Apple-derived extracellular vesicles modulate the expression of human intestinal bile acid transporter ASBT/SLC10A2 via downregulation of transcription factor RARα.

PURPOSE: Plant-derived extracellular vesicles (EVs) have been reported to exert biological activity on intestinal tissues by delivering their contents into intestinal cells. We previously reported that ASBT/SLC10A2 mRNA was downregulated by apple-derived extracellular vesicles (APEVs). ASBT downregulation is effective in the treatment of cholestasis and chronic constipation, similar to the beneficial effects of apples. Therefore, this study aimed to establish the mechanism of ASBT downregulation by APEVs, focusing on microRNAs present in APEVs. RESULTS: APEVs downregulated the expression of ASBT, but no significant effect on SLC10A2-3'UTR was observed. Proteomics revealed that APEVs decreased the expression of RARα/NR1B1. The binding of RARα to SLC10A2 promoter was also decreased by APEVs. The stability of NR1B1 mRNA was attenuated by APEVs and its 3'UTR was found to be a target for APEVs. Apple microRNAs that were predicted to interact with NR1B1-3'UTR were present in APEVs, and their mimics suppressed NR1B1 mRNA expression. CONCLUSIONS: Suppression of ASBT by APEVs was indirectly mediated by the downregulation of RARα, and its stability was lowered by microRNAs present in APEVs. This study suggested that macromolecules in food directly affect intestinal function by means of EVs that stabilize them and facilitate their cellular uptake.

The mechanism of thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides.

In this study, we investigated thrombocytopenia caused by cholesterol-conjugated antisense oligonucleotides (Chol-ASO). First, we evaluated platelet activation induced by Chol-ASO in mice by flow cytometry after administration of platelet-rich plasma (PRP). An increase in the number of large particle-size events with platelet activation was detected in the Chol-ASO-treated group. In a smear study, numerous platelets were observed to attach to nucleic acid-containing aggregates. A competition binding assay showed that the conjugation of cholesterol to ASOs increased their affinity for glycoprotein VI. Platelet-free plasma was then mixed with Chol-ASO to form aggregates. The assembly of Chol-ASO was confirmed by dynamic light scattering measurements in the concentration range in which the formation of aggregates with plasma components was observed. In conclusion, the mechanism by which Chol-ASOs causes thrombocytopenia is proposed to be as follows: (1) Chol-ASOs form polymers, (2) the nucleic acid portion of the polymers interacts with plasma proteins and platelets, which cross-links them to form aggregates, and (3) platelets bound to aggregates become activated, resulting in platelet aggregation, leading to a decrease in platelet count in vivo. The details of the mechanism revealed in this study could contribute to creating safer oligonucleotide therapies without the risk of thrombocytopenia.

α1-Acid Glycoprotein Enhances the Immunosuppressive and Protumor Functions of Tumor-Associated Macrophages.

Blood levels of acute-phase protein α1-acid glycoprotein (AGP, orosmucoid) increase in patients with cancer. Although AGP is produced from hepatocytes following stimulation by immune cell-derived cytokines under conditions of inflammation and tumorigenesis, the functions of AGP in tumorigenesis and tumor progression remain unknown. In the present study, we revealed that AGP contributes directly to tumor development by induction of programmed death ligand 1 (PD-L1) expression and IL6 production in macrophages. Stimulation of AGP induced PD-L1 expression in both human monocyte-derived macrophages through STAT1 activation, whereas AGP had no direct effect on PD-L1 expression in tumor cells. AGP also induced IL6 production from macrophages, which stimulated proliferation in tumor cells by IL6R-mediated activation of STAT3. Furthermore, administration of AGP to AGP KO mice phenocopied effects of tumor-associated macrophages (TAM) on tumor progression. AGP decreased IFNγ secretion from T cells and enhanced STAT3 activation in subcutaneous tumor tissues. In addition, AGP regulated PD-L1 expression and IL6 production in macrophages by binding with CD14, a coreceptor for Toll-like receptor 4 (TLR4), and inducing TLR4 signaling. These results provide the first evidence that AGP is directly involved in tumorigenesis by interacting with TAMs and that AGP might be a target molecule for anticancer therapy. SIGNIFICANCE: AGP-mediated suppression of antitumor immunity contributes to tumor progression by inducing PD-L1 expression and IL6 production in TAMs.

