Fructose suppresses uric acid excretion to the intestinal lumen as a result of the induction of oxidative stress by NADPH oxidase activation.

BACKGROUND: A high intake of fructose increases the risk for hyperuricemia. It has been reported that long-term fructose consumption suppressed renal uric acid excretion and increased serum uric acid level. However, the effect of single administration of fructose on excretion of uric acid has not been clarified. METHODS: We used male Wistar rats, which were orally administered fructose (5g/kg). Those rats were used in each experiment at 12h after administration. RESULTS: Single administration of fructose suppressed the function of ileal uric acid excretion and had no effect on the function of renal uric acid excretion. Breast cancer resistance protein (BCRP) predominantly contributes to intestinal excretion of uric acid as an active homodimer. Single administration of fructose decreased BCRP homodimer level in the ileum. Moreover, diphenyleneiodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox), recovered the suppression of the function of ileal uric acid excretion and the Bcrp homodimer level in the ileum of rats that received single administration of fructose. CONCLUSIONS: Single administration of fructose decreases in BCRP homodimer level, resulting in the suppression the function of ileal uric acid excretion. The suppression of the function of ileal uric acid excretion by single administration of fructose is caused by the activation of Nox. The results of our study provide a new insight into the mechanism of fructose-induced hyperuricemia.

Intestinal P-glycoprotein expression is multimodally regulated by intestinal ischemia-reperfusion.

PURPOSE: Reactive oxygen species (ROS) have multiple physiological effects that are amount-dependent. ROS are one of the causes of intestinal ischemia-reperfusion (I/R) injury. In this study, we investigated whether the amount of ROS and the degree of intestinal I/R injury affect the expression level of P-glycoprotein (P-gp). METHODS: . We used hydrogen peroxide (H2O2) as ROS in in vitro experiments. Intestinal I/R model rats, which were subjected 15-min ischemia (I/R-15), were used in in vivo experiments. RESULTS: P-gp expression in Caco-2 cells was increased in response to 1 µM of H2O2 but decreased upon exposure to 10 mM of H2O2. We previously reported that P-gp expression is decreased after intestinal I/R with 30-min ischemia (I/R-30), which time a large amount of ROS is generated. I/R-15 induced slightly less mucosal and oxidative injury than did I/R-30. P-gp expression in the jejunum was increased at 1 h after I/R-15, and ileal paracellular permeability was increased. The blood concentration of tacrolimus, a P-gp substrate, was lower during 0-20 min but was higher during 40-90 min post-administration compared with that in the sham-operated rats. P-gp expression in the ileum was decreased at 6 h after I/R-15, due to abnormal localization of P-gp, resulting in a high blood tacrolimus concentration in rats reperfused for 6 h. CONCLUSIONS: ROS multimodally regulate P-gp expression depending on its amount. This is important for understanding the pattern of P-gp expression after intestinal I/R.

Quercetin-3-rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1.

Quercetin-3-rhamnoglucoside (rutin) has a wide spectrum of biochemical and pharmacological activities. Rutin is absorbed mainly in its unmetabolized form. Organic anion transporting polypeptide (OATP) 2B1 is a major uptake transporter in the intestine. Thus, it is important for the prevention of adverse events to understand drug interactions mediated by OATP2B1 in the absorption process. This study assessed the effect of rutin on transport by OATP2B1. Rutin stimulated the uptake of estrone-3-sulfate (E-3-S), taurocholic acid (TCA), cholic acid (CA) and rosuvastatin by OATP2B1, but not p-coumaric acid or ferulic acid. The EC50 of rutin for transport by OATP2B1 was 2.32 µm. The Km value of E-3-S for OATP2B1 in the presence of rutin (9.21 µm) was almost the same as that in the absence of rutin (8.53 µm). On the other hand, the Vmax of E-3-S transport by OATP2B1 in the presence of rutin (270 pmol/mg protein/min) was 1.2-fold higher than that in the absence of rutin (218 pmol/mg protein/min). Moreover, the expression level of OATP2B1 on the cell membrane was increased by treatment with rutin for 5 min without alteration of the total OATP2B1 expression level. Moreover, the increase in the localization of OATP2B1 at the cell surface was detected by the immunocytochemistry. The stimulatory effect of rutin is a little weak but may affect the absorption of OATP2B1 substrates, because rutin is taken daily in foods and its intestinal concentration would reach the stimulatory range of OATP2B1.

