Sodium-dependent Bile Salt Transporter Research Articles

Transporter Proteins as Therapeutic Drug Targets-With a Focus on SGLT2 Inhibitors.

Membrane transporters interact not only with endogenous substrates but are also engaged in the transport of xenobiotics, including drugs. While the coordinated function of uptake (solute carrier family-SLC and SLCO) and efflux (ATP-binding cassette family-ABC, multidrug and toxic compound extrusion family-MATE) transporter system allows vectorial drug transport, efflux carriers alone achieve barrier functions. The modulation of transport functions was proved to be effective in the treatment strategies of various pathological states. Sodium-glucose cotransporter-2 (SGLT2) inhibitors are the drugs most widely applied in clinical practice, especially in the treatment of diabetes mellitus and heart failure. Sodium taurocholate co-transporting polypeptide (NTCP) serves as virus particles (HBV/HDV) carrier, and inhibition of its function is applied in the treatment of hepatitis B and hepatitis D by myrcludex B. Inherited cholestatic diseases, such as Alagille syndrome (ALGS) and progressive familial intrahepatic cholestasis (PFIC) can be treated by odevixibat and maralixibat, which inhibit activity of apical sodium-dependent bile salt transporter (ASBT). Probenecid can be considered to increase uric acid excretion in the urine mainly via the inhibition of urate transporter 1 (URAT1), and due to pharmacokinetic interactions involving organic anion transporters 1 and 3 (OAT1 and OAT3), it modifies renal excretion of penicillins or ciprofloxacin as well as nephrotoxicity of cidofovir. This review discusses clinically approved drugs that affect membrane/drug transporter function.

Atorvastatin Modulates Bile Acid Homeostasis in Mice with Diet-Induced Nonalcoholic Steatohepatitis.

Bile acids (BA) play a significant role in the pathophysiology of nonalcoholic steatohepatitis (NASH). The present study evaluates the modulation of bile acid metabolomics by atorvastatin, a cholesterol-lowering agent commonly used to treat cardiovascular complications accompanying NASH. NASH was induced in mice by 24 weeks of consuming a high–saturated fat, high-fructose, and high-cholesterol diet (F), with atorvastatin administered orally (20 mg/kg/day) during the last three weeks. Biochemical and histological analyses confirmed the effectiveness of the F diet in inducing NASH. Untreated NASH animals had significantly reduced biliary secretion of BA and increased fecal excretion of BA via decreased apical sodium-dependent bile salt transporter (Asbt)-mediated reabsorption. Atorvastatin decreased liver steatosis and inflammation in NASH animals consistently with a reduction in crucial lipogenic enzyme stearoyl–coenzyme A (CoA) desaturase-1 and nuclear factor kappa light chain enhancer of activated B-cell pro-inflammatory signaling, respectively. In this group, atorvastatin also uniformly enhanced plasma concentration, biliary secretion and fecal excretion of the secondary BA, deoxycholic acid (DCA). However, in the chow diet–fed animals, atorvastatin decreased plasma concentrations of BA, and reduced BA biliary secretions. These changes stemmed primarily from the increased fecal excretion of BA resulting from the reduced Asbt-mediated BA reabsorption in the ileum and suppression of synthesis in the liver. In conclusion, our results reveal that atorvastatin significantly modulates BA metabolomics by altering their intestinal processing and liver synthesis in control and NASH mice.

Characterization of Novel Fluorescent Bile Salt Derivatives for Studying Human Bile Salt and Organic Anion Transporters.

