Renal Cortical Brush-border Membrane Vesicles Research Articles

Inhibition of renal Na +/H + exchange in cadmium-intoxicated rats

Chronic exposure to cadmium (Cd) results in bicarbonaturia, leading to metabolic acidosis. To elucidate the mechanism(s) by which renal bicarbonate reabsorption is inhibited, we investigated changes in renal transporters and enzymes associated with bicarbonate reabsorption in Cd-intoxicated rats. Cd intoxication was induced by subcutaneous injections of CdCl 2 (2 mg Cd/kg per day) for 3 weeks. Cd intoxication resulted in a significant reduction in V max of Na +/H + antiport with no changes in K Na in the renal cortical brush-border membrane vesicles (BBMV). Western blotting of BBM proteins and indirect immunohistochemistry in renal tissue sections, using an antibody against Na +/H + exchange-3 (NHE3), showed a diminished expression of NHE3 protein in the BBM. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that NHE3 mRNA expression was reduced in the renal cortex. The activity of carbonic anhydrase IV (CA IV) in BBM was not changed. The protein abundance of Na +-HCO 3 − cotransporter-1 (NBC1) in whole kidney membrane fractions was slightly attenuated, whereas that of the Na +-K +-ATPase α-subunit was markedly elevated in Cd-intoxicated animals. These results indicate that Cd intoxication impairs NHE3 expression in the proximal tubule, thereby reducing the capacity for bicarbonate reabsorption, leading to bicarbonaturia in an intact animal.

The integrity of renal cortical brush-border and basolateral membrane vesicles is damaged in vitro by nephrotoxic heavy metals

Poisoning of experimental animals with cadmium (Cd), mercury (Hg), lead (Pb) or cis-diamminedichloroplatinum (cis-Pt) causes shortening and focal loss of microvilli in proximal tubule (PT) cells, thus indicating that the reduced reabsorptive surface due to damaged integrity of brush-border membrane (BBM) may contribute to the reabsorptive and secretory defects in these toxic states. In addition, in in vitro studies with isolated renal cortical BBM vesicles (BBMV), heavy metals (HM) inhibit transport of various compounds, and these data were interpreted as being a result of a direct inhibition of the respective membrane transporters. In this work we used a ΔpH-driven acridine orange fluorescence quench assay to test if various divalent cations affect in vitro the integrity of BBMV and basolateral membrane vesicles (BLMV) isolated from the rat renal cortex. In Cd-treated BBMV we found that: (a) the integrity of vesicles decreased with increasing concentrations of Cd; and (b) the loss of sealed vesicles was high at 37°C, intermediate at 25°C, and very low at 0°C. The loss of sealed BBMV was caused also by Hg, Cu, Pb and Zn (Hg⋙Cu=Cd>Pb=Zn). Cis-Pt, Al, Fe, Ba, Mg and Mn had no effect. BLMV were damaged by HM with an efficiency Hg⋙Cd=Pb=Cu, whereas other divalent cations, including Zn, were ineffective. An SH-group protector, dithiothreitol, prevented the loss of sealed vesicles in some (Hg, Pb, Cu) but not in all (Cd, Zn) cases. We conclude that the nephrotoxic HM directly damage the integrity of PT cell plasma membranes; this may cause shortening and loss of microvilli and basolateral invaginations in HM-treated experimental animals in vivo. The data also indicate that caution should be taken when effects of HM on various transports are studied in isolated membrane vesicles in vitro; an impaired transport may result from the loss of vesicle integrity, and not necessarily from the direct inhibition of a transporter.

Thyroid hormones modulate zinc transport activity of rat intestinal and renal brush-border membrane.

