Tubule Brush Border Membrane Vesicles Research Articles

Hypothyroidism increases osmotic water permeability (Pf) in the developing renal brush border membrane.

The osmotic water permeability (Pf) of the rabbit proximal tubule brush border membrane vesicles (BBMV) increases during maturation and is mediated by an increase in aquaporin-1 (AQP1) protein expression. Serum thyroid hormone levels increase after birth and have been shown to play a role in the maturation of other renal transport functions. We examined the hypothesis that thyroid hormone plays a role in the maturational increase in osmotic water permeability. Hypothyroidism was induced by addition of 0.1% propylthiouracil (PTU) to the drinking water of pregnant rabbits (starting 9 d before delivery) and was continued until the rabbits were studied as adults (9-11 wk). Some animals received thyroid hormone replacement by daily injection with triiodothyronine (T3; 10 microg/100 g body weight) for three days before study. Pf was found to be higher in BBMV from hypothyroid (82.7 +/- 5.5 microm/s) than from euthyroid (60.6 +/- 4.0 microm/s) and T3-replacement rabbits (69.0 +/- 5.0 microm/s) (p < 0.05). The activation energy (Ea; in kcal/deg.mol) of Pf was not different among the three experimental groups (euthyroid 5.6 +/- 0.9, hypothyroid 4.9 +/- 0.8, T3-replacement 5.0 +/- 1.0; p = NS), nor was the percentage mercury inhibition of Pf (euthyroid 66.5 +/- 5.3, hypothyroid 74.2 +/- 3.2 and T3-replacement 73.1 +/- 4.3; p = NS). AQP1 expression, measured by immunoblotting, was highest in BBMV from hypothyroid rabbits (p < 0.05). Membrane fluidity, measured as steady-state generalized polarization (GP) of Laurdan, which is inversely related to membrane fluidity, was significantly different between the three groups (GP: euthyroid 0.307 +/- 0.004, hypothyroid 0.271 +/- 0.004 and T3-replacement 0.287 +/- 0.003; for all p < 0.05). These data demonstrate that the maturational increase in thyroid hormone levels is not responsible for the maturational increase in water transport. Surprisingly, congenital hypothyroidism in rabbits is associated with an increased Pf when rabbits are studied as adults. The higher Pf in hypothyroid adult rabbits is due to a higher expression of AQP1 protein as well as a greater membrane fluidity than in euthyroid rabbits.

Renal brush border sodium-sulfate cotransport in guinea pig: effect of age and diet.

Renal adaptation to changes in inorganic sulfate intake and age was studied by comparing sulfate uptake by proximal tubule brush border membrane vesicles (BBMV) from guinea pigs of different ages on relatively high- or low-sulfate diets. Adult (> 60 days) or young guinea pigs (< 25 days) were fed either a control diet (0.28% sulfur content), a sulfur-free diet, or a high-sulfate diet. After 5 days on the diet, BBMV were obtained and kinetic analysis of 35sulfate uptake was determined. In adult guinea pigs, the low-sulfate diet produced a significant increase in apparent maximal velocity (Vmax). In young guinea pigs, a lower sulfate intake did not appreciably increase Vmax, but a high-sulfate intake produced a reduction in Vmax. The affinity for sulfate (Km) was not changed in either age group. The dietary sulfate intake did not alter sodium gradient dependent-D-glucose or 32phosphate Vmax. In conclusion, our data indicate that renal inorganic sulfate BBMV uptake is regulated and responds to conditions of increased need (i.e., during the growth phase in young animals and during periods of decreased sulfate availability in adult animals) by increasing BBMV Vmax.

Evidence from incorporation and patch-clamp experiments for a nonselective channel of large conductance at the luminal membrane of rabbit proximal tubule

The presence of nonselective ionic channels at the apical membrane of rabbit proximal tubule cells was investigated by fusion of proximal tubule brush border membrane vesicles onto a planar bilayer and by means of patch clamp measurements on primary cultured proximal tubule cells. The fusion of brush border membrane vesicles onto a phosphatidylethanolamine-phosphatidylcholine (1:1) planar bilayer led to the identification of a nonselective channel of 115 pS in 200 mM NaCl conditions with an ionic permeability sequence for Na+, K+, and Cl- corresponding to PNa/PK = 0.3 and PNa/PCl = 3, respectively. This channel showed an increased open probability at depolarizing potentials, but its activity was independent of the Ca2+ concentration in both the cis and trans compartments. These findings were subsequently confirmed in inside-out patch clamp experiments carried out on primary cultured proximal tubule cells. This channel was seldom seen in cell-attached recordings on resting cells, but its presence was observed with a higher probability in patch excised experiments performed following superfusion of the cells with a hypotonic solution. It is suggested on the basis of these observations that the apical membrane of rabbit proximal tubule cells contains a voltage-dependent nonselective channel, the activity of which may contribute to the regulation of the membrane potential.

