Biotin uptake by human proximal tubular epithelial cells: cellular and molecular aspects

Cellular and molecular regulation of renal biotin uptake in humans is not well defined. The contribution of the human Na(+)-dependent multivitamin transporter (hSMVT) to carrier-mediated biotin uptake by human proximal tubular epithelial cells is not clear. The aim of this study was to address these issues, with the human-derived proximal tubular epithelial HK-2 cells used as a model. First, we characterized the mechanism of biotin uptake by these cells and obtained evidence for involvement of an Na(+)-, temperature-, and energy-dependent carrier-mediated uptake system. This system was inhibited by the biotin structural analog desthiobiotin, pantothenic acid, and lipoate. These findings suggest involvement of the hSMVT system in the uptake process. This was confirmed by demonstrating that the hSMVT system is expressed in HK-2 cells at the protein and mRNA levels and by selective silencing of the hSMVT gene with the use of gene-specific small interfering RNAs, which led to specific and significant inhibition of carrier-mediated biotin uptake. Of the two recently cloned promoters of the hSMVT gene, promoter 1 was more active than promoter 2 in these cells. Pretreatment of HK-2 cells with modulators of PKC- and Ca(2+)/calmodulin-mediated pathways (but not those that modulate PKA-, protein tyrosine kinase-, or nitric oxide-mediated pathways) led to significant alterations in biotin uptake. Maintaining the HK-2 cells in a biotin-deficient growth medium led to a marked upregulation in biotin transport, which was associated with an increase in hSMVT protein and RNA levels and an increase in activity of the hSMVT promoters. These results demonstrate that biotin uptake by human renal epithelial cells occurs via the hSMVT system and that the process is regulated by intracellular PKC- and Ca(2+)/calmodulin-mediated pathways. The uptake process appears to be adaptively regulated by extracellular biotin level, which involves transcriptional regulatory mechanism(s).