Effects And Mechanisms Of Rifampicin Induced Bicarbonate-rich Choleresis In Rats

Rifampicn is one of the most widely used anti-tubercular drugs. Our previous results have shown that rifampicin induced liver cholestasis adaptive reaction in mice. However, studies have shown that rifampicin-induced choleresis in vivo and isolated perfused rat liver, these studies suggest that RIF may play a beneficial role in pathophysio logical statuses in which bile secretion is impaired; however, the mechanisms of choleresis have not been well described. The aim of this study was to investigate the mechanisms underlying in vivo rifampicin-induced choleresis.1. Rifampicin induced choleresis in chronic treatment rats and its molecular mechanismRats were treated with an oral gavage of RIF at doses of 50,100 or 250 mg/kg body wt for 1,3 or 7 days, respectively. The common bile duct was cannulated using a PE-10 catheter. Bile was collected for a further 60-min period in pre-weighed vials. At the end of each experiment, the rats were sacrificed to obtain serum and liver samples for the subsequent studies.Results showed that there was a marked increase in bile flow after chronic treatment with RIF (50 mg/kg, day 7; 100 and 250 mg/kg, days 1,3 and 7) (P<0.05) Biliary TBA and GSH outputs were not altered, whereas the HCO3- output was markedly increased (50 mg/kg, day 7; 100 and 250 mg/kg, days 1,3 and 7) (P<0.01), suggesting that increased biliary HCO3-output is a major factor in choleresis. Serum parameters showed that The levels of ALT (250 mg/kg, days 1,3 and 7), ALP (100 mg/kg, days 3; 250 mg/kg, days 3 and 7) and TBA (100 mg/kg, days 3 and 7; 250 mg/kg, days 1,3 and 7) were significantly increased (P<0.05), the levels of AST was not altered.Because high doses of RIF (250 mg/kg) noticeably increased ALT, we used the 100 mg/kg treatment dose of RIF for the subsequent studies in the chronic treatment group. Because the change in biliary components likely resulted from the altered expression and location of the transporters, we further studied mRNA and protein expression and the location of the main hepatic export transporters using real time-PCR, Western blotting and immuno fluorescence, respectively. Mrp2 mRNA was significantly increased compared with control on day 7 (P<0.05), whereas Mrp2 protein levels did not change. There was no change in Bsep mRNA levels. Although Bsep mRNA expression was not influenced by RIF, its protein level was significantly increased compared with control on days 1,3 and 7 (P<0.05), respectively. AE2 mRNA level tended to increase in response to RIF, although it did not reach the level of statistical significance. AE2 protein level increased to 284,231 and 216% that of the control on days 1,3 and 7, respectively, suggesting increased HCO3" excretion due to a increased AE2-mediated biliary excretion of HCO3". The tissue distributions of Bsep, Mrp2 and AE2 were examined by immunofluorescence after chronic treatment with RIF. The images demonstrated that Bsep, Mrp2 and AE2 were mainly localized in the liver plasma membrane.2. Rifampicin induced choleresis in acute infused rats and its molecular mechanismThe common bile duct and iliac vein were cannulated separately. After 10 min of equilibration, different doses (0.5,1,2.5,5 and 20 mg/ml) of RIF were continuously infused (1.5 ml/h) through the iliac vein. Twenty minutes after the RIF infusion, bile was collected for the subsequent studies.Infused rats with 0.5 mg/kg dose of RIF, bile flow and HCO3- output were not altered; infused rats with 1,2.5 and 5 mg/kg dose of RIF, bile flow and HCO3- output were significantly increased compared with control (P<0.05); whereas, infused rats with 20 mg/kg dose of RIF, bile flow and total bile acids output were significantly decreased compared with control (P<0.05). Biliary GSH output was not affected by the RIF infusion. High-performance liquid chromatography results showed that the amount of RIF in the bile significantly increased with the increase in the infused dose, but was not directly associated with increased bile flow, suggesting that the increased bile flow is not dependent on the RIF content in bile. Because 1 mg/ml RIF infused dose caused higher bile flow compared with other infused doses, we chose this infused dose for the subsequent studies in the acute infused treatment group.Next, we explore whether rifampicin induced bicarbonate-rich choleresis involvement of cholehepatic shunt. The portal vein was cannulated, infused with a single bolus of RIF or control (negative control:cholyltaurine; positive control:UDCA). Results showed that the trend of bile flow was induced by infused with a single bolus of RIF was almostly concordant with the trend of bile flow was induced by infused with a single bolus of cholyltaurine, suggesting that rifampicin induced bicarbonate-rich choleresis did not dependent on cholehepatic shunt.In order to further explore the choleretic mechanism, we performed the bile duct retrograde fluxes systerm. The common bile duct and iliac vein were cannulated. After a bile stabilization period of 10 min, intrabiliary solutions (0.2 ml) with 0.5 mmol/1 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS) were administered with retrograde fluxes system and closed bile cannula. Meanwhile,1 mg/ml doses of RIF were continuously infused (1.5 ml/h) through the iliac vein. Twenty minutes after the RIF infusion, opened bile cannula, and bile was collected in pre-weighed vials.Results showed that DIDS led to a significantly inhibition of the initial RIF-induced increase in bile flow compared with that of RIF-stimulated rats without DIDS (P<0.01). Similar to the effect on the bile flow, the effect of RIF on biliary HCO3- excretion was significantly inhibited by DIDS (P<0.01). These results indicate that increased bile flow involved of increased AE2 activity in cholangiocytes.In conclusion, (1) Rifampicin increased bile flow in normal rats. Increased biliary HCO3- output is the main cause of RIF-induced choleresis. (2) Increased bile flow is not associated with the amount of RIF in bile. (3) Rifampicin induced bicarbonate-rich choleresis did not dependent on cholehepatic shunt. (4) Rifampicin induce Ae2 activity in cholangiocytes, since we found DIDS primarily blocked the rifampicin-stimulated bile flow and HCO3- output.