MicroRNAs in Apple-Derived Nanoparticles Modulate Intestinal Expression of Organic Anion-Transporting Peptide 2B1/SLCO2B1 in Caco-2 Cells.

Plant-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo to intestinal tissues. We previously reported that apple-derived nanoparticles (APNPs) downregulate the mRNA of the human intestinal transporter organic anion-transporting peptide 2B1 (OATP2B1)/SLCO2B1 and that the 3'-untranslated region (3'UTR) is required for the response to APNPs. Here, we investigated the involvement of microRNAs (miRNAs) in APNPs in suppressing OATP2B1 expression to demonstrate that APNP macromolecules directly interact with intestinal tissues. Using in silico analysis, seven apple miRNAs were predicted as candidate miRNAs that interact with the SLCO2B1-3'UTR. The APNP-mediated decrease in luciferase activity of pGL3/SLCO2B1-3'UTR was abrogated by inhibitors of mdm-miR-160a-e, -7121a-c, or -7121d-h. Each miRNA mimic reduced the endogenous expression of SLCO2B1 mRNA in Caco-2 cells. The luciferase activity of the truncated pGL3/SLCO2B1-3'UTR, which contains approximately 200 bp around each miRNA recognition element (MRE), was decreased by the miR-7121d-h mimic but decreased little by the other mimics. APNP also reduced the luciferase activity of truncated pGL3/SLCO2B1-3'UTR containing an MRE for miR-7121d-h. Thus, we demonstrated that mdm-miR-7121d-h contributes to the APNP-mediated downregulation of intestinal OATP2B1. Accordingly, plant macromolecules, such as miRNAs, may directly interact with intestinal tissues via nanoparticles. SIGNIFICANCE STATEMENT: This study demonstrates that mdm-miR7121d-h contained in apple-derived nanoparticles downregulated the mRNA expression of SLCO2B1 by interacting with SLCO2B1-3'-untranslated region directly and that SLCO2B1 mRNA might also be decreased by mdm-miR160a-e and -7121a-c indirectly. This finding that the specific apple-derived microRNAs influence human intestinal transporters provides a novel concept that macromolecules in foods directly interact with and affect the intestinal function of the host.

An acute phase protein α1-acid glycoprotein mitigates AKI and its progression to CKD through its anti-inflammatory action.

The molecular mechanism for acute kidney injury (AKI) and its progression to chronic kidney disease (CKD) continues to be unclear. In this study, we investigated the pathophysiological role of the acute phase protein α1-acid glycoprotein (AGP) in AKI and its progression to CKD using AGP KO mice. Plasma AGP levels in WT mice were increased by about 3.5-fold on day 1-2 after renal ischemia-reperfusion (IR), and these values then gradually decreased to the level before renal IR on day 7-14. On day 1 after renal IR, the AGP KO showed higher renal dysfunction, tubular injury and renal inflammation as compared with WT. On day 14, renal function, tubular injury and renal inflammation in WT had recovered, but the recovery was delayed, and renal fibrosis continued to progress in AGP KO. These results obtained from AGP KO were rescued by the administration of human-derived AGP (hAGP) simultaneously with renal IR. In vitro experiments using RAW264.7 cells showed hAGP treatment suppressed the LPS-induced macrophage inflammatory response. These data suggest that endogenously induced AGP in early renal IR functions as a renoprotective molecule via its anti-inflammatory action. Thus, AGP represents a potential target molecule for therapeutic development in AKI and its progression CKD.

Uptake Pathway of Apple-derived Nanoparticle by Intestinal Cells to Deliver its Cargo.