Bile salts, such as cholate, glycocholate, taurocholate, and glycochenodeoxycholate, are taken up from the portal blood into hepatocytes via transporters, such as the Na+-taurocholate-cotransporting polypeptide (NTCP) and organic anion-transporting polypeptides (OATPs). These bile salts are later secreted into bile across the canalicular membrane, which is facilitated by the bile salt export pump (BSEP). Apart from bile salt transport, some of these proteins (e.g., OATPs) are also key transporters for drug uptake into hepatocytes. In vivo studies of transporter function in patients by using tracer compounds have emerged as an important diagnostic tool to complement classic liver parameter measurements by determining dynamic liver function both for diagnosis and monitoring progression or improvement of liver diseases. Such approaches include use of radioactively labeled bile salts (e.g., for positron emission tomography) and fluorescent bile salt derivatives or dyes (e.g., indocyanine green). To expand the list of liver function markers, we synthesized fluorescent derivatives of cholic and chenodeoxycholic acid by conjugating small organic dyes to the bile acid side chain. These novel fluorescent probes were able to block substrate transport in a concentration-dependent manner of NTCP, OATP1B1, OATP1B3, OATP2B1, BSEP, and intestinal apical sodium-dependent bile salt transporter (ASBT). Whereas the fluorescent bile acid derivatives themselves were transported across the membrane by OATP1B1, OATP1B3, and OATP2B1, they were not transport substrates for NTCP, ASBT, BSEP, and multidrug resistance-related protein 2. Accordingly, these novel fluorescent bile acid probes can potentially be used as imaging agents to monitor the function of OATPs. SIGNIFICANCE STATEMENT: Synthetic modification of common bile acids by attachment of small organic fluorescent dyes to the bile acid side chain resulted in bright, fluorescent probes that interact with hepatic and intestinal organic anion [organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1], bile salt uptake (Na+-taurocholate-cotransporting polypeptide, apical sodium-dependent bile salt transporter), and bile salt efflux (bile salt export pump, multidrug resistance-related protein 2) transporters. Although the fluorescent bile salt derivatives are taken up into cells via the OATPs, the efflux transporters do not transport any of them but one.

THU-027-The human apical sodium dependent bile salt transporter (ASBT) activates a bile salt-induced defense in human cholangiocytes

THU-027-The human apical sodium dependent bile salt transporter (ASBT) activates a bile salt-induced defense in human cholangiocytes

FXR agonist GW4064 improves liver and intestinal pathology and alters bile acid metabolism in rats undergoing small intestinal resection.

Mortality associated with liver disease has been observed in patients with short bowel syndrome (SBS); however, its mechanism remains unclear, but bile acid (BA) dysmetabolism has been proposed as a possible cause. The farnesoid X receptor (FXR) is the key regulator of BA synthesis. Here, we showed that, in a rat model of short bowel resection associated with liver disease (SBR-ALD), the BA composition of hepatic tissues reflected a larger proportion of primary and secondary unconjugated BAs, whereas that of the colon contents and serum showed an increased ratio of secondary unconjugated BAs. Both hepatic and intestinal regulation of BA synthesis was characterized by a blunted hepatic FXR activation response. The mRNA expression levels of cholesterol 7a-hydroxylase (CYP7A1), sterol 12a-hydroxylase (CYP8B1), and sterol 27 hydroxylase (CYP27A1), the key enzymes in BA synthesis, were upregulated. After intervention with the FXR agonist GW4064, both the liver histology and serum transaminase activity were improved, which demonstrated the attenuation of SBR-ALD. The BA compositions of hepatic tissue, the colon contents, and serum recovered and were closer to those of the sham group. The expression levels of hepatic FXR increased, and its target genes were activated. Consistent with this, the expression levels of CYP7A1, CYP8B1, and CYP27A1 were downregulated. Ileum tissue FXR and its target genes were slightly elevated. This study showed that the FXR agonist GW4064 could correct BA dysmetabolism to alleviate hepatotoxicity in SBR animals. GW4064 intervention resulted in a decrease in fecal bile excretion and elevated plasma/hepatic conjugated BA levels. GW4064 increased the reabsorption of conjugated BAs by inducing apical sodium-dependent bile salt transporter expression in the ileum. Concomitantly, FXR activation in the presence of GW4064 decreased BA production by repressing the expression of key synthetases, including CYP7A1, CYP8B1, and CYP27A1. These findings provide a clinical research direction for the prevention of liver disease in patients with SBS.NEW & NOTEWORTHY This study assessed the impact of treatment with GW4064, a farnesoid X receptor agonist, on the development of short bowel resection (SBR) associated with liver disease in a rat model of SBR. GW4064 was able to correct bile acid dysmetabolism and alleviate hepatotoxicity in SBR animals.

Organic Anion Transporting Polypeptides Contribute to the Disposition of Perfluoroalkyl Acids in Humans and Rats.

