Combined ursodeoxycholic acid/secretin treatment reduces biliary senescence and liver fibrosis in a murine model of late stage primary biliary cholangitis

Abstract

IntroductionLate PBC (Stage III/IV) is characterized by ductopenia, inflammation and bridging fibrosis. UDCA is used to treat PBC, but ~30% of patients are unresponsive. The “bicarbonate umbrella” protects cholangiocytes from toxic bile acids and is mediated by Sct/Sct receptor (SR) activation of cystic fibrosis transmembrane conductance regulator (CFTR) and opening of anion exchange protein 2 (AE2); AE2 expression is reduced in PBC patients. The dominant‐negative transforming growth factor beta receptor II (dnTGFbRII) mouse at 32 wks of age displays some phenotypes of late stage PBC; including, lymphocytic infiltration, serum antimitochondrial antibodies (AMA) and ductopenia. The Sct/SR axis, only expressed by cholangiocytes, promotes biliary proliferation. UDCA modulates Sct/SR signaling in cholestatic models. We aimed to evaluate the therapeutic potential of Sct/UDCA treatment in a mouse model of PBC.MethodsHuman female liver samples were obtained from normal controls, untreated stage I/II (early stage) PBC, untreated stage III/IV (late stage) PBC and UDCA treated late stage PBC patients. Sct/SR/CFTR/AE2 expression was shown by immunostaining. Sct serum levels were assessed by EIA. Female wild‐type (WT) and dnTGFbRII mice at 32 wks of age were treated with vehicle, Sct (2.5 nmoles/kg BW/day), 1% UDCA (275 mmol/day) or Sct/UDCA combination for 1 wk. Sct/SR/CFTR/AE2 axis was shown by immunostaining. Liver damage was evaluated by H&E. Ductular reaction was determined by immunostaining for CK‐19. Senescence was imaged by SA‐b‐Galactosidase activity and p18/CK‐19 double immunostaining. Liver inflammation was evaluated by immunostaining for F4/80 (Kupffer cell marker). Liver fibrosis was assessed by Sirius Red staining and qPCR for collagen‐1a and a‐SMA. Hepatic stellate cell (HSC) activation was evaluated by synaptophysin‐9 staining.ResultsLate stage PBC patients had decreased biliary Sct/SR/CFTR/AE2 expression compared to early stage PBC patients. Late stage PBC patients had reduced serum Sct levels compared to early stage, but Sct levels were restored in UDCA treated advanced stage PBC patients. dnTGFbRII mice had decreased biliary Sct/SR/CFTR/AE2 expression, but mice treated with Sct, UDCA or a combination had restoration of this axis. dnTGFbRII mice treated with Sct, UDCA or a combination had an amelioration of liver injury, ductopenia, inflammation, liver fibrosis and HSC activation compared to controls.ConclusionStimulation of the Sct/SR axis increases bile duct mass and reduces liver injury in a model of late stage PBC via activation of Sct/SR/CFTR/AE2. UDCA may work synergistically with Sct to enhance the bicarbonate umbrella. A combination of Sct/UDCA treatment may be a promising therapeutic for PBC patients with inadequate response to UDCA.Support or Funding InformationNIH NIDDK R01s, VA Merits, IU School of Medicine, Texas A&M University