Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver

Abstract

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

Figure 1. Increased plasma bilirubin glucuronide in Slco1a/1b^–/– mice is in part dependent on Abcc3.

(A) BMG, (B) BDG, and (C) UCB levels in plasma of male wild-type, Abcc3^–/–, Abcc2^–/–, Abcc2^–/–Abcc3^–/–, Slco1a/1b^–/–, Slco1a/1b;Abcc3^–/–, Slco1a/1b;Abcc2^–/–, and Slco1a/1b;Abcc2;Abcc3^–/– mice (n = 4–7). +Oatp1a/1b denotes strains possessing Oatp1a/1b proteins, and –Oatp1a/1b denotes strains lacking Oatp1a/1b proteins. Data are mean ± SD. *P < 0.05, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001. ND, not detectable; detection limit was 0.1 μM.

Figure 2. In the presence of Oatp1a/1b, but not in its absence, Abcc3 enhances biliary excretion of bilirubin glucuronides.

(A) BMG, (B) BDG, and (C) UCB output in bile of male wild-type, Abcc3^–/–, Abcc2^–/–, Abcc2^–/–Abcc3^–/–, Slco1a/1b^–/–, Slco1a/1b;Abcc3^–/–, Slco1a/1b;Abcc2^–/–, and Slco1a/1b;Abcc2;Abcc3^–/– mice. Bile collected during the first 15 minutes after gall bladder cannulation was analyzed. +Oatp1a/1b denotes strains possessing Oatp1a/1b proteins, and –Oatp1a/1b denotes strains lacking Oatp1a/1b proteins. Data are shown as mean ± SD (n = 4–7). **P < 0.01, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†P < 0.05, ^††P < 0.01.

Figure 3. Increased plasma bilirubin glucuronide in Slco1a/1b^–/– mice is reversed by human OATP1B1 and OATP1B3.

(A) BMG, (B) BDG, and (C) UCB levels in plasma of male wild-type and Slco1a/1b^–/– mice, and of the derived OATP1B1- and OATP1B3-transgenic strains (Slco1a/1b^–/–;1B1^tg and Slco1a/1b^–/–1B3^tg, respectively) (n = 5–8). Data are mean ± SD. **P < 0.01, ***P < 0.001 compared with wild-type mice. Bracketed comparisons: ^†††P < 0.001. Detection limit was 0.1 μM.

Figure 4. RS families display deficiencies in SLCO1B1 and SLCO1B3.

(A) Pedigrees of the investigated families. Black symbols denote RS index subjects. Parents in family CE4 had a documented common ancestor. Families CE1–CE3 (only single individuals analyzed) are not shown. (B) Homozygosity regions in 8 RS index subjects and overview of detected mutations and polymorphisms. The genome map shows number and location of overlapping homozygosity regions in RS index subjects, gene content of the top candidate region on chromosome 12, and the genotypes forming all 3 identified RS haplotypes. Mutations crucial for RS are shown in red. chr, chromosome. (C) Sequences and electropherograms of the R1 and R2 deletion breakpoints. (D) Pathogenic point mutations in R1 and R3 haplotypes. Electropherograms indicate the c.1738C→T (p.R580X) mutation in SLCO1B1 in probands CE1, CE2, and CE4 II.1 and the c.757C→T (p.R253X) and c.1747+1G→A mutations in SLCO1B1 and SLCO1B3, respectively, in family P1.

Figure 5. Liver expression of OATP1B proteins in RS subjects and control.

With an anti-OATP1B1/3 antibody, basolateral membrane immunostaining of hepatocytes in centrilobular areas was intense in control. Asterisks indicate central veins, arrowheads bile canaliculi, and crosses sinusoids. OATP1B proteins were not detectable in RS subjects CE1 (haplotype R1), CE3 (haplotype R2), and P1 II.1 (haplotype R3). Scale bars: 25 μm (original magnification of CE1 and CE3, ×400; original magnification of P1 II.1 and control, ×200); inset: 5 μm (original magnification, ×1,000).

Figure 6. Hepatocyte hopping distributes the biliary excretion load of bilirubin glucuronides across the liver lobule.

(A) Schematic of liver lobule. Hepatocytes are organized around portal tracts, with branches of the portal vein (PV), hepatic artery (HA), and bile ducts (BD). The PV and HA deliver nutrient- and oxygen-rich blood, respectively, which flows through the sinusoids toward the central vein (CV). Basolateral (sinusoidal) membranes of hepatocytes are flushed with perisinusoidal plasma. Bile flows in the opposite direction toward bile ducts through canaliculi lined by canalicular membranes of hepatocytes. (B) Hepatocyte hopping cycle. UCB enters the hepatocytes via passive diffusion and/or transporters, which may include OATP1B1 and/or OATP1B3 in non-Rotor subjects. Conjugation with glucuronic acid by UGT1A1 to bilirubin glucuronides (BG) takes place in endoplasmic reticulum. BG is secreted into bile mainly by ABCC2. ABCG2 also can contribute to this process. Even under physiological conditions, a substantial fraction of the intracellular BG is rerouted by ABCC3 to the blood, from which it can be taken up by downstream hepatocytes via OATP1B1/3 transporters. This flexible off-loading of BG to downstream hepatocytes prevents saturation of biliary excretion capacity in upstream hepatocytes. Relative type sizes of UCB and BG represent local concentrations. Schematic modified, with permission, from ref. .