Ischemia–reperfusion injury in rat steatotic liver is dependent on NFκB P65 activation

doi:10.1016/j.trim.2012.01.001

Transplant Immunology

Volume 26, Issue 4, June 2012, Pages 201-206

https://doi.org/10.1016/j.trim.2012.01.001 Get rights and content

Abstract

Background

Steatotic liver grafts tolerate ischemia–reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury.

Methods

We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury.

Results

I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p < 0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58 ± 0.18 vs. 1.37 ± 0.06 O.D./min/10 μg protein, p < 0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106 ± 17.5 vs. 443.3 ± 49.9 vs. 176 ± 10.6 pg/mL, p = 0.02), TNF-α (223.8 ± 29.9 vs. 518.5 ± 66.5 vs. 264.5 ± 30.1 pg/2 μg protein, p = 0.003) and IL-1β (6.0 ± 0.91 vs. 19.8 ± 5.2 vs. 5 ± 1.7 pg/10 μg protein, p = 0.02) along with a significant reduction in ALT levels (715 ± 71 vs. 3712.5 ± 437.5 vs. 606 ± 286 U/L, p = 0.01).

Conclusion

These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.

Highlights

► Steatotic livers exhibit exacerbated I/R injury. ► I/R injury in steatotic liver results in increased activation of NF-κB p65 subunit. ► Proteasome inhibition in steatotic liver reduces NFκB p65 activation and I/R injury. ► Bortezomib reduces expression of inflammatory cytokines in steatotic and lean liver.

Introduction

Liver transplantation is an effective treatment option for a variety of end stage liver diseases. Though the number of patients on waiting lists for liver transplantation has increased the number of organs available for transplantation has remained stagnant. Hepatic steatosis remains one of the major reasons accounting for liver organ discards.

Hepatic steatosis, especially macrosteatosis, is associated with increased rates of primary graft non-function and operative mortality post-transplantation [1], [2], [3], [4]. Transplant centers routinely discard 25% of the grafts offered that exhibit greater than 30% macrosteatosis, and nearly all decline grafts with > 50% steatosis [5]. It has been hypothesized that steatotic grafts are particularly vulnerable to I/R injury. However the mechanisms that contributes to increased susceptibility of steatotic grafts to I/R injury remains poorly defined. In this study we transplanted livers from lean and obese Zucker rats following 2 h of cold storage into lean recipients and examined at 2 and 24 h post transplantation to define the underlying mechanisms that contribute to the enhanced I/R injury. We utilized a short cold ischemia time to define the signaling events since steatotic livers exhibit severe I/R injury and high mortality.

Transcription factor NF-κB is central in mediating hepatic I/R injury through increased expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8), cell adhesion molecules (ICAM-1 and VCAM-1) and acute phase proteins [6], [7]. Increased levels of TNF-α and IL-1β in circulation induces necrosis in several in vitro and in vivo models. Fatty livers have also been shown to develop massive necrosis following I/R insults [8], whereas lean livers display apoptosis. In the current study we used the Leptin deficient Zucker rat model which exhibits relatively uniform steatosis to test our hypothesis. Macrovesicular steatosis is the major reason for discarding potential livers for transplantation. Previous studies have shown that animals in the Zucker model develop macrovesicular steatosis that closely resembles the clinical setting and was used for this study. Moreover, the mechanisms that mediate I/R injury are similar in the Zucker rat and the dietary induced model. Based on its key role in mediating I/R injury, we postulated that activation of the NF-κB inflammatory pathway might be a major mechanism responsible for the altered pathophysiology observed in the steatotic liver. We further hypothesized that inhibition of NF-κB activation might improve results in steatotic liver grafts by decreasing acute inflammation and attenuating I/R injury. In this study we demonstrate that transplantation of steatotic liver results in increased activation of NF-κB resulting in a significant increase in levels of pro-inflammatory cytokines TNF-α, IL-1β and MIP-2 that are associated with necrosis of transplanted steatotic liver and increased serum transaminases. Bortezomib (PS-341), a selective inhibitor of 26S proteasome is an FDA approved drug for NF-κB inhibition, has been successfully employed to treat multiple myeloma and mantle cell lymphoma. PS-341 mediated blockade of NF-κB activation has been shown to induce immunomodulatory changes that inhibit the functions of CD4 T cells, dendritic cells, and plasma cells through the modulation of cytokine secretion (IFN-g, TNF-a, IL-6, IL-5, SDF-1 etc.). We had selected PS-341 to use in this study because of its significant effect of apoptosis and immune functions, the two major contributors of I/R injury. Using the proteasome inhibitor, bortezomib we further demonstrate that donor pretreatment results in significant reduction in levels of activated NF-κB, leading to decreased pro-inflammatory cytokines. We also demonstrate markedly improved histology in transplanted steatotic grafts, without evidence of necrosis, and improvement in serum transaminase levels.

Section snippets

Objective

The overall objective of the study is to define the early molecular events that contribute to the exacerbated I/R injury in the steatotic liver following transplantation and to determine the ability of NF-κB inhibition to ameliorate I/R injury in the steatotic liver transplantation model.