Cadmium-induced nephrotoxicity in rhesus monkeys ( Macaca, mulatta) in relation to protein calorie malnutrition

In this study, we compared results obtained in protein calorie malnourished (PCM) monkeys and controls given Cd 2+ (5 mg Cd 2+/kg body wt./day) orally for 24 weeks. After 16 weeks of Cd 2+ exposure, an indolent renal failure develops in PCM monkeys which resulted in significant increase in urinary excretion of total protein, Cd 2+, Zn 2+ and Ca 2+ as compared to corresponding Cd 2+-treated control group. In isolated proximal tubule brush border membrane vesicles (BBMV), Cd 2+, Zn 2+ and Ca 2+ transport were significantly impaired in Cd 2+-exposed PCM monkeys as compared to Cd 2+-treated controls. The mechanism of higher urinary excretion of Cd 2+, Zn 2+ and Ca 2+ was examined by analyzing the kinetic parameters of transport systems. The kinetic studies of Cd 2+, Zn 2+ and Ca 2+ transport systems in the BBMV preparations of Cd 2+-exposed PCM monkeys exhibited a significant decrease in V max and an appreciable increase in K m as compared to Cd 2+-treated controls. These findings suggested that Cd 2+ treatment of PCM monkeys caused either a decrease in the number of transporters in the brush border membrane or an increase in the number of less active transporters for Cd 2+, Zn 2+ and Ca 2+. Furthermore, brush border membrane-bound enzymes, viz. alkaline phosphatase and leucine aminopeptidase, activities were significantly impaired in Cd 2+-exposed PCM monkeys. Cadmium content in kidney cortex of Cd 2+-exposed PCM monkeys was 3.34-fold higher than Cd 2+-exposed controls. These findings also established that Cd 2+ not bound to metallothionein (MT) was significantly higher in Cd-exposed PCM monkeys, which may be an important determinant in renal toxicity by interacting with sensitive sites in the renal cells and causing renal damage in Cd-exposed PCM monkeys.

Chloride and membrane potential dependence of sodium ion-proline symport.

Proline accumulation by renal proximal tubule brush border membrane vesicles is Na+ dependent, but little is known about the role of anions or membrane potential on proline uptake. Recent studies in a variety of transport systems, including rat renal brush border membrane vesicles, indicate that halide anions chloride (Cl-) and bromide (Br-) are essential for glycine, beta-alanine, gamma-aminobutyric acid, and taurine uptake, so the possibility that Na(+)-proline symport is Cl- dependent was explored. Also, the role of membrane potential on transport was assessed by determining the effect of external anions with different membrane permeabilities. The ratio of initial rate Cl- stimulated to thiocyanate (SCN)(-)-stimulated uptake values serves to measure Cl- dependence. The initial rate of proline uptake to equilibrium value was 3.11 +/- 0.5 (SE) in the presence of Cl- versus SCN-. The ratio for D-glucose, whose uptake is governed only by electrogenic status of the membrane, was 0.61 +/- 0.47 (P less than 0.001 versus proline). In another series of experiments, uptake values for various anions as a percent of equilibrium (I/E x 100) were: SCN-, 84.9 +/- 10.9; NO3, 49.9 +/- 11.0; SO4(2-), 27.3 +/- 4.4; F-, 68.5 +/- 18.3; Cl-, 164.1 +/- 44.6; Br-, 150.6 +/- 30.2; I-, 56.7 +/- 13.5. The stoichiometry of uptake by Hill plot analysis of proline uptake in the presence of varying concentrations of Na+ (0 to 100 mM) and Cl- (0 to 100 mM) was 2Na+:1Cl-:1 proline.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary Acid and Alkali Loading Do Not Alter Taurine Uptake by Renal Proximal Tubule Brush Border Membrane Vesicles in Kittens

Kittens adapted to a purified control diet containing 43.5% soy protein plus 0.15% taurine were randomly divided into three groups: control, acid-loaded or alkali-loaded. For dietary acid or alkali loadings, 2% NH4Cl or 0.87% NaHCO3 plus 0.87% KHCO3 were added to the control diet. Acid-loaded, control and alkali-loaded kittens had venous blood pH of 7.33 +/- 0.01, 7.37 +/- 0.02 and 7.39 +/- 0.02, and urine pH of 5.5 +/- 0.1, 7.2 +/- 0.1 and 8.1 +/- 0.1, respectively. After 6 wk of the dietary treatment, the plasma taurine concentrations of acid-loaded, control and alkali-loaded kittens were 74 +/- 4, 77 +/- 7 and 87 +/- 8 mumol/L (P greater than 0.05) respectively. Compared with the control group, dietary acid or alkali loading did not significantly change the taurine uptake by brush border membrane vesicles (BBMV) when BBMV were at the same in vitro pH (7.35). However, when the pH of the medium was changed to 8.0 for the alkali-loaded group or to 5.5 for the acid-loaded group, the initial taurine uptake was significantly elevated (55%) or decreased (31%) (P less than 0.05), respectively, compared with the values for the control group at pH 7.35. When BBMV prepared from the same group were tested at different pH levels (8.0 vs. 7.35 vs. 5.5), the initial uptake significantly increased (30%) at pH 8.0 and decreased (37%) at pH 5.5 compared with that observed at pH 7.35 (P less than 0.05), regardless of the acidity of the diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and characterization of a new fluorescent probe for measuring potassium

