Rat Renal Cortical Brush Border Research Articles

Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491–5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069–75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na + and d-glucose. Using α- 14 C -methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant ( K 0.5∼2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.

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 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.

Study of rat renal brush border membrane vesicles by flow cytometry

The rich heterogeneity of renal tubular membranes and cells continues to provide formidable challenges in the isolation of homogeneous membrane vesicle populations for study. The present study applies flow cytometry, the technique of fluorescence-activated cell sorting, to the study of brush border membrane vesicles. Direct comparison was made of enzymatic marker purity of rat renal cortical brush border membrane vesicles prepared by divalent ion precipitation, or flow cytometry sorting. Flow cytometry sorted membrane vesicles were characterized by greater brush border membrane markers, no detectable mitochondrial or basolateral markers, and greatly reduced Golgi and lysosomal markers. The flow sorted membrane vesicles were functional for transport studies as they took up at least as much 3H-proline and 3H-glucose per mg protein as divalent ion precipitation membrane vesicles. Preparation of membrane vesicles from superficial and deep cortex allowed us to image the different distributions of gamma-glutamyl transferase in membrane vesicles from these areas. Hence, membrane vesicle populations of exceptional purity can be separated according to fluorescent markers using flow cytometry. High speed observations on large numbers of individual vesicles allows identification of subpopulations, and statistical comparison, within a single heterogeneous sample.

Oxalate:OH exchange across rat renal cortical brush border membrane

We demonstrated the presence of oxalate:OH exchange in rat renal brush border membrane. Transient concentrative uptake of oxalate ("overshoot") was observed in the presence of an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient, but this pH gradient-stimulated oxalate uptake was abolished by 1 mM DIDS, indicating that DIDS-sensitive and -insensitive oxalate uptake mechanisms were present. The DIDS-sensitive oxalate uptake was temperature-dependent and saturable with a Km of 0.0365 mM and a Vmax of 1.38 nmol.30 second-1.mg protein-1. In addition, oxalate was transported into the osmotically active internal space. In the presence of the pH gradient, a change in transmembrane potential had no effect on pH gradient-stimulated oxalate uptake. Oxalate was exchanged for OH and this exchange was sensitive to inhibition by DIDS. Inhibition by DIDS, furosemide and probenecid facilitated the distinction of oxalate:OH exchange from formate:Cl and oxalate:Cl exchange. In preparation of our brush border membrane vesicles, no apparent SO4:HCO3 exchange was present. These data indicate that oxalate:OH exchange occurs on the brush border membrane.

Structural requirement of monophosphates for inhibition of Na +-P i cotransport in renal brush border membrane

Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for competitive inhibition of Na +-P i cotransport in rat renal cortical brush border membrane (BBM) vesicles (BBMV). The effects of compounds on [Na + 0 >Na i +]-gradient-dependent 32P i uptake by BBMV were examined using various inhibitor-to- 32P i concentration ratios in the transport assay medium. The replacement of a phosphono-group with an arsono-group in PAA, or the substitution of a carboxylic group in PAA by an amino or hydroxyl group, totally abolished the inhibitory action on Na +-P i cotransport. Decreased electro-negativity of carboxyl in PAA by coupling with hydrazine or hydroxylamine lowered the inhibitory potenty of PAA. Substitution of H at the α-carbon of PAA with ethyl or p-Cl-phenyl groups completely abolished the inhibitory activity, whereas α-halogenation with Br greatly increased the inhibitory potency of PAA, close to that of phosphonoformic acid (PFA). The inhibition by all the active tested monophosphates was strictly competitive. The tested compounds displaced [ 14C]PFA pre-bound onto BBMV in the presence of 100 mM NaCl. The ability of monophosphates to inhibit Na +-P i cotransport across BBM and the binding of [ 14C]PFA were closely correlated ( r = 0.925; P < 0.001). These results show that: (a) strong electronegativity at both ends of the PAA molecule is needed for inhibitory action, (b) an α-aliphatic or aromatic substituent at the α-carbon probably hinders the acess of the inhibitor to the P i-binding site of the Na +-P i cotransporter in BBM, whereas (c) an α-electrophilic substituent—Br—enhances the inhibitory potency of PAA. The tested compounds inhibited Na +-P i cotransport by binding, in the presence of Na +, on the same site on the luminal surface of BBM as did PFA and, by extension, P i.

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.

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: role of cholesterol.

Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104 vs. 0.60, P less than 0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35 degrees C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r infinity) (0.212 vs. 0.154, P less than 0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-AS probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vitamin D-induced chronic hypercalcemia on rat renal cortical plasma membranes and mitochondria.

Increases in intracellular and mitochondrial calcium content that accompany ischemic and toxic acute renal failure have been suggested to mediate renal tubular cell injury and dysfunction, but the mechanism(s) are unknown. We studied the effects of in vivo vitamin D-induced chronic hypercalcemia on rat renal cortical brush-border and basolateral membranes and mitochondria. In the brush-border membrane, hypercalcemia caused significant decreases in alkaline phosphatase-specific activity, total phospholipid molar content, and phosphatidylserine percent molar composition and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol percent molar composition. In the basolateral membrane, hypercalcemia caused significant decreases in Na+-K+-ATPase-specific activity and total phospholipid molar content and increases in the cholesterol-to-total phospholipid molar ratio and phosphatidylinositol 4,5-bisphosphate percent molar composition. In the mitochondria, hypercalcemia caused a mild increase in the mitochondrial calcium content, but no alterations in succinic dehydrogenase-specific activity, succinate-, ADP-, or uncoupler-induced respiration. Thus hypercalcemia caused alterations in brush-border and basolateral membrane enzyme activity and lipid composition, but no functional changes were detected in mitochondria. These hypercalcemia-induced plasma membrane biochemical alterations may be markers of early cell injury and suggest a role for calcium in causing or predisposing to renal tubular cell injury.

Maintenance of epithelial surface membrane lipid polarity: a role for differing phospholipid translocation rates.

Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using 32P and either [3H]-glycerol or [2-3H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies, again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 X faster than into the brush border membrane and its relative composition was 2.5 X greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.

Procainamide transport in rabbit renal cortical brush border membrane vesicles.

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