Single-cell analysis reveals different age-related somatic mutation profiles between stem and differentiated cells in human liver.

Accumulating somatic mutations have been implicated in age-related cellular degeneration and death. Because of their random nature and low abundance, somatic mutations are difficult to detect except in single cells or clonal cell lineages. Here, we show that in single hepatocytes from human liver, an organ exposed to high levels of genotoxic stress, somatic mutation frequencies are high and increase substantially with age. Considerably lower mutation frequencies were observed in liver stem cells (LSCs) and organoids derived from them. Mutational spectra in hepatocytes showed signatures of oxidative stress that were different in old age and in LSCs. A considerable number of mutations were found in functional parts of the liver genome, suggesting that somatic mutagenesis could causally contribute to the age-related functional decline and increased incidence of disease of human liver. These results underscore the importance of stem cells in maintaining genome sequence integrity in aging somatic tissues.

A pre-neoplastic epigenetic field defect in HCV-infected liver at transcription factor binding sites and polycomb targets.

The predisposition of patients with Hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) involves components of viral infection, inflammation and time. The development of multifocal, genetically distinct tumours is suggestive of a field defect affecting the entire liver. The molecular susceptibility mediating such a field defect is not understood. One potential mediator of long-term cellular reprogramming is heritable (epigenetic) regulation of transcription, exemplified by DNA methylation. We studied epigenetic and transcriptional changes in HCV-infected livers in comparison with control, uninfected livers and HCC, allowing us to identify pre-neoplastic epigenetic and transcriptional events. We find the HCV-infected liver to have a pattern of acquisition of DNA methylation targeted to candidate enhancers active in liver cells, enriched for the binding sites of the FOXA1, FOXA2 and HNF4A transcription factors. These enhancers can be subdivided into those proximal to genes implicated in liver cancer or to genes involved in stem cell development, the latter distinguished by increased CG dinucleotide density and polycomb-mediated repression, manifested by the additional acquisition of histone H3 lysine 27 trimethylation (H3K27me3). Transcriptional studies on our samples showed that the increased DNA methylation at enhancers was associated with decreased local gene expression, results validated in independent samples from The Cancer Genome Atlas. Pharmacological depletion of H3K27me3 using the EZH2 inhibitor GSK343 in HepG2 cells suppressed cell growth and also revealed that local acquired DNA methylation was not dependent upon the presence of polycomb-mediated repression. The results support a model of HCV infection influencing the binding of transcription factors to cognate sites in the genome, with consequent local acquisition of DNA methylation, and the added repressive influence of polycomb at a subset of CG-dense cis-regulatory sequences. These epigenetic events occur before neoplastic transformation, resulting in what may be a pharmacologically reversible epigenetic field defect in HCV-infected liver.

A novel casein kinase 2 alpha-subunit regulates membrane protein traffic in the human hepatoma cell line HuH-7.

A previously isolated endocytic trafficking mutant (TRF1) isolated from HuH-7 cells is defective in the distribution of subpopulations of cell-surface receptors for asialoorosomucoid (asialoglycoprotein receptor (ASGR)), transferrin, and mannose-terminating glycoproteins. The pleiotropic phenotype of TRF1 also includes an increased sensitivity to Pseudomonas toxin and deficient assembly and function of gap junctions. HuH-7xTRF1 hybrids exhibited a normal subcellular distribution of ASGR, consistent with the TRF1 mutation being recessive. A cDNA expression library derived from HuH-7 mRNA was transfected into TRF1 cells, which were subsequently selected for resistance to Pseudomonas toxin. Sequence analysis of a recovered cDNA revealed a unique isoform of casein kinase 2 (CK2), CK2alpha". Western blot analysis of TRF1 proteins revealed a 60% reduction in total CK2alpha expression. Consistent with this finding, the hybrids HuH-7xHuH-7 and HuH-7xTRF1 expressed equivalent amounts of total CK2alpha. Immunoblots using antibodies against peptides unique to the previously described CK2 isoforms CK2alpha and CK2alpha' and the novel CK2alpha" isoform showed that, although TRF1 and parental HuH-7 cells expressed comparable amounts of CK2alpha and CK2alpha', the mutant did not express CK2alpha". Based on the genomic DNA sequence, RNA transcripts encoding CK2alpha" apparently originate from alternative splicing of a primary transcript. Protein overexpression following transfection of TRF1 cells with cDNAs encoding either CK2alpha or the newly cloned CK2alpha" restored the parental HuH-7 phenotype, including Pseudomonas toxin resistance, cell-surface ASGR binding activity, phosphorylation, and the assembly of gap junctions. This study suggests that HuH-7 cells express at least three CK2alpha isoforms and that the pleiotropic TRF1 phenotype is a consequence of a reduction in total CK2 expression.

