Functional expression of organic anion transporters in hepatic organoids reconstructed by rat small hepatocytes

Abstract

Small hepatocytes (SHs) are hepatic progenitor cells with hepatic characteristics. They can proliferate to form colonies in culture and change their morphology from flat to rising/piled-up with bile canaliculi (BC), which results in maturation. In this study, we examined whether SHs could express hepatic transporters with polarity, whether the transporters could transport organic anion substrates into BC, and whether the secreted substances could be recovered from BC. Immunocytochemistry and RT-PCR were carried out. [(3)H]-labeled estrogen derivatives were used to measure the functions of the transporters in SHs isolated from normal and multidrug resistance-associated protein (Mrp) 2-deficient rats. The results showed that organic anion-transporting proteins (Oatps) 1 and 2, Na(+)-dependent taurocholate co-transporting polypeptide (Ntcp), Mrp2, and bile-salt export pump (Bsep) were well expressed in rising/piled-up cells and that their expression was correlated to that of hepatocyte nuclear factor 4alpha. Although small SHs expressed not Oatps and Mrp2 but Mrp3, rising/piled-up SHs expressed Oatp1 and 2 and Mrp2 proteins in the sinusoidal and BC membranes, respectively. On the other hand, breast cancer resistant protein (Bcrp) and Mrp3 expression decreased as SHs matured. The substrate transported via Oatps and Mrp2 was secreted into BC and it accumulated in both BC and cyst-like structures. The secreted substrate could be efficiently recovered from BC reconstructed by SHs derived from a normal rat, but not from an Mrp2-deficient rat. In conclusion, SHs can reconstitute hepatic organoids expressing functional organic anion transporters in culture. This culture system may be useful to analyze the metabolism and excretion mechanisms of drugs.

The Role of the Sodium-Taurocholate Cotransporting Polypeptide (NTCP) and of the Bile Salt Export Pump (BSEP) in Physiology and Pathophysiology of Bile Formation

“…Functionally, sodium-dependent bile salt transport is observed at day 20 of gestation [304] coinciding with the first basolateral protein expression of Ntcp in fetal liver. This relatively late appearance of Ntcp during ontogenesis is reproduced in primary cultures of small hepatocytes, which expand and differentiate into mature hepatocytes [243,280]. Small hepatocytes are hepatocyte progenitor cells found in adult liver [44].…”

Evaluation of Concanavalin A-induced Acute Liver Injury in Rats using an Empirical Mathematical Model and Dynamic Contrast-enhanced MR Imaging with Gd-EOB-DTPA

“…The estimated parameter values, AST values, and ALT values were expressed as mean ± standard error (SE) Figure 1 shows examples of curveˆtting in the normal control (a), Con A (10) (b), and Con A (20) groups (c). After Gd-EOB-DTPA administration, a continuous increase and slow reduction of RE(t ) after peaking at T max were induced in all groups, but signal proˆles diŠered among the 3 groups.…”

“…The rats were allocated to control (n＝6), Con A [10] (n＝6) and Con A [20] groups (n＝6). The rats in the Con A [10] and Con A [20] groups were injected intravenously from the tail vein with 10 mg/kg body weight (BW) and 20 mg/kg BW of Con A (Sigma Chemical Co., St. Louis, MO, USA), respectively, dissolved in normal saline (4 mL/kg BW).…”

“…The rats in the Con A [10] and Con A [20] groups were injected intravenously from the tail vein with 10 mg/kg body weight (BW) and 20 mg/kg BW of Con A (Sigma Chemical Co., St. Louis, MO, USA), respectively, dissolved in normal saline (4 mL/kg BW). The rats in the control group were injected intravenously with saline of the same volume as that of Con A. Twenty-four hours after Con A or saline injection, we performed the DCE-MRI studies,ˆrst anaesthetizing the rats by intraperitoneal injection of chloral hydrate solution (4z, 400 mg/kg BW) and immobilizing each animal with surgical tape in the supine position to prevent unnecessary motion.…”

“…These results may suggest greater damage of the MRP2 than OATP1 in the Con Ainduced acute liver injury. Immunocytochemistry 20 and RNA extraction and real-time polymerase chain reaction (RT-PCR) 21 enabled assessments of the expression of transporters, such as OATP1 and MRP2. Therefore, further study was needed to clarify fully the relationship between the uptake and washout of Gd-EOB-DTPA and expression of transporters in the hepatic injury.…”