Cortical Brush-border Membrane Vesicles Research Articles

Phenol injury-induced hypertension stimulates proximal tubule Na+/H+ exchanger activity

Injection of 50 microl 10% phenol into rat renal cortex activates renal sympathetic nerve activity which provokes acute hypertension that persists for weeks. We have previously shown with membrane fractionation that phenol injury caused a redistribution of the main proximal tubule (PT) apical transporter NHE3 (Na+/H+ exchanger isoform 3) to low density membranes enriched in apical microvilli. The aim of this study was to determine whether phenol injury increases PT apical Na+/H+ exchanger (NHE) activity. NHE activity was measured in vivo as the initial rate of change in intracellular pH (dpH(i)/dt) during luminal Na+ removal in PT preloaded with the pH-sensitive fluorescence dye BCECF. Injection of 50 microl 10% phenol increased blood pressure from 113 +/- 5.2 to 130 +/- 4.6 mmHg without changing glomerular filtration rate or urine output. NHE activity increased 2.6-fold by 70 min after phenol injury. The increase of NHE activity was accompanied with an increase of tubular reabsorption. Total NHE activity/NHE3 protein in cortical brush-border membrane (BBM) vesicles, measured by acridine orange quench and immunoblot, respectively, was unchanged by phenol injury. In conclusion, acute phenol injury provokes coincident increases in PT apical NHE activity, redistribution of NHE3 into low density apical membranes, and hypertension. The increase in NHE activity may contribute to the lack of pressure-diuresis and the maintenance of chronic hypertension in this model.

Kinetic characteristics of folate binding to rat renal brush border membrane in chronic alcoholism

The binding of folic acid to the plasma membrane is an important event for its reabsorption and conservation by renal epithelium. We studied [3H] folic acid binding to the renal cortical brush border membrane vesicles (BBMV) in rats after 12 weeks of chronic ethanol ingestion. Our results demonstrated that after chronic ethanol intake, the binding of folic acid to the membrane vesicles: (1) was decreased by a mechanism that decreased B(max) without affecting K(d), (2) was insensitive to Na+ ions in the medium (3) behaved differently to divalent cations in the medium in contrast to control group. However under such conditions there was no significant effect of ethanol on pH optimum of the process. Binding was reduced at pH less than 6 but there was no effect of ethanol on binding in pH range from 6 to 8. Increasing the osmolarity at pH 5.5 had no effect on the binding of folate to BBMV, thus confirming that the observed changes in B(max) values were due to site-specific binding in the two groups. Moreover, ethanol does not impart its effect on involvement of sulfhydryl group at the binding site of folate transport system. These findings highlight the possible mechanism of perturbed renal conservation of folate during chronic alcoholism.

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 acute increases in filtered [formula omitted] on renal hydrogen transporters: II. H+-ATPase

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-ATPase activity was assessed in BBMV obtained from hydropenic and plasma expanded Munich-Wistar rats, using a NADH-linked ATPase assay. H(+)-ATPase activity was measured as the ouabain and oligomycin-insensitive, bafilomycin A1-sensitive component of total ATPase activity. Acute plasma expansion doubled single nephron FLHCO3, and this change was associated with a 64% increase in the Vmax for H(+)-ATPase activity, with no change in apparent Km. The Vmax for H(+)-ATPase activity correlated directly with whole kidney GFR and FLHCO3 (r = 0.68 and 0.72, respectively), and with single nephron GFR and FLHCO3 (r = 0.76 and 0.80, respectively). Thus, the mechanism for the adaptive increase in proximal tubular HCO3- reabsorption that occurs in response to acute increases in FLHCO3 appears to be related to increased activity of both H(+)-ATPase and Na+/H+ exchange in the apical membrane of the proximal tubule epithelium.

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 type II Na/Pi-cotransporter is strongly impaired whereas the Na/sulphate-cotransporter and aquaporin 1 are unchanged in cadmium-treated rats.

The cellular mechanisms of cadmium (Cd) nephrotoxicity are poorly understood. In this study we investigated the cellular causes of the Cd-induced phosphaturia in the rat. Compared to controls, Cd-treated rats (2 mg Cd/kg body weight, s.c. for 14 days) showed a marked polyuria, proteinuria and phosphaturia. As studied by the rapid filtration technique in isolated cortical brush-border membrane vesicles (BBMV), Na+-gradient-driven uptake of phosphate ([32Pi]) and of [3H] glucose were markedly decreased in Cd-treated rats, whereas uptake of sulphate ([35S]) remained unchanged. By Western blotting of BBMV proteins and by indirect immunocytochemistry in 4-micron thick frozen fixed kidney sections, using an antibody against the type II Na/Pi-cotransporter (NaPi-2), we found a diminished expression of this protein in the brush-border membrane from Cd-treated rats. How ever, the expression of the water channel aquaporin 1, estimated from the specific antibody staining in brush-border membranes, remained unchanged by Cd. Northern blot analysis showed a strong reduction of 2.7 kb NaPi-2-related mRNA in Cd-affected kidneys. Our data indicate that: (1) Cd may reduce reabsorption of Pi in proximal tubules by affecting the expression of the functional Na/Pi-cotransporters in the luminal membrane, and (2) Cd effects on brush-border transporters are selective.

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.

