassium ion and two or more sodium ions in the L-glutamate transport event. Efflux studies indicated that sodium and potassium act at different steps in the transport cycle, sodium to facilitate the translocation of the glutamate-carrier complex, and potassium to facilitate the return of the unloaded carrier. A model for renal Na/K-dependent L-glutamate transport is suggested on the basis of these results.

Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes.

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.

Evidence for electroneutral chloride transport in rabbit renal cortical brush border membrane vesicles.

Controversial evidence exists as to whether electroneutral Cl-OH exchange occurs across the proximal tubular brush border membrane of the mammalian kidney. To help resolve this controversy, we carried out a series of experiments using rabbit renal cortical brush border membrane vesicles to determine the effect of changes in pH on 36Cl uptake across this membrane. Reducing pH equally inside and outside of the vesicle stimulated Cl-Cl exchange as well as chloride uptake. A proton gradient (out greater than in) stimulated and caused an overshoot in unidirectional Cl uptake. All three of these processes were inhibited by 4-acetoamido-4-isothiocyano-2,2-disulfonic stilbene. Valinomycin with K+ out-in added to shunt any proton diffusion potential, minimally inhibited pH gradient-dependent Cl uptake, and carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP), added to increase the proton diffusion potential, minimally stimulated pH gradient-dependent Cl uptake. Sodium-glucose cotransport (an electrogenic system) was used to assess the efficacy of these ionophores. FCCP markedly enhanced and valinomycin markedly inhibited pH gradient stimulated sodium-glucose cotransport. Valinomycin added to vesicles with a 180/0 meq K+ out greater than in gradient stimulated less Cl uptake than a 6.0/7.5 pH out greater than in gradient. We conclude that these results provide strong evidence for the existence of an electroneutral Cl-OH exchanger or Cl-H symporter in this membrane.

Sodium-dependent sulfate transport in renal outer cortical brush border membrane vesicles.

The transport of sulfate into outer cortical brush border membrane vesicles (early proximal tubule) was studied. Sulfate uptake was markedly enhanced by sodium and slightly enhanced by lithium but showed no stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of sulfate flux indicated a single transport system obeying Michaelis-Menten kinetics. Studies of the dependence of sulfate flux on sodium concentration indicated that more than one sodium ion was involved in the sulfate transport event. Although sulfate influx was found to be stimulated by negative intravesicular potentials, evidence is presented that sodium/sulfate cotransport is nevertheless an electroneutral event presumably involving 2 Na+ per SO2-4. Under zero trans sodium conditions sodium-dependent sulfate influx was stimulated by the presence of intravesicular unlabeled sulfate (trans stimulation); however, a similar stimulation of sulfate efflux by the presence of unlabeled extravesicular sulfate was not observed. These latter results are consistent with an ordered binding scheme for the transporter, with sulfate binding before sodium on the extravesicular (urine) surface and dissociating before sodium on the intravesicular (cytosolic) surface.

Sodium-dependent succinate transport in renal outer cortical brush border membrane vesicles.

The transport of succinate into outer cortical brush border membrane vesicles (early proximal tubule) was studied. Succinate is taken up into an osmotically active space and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as D-glucose. Succinate uptake is markedly enhanced by sodium and slightly enhanced by lithium but shows no stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of succinate flux indicates a single transport site obeying Michaelis-Menten kinetics (Km = 1 mM and Vmax = 50 nmol X min -1 X mg protein -1 as measured under zero trans conditions at 100 mM NaCl and 28 degrees C with delta psi = 0). Direct evidence is given that succinate transport is coupled to sodium and is rheogenic, involving the net transfer of positive charge. The sodium:succinate coupling stoichiometry is found to be 2:1 by two independent methods.

Effect of metabolic acidosis on renal brushborder membrane adaptation to low phosphorus diet

