KEAP1-NRF2 complex in ischemia-induced hepatocellular damage of mouse liver transplants

Abstract

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.

Keywords: ARE; H(2)O(2); HIF-1; HKO; HO-1; HRE; IRI; Keap1; Keap1-Nrf2 redox system; Kelch-like ECH-associated protein 1; LDH; Liver ischemia/reperfusion injury; Liver transplantation; Nrf2; OLT; PI3K; TUNEL; Trx1; anti-oxidant response element; heme-oxygenase-1; hepatocyte knockout; hydrogen peroxide; hypoxia inducible factor-1; hypoxia response element; ischemia/reperfusion injury; lactate dehydrogenase; nuclear factor erythroid 2-related factor 2; orthotopic liver transplantation; phosphoinositide 3-kinase; sALT; serum alanine aminotransferase; terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling; thioredoxin 1.