Thyroid hormone status influences the Zn2+ and metallothionein levels in intestine, liver, and kidney. To evaluate the impact of thyroid hormones on Zn2+ metabolism, Zn2+ uptake studies were carried out in intestinal and renal brush-border membrane vesicles (BBMV). Steady-state Zn2+ transport in intestinal and renal cortical BBMV was increased in hyperthyroid (Hyper-T) rats and decreased in the hypothyroid (Hypo-T) rats relative to euthyroid (Eu-T) rats. In both the intestinal and renal BBMV, Hyper-T rats showed a significant increase in maximal velocity compared with Eu-T and Hypo-T rats. Apparent Michaelis constant was unaltered in intestinal and renal BBMV prepared from the three groups. Fluorescence anisotropy of diphenyl hexatriene was decreased significantly in intestinal and renal brush-border membrane (BBM) isolated from Hyper-T rats compared with Hypo-T and Eu-T rats. A significant reduction in the microviscosity and transition temperature for Zn2+ uptake in intestinal and renal BBM from Hyper-T rats is in accordance with the increased fluidity of these BBMs. These findings suggest that the increased rate of Zn2+ transport in response to thyroid hormone status could be associated with either an increase in the number of Zn2+ transporters or an increase in the active transporters due to alteration in the membrane fluidity. Thus the thyroid hormone-mediated change in membrane fluidity might play an important role in modulating Zn2+ transport activity of intestinal and renal BBM.

Proton-coupled oligopeptide transport by rat renal cortical brush border membrane vesicles: a functional analysis using ACE inhibitors to determine the isoform of the transporter

We demonstrate that the angiotensin-converting enzyme inhibitors enalapril and captopril inhibit the transport of D-Phe-L-Gln into PepT1-expressing Xenopus oocytes and into rat renal cortical brush border membrane vesicles (BBMV). The kinetics of inhibition are competitive. Enalapril and captopril are not substrates for PepT2 (Boll et al., Proc. Natl. Acad. Sci. 93 (1996) 284–289). Therefore we conclude that in rat renal cortical BBMV this neutral dipeptide is transported via PepT1.

Effect of Cadmium on Na–PiCotransport Kinetics in Rabbit Renal Brush-Border Membrane Vesicles

Chronic exposure to cadmium impairs various renal functions, including phosphate (Pi) transport. To further investigate the mechanism of cadmium-induced alterations in renal Pitransport, kinetics of Na+-dependent Piuptake were studied in renal cortical brush-border membrane vesicles (BBMV) exposed to CdCl2in vitro.BBMVs isolated from rabbit renal outer cortex were preincubated in a buffer containing CdCl2(50 μmin most cases) for 60 min at 37°C and then tested for Piuptake at 25°C. CdCl2treatment resulted in a marked attenuation of Na+-dependent Piuptake with no changes in Na+-independent Piuptake and membrane permeability to Na+. CdMt treatment induced no changes in Pitransport. The inhibition required preincubation of vesicles with CdCl2for more than 30 min and was not reversed by extravesicular EDTA, suggesting that cadmium affects the transport system at the internal side of the membrane. Kinetic analysis indicated that two sodium ions and one phosphate ion interact with a carrier, and this stoichiometry was not altered by cadmium treatment. Cadmium treatment did not change the apparentKmfor Na+(KNa) and that for phosphate (KPi), but it markedly reduced theVmaxof the Na+-dependent Pitransport. These results indicate that exposure of proximal tubular brush border membranes to cadmium impairs the Pitransport capacity, probably by reducing the effective number of Na–Picotransporter units without altering substrate affinities of the carrier.

Human stanniocalcin inhibits renal phosphate excretion in the rat.

Stanniocalcin (STC) is a glycoprotein hormone first identified in bony fishes where it counteracts hypercalcemia by inhibiting gill calcium uptake and stimulating renal inorganic phosphate (Pi) reabsorption. Human STC (hSTC) has recently been cloned and sequenced and is highly homologous to the fish hormone at the amino acid level. The objective of this study was to examine the possible effects of hSTC on electrolyte homeostasis and renal function in the rat. Recombinant hSTC was expressed in bacteria and purified by metal-ion affinity chromatography and reverse-phase high performance liquid chromatography. Anesthetized animals were given bolus infusions of 1, 5, or 10 nmol hSTC per kilogram of body weight. Control animals received solvent alone. The most effective dosage was 5 nmol/kg, which caused significant reductions in both absolute and fractional phosphate excretion in comparison with control rats. The hSTC had no effect on the renal excretion of other ions, the glomerular filtration rate, renal blood flow, blood pressure, or plasma electrolytes (Na+, K+, Ca2+, Pi, Mg/+). The maximum effect of hSTC on phosphate excretion was observed 60-80 minutes postinjection. Lesser effects were obtained with higher and lower dosages of hormone. When renal cortical brush-border membrane vesicles were isolated from control and hormone-treated animals 80 minutes postinjection, the rate of Na+/Pi cotransport was found to be 40% higher in vesicles from hormone-treated animals (p < 0.01; 5 nmol hSTC/kg). Together, the renal clearance and membrane vesicle data indicate that hSTC participates in the renal regulation of Pi homeostasis in mammals.

