Search

We isolated cDNA coding a new human riboflavin transporter (hRFT)3, which exhibits 86.7 and 44.1% amino acid identity with hRFT1 and hRFT2, respectively. It was predicted to have 10 putative membrane-spanning domains. The functional characteristics of hRFT3 were examined and compared with those of its isoforms, hRFT1 and hRFT2. Real-time PCR revealed that hRFT3 mRNA was strongly expressed in the brain and salivary gland, hRFT1 mRNA was strongly expressed in the placenta and small intestine, whereas hRFT2 mRNA was most abundantly expressed in the testis and strongly in the small intestine and prostate, hRFT-mediated uptake of [[sup.3]H]riboflavin was evaluated using human embryonic kidney 293 cells transiently transfected with the cDNA coding each hRFT. The apparent Michaelis-Menten constants of hRFT1, hRFT2, and hRFT3 for riboflavin were 1.38, 0.98, and 0.33 [micro]mol/L, respectively. The hRFT-mediated [[sup.3]H]ribofiavin uptake was independent of extracellular [Na.sup.+] and [CI.sup.-] . Specific uptake of [[sup.3]H]ribofiavin by hRFT2, but not hRFT1 and hRFT3, decreased as extracellular pH was changed from 5.4 to 8.4. The substrate specificities of the hRFT family were similar, hRFT-mediated uptake of [[sup.3]H]riboflavin was inhibited by some riboflavin analogs, but not D-ribose, organic ions, or other vitamins. The newly isolated hRFT3 may play an important role in brain riboflavin homeostasis. Its amino acid sequence and functional characteristics are similar to those of hRFT1, but not hRFT2. J. doi: 10.3945/jn.110.122911

To clarify the specific molecular events of progressive tubular damage in chronic renal failure (CRF), we conducted microarray analyses using isolated proximal tubules from subtotally nephrectomized (Nx) rats as a model of CRF. Our results clearly demonstrated time-dependent changes in gene expression profiles localized to proximal tubules. The expression of mitosis-specific genes Cyclin B2 and Cell division cycle 2 (Cdc2) was significantly and selectively increased in the proximal tubules during the compensated period but decreased to basal level in the end-stage period. Administration of everolimus, a potent inhibitor of mammalian target of rapamycin, markedly reduced compensatory hypertrophy and hyperplasia of epithelial cells, which was accompanied by complete abolishment of the expression of Cyclin B2 and Cdc2 enhancement; renal function was then severely decreased. Treatment with the Cdc2 inhibitor 2-cyanoethyl alsterpaullone clearly decreased epithelial cell hyperplasia, based on staining of phosphorylated histone H3 and Ki-67, while hypertrophy was not inhibited. In conclusion, we have demonstrated roles of Cyclin B2 and Cdc2 in the epithelial hyperplasia in response to Nx. These results advance the knowledge of the contribution of cell cycle regulators, especially M phase, in pathophysiology of tubular restoration and/or degeneration, and these two molecules are suggested to be a marker for the proliferation of proximal tubular cells in CRF. proximal tubule; microarray; cell cycle; [G.sub.2]-M; hypertrophy doi: 10.1152/ajprenal.00567.2009.

Human organic cation transporter 1 (OCT l/SLC22A1) plays important roles in the hepatic uptake of cationic drugs. The functional characteristics of this transporter have been well evaluated, but molecular information regarding transcriptional regulation is limited. In the present study, therefore, we examined the gene regulation of OCT1 gene focusing on basal core expression. An ~2.5-kb fragment of the OCT1 promoter region was isolated, and promoter activity was measured by luciferase assay in the human liver cell lines Huh7 and HepG2. Deletion analysis suggested that the region spanning -141/-69 was essential for the basal core transcriptional activity and that this region contained the sequence of a cognate E-box (CACGTG). The E-box is known to be bound by the basal transcription factors, upstream stimulating factors (USFs), and the functional involvements of USF1 and USF2 were confirmed by a transactivation effect, a mutational analysis of the E-box, and an electrophoretic mobility shift assay. The transactivation effect of USFs on the OCT1 promoter was further stimulated by hepatocyte nuclear factor 4[alpha], a liver-enriched transcription factor. There were no polymorphisms in the proximal promoter region (about 400 bp) of OCT1 gene (n = 109). These findings indicated that both USF1 and USF2 bind to an E-box sequence located in the OCT1 core promoter region and are required for the basal gene expression of this transporter. liver; E-box

Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, the novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified on the basis of our rat kidney mRNA expression database (Horiba N, Masuda S, Takeuchi A, Saito H, Okuda M, Inui K. Kidney Int 66: 29-45, 2004). hRFT1 and rRFT1 cDNAs have an open reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to one another. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFF1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in human embryonic kidney (HEK)-293 and Caco-2 cells. HEK-293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFF1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [[sup.3]H]riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [[sup.3]H]riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is [Na.sup.+], potential, and pH independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK-293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family. vitamin B2; intestine; absorption; kidney; human embryonic kidney-293 cells