Perfluoroalkyl sulfonates (PFSAs) such as perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS) have very long serum elimination half-lives in humans, and preferentially distribute to serum and liver. The enterohepatic circulation of PFHxS and PFOS likely contributes to their extended elimination half-lives. We previously demonstrated that perfluorobutane sulfonate (PFBS), PFHxS, and PFOS are transported into hepatocytes both in a sodium-dependent and a sodium-independent manner. We identified Na+/taurocholate cotransporting polypeptide (NTCP) as the responsible sodium-dependent transporter. Furthermore, we demonstrated that the human apical sodium-dependent bile salt transporter (ASBT) contributes to the intestinal reabsorption of PFOS. However, so far no sodium-independent uptake transporters for PFSAs have been identified in human hepatocytes or enterocytes. In addition, perfluoroalkyl carboxylates (PFCAs) with 8 and 9 carbons were shown to preferentially distribute to the liver of rodents; however, no rat or human liver uptake transporters are known to transport these PFCAs. Therefore, we tested whether PFBS, PFHxS, PFOS, and PFCAs with 7-10 carbons are substrates of organic anion transporting polypeptides (OATPs). We used CHO and HEK293 cells to demonstrate that human OATP1B1, OATP1B3, and OATP2B1 can transport PFBS, PFHxS, PFOS, and the 2 PFCAs (C8 and C9). In addition, we show that rat OATP1A1, OATP1A5, OATP1B2, and OATP2B1 transport all 3 PFSAs. In conclusion, our results suggest that besides NTCP and ASBT, OATPs also are capable of contributing to the enterohepatic circulation and extended human serum elimination half-lives of the tested perfluoroalkyl acids.

Drug Transport by the Blood-Aqueous Humor Barrier of the Eye.

The ocular barriers (cornea, blood-retinal barrier, and blood-aqueous humor barrier) make treating eye diseases with therapeutic drugs challenging. The tight capillary endothelium of the iris and the ciliary body epithelium form the blood-aqueous humor barrier. The iris and ciliary body (iris-ciliary body) express a variety of drug transporters in the ATP-binding cassette and solute carrier (SLC) families. ATP-binding cassette family drug transporters that are present in the iris-ciliary body include P-glycoprotein, breast cancer resistance protein, and several multidrug resistance-associated proteins. SLC family drug transporters that are present in the iris-ciliary body include organic anion transporters, organic anion transporting polypeptides, bile acid transporters (apical sodium-dependent bile salt transporter and sodium taurocholate cotransporter), organic cation transporters (novel organic cation transporter and multidrug and toxin extrusion transporter) and peptide transporters. Freshly dissected iris-ciliary body preparations actively accumulate a variety of substrates of SLC drug transporters that are expressed in the tissue. The ciliary body in vitro supports active transport in the aqueous humor-to-blood direction of several substrates of organic anion transporters and multidrug resistance-associated proteins, consistent with the subcellular localization of these transporters in the ciliary body epithelium. In vivo data suggest that drug transporters in the iris-ciliary body reduce the permeation of drugs in the direction of blood-to-aqueous humor, thereby reducing ocular drug bioavailability, and are also involved in active drug elimination from the aqueous humor. An understanding of the influence on pharmacokinetics of drug transporters in the blood-aqueous humor barrier should help improve drug delivery and efficacy in the eye.

Na+/Taurocholate Cotransporting Polypeptide and Apical Sodium-Dependent Bile Acid Transporter Are Involved in the Disposition of Perfluoroalkyl Sulfonates in Humans and Rats.

Among the perfluoroalkyl sulfonates (PFASs), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) have half-lives of several years in humans, mainly due to slow renal clearance and potential hepatic accumulation. Both compounds undergo enterohepatic circulation. To determine whether transporters involved in the enterohepatic circulation of bile acids are also involved in the disposition of PFASs, uptake of perfluorobutane sulfonate (PFBS), PFHxS, and PFOS was measured using freshly isolated human and rat hepatocytes in the absence or presence of sodium. The results demonstrated sodium-dependent uptake for all 3 PFASs. Given that the Na(+)/taurocholate cotransporting polypeptide (NTCP) and the apical sodium-dependent bile salt transporter (ASBT) are essential for the enterohepatic circulation of bile acids, transport of PFASs was investigated in stable CHO Flp-In cells for human NTCP or HEK293 cells transiently expressing rat NTCP, human ASBT, and rat ASBT. The results demonstrated that both human and rat NTCP can transport PFBS, PFHxS, and PFOS. Kinetics with human NTCP revealed Km values of 39.6, 112, and 130 µM for PFBS, PFHxS, and PFOS, respectively. For rat NTCP Km values were 76.2 and 294 µM for PFBS and PFHxS, respectively. Only PFOS was transported by human ASBT whereas rat ASBT did not transport any of the tested PFASs. Human OSTα/β was also able to transport all 3 PFASs. In conclusion, these results suggest that the long half-live and the hepatic accumulation of PFOS in humans are at least, in part, due to transport by NTCP and ASBT.

