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Use of a Hanging-weight System for Liver Ischemia in Mice
Published on: August 7, 2012
KEAP1-NRF2 complex in ischemia-induced hepatocellular damage of mouse liver transplants
Bibo Ke1, Xiu-Da Shen, Yu Zhang
1Dumont-UCLA Transplant Center, Department of Surgery, Division of Liver and Pancreas Transplantation, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States.
Background & Aims:
The Keap1-Nrf2 signaling pathway regulates host cell defense responses against oxidative stress and maintains the cellular redox balance.
Methods:
We investigated the function/molecular mechanisms by which Keap1-Nrf2 complex may influence liver ischemia/reperfusion injury (IRI) in a mouse model of hepatic cold storage (20h at 4°C) followed by orthotopic liver transplantation (OLT).
Results:
The Keap1 hepatocyte-specific knockout (HKO) in the donor liver ameliorated post-transplant IRI, evidenced by improved hepatocellular function and OLT outcomes (Keap1 HKO→Keap1 HKO; 100% survival), as compared with controls (WT→WT; 50% survival; p<0.01). By contrast, donor liver Nrf2 deficiency exacerbated IRI in transplant recipients (Nrf2 KO→Nrf2 KO; 40% survival). Ablation of Keap1 signaling reduced macrophage/neutrophil trafficking, pro-inflammatory cytokine programs, and hepatocellular necrosis/apoptosis, while simultaneously promoting anti-apoptotic functions in OLTs. At the molecular level, Keap1 HKO increased Nrf2 levels, stimulated Akt phosphorylation, and enhanced expression of anti-oxidant Trx1, HIF-1α, and HO-1. Pretreatment of liver donors with PI3K inhibitor (LY294002) disrupted Akt/HIF-1A signaling and recreated hepatocellular damage in otherwise IR-resistant Keap1 HKO transplants. In parallel in vitro studies, hydrogen peroxide-stressed Keap1-deficient hepatocytes were characterized by enhanced expression of Nrf2, Trx1, and Akt phosphorylation, in association with decreased release of lactate dehydrogenase (LDH) in cell culture supernatants.
Conclusions:
Keap1-Nrf2 complex prevents oxidative injury in IR-stressed OLTs through Keap1 signaling, which negatively regulates Nrf2 pathway. Activation of Nrf2 induces Trx1 and promotes PI3K/Akt, crucial for HIF-1α activity. HIF-1α-mediated overexpression of HO-1/Cyclin D1 facilitates cytoprotection by limiting hepatic inflammatory responses, and hepatocellular necrosis/apoptosis in a PI3K-dependent manner.
Insights
The Keap1-Nrf2 pathway protects against liver transplant injury. Blocking Keap1 enhances Nrf2, reducing inflammation and cell death, improving transplant outcomes.
Area of Science:
- Hepatology
- Transplantation Immunology
- Molecular Biology
Background:
- The Keap1-Nrf2 pathway is crucial for cellular defense against oxidative stress and redox balance.
- Liver ischemia/reperfusion injury (IRI) is a significant complication in liver transplantation.
Purpose of the Study:
- To investigate the role of the Keap1-Nrf2 signaling pathway in liver IRI during orthotopic liver transplantation (OLT).
- To elucidate the molecular mechanisms by which Keap1 influences IRI and OLT outcomes.
Main Methods:
- Utilized a mouse model of hepatic cold storage followed by OLT.
- Generated Keap1 hepatocyte-specific knockout (HKO) and Nrf2 knockout (KO) mouse models.
- Assessed hepatocellular function, survival rates, inflammatory markers, and molecular signaling pathways (Akt, HIF-1α, HO-1, Trx1).
Main Results:
- Donor liver Keap1 HKO significantly ameliorated post-transplant IRI, improving OLT outcomes and survival rates (100% vs. 50% in controls).
- Conversely, donor liver Nrf2 deficiency exacerbated IRI (40% survival).
- Keap1 ablation reduced inflammation and apoptosis, while promoting anti-apoptotic functions, by increasing Nrf2, Akt phosphorylation, and antioxidant/cytoprotective gene expression (Trx1, HIF-1α, HO-1).
Conclusions:
- The Keap1-Nrf2 complex negatively regulates Nrf2 signaling, protecting against oxidative injury in IR-stressed OLTs.
- Nrf2 activation, via Keap1 inhibition, promotes PI3K/Akt and HIF-1α activity, leading to HO-1 and Cyclin D1 overexpression.
- This pathway limits hepatic inflammation and hepatocellular damage in a PI3K-dependent manner, enhancing cytoprotection during OLT.