PURPOSE: Food-derived nanoparticles exert cytoprotective effects on intestinal cells by delivering their cargo, which includes macromolecules such as microRNAs and proteins, as well as low-molecular weight compounds. We previously reported that apple-derived nanoparticles (APNPs) downregulate the expression of human intestinal transporter OATP2B1/SLCO2B1 mRNA. To verify the involvement of the cargo of APNPs in affecting the expression of transporters, we characterized the uptake mechanism of APNPs in intestinal cells. METHODS: The uptake of fluorescent PKH26-labeled APNPs (PKH-APNPs) into Caco-2, LS180, and HT-29MTX cells was evaluated by confocal microscopy and flow cytometry. RESULTS: The uptake of PKH-APNPs was prevented in the presence of clathrin-dependent endocytosis inhibitors, chlorpromazine and Pitstop2. Furthermore, PKH-APNPs were incorporated by the HT29-MTX cells, despite the disturbance of the mucus layer. Additionally, the decrease in SLCO2B1 mRNA by APNPs was reversed by Pitstop 2 in Caco-2 cells, indicating that APNPs decrease SLCO2B1 by being incorporated via clathrin-dependent endocytosis. CONCLUSIONS: We demonstrated that clathrin-dependent endocytosis was mainly involved in the uptake of APNPs by intestinal cells, and that the cargo in the APNPs downregulate the mRNA expression of SLCO2B1. Therefore, APNPs could be a useful tool to deliver large molecules such as microRNAs to intestinal cells.

Biological Distribution of Orally Administered [123I]MIBG for Estimating Gastrointestinal Tract Absorption.

Gastrointestinal tract absorption of cationic anticancer drugs and medicines was estimated using whole-body imaging following oral [123I]MIBG administration. [123I]MIBG was added to cultures of human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)2B1, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)1, OCT2, and OCT3 with and without cimetidine (an OCTN and OCT inhibitor) and L-carnitine (an OCTN inhibitor). Biodistribution analyses and single-photon emission computed tomography (SPECT) imaging in normal and dextran sodium sulfate (DSS)-induced experimental colitis mice were conducted using [123I]MIBG with and without cimetidine. [123I]MIBG uptake was significantly higher in HEK293/OCTN1, 2, and OCT1-3 cells than in mock cells. Uptake via OCTN was inhibited by L-carnitine, whereas OCT-mediated uptake was inhibited by cimetidine. Biodistribution analyses and SPECT imaging studies showed significantly lower accumulation of [123I]MIBG in the blood, heart, liver, and bladder in DSS-induced experimental colitis mice and mice with cimetidine loading compared with normal mice, whereas significantly higher accumulation in the stomach and kidney was observed after [123I]MIBG injection. [123I]MIBG imaging after oral administration can be used to estimate gastrointestinal absorption in the small intestine via OCTN and/or OCT by measuring radioactivity in the heart, liver, and bladder.

A Novel Fluorescence-Based Method to Evaluate Ileal Apical Sodium-Dependent Bile Acid Transporter ASBT.

This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5ß-cholestan-26-oyl)-2'-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed in Xenopus laevis oocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 µM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 µM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 µM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na+ free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na+-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.

α1-Acid Glycoprotein Attenuates Adriamycin-Induced Nephropathy via CD163 Expressing Macrophage Induction.

Background: Recent clinical studies have shown that proteinuria is a critical factor in the progression of CKD and onset of cardiovascular disease. Inflammation and infiltration of macrophages into renal tissue are implicated as causes of proteinuria. α1-Acid glycoprotein (AGP), an acute-phase plasma protein, is leaked into the urine in patients with proteinuria. However, the relationship between urinary leakage of AGP, renal inflammation, and proteinuria remains unclear. Methods: Human AGP (hAGP) was exogenously administrated for 5 consecutive days to adriamycin-induced nephropathy model mice. Results: Adriamycin treatment increased urinary AGP, accompanied by decreased plasma AGP in mice. Exogenous hAGP administration to adriamycin-treated mice suppressed proteinuria, renal histologic injury, and inflammation. hAGP administration increased renal CD163 expression, a marker of anti-inflammatory macrophages. Similar changes were observed in PMA-differentiated THP-1 cells treated with hAGP. Even in the presence of LPS, hAGP treatment increased CD163/IL-10 expression in differentiated THP-1 cells. Conclusions: AGP alleviates proteinuria and renal injury in mice with proteinuric kidney disease via induction of CD163-expressing macrophages with anti-inflammatory function. The results demonstrate that endogenous AGP could work to protect against glomerular disease. Thus, AGP supplementation could be a possible new therapeutic intervention for patients with glomerular disease.

Changes of drug pharmacokinetics mediated by downregulation of kidney organic cation transporters Mate1 and Oct2 in a rat model of hyperuricemia.