Fibrates and cholestasis.

Cholestasis, including primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), results from an impairment or disruption of bile production and causes intracellular retention of toxic bile constituents, including bile salts. If left untreated, cholestasis leads to liver fibrosis and cirrhosis, which eventually results in liver failure and the need for liver transplantation. Currently, the only therapeutic option available for these patients is ursodeoxycholic acid (UDCA), which slows the progression of PBC, particularly in stage I and II of the disease. However, some patients have an incomplete response to UDCA therapy, whereas other, more advanced cases often remain unresponsive. For PSC, UDCA therapy does not improve survival, and recommendations for its use remain controversial. These considerations emphasize the need for alternative therapies. Hepatic transporters, located along basolateral (sinusoidal) and apical (canalicular) membranes of hepatocytes, are integral determinants of bile formation and secretion. Nuclear receptors (NRs) are critically involved in the regulation of these hepatic transporters and are natural targets for therapy of cholestatic liver diseases. One of these NRs is peroxisome proliferator-activated receptor alpha (PPARα), which plays a central role in maintaining cholesterol, lipid, and bile acid homeostasis by regulating genes responsible for bile acid synthesis and transport in humans, including cytochrome P450 (CYP) isoform 7A1 (CYP7A1), CYP27A1, CYP8B1, uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, 1A4, 1A6, hydroxysteroid sulfotransferase enzyme 2A1, multidrug resistance protein 3, and apical sodium-dependent bile salt transporter. Expression of many of these genes is altered in cholestatic liver diseases, but few have been extensively studied or had the mechanism of PPARα effect identified. In this review, we examine what is known about these mechanisms and consider the rationale for the use of PPARα ligand therapy, such as fenofibrate, in various cholestatic liver disorders.

Hypolipidemic Effect and Mechanism of Palmatine from Coptis chinensis in Hamsters Fed High-Fat diet.

Palmatine (PAL) is one of the main alkaloids in Coptis chinensis. The present aim was to investigate the hypolipidemic effect and mechanism of palmatine in hamsters fed with high-fat diet (HFD). PAL treatment decreased serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels, as well as increased fecal excretion of TC and total bile acids (TBA) in hyperlipidemic hamsters. Furthermore, PAL treatment up-regulated low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein expression and down-regulated apical sodium-dependent bile salt transporter (ASBT) mRNA and protein expression. These results demonstrated that PAL as a potential natural cholesterol lowering agent works by up-regulating LDLR and CYP7A1 mRNA and protein expression, down-regulating ASBT mRNA and protein expression, as well as enhancing fecal excretion of TC and TBA. The findings in our study suggest that palmatine could be a potential natural agent for treating hyperlipidemia.

Dioxin-Produced Alteration in the Profiles of Fecal and Urinary Metabolomes: A Change in Bile Acids and Its Relevance to Toxicity.

This study investigated dioxin-induced changes in metabolomes in pubertal rat excrement. The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or restricting dietary intake (pair-fed group) markedly altered the metabolomic profile including lipids, hormones, and vitamins in the urine and feces. TCDD caused an increase in the fecal chenodeoxycholic acid and taurocholic acid content and in urinary adrenaline and 17β-estradiol, while the urinary melatonin level was reduced by TCDD. These changes were not observed in the pair-fed group. In accordance with the elevated level of fecal bile acids, TCDD reduced the intestinal expression of the apical sodium-dependent bile salt transporter, which plays a role in resorbing bile acids from the bile duct. In addition, CYP7A1, a rate-limiting enzyme for bile acid biosynthesis, was attenuated by TCDD treatment, although TCDD induced hepatic CYP8B1, an enzyme essential for cholic acid synthesis. Supplying cholic acid or chenodeoxycholic acid to TCDD-exposed rats tended to restore the TCDD-produced reduction in serum triglycerides, whereas no similar trend was observed in wasting syndrome and lipid accumulation in the liver. These results suggest that: 1) TCDD alters the circulating levels of bile acids and hormones via a mechanism distinct from an attenuation in dietary intake, although the majority of TCDD-induced changes in nutrient contents in the excrement is due to a reduction in food intake; and 2) TCDD facilitates the excretion of bile acids and disrupts their biosynthesis, resulting in the disturbance of lipid homeostasis.

