Na+‐taurocholate cotransporting polypeptide inhibition has hepatoprotective effects in cholestasis in mice

Abstract

Accumulation of bile salts (BSs) during cholestasis leads to hepatic and biliary injury, driving inflammatory and fibrotic processes. The Na+‐Taurocholate Cotransporting Polypeptide (NTCP) is the major hepatic uptake transporter of BSs, and can be specifically inhibited by myrcludex B. We hypothesized that inhibition of NTCP dampens cholestatic liver injury. Acute cholestasis was induced in mice by a 3.5‐diethoxycarbonyl‐1.4‐dihydrocollidine (DDC) diet or by bile duct ligation (BDL). Chronic cholestasis was investigated in Atp8b1‐G308V and Abcb4/Mdr2 deficient mice. Mice were injected daily with myrcludex B or vehicle. Myrcludex B reduced plasma alkaline phosphatase (ALP) levels in DDC‐fed, Atp8b1‐G308V and BDL mice by 39%, 27% and 48% respectively. Expression of genes involved in fibrosis, proliferation and inflammation was reduced by myrcludex B treatment in DDC‐fed and Atp8b1‐G308V mice. NTCP‐inhibition increased plasma BS levels from 604±277 to 1746±719 μm in DDC‐fed mice, 432±280 to 762±288 μm in Atp8b1‐G308V mice and from 522±130 to 3625±378 μm in BDL mice. NTCP‐inhibition strongly aggravated weight loss in BDL mice, but not in other cholestatic models studied. NTCP‐inhibition reduced biliary BS output in DDC‐fed and Atp8b1‐G308V mice by ∼50% while phospholipid (PL) output was maintained, resulting in a higher PL/BS ratio. Conversely, liver injury in Abcb4 deficient mice, lacking biliary phospholipid output, was aggravated after myrcludex B treatment. Conclusion: NTCP‐inhibition by myrcludex B has hepatoprotective effects, by reducing BS load in hepatocytes and increasing the biliary PL/BS ratio. High micromolar plasma BS levels after NTCP‐inhibition were well tolerated. NTCP‐inhibition may be beneficial in selected forms of cholestasis. (Hepatology 2018).

Blocking Sodium‐Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice

“…Biliary phospholipid secretion relies completely on ABCB4 activity, and we previously showed that NTCP inhibition does not stimulate phospholipid or cholesterol secretion in Abcb4-deficient mice, indicating that the induction of lipid secretion by Myrcludex B completely relies on ABCB4-mediated phospholipid translocation. (9) Hence, these experiments demonstrate that induction of phospholipid and cholesterol secretion by Myrcludex B requires activity of ABCB4, but increased activity of this transporter is unlikely to drive this effect.…”

“…As such, Myrcludex B is a potent tool to investigate the physiological consequences of interrupting enterohepatic cycling of bile salts by targeting hepatic bile salt (re)uptake. Previously, we have already shown that NTCP inhibition by Myrcludex B reduces cholestatic liver injury in mice by lowering the bile salt load on the liver and altering the phospholipid to bile salt ratio in bile, thereby reducing bile salt toxicity …”

“…Previously, we have already shown that NTCP inhibition by Myrcludex B reduces cholestatic liver injury in mice by lowering the bile salt load on the liver and altering the phospholipid to bile salt ratio in bile, thereby reducing bile salt toxicity. (9) In this study, we set out to evaluate whether NTCP inhibition by Myrcludex B specifically affects the bile formation process and hepatobiliary output of major bile components in healthy, noncholestatic mice. Surprisingly, NTCP inhibition increased biliary phospholipid and cholesterol output, whereas output of endogenous bile salts remained unaffected.…”

Calnexin Depletion by Endoplasmic Reticulum Stress During Cholestasis Inhibits the Na+‐Taurocholate Cotransporting Polypeptide

“…We therefore propose that this mechanism contributes to the hepatoprotective repression of hepatic bile acid uptake in cholestatic conditions, complementing FXR‐dependent transcriptional down‐regulation of NTCP mRNA . We recently demonstrated that pharmacologic inhibition of NTCP by myrcludex B injection reduced the level of liver damage in specific mouse models of cholestasis, illustrating the relevance of such an additional posttranscriptional fine tuning of NTCP regulation . The control of NTCP by ER stress‐induced calnexin down‐regulation could act as a naturally occurring (patho)physiologic mechanism to dampen hepatic bile acid uptake that can be further improved by pharmacologic treatments.…”

“…Two‐month‐old male wild‐type (WT) C57BI6/J mice were purchased from Envigo (Venray, the Netherlands). Cholestasis was induced by feeding the mice a chow diet (D12450B1, OpenSource Diets; Research Diets, Inc.), supplemented with 0.1% DDC (Sigma) for 7 days . In FXR –/– mice and WT counterparts, cholestasis was induced by 3 days of bile duct ligation .…”