The effects of hyperuricemia on the expression of kidney drug transporters and on the pharmacokinetics of several substrate drugs were examined. We first established a rat model of hyperuricemia without marked symptoms of chronic kidney failure by 10-day co-administration of oxonic acid (uricase inhibitor) and adenine (biosynthetic precursor of uric acid). These hyperuricemic rats showed plasma uric acid concentrations of up to 6 mg/dL, which is similar to the serum uric acid level in hyperuricemic humans, with little change of inulin clearance. The mRNA levels of multidrug and toxin extrusion 1 (Mate1, Slc47a1), organic anion transporter 1 (Oat1, Slc22a6), organic cation transporter 2 (Oct2, Slc22a2), urate transporter 1 (Urat1, Slc22a12) and peptide transporter 1 (Pept1, Slc15a1) were significantly decreased in kidney of hyperuricemic rats. Since Oct2, Mate1 and Oat1 are important for renal drug elimination, we next investigated whether the pharmacokinetics of their substrates, metformin, cephalexin and creatinine, were altered. The plasma concentration of metformin was not affected, while its kidney tissue accumulation was significantly increased. The plasma concentration and kidney tissue accumulation of cephalexin and the plasma concentration of creatinine were also increased. Furthermore, the protein expression of kidney Mate1 was decreased in hyperuricemic rats. Accordingly, although multiple factors may influence renal handling of these drugs, these observations can be accounted for, at least in part, by downregulation of Mate1-mediated apical efflux from tubular cells and Oct2-mediated basolateral uptake. Our results suggest that hyperuricemia could alter the disposition of drugs that are substrates of Mate1 and/or Oct2.

Experimental Evidence for Resecretion of PGE2 across Rat Alveolar Epithelium by OATP2A1/SLCO2A1-Mediated Transcellular Transport.

Prostaglandin transporter Oatp2a1/Slco2a1 is expressed at the apical (AP) membranes of type-1 alveolar epithelial (AT1) cells. To investigate the role of OATP2A1 in prostaglandin E2 (PGE2) handling by alveolar epithelium, we studied PGE2 transport across and secretion from monolayers of rat AT1-like (AT1-L) cells obtained by trans-differentiation of type-2 alveolar epithelial cells isolated from male Wistar rats. Rat AT1-L cells expressed Oatp2a1/Slco2a1, together with smaller amounts of Mrp4/Abcc4 and Oct1/Slc22a1 PGE2 uptake was saturable with Km 43.9 ± 21.9 nM. Transcellular transport of PGE2 across AT1-L cells grown on permeable filters in the AP-to-basolateral (BL) direction was 5-fold greater than that in the reverse direction and was saturable with Km 118 ± 26.8 nM; it was significantly inhibited by OATP inhibitors bromosulfophthalein (BSP) and suramin, and an MRP4 inhibitor, Ceefourin 1. We simultaneously monitored the effects of BSP on the distribution of PGE2 produced by bradykinin-treated AT1-L cells and PGE2-d4 externally added on the AP side of the cells. In the presence of BSP, PGE2 increased more rapidly on the AP side, whereas PGE2-d4 decreased more slowly on the AP side. The decrease in PGE2-d4 from the AP side corresponded well to the increase on the BL side, indicating that intracellular metabolism did not occur. These results suggest that Oatp2a1 and Mrp4 mediate transepithelial transport of PGE2 in the AP-to-BL direction. Therefore, OATP2A1 may be an important regulator of PGE2 in alveolar epithelium by reducing secretion of PGE2 and facilitating "resecretion" of PGE2 present in the alveolar lumen to the interstitial space or blood.

A downstream molecule of 1,25-dihydroxyvitamin D3, alpha-1-acid glycoprotein, protects against mouse model of renal fibrosis.

