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Patients who undergo a percutaneous liver biopsy as outpatients should be monitored closely for the development of complications. A percutaneous liver biopsy is a procedure used to diagnosis patients with suspected liver disease or to plan their treatment. This procedure was first used in the late 1800s by a German physician. It did not gain widespread acceptance until after the development of a safer technique in 1957. Physicians first started performing the percutaneous liver biopsy on an outpatient basis in 1966. Patients who undergo a liver biopsy as outpatients are closely monitored for the first six hours following the procedure. A research study found that 3% of patients who underwent a liver biopsy as outpatients developed complications. The complication rate among outpatients may vary depending on the technique used to do the biopsy. Outpatients in more advanced stages of liver disease may have a higher risk of complications than other patients.

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.

Breast cancer resistance protein (Bcrp) is a member of the ATP-binding cassette membrane transporter family, which is expressed apically in liver, kidney, and intestine epithelium. Recent reports suggest that in addition to xenobiotics, porphyrins, and food toxins, Bcrp can also transport bile acids and, therefore, may participate in the adaptive response to cholestasis. Bile duct ligation (BDL), an experimental model of obstructive cholestasis, was performed in male wild-type (WT) and Bcrp knockout (KO) mice. An initial time course of 3, 7, and 14 days of BDL in WT mice revealed that Bcrp expression was significantly reduced in liver but increased in ileum by 7 days. Subsequent experiments using 7-day BDL in WT and Bcrp KO mice demonstrated that there was no difference in liver necrosis, serum glutamic pyruvate aminotransferase, bilirubin, or bile acid levels in serum, hepatic tissue, bile, urine, or feces between the two groups. Protein expression levels for liver organic solute transporter (Ost) α and multidrug resistance protein 1 and kidney multidrug resistance-associated protein (Mrp) 2, Mrp3, and Mrp4 were significantly greater in the sham Bcrp KO versus sham WT mice. The expression of Mrp2 and Mrp4 in KO kidneys was further increased after BDL. In contrast, the adaptive response of transporters to BDL in the liver was similar in KO and WT BDL mice, including Ostα and Ostβ expression, which increased in liver and kidney but decreased in the ileum. These findings suggest that Bcrp does not have a significant role in the adaptive response to cholestasis in the liver but may be more important for solute export in the kidney and intestine.

Cholesterol 7α-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRα by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1α plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.

The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 µmol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of γ-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (–39% and –33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases α, µ, and π, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.

The nuclear farnesoid X receptor (FXR) plays a pivotal role in maintaining bile acid homeostasis by regulating key genes involved in bile acid synthesis, metabolism and transport, including CYP7A1, UGT2B4, BSEP, MDR3, MRP2, ASBT, I-BABP, NTCP and OSTα-OST in humans. Altered expression or malfunction of these genes has been described in patients with cholestatic liver diseases. This review examines the rationale for the use of FXR ligand therapy in various cholestatic liver disorders and includes potential concerns.

Since the origination of the liver biopsy, the technique has evolved into an essential diagnostic tool, with very few complications. In addition to the percutaneous approach, a liver biopsy can also be obtained via transjugular, laparoscopic, or intraoperative approach. While in the early 1960s and 1970s the liver biopsy was used for making a diagnosis in cases of clinically suspected medical liver disease, today it is more often performed to assess disease prognosis and evaluate therapeutic strategies. As a result, indications for the liver biopsy have evolved over the past 2 decades. However with advances in serologic diagnosis of viral/autoimmune hepatitis and laboratory tests for genetic disorders, the role of liver biopsy in certain clinical settings is currently debated. This review discusses the technique, indications, contraindications, and the changing role of liver biopsy in some of the common disorders and the associated controversies.

This review highlights recent developments in the molecular pathogenesis of cholestasis as well new aspects of pathogenesis and management of clinical cholestatic disorders.