Animal model

Zucker rats represent a well-characterized model of leptin receptor deficiency induced obesity [9], [10], [11], [12] and were used in this study. Animals were housed in pathogen-free conditions with 12-hour diurnal light cycle and access to standard rodent chow and water ad libitum. Surgical procedures were performed under aseptic conditions approved by the Washington University Animals Studies Committee and in accordance with the National Institutes of Health guidelines in “The Guide for the

Activated NFκB p65 subunit is significantly increased in steatotic grafts

Analysis of the steatotic and lean liver grafts demonstrated a significant increase in NF-κB activation at both 2 and 24 h post-reperfusion in steatotic grafts (Fig. 1A lanes1 and 4) compared to lean. Activation was markedly decreased by donor treatment with bortezomib (Fig. 1A, lanes 2 and 5), resulting in levels comparable to lean grafts.

Analysis of individual members of NF-κB family using a NF-κB profile ELISA revealed that NFκB activation in steatotic grafts was due to the p65 subunit (Fig. 1

Discussion

Hepatic steatosis remains one of the major impediments in the under-utilization of marginal livers for transplantation. Hepatic steatosis and its associated higher rates of primary graft non-function, morbidity and mortality are increasingly complicating the current practice of liver surgery and transplantation [2], [3], [15], [16], [17]. The lack of understanding of the mechanisms of injury in the steatotic liver results in the inability to initiate specific therapeutic targeting of the

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It has been reported that activation of p65 stimulates downstream genes (interleukin-1β [IL-1β], IL-6 and tumor necrosis factor-α [TNF-α]), which induces leukocytes infiltration and cytokine secretion to further promote the inflammatory response.7 Activation of the NF-κB pathway increased expression of several proinflammatory cytokines, including macrophage inflammatory protein-2, IL-1β, and TNF-α, leading to exacerbation of I/R injuries in rat liver transplants.8 Moreover, the central subunit of the NF-κB signaling pathway, p65, could induce expression of apoptosis-related proteins and further exacerbate the progression of renal tubule injury and eventual cell death.9
Renal ischemia/reperfusion (I/R)-induced acute kidney injury remains to be a troublesome condition in clinical practice. Although the exact molecular mechanisms underlying renal I/R injury are incompletely understood, the deleterious progress of renal I/R injury involves inflammation, apoptosis, and oxidative stress. Diosmetin is a member of the flavonoid glycosides family, which suppresses the inflammatory response and cellular apoptosis and enhances antioxidant activity. The purpose of this study was to investigate the protective effect of diosmetin on I/R-induced renal injury in mice.
Thirty BALB/c mice were randomly divided into five groups. Four groups of mice received diosmetin (0.25, 0.5, and 1 mg/kg) or vehicle (I/R group) before ischemia. Another group received vehicle without ischemia to serve as a negative control (sham-operated group). Twenty-four hours after reperfusion, serum and renal tissues were harvested to evaluate renal function and histopathologic features. In addition, the expression of inflammation-related proteins, apoptotic molecules, and antioxidant enzymes was analyzed.
Compared with sham mice, the I/R group significantly exacerbated renal function and renal tube architecture and increased the inflammatory response and renal tubule apoptosis. Nevertheless, pretreatment with diosmetin reversed these changes. In addition, diosmetin treatment resulted in a marked increase in antioxidant protein expression compared with I/R mice.
The renoprotective effects of diosmetin involved suppression of the nuclear factor-κB and mitochondrial apoptosis pathways, as well as activation of the nuclear factor erythroid 2–related factor 2/heme oxygenase-1 pathway. Diosmetin has significant potential as a therapeutic intervention to ameliorate renal injury after renal I/R.

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^☆: Financial support: This work was supported by NIH P01 HL66196, DDRCC P30 DK052574 (WC) and NIH DK065982 (TM).

¹: Equal contribution by SR and JL.

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Ischemia–reperfusion injury in rat steatotic liver is dependent on NFκB P65 activation☆

Abstract

Background

Methods

Results

Conclusion

Highlights

Introduction

Section snippets

Objective

Animal model

Activated NFκB p65 subunit is significantly increased in steatotic grafts

Discussion

Lancet

Hepatology

Prog Lipid Res

Hepatology

J Gastrointest Surg

J Hepatol

J Hepatol

Hepatology

Cell

Gastroenterology

The outcome of steatotic grafts in liver transplantation

Transplant Proc

Use of fatty donor liver is associated with diminished early patient and graft survival

Transplantation

Primary nonfunction of hepatic allografts with preexisting fatty infiltration

Transplantation

The predictive value of donor liver biopsies on the development of primary nonfunction after orthotopic liver transplantation

Transplant Proc

Essential role for nuclear factor kappaB in ischemic preconditioning for ischemia–reperfusion injury of the mouse liver

Transplantation

Dual role of tumor necrosis factor-alpha in hepatic ischemia–reperfusion injury: studies in tumor necrosis factor-alpha gene knockout mice

Hepatology

The Zucker-fatty rat: a review

Fed Proc

Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor

Science