Control Brush Border Membrane Vesicles Research Articles

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Effect of platinum coordination complex (PtCx) on citrate uptake by rat renal brush border membrane vesicles (BBMV): direct effect of carboplatin.

Inhalation of platinum, as soluble salts, is known to cause respiratory distress and severe dermatitis in workers. Platinum coordination complexes are widely used in the treatment of a variety of solid tumors. However, the clinical use of cisplatin (CDDP) (the most useful agent) is limited by the development of nephrotoxicity. High dose accidental exposure to soluble platinum in platinum refineries and pharmaceutical factories could induce occupational nephrotoxicity. Carboplatin (CBDCA), a second-generation platinum coordination complex, is highly effective against a variety of malignancies at doses five- to ten-times higher than CDDP. At therapeutic doses, CBDCA is less nephrotoxic than CDDP. Additionally, urinary citrate is freely filtered at the glomerulus, and its reabsorption in the proximal tubule is the major determinant of the rate of renal excretion. In our previous study, the preincubation of rat renal brush border membrane vesicles (BBMV) with 5 mM cisplatin for 4 and 8 hours significantly inhibited the citrate uptake compared with that of the control BBMV. In this study, we exposed BBMV to 100 mM carboplatin (twenty-times higher concentration than cisplatin) and examined the citrate uptake characteristics to clarify the toxic mechanism of platinum coordination complexes. The preincubation of BBMV with 100 mM carboplatin for 8 hours also significantly inhibited the citrate uptake compared with that of the control BBMV, but the alterations were not as severe as those with 5 mM cisplatin.

Effect of platinum coordination complex (PtCx) on citrate uptake by rat renal brush border membrane vesicles (BBMV): cisplatin-intoxicated rats.

Platinosis with severe dermatitis and/or asthma is broadly defined as the effects of soluble platinum salts on people exposed to them occupationally. Platinum coordination complexes are widely used in the treatment of a variety of solid tumors. However, the clinical use of cisplatin, the most useful agent, is limited by the development of nephrotoxicity. Thus, an accidental exposure to soluble platinum at a high dose in platinum refineries and pharmaceutical factories could induce occupational nephrotoxicity. Urinary citrate is freely filtered at the glomerulus, and its reabsorption in the proximal tubule is the major determinant of the rate of renal excretion. In our previous studies, we found that the preincubation of rat renal brush border membrane vesicles (BBMV) with cisplatin and carboplatin, a second-generation platinum coordination complex, significantly inhibited the citrate uptake compared with that of the control BBMV. In this study, we performed in vivo experiments in cisplatin-intoxicated rats to elucidate the toxic mechanism of cisplatin. And our results showed that the citrate uptake was significantly inhibited in cisplatin-intoxicated rats at 1 min compared with that of control rats.

Effect of platinum coordination complex (PtCx) on citrate uptake by rat renal brush border membrane vesicles (BBMV): direct effect of cisplatin.

Platinosis (severe dermatitis and/or asthma) may be broadly defined as the effects of soluble platinum salts on people exposed to them occupationally. Platinum coordination complexes are widely used in the treatment of a variety of solid tumors. However, the clinical use of cisplatin (the most useful agent) is limited by the development of nephrotoxicity. High dose exposure of soluble platinum could possibly induce nephrotoxicity, occupationally. Urinary citrate is freely filtered at the glomerulus, and its reabsorption in the proximal tubule is the major determinant of rate. In this study, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cisplatin and examined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. The preincubation of BBMV with 5 mM of cisplatin for 4 and 8 hours significantly inhibited citrate uptake compared with that of the control BBMV. These findings might contribute to nephrotoxicity in cisplatin therapy.

Direct effect of inorganic mercury on citrate uptake by isolated rat renal brush border membrane vesicles.

Occupational exposure to mercury has long been associated with renal proximal injury and an increased incidence of proteinuria, as has such exposure to cadmium. Renal citrate excretion is very important with respect to acid-base balance since the metabolism of citrate generates three bicarbonate ions. In this study, we exposed isolated rat renal brush border membrane vesicles (BBMV) to mercury (Hg2+) and examined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration method. The preincubation of BBMV with 0.5 and 2 mM HgCl2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Hg preincubation. The analysis of the time course of citrate uptake during a 30-min preincubation of BBMV with 0.1 mM Hg2+ also revealed a significant reduction in the uptake compared with that of the control BBMV without preincubation. These findings indicate that the preincubation of BBMV with mercury results in a time- and concentration-dependent inhibition of citrate uptake.