Further insights into the cellular and molecular mechanisms underlying hepatobiliary transport function and its regulation now permit a better understanding of the pathogenesis and treatment options of cholestatic liver diseases. Identification of the molecular basis of hereditary cholestatic syndromes will result in an improved diagnosis and management of these conditions. New insights into the pathogenesis of extrahepatic manifestations of cholestasis (eg, pruritus) have facilitated new treatment strategies. Important new studies have been published about the pathogenesis, clinical features, diagnosis, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, cholestasis of pregnancy, total parenteral nutrition-induced cholestasis, drug-induced cholestasis, and viral cholestatic syndromes.

Background & Aims:Obstructive cholestasis is associated with adaptive changes in expression of hepatocyte transport proteins. These include a significant reduction in hepatic expression of Mrp2 (Abcc2), the principal canalicular multispecific organic anion transporter. Renal Mrp2 expression is preserved under these conditions. We have recently reported that the rat Mrp2 promoter is activated by RARα:RXRα, and that interleukin 1β (IL-1β) repressed promoter activity via this element. We hypothesized that cytokines, which are up-regulated in obstructive cholestasis, would reduce nuclear RARα:RXRα levels, and that this would be associated with suppression of hepatic Mrp2 expression. Methods:Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham surgery, and liver and kidney RNA and protein were isolated. Primary rat hepatocytes were treated with bile acids, retinoids, or cytokines, and RNA and protein were isolated. Mrp2 and RARα:RXRα protein abundance and activity were assessed by using electrophoretic mobility shift assays (EMSA) and immunoblots. IL-1β abundance was determined by enzyme-linked immunosorbent assay. RARα, RXRα, and Mrp2 RNA levels were determined by using ribonuclease protection assays (RPA). Results:Mrp2 down-regulation and IL-1β up-regulation were observed in liver after BDL. This was temporally associated with down-regulation of liver RARα:RXRα nuclear protein levels and binding to the Mrp2 promoter cis element. Renal RARα:RXRα and Mrp2 expression were preserved under these conditions. IL-1β treatment of primary hepatocytes reduced Mrp2 and RXRα expression. Conclusions:Organ-specific regulation of Mrp2 expression in obstructive cholestasis is associated with cytokine-dependent alterations in RARα:RXRα nuclear receptors. Preservation of renal Mrp2 expression may permit urinary excretion of toxic organic anions and xenobiotics under conditions in which biliary excretion is impaired.

Further insights into the molecular regulation of bile acid transport and metabolism have provided the basis for a better understanding of the pathogenesis of cholestatic liver diseases. Novel insights into the mechanisms of action of ursodeoxycholic acid should advance our understanding of the treatment of cholestatic liver diseases. Mutations of transporter genes can cause hereditary cholestatic syndromes in both infants and adults as well as cholesterol gallstone disease. Important studies have been published on the pathogenesis, clinical features, and treatment of primary biliary cirrhosis, drug-induced cholestasis, and cholestasis of pregnancy.

New insights into the regulation of hepatobiliary transport proteins have provided the basis for a better understanding of the pathogenesis of cholestatic liver diseases. Mutations of transporter genes can cause hereditary cholestatic syndromes, the study of which has shed much light on the basic mechanisms of bile secretion and cholestasis. Important new studies have been published about the pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, cholestasis of pregnancy, total parenteral nutrition–induced cholestasis, and drug-induced cholestasis.

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinaceaexpress a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61–64% similarity to P2Y1receptors and 21–37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopusoocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y1receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.

Continued advances in the field of liver cell biology and molecular biology have provided further insights into the normal physiology of bile secretion and the pathogenesis and therapy for cholestatic syndromes. Important new data have also been published about pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, drug-induced cholestasis, and cholestatic syndromes caused by viral infections.

