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3 Medical Research Service,
Riboflavin (RF), a water-soluble vitamin, is essential for
normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the
human situation, are poorly understood. The aim of this study was
therefore to address these issues, using a recently established human
normal renal epithelial cell line, HK-2, as a model. Uptake of RF by
HK-2 cells was found to be 1) linear
with time for 5 min of incubation and occurring with minimal metabolic
alterations, 2) temperature
dependent, 3)
Na+ independent,
4) saturable as a function of
concentration [apparent Michaelis constant
(Km) of 0.67 ± 0.21 µM and maximal velocity (Vmax) of 10.05 ± 0.87 pmol · mg
protein
1 · 3 min1],
5) inhibited by structural analogs
and anion transport inhibitors, and
6) energy dependent. Protein kinase
C-, protein kinase A-, and protein tyrosine kinase-mediated pathways
were found to have no role in regulating RF uptake. On the other hand,
a Ca2+/calmodulin-mediated pathway
appeared to play a role in the regulation of RF uptake by HK-2 cells
via an effect on the
Vmax, as well as
on the apparent
Km of the RF
uptake process. The uptake process of RF was also found to be
adaptively regulated by the level of the substrate in the growth
medium, with the effect being mediated through changes in the apparent
Km and the
Vmax of the
uptake process. These results demonstrate that RF uptake by the
human-derived renal epithelial cell line HK-2 is via a carrier-mediated
system that is temperature and energy dependent and appears to be under the regulation of a
Ca2+/calmodulin-mediated pathways
and substrate level in the growth medium.
uptake regulation
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