In previous in vitro studies the level of oxidized nicotinamide adenine dinucleotide (NAD+) in renal cortex changed parallel to changes in gluconeogenesis and NAD+ inhibited phosphate transport by renal cortical brushborder membrane (BBM) vesicles. To determine whether or not changes in renal gluconeogenesis in vivo were accompanied by altered renal handling of phosphate, possibly related to NAD+ action on BBM phosphate transport in vivo, renal gluconeogenesis was stimulated in rats by metabolic acidosis. Chronic acidosis in rats previously adapted to low phosphorus diet was associated with increased UPiV (controls, 68 +/- 19; acidotic, 1055 +/- 428 nmoles/mg creatinine; P less than 0.05) without changes in plasma phosphate and creatinine (Cr) and in UCrV compared to controls. The initial rate of sodium gradient-dependent transport of phosphate by renal cortical BBM vesicles was lower in acidotic TPTX rats compared to TPTX controls (controls, 3.10 +/- 0.16; acidotic, 1.50 +/- 0.06 nmole/mg protein/0.5 min; P less than 0.001), attributed to a decrease in the apparent Vmax. In renal cortex, gluconeogenesis and the NAD+/NADH ratio were increased in acidosis. Decreased BBM transport of phosphate in proximal tubules of acidotic rats may explain the increased UPiV. This change indicates reversal of the adaptation of the BBM phosphate transport system to dietary phosphorus deprivation and may be related to increases in gluconeogenesis and the NAD+/NADH ratio in renal cortex.

Effects of metabolic intermediates on sugar and amino acid uptake in rabbit renal tubules and brush border membranes.

1. The effects of tricarboxylic acid cycle intermediates on the renal transport of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid were examined using separated renal tubules of the rabbit. 2. The effect of citrate on alpha-methyl-D-glucoside and alpha-amino-isobutyric acid uptake was markedly biphasic with maximum stimulation of transport occurring at a citrate concentration of 0.64 mM. Biphasic effects were also apparent for L-malate, succinate, fumarate, alpha-ketoglutarate and oxaloacetate. 3. The route of uptake of alpha-methyl-D-glucoside into separated renal tubules is primarily across the brush border (luminal) membrane. 4. Tricarboxylic acid cycle intermediates produced significant stimulation of renal O2 consumption; however, the effects on O2 consumption were not biphasic suggesting that reduced stimulation of transport at high substrate concentration was not caused by a reduction in the supply of metabolic energy. 5. In purified renal cortical brush border membrane vesicles, citrate and alpha-ketoglutarate inhibited the uptake of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid indicating that inhibition of their transport in respiring renal tubules by high concentrations of tricarboxylic acid cycle intermediates occurs via an effect at the membrane level.

Renal brush border membrane adaptation to phosphorus deprivation: Effects of fasting versus low-phosphorus diet

Alimentary phosphorus deprivation due to a low-phosphorus diet (LPD) elicits a profound antiphosphaturia and an increase in sodium-dependent inorganic phosphate (Pi) uptake by renal cortical brush border membrane (BBM) vesicles. But, in alimentary phosphorus deprivation due to total fasting, high urinary excretion of Pi persists. In the present study, we determined whether low tubular reabsorption of Pi in fasting is due to a diminished capacity of the specific Pi transport system with the renal cortical luminal BBM or whether it is due to a reduced transepithelial reabsorption of Pi because of metabolic conditions occurring in proximal tubule cells during fasting. Sodium-dependent Pi transport in compared with fasted rats or rats fed a normal phosphorus diet. Sodium-dependent uptake of D-glucose was significantly lower in LPD rats, compared with fast animals or animals fed a normal diet. Thus, in contrast to LPD, fasting does nt elicit an increase in Pi transport and a decrease in D-glucose transport across the isolated renal BBM. The same differences in BBM transport of Pi were present also in thyroparathyroidectomized rats. Further experiments demonstrated that the adaptation of renal function and the renal BBM transport to LPD are overridden by a subsequent period of total fasting. Results of the present study show that fasting both prevents and reverses the renal response of rats to alimentary phosphorus deprivation. The differences in Pi excretion between fasted rats, LPD rats, and LPD rats subsequently fasted are attributed, at least in part, to specific adaptive changes in sodium-dependent Pi transport across the luminal BBM, rather than to alterations in other cellular (metabolic) components of transepithelial Pi reabsorption in the proximal tubule.

Phosphate transport across renal cortical brush border membrane vesicles from rats stabilized on a normal, high or low phosphate diet

Phosphate uptake measured at 0.25, 0.5 and 1.0 min time intervals was 2-fold higher in renal brush border membrane vesicles from rats fed a low phosphate diet compared to control rats given a normal phosphate diet. In rats on a high phosphate diet the uptake of phosphate at the same time intervals was 2-fold lower compared to controls. D-glucose uptake by the same vesicle preparations was not significantly different between the three groups at 0.25, 0.5 and 1.0 min. At each of these time points there was a positive correlation between phosphate uptake and the activity of alkaline phosphatase which was measured in the vesicle preparations from each group of rats. There was no correlation between phosphate uptake and the activities of three other brush border enzymes.