Renal fibrosis, the characteristic feature of progressive chronic kidney disease, is associated with unremitting renal inflammation. Although it is reported that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, elicits an anti-renal fibrotic effect, its molecular mechanism is still unknown. In this study, renal fibrosis and inflammation observed in the kidney of unilateral ureteral obstruction (UUO) mice were reduced by the treatment of 1,25(OH)2D3. The plasma protein level of alpha-1-acid glycoprotein (AGP), a downstream molecule of 1,25(OH)2D3, was increased following administration of 1,25(OH)2D3. Additionally, increased mRNA expression of ORM1, an AGP gene, was observed in HepG2 cells and THP-1-derived macrophages that treated with 1,25(OH)2D3. To investigate the involvement of AGP, exogenous AGP was administered to UUO mice, resulting in attenuated renal fibrosis and inflammation. We also found the mRNA expression of CD163, a monocyte/macrophage marker with anti-inflammatory potential, was increased in THP-1-derived macrophages under stimulus from 1,25(OH)2D3 or AGP. Moreover, AGP prevented lipopolysaccharide-induced macrophage activation. Thus, AGP could be a key molecule in the protective effect of 1,25(OH)2D3 against renal fibrosis. Taken together, AGP may replace vitamin D to function as an important immune regulator, offering a novel therapeutic strategy for renal inflammation and fibrosis.

Apple-Derived Nanoparticles Modulate Expression of Organic-Anion-Transporting Polypeptide (OATP) 2B1 in Caco-2 Cells.

Interaction of foods with intestinal transporters has generally been ascribed to small molecules, but recently, edible-plant-derived nanoparticles (NPs) have been suggested to affect intestinal function. Here, we examined the effects of NPs contained in edible fruits on intestinal transporters. Apple-derived NPs (APNPs) were isolated by ultracentrifugation and characterized by measurement of particle size distribution and electron microscopy. Human epithelial colorectal adenocarcinoma (Caco-2) cells internalized fluorescently labeled APNPs, suggesting that fruit-derived NPs would be internalized into intestinal epithelial cells in vivo. We found that the mRNA expression levels of several transporters, including organic-anion-transporting polypeptide (OATP) 2B1, were changed in APNP-treated Caco-2 cells. The protein expression and activity of OATP2B1 were also decreased by APNP exposure, as determined by Western blotting and measurements of [3H]estrone-3-sulfate uptake by Caco-2 cells, respectively. These actions required intact APNPs, because sonication or boiling abrogated the effects. Since the content of apple-derived small molecules in APNPs was negligible, the observed decrease of OATP2B1 expression appears to be mediated by large molecules in the APNPs. We further found that the 3'-untranslated region of the OATP2B1 gene was required for the response to APNPs, suggesting that microRNA in the APNPs might be involved. These results propose a novel mechanism, in which large molecules such as microRNA in food could affect intestinal transporters through food-derived NPs, which also demonstrates that food-derived NPs should be useful for delivery of biologically active large molecules to intestinal tissues.

Hyperuricemia enhances intracellular urate accumulation via down-regulation of cell-surface BCRP/ABCG2 expression in vascular endothelial cells.

Hyperuricemia has been recognized as an independent risk factor for cardiovascular disease. Urate stimulates NADPH oxidase and induces production of reactive oxygen species (ROS); consequently, intracellular urate accumulation can induce oxidative stress leading to endothelial dysfunction. Here, we studied the mechanism involved, using human umbilical vascular endothelial cells (HUVEC) as a model. Pretreatment with 15 mg/dL unlabeled uric acid (corresponding to hyperuricemia) resulted in increased uptake of [14C]uric acid at steady-state by HUVEC, whereas pretreatment with 5 mg/dL uric acid (in the normal serum concentration range) did not. However, the initial uptake rate of [14C]uric acid was not affected by uric acid at either concentration. These results suggest that efflux transport of uric acid is decreased under hyperuricemic conditions. We observed a concomitant decrease of phosphorylated endothelial nitric oxide synthase. Plasma membrane expression of breast cancer resistance protein (BCRP), a uric acid efflux transporter, was decreased under hyperuricemia, though the total cellular expression of BCRP remained constant. Uric acid did not affect expression of another uric acid efflux transporter, multidrug resistance associated protein 4 (MRP4). Moreover, phosphorylation of Akt, which regulates plasma membrane localization of BCRP, was decreased. These uric acid-induced changes of BCRP and Akt were reversed in the presence of the antioxidant N-acetylcysteine. These results suggest that in hyperuricemia, uric acid-induced ROS generation inhibits Akt phosphorylation, causing a decrease in plasma membrane localization of BCRP, and the resulting decrease of BCRP-mediated efflux leads to increased uric acid accumulation and dysregulation of endothelial function.

