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.

Figure 1

Hepatocyte-specific Keap1 deficiency (HKO) ameliorates hepatic IRI in OLTs. Donor livers were stored in UW solution (4 C) for 20h prior to the transplant. Experimental groups: (a; □) sham; (b; ) WT WT; (c; ) Keap1HKO Keap1HKO; (d; ) Nrf2KO Nrf2KO. (A) sALT levels. Mean SD; n=4 mice/group. *p<0.05; **p<0.01; ***p<0.0005. (B) Representative OLT histology (H&E; magnification x100); (C) Suzuki's histological grading of IRI (24h). Mean SD; n=4-6 mice/group. *p<0.05, **p<0.001. (D) OLT survival: (■) Keap1HKO Keap1HKO (100%); (◆) WT WT (50%); (▲) Nrf2KO Nrf2KO (40%; p<0.01). N=10 mice/group.

Figure 2

Hepatocyte-specific Keap1 deficiency (HKO) reduces macrophage and neutrophil trafficking/activation in IR-stressed OLTs: (a; ) WT WT; (b; ) Keap1HKO Keap1HKO; (c; ) Nrf2KO Nrf2KO. (A/B) Immunohistochemical staining for CD68+ cells. Representative of 4 mice/group; magnification ×400; *p<0.005, **p<0.0001. (C) Quantitative RT-PCR-assisted cytokine/chemokine gene expression; Mean±SD; n=3-4/group; *p<0.05, **p<0.005. (D/E) Immunohistochemical staining for LY6G+ cells. Representative of 4 mice/group; magnification ×400; *p<0.001, **p<0.0001. (F) Neutrophil MPO activity (U/gm). Mean±SD; n=3-4/group; *p<0.05, **p<0.01. (G) Quantitative RT-PCR-assisted detection of CXCL-1. Mean±SD; n=3-4/group; *p<0.05, **p<0.01.

Figure 3

Hepatocyte-specific Keap1 deficiency (HKO) promotes anti-apoptotic functions, reduces apoptosis and activates Nrf2-mediated Trx1/Akt/HIF-1 in IR-stressed OLTs. (A) Western analysis of cleaved caspase-3 and Bcl-2/Bcl-xl. Representative of three experiments. (B). Caspase-3 activity. Mean±SD; n=3-4/group; *p<0.005; **p<0.0005. (C/D) TUNEL staining: (a; ) WT WT; (b; ) Keap1HKO Keap1HKO; (c; ) Nrf2KO Nrf2KO. Representative of 4 mice/group; magnification ×200 *p<0.05; **p<0.001. (E) Western analysis of Keap1, Nrf2, Trx1, p-Akt, HIF-1 , and HO-1 in OLTs. β-actin served as an internal control. Representative of three experiments. (F) Quantitative RT-PCR-assisted detection of mRNA coding for Trx1, Nqo1 and Gclc. Data were normalized to HPRT gene expression. Mean±SD; n=3-4/group; *p<0.05, **p<0.01.

Figure 4

Inhibition of PI3K disrupts Akt/HIF-1 signaling and recreates liver IRI in Keap1HKO OLTs. Groups of WT or Keap1HKO liver donor mice were pre-treated with Ly294002 or DMSO (-1 h). (A) sALT levels (IU/L): (□) sham; () WT+DMSO; () WT+ Lly294002; () Keap1 HKO+DMSO; () Keap1 HKO+Ly294002. Mean±SD; n=4 mice/group; *p<0.05, **p<0.01. (B) Western analysis of p-Akt and HIF-1 in OLTs. β-actin served as an internal control. Representative of three experiments. (C) Representative H&E staining of OLTs (n=4) at 24h: Panel (a) WT+DMSO; (b) WT+Ly294002; (c) Keap1HKO+DMSO; (d) Keap1HKO+Ly294002 (magnification x100). Keap1-dependent Nrf2 activation promoted Trx1/Akt/HIF-1 signaling in mouse hepatocytes in vitro. (D) Western blot expression of Nrf2, Trx1 and p-Akt in primary H₂O_2-stressed hepatocyte (WT, Keap1HKO or Nrf2KO) cultures. Representative of three experiments. (E) Primary H₂O_2-stressed hepatocytes were pretreated with PI3K inhibitor (LY294002) or DMSO; p-Akt/HIF-1 expression was analyzed by Western blots. Representative of three experiments. (F) Quantitative RT-PCR-assisted detection of HIF-1 , HO-1, and Cyclin D1 in LY294002/DMSO-pretreated H₂O_2-stressed hepatocyte cultures. (G) LDH release (U/L) in LY294002/DMSO-treated hepatocytes. (F-G): (□) WT cells; () WT cells+DMSO; () WT cells+Ly294002; () Keap1 HKO cells; () Keap1 HKO cells+DMSO; () Keap1 HKO cells+Ly294002. Mean±SD; n=3-4/group; *p<0.005, **p<0.0005.