Direct effect of cadmium on citrate uptake by isolated rat renal brush border membrane vesicles

High incidence of multiple kidney stone formation has been observed among workers exposed to cadmium (Cd). Citrate is known to be a protective factor against renal stone formation. To study the direct effect of cadmium on citrate uptake by the renal brush border membrane, we exposed isolated rat renal brush border membrane vesicles (BBMV) to cadmium and determined their citrate uptake characteristics. BBMV were prepared by the divalent cation precipitation method. Citrate uptake was measured by the Millipore rapid membrane filtration technique. Preincubation of BBMV with 2 and 10 mM CdCl 2 for 1 min significantly inhibited citrate uptake compared with that of BBMV without Cd. Analysis of the time course of citrate uptake during 30-min preincubation of BBMV with 0.5 mM Cd also revealed significant reduction of the uptake compared with that of the control BBMV without preincubation. These findings indicate that preincubation of BBMV with cadmium results in time-dependent and concentration-dependent inhibition of citrate uptake.

Phosphate transport in brush border membranes from uremic rats. Response to phosphonoformic acid.

Phosphate retention in chronic renal failure may result in the development of secondary hyperparathyroidism and renal osteodystrophy. Thus, the addition of a specific phosphaturic agent may be beneficial in the treatment of chronic renal failure. Phosphonoformic acid (PFA), a specific and competitive inhibitor of Na(+)-Pi cotransport in renal and intestinal brush border membrane vesicles (BBMV), can induce phosphaturia in thyroparathyroidectomized (TPTX) rats. The aim of this study was to determine if PFA retains its inhibitory activity in uremic intestine and kidney. The effect of PFA, and its derivative phosphonoacetic acid (PAA), added in vitro, on Pi transport in BBMV prepared from the intestine and the remnant kidney of 5/6 nephrectomized (NX) rats was studied. In intestinal BBMV, the time course of Pi transport was not significantly different between NX and sham-operated (SH) control rats. Compared with SH, Na(+)-dependent Pi transport was reduced in BBMV from remnant kidney of NX, with no difference in Na(+)-independent or equilibrium uptakes. The reduced transport was specific for Pi with no change in Na+ gradient-dependent L-proline uptake, suggesting a normal Na+ conductance in uremic BBM. PFA and PAA produced a marked inhibition of Na(+)-Pi cotransport in intestinal and renal BBMV from NX and SH with similar relative inhibitory potency in uremic and control BBMV. It was concluded that the relative inhibitory potency of PFA or PAA on intestinal and renal Na(+)-Pi cotransport is preserved in uremia.

60 ENTEROPATHOGENIC E. COLI (EPEC) DISRUPT SMALL INTESTINAL BRUSH BORDER MEMBRANCE Na+/H* EXCHANGE

Despite a highly specific morphological lesion of the small intestinal brush border membrane, the mechanisms whereby EPEC produce diarrhoea are unknown; mucosal invasion and toxin production are not thought to be important factors. We have therefore studied the effects of the rabbit EPEC strain, RDEC-1, upon Na+/H+ exchange at 60 sec in rabbit small intestinal brush border membrane vesicles (BBMV), following incubation at 37°C with RDEC-1. Before incubation, BBMV showed a 3.4-fold enhancement of Na+ uptake in the presence of an outwardly directed H+ gradient (p<0.0001), indicating intact Na+/H+ exchange. However, after 60 min incubation with RDEC-1; H+-stimulated Na+ uptake was markedly reduced to 43% of original enhancement (p<0.01) but reduced to only 90% in control BBMV (NS).

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, amiloride analogues, and renal Na+/H+ antiporter.

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a carboxyl-activating agent and has been shown to inhibit the renal Na+/H+ antiporter. The purposes of the present studies were to characterize the kinetics of inhibition of the Na+/H+ antiporter by EEDQ and to determine whether amiloride analogues affect the ability of EEDQ to inhibit the rate of Na+/H+ exchange. Brush-border membrane vesicles (BBMV) were prepared from rabbit kidneys; Na+/H+ exchange rate was assessed by the fluorescence quenching of acridine orange. EEDQ produced a concentration-dependent inhibition of Na+/H+ exchange; the effect was to decrease the maximum activity (Vmax) from 5.51 to 2.03 fluorescence units X mg protein-1 X S-1) and Km (from 14.1 to 8.7 mM) compared with control BBMV. Pretreatment of BBMV with amiloride before the addition of EEDQ maintained both Vmax and Km at values that were not significantly different from those for control BBMV. Compared with a series of analogues, amiloride was only the third most potent inhibitor of the rabbit renal Na+/H+ antiporter; amiloride, however, provided the greatest protection against inhibition of the antiporter by the subsequent addition of EEDQ. These findings suggest that the 2-carbonylguanidininum moiety and 6-chloro atom are important for binding of amiloride to sites at or near the antiporter; the group at position 5 is important in determining the ability of amiloride to protect against inhibition of the Na+/H+ antiporter by EEDQ. Finally, the ability of amiloride to protect against inactivation of the renal Na+/H+ antiporter by EEDQ is reversible.

