BACKGROUND: Hepatic steatosis is associated with significant morbidity and mortality after transplantation. Autophagy was known as a protective response to ischemia and reperfusion (I/R) injury. We investigated the role of autophagy in the steatosis dependence of sensitivity to I/R injury.

METHODS: Hepatocytes and livers from normal and genetic(ob/ob mice)/dietary models of nonalcoholic steatohepatitis (NASH) were subjected to in vitro and in vivo I/R, respectively. We analyzed changes in autophagy-related proteins (Atg). Autophagy dysfunction was visualized using confocal and intravital multi-photon microscopy of isolated hepatocytes and livers, respectively.

RESULTS: Quantitative immunohistochemistry for donor liver biopsy at 1 hour after reperfusion showed the decrement of autophagy maker LC3 significantly correlates with degree of steatosis and poor survival of liver transplantation recipients. Western blot and imaging analyses associated the increase in sensitivity to I/R injury with NASH livers duo to severe autophagyic flux impairment . Autophagy associated proteins screening showed Atg3 and Atg7 was dramatically decreased during I/R in NASH livers as the result of Calpain 2 mediated cleavage. Calpain 2 inhibition or knockdown enhance autophagy and suppressed cell death during NASH livers I/R. Further point mutation experiments revealed that cleavage sites was at Atg3δ92-97 and Atg7δ344-349, respectively. Overexpression of Atg3 or Atg7 increased autophagy and suppressed cell death after I/R in NASH livers.

CONCLUSIONS: Calpain 2 mediated degradation Atg3 and Atg7 in fatty livers increases their sensitivity to I/R injury. Increasing autophagy might ameliorate fatty liver damage and represent a valuable method to expand the liver donor pool.

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