The safety and anti-hypercholesterolemic effect of coptisine in Syrian golden hamsters.

Current work was conducted to evaluate the cholesterol-lowering effect of coptisine extracted from Rhizoma coptidis in Syrian golden hamsters. The safety results indicated that coptisine was a safe and low-toxic compound. Coptisine showed a beneficial effect in the abnormal serum lipid levels induced by a high-fat and high-cholesterol diet (HFHC): at a concentration of 70.05 mg/kg, coptisine significantly led to a decrease in total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-c) levels by 26.70, 15.38, and 22.22 %, respectively, and high-density lipoprotein cholesterol (HDL-c) was increased by 41.74 % in serum of hamsters (p < 0.01). In addition, total bile acid (TBA) levels in feces of hamsters were elevated after coptisine administration. Further investigation has suggested that the mRNA and protein expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) in the liver of hamsters was down-regulated by high-dosage coptisine treatment (p < 0.05); mRNA and protein expression of low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1) were dramatically up-regulated by coptisine administration. The apical sodium-dependent bile salt transporter expression was down-regulated in the coptisine-treated animals, but showed no significant differences from the HFHC groups. Taken together, our results demonstrate that a high dosage of coptisine could inhibit cholesterol synthesis via suppressing the HMGCR expression and promoting the use and excretion of cholesterol via up-regulating LDLR and CYP7A1 expression. These findings suggest a critical role for coptisine in anti- hypercholesterolemia, and thus it needs to be considered as a potential natural cholesterol lowering agent.

The lipid flippase heterodimer ATP8B1–CDC50A is essential for surface expression of the apical sodium-dependent bile acid transporter (SLC10A2/ASBT) in intestinal Caco-2 cells

Deficiency of the phospholipid flippase ATPase, aminophospholipid transporter, class I, type 8B, member 1 (ATP8B1) causes progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1). Apart from cholestasis, many patients also suffer from diarrhea of yet unknown etiology. Here we have studied the hypothesis that intestinal ATP8B1 deficiency results in bile salt malabsorption as a possible cause of PFIC1/BRIC1 diarrhea.Bile salt transport was studied in ATP8B1-depleted intestinal Caco-2 cells. Apical membrane localization was studied by a biotinylation approach. Fecal bile salt and electrolyte contents were analyzed in stool samples of PFIC1 patients, of whom some had undergone biliary diversion or liver transplantation.Bile salt uptake by the apical sodium-dependent bile salt transporter solute carrier family 10 (sodium/bile acid cotransporter), member 2 (SLC10A2) was strongly impaired in ATP8B1-depleted Caco-2 cells. The reduced SLC10A2 activity coincided with strongly reduced apical membrane localization, which was caused by impaired apical membrane insertion of SLC10A2. Moreover, we show that endogenous ATP8B1 exists in a functional heterodimer with transmembrane protein 30A (CDC50A) in Caco-2 cells. Analyses of stool samples of post-transplant PFIC1 patients demonstrated that bile salt content was not changed, whereas sodium and chloride concentrations were elevated and potassium levels were decreased.The ATP8B1–CDC50A heterodimer is essential for the apical localization of SLC10A2 in Caco-2 cells. Diarrhea in PFIC1/BRIC1 patients has a secretory origin to which SLC10A2 deficiency may contribute. This results in elevated luminal bile salt concentrations and consequent enhanced electrolyte secretion and/or reduced electrolyte resorption.

