A Novel, Cell-Based Assay Allows Direct Measurement of Bile Salt Transport and Serves as Diagnostic Tool for Antibody-Induced BSEP Deficiency (AIBD)

Background:

Antibody-induced bile salt export pump deficiency (AIBD) is an aquired type of intrahepatic cholestasis that may arise in patients lacking native Bile Salt Export Pump BSEP expression after orthotopic liver transplantation for severe BSEP deficiency (PFIC-2). Here, antibodies are formed against the transplanted allo-antigen BSEP and can bind and inhibit the canalicular bile salt transporter from the extracellular (biliary) side (trans-inhibition). In order to confirm diagnosis of this severe condition, we developed a robust and easy assay that directly measures trans-inhibition of BSEP by patients' serum antibodies.

Methods:

Patient serum was tested for the presence of BSEP antibodies by immunoblotting experiments as well as immunofluorescence staining of BSEP-EYFP-expressing cells. Human embryonal kidney (HEK) 293 cell lines stably expressing either human NTCP-mCherry, BSEP-EYFP, or both transporters were generated by lentiviral transduction and clonal isolation. With these, we established an assay procedure consisting of preloading cells via NTCP with [3 H]-Taurocholate (TC) followed by its BSEP-mediated export into fresh medium. We then tested for antibody-mediated BSEP inhibition by preincubating NTCP/BSEP-expressing cells either with AIBD or naive control sera depleted of free bile salts or with antibodies purified from these.

Results:

Comparison of HEK293 and their transporter-expressing derivatives showed specific, NTCP-mediated [3 H]-TC uptake and export by BSEP. The assay is divided into an uptake phase dominated by NTCP and an export phase dominated by BSEP. In the latter, re-import of [3 H]-TC by the sodium-dependent NTCP is prevented by substituting choline for the extracellular sodium. Applying this assay, we demonstrate BSEP inhibition by several bile salt-free AIBD serum samples as well as purified AIBD antibodies. For non-radioactive measurements, we adapted assay conditions to accommodate the fluorescent substrate Tauro-nor-THCA-24-DBD.

Conclusion:

Binding their target from the extracellular side, BSEP-reactive antibodies effectively impair bile salt transport into the canalicular lumen, thus causing AIBD. Our novel, cell-based transport assay represents a direct test for extracellular transport inhibition by BSEP antibodies in AIBD serum samples, thus providing a crucial information for the conclusive diagnosis of AIBD. Furthermore, its application may also improve our understanding of AIBD disease onset and progression.