Intestinal ischemia-reperfusion suppresses biliary excretion of hepatic organic anion transporting polypeptides substrate.

PURPOSE: Intestinal ischemia-reperfusion (I/R) causes gut dysfunction and promotes multi-organ failure. The liver and kidney can be affected by multi-organ failure after intestinal I/R. Organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs) are recognized in a broad spectrum from endogenous compounds to xenobiotics, including clinically important drugs. Therefore, it is important for understanding the pharmacokinetics to obtain evidence of alterations in OATPs and OATs expression and transport activities. In the present study, we investigated the expression of rat Oatps and Oats after intestinal I/R. METHODS: We used intestinal ischemia-reperfusion (I/R) model rats. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Plasma concentration and biliary excretion of sulfobromophthalein (BSP), which is used as a model compound of organic anion drugs, were measured after intravenous administration in intestinal I/R rats. RESULTS: Although Oat1 and Oat3 mRNA levels were not altered in the kidney, Oatp1a1, Oatp1b2 and Oatp2b1 mRNA levels in the liver were significantly decreased at 1-6 h after intestinal I/R. Moreover, Oatp1a1 and Oatp2b1 protein expression levels were decreased at 1 h after intestinal I/R. Plasma concentration of BSP, which is a typical substrate of Oatps, in intestinal I/R rats reperfused 1 h was increased than that in sham-operated rats. Moreover, the area under the concentration-time curve (AUC0₋90) in intestinal I/R rats reperfused 1 h was significantly increased than that in sham-operated rats. The total clearance (CL(tot)) and the biliary clearance (CL(bile)) in intestinal I/R rats reperfused 1 h were significantly decreased than those in sham-operated rats. CONCLUSIONS: Oatp1a1 and Oatp2b1 expression levels are decreased by intestinal I/R. The decreases in these transporters cause alteration of pharmacokinetics of organic anion compound. The newly found influence of intestinal I/R on the expression and function of Oatps may be a key to perform appropriate drug therapy.

The decrease in farnesoid X receptor, pregnane X receptor and constitutive androstane receptor in the liver after intestinal ischemia-reperfusion.

PURPOSE: Intestinal ischemia-reperfusion (I/R) damages remote organs, including the liver, and promotes multi-organ failure (MOF). However, the molecular mechanisms underlying acute liver injury after intestinal I/R have not been completely elucidated. Farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate metabolizing enzymes and transporters, and coordinately prevent hepatotoxicity reflecting an inability of appropriate excretion of endogenous toxic compounds. In this study, we assessed FXR, PXR and CAR expression levels and their localization levels in nuclei in the liver after intestinal I/R. We also investigated the effect of IL-6 on FXR, PXR and CAR expression levels and their localization levels in nuclei in in vitro experiments. METHODS: We used intestinal I/R model rats. Moreover, HepG2 cells were used in in vitro study. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Nuclear receptor localization in nuclei was analyzed by Western blotting using nuclear extracts. RESULTS: FXR and PXR expression levels began to be decreased at 3 h, and FXR, PXR and CAR expression levels were decreased at 6 h after intestinal I/R. The localization levels of FXR, PXR and CAR in nuclei began to be decreased at 3 h, and all of them were decreased at 6 h after intestinal I/R. In HepG2 cells, FXR, PXR and CAR expression levels were decreased by 0.5-1 ng/mL, 0.5-100 ng/mL and 100 ng/mL IL-6 treatment for 24 h, respectively. FXR, PXR and CAR localization levels in nuclei were suppressed by 0.5-10 ng/mL, 10-100 ng/mL and 10-100 ng/mL IL-6 treatment for 24 h, respectively. CONCLUSIONS: FXR, PXR and CAR expression levels are decreased in the liver after intestinal I/R. IL-6 is one of main causes the decreases in expressions of these receptors.

Intestinal ischemia-reperfusion increases efflux for uric acid via paracellular route in the intestine, but decreases that via transcellular route mediated by BCRP.

