Thiamin uptake by the human-derived renal epithelial (HEK-293) cells: cellular and molecular mechanisms.
Thiamin (vitamin B(1)) is essential for normal cellular functions. The kidneys play a critical role in regulating body thiamin homeostasis, by salvaging the vitamin via reabsorption from the glomerular filtrate, but little is known about the mechanism(s) and regulation of thiamin transport in the human renal epithelia at cellular and molecular levels. Using the human-derived renal epithelial HEK-293 cells as a model, we have addressed these issues. Our results showed [(3)H]thiamin uptake to be 1) temperature and energy dependent but Na(+) independent, 2) pH dependent with higher uptake at alkaline/neutral buffer pH compared with acidic pH, 3) saturable as a function of concentration over the nanomolar (apparent K(m) = 70.0 +/- 18.4 nM) and micromolar (apparent K(m) = 2.66 +/- 0.18 microM) ranges, 4) cis-inhibited by unlabeled thiamin and its structural analogs but not by unrelated organic cations, 5) trans-stimulated by unlabeled thiamin, and 6) competitively inhibited by amiloride with an apparent K(i) of 0.6 mM. Using a gene-specific small-interference RNAs (siRNAs) approach, human thiamin transporters 1 and 2 (hTHTR-1 and hTHTR-2) were both found to be expressed and contributed toward total carrier-mediated thiamin uptake. Maintaining the cells in thiamin-deficient medium led to a significant (P < 0.01) and specific upregulation in [(3)H]thiamin uptake, which was associated with an increase in hTHTR-1 and hTHTR-2 protein and mRNA levels as well as promoter activities. Uptake of thiamin by HEK-293 cells also appeared to be under the regulation of an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time that thiamin uptake by HEK-293 cells is mediated via a specific pH-dependent process, which involves both the hTHTR-1 and hTHTR-2. In addition, the uptake process appears to be under the regulation of an intracellular Ca(2+)/CaM-mediated pathway and also adaptively upregulated in thiamin deficiency via transcriptional regulatory mechanism(s) that involves both the hTHTR-1 and hTHTR-2.
Ashokkumar
Balasubramaniem
B
Department of Medicine, University of California, Irvine, CA, USA.
Vaziri
Nosratola D
ND
Said
Hamid M
HM
D001483:Base Sequence; D001692:Biological Transport; D002455:Cell Division; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics; D013831:Thiamine / Q000378:metabolism*; D019459:Urothelium
D017931:DNA Primers; D026901:Membrane Transport Proteins; D034741:RNA, Small Interfering; C120043:SLC19A2 protein, human; C492291:SLC19A3 protein, human; D013831:Thiamine