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Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease.

ABSTRACT: Organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1/3) as important uptake transporters play a fundamental role in the transportation of exogenous drugs and endogenous substances into cells. Rat OATP1B2, encoded by the Slco1b2 gene, is homologous to human OATP1B1/3. Although OATP1B1/3 is very important, few animal models can be used to study its properties. In this report, we successfully constructed the Slco1b2 knockout (KO) rat model via using the CRISPR/Cas9 technology for the first time. The novel rat model showed the absence of OATP1B2 protein expression, with no off-target effects as well as compensatory regulation of other transporters. Further pharmacokinetic study of pitavastatin, a typical substrate of OATP1B2, confirmed the OATP1B2 function was absent. Since bilirubin and bile acids are the substrates of OATP1B2, the contents of total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids in serum are significantly higher in Slco1b2 KO rats than the data of wild-type rats. These results are consistent with the symptoms caused by the absence of OATP1B1/3 in Rotor syndrome. Therefore, this rat model is not only a powerful tool for the study of OATP1B2-mediated drug transportation, but also a good disease model to study hyperbilirubinemia-related diseases.

SUBMITTER: Ma X

PROVIDER: S-EPMC7276679 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and-OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50-250 nM, 2-7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217?G and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.

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Characterization of organic anion transporting polypeptide 1b2 knockout rats generated by CRISPR/Cas9: a novel model for drug transport and hyperbilirubinemia disease.

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