Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.

Active removal of inorganic phosphate from cerebrospinal fluid by the choroid plexus.

The P(i) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF P(i) concentration. The large sheet-like fourth CP of the shark was mounted in Ussing chambers where unidirectional (33)P(i) fluxes revealed potent active transport from CSF to the blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 Ω·cm(2) and electrical potential difference of +0.9 ± 0.17 mV (CSF side positive). Active P(i) absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na(+)-free medium, and increasing the K(+) concentration from 5 to 100 mM. Li(+) stimulated transport twofold compared with Na(+)-free medium. Phosphonoformic acid (1 mM) had no effect on active P(i) transport. RT-PCR revealed both P(i) transporter (PiT)1 and PiT2 (SLC20 family) gene expression, but no Na(+)-P(i) cotransporter II (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot analysis and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both the shark and rat. PiT1 appeared to be localized primarily to vascular endothelial cells. Taken together, these data indicate that the CP actively removes P(i) from CSF. This process has transport properties consistent with a PiT2, Na(+)-dependent transporter that is located in the apical region of the CP epithelium.

Avian renal proximal tubule urate secretion is inhibited by cellular stress-induced AMP-activated protein kinase.

Urate is a potent antioxidant at high concentrations but it has also been associated with a wide variety of health risks. Plasma urate concentration is determined by ingestion, production, and urinary excretion; however, factors that regulate urate excretion remain uncertain. The objective of this study was to determine whether cellular stress, which has been shown to affect other renal transport properties, modulates urate secretion in the avian renal proximal tubule. Chick kidney proximal tubule epithelial cell primary culture monolayers were used to study the transepithelial transport of radiolabeled urate. This model allowed examination of the processes, such as multidrug resistance protein 4 (Mrp4, Abcc4), which subserve urate secretion in a functional, intact, homologous system. Our results show that the recently implicated urate efflux transporter, breast cancer resistance protein (ABCG2), does not significantly contribute to urate secretion in this system. Exposure to a high concentration of zinc for 6 h induced a cellular stress response and a striking decrease in transepithelial urate secretion. Acute exposure to zinc had no effect on transepithelial urate secretion or isolated membrane vesicle urate transport, suggesting involvement of a cellular stress adaptation. Activation of AMP-activated protein kinase (AMPK), a candidate modulator of ATP-dependent urate efflux, by 5'-aminoimidazole-4-carboxamide 1-ß-d-ribo-furanoside caused a decrease in urate secretion similar to that seen with zinc-induced cellular stress. This effect was prevented with the AMPK inhibitor compound C. Notably, the decrease in urate secretion seen with zinc-induced cellular stress was also prevented by compound C, implicating AMPK in regulation of renal uric acid excretion.

Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, NAD(P)H: quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention.

Avian renal proximal tubule epithelium urate secretion is mediated by Mrp4.

Birds are uricotelic and, like humans, maintain high plasma urate concentrations (approximately 300 microM). The majority of their urate waste, as in humans, is eliminated by renal proximal tubular secretion; however, the mechanism of urate transport across the brush-border membrane of the intact proximal tubule epithelium during secretion is uncertain. The dominance of secretory urate transport in the bird provides a convenient model for examining this process. The present study shows that short hairpin RNA interference (shRNAi) effectively knocked down gene expression of multidrug resistance protein 4 (Mrp4; 25% of control) in primary monolayer cultures of isolated chicken proximal tubule epithelial cells (cPTCs). Control and Mrp4-shRNAi-treated cPTCs were mounted in Ussing chambers and unidirectional transepithelial fluxes of urate were measured. To detect nonspecific effects, transepithelial electrical resistance (TER) and sodium-dependent glucose transport (Iglu) were monitored throughout experiments. Knocking down Mrp4 expression resulted in a reduction of transepithelial urate secretion to 35% of control with no effects on TER or Iglu. Although electrical gradient-driven urate transport in isolated brush-border membrane vesicles was confirmed, potassium-induced depolarization of the plasma membrane in intact cPTCs failed to inhibit active transepithelial urate secretion. However, electrical gradient-dependent vesicular urate transport was inhibited by the MRP4 inhibitor MK-571 also known to inhibit active transepithelial urate transport by cPTCs. Based on these data, direct measure of active transepithelial urate secretion in functional avian proximal tubule epithelium indicates that Mrp4 is the dominant apical membrane exit pathway from cell to lumen.