The intestinal [H.sup.+/peptide cotransporter 1 (PEPT1) plays important roles as a nutrient and drug transporter. Previously, we reported that rat intestinal PEPT1 showed a diurnal rhythm and that this rhythm is closely related to the feeding schedule. Furthermore, we also demonstrated that transcription factors, Spl, Cdx2, and peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha]) contribute to the basal, intestine-specific, and fasting-induced expression of PEPT1, respectively. In this study, to clarify the molecular mechanism governing the diurnal rhythm of PEPT1 expression, we compared expression profiles of these transcription factors under two kinds of feeding schedules. The intestinal Sp 1 and Cdx2 did not show a circadian accumulation of mRNA or response to the daytime feeding regimen. Plasma free fatty acids, endogenous PPAR-[alpha] ligands, exhibited a robust circadian fluctuation in phase with that of PEPT1. However, subsequent experiments using PPAR-[alpha]-null mice revealed the absence of any association between the circadian rhythm of PEPT1 and PPAR-[alpha]. We then focused on the clock genes (Clock, Bmal 1, Per 1-2, and Cry 1) and clock-controlled gene, albumin D site-binding protein (DBP). A robust and coordinated circadian expression of the clock genes was observed, and daytime feeding entirely inverted the phase except for Clock. The expression of DBP was in phase with that of PEPT1 in both groups. Electrophoretic mobility shift assays and reporter assays revealed that DBP has the ability to bind the DBP binding site located in the distal promoter region of the rat PEPT1 gene and induce the transcriptional activity. These findings indicate that DBP plays pivotal roles in the circadian oscillation of PEPT1. SLC15A1; albumin D site-binding protein; clock gene; restricted feeding; small intestine

During cholestasis, bile acids are mainly excreted into the urine, but adaptive renal responses to cholestasis, especially molecular mechanisms for renal secretion of bile acids, have not been well understood. Organic anion transporters (OAT1 and OAT3) are responsible for membrane transport of anionic compounds at the renal basolateral membranes. In the present study, we investigated the pathophysiological roles of OAT1 and OAT3 in terms of renal handling of bile acids. The Eisai hyperbilirubinemic rats (EHBR), mutant rats without multidrug resistance-associated protein 2, showed higher serum and urinary concentrations of bile acids, compared with Sprague-Dawley (SD) rats (wild type). The protein expression level of rat OAT3 was significantly increased in EHBR compared with SD rats, whereas the expression of rat OAT1 was unchanged. The transport activities of rat and human OAT3, but not OAT1, were markedly inhibited by various bile acids such as chenodeoxycholic acid and cholic acid. Cholic acid, glycocholic acid, and taurocholic acid, which mainly increased during cholestasis, are transported by OAT3. The plasma concentration of [beta]-lactam antibiotic cefotiam, a specific substrate for OAT3, was more increased in EHBR than in SD rats despite upregulation of OAT3 protein. This may be due to the competitive inhibition of cefotiam transport by bile acids via OAT3. In conclusion, the present study clearly demonstrated that OAT3 is responsible for renal secretion of bile acids during cholestasis and that the pharmacokinetic profile of OAT3 substrates may be affected by cholestasis. Eisai hyperbilirubinemic rat; renal secretion; SLC22A8

Human organic cation transporter 2 (OCT2/SLC22A2), which is specifically expressed in the kidney, plays critical roles in the renal secretion of cationic compounds. Tissue expression and membrane localization of OCT2 are closely related to the tissue distribution, pharmacological effects, and/or adverse effects of its substrate drugs. However, the molecular mechanisms underlying the kidney-specific expression of OCT2 have not been elucidated. In the present study, therefore, we examined the contribution of DNA methylation of the promoter region for the OCT2 gene to its tissue-specific expression using human tissue samples. In vivo methylation status of the proximal promoter region of OCT2 and that of OCT1, a liver-specific organic cation transporter, were investigated by bisulfite sequencing using human genomic DNA extracted from the kidney and liver. All CpG sites in the OCT2 proximal promoter were hypermethylated in the liver, while hypomethylated in the kidney. On the other hand, the promoter region of OCT1 was hypermethylated in both the kidney and liver. The level of methylation of the OCT2 promoter was especially low at the CpG site in the E-box, the binding site of the basal transcription factor upstream stimulating factor (USF) 1. In vitro methylation of the OCT2 proximal promoter dramatically reduced the transcriptional activity, and an electrophoretic mobility shift assay showed that methylation at the E-box inhibited the binding of USF1. These results indicate that kidney-specific expression of human OCT2 is regulated by methylation of the proximal promoter region, interfeting with the transactivation by USF1. drug transporter; renal secretion; tissue-specifc gene expression; epigenetics