PURPOSE: Uric acid is thought to be one of the most important antioxidants in human biological fluids. Intestinal ischemia-reperfusion (I/R) is an important factor associated with high rates of morbidity and mortality. Reactive oxygen species (ROS) are responsible for intestinal I/R injury. The aim of this study was to clarify the efflux for uric acid from the intestine after intestinal I/R. METHODS: We used intestinal ischemia-reperfusion (I/R) model rats. Serosal to mucosal flux for [¹4C]-uric acid was assessed by using Ussing-type diffusion chambers. BCRP/Bcrp expression was assessed by Western blot analysis. Caco-2 cells were used for a model of the intestinal epithelium, and rotenone was used as a mitochondrial dysfunction inducer. RESULTS: Serosal to mucosal flux for uric acid was increased after intestinal I/R, and that for mannitol was also increased. Ko143, which is a BCRP inhibitor, did not affect the uric acid transport. The decreasing uric acid transport mediated by Bcrp was caused by decrease in the level of Bcrp homodimer, bridged by an S-S bond. The suppression of Bcrp S-S bond formation was associated with mitochondrial dysfunction. Moreover, BCRP S-S bond formation activity was decreased by rotenone in Caco-2 cells. CONCLUSIONS: Serosal to mucosal flux for uric acid is significantly increased via the paracelluler route, but that via the transcellular route mediated by Bcrp is decreased after intestinal I/R. The decreasing uric acid flux mediated by Bcrp is caused by suppression of Bcrp S-S bond formation. This suppression of Bcrp S-S bond formation may be related to mitochondrial dysfunction.

Reactive oxygen species derived from xanthine oxidase interrupt dimerization of breast cancer resistance protein, resulting in suppression of uric acid excretion to the intestinal lumen.

The prevalence of hyperuricemia/gout increases with aging. However, the effect of aging on function for excretion of uric acid to out of the body has not been clarified. We found that ileal uric acid clearance in middle-aged rats (11-12 months) was decreased compared with that in young rats (2 months). In middle-aged rats, xanthine oxidase (XO) activity in the ileum was significantly higher than that in young rats. Inosine-induced reactive oxygen species (ROS), which are derived from XO, also decreased ileal uric acid clearance. ROS derived from XO decreased the active homodimer level of breast cancer resistance protein (BCRP), which is a uric acid efflux transporter, in the ileum. Pre-administration of allopurinol recovered the BCRP homodimer level, resulting in the recovering ileal uric acid clearance. Moreover, we investigated the effects of ROS derived from XO on BCRP homodimer level directly in Caco-2 cells using hypoxanthine. Treatment with hypoxanthine decreased BCRP homodimer level. Treatment with hypoxanthine induced mitochondrial dysfunction, suggesting that the decreasing BCRP homodimer level might be caused by mitochondrial dysfunction. In conclusion, ROS derived from XO decrease BCRP homodimer level, resulting in suppression of function for uric acid excretion to the ileal lumen. ROS derived from XO may cause the suppression of function of the ileum for the excretion of uric acid with aging. The results of our study provide a new insight into the causes of increasing hyperuricemia/gout prevalence with aging.

Effect of oxidative stress on expression and function of human and rat organic anion transporting polypeptides in the liver.

Reactive oxygen species (ROS) have physiological function and involve alteration of physical state. However, it is not clear effect of oxidative stress on pharmacokinetics. Organic anion transporting polypeptides (human: OATPs, rodent: Oatps) are important for uptake of endogenous and exogenous compounds into hepatocytes. Thus, alteration of OATPs/Oatps expression level may affect pharmacokinetics of various drugs. In this study, we investigated the alteration of OATPs/Oatps expression levels and function by oxidative stress, and the effect of alteration of those on pharmacokinetics of a typical OATPs/Oatps substrate pravastatin. OATPs/Oatps expression levels and function were altered by H2O2-induced oxidative stress in in vitro experiments. The alteration of Oatps expression by oxidative stress also occurred in in vivo experiments. Oatp1a1, Oatp1a4 and Oatp1b2 expression in the liver were decreased in rats fed powdery diet containing 2% inosine, which induces oxidative stress through activation of xanthine oxidase, for 1 day. The decrease in Oatps expression levels by oxidative stress caused the suppression of pravastatin uptake to the liver, and resulted in high plasma concentration of pravastatin and low biliary excretion. In conclusion, oxidative stress induces alteration of OATPs/Oatps expression and function in hepatocytes, resulting in alteration of pharmacokinetics of their substrates.