Microcystin-induced ser/thr protein phosphatase (PP) inhibition and toxicity were examined in the little skate (Raja erinacea), an evolutionarily primitive marine vertebrate. As in mammals, PP inhibition and toxicity were exclusively hepatocellular, but were much more persistent in the skate. A dose of 63 μg/kg given iv to adult male skates resulted in the near complete inhibition of hepatic PP activity at 24 h. PP activity was still 95% inhibited 7 days after dosing in skates given 125 μg/kg microcystin. Mortality occurred at doses of 500 μg/kg or more. Hepatic lesions were only seen in animals with fully inhibited PP activity in liver. The histological changes seen at 125 μg/kg were mild periportal inflammatory changes increasing in severity together with hepatocyte necrosis at higher doses of microcystin. Microcystin persisted and could be detected in plasma up to 7 days after dosing. This finding shows that, in the skate, as in mammals, the liver is the only organ capable of uptake of microcystin, since there was no significant inhibition of PP activity in the rectal gland and small decreases in PP activity of the kidney that were not time or dose dependent. In vitromicrocystin caused dose-dependent inhibition of PP activity in isolated skate hepatocytes, while it was without effect in cultured rectal glands. Uptake of microcystin and the accompanying inhibition of PP activity in skate hepatocytes was prevented by the addition of a series of organic dyes and bile acids. The spectrum of inhibitors of microcystin uptake in skate is similar to that seen in the rat, indicating common features of the carrier(s) in these diverse species.

Basolateral to apical vesicular transcytosis in the hepatocyte is an essential pathway for the delivery of compounds from the sinusoidal blood to the bile and to traffic newly synthesized resident apical membrane proteins to their site of function at the canalicular membrane front. To characterize this pathway better, microtubules in a hepatocyte homogenate were polymerized by addition of taxol, and associated membrane-bound vesicles were isolated. This fraction was enriched in polymeric immunoglobulin A receptor and contained apical membrane proteins. Immunoelectron microscopy demonstrated that polymeric immunoglobulin A receptor was localized predominantly on vesicles ranging from 100 to 160 nm and that the multidrug resistance protein 2 and the bile salt export pump co-localized on these vesicles. The minus-ended microtubule motor, dynein, was highly enriched in the fraction, and its intermediate chain could be released effectively by incubation with 1 mmATP or GTP. However, the association of the transcytotic vesicles with the microtubules was not sensitive to hydrolyzable or non-hydrolyzable nucleotides. This study characterizes a fraction of microtubule-associated vesicles from rat hepatocytes and demonstrates that several resident apical membrane transport proteins and the polymeric immunoglobulin A receptor traffic on the same vesicle.

Recent advances in cholestatic liver disease have occurred in several areas. Molecular cloning of hepatobiliary transport systems has resulted in the identification of the molecular basis of hereditary and acquired cholestatic syndromes. Apoptosis has been identified as an important mechanism of cholestatic liver injury and bile duct loss. New insights into the pathogenesis of pruritus and fatigue have resulted in new treatment strategies for these debilitating symptoms. Important new studies have been published about pathogenesis, clinical features, and treatment of primary biliary cirrhosis, primary sclerosing cholangitis, cholestasis of pregnancy, and drug-induced cholestasis.

Background & Aims:Vasoactive intestinal polypeptide (VIP) is a neuropeptide with diverse biological functions including stimulatory effects on bile secretion. The effects of VIP on bile secretion and its site of action were examined. Methods:Choleretic effects of VIP were examined using isolated perfused livers, hepatocyte couplets, isolated bile duct units, and cholangiocytes from rat liver. Results:VIP (100 nmol/L) produced a small increase in bile flow and bile salt output in taurocholate-supplemented isolated perfused livers but had no significant effect on bile flow in the absence of bile salt supplements or on fluid secretion in isolated hepatocyte couplets. In addition, VIP significantly increased bile pH, bicarbonate concentration, and output in the isolated perfused livers from both normal and 2 week bile duct–ligated rats, although bile flow increased only in the bile duct–ligated model. VIP also produced a dose-dependent increase in fluid secretion in isolated bile duct units, which was inhibited significantly by VIP antagonist, a specific VIP receptor inhibitor. This VIP-stimulated secretory response in isolated bile duct units was more potent than those produced by bombesin or secretin. Neither somatostatin nor substance P inhibited the VIP response in isolated bile duct units. In contrast to secretin, VIP had no significant effect on adenosine 3',5'-cyclic monophosphate (cAMP) levels in isolated cholangiocytes. Conclusions:VIP is a potent stimulant of fluid and bicarbonate secretion from cholangiocytes via cAMP-independent pathways, suggesting that this neuropeptide plays a major regulatory role in biliary transport and secretion.

