Fig 2: Digital analyses for the DAB positive area to compare the size of the IHC staining for HMTs in viable and necrotic areas from the same sample. Four to 10 regions of interest were selected in the representative samples from IR90. The staining was generally crisp in viable areas, whereas it was dispersed and granular in areas of necrosis. (A) The sizes of the DAB positive area for MRP2 were smaller in the area of necrosis, and the difference was significant only in the samples from animals with BARD administration. (B) The sizes of the DAB positive areas for BSEP were smaller in the area of necrosis. (C) The sizes of the DAB positive areas for MRP3 were larger in the area of necrosis, and the difference was significant only in the sample from animals with BARD administration. (D) The sizes of the DAB positive areas for NTCP were smaller in the area of necrosis, and the difference was significant only in the sample from animals with BARD administration. (E) The sizes of the DAB positive areas for OATP1 were larger in the area of necrosis. *P < 0.05 based on the Mann-Whitney test. BARD, bardoxolone methyl; BSEP, bile salt export pump; DAB, 3,3’Diaminobenzidine; HMT, hepatocyte membrane transporters; IHC, immunohistochemistry; IR60, 60 min of ischemia followed by 24 h of reperfusion; IR90, 90 min of ischemia followed by 24 h of reperfusion; MRP2, multidrug resistance-associated protein 2; MRP3, multidrug resistance-associated protein 3; NTCP, sodium-taurocholate cotransporter; OATP1, organic anion-transporting polypeptide 1.

Fig 3: The effect of BARD administration on the HMT protein level. The protein levels were expressed as values relative to GAPDH level. (A) MRP2 levels were not significantly different among the groups where the vehicle was administered; however, MRP2 levels were significantly higher in the IR90 group among BARD groups. (B) BSEP levels were not significantly different among the vehicle or BARD groups. (C) MRP3 levels were not significantly different among the vehicle groups; however, MRP3 levels were significantly lower in the IR60 group and higher in the IR90 group among BARD groups. (D) Among the vehicle groups, NTCP levels of IR90 were significantly higher than those of the sham group. Among the BARD groups, NTCP levels of IR90 were significantly higher than those of both the sham and IR60 groups. (E) OATP1 levels were not significantly different among the vehicle or BARD groups. The different symbols on the bars (*, †) indicate significant differences between the groups by the post hoc Mann-Whitney test with Bonferroni’s correction after the Kruskal-Wallis test. N = 5 in each group. BARD, bardoxolone methyl; BSEP, bile salt export pump; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMT, hepatocyte membrane transporters; IR60, 60 min of ischemia followed by 24 h of reperfusion; IR90, 90 min of ischemia followed by 24 h of reperfusion; IRI, ischemia-reperfusion injury; MRP2, multidrug resistance-associated protein 2; MRP3, multidrug resistance-associated protein 3; NTCP, sodium-taurocholate cotransporter; OATP1, organic anion-transporting polypeptide 1.

Fig 4: The effect of BARD administration on the mRNA levels of HMT genes in hepatic tissue. The mRNA levels were expressed as values relative to Gapdh levels. Significant decreases of mRNA levels of target genes after IRIs are shown by the diminished maximum ranges of the Y axis in each graph. (A) The mRNA levels of Mrp2 were significantly increased by BARD administration in the IR60 group but not in the sham or IR90 groups. (B) The mRNA levels of Bsep were significantly decreased by BARD administration in the IR90 group but not in the sham or IR60 groups. (C) The mRNA levels of Mrp3 were significantly increased by BARD administration in the sham and IR60 groups but not in the IR90 group. (D) The mRNA levels of Ntcp were not significantly changed by BARD administration in any group. (E) The mRNA levels of Oatp1 were not significantly changed by BARD administration in any group. *P < 0.05 by Mann-Whitney test. N = 5 in each group. BARD, bardoxolone methyl; Bsep, bile salt export pump; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; HMT, hepatocyte membrane transporter; IR60, 60 min of ischemia followed by 24 h of reperfusion; IR90, 90 min of ischemia followed by 24 h of reperfusion; IRI, ischemia-reperfusion injury; Mrp, multidrug resistance-associated protein; Ntcp, sodium-taurocholate; Oatp, organic anion-transporting polypeptide 1.