Identification of two unique polypeptides from dog kidney outer cortex and outer medulla that exhibit different Na +/ d-glucose cotransport functional properties

The cloned Na +/ d-glucose cotransporter SGLT1 and an additional recently isolated human kidney cDNA Hu14/K15 belong to a family of similar cotransport proteins including the Na +-dependent nucleoside and Na +-dependent myo-inositol carrier SMIT1. For the present study we used two different polyclonal antibodies raised against the amino acid sequence 402–420 (Ab-E) and 565–574 (Ab-P) of SGLT1 to probe brush-border membrane fractions from different regions ( outer cortex a ̊ outer medulla ) of dog kidney. In Western blots both Ab-E and Ab-P react specifically (peptide blockable) with two distinct bands migrating on SDS-PAGE under reducing conditions at 75.5 kDa and 72.5 kDa. The higher molecular mass polypeptide is greatly enriched (13:1) in outer cortex and diminishes progressively towards outer medulla, whereas the lower molecular mass band is barely detectable in outer cortex but is enriched in outer medulla (4:1). Brush-border membrane vesicles (BBMV) prepared from the same outer cortical and outer medullary regions that were probed with Ab-E and Ab-P exhibit strikingly different Na +/ d-glucose functional transport behavior. The Na +/ d-glucose cotransport activity in outer cortical BBMV is a low-affinity system with K m  5.98 ± 1.01 mM, V max  13.05 ± 0.55 nmol/mg protein per min, and with 1:1 Na +: d-glucose stoichiometry. Outer medulla BBMV exhibit high-affinity K m  0.27 ± 0.03 mM V max  0.97 ± 0.04 nmol/mg protein per min and 2:1 Na +: d-glucose stoichiometry. Comparison of SGLT1, Hu14/K15, SNST1 and SMIT indicates that Ab-E could cross react with all four, but Ab-P would recognize SGLT1, Hu14/K15, SNST1 but not SMIT. Also SNST1 is not expressed in outer cortex. Based on currently available sequence data, and its marked enrichment in outer cortex, the 75.5 kDa band is a likely candidate protein responsible for low-affinity and 1:1 Na +: d-glucose stoichiometric Na +/ d-glucose cotransport activity (Hu14/K15) while the minor 72.5 kDa band in outer cortex is probably SGLT1. In outer medulla, the predominant band recognized by both Ab-E and Ab-P is the 72.5 kDa protein and this could be either SGLT1 or SNST1.

Effect of thiazide on urinary citrate excretion

In order to study the effect of thiazide on urinary citrate excretion, we measured sodium dependent citrate uptake by rat renal cortical brush border membrane vesicles (BBMVs) and analyzed acid-base balance with or without thiazide treatment. 5-weeks male Wistar rats (Jcl) were housed in metabolic cages, given 0.1 mg/100 g BW/day hydrochlorothiazide (HCTZ) by a gastric tube once a day for 3 weeks (thiazide treated rats). Arterial blood was taken from aorta and BBMVs were prepared with divalent cation precipitation method. Citrate uptake was measured by a Millipore rapid membrane filtration technique. Oral treatment of HCTZ for 3 weeks showed a significant increase of urinary citrate excretion in both concentration and daily excretion. The sodium dependent citrate transport was observed in rat renal brush border membrane (BBM). But no significant effect of oral HCTZ was observed on acid base balance and sodium dependent citrate uptake in rat renal BBM. These data suggested that the stimulative effect of thiazide on citrate excretion was not via sodium dependent citrate transport in rat renal BBM.

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.

Nucleoside transport in cultured LLC-PK epithelia

The transport of nucleosides by LLC-PK 1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent K m ≈34 μM at 22°C) and inhibited by > 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK 1 cells exhibited both a high affinity for cytidine (apparent K i ≈65 μM for influx) and differential ‘mobility’ of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK 1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 μM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the NI sodium-dependent nucleoside carrier in bovine rental outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27–33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na +-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK 1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.

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.

Comparison of GABA uptake by brain and kidney preparations

The uptake ofγ-aminobutyric acid (GABA) was measured in rat cerebral cortical synaptosomes and rat kidney brush-border membrane vesicles (BBMV). Three GABA uptake systems (Km = 1.3, 50 and 3246µM respectively) were present in synaptosomes, but only two uptake systems (Km = 11 and 1203µM respectively) were detectable in BBMV. The uptake systems in the two types of tissue preparations were similar in that every system was inhibited byp-hydroxymercuribenzoate and by the action of neuraminidase, thereby indicating that, irrespective of the tissue, both sulfhydryl and sialyl groups were necessary components of all the GABA uptake systems. In contrast, differences were observed between synaptosomal and BBMV uptake systems with respect to their sensitivity to inhibition by GABA structural analogs. While the synaptosomal GABA uptake systems were strongly inhibited by nipecotic acid, diaminobutyric acid,β-alanine and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), none of these compounds significantly inhibited the BBMV uptake systems. It is therefore concluded that the GABA uptake systems in brain and kidney tissues are quite different entities. While the role of GABA transport in the inactivation of the neurotransmitter function of GABA is well documented, the role of the kidney GABA uptake system is unclear, and the possibility exists that the latter systems are present in kidney tissues primarily to transport compounds other than GABA. The present study does however highlight a difference in drug susceptibility of the brain and kidney uptake systems, a phenomenon which may have some therapeutic relevance with respect to the use of GABA analogs as anticonvulsant agents.

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.