Severe burn-induced endoplasmic reticulum stress and hepatic damage in mice

Abstract

Severe burn injury results in liver dysfunction and damage, with subsequent metabolic derangements contributing to patient morbidity and mortality. On a cellular level, significant postburn hepatocyte apoptosis occurs and likely contributes to liver dysfunction. However, the underlying mechanisms of hepatocyte apoptosis are poorly understood. The endoplasmic reticulum (ER) stress response/unfolded protein response (UPR) pathway can lead to hepatocyte apoptosis under conditions of liver dysfunction. Thus, we hypothesized that ER stress/UPR may mediate hepatic dysfunction in response to burn injury. We investigated the temporal activation of hepatic ER stress in mice after a severe burn injury. Mice received a scald burn over 35% of their body surface and were killed at 1, 7, 14, and 21 d postburn. We found that severe burn induces hepatocyte apoptosis as indicated by increased caspase-3 activity (P < 0.05). Serum albumin levels decreased postburn and remained lowered for up to 21 d, indicating that constitutive secretory protein synthesis was reduced. Significantly, upregulation of the ER stress markers glucose-related protein 78 (GRP78)/BIP, protein disulfide isomerase (PDI), p-protein kinase R-like endoplasmic reticulum kinase (p-PERK), and inositol-requiring enzyme 1alpha (IRE-1alpha) were found beginning 1 d postburn (P < 0.05) and persisted up to 21 d postburn (P < 0.05). Hepatic ER stress induced by burn injury was associated with compensatory upregulation of the calcium chaperone/storage proteins calnexin and calreticulin (P < 0.05), suggesting that ER calcium store depletion was the primary trigger for induction of the ER stress response. In summary, thermal injury in mice causes long-term adaptive and deleterious hepatic function alterations characterized by significant upregulation of the ER stress response.

Figure 1

Burn injury causes hepatic damage and liver dysfunction. (A) Caspase-3 activity in liver cytosolic fractions after burn injury, as determined by caspase-3 activity assay. (B) The expression of liver-secreted albumin after burn injury. Time after injury (in d) is indicated. Data represent the mean ± SEM. ^*P < 0.05, value versus control for each time point after burn injury pooled from four control and six burned animals at each time point.

Figure 2

ER chaperone levels associated with the UPR are increased 24 h after a burn injury in mice. The expression of GRP78/BIP, PDI, calnexin, and calreticulin among the normal (n = 3), sham (n = 3), and burned (n = 3) groups normalized to GAPDH. Data are the mean ± SEM. ^*P < 0.05, burned versus normal. ^†P < 0.05, burned versus sham pooled from three animals at each time point.

Figure 3

Time course of activation of the canonical ER stress pathway after burn injury. Protein levels of (A) p-PERK, (B) p-IRE-1, (C) cleaved ATF6, and (D) CHOP during the postburn time course. Protein levels are normalized to GAPDH. Data represent the mean ± SEM. ^*P < 0.05, value versus control for each time point after burn injury pooled from four control and six burned animals at each time point.

Figure 4

ER chaperone levels are increased up to 21 d after burn injury. Protein expressions of (A) GRP78/BIP, (B) PDI, (C) calreticulin, and (D) calnexin during the postburn time course. Protein levels are normalized to GAPDH. Data represent the mean ± SEM. ^*P < 0.05, value versus control for each time point after burn injury pooled from four control and six burned animals at each time point.