Renal handling of carnitine in experimental vitamin C deficiency

Experimental vitamin C deficiency is associated with low carnitine concentrations in blood and some tissues, but is not due to a decreased ability of scorbutic animals to synthesize carnitine. The effect of experimental vitamin C deficiency on urinary carnitine excretion in vivo and carnitine transport into renal cortical brush-border membrane vesicles in vitro was investigated in guinea pigs fed normal and vitamin C-deficient diets for 24 days. Excretion of free and total carnitine was approximately fourfold greater in scorbutic animals as compared with normal guinea pigs during the last 6 days of the experimental regiment. The rate of carnitine transport into renal cortical brush-border membrane vesicles prepared from scorbutic animals was approximately 36% lower than the corresponding rate for vesicles prepared from normal animals. However, this effect was not specific, since rates of sodium gradient-dependent transport of glucose, lysine, and taurine (but not alanine) were also lower vesicles prepared from scorbutic animals, although the magnitude of the decrease was less than for carnitine. results are consistent with the hypothesis that carnitine depletion in vitamin C deficiency is due to decreased efficiency of carnitine reabsorption.

Transport of Inorganic Phosphate in Renal Cortical Brush-Border Membrane Vesicles of Cadmium-Intoxicated Rats

The effect of cadmium intoxication on the renal proximal tubular phosphate transport system was studied in adult male Sprague-Dawley rats. Subcutaneous injections of CdCl2 at a dose of 2 mg Cd/kg body wt per day for 2 weeks induced marked polyuria, glycosuria, proteinuria, and phosphaturia, which are characteristics of chronic cadmium intoxication. In the renal cortical brush-border membrane vesicles prepared from cadmium-intoxicated rats, the cadmium content was drastically increased and the Na(+)-dependent phosphate uptake was markedly attenuated. Similar results were obtained in normal membrane vesicles directly exposed to free cadmium. These results indicate that cadmium intoxication impairs the Na(+)-phosphate cotransport system in the proximal tubular brush-border membrane, which may lead to phosphaturia in intact animals.

Characterization of Na(+)-phosphate cotransporters in renal cortical endosomes

To determine the role of membrane recycling in proximal tubular P(i) transport, we studied the transport functions of simultaneously prepared rat renal cortical endosomal vesicles (EV) and brush-border membrane vesicles (BBMV). Initial P(i) uptake was Na+ gradient-dependent in both vesicles. Kinetic studies showed a lower apparent maximal rate (Vmax) for P(i) uptake in EV compared with BBMV (446 +/- 69 vs. 1493 +/- 117 pmol.mg protein-1 x 15 s-1, respectively; n = 4, P < 0.05), with no difference in the apparent Michaelis constant (Km). Endosomal Na(+)-P(i) cotransport was inhibited by phosphate analogues, phosphonoformic acid and arsenate, but not by the anion-exchange inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Na(+)-P(i) cotransport was electroneutral in both vesicles, with a stoichiometry of 2 for BBMV and 1 for EV. The nonpermeant sulfhydryl reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), produced a marked inhibition of Na(+)-P(i) cotransport in EV but not in BBMV, suggesting accessible sulfhydryl groups, an "inside-out" orientation in EV, and "right-side-out" orientation in BBMV. The EV and the BBMV differed significantly in their phospholipid composition and lipid fluidity. The Na(+)-P(i) cotransporter protein (NaPi-2) abundance, determined by Western blots, was five times lower in EV than in BBMV (0.25 +/- 0.05 vs. 1.36 +/- 0.20 arbitrary units). Renal cortical endosomes contain Na(+)-P(i) cotransporters, albeit at a lower density, suggesting involvement of membrane recycling in the regulation of proximal tubular P(i) transport.

