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. 2022 Jul 11;23(14):7666.
doi: 10.3390/ijms23147666.

Bile Duct Ligation Impairs Function and Expression of Mrp1 at Rat Blood-Retinal Barrier via Bilirubin-Induced P38 MAPK Pathway Activations

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Bile Duct Ligation Impairs Function and Expression of Mrp1 at Rat Blood-Retinal Barrier via Bilirubin-Induced P38 MAPK Pathway Activations

Ping Li et al. Int J Mol Sci. .

Abstract

Liver injury is often associated with hepatic retinopathy, resulting from accumulation of retinal toxins due to blood-retinal barrier (BRB) dysfunction. Retinal pigment epithelium highly expresses MRP1/Mrp1. We aimed to investigate whether liver injury affects the function and expression of retinal Mrp1 using bile duct ligation (BDL) rats. Retinal distributions of fluorescein and 2,4-dinitrophenyl-S-glutathione were used for assessing Mrp1 function. BDL significantly increased distributions of the two substrates and bilirubin, downregulated Mrp1 protein, and upregulated phosphorylation of p38 and MK2 in the retina. BDL neither affected the retinal distribution of FITC-dextran nor expressions of ZO-1 and claudin-5, demonstrating intact BRB integrity. In ARPE-19 cells, BDL rat serum or bilirubin decreased MRP1 expression and enhanced p38 and MK2 phosphorylation. Both inhibiting and silencing p38 significantly reversed the bilirubin- and anisomycin-induced decreases in MRP1 protein. Apparent permeability coefficients of fluorescein in the A-to-B direction (Papp, A-to-B) across the ARPE-19 monolayer were greater than Papp, B-to-A. MK571 or bilirubin significantly decreased Papp, A-to-B of fluorescein. Bilirubin treatment significantly downregulated Mrp1 function and expression without affecting integrity of BRB and increased bilirubin levels and phosphorylation of p38 and MK2 in rat retina. In conclusion, BDL downregulates the expression and function of retina Mrp1 by activating the p38 MAPK pathway due to increased bilirubin levels.