Different Involvement of OAT in Renal Disposition of Oral Anticoagulants Rivaroxaban, Dabigatran, and Apixaban.

This study aimed to investigate the interactions of 3 anticoagulants, rivaroxaban, apixaban, and dabigatran, with 5 human solute carrier transporters, hOAT1, hOAT3, hOCT2, hOATP1B1, and hOATP1B3. Apixaban inhibited hOAT3, hOATP1B1, and hOATP1B3, and rivaroxaban inhibited hOAT3 and hOATP1B3, with IC50 values of >20 and >5 µM, respectively. The effect of dabigatran was negligible or very weak, so significant drug interactions at therapeutic doses are unlikely. Specific uptake of rivaroxaban was observed only in human and mouse OAT3-expressing cells. The Km for mouse Oat3 (mOat3) was 1.01 ± 0.70 µM. A defect in mOat3 reduced the kidney-to-plasma concentration ratio of rivaroxaban by 38% in mice. Probenecid treatment also reduced the kidney-to-plasma concentration ratio of rivaroxaban in rats by 73%. Neither mOat3 defect nor probenecid administration in rats reduced the renal clearance of rivaroxaban. The uptake of rivaroxaban by monkey kidney slices was temperature dependent and inhibited by probenecid but not by tetraethylammonium. Taken together, organic anion transporters, mainly OAT3, may mediate basolateral uptake of rivaroxaban in kidneys. hOAT3 could be an additional factor that differentiates the potential drug-drug interactions of the 3 anticoagulants in the urinary excretion process in clinical settings.

The change of pharmacokinetics of fexofenadine enantiomers through the single and simultaneous grapefruit juice ingestion.

The stereoselective pharmacokinetics of fexofenadine are associated with OATP2B1-mediated transport, and grapefruit juice (GFJ) is an inhibitor of OATP2B1. Therefore, in this study, we aimed to investigate whether and to what extent GFJ ingestion affected the pharmacokinetics of fexofenadine enantiomers in healthy subjects. In a randomized, two-phase, open-label, crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or GFJ. Ingestion of GFJ significantly decreased the areas under the plasma concentration-time curve (AUC0-24) for (R)- and (S)-fexofenadine by 39% and 52%, respectively. Subsequently, GFJ increased the mean R/S ratio of the AUC0-24 from 1.58 to 1.96 (P < 0.05). Although GFJ greatly reduced the amounts of (R)- and (S)-fexofenadine excreted into the urine (Ae0-24) by 52% and 61%, respectively, the mean R/S ratios of Ae0-24 and the renal clearances of both enantiomers were unchanged between the control and GFJ phases. GFJ, an OATP2B1 inhibitor, significantly reduced the plasma concentrations of fexofenadine enantiomers, exhibiting clinically moderate effects. The present results suggested that changes in OATP2B1 activity by GFJ may alter the stereoselective pharmacokinetics of fexofenadine and that reduced intestinal OATP2B1 activity may affect the stereoselectivity of fexofenadine.

Prostaglandin Transporter (PGT/SLCO2A1) Protects the Lung from Bleomycin-Induced Fibrosis.