Thirty-six patients presenting with asymptomatic primary biliary cirrhosis have been followed for a median period of 11.4 yr, extending by 5 yr a previously reported median follow-up study of 6 yr. Life table survival analysis indicates that the overall survival of this subgroup of patients with primary biliary cirrhosis continues to remain similar to that of the general population (p = 0.91). Over this period, 15 patients developed symptoms and 8 patients died, 6 from liver disease; 21 patients remained in an asymptomatic state. Portal granulomas on initial liver biopsy were the only finding that correlated with a normal survival and a continued asymptomatic state (p = 0.03). In contrast, associated autoimmune disorders (thyroiditis, sicca syndrome, CRST syndrome, Raynaud's phenomenon) correlated with decreased survival (p = 0.01). No other clinical, laboratory, or histologic features correlated with survival or the development of symptoms. This extended follow-up study (median 11.4 yr) indicates that many patients with asymptomatic primary biliary cirrhosis have a benign outcome. Although 42% developed signs or symptoms of progressive disease at variable times up to 14 yr from presentation, the group survival remained similar to the general population.

The clinicopathologic features and natural history of primary sclerosing cholangitis were reviewed in 53 patients followed at the Yale Liver Center during the past 30 yr. At presentation, the mean age of patients was 46 yr, and the male to female ratio was 1.4:1. Biliary sclerosis was limited to the intrahepatic ductal system in 21% of the patients. Fiftythree percent of the patients had mild disease without portal hypertension at presentation, and 25% had no symptoms attributable to their liver disease. Long-term follow-up was available for 42 patients and averaged 56 mo. Over this period, 16 patients remained mildly symptomatic, and 11 were asymptomatic. Survival was calculated by a Kaplan-Meier life-table analysis and demonstrated that 75% of the patients were alive 9 yr after the diagnosis of primary sclerosing cholangitis. A multivariate analysis of clinical features revealed that hepatomegaly and a serum bilirubin level >1.5 mg/dl at the onset of disease were independent discriminators of a poor prognosis. Patients referred to this university medical center displayed different clinical characteristics than previously reported in primary sclerosing cholangitis. A higher percentage were older, female, and asymptomatic, and more had disease limited to the intrahepatic ductal system. Survival was also considerably improved in this group of patients and suggests that the long-term prognosis for patients with primary sclerosing cholangitis may be considerably better than previously believed.

Chlorpromazine (2.5 × 10-4M) significantly diminished the rate of bile acid excretion when added to the perfusate of the isolated perfused rat liver during a constant infusion of sodium taurocholate (40 μmol/hr). The inhibition of bile acid excretion was associated with a significant reduction of the clearance and extraction efficiency of bile acids and with an immediate and progressive increase in the perfusate concentrations of both glutamic pyruvic and glutamic oxalacetic transaminase activities. The reduced hepatic perfusion which followed the drug administration only partly accounted for the diminished bile acid excretion, as these effects of chlorpromazine on bile acid extraction and transaminase release could not be reproduced by comparable mechanical restriction of portal perfusate flow in control livers. The present findings together with previous observations demonstrating alterations in hepatic function and ultrastructure (12, 31), suggest that the inhibition of bile acid excretion by chlorpromazine in the isolated perfused rat liver is related to a generalized effect of the drug on hepatocyte plasma membranes, resulting in impairment of the uptake and eventual excretion of bile acids.

Hypotonic swelling of isolated skate hepatocytes activates a regulatory volume decrease (RVD) which is achieved in part by the release of taurine and other intracellular organic osmolytes. Volume-activated taurine efflux appears to be mediated by an anion channel that exhibits a taurine/chloride permeability ratio of approximately 0.2. Of significance, this channel was shown to be regulated by intracellular nucleotide. When intracellular ATP was decreased to about 50% of control levels, channel opening was completely prevented. Many putative ion channel blockers were found to inhibit the channel indirectly, by depleting intracellular ATP, rather than by directly interacting with the channel. Investigators using these channel blockers in whole cell preparations should be aware of this alternative mechanism. Cell swelling-activated taurine efflux was also inhibited by HgCl2, DIDS, and pyridoxal 5-phosphate, at concentrations of these agents that had no effect on intracellular ATP levels, suggesting additional mechanisms of inhibition and regulation of the volume-sensitive osmolyte channels. J. Exp. Zool. 279:471–475, 1997. © 1997 Wiley-Liss, Inc.

