TY - JOUR
T1 - Transport of thiamin in rat renal brush border membrane vesicles
AU - Gastaldi, Giulia
AU - Cova, Emanuela
AU - Verri, Anna
AU - Laforenza, Umberto
AU - Faelli, Alide
AU - Rindi, Gianguido
PY - 2000
Y1 - 2000
N2 - Background. Unlike intestinal absorption, renal transport of thiamin has received little attention. This study was designed to investigate the reabsorptive steps of thiamin transport in brush border membrane vesicles (BBMVs) from rat kidney proximal tubules using tritiated thiamin with a high specific activity. Methods. BBMVs prepared from the cortex kidney of rats were suspended in different media, controlling the composition of the intravesicular fluid by prolonged equilibration at 4°C in preincubation buffers of desired composition. Then they were held on ice until used, when they were warmed at 25°C for the uptake experiments. The amount of radioactivity taken up by the vesicles was measured radiometrically after separation with a rapid-filtration procedure. Results. The time course profile of thiamin uptake was Na+ independent; 53% of thiamin taken up was membrane bound. The concentration curve had a biphasic course that was nonlinear (saturable) at physiological concentrations (+ gradient (pH(in) 6:pH(out) 7.5), which caused a transient accumulation of thiamin inside BBMVs against a concentration gradient. The enhanced thiamin uptake was only due to the H+ gradient, which made thiamin binding virtually negligible compared with translocation, and maintained the biphasic course of the concentration curve. The saturable component, however, had kinetic constants 12-fold higher than those in the absence of gradient. Moreover, the saturable component was inhibited by nonlabeled thiamin and its structural analogues, by inhibitors of intestinal thiamin/H+, renal guanidine/H+, and Na+/H+ antiporters, while it remained unmodified by some typical organic cations transported in renal BBMVs. Conclusion. The results provide strong evidence for the presence in renal BBMVs of a thiamin/H+ antiport having a 1:1 stoichiometric ratio.
AB - Background. Unlike intestinal absorption, renal transport of thiamin has received little attention. This study was designed to investigate the reabsorptive steps of thiamin transport in brush border membrane vesicles (BBMVs) from rat kidney proximal tubules using tritiated thiamin with a high specific activity. Methods. BBMVs prepared from the cortex kidney of rats were suspended in different media, controlling the composition of the intravesicular fluid by prolonged equilibration at 4°C in preincubation buffers of desired composition. Then they were held on ice until used, when they were warmed at 25°C for the uptake experiments. The amount of radioactivity taken up by the vesicles was measured radiometrically after separation with a rapid-filtration procedure. Results. The time course profile of thiamin uptake was Na+ independent; 53% of thiamin taken up was membrane bound. The concentration curve had a biphasic course that was nonlinear (saturable) at physiological concentrations (+ gradient (pH(in) 6:pH(out) 7.5), which caused a transient accumulation of thiamin inside BBMVs against a concentration gradient. The enhanced thiamin uptake was only due to the H+ gradient, which made thiamin binding virtually negligible compared with translocation, and maintained the biphasic course of the concentration curve. The saturable component, however, had kinetic constants 12-fold higher than those in the absence of gradient. Moreover, the saturable component was inhibited by nonlabeled thiamin and its structural analogues, by inhibitors of intestinal thiamin/H+, renal guanidine/H+, and Na+/H+ antiporters, while it remained unmodified by some typical organic cations transported in renal BBMVs. Conclusion. The results provide strong evidence for the presence in renal BBMVs of a thiamin/H+ antiport having a 1:1 stoichiometric ratio.
KW - BBMV
KW - Proximal tubules
KW - Renal transport
KW - Thiamin/H antiport
KW - Vitamin
UR - http://www.scopus.com/inward/record.url?scp=0034062304&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034062304&partnerID=8YFLogxK
U2 - 10.1046/j.1523-1755.2000.00053.x
DO - 10.1046/j.1523-1755.2000.00053.x
M3 - Article
C2 - 10792623
AN - SCOPUS:0034062304
VL - 57
SP - 2043
EP - 2054
JO - Kidney International
JF - Kidney International
SN - 0085-2538
IS - 5
ER -