Published papers

We retrospectively reviewed our 10-year experience with living donor liver transplantation (LDLT) in 30 consecutive patients with end-stage primary sclerosing cholangitis (PSC) to determine long-term patient and graft survival and risk factors for recurrence of PSC. For strict diagnosis of recurrence, patients with hepatic artery thrombosis (n = 2), ABO blood type incompatible transplantation (n = 3), and postoperative survival shorter than 1 year (n = 5) were excluded from the study, leaving 20 patients for analysis. Recurrence was diagnosed in 11 patients 26-71 months after transplantation. Multivariate analysis showed that cytomegalovirus diseases within 3 months after transplantation and related donors were independent risk factors for recurrence. When the effects on recurrence were compared among donor-recipient relationships, there were significant differences, especially between nonrelated donors and parents. Multivariate analysis showed that age was an independent risk factor for time to graft loss. Cytomegalovirus prophylaxis and avoidance of related donors are important in reducing PSC recurrence, although this is a preliminary report with limitations due to the small number of patients. LDLT for young patients with PSC using grafts from their parents might have to be avoided where deceased donor liver transplantation is available.

As a form of therapeutic angiogenesis, we sought to investigate the safety and efficacy of a sustained-release system of basic fibroblast growth factor (bFGF) using biodegradable gelatin hydrogel in patients with critical limb ischemia (CLI). We conducted a phase I-IIa study that analyzed 10 CLI patients following a 200-μg intramuscular injection of bFGF-incorporated gelatin hydrogel microspheres into the ischemic limb. Primary endpoints were safety and transcutaneous oxygen pressure (TcO2) at 4 and 24 weeks after treatment. During the follow-up, there was no death or serious procedure-related adverse event. After 24 weeks, TcO2 (28.4 ± 8.4 vs. 46.2 ± 13.0 mmHg for pretreatment vs after 24 weeks, p < 0.01) showed significant improvement. Regarding secondary endpoints, the distance walked in 6 min (255 ± 105 vs. 318 ± 127 m, p = 0.02), the Rutherford classification (4.4 ± 0.5 vs. 3.1 ± 1.4, p = 0.02), the rest pain scale (1.7 ± 1.0 vs. 1.2 ± 1.3, p = 0.03), and the cyanotic scale (2.0 ± 1.1 vs. 0.9 ± 0.9, p < 0.01) also showed improvement. The blood levels of bFGF were within the normal range in all patients. A subanalysis of patients with arteriosclerosis obliterans (n = 7) or thromboangiitis obliterans (Buerger's disease) (n = 3) revealed that TcO2 had significantly improved in both subgroups. TcO2 did not differ between patients with or without chronic kidney disease. The sustained release of bFGF from biodegradable gelatin hydrogel may offer a safe and effective form of angiogenesis for patients with CLI.

The neuroinflammatory responses to human immunodeficiency virus type 1 (HIV-1) coat proteins, such as glycoprotein 120 (gp120), are considered to be responsible for the HIV-associated distal sensory neuropathy. Accumulating evidences suggest that T-cell line tropic X4 gp120 increases macrophage infiltration into the peripheral nerves, and thereby induces neuroinflammation leading to pain. However, the mechanisms underlying X4 gp120-induced macrophage recruitment to the peripheral nervous systems remain unclear. Here, we demonstrated that perineural application of X4 gp120 from HIV-1 strains IIIB and MN elicited mechanical hypersensitivity and spontaneous pain-like behaviors in mice. Furthermore, flow cytometry and immunohistochemical studies revealed increased infiltration of bone marrow-derived macrophages into the parenchyma of sciatic nerves and dorsal root ganglia (DRG) 7 days after gp120 IIIB or MN application. Chemical deletion of circulating macrophages using clodronate liposomes markedly suppressed gp120 IIIB-induced pain-like behaviors. In in vitro cell infiltration analysis, RAW 264.7 cell (a murine macrophage cell line) was chemoattracted to conditioned medium from gp120 IIIB- or MN-treated cultured Schwann cells, but not to conditioned medium from these gp120-treated DRG neurons, suggesting possible involvement of Schwann cell-derived soluble factors in macrophage infiltration. We identified using a gene expression array that CXCL1, a chemoattractant of macrophages and neutrophils, was increased in gp120 IIIB-treated cultured Schwann cells. Similar to gp120 IIIB or MN, perineural application of recombinant CXCL1 elicited pain-like behaviors accompanied by macrophage infiltration to the peripheral nerves. Furthermore, the repeated injection of CXCR2 (receptor for CXCL1) antagonist or CXCL1 neutralizing antibody prevented both pain-like behaviors and macrophage infiltration in gp120 IIIB-treated mice. Thus, the present study newly defines that Schwann cell-derived CXCL1, secreted in response to X4 gp120 exposure, is responsible for macrophage infiltration into peripheral nerves, and is thereby associated with pain-like behaviors in mice. We propose herein that communication between Schwann cells and macrophages may play a prominent role in the induction of X4 HIV-1-associated pain.

