Accumulating bile salts promote liver fibrosis in cholestasis: in vivo evidence in a mouse model

Background:

Despite countless efforts over the past decades, the pathogenesis of liver fibrosis in chronic cholestatic liver disease such as Primary Biliary Cholangitis (PBC) remains elusive. Since the 1970ies, it has been an accepted concept that hydrophobic bile salts accumulating during cholestasis promote liver fibrosis. However, experimental evidence to support this view remains scarce. No animal model exists to demonstrate cholestasis-induced liver fibrosis. Genetically cholestatic animal models, e.g. by knockout of bile salt export pump (BSEP), do not develop liver fibrosis and in the most widely used cholestatic animal model, the multidrug resistance protein 2 knockout (Mdr2-/-), both cholestasis and fibrosis are secondary to biliary inflammation and sclerosis. Therefore, we tested the hypotheses (i) that bile salts can induce a pro-fibrotic response in vitro and (ii) that chronic, hepatocellular cholestasis leads to liver fibrosis in vivo.

Methods:

Hepatic stellate cells were isolated from wild type mouse livers and stimulated with hydrophobic and hydrophilic bile salts. Activation (expression of aSMA as determined by western blotting), proliferation (BrdU assays) and cell number (absolute cell count per cm2) were determined. Atp8b1-/- mice were subjected to cholate feeding to induced chronic cholestasis and measures of liver fibrosis were determined.

Results:

In primary murine HSC, we have identified a EGFR- and MEK1/2-dependent pro-proliferative effect of hydrophobic bile salts, as determined by BrdU assays. This resulted in an increased number of myofibroblasts during culture. In contrast, hydrophilic bile salts did not result in increased numbers of myofibroblasts. In vivo, we utilized the Atp8b1-/- mouse model, in which cholestasis can be triggered by feeding of cholate. We induced cholestasis chronically, as evidenced by increased serum alkaline phosphatase and bilirubin levels. Liver fibrosis was evidenced by increased collagen deposition in the liver and increased spleen size as a surrogate of portal hypertension.

Conclusion:

In summary, we provide evidence that cholestasis per se, independently of an inflammatory trigger, can induce and promote liver fibrosis in vivo.