Expression and localization of hepatobiliary transport proteins in progressive familial intrahepatic cholestasis

Abstract

Mutations of the bile salt export pump (BSEP) or the multidrug resistance P-glycoprotein 3 (MDR3) are linked to impaired bile salt homeostasis and lead to progressive familial intrahepatic cholestasis (PFIC)-2 and -3, respectively. The regulation of bile salt transporters in PFIC is not known. Expression of hepatobiliary transporters in livers of ten patients with a PFIC phenotype was studied by quantitative reverse transcription polymerase chain reaction, Western blotting, and immunofluorescence microscopy. PFIC was diagnosed by clinical and laboratory findings. All patients could be assigned to PFIC-2 or PFIC-3 by the use of BSEP-and MDR3-specific antibodies and by MDR3 gene-sequencing. Whereas in all PFIC-2 patients, BSEP immunoreactivity was absent from the canalicular membrane, in three PFIC-3 livers, canalicular MDR3 immunoreactivity was detectable. Serum bile salts were elevated to 276 ؎ 233 and to 221 ؎ 109 mol/L in PFIC-2 and PFIC-3, respectively. Organic anion transporting polypeptide OATP1B1, OATP1B3, and MRP2 mRNA and protein levels were reduced, whereas sodium taurocholate cotransporting polypeptide (NTCP) was only reduced at the protein level, suggesting a posttranscriptional NTCP regulation. Whereas MRP3 mRNA and protein were not significantly altered, MRP4 messenger RNA and protein were significantly increased in PFIC. In conclusion, PFIC-2 may be reliably diagnosed by immunofluorescence, whereas the diagnosis of PFIC-3 requires gene-sequencing. Several mechanisms may contribute to elevated plasma bile salts in PFIC: reduced bile salt uptake via NTCP, OATP1B1, and OATP1B3, decreased BSEP-dependent secretion into bile, and increased transport back into plasma by MRP4. Upregulation of MRP4, but not of MRP3, might represent an important escape mechanism for bile salt extrusion in PFIC. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/ suppmat/index.html). (HEPATOLOGY 2005;41:1160-1172

Multilabel immunofluorescence and antigen reprobing on formalin-fixed paraffin-embedded sections: novel applications for precision pathology diagnosis

“…It should be noted that immunostaining for BESP is a useful marker in the diagnosis of congenital cholestasis syndromes. 15 In sections of liver with normal histology shown in HE stains (Figure 4a), as expected, BSEP was localized to the bile canalicular membrane with sharp contrast and clear definition (Figures 4b and 3d reconstruction in Figure 4c). In contrast, in a case of PSC, HE hematoxilin and eosin staining showed cholestasis and immunostaning for BESEP protein revealed discontinuous, focally beaded staining pattern (Figure 4d and e, and Figure 3d reconstruction in Figure 4f).…”

The bile salt export pump

“…The majority of these mutations affect evolutionary conserved amino acid residues, and the affected amino acids tend to be located in the large cytoplasmic loops of BSEP. In many patients with severe BSEP deficiency syndrome, BSEP staining was absent on liver sections [45,46]. Direct investigation of the functional consequences of the BSEP mutations in human liver is impossible.…”

“…Since the affinity of Mdr1 for bile salts is much lower, intracellular bile salts may rise to toxic levels in hepatocytes and cause liver injury (see article by V. Ling, this issue). The situation in patients with PFIC2 may differ to some extent from the knockout animals since analysis of four PFIC2 patients showed no significant upregulation of MDR1 mRNA [46]. Overexpression of Bsep in the liver transgenic animals leads to an increased biliary bile salt output concomitant with an increase in bile flow rate and biliary lipid secretion [26].…”

Age‐dependent glycosylation of the sodium taurocholate cotransporter polypeptide: From fetal to adult human livers

“…Patients with primary hypercholanemia have been described with relatively mild clinical phenotypes, which have also been associated with growth retardation and vitamin deficiency 11, 12. Interestingly, down‐regulation of plasma membrane NTCP was reported in cases of progressive familial intrahepatic cholestasis‐2 and ‐313 and was thought to be linked to NTCP posttranslational modifications.…”