The effect of oxaliplatin against colorectal cancer is superior to that of cisplatin, but the molecular mechanism(s) involved is not clear. We found previously that oxaliplatin, but not cisplatin, was transported by human (h) and rat organic cation transporter 3 (OCT3)/SLC22A3. In the present study, we examined whether hOCT3 was significantly involved in the oxaliplatin-induced cytotoxicity and accumulation of platinum in colorectal cancer. The level of hOCT3 mRNA in the colon was 9.7-fold higher in cancerous than in normal tissues in six Japanese patients (P = 0.0247). In human colorectal cancer-derived cell lines, the mRNA of hOCT3 was highly expressed compared with that of other organic cation transporters. The release of lactate dehydrogenase (LDH) and accumulation of platinum with oxaliplatin treatment were increased in SW480 cells transfected with hOCT3 cDNA compared with empty vector-transfected cells. T84 and SW837 cells, with high levels of hOCT3, released more LDH and accumulated more platinum after oxaliplatin treatment than low hOCT3-expressing cells such as SW480, HCT116, HT29, and Lovo. However, the amount of platinum accumulated after cisplatin treatment did not differ among these six cell lines. The levels of hOCT3 expression in colon and rectum were also higher in cancerous than in normal tissues in Caucasian patients as determined by dot blotting. In conclusion, the hOCT3-mediated uptake of oxaliplatin into the cancers was suggested to be important for its cytotoxicity, and hOCT3 expression may be a marker for cancer chemotherapy including oxaliplatin.

Paraquat is a commonly used herbicide; however, it is highly toxic to humans and animals. Exposure to paraquat causes severe lung damage, leading to pulmonary fibrosis. However, it has not been well clarified as how paraquat causes cellular damage, and there is no established standard therapy for paraquat poisoning. Meanwhile, endoplasmic reticulum stress (ERS) is reported to be one of the causative factors in many diseases, although mammalian cells have a defense mechanism against ERS-induced apoptosis (unfolded protein response). Here, we demonstrated that paraquat changed the expression levels of unfolded protein response-related molecules, resulting in ERS-related cell death in human lung epithelial A549 cells. Moreover, treatment with sodium tauroursodeoxycholate (TUDCA), a chemical chaperone, crucially rescued cells from death caused by exposure to paraquat. These results indicate that paraquat toxicity may be associated with ERS-related molecules/events. Through chemical chaperone activity, treatment with TUDCA reduced paraquat-induced ERS and mildly suppressed cell death. Our findings also suggest that TUDCA treatment represses the onset of pulmonary fibrosis caused by paraquat, and therefore chemical chaperones may have novel therapeutic potential for the treatment of paraquat poisoning. (C) 2013 Elsevier Inc. All rights reserved.

Telaprevir (TVR) is a protease inhibitor used in combination with pegylated interferon alfa-2b and ribavirin for hepatitis C, and TVR strongly inhibits CYP3A4 and CYP3A5. We reported successful TVR treatment of liver transplant patients with recurrence of hepatitis C during receiving immunosuppressive therapy. Before initiation of triple therapy, all patients switched from tacrolimus to cyclosporine, which has a lower inhibitory effect on CYP3A4 and CYP3A5 than tacrolimus. To avoid graft failure, we measured the cyclosporine blood concentrations at 0, 2, and 6 h after administration to maintain the target level (150-200 ng/mL) within 1 week after initiation of TVR and adjusted the dose of cyclosporine. The dose of cyclosporine was decreased 0.24-0.40 fold in all patients after initiation of TVR treatment. In 3 patients, the dose of TVR was decreased two-thirds of starting dose because of adverse effects, including anorexia and skin rash. However, the HCV RNA level rapidly decreased to undetectable levels within 1 month. Furthermore, all patients completed the TVR therapy in 12 weeks and did not experience liver graft rejection. In addition, we found the rapid elimination of inhibitory effect of TVR on the disposition of cyclospirne in the all four cases and therefore, rapid increase in the dosage of cyclosporine would be required immediately after the end of TVR administration. These results suggest that frequent measurement of cyclosporine levels was important for successful TVR triple therapy and prevention of rejection.