Prostaglandin (PG) E2 exhibits an anti-fibrotic effect in the lung in response to inflammatory reactions and is a high-affinity substrate of PG transporter (SLCO2A1). The present study aimed to evaluate the pathophysiological relevance of SLCO2A1 to bleomycin (BLM)-induced pulmonary fibrosis in mice. Immunohistochemical analysis indicated that Slco2a1 protein was expressed in airway and alveolar type I (ATI) and II (ATII) epithelial cells, and electron-microscopic immunohistochemistry further demonstrated cell surface expression of Slco2a1 in ATI cells in wild type (WT) C57BL/6 mice. PGE2 uptake activity was abrogated in ATI-like cells from Slco2a1-deficient (Slco2a1-/-) mice, which was clearly observed in the cells from WT mice. Furthermore, the PGE2 concentrations in lung tissues were lower in Slco2a1-/- than in WT mice. The pathological relevance of SLCO2A1 was further studied in mouse BLM-induced pulmonary fibrosis models. BLM (1 mg/kg) or vehicle (phosphate buffered saline) was intratracheally injected into WT and Slco2a1-/- mice, and BLM-induced fibrosis was evaluated on day 14. BLM induced more severe fibrosis in Slco2a1-/- than in WT mice, as indicated by thickened interstitial connective tissue and enhanced collagen deposition. PGE2 levels were higher in bronchoalveolar lavage fluid, but lower in lung tissues of Slco2a1-/- mice. Transcriptional upregulation of TGF-ß1 was associated with enhanced gene transcriptions of downstream targets including plasminogen activator inhitor-1. Furthermore, Western blot analysis demonstrated a significant activation of protein kinase C (PKC) δ along with a modest activation of Smad3 in lung from Slco2a1-/- mice, suggesting a role of PKCδ associated with TGF-ß signaling in aggravated fibrosis in BLM-treated Slco2a1-/- mice. In conclusion, pulmonary PGE2 disposition is largely regulated by SLCO2A1, demonstrating that SLCO2A1 plays a critical role in protecting the lung from BLM-induced fibrosis.

In-vitro evidence of enhanced breast cancer resistance protein-mediated intestinal urate secretion by uremic toxins in Caco-2 cells.

OBJECTIVES: It has been reported that intestinal urate excretion is increased at chronic kidney disease (CKD) state. In this report, whether uremic toxins are involved in the upregulation of intestinal breast cancer resistance protein (BCRP), an intestinal urate exporter, was examined. METHODS: Uremic toxins that were increased at least 15-fold at CKD state were selected for investigation. Caco-2 cells were exposed to these uremic toxins at clinically relevant concentrations. mRNA was quantified by real-time PCR, and flow cytometry was utilized to measure BCRP protein and function in Caco-2 cells. Transcellular secretory transport of [(14) C]urate was determined utilizing Transwell studies after uremic toxin exposure. KEY FINDINGS: Indoxyl sulfate (IS) treatment alone resulted in ∼ 3-fold increase in BCRP mRNA in Caco-2 cells. Membrane protein expression of BCRP in Caco-2 cells also was increased by 1.8-fold after treatment with IS. Intracellular accumulation of pheophorbide A, a selective BCRP substrate, was decreased by 22% after IS treatment for 3 days. Consistent with these findings, transcellular secretory transport of urate across Caco-2 cell monolayers was increased by 22%. CONCLUSION: Intestinal urate secretion may be increased at CKD state partially by upregulation of intestinal BCRP by uremic toxins such as IS.

Effects of one-time apple juice ingestion on the pharmacokinetics of fexofenadine enantiomers.

PURPOSE: We examined the effect of a single apple juice intake on the pharmacokinetics of fexofenadine enantiomers in healthy Japanese subjects. METHODS: In a randomized two phase, open-label crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or apple juice. For the uptake studies, oocytes expressing organic anion-transporting polypeptide 2B1 (OATP2B1) were incubated with 100 µM (R)- and (S)-fexofenadine in the presence or absence of 10 % apple juice. RESULTS: One-time ingestion of apple juice significantly decreased the area under the plasma concentration-time curve (AUC0-24) for (R)- and (S)-fexofenadine by 49 and 59 %, respectively, and prolonged the time to reach the maximum plasma concentration (t max) of both enantiomers (P < 0.001). Although apple juice greatly reduced the amount of (R)- and (S)-fexofenadine excretion into urine (Ae0-24) by 54 and 58 %, respectively, the renal clearances of both enantiomers were unchanged between the control and apple juice phases. For in vitro uptake studies, the uptake of both fexofenadine enantiomers into OATP2B1 complementary RNA (cRNA)-injected oocytes was significantly higher than that into water-injected oocytes, and this effect was greater for (R)-fexofenadine. In addition, apple juice significantly decreased the uptake of both enantiomers into OATP2B1 cRNA-injected oocytes. CONCLUSIONS: These results suggest that OATP2B1 plays an important role in the stereoselective pharmacokinetics of fexofenadine and that one-time apple juice ingestion probably inhibits intestinal OATP2B1-mediated transport of both enantiomers. In addition, this study demonstrates that the OATP2B1 inhibition effect does not require repeated ingestion or a large volume of apple juice.