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Mechanism and regulation of thiamin uptake in human‐derived renal epithelial cells
Author(s) -
Balasubramaniem Ashokkumar,
Vaziri Nosratola D,
Said Hamid M
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a841-b
Subject(s) - thiamine , chemistry , intracellular , small interfering rna , vitamin , transporter , amiloride , biochemistry , microbiology and biotechnology , sodium , transfection , gene , biology , organic chemistry
Thiamin (vitamin B1) is essential for cellular function, growth and development. Little is known about the molecular mechanism(s) and regulation of renal proximal tubular thiamin uptake. Using the human‐derived renal epithelial HEK‐293 cells we addressed these issues. The results showed 3 H‐thiamin uptake to be: 1) Na + ‐independent, temperature and energy‐ dependent 2) pH dependent 3) saturable as a function of concentration over the nano‐molar (apparent K m = 70.0±18.4 nM) and micro‐molar (apparent K m = 2.66±0.2 μM) ranges 4) cis‐inhibited by unlabeled thiamin and its structural analogues but trans‐stimulated by unlabeled thiamine 5) amiloride‐sensitive (Ki of = 0.6 mM), and 6) under the regulation of Ca 2+ /calmodulin (Ca 2+ /CaM) ‐ mediated pathway. The human thiamin transporters 1 and 2 ( hTHTR‐1 & 2) were both found to be expressed in these cells with hTHTR‐1 being the predominant form. Using gene‐specific small interfering RNAs (siRNAs), hTHTR‐1 & 2 were found to be involved in thiamin uptake. Maintaining cells under thiamin‐deficient condition lead to a significant (p <0.05) and specific up‐regulation in thiamin uptake that was associated with an increase in hTHTR‐1 & 2 protein and mRNA levels as well in the level of activity of hTHTR‐1 & 2 promoters. These results demonstrate that renal cells possess specific uptake mechanism for thiamine uptake, which is under the regulation of an intracellular Ca 2+ /CaM‐ mediated pathway. Thiamin uptake also appears to be adaptively regulated in thiamin deficiency via transcriptional regulation mechanism(s) that involves both the hTHTR‐1 & 2 [Supported by the grants from DVA and NIH (DK58057 and DK 56061)].