Impairment of peripheral neurons by anti-cancer agents, including taxanes and platinum derivatives, has been considered to be a major cause of chemotherapy-induced peripheral neuropathy (CIPN), however, the precise underlying mechanisms are not fully understood. Here, we examined the direct effects of anti-cancer agents on Schwann cells. Exposure of primary cultured rat Schwann cells to paclitaxel (0.01 mu M), cisplatin (1 mu M), or oxaliplatin (3 mu M) for 48 h induced cytotoxicity and reduced myelin basic protein expression at concentrations lower than those required to induce neurotoxicity in cultured rat dorsal root ganglion (DRG) neurons. Similarly, these anti-cancer drugs disrupted myelin formation in Schwann cell/DRG neuron co-cultures without affecting nerve axons. Cisplatin and oxaliplatin, but not paclitaxel, caused mitochondrial dysfunction in cultured Schwann cells. By contrast, paclitaxel led to dedifferentiation of Schwann cells into an immature state, characterized by increased expression of p75 and galectin-3. Consistent with in vitro findings, repeated injection of paclitaxel increased expression of p75 and galectin-3 in Schwann cells within the mouse sciatic nerve. These results suggest that taxanes and platinum derivatives impair Schwan cells by inducing dedifferentiation and mitochondrial dysfunction, respectively, which may be important in the development of CIPN in conjunction with their direct impairment in peripheral neurons.

Therapeutic monoclonal antibodies (mAbs) have heterogeneities in their structures. Multiple studies have reported that the variety of post-translational modifications could affect the pharmacokinetic profiles or pharmacological potencies of therapeutic mAbs. Taking into the account that the structural modification of mAbs would affect the efficacy, it is worth investigating the structural alteration of therapeutic mAbs in the blood and the relationship between their structures and pharmacological effects. Herein, we have developed the method to isolate rituximab from plasma in which endogenous IgGs interfere the detection of rituximab, and successfully developed the analytical method with a liquid chromatograph time-of-flight mass spectrometer to detect the structure of rituximab in plasma with errors less than 30 parts per millions. Eight types of carbohydrate chains in rituximab were detected by this method. Interestingly, time-dependent changes in carbohydrate chains such as AAF (G2F) and GnGn (G0) were observed in rats, although the amino acids were stable. Additionally, these structural changes were observed via incubation in plasma as in the rat experiment, suggesting that a certain type of enzyme in plasma caused the alterations of the carbohydrate chains. The present analytical methods could clarify the actual pharmacokinetics of therapeutic mAbs, and help to evaluate the interindividual variations in pharmacokinetics and efficacy.

Background: Sudden sensorineural hearing loss (SSHL) is a common condition in which patients lose the hearing in one ear within 3 days. Systemic glucocorticoid treatments have been used as standard therapy for SSHL; however, about 20% of patients do not respond. We tested the safety and efficacy of topical insulin-like growth factor 1 (IGF1) application using gelatin hydrogels as a treatment for SSHL. Methods: Patients with SSHL that showed no recovery to systemic glucocorticoid administration were recruited. We applied gelatin hydrogels, impregnated with recombinant human IGF1, into the middle ear. The primary outcome measure was the proportion of patients showing hearing improvement 12 weeks after the test treatment. The secondary outcome measures were the proportion of patients showing improvement at 24 weeks and the incidence of adverse events. The null hypothesis was that 33% of patients would show hearing improvement, as was reported for a historical control after hyperbaric oxygen therapy. Results: In total, 25 patients received the test treatment at a median of 23 days (range 15 32) after the onset of SSHL, between 2007 and 2009. At 12 weeks after the test treatment, 48% (95% CI 28% to 69%; P = 0.086) of patients showed hearing improvement, and the proportion increased to 56% (95% CI 35% to 76%; P = 0.015) at 24 weeks. No serious adverse events were observed. Conclusions: Topical IGF1 application using gelatin hydrogels is well tolerated and may be efficacious for hearing recovery in patients with SSHL that is resistant to systemic glucocorticoids.