A new fluorescent probe, 6,7-(4-methyl)coumaro-[2.2.2] cryptand, has been developed for measuring K+. This compound was made by fusing [2.2.2] cryptand with 4-methylcoumarin through 2 ethoxy bridges at the 6 and 7 positions. The probe has a fluorescent excitation peak at 340 nm and an emission peak at 420 nm. In aqueous solutions, increasing the K+ concentration from 0 to 10 mM causes the fluorescence intensity to increase by 143%. The dissociation constant (Kd) for K+ in aqueous solutions is 1.9 mM. In 100% methanol, the Kd for K+ decreases to 0.012 mM, making it possible to measure K+ concentrations in the micromolar range. Na+, tetramethylammonium, NH4+, Ca2+, and Mg2+ have a minimal affect on the fluorescence of the probe in the absence of K+. The coefficient of variation for the measurement of K+ by use of this new dye is less than 1%. In this report, the synthetic procedure is described and the spectral properties of the probe are characterized. Experiments are described demonstrating the use of this probe 1) in measuring K+ in aqueous solutions from 0 to 10 mM and in a microfluorometric assay to measure K+ from 0.0005 to 0.003 mM and 2) in monitoring K+ transport in rabbit proximal tubule brush-border membrane vesicles.

Effect of Dietary Taurine on Renal Taurine Transport by Proximal Tubule Brush Border Membrane Vesicles in the Kitten

Renal adaptation of the kitten to altered dietary taurine intake was assessed using proximal tubule brush border membrane (BBM) vesicles. Three groups of kittens were adapted to purified diets containing 43.5% soy protein that were either taurine-free (OT) or contained 0.15% taurine (NT) or 1.0% taurine (HT). The plasma taurine concentration of the kittens fed OT decreased from 104 +/- 25 microM to 16 +/- 5 microM and 1.7 +/- 0.5 microM in 1 and 6 wk, respectively. Feeding HT increased plasma taurine concentration to 350 +/- 116 microM in 1 wk. Compared to NT, taurine accumulation by BBM vesicles was significantly elevated after 4 wk of feeding OT and decreased after 2 wk or less of feeding HT (P less than 0.05). Maximum renal adaptation occurred by 6 wk of feeding OT (206% increase in taurine uptake/15 s compared to NT) and by 2 wk or less of feeding HT (43% decrease in taurine uptake/15 s compared to NT). Evaluation of transport kinetics using renal cortex from groups of four kittens (16 determinations) fed NT, OT (12 wk) or HT (10 wk) revealed a Vmax of 55 +/- 10, 123 +/- 24 or 39 +/- 7 pmol.mg protein-1.10 s-1 and a Km of 32 +/- 7, 16 +/- 2 or 37 +/- 8 microM, respectively. The differences in Vmax and Km were significant between NT and OT (P less than 0.05), but not significant between NT and HT (P greater than 0.05). Our results suggest that renal adaptation of the kitten to changes in dietary taurine occurs with modifications of both Vmax and Km of the taurine transport system.

Ionic requirements for amino acid transport.

The reabsorption of amino acids by the proximal tubule is remarkably efficient. Current evidence indicates that this process occurs by Na+-amino acid cotransport or symport. The energy for amino acid entry is derived from the chemical and voltage gradient for Na+ entry across the apical surface of the renal cell maintained by pumping Na+ out of the cell by Na+-K+-adenosine triphosphatase (ATPase) activity at the basolateral membrane. We chose the beta-amino acid taurine to study the anionic requirements as well as voltage- and pH-dependence of Na+-taurine symport into rat proximal tubule brush border membrane vesicles. Maximal uptake was found when Cl- or Br- were the anions. The addition of various ionophores (amiloride, carbonyl cyanide-n chlorophenyl-hydrazone, and valinomycin) under pH-equilibrated conditions did not change taurine entry into the vesicle. Hill equation analysis of the initial rate of taurine uptake into vesicles indicates that transport operates by means of a 2 Na+:1 Cl-:1 taurine-carrier complex. Because taurine is a zwitterion, this complex has a net positive charge. Its entry into the vesicle is favored by the imposition of an outwardly directed K+ gradient in the presence of valinomycin. The movement of a quaternary complex of this type across the apical surface of the proximal tubular cell would assure that the movement of both Cl- and the amino acid is energized by the Na+ gradient. Because most amino acids are zwitterions at physiologic pH this complex would be positively charged, favoring entry into the voltage negative renal cell interior.

