Significant lethality following liver resection in A20 heterozygous knockout mice uncovers a key role for A20 in liver regeneration

Hepatic expression of A20, including in hepatocytes, increases in response to injury, inflammation and resection. This increase likely serves a hepatoprotective purpose. The characteristic unfettered liver inflammation and necrosis in A20 knockout mice established physiologic upregulation of A20 as integral to the anti-inflammatory and anti-apoptotic armamentarium of hepatocytes. However, the implication of physiologic upregulation of A20 in modulating hepatocytes' proliferative responses following liver resection remains controversial. To resolve the impact of A20 on hepatocyte proliferation and the liver's regenerative capacity, we examined whether decreased A20 expression, as in A20 heterozygous knockout mice, affects outcome following two-third partial hepatectomy. A20 heterozygous mice do not demonstrate a striking liver phenotype, indicating that their A20 expression levels are still sufficient to contain inflammation and cell death at baseline. However, usually benign partial hepatectomy provoked a staggering lethality (>40%) in these mice, uncovering an unsuspected phenotype. Heightened lethality in A20 heterozygous mice following partial hepatectomy resulted from impaired hepatocyte proliferation due to heightened levels of cyclin-dependent kinase inhibitor, p21, and deficient upregulation of cyclins D1, E and A, in the context of worsened liver steatosis. A20 heterozygous knockout minimally affected baseline liver transcriptome, mostly circadian rhythm genes. Nevertheless, this caused differential expression of >1000 genes post hepatectomy, hindering lipid metabolism, bile acid biosynthesis, insulin signaling and cell cycle, all critical cellular processes for liver regeneration. These results demonstrate that mere reduction of A20 levels causes worse outcome post hepatectomy than full knockout of bona fide liver pro-regenerative players such as IL-6, clearly ascertaining A20's primordial role in enabling liver regeneration. Clinical implications of these data are of utmost importance as they caution safety of extensive hepatectomy for donation or tumor in carriers of A20/TNFAIP3 single nucleotide polymorphisms alleles that decrease A20 expression or function, and prompt the development of A20-based liver pro-regenerative therapies.

The Gut Microbiome and Cirrhosis: Basic Aspects

In this chapter the basic aspects helping to understand the microbiome in terms of quantity, diversity, complexity, function, and interaction with the host are discussed. First the nomenclature, definitions of taxa, and measures of diversity as well as methods to unravel this kingdom are outlined. A brief summary on its physiological relevance for general health and the functions exerted specifically by the microbiome is presented. Differences in the composition of the microbiome along the gastrointestinal tract and across the gut wall and its interindividual variations, enterotypes, and stability are highlighted. The reader will be familiarized with all different modulators impacting on the microbiome, namely, intrinsic and extrinsic factors. Intrinsic factors include gastrointestinal secretions (gastric acid, bile, pancreatic juice, mucus), antimicrobial peptides, motility, enteric nervous system, and host genotype. Extrinsic factors are mainly dietary choices, hygiene, stress, alcohol consumption, exercise, and medications. The second part of the chapter focuses on quantitative and qualitative changes in microbiome in liver cirrhosis. The mechanisms contributing to dysbiosis, small intestinal bacterial overgrowth, and bacterial translocation are delineated underscoring their role for the liver-gut axis.

ENaC activity in collecting ducts modulates NCC in cirrhotic mice.

Cirrhosis is a frequent and severe disease, complicated by renal sodium retention leading to ascites and oedema. A better understanding of the complex mechanisms responsible for renal sodium handling could improve clinical management of sodium retention. Our aim was to determine the importance of the amiloride-sensitive epithelial sodium channel (ENaC) in collecting ducts in compensate and decompensate cirrhosis. Bile duct ligation was performed in control mice (CTL) and collecting duct-specific αENaC knockout (KO) mice, and ascites development, aldosterone plasma concentration, urinary sodium/potassium ratio and sodium transporter expression were compared. Disruption of ENaC in collecting ducts (CDs) did not alter ascites development, urinary sodium/potassium ratio, plasma aldosterone concentrations or Na,K-ATPase abundance in CCDs. Total αENaC abundance in whole kidney increased in cirrhotic mice of both genotypes and cleaved forms of α and γ ENaC increased only in ascitic mice of both genotypes. The sodium chloride cotransporter (NCC) abundance was lower in non-ascitic KO, compared to non-ascitic CTL, and increased when ascites appeared. In ascitic mice, the lack of αENaC in CDs induced an upregulation of total ENaC and NCC and correlated with the cleavage of ENaC subunits. This revealed compensatory mechanisms which could also take place when treating the patients with diuretics. These compensatory mechanisms should be considered for future development of therapeutic strategies.

Towards science-based sediment quality standards-Effects of field-collected sediments in rainbow trout (Oncorhynchus mykiss).

Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events. Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.