Cellular and molecular biology of the liver.

Recently, several lines of investigation focused on basic mechanisms governing cellular and molecular aspects of liver biology have intersected at the study of the hepatic stem cell. Despite years of study, the very question of the existence of the hepatic stem cell has yet to be unequivocally established. A second field of investigation into the cellular and molecular aspects of liver biology is aimed at liver-directed gene therapy in which several new vehicles have been devised to mediate gene transfer. Gene therapy is no longer thought of in the limited framework of a means to correct inherited disorders; it is now expanding into new therapeutic applications. A third major area of investigation includes studies of mechanisms that regulate membrane protein traffic necessary to maintain the integrity of differentiated liver cell function. In this review, some of the most recent advances and applications in these three areas are highlighted, and, where appropriate, points of interaction and potential therapeutic importance are emphasized.

Microtubule and motor-dependent endocytic vesicle sorting in vitro.

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor-ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by approximately 300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end-directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 microM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Uptake of enalapril and expression of organic anion transporting polypeptide 1 in zonal, isolated rat hepatocytes.

Sinusoidal entry is the first obligatory process preceding intracellular drug removal in liver. Transport of the angiotensin converting enzyme inhibitor enalapril (1-750 microM with [(3)H]enalapril), a substrate of Oatp1, the sodium-independent organic anion transporting polypeptide 1 cloned from rat liver, was studied in rat hepatocytes isolated from all zones of the liver (homogeneous) and from enriched periportal (PP) and perivenous (PV) hepatocytes prepared by collagenase perfusion and zone-selective destruction with digitonin, respectively. Uptake was linear over 1 min and was concentration-dependent. Transport by the homogeneous hepatocytes (in the presence and absence of Na(+)) and PP and PV cells was described by single saturable components of similar kinetic constants (K(m) values of 344-461 microM and V(max) values of 9.5-11.6 nmol/min/10(6) cells; P >.05, ANOVA). The K(m) value for enalapril uptake in hepatocytes was of the same order of magnitude compared with that for Oatp1 expressed in HeLa cells transfected with cDNA-Oatp1 and Western blot analysis revealed similar levels of immunoreactive Oatp1 expression in PP and PV hepatocytes. However, enalapril was not taken up by Oatp2 nor by the human OATP expressed in recombinant vaccinia systems.

Reconstitution of ATP-dependent movement of endocytic vesicles along microtubules in vitro: an oscillatory bidirectional process.

We have previously used the asialoglycoprotein receptor system to elucidate the pathway of hepatocytic processing of ligands such as asialoorosomucoid (ASOR). These studies suggested that endocytic vesicles bind to and travel along microtubules under the control of molecular motors such as cytoplasmic dynein. We now report reconstitution of this process in vitro with the use of a microscope assay to observe the interaction of early endocytic vesicles containing fluorescent ASOR with fluorescent microtubules. We find that ASOR-containing endosomes bind to microtubules and translocate along them in the presence of ATP. This represents the first time that mammalian endosomes containing a well-characterized ligand have been directly observed to translocate on microtubules in vitro. The endosome movement does not require cytosol or exogenous motor protein, is oscillatory, and is directed toward the plus and minus ends at equal frequencies. We also observe endosomes being stretched in opposite directions along microtubules, suggesting that microtubules could provide a mechanical basis for endocytic sorting events. The movement of endosomes in vitro is consistent with the hypothesis that microtubules actively participate in the sorting and distribution of endocytic contents.

Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1.

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7.

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca(2+) waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.

Effects of LPS on transport of indocyanine green and alanine uptake in perfused rat liver.

Lipopolysaccharide (LPS) initiates cholestasis. Whether this process is mediated by tumor necrosis factor-alpha (TNF-alpha) and whether the cholestatic response to LPS is associated with intrahepatic accumulation of possibly toxic substances are under debate. To study these questions the hepatic uptake and biliary excretion of indocyanine green (ICG) was examined in the isolated perfused rat liver 18 h after intravenous treatment of rats with either saline, 1 mg/kg body wt LPS, or LPS and intraperitoneal pentoxifylline (POF) (n = 6 in each group). POF inhibits TNF-alpha release after LPS administration. LPS induced a typical acute-phase response with increased mRNA for acute-phase proteins, reduced albumin mRNA, and increased hepatic uptake of alanine. Intrinsic hepatic clearance of ICG in controls (1.01 +/- 0.05 ml. min(-1). g liver(-1)) was similarly decreased by LPS alone (0.62 +/- 0.04 ml. min(-1). g(-1); P = 0.002 vs. control) or combined with POF (0.66 +/- 0.06 ml. min(-1). g(-1)). A kinetic analysis indicated that LPS reduced both uptake and excretion processes in a balanced manner, so that intrahepatic ICG content was unaffected or even slightly reduced, as confirmed by measurement of ICG contents in the perfused livers. In livers from parallel-treated nonperfused rats, mRNA for the organic anion transporting protein-1 (Oatp1, which is likely to mediate ICG uptake) was unaffected by LPS, whereas the concentration of Oatp1 protein was reduced. Thus LPS induced an acute-phase response that included downregulation of ICG uptake by reduction of Oatp1 protein concentration, possibly at a posttranscriptional level. TNF-alpha appears not to be the mediator because POF did not modify these LPS effects.

The modified dipeptide, enalapril, an angiotensin-converting enzyme inhibitor, is transported by the rat liver organic anion transport protein.

Oatp1, the organic anion transport polypeptide, is an integral membrane protein cloned from rat liver that mediates the uptake of various organic anions such as bromosulfophthalein (BSP) and taurocholate (TCA). Recent studies by others revealed that the thrombin inhibitor, CRC 220, a modified dipeptide, was transported by oatp1. The present study was designed to examine whether another modified peptide, enalapril, an angiotensin-converting enzyme inhibitor, was also a substrate. Transport was studied with enalapril (1 to 800 micromol/L, with [3H]enalapril) in a HeLa cell line stably transfected with oatp1-cDNA under the regulation of a Zn2+-inducible promoter. Noninduced transfected cells (without zinc) that did not express oatp1 failed to take up enalapril. In contrast, cells expressing oatp1 transported enalapril, estrone sulfate (E1S), taurolithocholic acid sulfate (TLCAS), and the glutathione conjugate of BSP (BSPGSH). Uptake of enalapril by oatp1 at 37 degreesC was substantially higher than that at 4 degreesC. The rate at 37 degreesC (uptake rates for induced - noninduced, transfected cells) was linear over 5 minutes and was concentration-dependent, characterized by a Km of 214 +/- 67 micromol/L and a Vmax of 0.51 +/- 0.15 nmol/min/mg protein. Enalapril uptake was inhibited competitively by BSP (at 1, 5, 10, and 50 micromol/L) and TCA (at 5, 25, and 100 micromol/L) with inhibition constants (Ki) of 2 and 32 micromol/L, respectively. The metabolite enalaprilat was, however, not transported by oatp1. That oatp1 is not a general transporter of anionic compounds was further shown by the lack of transport of harmol sulfate, benzoate, and hippurate. These observations attest to the role of oatp1 as a specific transporter for at least two classes of pharmacologically important peptides.