Altered membrane ionic permeability in a rat model of chronic renal failure.

Acute elevations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentrations are known to increase ionic chloride permeability in diverse tissues. To determine if chronic endogenous increases in cAMP are associated with sustained alterations in membrane ionic permeabilities, renal cortical brush border membrane vesicles (BBMV) were prepared and red blood cells were harvested in a model of chronic renal failure, the 75% nephrectomized rat. Relative ionic permeabilities were determined using the potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. These studies demonstrate that renal cortical homogenate and RBC cAMP concentrations are increased in chronic renal failure animals. In the same animals relative ionic chloride permeability (PCl/PK) was significantly increased in renal cortical BBMV and RBC ghosts: PNa/PK was not affected. This selective change in permeability results in a significant increase in PCl/PNa and hyperpolarization of BBMV of sufficient magnitude to stimulate Na(+)-dependent glutamine transport. The change in glutamine uptake was not consequent to an alteration in the kinetics of glutamine transport or delayed dissipation of the inward Na+ gradient. Renal hypertrophy per se did not effect renal homogenate cAMP concentration or relative ionic permeability of renal cortical BBMV prepared from kidneys of uninephrectomized animals fed a 40% protein diet. These studies demonstrate that relative ionic chloride permeability and tissue [cAMP] are chronically increased in diverse cells (renal proximal tubule and RBCs) in a rat model of renal failure. These findings suggest that membrane ionic permeability may be altered and electrogenic transport secondarily perturbed in renal failure in association with hormonally-induced chronic elevations of intracellular cAMP concentrations.

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.

The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranes

Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg 2+ < Ca 2+ < Mn 2+ < Ba 2+ < Zn 2+. At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg 2+-stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity ( K′ m = 0.16 mM) and low-affinity site ( K′ m = 9.0 mM, respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent K m = 0.38 ± 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent K m = 76 ± 9 μM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems.

Dopamine inhibits Na(+)-H+ exchanger activity in renal BBMV by stimulation of adenylate cyclase

To determine a renal tubular mechanism for the natriuretic effect of dopamine (DA) and DA-1 agonists, we measured Na(+)-H+ exchange activity (amiloride sensitive) in rat renal cortical brush-border membrane vesicles (BBMV). Renal cortical tissues were preincubated with ligands before BBMV preparation to study Na(+)-H+ exchange activity in the absence of the added ligands that may compete for ion binding sites of the exchanger. DA and DA-1 agonist-inhibited Na(+)-H+ exchange activity was concentration and time dependent. The inhibitory effect was not due to increased permeability, collapse of the proton gradient, or change in vesicle size and did not extend to Na(+)-glucose symport. DA-2 agonists had no effect, whereas alpha-adrenergic agonists increased Na(+)-H+ exchange activity. Kinetic analysis revealed that the DA-1 agonist inhibited Na(+)-H+ exchange activity by a noncompetitive process. 2',5'-Dideoxyadenosine inhibited adenylate cyclase activity and reversed the inhibitory effect of DA-1 agonist on the exchanger. H4, an isoquinoline sulfonamide, which inhibits protein kinase A, also reversed the inhibitory effect of DA-1 agonist on the exchanger. Thus the DA-1 agonist-mediated inhibition of Na(+)-H+ exchange activity in BBMV is a receptor-mediated adenylate cyclase-linked process.