To clarify the transcellular transport of organic cations via basolateral and apical. transporters, we established double-transfected Madin-Darby canine kidney (MDCK) cells expressing both human organic cation transporter hOCT1 and hMATE1 (MDCK-hOCT1/hMATE1), and hOCT2 and hMATE1 (MDCK-hOCT2/hMATE1) as models of human hepatocytes and renal epithelial cells, respectively. Using the specific antibodies, hOCT1 and hMATE1 or hOCT2 and hMATE1 were found to be localized in the basolateral and apical membranes of MDCK-hOCT1/hMATE1 or MDCK-hOCT2/hMATE1 cells, respectively. A representative substrate, [[C-14]tetraethylammonium, was transported unidirectionally from the basolateral to apical side in these double transfectants. The optimal pH was showed to be 6.5 for the transcellular transport of [C-14]tetraethylammonium, when the pH of the incubation medium on the apical side was varied from 5.5 to 8.5. The basolateral-to-apical transport also decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium or 1 mM levofloxacin on the basolateral side of both double transfectants. In MDCK-hOCT2/hMATE1 cell monolayers, but not in MDCK-hOCT1/hMATE1 cell monolayers, the accumulation of [14C] tetra ethylammonium was decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium, but significantly increased in the presence of 1 mM levofloxacin. The uptake of [C-14]tetraethylammonium, [H-3]1-methyl-4-phenylpyridinium, [C-14]metformin and [H-3]cimetidine, but not of [C-14]procainamide and [H-3]quinidine, by HEK293 cells was stimulated by expression of the hOCT1, hOCT2 or hMATE1 compared to control cells. However, transcellular transport of [C-14]procainamide and [H-3]quinidine was clearly observed in both double-transfectants. These cells could be useful for examining the routes by which compounds are eliminated, or predicting transporter-mediated drug interaction. (c) 2008 Elsevier Inc. All rights reserved.

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

We previously reported that donepezil, a central-type acetylcholinesterase inhibitor, showed neuroprotective action via alpha 4-and alpha 7-nicotinic acetylcholine receptors against glutamate neurotoxicity in rat cortical culture. The present study was performed to investigate whether the neuroprotective action of acetylcholinesterase inhibitors is accompanied by the alteration of expression and function of nicotinic receptors. Four days treatment with acetylcholinesterase inhibitors (10 mu M) enhanced the nicotine-induced increase of the intracellular calcium concentration. Immunoblot analysis revealed that donepezil increased both alpha 4 and alpha 7 subunit proteins. Donepezil and galanthamine increased the number of cells expressing alpha 4- and alpha 7-nicotinic receptors in immunocytochemical analysis. We examined whether up-regulation of nicotinic receptors affected the neuroprotective action of acetylcholinesterase inhibitors. Under up-regulating conditions, donepezil and galanthamine exerted neuroprotective action at lower concentrations. These results suggest that donepezil and galanthamine up-regulate nicotinic receptors in cortical neurons, and that the up-regulation of nicotinic receptors may make cortical neurons more sensitive to the neuroprotective action of donepezil and galanthamine. (c) 2005 Elsevier B.V. All rights reserved.

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5 mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2 mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and. serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury. (C) 2012 Elsevier Inc. All rights reserved.

Serum leucine-rich alpha-2 glycoprotein (LRG) can monitor disease activities during biologics treatment in patients with inflammatory bowel disease (IBD). It is unclear whether the pretreatment serum LRG level can predict clinical effectiveness including serum trough levels of ustekinumab in patients with IBD. This multicenter prospective cohort study included 184 patients (Crohn’s disease, 104; ulcerative colitis, 80) who received ustekinumab (n = 119) or anti-tumor necrosis factor (n = 65) between January 2019 and March 2023. Multivariate logistic regression analysis revealed serum LRG level at week 0 (0w-LRG, odds ratio 0.12, 95% confidence interval 0.02–0.68) as one of significant factors for clinical remission at week 8. We divided patients into the low- and the high-LRG groups by the median 0w-LRG (18.2 µg/mL) and compared the effectiveness. In patients who received ustekinumab, the proportion of clinical remission at week 8 was significantly different between in the low- (76.9%) and in the high-LRG group (59.3%, P = 0.038), and median serum trough level at week 8 was significantly different between in the low- (10.9 µg/mL, interquartile range 6.7–13.4) and the high-LRG group (5.3 µg/mL, interquartile range 2.4–8.3, P < 0.001). The 0w-LRG can predict the effectiveness including serum trough levels of ustekinumab during induction treatment for patients with IBD.