The effect of taurocholate on transcytotic vesicular pathways labeled with horseradish peroxidase was assessed in isolated perfused rat liver preparations. Forty-five minutes after a horseradish peroxidase load in a recirculating system, continuous infusion of taurocholate but not taurodehydrocholate significantly increased horseradish peroxidase excretion in bile by 50% compared with controls. When horseradish peroxidase (25 mg) was pulse loaded for 1 minute in control perfusions, it appeared in bile in early (4–6 minutes) and late (20–25 minutes) peaks, the latter accounting for 90% of total horseradish peroxidase output. Taurocholate infusion significantly increased horseradish peroxidase output in both early and late peaks, whereas only a small increase in the early peak was observed with taurodehydrocholate. Colchicine pretreatment increased the early peak in bile but abolished the second peak. Electron micrographs from control livers revealed the accumulation of horseradish peroxidase-containing vesicles in pericanalicular regions at early (2 minutes) as well as late (18 minutes) periods. When a morphometric analysis of electron micrographs was performed from pericanalicular regions 2 minutes after a 1-minute pulse of horseradish peroxidase (500 mg), taurocholate but not taurodehydrocholate increased both the density and percent area of horseradish peroxidase-containing vesicles compared with controls. In contrast, colchicine pretreatment had no effect on the density of the early-appearing vesicles, although their individual sizes were reduced. Taurocholate but not taurodehydrocholate also increased the percent of tubular structures in the pericanalicular region. These findings indicate that taurocholate stimulates both early and late transcytotic vesicle pathways and that a rapid transcellular-colchicine-insensitive and therefore probably microtubule-independent vesicle pathway is present in hepatocytes that must be distinguished from paracellular routes.

Laboratory-prepared and commercially obtained fluorescein-labeled rabbit antihuman IgG were compared in performing the antimitochondrial antibody (AMA) assay. Identical results were obtained using either of the fluorescent antisera at protein concentrations of 1.5 mg/ml and 1:10 dilutions of patients' sera. Positive AMA tests with either antisera were observed in each of 7 patients with primary biliary cirrhosis (PBC), 2 of 83 patients with miscellaneous hepatic diseases, and in 1 of 24 patients with extrahepatic biliary obcent antiserum (15.8 mg protein/ml) was substituted in the assay, sera from 11 of 23 AMA-negative patients with EBO and 12 of 15 with miscellaneous liver diseases demonstrated an atypical fluorescence located primarily along the periphery of the rat renal tubules. Thus, if the conjugated antibody is not adjusted to an optimal protein concentration, this atypical fluorescence could be interpreted as a positive AMA test and lead to diagnostic error.

The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions. This transport function is markedly diminished in mutant rats that lack the transport protein. To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity. The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted. The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein. The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane. Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane. The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference. These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane. The relevance of this phenomenon for the intact liver is discussed.

Background:Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effects of TUDCA on cytosolic free calcium [(Ca2+)i], which regulates hepatocyte secretion, are unknown. Methods:The effect of TUDCA on (Ca2+)iwas investigated in groups of isolated rat hepatocytes by microspectrofluorometry and in single cells by confocal line scanning microscopy. Results:Administration of TUDCA (5–50 μmol/L) induced a nearly fourfold increase of basal levels of (Ca2+)i. After a 15 minute treatment period, the TUDCA (10 μmol/L)-induced change in (Ca2+)iwas higher than that of other monodi-, and trihydroxy bile acids at equimolar concentrations. Pretreatment with TUDCA (10 μmol/L) markedly reduced or abolished increases in (Ca2+)iinduced by phenylephrine (1 μmol/L), the microsomal Ca2+translocase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (25 μmol/L), or taurolithocholic acid (10–25 μmol/L). In Ca2+-free medium, TUDCA caused only a reduced and transient increase in (Ca2+)i. TUDCA (10 μmol/L) induced Ca2+oscillations in all single cells that responded. However, levels of inositol-1,4,5-trisphosphate (IP3) in hepatocytes were not increased by treatment with TUDCA (10 μmol/L). Conclusions:TUDCA at physiological concentrations potently modulates (Ca2+)isignals in hepatocytes by (1) mobilizing microsomal IP3-sensitive Ca2+stores by an IP3-independent mechanism, (2) initiating Ca2+oscillations, and (3) inducing influx of extracellular Ca2+.