Sodium-dependent bile salt transporters of the SLC10A transporter family: more than solute transporters

The SLC10A transporter gene family consists of seven members and substrates transported by three members (SLC10A1, SLC10A2 and SLC10A6) are Na(+)-dependent. SLC10A1 (sodium taurocholate cotransporting polypeptide [NTCP]) and SLC10A2 (apical sodium-dependent bile salt transporter [ASBT]) transport bile salts and play an important role in maintaining enterohepatic circulation of bile salts. Solutes other than bile salts are also transported by NTCP. However, ASBT has not been shown to be a transporter for non-bile salt substrates. While the transport function of NTCP can potentially be used as liver function test, interpretation of such a test may be complicated by altered expression of NTCP in diseases and presence of drugs that may inhibit NTCP function. Transport of bile salts by NTCP and ASBT is inhibited by a number of drugs and it appears that ASBT is more permissive to drug inhibition than NTCP. The clinical significance of this inhibition in drug disposition and drug-drug interaction remains to be determined. Both NCTP and ASBT undergo post-translational regulations that involve phosphorylation/dephosphorylation, translocation to and retrieval from the plasma membrane and degradation by the ubiquitin-proteasome system. These posttranslational regulations are mediated via signaling pathways involving cAMP, calcium, nitric oxide, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) and protein phosphatases. There appears to be species difference in the substrate specificity and the regulation of plasma membrane localization of human and rodent NTCP. These differences should be taken into account when extrapolating rodent data for human clinical relevance and developing novel therapies. NTCP has recently been shown to play an important role in HBV and HDV infection by serving as a receptor for entry of these viruses into hepatocytes.

Homologue gene of bile acid transporters ntcp, asbt, and ost-alpha in rainbow trout Oncorhynchus mykiss: tissue expression, effect of fasting, and response to bile acid administration

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.

Arylsulfonylamino-Benzanilides as Inhibitors of the Apical Sodium-Dependent Bile Salt Transporter (SLC10A2)

The apical sodium-dependent bile salt transporter (ASBT) plays a pivotal role in maintaining bile acid homeostasis. Inhibition of ASBT would reduce bile acid pool size and lower cholesterol levels. In this report, a series of novel arylsulfonylaminobenzanilides were designed and synthesized as potential inhibitors of ASBT. Most of them demonstrated great potency against ASBT’s bile acid transport activity. In particular, compound 5g2 inhibited ASBT activity with an IC50 value of 0.11 μM. These compounds represent potential cholesterol-lowering drugs.

Alterations in bile acid synthesis in carriers of hepatocyte nuclear factor 1α mutations

Heterozygous mutations in hepatocyte nuclear factor 1α (HNF1α) cause maturity onset diabetes of the young 3 (MODY3), an autosomal dominant form of diabetes. Deficiency of HNF1α in mice results in diabetes, hypercholesterolaemia and increased bile acid (BA) and cholesterol synthesis. Little is known about alterations in lipid metabolism in patients with MODY3. The aim of this study was to investigate whether patients with MODY3 have altered cholesterol and BA synthesis and intestinal cholesterol absorption. A secondary aim was to investigate the effects of HNF1α mutations on the transcriptional regulation of BA metabolism. Plasma biomarkers of BA and cholesterol synthesis and intestinal cholesterol absorption were measured in patients with MODY3 (n = 19) and in matched healthy control subjects (n = 15). Cotransfection experiments were performed with several promoters involved in BA metabolism along with expression vectors carrying the mutations found in these patients. Plasma analysis showed higher levels of BA synthesis in patients with MODY3. No differences were observed in cholesterol synthesis or intestinal cholesterol absorption. Cotransfection experiments showed that one of the mutations (P379A) increased the induction of the cholesterol 7α-hydroxylase promoter compared with HNF1α, without further differences in other studied promoters. By contrast, the other four mutations (L107I, T260M, P291fsinsC and R131Q) reduced the induction of the farnesoid X receptor (FXR) promoter, which was followed by reduced repression of the small heterodimer partner promoter. In addition, these mutations also reduced the induction of the apical sodium-dependent bile salt transporter promoter. BA synthesis is increased in patients with MODY3 compared with control subjects. Mutations in HNF1α affect promoters involved in BA metabolism.

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

Effect of the dimeric bile acid analogue S 0960, a specific inhibitor of the apical sodium-dependent bile salt transporter in the ileum, on the renal handling of taurocholate.

