Ghallab Ahmed, Kunz Sebastian, Drossel Celine, Billo Veronica, Friebel Adrian, Georg Mats, Göttlich Richard, Hobloss Zaynab, Hassan Reham, Myllys Maiju, Seddek Abdel-Latief, Abdelmageed Noha, Dawson Paul A, Lindström Erik, Hoehme Stefan, Hengstler Jan G, Geyer Joachim
Department of Toxicology, Leibniz Research Centre for Working Environment and Human Factors, Technical University Dortmund, Ardeystr. 67, 44139 Dortmund, Germany.
Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, South Valley University, 83523 Qena, Egypt.
EXCLI J. 2024 Oct 30;23:1330-1352. doi: 10.17179/excli2024-7707. eCollection 2024.
Fluorophore-coupled bile acids (BA) represent an important tool for intravital analysis of BA flux in animal models of cholestatic diseases. However, addition of a fluorophore to a BA may alter transport properties. We developed and validated a 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-coupled taurocholic acid (3β-NBD-TCA) as a probe for intravital analysis of BA homeostasis. We compared transport of 3β-NBD-TCA to [3H]-TCA in HEK293 cells stably expressing the mouse hepatic or renal BA carriers mNtcp or mAsbt, respectively. We also studied distribution kinetics intravitally in livers and kidneys of anesthetized wildtype and mOatp1a/1b cluster knockout mice (OatpKO) with and without administration of the Ntcp inhibitor Myrcludex B and the ASBT inhibitor AS0369. In vitro, 3β-NBD-TCA and [3H]-TCA showed comparable concentration- and time-dependent transport via mNtcp and mAsbt as well as similar inhibition kinetics for Myrcludex B and AS0369. Intravital analysis in the livers of wildtype and OatpKO mice revealed contribution of both mNtcp and mOatp1a/1b in the 3β-NBD-TCA uptake from the sinusoidal blood into hepatocytes. Combined deletion of mOatp1a/1b and inhibition of mNtcp by Myrcludex B blocked the uptake of 3β-NBD-TCA from sinusoidal blood into hepatocytes. This led to an increase of 3β-NBD-TCA signal in the systemic circulation including renal capillaries, followed by strong enrichment in a subpopulation of proximal renal tubular epithelial cells (TEC). The enrichment of 3β-NBD-TCA in TEC was strongly reduced by the systemic ASBT inhibitor AS0369. NBD-coupled TCA has similar transport kinetics as [3H]-TCA and can be used as a tool to study hepatorenal BA transport. See also the graphical abstract(Fig. 1).
荧光团偶联胆汁酸（BA）是胆汁淤积性疾病动物模型中活体分析BA通量的重要工具。然而，向BA添加荧光团可能会改变其转运特性。我们开发并验证了一种4-氯-7-硝基苯并-2-恶唑-1,3-二唑偶联牛磺胆酸（3β-NBD-TCA）作为BA体内稳态活体分析的探针。我们分别比较了3β-NBD-TCA与[3H]-TCA在稳定表达小鼠肝脏或肾脏BA转运体mNtcp或mAsbt的HEK293细胞中的转运情况。我们还在麻醉的野生型和mOatp1a/1b簇敲除小鼠（OatpKO）的肝脏和肾脏中进行了活体分布动力学研究，分别给予和不给予Ntcp抑制剂Myrcludex B和ASBT抑制剂AS0369。在体外，3β-NBD-TCA和[3H]-TCA通过mNtcp和mAsbt表现出相似的浓度和时间依赖性转运，以及对Myrcludex B和AS0369相似的抑制动力学。在野生型和OatpKO小鼠肝脏中的活体分析显示，mNtcp和mOatp1a/1b在从窦状隙血液摄取3β-NBD-TCA进入肝细胞过程中均有作用。mOatp1a/1b的联合缺失以及Myrcludex B对mNtcp的抑制作用阻断了3β-NBD-TCA从窦状隙血液摄取进入肝细胞。这导致包括肾毛细血管在内的体循环中3β-NBD-TCA信号增加，随后在近端肾小管上皮细胞（TEC）亚群中强烈富集。全身给予ASBT抑制剂AS0369可显著降低TEC中3β-NBD-TCA的富集。NBD偶联的TCA具有与[3H]-TCA相似的转运动力学，可作为研究肝肾BA转运的工具。另见图1（图形摘要）。