Two women, aged 41 and 51 years, developed jaundice, encephalopathy, and hypoprothrombinemia during rapid weight loss four and 12 months after jejunoileal bypass for refractory obesity. Both were treated for liver failure and received a prolonged course of nutrition parenterally and orally. Serial liver biopsy specimens demonstrated extensive alcoholic-like hepatitis and cirrhosis that improved with nutritional repletion and reanastomosis. Postoperative biopsy specimens later demonstrated minimal portal fibrosis in one patient and inactive mild cirrhosis in the other. Although previous reports indicate that patients usually die when they develop liver failure of this severity after jejunoileal bypass, prolonged intensive nutritional repletion was associated with sufficient clinical and histologic improvement in these two patients so that intestinal reanastomosis could be performed safely.(Arch Intern Med 139:289-292, 1979)

The mechanism by which mercury leads to cell swelling and impairs the normal regulatory volume decrease (RVD) in cells swollen in hypotonic media was examined in hepatocytes isolated from the little skate,Raja erinacea,an osmoconforming marine elasmobranch. Skate hepatocytes treated with 50 μMHgCl2in isotonic medium swelled to volumes double those of control cells, and this was associated with an increase in Na+and K+permeability. The gain in intracellular Na+exceeded the K+loss by 0.27 μEq/mg protein, accounting in large part for the observed cell swelling. The effects of mercury were blunted when hepatocytes were incubated in medium in which the Na+was replaced with K+, and were essentially absent when Na+was replaced with choline+, indicating an important role of Na+influx in mediating mercury's effects on cell volume regulation. The inhibition of RVD by mercury was prevented if the metal was administered as a mercaptide with dithiothreitol or glutathione. However, when these chelating agents were added after the mercury, only the membrane permeant dithiothreitol was able to reverse the inhibition of RVD, suggesting an intracellular site of action. Mercuric chloride also produced a concentration-dependent inhibition of the ATP-sensitive volume-regulatory osmolyte channel in skate hepatocytes, as assessed by inhibition of swelling-activated [14C]taurine efflux. [14C]Taurine efflux was inhibited at mercury concentrations (20–40 μM) that had no effect on intracellular ATP levels or ATP/ADP ratios, consistent with a direct interaction with the channel. These findings indicate that mercury impairs cell volume regulation in skate hepatocytes at multiple sites, including the volume-regulatory osmolyte channels, and Na+and K+permeability pathways. The combined effects of increased Na+influx and the inability to extrude organic osmolytes may account for the inhibition of RVD.

Pathological activation of TGF-β signaling is universal in fibrosis. Aberrant TGF-β signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-β pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-β1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-β1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases.

To the Editor.—Through their comparative group selection, Ryback and co-workers have compromised their sophisticated analysis of biochemical and hematologic correlates of alcoholism and liver disease. These correlates would be more meaningful if they would allow distinction between alcoholics with alcoholic liver disease and alcoholics with nonalcoholic liver disease. Distinguishing alcoholic patients with liver disease often can be intimated by taking a history, as Ryback and associates have done in part by choosing their two major groups for study. They, in fact, may be identifying a pattern that reflects simultaneously both nonspecific liver disease and alcoholism rather than alcoholic liver disease per se. Moreover, their third group—patients in the alcoholic treatment program— may have had serious alcoholic or nonalcoholic hepatic disease without dramatic abnormalities in their laboratory test results.1 Although the authors may not have felt justified in performing liver biopsies in these patients, this group remains a weak