OATP1B1/1B3 deficiency exacerbates hyperbilirubinemia in erythropoietic protoporphyria

Abstract

Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in ferrochelatase (FECH), leading to the accumulation of its substrate, protoporphyrin IX (PPIX). PPIX is primarily produced in the bone marrow and transported to the liver for excretion. Because PPIX is hydrophobic, its elevated levels can cause bile duct blockage, cholestatic liver injury, and even liver failure. However, the specific transporter responsible for PPIX uptake into hepatocytes remains unclear. The OATP1B1/1B3 transporters, which are expressed in hepatocytes, facilitate the uptake of coproporphyrin III, a structural analog of PPIX. Additionally, OATP1B1/1B3 mediates the uptake of bilirubin, a biomarker of liver injury, from plasma into the liver for excretion. Therefore, we aimed to determine the role of OATP1B1/1B3 in regulating PPIX and bilirubin homeostasis under EPP conditions. A mouse strain carrying a Fech mutation was used as an EPP model. Building on this, we generated a new EPP mouse model with Oatp1a/1b deficiency. Using these EPP mouse models, along with OATP1B1/1B3-overexpressing cells, our study revealed that PPIX is not a substrate of OATP1B1/1B3. Notably, our work found that genetic deficiency or pharmacologic suppression of Oatp1a/1b exacerbates hyperbilirubinemia in EPP mice without worsening liver injury. Mechanistically, Oatp1a/1b deficiency impairs bilirubin uptake from plasma, while Fech deficiency leads to PPIX-mediated bile duct blockage and reduced bilirubin excretion, synergistically exacerbating hyperbilirubinemia. In summary, our work demonstrated that deficiency or suppression of Oatp1a/1b exacerbates hyperbilirubinemia in EPP mouse models, suggesting that assessment of OATP1B1/1B3 function is crucial in EPP patients with EPP with hyperbilirubinemia. SIGNIFICANCE STATEMENT: This work revealed that serum bilirubin levels are not paralleled with liver damage in the erythropoietic protoporphyria mouse models with Oatp1a/1b deficiency. Our findings suggest that assessment of OATP1B1/1B3 function is crucial in patients with erythropoietic protoporphyria with hyperbilirubinemia.

Keywords: Bilirubin; Liver diseases; OATPs; Protoporphyrin IX.

Fig. 1

Characterization of Fech-mut/Oatp1a/1b-KO mice. (A) Genotyping results of WT, Fech-mut, Oatp1a/1b-KO, and Fech-mut/Oatp1a/1b-KO mice. (B) Hepatic expression of Oatp1a/1b genes, quantified by qPCR. Expression levels in Fech-mut mice are set to 1, with data presented as mean ± SD (n = 4–5). (C) Gross appearance of mouse ears. (D) Serum samples from Fech-mut and Fech-mut/Oatp1a/1b-KO mice. The ear skin and serum of Fech-mut/Oatp1a/1b-KO mice (right) appear more yellow compared to Fech-mut mice (left).

Fig. 2

Metabolomic analysis reveals hyperbilirubinemia in Fech-mut/Oatp1a/1b-KO mice. (A, B) Metabolomic analysis of serum samples from Fech-mut and Fech-mut/Oatp1a/1b-KO mice. (A) Score plot illustrating the separation between Fech-mut and Fech-mut/Oatp1a/1b-KO groups. (B) Loading S-plot highlighting metabolites contributing to group separation. The x-axis represents the relative abundance of metabolites, while the y-axis indicates the correlation of each metabolite to the model. UCB, BMG1, BMG2, and BDG were among the top-ranking metabolites in Fech-mut/Oatp1a/1b-KO mouse sera. (C–F) Relative quantification of UCB, BMG1, BMG2, and BDG in serum. Data are presented as mean ± SD (n = 4). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ND, not detected; ns, not significant.

Fig. 3

Pharmacological inhibition of Oatp1a/1b increases serum bilirubin levels in Fech-mut mice. Blood samples were collected before and 2 hours after RIF treatment. (A) Relative quantification of serum UCB, BMG1, BMG2, and BDG in Fech-mut mice (n = 8) before and after RIF treatment. (B) Relative quantification of serum UCB, BMG1, BMG2, and BDG in Fech-mut/Oatp1a/1b-KO mice (n = 4) before and after RIF treatment. For each group, baseline serum levels of UCB, BMG1, BMG2, and BDG before RIF treatment are set to 1. Data were analyzed using a paired t test. ∗∗P < .01; ∗∗∗P < .001; ∗∗P < .0001.

Fig. 4

PPIX is not a substrate of OATP1A/1B. (A, B) PPIX levels in the serum (A) and liver (B) of WT, Oatp1a/1b-KO, Fech-mut, and Fech-mut/Oatp1a/1b-KO mice (n = 4 per group). PPIX was analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer. (C, D) The uptake of PPIX by cells overexpressed with OATP1B1 (C) or OATP1B3 (D). The cells (n = 3 per group) were incubated with PPIX (10 μM) for 10 or 30 minutes. CopIII (10 μM), a known substrate of OATP1B1 and 1B3, was used as a positive control. CopIII and PPIX were determined by fluorescence analysis. The data in control groups (C, D) are set as 1. All data are expressed as mean ± SD. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant.

Fig. 5

Oatp1a/1b deficiency does not potentiate liver injury in EPP mice. Blood and liver samples were collected from WT, Fech-mut, Oatp1a/1b-KO, and Fech-mut/Oatp1a/1b-KO mice. (A–C) Serum activities of ALT, AST, and ALP. The data are expressed as mean ± SD (n = 4–9). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (D) Representative liver sections with H&E staining. Scale bars, 20 μm. ns, not significant; PV, portal vein.

Fig. 6

Deficiency of Oatp1a/1b exacerbates hyperbilirubinemia in EPP mouse models. (A) Total bilirubin levels in the serum of WT, Oatp1a/1b-KO, Fech-mut, and Fech-mut/Oatp1a/1b-KO mice. (B) qPCR analysis of Ugt1a1 mRNA in the liver of Fech-mut and Fech-mut/Oatp1a/1b-KO mice. (C) Western blot of Ugt1a1 protein in the liver of Fech-mut and Fech-mut/Oatp1a/1b-KO mice. (D) PPIX levels in the bile of WT, Fech-mut, and Fech-mut/Oatp1a/1b-KO mice. Data are expressed as mean ± SD (n = 3 or 4). ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (E) A proposed mechanism of hyperbilirubinemia in EPP mice with Oatp1a/1b deficiency: deficiency of Oatp1a/1b impairs the uptake of both UCB and CBs from the plasma. At the same time, PPIX-mediated bile duct blockage through ABCG2 reduces the hepatobiliary excretion of CBs, resulting in a synergistic effect that leads to hyperbilirubinemia. ns, not significant.