Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function

Abstract

During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.

Fig. 1.

Depletion of Kupffer cells by clodronate liposome treatment. Mice were injected with empty liposomes (A and C) or clodronate liposomes (B and D). After 48 h some mice were euthanized 20 min after injection of India ink for the determination of carbon particle uptake in hematoxylin and eosin-stained liver sections (A and B). Other mice were euthanized at 48 h after liposome treatment and liver sections were stained with F4/80 antibody (C and D). Images were taken at ×20 and ×40 (insets) magnification.

Fig. 2.

Plasma alanine aminotransferase (ALT) activity after acetaminophen (APAP) treatment. Plasma was isolated from empty or clodronate liposome-pretreated mice 6, 24, 48, and 72 h following dosing with APAP (500 mg/kg) or vehicle. The data are presented as mean plasma ALT (U/l) ± SE (n = 3–12 animals). *Statistical difference (P < 0.05) from pooled control mice of the same liposome treatment (0 h); †statistical difference (P < 0.05) from empty liposome APAP-treated mice.

Fig. 3.

Analysis of APAP bioactivation pathways in Kupffer cell-depleted and nondepleted mice. Mice were injected with empty liposomes (EL) or clodronate liposomes (CL) and euthanized after 48 h. Western blots were performed by using liver membrane fractions. The data are presented as representative blots with each lane representing an individual mouse (A) and as mean relative Cyp2e1, Cyp1a2, or Cyp3a11 protein expression ± SE (n = 3) as quantified from the blots in A (B). Microsomes were prepared and assayed for APAP bioactivation (C), with data presented as means ± SE (n = 3).

Fig. 4.

Analysis of APAP detoxification pathways in Kupffer-cell depleted and nondepleted mice. Mice were injected with empty liposomes or clodronate liposomes and euthanized after 48 h. Western blots were performed by using cytosolic fractions (for Gclc and Gclm) or liver membrane fractions (for Ugt1a6). The data are presented as representative blots with each lane representing an individual mouse (A) and as mean relative Gclc, Gclm, or Ugt1a6 protein expression ± SE (n = 3) as quantified from the blots in A (B). Hepatic nonprotein sulfhydryl (NPSH) content was determined (C), and microsomes were prepared and assayed for APAP glucuronidation (D). The NPSH and glucuronidation data are presented as means ± SE (n = 3).

Fig. 5.

Western blot analysis of hepatic uptake transport proteins. Western blots were performed by using liver membrane fractions from liposome-pretreated mice at 6, 24, 48, and 72 h after APAP (500 mg/kg) or vehicle treatment (control). The data are presented as mean relative Oatp1a1, Oatp1a4, Oatp1b2, or Ntcp protein expression ± SE (n = 4–9) (A) and as representative blots at 48 h, with each lane representing an individual mouse (B). *Statistical difference (P < 0.05) from pooled control (0 h) mice of the same liposome treatment; †statistical difference (P < 0.05) from empty liposome APAP-treated mice; ‡statistical difference (P < 0.05) from empty liposome control mice.

Fig. 6.

Western blot analysis of hepatic efflux transport proteins. Western blots were performed by using liver membrane fractions from liposome-pretreated mice at 6, 24, 48, and 72 h after APAP (500 mg/kg) or vehicle treatment (control). The data are presented as mean relative Mrp1, Mrp2, Mrp3, Mrp4, or P-gp protein expression ± SE (n = 4–9) (A) and as representative blots at 48 h with each lane representing an individual mouse (B). *Statistical difference (P < 0.05) from pooled control (0 h) mice of the same liposome treatment; †statistical difference (P < 0.05) from empty liposome APAP-treated mice; ‡statistical difference (P < 0.05) from empty liposome control mice.

Fig. 7.

Immunohistochemical analysis of Mrp4. Mice pretreated with empty liposomes (A, C, E, and G) and clodronates liposome (B, D, F, and H) were challenged with APAP (500 mg/kg) or vehicle (A and B) and euthanized at 24 (C and D), 48 (E and F), and 72 (G and H) h. Mrp4 staining was performed on formalin fixed paraffin-embedded liver sections. Tissues were counterstained with hematoxylin and eosin. Images were taken at ×40 magnification.

Fig. 8.

Effect of Kupffer cell depletion on APAP-induced cytokine and chemokine plasma levels. Mice were pretreated with either empty or clodronate liposomes and challenged with APAP (500 mg/kg) or vehicle. Plasma was collected at all time points after APAP administration (0–72 h). Plasma levels of IL-1β, IL-4, IL-6, IL-10, IL-13, GM-CSF, IFN-γ, KC, MCP-1, and TNF-α were measured by Bio-plex Cytokine Assays. The data are presented as means ± SE (n = 3–6 animals). ND, not detectable. *Statistical difference (P < 0.05) from pooled control mice of the same liposome treatment (0 h).