Dirk R. de Waart

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

Growth factors VEGF and TGF-β1 in peritoneal dialysis ☆ ☆☆ ★ ★★

The morphologic alterations in the kidney and the retina that can be present in patients with diabetic microangiopathy are mediated by growth factors. Vascular endothelial growth factor (VEGF) is a mediator of neoangiogenesis in diabetic retinopathy. Transforming growth factor-β (TGF-β) is involved in the extracellular matrix proliferation in diabetic nephropathy. The aim of the present study was to investigate the presence of VEGF and TGF-β1 in peritoneal effluents of patients undergoing continuous ambulatory peritoneal dialysis who are being treated with glucose-containing dialysis solutions in relation to parameters of peritoneal transport. Standard peritoneal permeability analyses with 3.86% glucose dialysate were performed in 16 stable patients undergoing peritoneal dialysis (PD) (median duration of PD 39 months, range 1 to 104 months). The power relationship that is present between dialysate/serum (D/S) ratios of serum proteins that are transported only across the peritoneal membrane and their molecular weights was used to predict the D/S ratios when diffusion would be the only explanation for the measured dialysate concentration. It was assumed that all TGF-β1 in the circulation was bound to α2-macroglobulin. The D/S ratios of VEGF (P < .0005) and TGF-β1 (P < .015) were significantly higher than expected when VEGF and TGF-β1 would have been transported from the circulation only by diffusion. No relationship was present between the effluent concentration attributed to the local production of VEGF (LVEGF) and that of TGF-β1 (LTGF-β1). LVEGF correlated with the mass transfer area coefficient (MTAC) creatinine value (r = 0.69, P < .007), MTAC urate value (r = 0.60, P < .02), and glucose absorption value (r = 0.75, P < .004), all reflections of the peritoneal vascular surface area. A negative correlation was observed between the transcapillary ultrafiltration (926 mL/4 h, 394 to 1262 mL/4 h) and LVEGF (r = –0.52, P < .045). This negative tendency was also observed between the net ultrafiltration (622 mL/4 h, –43 to 938 mL/4 h) and LVEGF (r = –0.48) but did not reach significance. LVEGF and the duration of treatment did not correlate, possibly because of the relatively small number of patients. LTGF-β1 showed no relationship with transport parameters or duration of treatment. In conclusion, we found evidence for the local production of both VEGF and TGF-β1 in the peritoneal membrane of patients undergoing long-term peritoneal dialysis with glucose-based dialysate solutions. The analogy with VEGF in diabetic retinopathy suggests a pathogenetic role of high dialysate glucose concentrations in the development of these alterations in the peritoneal membrane. (J Lab Clin Med 1999;134:124-32)

Role of MRP2 and GSH in intrahepatic cycling of toxins

MRP2 is a canalicular transporter in hepatocytes mediating the transport of a wide spectrum of amphipathic compounds. This includes organic anions but also compounds complexed with GSH as, e.g. alpha-naphthylisothiocyanate (ANIT) and arsenite. These reversible complexes may fall apart in bile after MRP2-mediated transport, which induces high concentrations of the toxic compound in the biliary tree. To further investigate the role of MRP2 in transport and toxicity of both compounds, we conducted experiments in transduced polarized epithelial cells and in vivo, using the Mrp2-deficient TR(-) rat as a model. Our results show, that in MRP2-transduced MDCK II cells both compounds induce disproportionally strong apical GSH secretion. This induction of GSH secretion was not observed in the parent cells lacking MRP2 expression. This indicated that after transport via MRP2 both complexes released GSH upon which the compound could re-enter the cells. The resulting cycling of both toxins led to concentration dependent GSH depletion of the cells. To further test our hypothesis we administered arsenite (12.5 micromol absolute i.v.) to Wistar and Mrp2-deficient TR(-) rats and collected bile. While both arsenite and GSH secretion were absent in TR(-) rats, the total secretion of arsenite into Wistar bile (2.91 micromol) was accompanied by a excess secretion of 24 micromol GSH, indicating that arsenite undergoes multiple cycles of GSH complexation. We also administered ANIT to both animal models and could show that TR(-) rats are protected from ANIT induced cholestasis. This indicates that Mrp2-mediated biliary secretion of GS-ANIT is a prerequisite for development of cholestasis in rats. We hypothesize that the toxic parent compound ANIT is regenerated in the biliary tree where it can exert its toxic properties on bile duct epithelial cells.