Importance of cholesterol and cholesterol transporters in the placental trophoblast during pregnancy

Membrane transporters are essential during pregnancy, being a core component of the exchange of nutrients, gases, and metabolic products between the mother and the developing fetus. Important compounds to be transported include vitamins and minerals, amino acids, glucose, as well as cholesterol. Cholesterol transport across the plasma membrane is mediated mainly by members of the ATP-binding cassette (ABC) transporter family. Cholesterol is present in every cell of the body, where it helps maintain the integrity of cell membranes and also plays an important role in cell signaling events. Cholesterol also acts as a precursor for the biosynthesis of steroids that include sex hormones, glucocorticoids, mineralcorticoids, as well as bile acids and oxysterols. Cholesterol transport is therefore crucial for a host of different physiological processes. The following chapter addresses the involvement and importance of ABC transporters in these different processes. The critical role that ABC transporters Play for a successful pregnancy outcome is highlighted by pathological processes that result malfunction of cholesterol transport during pregnancy. Avenues of future research are also described, which may help to further delineate the function and mechanism of action of ABC transporters.

The heparan sulfate-fibroblast growth factor signaling system in liver growth and function

The heparan sulfate (HS)-fibroblast growth factor (FGF) signaling system is a ubiquitous regulator that senses local environmental changes and mediates cell-to-cell communication. This system consists of three mutually interactive components. These are regulatory polypeptides (FGF), FGF receptor (FGFR) and heparan sulfate proteoglycans (FGFRHS). All four FGFR genes are expressed in the adult liver. Expression of the FGFR1–3 genes is generally associated with non-parenchymal cells while expression of the FGFR4 gene is associated with parenchymal hepatocytes. We showed that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (−/−) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7α-hydroxylase (Cyp7a), the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. These results indicated that FGFR4 was not directly involved in liver growth but exerted negative control on liver bile acid synthesis. This was confirmed in transgenic mice overexpressing the constitutively active human FGFR4 in livers. The transgenic mice exhibited decreased fecal bile acid excretion, bile acid pool size, and expression of Cyp7a. Introduction of this constitutively active human FGFR4 into FGFR4 (−/−) mice restored the inhibition of bile acid synthesis. Activation of the c-Jun N-terminal Kinase (JNK) pathway by FGFR4 correlated with the repressive effect on bile acid synthesis. ^ To determine whether FGFR4 played a broader role in liver-specific metabolic function, we examined the impact of both acute and chronic exposure to CCl 4 in FGFR4 (−/−) mice. Following acute CCl4 exposure, the FGFR4 (−/−) mice exhibited accelerated liver injury, a significant increase in liver mass and delayed hepatolobular repair, with no apparent effect on liver cell proliferation and restoration of cellularity. Chronic CCl4 exposure resulted in severe fibrosis in livers of FGFR4 (−/−) mice compared to normal mice. Analysis at both mRNA and protein levels indicated an 8 hr delay in FGFR4-deficient mice in the down-regulation of cytochrome P450 2E1 (CYP2E1) protein, the major enzyme whose products underlie CCl 4-induced injury. These results show that hepatocyte FGFR4 protects against acute and chronic insult to the liver and prevents accompanying fibrosis. ^ Of the 23 FGF polypeptides, FGF1 and FGF2 are present at significant levels in the liver. To determine whether FGF1 and FGF2 played a role in CCl 4-induced liver injury and fibrosis, we examined the impact of both acute and chronic exposure to CCl4 in both wild-type and FGF1-FGF2 double-knockout mice. Following acute CCl4 exposure, FGF1(−/−)FGF2(−/−) mice exhibited accelerated liver injury, overall normal liver growth and repair, and decreased liver collagen α1(I) induction. Liver fibrosis resulting from chronic CCl4 exposure was markedly decreased in livers of FGF1(−/−)FGF2(−/−) mice compared to wild-type mice. This study suggests a role for FGF1 and FGF2 in hepatic fibrogenesis. ^ In summary, our three part study shows that specific components of the ubiquitous HS-FGF signaling family in the liver context interfaces with metabolite- and xenobiotic-controlled networks to regulate liver function, but has no apparent direct effect on liver cell growth. ^

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

(Table 3) Liver weight, blood liver parameters, and blood plasma PCB concentrations in 14 sledge dog bitches and their 14 pups in Aasiaat, west Greenland

We assessed the relationship between exposure to organohalogen polluted minke whale (Balaenoptera acutorostrata) blubber and liver morphology and function in a generational controlled study of 28 Greenland sledge dogs (Canis familiaris). The prevalence of portal fibrosis, mild bile duct hyperplasia, and vascular leukocyte infiltrations was significantly higher in the exposed group (all Chi-square: p<0.05). In case of granulomas, the frequency was significantly highest in the bitches (P generation) while the prevalence of portal fibrosis was highest in the F generation (pups) (both Chi-square: p<0.05). No significant difference between exposed and controls was found for bile acid, ALAT, and ALKP, while ASAT and LDH were significantly highest in the control group (both ANOVA: p<0.05). We therefore suggest that a daily intake of 50-200 g environmentally organohalogen polluted minke whale blubber can cause liver lesions in Greenland sledge dogs. It is reasonable to infer that other apex predators such as polar bears (Ursus maritimus) and humans may suffer from similar impacts.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Design of compounds that increase the absorption of polar molecules

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.