PPARγ ligand attenuates portal inflammation in the MRL-lpr mouse: a new strategy to restrain cholangiopathy in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterized by chronic destructive cholangitis, which is associated with the reduced expression of an anti-inflammatory molecule, peroxisome proliferator-activated receptor-γ (PPARγ), in intrahepatic bile ducts. We previously demonstrated the anti-inflammatory effects of PPARγ ligands using cultured human biliary epithelial cells. In this study, we evaluated the effectiveness of PPARγ ligand against peribiliary inflammation in vivo. As an animal model of PBC, we used MRL/lpr mice in which a PBC-like cholangitis occurs naturally. Anti-inflammatory effects of the intraperitoneal administration of a PPARγ ligand, the prostaglandin D metabolite 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2), were evaluated. In untreated mice, portal inflammation including cholangitis was found to some degree in the majority of portal tracts. In mice given a high-dose group, the degree of portal inflammation was significantly reduced and mice mostly lacking portal inflammation and cholangitis were also found. T cell numbers in portal tracts were markedly decreased in the high-dose group, compared with controls, whereas there was no significant difference in terms of B cells and macrophages. This study is the first to assess the therapeutic potential of a PPARγ ligand against portal inflammation including cholangitis. Anti-inflammatory effects of PPARγ ligands may prevent the progression of cholangiopathy in PBC patients.

Matrix proteins of basement membrane of intrahepatic bile ducts are degraded in congenital hepatic fibrosis and Caroli's disease.

Congenital hepatic fibrosis (CHF) and Caroli's disease are though to result from ductal plate malformation, and the basal laminar components play important roles in biliary differentiation during development. To clarify the involvement of basal laminar components in the ductal plate malformation, this study examined the immunohistochemical expression of laminin and type IV collagen in the livers of CHF and Caroli's disease. Using the polycystic kidney (PCK) rat, an animal model of Caroli's disease with CHF, in vivo and in vitro experiments were also performed. Immunostaining showed that basement membrane expression of laminin and type IV collagen around intrahepatic bile ducts was degraded in CHF, Caroli's disease, and the PCK rats. The degradation of laminin and type IV collagen around bile ducts was also observed in foci of cholangiocarcinoma in situ of Caroli's disease. In vitro, PCK cholangiocytes were found to overexpress plasminogen and a serine proteinase, the tissue-type plasminogen activator (tPA). When PCK cholangiocytes were cultured in Matrigel, the amounts of laminin and collagen in the gel were significantly reduced, and addition of alpha2-antiplasmin in the culture medium inhibited the degradation of laminin and collagen in Matrigel. These results suggest that biliary overexpression of plasminogen and tPA leads to the generation of excessive amounts of plasmin, and subsequent plasmin-dependent lysis of the extracellular matrix molecules may contribute to the biliary dysgenesis in CHF and Caroli's disease, including progressive cystic dilatation of the intrahepatic bile ducts in Caroli's disease. In addition, it is suggested that once cholangiocarcinoma in situ develops in the biliary epithelium of CHF and Caroli's disease, it tends to transform into invasive carcinoma, due to instability of the basement membrane of the bile ducts.

Comparative articular toxicity of garenoxacin, a novel quinolone antimicrobial agent, in juvenile beagle dogs.

The articular toxicity of garenoxacin (formerly T-3811 or BMS-284756) was experimentally examined utilizing juvenile beagle dogs. Garenoxacin and two other reference quinolones were administered at intravenous dosages of 30 and 60 mg/kg. Each group consisted of 3 male dogs (Experiment I). Oral dosages of 50 mg/kg of 3 compounds were also given daily to male only and female only groups (Experiment II) over a period of 7 days. We evaluated the articular toxicity of garenoxacin compared to ciprofloxacin and norfloxacin. In Experiment I, no articular toxicity was detected in the 30 mg/kg garenoxacin group. One animal from the 60 mg/kg garenoxacin group developed detectable histopathological lesions in the articular cartilages of the shoulder, elbow and knee joints. In the 30 mg/kg ciprofloxacin group and the 30 and 60 mg/kg norfloxacin groups, histopathological articular cartilage lesions of the shoulder, elbow, carpus, hip, knee and tarsus joints were observed in all of the dogs. The area under the plasma concentration-time curve (AUC0-->infinity) values, after the first dose was administered, for the 30 mg/kg groups given garenoxacin, ciprofloxacin and norfloxacin were 164, 68.1 and 65.7 micrograms.hr/mL, respectively. In Experiment II, the degree of histopathological change was most significant in the ciprofloxacin group, followed by the norfloxacin group, and with comparatively the least changes in the garenoxacin group. The AUC0-->infinity values, obtained after the 6th day of antimicrobial administration, were 202 and 173 micrograms.hr/mL for male and female dogs, respectively, from the 50 mg/kg garenoxacin group. The AUC0-->infinity values for the garenoxacin group after the 6th daily administration were 7.8 to 17.0 times greater for male dogs and 3.8 to 13.2 times greater for female dogs than those obtained from the ciprofloxacin and norfloxacin groups. The concentrations of garenoxacin in the synovia, articular cartilage and the synovialis 4 hr following the last garenoxacin administration were 2.0 to 6.5 times higher for male dogs and 1.5 to 3.3 times higher for female dogs than the antimicrobial levels measured in the ciprofloxacin and norfloxacin groups. As discussed above, although the garenoxacin concentrations in plasma and joint tissue were higher than those for ciprofloxacin and norfloxacin, however, the articular toxicity of garenoxacin was much less than that of the other two antimicrobials.

[Attenuation of arbekacin-induced nephrotoxicity in rats by pazufloxacin mesilate].