Organic anion transporting polypeptide 1a/1b–knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

Altered disposition of acetaminophen in mice with a disruption of the Mrp3 gene

MRP3 is an ABC transporter localized in the basolateral membrane of epithelial cells such as hepatocytes and enterocytes. In this study, the role of Mrp3 in drug disposition was investigated. Because Mrp3 preferentially transports glucuronide conjugates, we investigated the in vivo disposition of acetaminophen (APAP) and its metabolites. Mrp3+/+ and Mrp3−/− knockout mice received APAP (150 mg/kg), and bile was collected. Basolateral and canalicular excretion of APAP was also assessed in the isolated perfused liver. In separate studies, mice received 400 mg APAP/kg for assessment of hepatotoxicity. No differences were found in the biliary excretion of APAP, APAP‐sulfate, and APAP‐glutathione between Mrp3+/+ and Mrp3−/− mice. However, 20‐fold higher accumulation of APAP‐glucuronide (APAP‐GLUC) was found in the liver of Mrp3−/− mice. Concomitantly, plasma APAP‐GLUC content in Mrp3−/− mice was less than 10% of that in Mrp3+/+ mice. In addition, APAP‐GLUC excretion in bile of Mrp3−/− mice was tenfold higher than in Mrp3+/+ mice. In the isolated perfused liver, we also found a strong decrease of APAP‐GLUC secretion into the perfusate of Mrp3−/− livers. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathology showed that Mrp3−/− mice are more resistant to APAP hepatotoxicity than Mrp3+/+ mice, which is most likely a result of the faster repletion of hepatic GSH. In conclusion, basolateral excretion of APAP‐GLUC in mice is nearly completely dependent on the function of Mrp3. In its absence, sufficient hepatic accumulation occurs to redirect some of the APAP‐GLUC to bile. This altered disposition in Mrp3−/− mice is associated with reduced hepatotoxicity. (HEPATOLOGY 2005;42:1091–1098.)

Hepatic transport mechanisms of Cholyl-L-Lysyl-Fluorescein.

Cholyl-l-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(−/−), and Abcc3(−/−) mice. Human Na+-dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with Km values of 4.6 ± 2.7, 3.3 ± 2.0, and 3.7 ± 1.0 μM, respectively. In Abcc2(−/−) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(−/−) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.

Functionally Overlapping Roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the Elimination of Methotrexate and Its Main Toxic Metabolite 7-Hydroxymethotrexate In vivo

Purpose: ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX). Experimental Design: We generated and characterized Abcc2;Abcg2-/- mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results: Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2-/- and Abcc2-/- mice, respectively, and 3.3-fold increased in Abcc2;Abcg2-/- mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2-/- mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2-/- mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2-/- and Abcc2;Abcg2-/- mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2-/- mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX. Conclusions: Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Effect of an Increase in C-Reactive Protein Level during a Hemodialysis Session on Mortality

The prevalence of chronic inflammation is high in dialysis patients. Moreover, it is associated with an increased mortality risk, yet the origin of chronic inflammation in dialysis patients remains unclear. The aim of this study was to determine the effect of a hemodialysis session (HD) on C-reactive protein (CRP) levels and to study the relation with survival. As part of a large, prospective, multicenter study in the Netherlands (Netherlands Cooperative Study on the Adequacy of Dialysis), patients who were started on dialysis treatment between September 1997 and May 1999 were included. Demographic data, clinical data, and serum samples were collected at regularly timed intervals. From this cohort, a random sample of patients was taken. CRP levels were determined before and after an HD session and before the next session. Date of death or censoring was recorded until September 2002. A total of 186 HD patients were included. Mean age was 65 yr (SD, 13); 56% were male. A total of 71 patients had a CRP level below the detection limit (3 mg/L), 68 patients showed no increase in CRP during an HD session (no-increase group), and 47 (25%) patients showed an increase in CRP level during an HD session (increase-group). No statistically difference in mean CRP levels before the dialysis session was found between the increase group (22.3 mg/L) and the no-increase group (19.4 mg/L). In the subsequent interdialytic period, CRP levels returned to the levels of the initial CRP value. Two-year survival was 44% in the increase group and 66% in the no-increase group (P = 0.09). Independent of CRP level before the session and adjusted for age, comorbidity, nutritional status, and primary kidney disease, a raise of 1 mg/L CRP during a session was associated with a 9% increased mortality risk (adjusted hazard ratio, 1.09; 95% CI, 1.02 to 1.16). The present study showed an increase in CRP level during a single dialysis session in 25% of the patients; during the succeeding interdialytic period, CRP level returned to its original value. More important, however, an increase in CRP level during an HD session was independently associated with a higher mortality risk.

Impaired uptake of conjugated bile acids and hepatitis b virus pres1‐binding in na+‐taurocholate cotransporting polypeptide knockout mice

The Na+‐taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte‐specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1‐knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1−/− hepatocytes showed absence of sodium‐dependent taurocholic acid uptake, whereas sodium‐independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1−/− mice, explained by increased Asbt and Ostα/β expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down‐regulation of OATP1A1 and up‐regulation of OATP1A4. Furthermore, sodium‐dependent taurocholic acid uptake was inhibited by Myrcludex B in wild‐type hepatocytes, while Slc10a1−/− hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1‐/‐ mice. Conclusion: The Slc10a1‐knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP‐independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation. (Hepatology 2015;62:207–219)

Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the In vivo Elimination of Methotrexate and its Main Toxic Metabolite 7-hydroxymethotrexate

Purpose: ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistance–associated protein 2 (MRP2)] and ABCC3 (MRP3) mediate the elimination of toxic compounds, such as drugs and carcinogens, and have a large overlap in substrate specificity. We investigated the roles of Abcc2 and Abcc3 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) in vivo. Experimental Design:Abcc2;Abcc3−/− mice were generated, characterized, and used to investigate possibly overlapping or complementary roles of Abcc2 and Abcc3 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results:Abcc2;Abcc3−/− mice were viable and fertile. In Abcc2−/− mice, the plasma area under the curve (AUCi.v.) for MTX was 2.0-fold increased compared with wild type, leading to 1.6-fold increased urinary excretion, which was not seen in Abcc2;Abcc3−/− mice. Biliary excretion of MTX was 3.7-fold reduced in Abcc2−/− but unchanged in Abcc2;Abcc3−/− mice. The plasma AUCi.v.s of 7OH-MTX were 6.0-fold and 4.3-fold increased in Abcc2−/− and Abcc2;Abcc3−/− mice, respectively, leading to increased urinary excretion. The biliary excretion of 7OH-MTX was 5.8-fold reduced in Abcc2−/− but unchanged in Abcc2;Abcc3−/− mice. 7OH-MTX accumulated substantially in the liver of Abcc2−/− and especially Abcc2;Abcc3−/− mice. Conclusions: Abcc2 is important for (biliary) excretion of MTX and its toxic metabolite 7OH-MTX. When Abcc2 is absent, Abcc3 transports MTX and 7OH-MTX back from the liver into the circulation, leading to increased plasma levels and urinary excretion. Variation in ABCC2 and/or ABCC3 activity may therefore have profound effects on the elimination and severity of toxicity of MTX and 7OH-MTX after MTX treatment of patients.