The effect of the dimeric bile acid analogue S 0960 (CAS 142974-51-4), a specific inhibitor of the apical sodium-dependent bile salt transporter (ASBT) in the ileum, on kidney function was studied by clearance experiments in anesthetized rats. Additional experiments were performed on proximal tubular cells freshly isolated from rat kidney cortex and enriched by nycodenz density gradient centrifugation. The clearance studies, which were performed after a 5 h bile duct ligation, revealed a marked rise of the 3H-taurocholate clearance (from 85.4 +/- 15.7 to 371.1 +/- 86.0 microliters/min 100 g b.w., p < 0.05) and a considerable fall of the fractional tubular 3H-taurocholate reabsorption (from 90.2 +/- 1.72 to 68.2 +/- 7.50%, p < 0.05) after S 0960 at a dose of 10 mg/kg i.v. whereas the glomerular filtration rate did not significantly change (from 919 +/- 165 to 1055 +/- 162 microliters/min/100 g b.w.). Isolated proximal tubular cells showed a significant accumulation of 3H-taurocholate. The 3H-taurocholate cell/bath concentration ratio amounted to 3.34 +/- 0.17 at a 3H-taurocholate bath concentration of 3 x 10(-7) mol/l. LiCl (10(-3) mol/l), which is known to inhibit sodium-dependent transport processes in the kidney, markedly diminished cellular 3H-taurocholate uptake (by 65.8%) whereas probenecid (CAS 57-66-9, 10(-4) mol/l), the classical inhibitor of the basolateral organic acid transporter in the kidney, did not significantly affect 3H-taurocholate uptake. This finding indicates that transport of taurocholate by the basolaterally located organic acid transporter is not involved in the uptake process. The kinetic studies revealed an apparent K(m) value of 31 mumol/l and a Vmax value of 6.7 mumol/l cell water/min for tubular 3H-taurocholate uptake. At concentrations > 30 mumol/l S 0960 virtually completely inhibited cellular 3H-taurocholate uptake. 3H-taurocholate uptake was half-maximally inhibited at a S 0960 concentration of 5.8 mumol/l. The results of this functional study are in line with recent molecular evidence that the apical sodium-dependent bile salt transporters in kidney and ileum are identical and demonstrate that S 0960 is a potent inhibitor of the apical sodium-dependent taurocholate transporter in the kidney which augments the renal clearance of 3H-taurocholate. Compounds such as S 0960 may be of special therapeutical value in patients with extrahepatic cholestasis and elevated levels of plasma bile acids.

Comparative Effects of Doxercalciferol (1α-Hydroxyvitamin D2) Versus Calcitriol (1α,25-Dihydroxyvitamin D3) on the Expression of Transporters and Enzymes in the Rat In Vivo

Effects of 1.28 nmol/kg doxercalciferol [1α(OH)D₂], a synthetic vitamin D₂ analog that undergoes metabolic activation to 1α,25-dihydroxyvitamin D₂, the naturally occurring, biologically active form of vitamin D₂, on rat transporters and enzymes were compared with those of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, active form of vitamin D₃; 4.8 and 6.4 nmol/kg] given on alternate days intraperitoneally for 8 days. Changes were mostly confined to the intestine and kidney where the vitamin D receptor (VDR) was highly expressed: increased intestinal Cyp24 and Cyp3a1 messenger RNA (mRNA) and a modest elevation of apical sodium-dependent bile salt transporter (Asbt) and P-glycoprotein (P-gp) protein; increased renal VDR, Cyp24, Cyp3a9, Mdr1a, and Asbt mRNA, as well as Asbt and P-gp protein expression; and decreased renal PepT1 and Oat1 mRNA expression. In comparison, 1α(OH)D₂ treatment exerted a greater effect than 1,25(OH)₂D₃ on Cyp3a and Cyp24 mRNA. However, the farnesoid X receptor -related repressive effects on liver Cyp7a1 were absent because intestinal Asbt, FGF15 and portal bile acid concentrations were unchanged. Rats on the alternate day regimen showed milder changes and lessened signs of hypercalcemia and weight loss compared with rats receiving daily injections (similar or greater amounts of 0.64-2.56 nmol/kg daily ×4) described in previous reports, showing that the protracted pretreatment regimen was associated with milder inductive and lesser toxic effects in vivo.