Hyperglycemia and liver ischemia reperfusion injury: a role for the advanced glycation endproduct and its receptor pathway

Abstract

Although pretransplant diabetes is a risk factor for mortality post-liver transplant, the underlying mechanism has not been fully defined. In a murine liver partial warm ischemia model, we addressed the question of how diabetes/hyperglycemia impacted tissue inflammatory injuries against ischemia reperfusion (IR), focusing on the advanced glycation endproduct (AGE) and its receptor (RAGE) pathway. Our results showed that hepatocellular injury was exacerbated in streptozotocin-induced diabetic mice against IR, in association with hyper-inflammatory immune activation in livers. Serum levels of AGEs, but not HMGB1, were increased in diabetic mice in response to liver IR. Both RAGE antagonist peptides and small interfering RNA alleviated liver injuries and inhibited inflammatory immune activation against IR in diabetic, but not normal, mice. Kupffer cells (KCs)/macrophages, but not hepatocytes, from diabetic mice expressed significantly higher levels of RAGE, leading to their hyper-inflammatory responsiveness to both TLR ligands and AGEs. In vitro, hyperglycemia increased macrophage RAGE expression and enhanced their TLR responses. Our results demonstrated that activation of the AGE-RAGE signaling pathway in KCs was responsible for hyper-inflammatory immune responses and exacerbated hepatocellular injuries in diabetic/hyperglycemic hosts against liver IR.

Keywords: cytokines/cytokine receptors; diabetes; immune/inflammatory; ischemia reperfusion injury (IRI); liver disease; macrophage/monocyte biology: activation.

Figure 1.

Liver IRI in streptozotocin-induced type I diabetic mice. Diabetic (STZ) and control mice were prepared as described in the Materials and Methods. (a) Blood glucose levels were measured at 14 days post 1^st STZ injection prior to the start of liver ischemia experiments. (b) Serum ALT levels in control and diabetic mice after either sham, or 90m liver ischemia and 0, 6, 24h reperfusion; or after 40 or 60m of ischemia and 6h of reperfusion. (c) Representative liver histological (H/E staining) pictures and (d) Average Suzuki scores of the same 90m liver ischemia groups of mice as in (b). Liver partial warm IR was performed as described in the Materials and Methods. Representative results of 3 independent experiments; n=3-4 mice/group, *p<0.05.

Figure 2.

Liver immune activation against IR in diabetic mice. Diabetic (STZ) and control mice were prepared and liver partial warm IR was performed as described in the Materials and Methods. IR liver tissues and serum samples were collected from control and diabetic mice after either sham operation, or 90m liver ischemia and 0, 6, 24h reperfusion. (a) Liver inflammatory gene expressions were measured by qRT-PCR, and (b) Serum cytokine levels by ELISA. Average target gene/HPRT ratios and cytokine levels of each experimental groups were plotted. Representative results of 2 independent experiments; n=3 mice/group, *p<0.05.

Figure 3.

The activation of the AGE-RAGE pathway by IR in diabetic mice. Diabetic (STZ) and control mice were prepared and liver partial warm IR was performed as described in the Materials and Methods. IR liver tissues and serum samples were collected from control and diabetic mice after either sham operation, or 90m liver ischemia and 0, 6, 24h reperfusion. (a) RAGE, NOD1 and TLR4 protein expressions in IR liver tissues were measured by Western blot analysis (2 samples/group). (b) RAGE gene expressions in IR liver tissues were determined by qRT-PCR (ratios of target gene/HPRT). AGE levels in liver tissues (c) and sera (d) were measured by ELISA. (e) HMGB1 levels in sera in sham or liver IR mice were measured by ELISA. Representative results of 2 separate experiments; n=3 mice/group, *p<0.05.

Figure 4.

Alleviation of liver IRI by RAGE antagonist peptides (RAP) in diabetic mice. Diabetic (STZ) and control mice were prepared as described in the Materials and Methods. RAGE antagonistic peptides were administered in groups of diabetic and control mice prior to the start of liver ischemia. Liver IRI was measured at 6h by (a) sALT, (b) liver histology with Suzuki scores, (c) liver tissue MPO activities, and (d) liver inflammatory gene inductions (Target gene/HPRT ratios). Representative results of 2 separate experiments; n=3 mice/group, *p<0.05.

Figure 5.

Alleviation of liver IRI in diabetic mice by RAGE siRNA. Diabetic (STZ) mice were prepared as described in the Materials and Methods. Non-specific (NS) or RAGE-specific siRNA were first transfected in macrophages in vitro and RAGE protein expressions were determined by Western blot (a). In vivo, NS or RAGE-specific siRNA was administered in separate groups of diabetic mice prior to the start of liver ischemia. Liver RAGE gene expressions were determined by qRT-PCR (b). Liver IRI was measured at 6h post reperfusion by sALT (c), liver tissue MPO activities (d), liver histology with Suzuki scores (e), and liver inflammatory gene inductions (f). Representative results of 2 separate experiments; n=3 mice/group, *p<0.05.

Figure 6.

RAGE expressions and functions in KCs/macrophages. KCs were isolated from control and diabetic (STZ) mice. RAGE, NOD1 and TLR4 protein expressions were measured by Western blots (a). KCs isolated from Ctl. or STZ mice were stimulated in vitro with either LPS or NECML, as described in the Materials and Methods. Cytokines in culture supernatants were measured by ELISA 24h after stimulation (b). Peritoneal macrophages were isolated from control or diabetic mice and stimulated in vitro with low doses of LPS (1 or 10ng/ml), or AGE-BSA (10-100μg/ml), or both. TNF-α productions in culture supernatants were measured by ELISA at 24h (c). Peritoneal macrophages were transfected with either non-specific (NS) or RAGE siRNA, or treated with RAP, as described in the Materials and Methods, followed by the stimulation of LPS. TNF-α and IL-6 productions in culture supernatants were measured by ELISA at 24h (d) Representative results of 2 separate experiments; n=3/group, *p<0.05.

Figure 6.

RAGE expressions and functions in KCs/macrophages. KCs were isolated from control and diabetic (STZ) mice. RAGE, NOD1 and TLR4 protein expressions were measured by Western blots (a). KCs isolated from Ctl. or STZ mice were stimulated in vitro with either LPS or NECML, as described in the Materials and Methods. Cytokines in culture supernatants were measured by ELISA 24h after stimulation (b). Peritoneal macrophages were isolated from control or diabetic mice and stimulated in vitro with low doses of LPS (1 or 10ng/ml), or AGE-BSA (10-100μg/ml), or both. TNF-α productions in culture supernatants were measured by ELISA at 24h (c). Peritoneal macrophages were transfected with either non-specific (NS) or RAGE siRNA, or treated with RAP, as described in the Materials and Methods, followed by the stimulation of LPS. TNF-α and IL-6 productions in culture supernatants were measured by ELISA at 24h (d) Representative results of 2 separate experiments; n=3/group, *p<0.05.

Figure 7.

The impact of hyperglycemia on macrophages in vitro. BMMs were differentiated under low or high glucose conditions, as described in the Materials and Methods. These cells were stimulated with LPS (100ng) for 24h and cytokine levels in culture supernatants were measured by ELISA (a). Peritoneal macrophages were cultured overnight in low glucose condition. RAGE induction by hyperglycemia (by changing to high glucose media) was determined by Western blot (b).