Keywords: bile duct ligation; bilirubin; blood–retinal barrier; liver injury; multidrug resistance-associated protein 1; p38 MAPK.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of BDL on the function of Mrp1 in rat retina. (a) Retina fluorescein levels, (b) plasma fluorescein levels, and (c) retina-to-plasma ratio (Kr/p) of fluorescein at 30 min following intravenous (i.v.) fluorescein (2 mg/kg) to rats. (d) Retina DNP-SG levels, (e) plasma DNP-SG levels, and (f) Kr/p of DNP-SG at 15 min following i.v. 1-chloro-2,4-dinitrobenzene (5 mg/kg) to rats. (g) Plasma UCB levels and (h) retina UCB levels in Sham and BDL rats. Lower limit of quantitation (LLOQ) = 0.67 pmol/mg protein. Data are expressed as means ± S.D. (n = 6). ** p < 0.01 vs. Sham rats.
Figure 2
Figure 2
Effect of BDL on expression of Mrp1 in rat retina. (a) The mRNA levels of Abcc1~6, Abcb1a/1b, and Abcg2 in eyecups of normal rats were measured by qRT-PCR and normalized to Actb (2−ΔCt). (b) Expression of Mrp1 protein in eyecups of Sham and BDL rats (n = 6). (c) The location of Mrp1 (green) and Glut1 (red) in the eyecup of rats (Scale bar = 100 μm). Nuclei are DAPI-stained (blue). Regions marked 1 and 2 indicate microvessel and RPE, respectively, which are detailed in the upper right and lower right (Scale bar = 20 μm). Ap, apical membrane; bl, basolateral membrane. (d) Immunostaining analysis of Mrp1 and Glut1 in eyecups (Scale bar = 100 μm) and (e) its integrated fluorescence intensity of Mrp1 normalized by RPE length (n = 3). (f) Plasma FITC-dextran levels, (g) retina FITC-dextran levels, and (h) Kr/p of FITC-dextran in Sham and BDL rats at 30 min following i.v. 50 mg/kg FITC-dextran (n = 5). (i) Expressions of ZO-1 and claudin-5 (n = 6). (j) Immunoblots for pp38, p38, pAkt, Akt, pERK, ERK, pp65, p65, pJNK, and JNK and ratios of phosphorylated proteins to total protein levels (n = 6). (k) Expressions of pMK2 and total MK2 levels (n = 6). (l) Expression of Brn-3a protein (n = 6). Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01 vs. Sham rats.
Figure 3
Figure 3
Effect of UCB on the function and expression of MRP1 in ARPE-19 cells. (a) The mRNA levels of efflux transporters in ARPE-19 cells were normalized to the ACTB (2−ΔCt) (n = 3). Effects of MRP1 inhibitor MK571 on cellular uptakes of (b) fluorescein (n = 6), (c) calcein (n = 6), and (d) UCB (n = 4) in ARPE-19 cells. (e) Effect of serum from BDL rats on MRP1 protein expression (n = 6). Effects of (f) UCB, (g) NH4Cl, (h) ADMA, and (i) bile salt cocktail on MRP1 protein expressions (n = 6). (j) TEER values across ARPE-19 monolayer after seeding (n = 4). (k) Apical (A) -to-basolateral (B) transport of FITC-dextran across ARPE-19 monolayer and control insert membranes (n = 4). (l) A-to-B and B-to-A transport of fluorescein with or without MK571 (n = 6). (m) Cross-sectional view of the reconstructed z-stack images in x-z plane. Ap, apical; bl, basolateral. (n) A-to-B transport of fluorescein after pre-treatment with UCB for 72 h (n = 4). (o) Protein levels of MRP1 were accessed by immunofluorescence after incubation with UCB (Scale bar = 50 μm). (p) The mean MRP1 fluorescence intensity was normalized to the mean DAPI fluorescence intensity (n = 3). Data are expressed as the mean ± S.D. * p < 0.05, ** p < 0.01 vs. Sham serum, control (Ctrl) cells or Papp, A-to-B of Ctrl.
Figure 4
Figure 4
Roles of signal pathways in impairment of MRP1 protein expression by UCB in APRE-19 cells. Effects of (a) UCB and (b) BDL rat serum on expressions of pp38/p38, pMK2/MK2, pAkt/Akt, pERK/ERK1/2, pp65/p65, and pJNK/JNK levels. Effects of (c) p38 inhibitors SB203580 (SB203) and SB202190 (SB202), (d) Akt inhibitor LY294002 (LY294), and ERK1/2 inhibitor U0126 on UCB-induced downregulation of MRP1 protein. Effects of SB203 and SB202 on increases in phosphorylation of (e) p38 and (f) MK2 levels by UCB. (g) Effects of SB203 and SB202 on anisomycin (ANI)-induced impairment of MRP1 protein expression and increases in phosphorylation of p38, MK2. (h) Validation of p38 siRNA. (i) Effects of p38 siRNA on UCB- and ANI-induced decreases in MRP1 protein expressions. (j) Effects of UCB on H2O2 -induced ROS formation. (k) Effects of NAC on UCB-induced ROS formation. (l) Effect of H2O2 on MRP1 protein levels. Data are expressed as the mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01 vs. control (Ctrl) cells, Sham serum, si-negative control (si-NC) + Ctrl. # p < 0.05, ## p< 0.01 vs. UCB (50 μM), ANI or si-NC + UCB. $$ p < 0.01 vs. si-NC + ANI or H2O2.
Figure 5
Figure 5
Effects of bilirubin (UCB) treatment on function and expression of Mrp1 in rat retina. (a) Levels of UCB in plasma of control (Ctrl) and hyperbilirubinemia (HB) rats. (b) Plasma fluorescein levels, (c) retina fluorescein levels, and (d) Kr/p of fluorescein at 30 min following i.v. fluorescein (2 mg/kg) to Ctrl and HB rats. (e) Plasma DNP-SG levels, (f) retina DNP-SG levels, and (g) Kr/p of DNP-SG at 15 min following i.v. 1-chloro-2,4-dinitrobenzene (5 mg/kg) to Ctrl and HB rats. (h) Retina UCB levels in Ctrl and HB rats. LLOQ = 0.67 pmol/mg protein. (i) Expressions of Mrp1, pp38/p38, and pMK2/MK2 levels in eyecups of Ctrl and HB rats. (j) Brn-3a protein expression in retina. Data are expressed as the mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01 vs. Ctrl.

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