Effect of ethanol on intestinal calcium transport in chicks

To test the hypothesis that chronic ethanol exposure would alter the membrane fluidity of the intestinal brush-border membrane, which would lead to calcium malabsorption, we gave chicks 15% ethanol in their drinking water from hatching to 3 or 4 wk of age. Although such chicks grew less quickly than their hatchmates not ingesting ethanol, their ability to absorb calcium from the duodenum in vivo was unimpaired. However, when calcium accumulation by isolated duodenal brush-border membrane vesicles (BBMVs) was assessed, the BBMVs from chicks ingesting ethanol for 1–3 wk had a lower rate of uptake than the BBMVs from the controls at all calcium concentrations evaluated (0.1–25 mM). This difference could not be explained by differences in membrane fluidity as assessed by fluorescence depolarization, or by changes in intravesicular volume. Glucose uptake was not affected. The acute addition of ethanol (up to 1 M) in vitro to the control BBMVs increased both membrane fluidity and calcium accumulation. No difference in the fluidizing effect of ethanol in BBMVs between ethanol-ingesting chicks and control chicks could be demonstrated, although the acute effect of ethanol on calcium accumulation was blunted in the BBMVs from chicks ingesting ethanol. Increasing the temperature of the incubation medium also increased membrane fluidity and calcium accumulation in BBMVs from control and ethanol-ingesting chicks, with a greater increase in calcium uptake by the control BBMVs despite comparable increases in fluidity in BBMVs from the control and ethanol-ingesting chicks. We conclude that the chronic ingestion of ethanol by chicks, although markedly altering growth rates, has minimal impact on the intestinal absorption of calcium when assessed in vivo. However, chronic ethanol ingestion does appear to alter the intestinal brush-border membrane to make it less permeable to calcium and less susceptible to the stimulation by ethanol of calcium flux across this membrane; this adaptation may prevent increased flux of calcium across the brush-border membrane into the cell in the presence of ethanol.

Phosphate transport by jejunal brush border membrane vesicles of the streptozocin-diabetic rat.

We studied the effects of acute diabetes mellitus on jejunal transport of phosphate (32P) by rat brush border membrane vesicles (BBMV) using a Millipore filtration technique. Diabetes was induced by an intravenous (i.v.) injection of 50 mg/kg of streptozocin (STZ). Control and diabetic rats were studied 4 days after the induction of diabetes. In both control and diabetic rats, the presence of a sodium gradient significantly enhanced the uptake of 32P at 20 s and at 1, 2, 5, and 60 min as compared with potassium gradient conditions. Na+-dependent 32P uptake at 20 s and at 1 and 2 min was significantly greater in the diabetic BBMV compared with controls. Na+-independent 32P uptake in both diabetic and control BBMV was similar. To determine whether the enhancement of Na+-dependent 32P uptake in diabetic BBMV is due to an induction of Na+/phosphate cotransporter activity, or a change in Na+ permeability, two additional studies were conducted. Trans-stimulation studies nullifying all electrochemical gradients across the membranes were performed. In diabetic BBMV, 32P uptake at all time points was significantly greater than corresponding values in controls, indicating an increase in the activity of Na+/phosphate cotransporters. 22Na uptake into BBMV at 30 s and at 1, 2, and 60 min was not different between diabetics and controls, indicating that Na+ permeability is not altered in diabetes. Furthermore, kinetic studies using phosphate concentrations between 0.05 and 2.5 mM indicate a significant increase in Vmax capacity of diabetic rats compared with controls without a change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of decreased Na +-dependent d-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95–101) we reported that the Na +-dependent d-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. d-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of l-glucose efflux from brush-border membrane vesicles. d-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na + concentrations and zero potential. Kinetic analyses of Na +-dependent d-glucose flux indicate that V max in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 ± 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 ± 9.9 nmol/min per mg protein), while the K m values in the two cases are not different from each other (22.3 ± 0.9 and 27.4 ± 1.8 nM, respectively). These results suggest that Na +-dependent d-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na +-dependent d-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.