D-penicillamine and the transport of L-cystine by rat and human renal cortical brush-border membrane vesicles

Brush-border membrane vesicles (BBMV) were prepared from rat and human renal cortical tissue by magnesium aggregation and differential centrifugation, and the uptake of L-cystine, L-cysteine, and L-cysteine-D-penicillamine were assessed by a rapid-filtration technique. L-Cystine uptake was relatively sodium independent and associated with membrane binding. Sodium-stimulated uptake was sensitive to a cation but not anion diffusion potential. Both sodium-independent and sodium-stimulated uptake rates were inhibited by the cationic L-amino acids and by some neutral L-amino acids. The uptake rates of L-cysteine and L-cysteine-D-penicillamine were more sodium dependent, and sodium-stimulated uptake rates were more sensitive to cation and anion diffusion potentials. Neither the sodium-independent nor the sodium-stimulated uptake rates of L-cysteine or L-cysteine-D-penicillamine were inhibited by the cationic L-amino acids. L-Cysteine-D-penicillamine showed relatively little membrane binding. It is concluded that L-cystine is transported into renal cortical BBMV by pathways distinct from those concerned with the transport of L-cysteine and L-cysteine-D-penicillamine, and it is postulated that these differences may account for some of the effects of D-penicillamine in cystinuria.

Adaptation of rabbit renal cortical Na+-H+ exchange activity in chronic hypocapnia

We have examined the activity and kinetic characteristics of the Na+-H+ exchanger in renal cortical brush-border membrane vesicles (BBMV) prepared from rabbits adapted to chronic hypocapnia in order to address whether this transporter might contribute to the suppressed proximal bicarbonate reabsorption characteristic of this disorder. Chronic hypocapnia was induced by exposing animals to 9% O2 for a 5-day period. In comparison with paired, contemporaneous controls, an average delta PaCO2 of 13 mmHg and an average delta [HCO3-] of 7.3 meq/l were obtained. Chronic hypocapnia led to a significant suppression of the 22Na+ uptake by BBMV; at the 3-s mark, a 30% suppression was observed (chronic hypocapnia, 4.05 +/- 0.43 nmol/mg protein; control, 5.72 +/- 0.39 nmol/mg protein) (P less than 0.01). A significant decrease in the Vmax of the antiporter was noted (chronic hypocapnia, 622.7 +/- 86.8 nmol.mg protein-1.min-1; control 857.5 +/- 64.8 nmol.mg protein-1.min-1) (P less than 0.01), whereas the Km for sodium remained unaltered. The specificity of this adaptation was supported by showing that Na+-dependent uptake of D-[3H]glucose by BBMV was not significantly different between chronic hypocapnia and control. Chronic normocapnic hypoxemia left Na+-H+ exchange activity undisturbed. We conclude that the observed change in the BBMV Na+-H+ antiporter might be responsible, at least in part, for the suppressed renal bicarbonate reabsorption characteristic of chronic hypocapnia and that a consequence of the hypocapnic state itself mediates this adaptation.

Computer analysis reveals changes in renal Na+-glucose cotransporter in diabetic rats.

A novel, computer-assisted program was developed to analyze the time course of Na+-glucose cotransport by rat renal cortical brush-border membrane vesicles (BBMV). Transporter characteristics can be measured, which routine kinetic analyses fail to distinguish: cotransporter membrane density is derived from the picomoles of D-glucose bound per milligram of protein. Binding is stereospecific, blocked by phlorizin, and supported equally well by Na+ or K+ (but not Cs+). Quasi-first-order influx and efflux rate constants for the composite Na+-driven influx and the (presumed) Na+-independent efflux processes were highly dependent on glucose concentration. Either two Na+-glucose transporters exist in proximal tubules or a single mechanism abruptly changes rate when glucose falls to low levels. The major operation mode is slow, has a high capacity but low affinity, and may have a 2 Na+:2 glucose stoichiometry (Hill coefficient is unity). The minor system is a fast, smaller-capacity, higher-affinity operation with a 2 Na+:1 glucose stoichiometry that was not distinguishable when the same data were analyzed in conventional kinetic plots. Results with streptozocin-induced diabetic rats illustrate the method's utility. Low-glucose-affinity cotransporters were upregulated in hyperglycemic, but not in cachectic, ketoacidotic animals. Rate constants, especially for efflux, were decreased in diabetes.