Dichotomous development of the organic anion transport protein in liver and choroid plexus.

Both adult liver and choroid plexus express the organic anion transport protein (oatp1) and transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.

Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

Effect of fasting on the uptake of bilirubin and sulfobromophthalein by the isolated perfused rat liver.

BACKGROUND & AIMS: Occurrence of hyperbilirubinemia after fasting has been recognized for many years. The pathogenesis of this syndrome is unclear. Although recent studies suggest that increased intestinal deconjugation and reabsorption of bilirubin may play a major role in establishment of hyperbilirubinemia during fasting, other studies have suggested that fasting down-regulates intrinsic hepatocyte transport of bilirubin. The present study was designed to examine this possibility in the isolated perfused rat liver. METHODS: Transport of 3H-bilirubin and 35S-sulfobromophthalein (BSP) was examined in isolated perfused livers from 48-hour fasted or control rats using a multiple indicator dilution technique. Data were analyzed to quantify single-pass extraction (model-independent analysis) and were also fit by computer to the model of Goresky to quantify unidirectional fluxes. RESULTS: Fasting for 48 hours resulted in an approximately 40% reduction in liver weight but had no effect on model-dependent or model-independent parameters of transport. Despite the fact that the liver was smaller, single-pass extraction of bilirubin and BSP by livers from fasted animals did not differ from control, indicating a greater efficiency at uptake of bilirubin and BSP. CONCLUSIONS: Enhanced enterohepatic circulation of bilirubin, not altered hepatic transport, is a major factor in the pathogenesis of fasting-induced hyperbilirubinemia.

Organic anion transporting polypeptide mediates organic anion/HCO3- exchange.

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591-25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (Km = 19.4 +/- 3.3 microM; Vmax = 62.2 +/- 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3- extrusion, we compared the rates of taurocholate-dependent HCO3- efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi ( approximately 7.7) by transient exposure to a 50 mM HCO3-, Cl--free solution. In the absence of extracellular Cl- and taurocholate, isohydric reduction of superfusate HCO3- concentration from 50 to 25 mM resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 microM taurocholate to the superfusate led to a rapid fall in pHi in induced (-0.037 +/- 0.011 pH units/min to pHi of 7.41 +/- 0.14) but not in noninduced (0.003 +/- 0.006 pH units/min to pHi of 7.61 +/- 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3- exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function.

The choroid plexus epithelium is the site of the organic anion transport protein in the brain.

The mRNA for organic anion transport protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase-PCR techniques and in situ hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial transport of organic anions between the choroid plexus and the cerebrospinal fluid.

Immunologic distribution of an organic anion transport protein in rat liver and kidney.

A Na(+)-independent organic anion transport protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this protein is sufficient for cells to transport the organic anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane protein. This 80-kDa protein migrated as a 65-kDa protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the transporter migrated as an 83-kDa protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this transporter in liver and kidney may have important functional and regulatory consequences.

Hepatocellular sinusoidal membrane organic anion transport and transporters.

This review focuses on oatp, a rat liver protein that has been cloned on the basis of its ability to transport organic anions such as bilirubin and sulfobromophthalein (BSP). Although other proteins have been suggested as having bilirubin or BSP transport activity, these data have primarily been indirect, and their cloning and expression have not yet been accomplished. Although preliminary data suggest that organic anion transporting polypeptide (oatp) accounts for a significant amount of BSP transport into the hepatocyte, there is certainly the possibility that other transporters exist. In addition, oatp appears to be a member of a new family of integral membrane transport proteins that may have overlapping substrate specificities. Organic anions such as bilirubin and sulfobromophthalein (BSP) circulate bound tightly to albumin from which they are rapidly extracted by hepatocytes. This process of organic anion uptake has been the subject of extensive investigation over many years. Although details remain unresolved, much has been learned recently regarding the mechanisms of organic anion transport. This article will examine the history and current status of this field.