The protective effect of pazufloxacin (PZFX) mesilate, a parenteral quinolone antimicrobial agent, on arbekacin (ABK)-induced nephrotoxicity was evaluated with 8-week-old male Sprague-Dawley rats. Animals were injected with ABK at a dose of 32 mg/kg intramuscularly, or a combination of ABK in the same manner with PZFX mesilate at a dose of 208 mg/kg (160 mg/kg convert to PZFX, active principle of PZFX mesilate) intravenously once a day for 4 days. In consequent, ABK induced increases in protein, beta 2-microglobulin and N-acetyl-beta-(D)-glucosaminidase in urine, and histopathological phospholipidosis in kidneys. The extent of these changes was reduced when ABK was given in a combination with PZFX mesilate. Renal cortex level of ABK increased after an administration of ABK 1 hour to 4 hours; however, the increase was suppressed by coadministration of PZFX mesilate. Taken together, these results suggest that PZFX mesilate has the protective effect on ABK-induced nephrotoxicity, and that this was attributable to a suppression of uptake of ABK in cortical renal tubules.

Collaborative work on evaluation of ovarian toxicity. 6) Two- or four-week repeated-dose studies and fertility study of cisplatin in female rats.

The main aim of the present study is to determine the optimal administration period of cisplatin with regards to its toxic effects on ovarian morphology in the repeated-dose toxicity study. Cisplatin was administered to female SD rats intraperitoneally once daily at dose levels of 0.25, 0.5, 1.0 and 2.0 mg/kg for 2 weeks, or at dose levels of 0.125, 0.25 and 0.5 mg/kg for 4 weeks in the repeated-dose toxicity study. In the female fertility study, 0.25, 0.5 and 1.0 mg/kg of cisplatin were administered in the same manner from 14 days prior to mating to Day 7 of gestation. In the repeated-dose toxicity study, a decrease in large follicle, an increase in atresia of medium and large follicles, and/or a decrease in currently formed corpus luteum were observed in animals receiving 1.0 and 2.0 mg/kg for 2 weeks, and decreases in small and/or large follicles and an increase in atresia of large follicle were observed in animals receiving 0.25 and 0.5 mg/kg for 4 weeks on the histopathological examination of the ovaries. In the female fertility study, the copulation and fertility indices in the animals receiving 1.0 mg/kg tended to be lower than those in the control animals. In conclusion, histopathological changes in the ovary that were attributable to cisplatin dosing were detected by detailed observation of the ovary in the 2-week study; and therefore, a 2-week administration period is sufficient to evaluate the ovarian toxicity of cisplatin.

Cholangiocytes with mesenchymal features contribute to progressive hepatic fibrosis of the polycystic kidney rat.

The polycystic kidney (PCK) rat is an animal model of Caroli's disease with congenital hepatic fibrosis, in which the mechanism of progressive hepatic fibrosis remains unknown. This study aimed to clarify the mechanism of hepatic fibrosis of the PCK rat from the viewpoint of the contribution of pathological cholangiocytes. In liver sections of the PCK rats, intrahepatic bile ducts were constituted by two different phenotypes: bile ducts lined by cuboidal-shaped and flat-shaped cholangiocytes. The flat-shaped cholangiocytes showed reduced immunohistochemical expression of the biliary epithelial marker cytokeratin 19 and positive immunoreactivity for vimentin and fibronectin. When cultured cholangiocytes of the PCK rat were treated with transforming growth factor (TGF)-beta1, a potent inducer of epithelial-mesenchymal transition, induction of vimentin, fibronectin, and collagen expression occurred in the PCK cholangiocytes. Although the TGF-beta1 treatment reduced cytokeratin 19 expression, the epithelial cell features characterized by the expression of E-cadherin and zonula occludens-1 was maintained, and alpha-smooth muscle actin expression was not induced in the cholangiocytes. Cholangiocytes of the PCK rat may acquire mesenchymal features in response to TGF-beta1 and participate in progressive hepatic fibrosis by producing extracellular matrix molecules, which seems to be a different event from epithelial-mesenchymal transition.

Inhibition of intrahepatic bile duct dilation of the polycystic kidney rat with a novel tyrosine kinase inhibitor gefitinib.

The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli's disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney.

Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease.

Polycystic kidney (PCK) rats exhibit a multiorgan cyst pathology similar to human autosomal recessive polycystic kidney disease, and are proposed as an animal model of Caroli's disease with congenital hepatic fibrosis (CHF). This study investigated the expression and function of selected components of the mitogen activated protein kinase (MAPK) pathway in cultured intrahepatic biliary epithelial cells (BECs) of PCK rats. Compared to the proliferative activity of cultured BECs of control rats, those of the PCK rats were hyperresponsive to epidermal growth factor (EGF). The increase in BEC proliferation was accompanied by overexpression of MAPK/extracellular signal-regulated protein kinase (ERK) kinase 5 (MEK5), and subsequent phosphorylation of ERK5 in vitro. The increased proliferative activity was significantly inhibited by the transfection of short interfering RNA against MEK5 mRNA. An EGF receptor tyrosine kinase inhibitor, gefitinib ("Iressa", ZD1839), also significantly inhibited the abnormal growth of cultured BECs of PCK rats. By contrast, treatment with PD98059 and U0126, inhibitors for MEK1/2, was less effective. These results suggest that the activation of the MEK5-ERK5 cascade plays a pivotal role in the biliary dysgenesis of PCK rats, and also provide insights into the pathogenesis of Caroli's disease with CHF. As the MEK5-ERK5 interaction is highly specific, it may represent a potential target of therapy.