Differential permeabilities of rat renal brush-border and basolateral membrane vesicles.

Potassium chloride permeability and relative ionic conductances of rat renal cortical brush-border (BBMV) and basolateral (BLMV) membrane vesicles were examined using the fluorescent probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)]. Vesicles were simultaneously isolated and separated by free-flow electrophoresis. These studies demonstrated that neither BBMV nor BLMV equilibrated in 100 mM KCl despite prolonged incubation. In both, an inwardly directed KCl gradient was sustained for 3 h. The low intravesicular KCl concentration of BLMV was confirmed utilizing the response of electrogenic Na+-dependent [3H]glutamine transport to variations in the membrane potential. Chloride conductance was significantly less than potassium conductance in BBMV and BLMV. Consequently, an inside-positive potential was maintained across both membranes. BLMV were significantly more fluid, less permeable, had a lower relative chloride conductance, and maintained a greater inside-positive potential than BBMV. The KCl permeabilities of BBMV and BLMV were inversely related to endogenous membrane copper content and were significantly reduced by exogenous copper. Permeability did not correlate with membrane magnesium content, nor was it affected by exogenous magnesium. These studies suggest that endogenous copper as well as intracellular factors may regulate the permeabilities of the brush-border and basolateral membranes of proximal tubule cells in vivo.

Short-term and long-term stimulation of Na+-H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney.

The effect of unilateral nephrectomy on Na+-H+ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70-75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted to Vmax, whereas the Km for Na+ was similar in control and compensatory growing kidney. The increase of Na+-H+ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na+-H+ exchange. The late, but not the initial stimulations of Na+-H+ exchange were associated with elevated activities of cortical (Na++K+)-ATPase indicating that changes in antiporter activity precede those in the (Na++K+)-pump. The early stimulation of Na+-H+ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na+-H+ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na+-H+ exchanger may be regulated by phosphorylation-dephosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of increased electroneutral Na +-H + exchange in renal cortical brush-border membranes from hyperthyroid rats

The fluorescence quenching of acridine orange was used to compare Na +-H + exchange and ion conductances in renal cortical brush-border membrane vesicles (BBMV) isolated from euthyroid and hyperthyroid rats. In BBMV from euthyroid animals, Na +-H + exchange was entirely electroneutral. In BBMV from hyperthyroid rats, the total rates of Na +-H + exchange were about 30% higher than in BBMV from euthyroid animals. However, the electroneutral exchange in these membranes was similar to that in BBMV from euthyroid rats; the observed increase in exchange was due to electrically coupled Na + and H + movements through conductive pathways in the membranes. Ion conductances in isolated BBMV were tested with outwardly directed K + gradients in the presence of carbonyl cyanide m-chlorophenylhydrazone (K + conductance) or valinomycin (H + conductance). The K + conductance was negligible and similar in BBMV from both groups of rats. A significant H + conductance was present in both kinds of membrane preparations and was by 37% higher in BBMV from hyperthyroid animals. Therefore, our experiments failed to demonstrate an increased electroneutral Na +-H + exchange in BBMV from hyperthyroid rats. Instead, a finding of a significant electrically coupled Na +-H + antiport in the presence of increased H + conductance in BBMV from hyperthyroid rats indicates that these membranes may also have increased Na + conductance.

Taurine transport by rabbit kidney brush-border membranes: coupling to sodium, chloride, and the membrane potential.

Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na+-D-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na+ gradient (K+ diffusion potential) and could be completely inhibited by other beta-amino acids. It displayed a specific anion requirement (Cl- greater than or equal to Br- much greater than SCN- greater than I- greater than NO-3). At chemical Na+ equilibrium, Cl- gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl- gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl- as well as Br-. The Cl- stoichiometry was found to be one, whereas taurine transport, in the presence of Cl-, was sigmoidally related to the Na+ concentration, resulting in a coupling ratio of 2 to 3 Na+: 1 taurine. Upon Cl- replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na+ concentration (up to 150 mM). These results suggest a 2 to 3 Na+ :1 Cl- :1 taurine cotransport mechanism driven mainly by the Na+ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl- appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.