Renal adaptation to metabolic acidosis in senescent rats.

In this study, we compared results obtained in senescent rats with young rats given an equivalent acid load. We examined the renal changes by giving equivalent acid loads for 48 h to both 6- and 24-mo-old rats. The basal excretion of ammonium was the same in both groups, whereas titratable acids, phosphate, and Ca2+ excretions were increased in the senescent animal. After administration of the acid load, ammonium, phosphate, Ca2+, and titratable acid excretions increased in both age groups, but there were greater absolute increases in ammonium and titratable acid excretions in the young rats. The total acid excreted by the 24-mo rats was reduced 50 (day 1) and 25% (day 2) compared with the young rats, which was reflected by the more severe acidosis in those animals. The portion of total acid excreted as titratable acids in senescent animals was also increased during acidosis when compared with the young animals. In isolated proximal tubule brush-border membrane vesicles, acidosis increased Na+-H+ exchange and decreased Na+-dependent phosphate transport in both age groups. We also found that the basal activity of the Na+-H+ exchanger was not changed with age but the Na+-dependent phosphate transporter was less in the 24-mo rat. The results suggest that physiological regulation of these renal processes remains intact in the aged rat but the responses may be reduced or delayed in the senescent animal.

Phosphate uptake by renal membrane vesicles of rabbits adapted to high and low phosphorus diets.

Renal adaptation to changes in phosphate intake was studied by comparing phosphate uptake by proximal tubule brush border membrane vesicles from rabbits on a relatively high or low phosphorus diet. The low phosphorus diet increased Na+ gradient-dependent phosphate uptake. Uptake in the absence of Na+ and in the presence of Na+, but no gradient, was not significantly affected. The phosphorus diet did not alter Na+ gradient-dependent D-glucose and L-proline uptake. The low phosphorus diet increased Vmax; affinity for phosphate was not appreciably changed. At all concentrations of extravesicular Na+, phosphate uptake was higher in membrane vesicles from animals fed the low phosphorus diet; the kinetics of the phosphate uptake system, with respect to Na+, was also altered by the change in dietary phosphate. These findings suggest that adaptation involves an alteration in the rate of translocation of the Na+-phosphate carrier when energized by a Na+ gradient driving force rather than a change in the number of Na+-phosphate carrier sites. With membrane vesicles from rabbits fed a low phosphorus diet, phosphate uptake increased several-fold when the pH of the uptake medium was raised, whereas with membrane vesicles from animals fed a high phosphorus diet the enhancement of uptake with alkalinization was relatively small. Irrespective of the diet, divalent phosphate was the probable preferred species for transport. Dietary adaptation was associated, however, with an alteration in the pH dependency of the transport system per se. These findings provide evidence that the adaptation of the kidney phosphate transport system to dietary phosphate load involves an intrinsic change in the Na+-phosphate carrier.

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [ 3H]arachidonate-labeled lipids, Ca 2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca 2+, Mg 2+, and Mn 2+ stimulated diglyceride production while Ba 2+, Zn 2+, Hg 2+, and K + were ineffective. HgCl 2 inhibited Ca 2+-stimulated phosphatidylinositol phosphodiesterase. Mg 2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca 2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1–5 m m) inhibited Ca 2+- or Mg 2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.

Myo-Inositol transport in renal brush border vesicles and it inhibition by D-glucose.

We examined the mechanism of myo-inositol uptake by rabbit renal proximal tubule brush border membrane vesicles and characterized the relationship between the transports of myo-inositol and D-glucose. A 100 mM Na+ electrochemical gradient (extravesicular medium > intravesicular medium) stimulated the initial rate of myo-inositol uptake 20- to 60-fold. Other cation gradients were ineffective. The Na+ myo-inositol co-transport system was shown to be electrogenic. The Na+ electrochemical gradient-dependent uptake of myo-inositol saturated at about 1 mM myo-inositol, with an apparent Km of 94 micro M at an initial 100 mM Na+ gradient. D-Glucose was an inhibitor of the Na+ gradient-dependent uptake of myo-inositol. D-Glucose, but not L-glucose, elicited accelerative exchange diffusion of myo-inositol. myo-Inositol did not significantly inhibit the Na+ gradient-dependent transport of D-glucose. We suggest that D-glucose inhibits myo-inositol uptake by dissipating the membrane potential and sharing the myo-inositol carrier. The inhibition of myo-inositol transport across the brush border membrane by D-glucose explains how glycosuria could produce inosituria in patients with diabetes mellitus.