Mrp4−/− mice have an impaired cytoprotective response in obstructive cholestasis

Abstract

Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4؊/؊ mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4؊/؊ mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ost␣-Ost␤ were upregulated in Mrp4؊/؊ mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4؊/؊ mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury. ( M ultidrug resistance-associated protein 4 (MRP4) (ABCC4) is an ATP-dependent organic anion transporter with broad substrate specificity. [1][2][3] It is a member of the ABC transporter superfamily 4 and is expressed in a variety of epithelia, including the basolateral and apical plasma membranes of the liver and kidneys, respectively. 5-7 However, this array of substrates and specific tissue localization have not provided insight into Mrp4 function in vivo.Mrp4 was first suggested to play a role in the adaptive response to the hepatic overload of bile acids following the genetic deletion of farnesoid X receptor (FXR), a bile acid sensor. 8,9 A subsequent study suggested that Mrp4 was not elevated upon feeding the hydrophobic bile acid, lithocholic acid. 10 Despite this finding, other studies have demonstrated that hepatic Mrp4 is upregulated in both rats and mice after bile duct ligation 6,11 and in pediatric patients with progressive familial intrahepatic cholestasis. 12 Recent studies demonstrate that MRP4 functions as an efflux pump for bile acids together with glutathione. 13 Further support for a role for MRP4 in hepatic bile acid overload is the recent demonstration that Mrp4 is upregulated by the constitutive androstane receptor. 14 Constitutive androstane receptor is a member of the nuclear hormone receptor superfamily that is required to elevate serum bile acids during cholestatic injury. 15 Cholestatic injury in mice, rats, and humans can also result in adaptive responses in other basolateral transporters. Examples include Mrp3, which is primarily a bilirubin conjugate transporter and also a constitutive androstane receptor target, 15 and the recently described heterodi...

Variability in human hepatic MRP4 expression: influence of cholestasis and genotype

“…Studies in cell culture 14,15 and in animal models [15][16][17][18] have shown increased hepatic MRP4/Mrp4 mRNA and protein expression under cholestatic conditions. Moreover, Mennone et al 19 showed that serum bile acid levels are lower in Mrp4 knockout mice than in wild-type CBDL (common bile duct ligation) mice, indicating a role of murine Mrp4 in the adaptive response to obstructive cholestatic liver injury. Keitel et al 20 reported a significant increase in MRP4 mRNA and protein expression in a small number of liver samples from children diagnosed with PFIC-2 and -3.…”

“…13 Recently, in vitro experiments 14,15 as well as animal models [15][16][17][18] of cholestasis have demonstrated that human MRP4 and rodent Mrp4 are induced under cholestatic conditions. Mennone et al 19 showed that serum bile acid concentrations are lower in Mrp4 knockout mice than in wild-type CBDL (common bile duct ligation) mice presumably owing to impaired secretion of bile acids over the basolateral hepatocyte membrane. Moreover, the upregulation of MRP4 mRNA and protein in human liver has been reported for a small number of liver specimens from children diagnosed with progressive familial intrahepatic cholestasis (PFIC)-2 and 3.…”

IGF-1R Contributes to Stress-Induced Hepatocellular Damage in Experimental Cholestasis

“…Mrp4 plays an essential protective role in the adaptive response to obstructive cholestatic liver injury. 29 Therefore, we could not attribute the better tolerance of cholestasis in LIGFREKO mice to enhanced adaptive transport.…”

Constitutive Androstane Receptor-Mediated Changes in Bile Acid Composition Contributes to Hepatoprotection from Lithocholic Acid-Induced Liver Injury in Mice

“…Liver samples were homogenized in a 1:1 solution of t-butanol/water (approximately 200 mg of liver/ml) and extracted overnight as described previously (Mennone et al, 2006). Samples were centrifuged at 10,000g for 20 min, and the supernatant was removed and placed in a SpeedVac (Thermo Fisher Scientific, Waltham, MA) until dry.…”