In the pharmacotherapy of patients with Parkinson's disease (PD), entacapone reduces the peripheral metabolism of L-dopa to 3-O-methyldopa (3-OMD), thereby prolonging the half-life (t1/2) of L-dopa and increasing the area under the concentration curve (AUC). The effect of entacapone on the pharmacokinetics of L-dopa differs between patients with high-activity (H/H) and low-activity (L/L) catechol-O-methyltransferase (COMT) Val158Met polymorphisms, but the effects are unclear in heterozygous (H/L) patients. 3-OMD has a detrimental effect and results in a poor response to L-dopa treatment in patients with PD; however, the influence of this polymorphism on the production of 3-OMD remains unknown. Therefore, the present study aimed to clarify the effect of the COMT Val158Met polymorphism on the concentrations of L-dopa and 3-OMD in the presence of entacapone. We performed an open-label, single-period, single-sequence crossover study at two sites in Japan. The study included 54 Japanese patients with PD, who underwent an acute L-dopa administration test with and without 100 mg entacapone on two different days. Entacapone increased L-dopa AUC0-infinity by 1.59 ± 0.26-fold in the H/H group, which was significantly higher than that in the H/L (1.41 ± 0.36-fold) and L/L (1.28 ± 0.21-fold) groups (p < 0.05). The concurrent administration of L-dopa with entacapone suppressed the increase in 3-OMD levels compared with L-dopa alone in all genotypes. Our results suggest that the COMT Val158Met polymorphism may be an informative biomarker for individualized dose adjustment of COMT inhibitors in the treatment of PD.

Remdesivir plays a key role in the treatment of coronavirus disease in 2019 (COVID-19). Haemodialysis is sometimes required for hospitalised patients with COVID-19, and patients undergoing haemodialysis are at an increased risk of severe COVID-19. In the present study, we report the serum concentrations of GS-441524, the active metabolite of remdesivir, in four patients undergoing continuous renal replacement therapy (CRRT). Patient 1, a male aged 70s, received a loading dose of 200 mg remdesivir on day 1, followed by 100 mg remdesivir from day 2, according to the package insert as in non-haemodialysis patients. The mean trough serum concentration of GS-441524 was 783.5 ng/mL, which was approximately 7-fold higher than the mean for patients with an estimated glomerular filtration rate (eGFR) ≥ 60 mL/min. Patients 2-4 received a loading dose of 200 mg remdesivir on day 1, followed by 100 mg once every 2 days from day 2. The mean trough serum concentrations of GS-441524 were 302.2 ng/mL, 585.8 ng/mL and 677.3 ng/mL, respectively. These were 3 to 6-fold higher than the mean for patients with eGFR ≥60 mL/min. The target doses for patients 1, 2, 3, and 4 receiving CRRT were 13.6 mL/kg/h, 6.0-12.5 mL/kg/h, 20.1 mL/kg/h, and 15.1 mL/kg/h, respectively, using a polysulphone membrane. The package insert dose of remdesivir is an overdose for CRRT patients with a target dose of 10-20 mL/kg/h. In low-intensity CRRT, as in Japan, it may be necessary to extend the interval between the doses of remdesivir.

Multidrug and toxin extrusions (MATE1/SLC47A1 and MATE2-K/SLC47A2) play important roles in the renal excretion of metformin. We have previously identified the nonsynonymous MATE variants with functional defects at low allelic frequencies. The purpose of this study was to evaluate the effects of heterozygous MATE variants on the disposition of metformin in mice and humans. Pharmacokinetic parameters of metformin in Mate1(+/-) heterozygous mice were comparable with those in Mate1(+/+) wild-type mice. Among 48 Japanese diabetic patients, seven patients carried heterozygous MATE variant and no patient carried homozygous MATE variant There was no significant difference in oral clearance of metformin with or without heterozygous MATE variants. In addition, creatinine clearance, but not heterozygous MATE variants, significantly improved the model fit of metformin clearance by statistical analysis using the nonlinear mixed-effects modeling program. In conclusion, heterozygous MATE variants could not influence the disposition of metformin in diabetic patients. Pharmacogenetics and Genomics 20:135-138 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

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 [(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 mu mol/L, respectively. The hRFT-mediated[(3)H]riboflavin uptake was independent of extracellular Nal(+) and CI(-). Specific uptake of [(3)H]riboflavin 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 [(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. Nutr. 140: 1220-1226, 2010.

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 hRFT1sv 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 rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [H-3] riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [H-3] riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is Na+, 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.