Functional shift with maintained regenerative potential following portal vein ligation.

Selective portal vein ligation (PVL) allows the two-stage surgical resection of primarily unresectable liver tumours by generating the atrophy and hypertrophy of portally ligated (LL) and non-ligated lobes (NLL), respectively. To evaluate critically important underlying functional alterations, present study characterised in vitro and vivo liver function in male Wistar rats (n = 106; 210-250 g) before, and 24/48/72/168/336 h after PVL. Lobe weights and volumes by magnetic resonance imaging confirmed the atrophy-hypertrophy complex. Proper expression and localization of key liver transporters (Ntcp, Bsep) and tight junction protein ZO-1 in isolated hepatocytes demonstrated constantly present viable and well-polarised cells in both lobes. In vitro taurocholate and bilirubin transport, as well as in vivo immunohistochemical Ntcp and Mrp2 expressions were bilaterally temporarily diminished, whereas LL and NLL structural acinar changes were divergent. In vivo bile and bilirubin-glucuronide excretion mirrored macroscopic changes, whereas serum bilirubin levels remained unaffected. In vivo functional imaging (indocyanine-green clearance test; 99mTc-mebrofenin hepatobiliary scintigraphy; confocal laser endomicroscopy) indicated transitionally reduced global liver uptake and -excretion. While LL functional involution was permanent, NLL uptake and excretory functions recovered excessively. Following PVL, functioning cells remain even in LL. Despite extensive bilateral morpho-functional changes, NLL functional increment restores temporary declined transport functions, emphasising liver functional assessment.

A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca2+ oscillation and sustained Ca2+ signals at 1µM and10 µM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures.

INTRODUCTION: Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. METHODS: CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. RESULTS: KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. DISCUSSION: Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures.

A Modified Procedure for Estimating the Impact of the Uptake on the Overall Biliary Clearance in Sandwich Culture of Rat Hepatocytes.

Sandwich culture of hepatocytes is commonly applied for the prediction of in vivo biliary clearance (CLbil). In this paper, we present a modified procedure for the determination of in vitro CLbil in sandwich culture of rat hepatocytes, which allows the estimation of the impact of uptake processes on the overall CLbil. The main point of this modification is the separation of uptake and efflux processes. Ten drugs from four biopharmaceutics drug disposition classification system classes were chosen in order to demonstrate the advantages of this method: 1) the uptake is performed identically before the canaliculi are opened, thus the efflux starts at the same intracellular concentration of the drugs and the effect of Ca2+/Mg2+ depletion on the uptake is excluded; 2) exact intracellular concentrations can be measured at the start and at the end of the efflux; 3) the biliary clearance can be determined irrespective of the uptake; 4) the canalicular and the sinusoidal transport can be measured simultaneously; 5) drug-drug interactions concerning uptake and efflux transporters can be estimated independently. Depending on the degree of uptake, CLbil,app (calculated using the concentration of drugs in the medium) was significantly higher (sulfasalazine, fluvastatin, rosuvastatin, atorvastatin) or lower (pravastatin, procainamide) than CLbil,int (calculated using the intracellular concentration of drugs). When the uptake had no impact on the CLbil, the apparent and intrinsic CLbil did not differ significantly (lovastatin, rifampicin, quetiapine). Our results confirm that transporters may play a significant role in the uptake of drugs both with high and poor permeability and solubility.

Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes.

Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects.