Inhibition of inducible nitric oxide synthase prevents mitochondrial damage and improves survival of steatotic partial liver grafts

Abstract

Background: Steatotic liver grafts are excluded for partial liver transplantation because of increased risk of primary nonfunction. Mechanisms underlying the failure of fatty partial liver grafts (FPG) remain unknown. This study investigated whether inducible nitric oxide synthase (iNOS) plays a role in failure of FPG.

Methods: Fatty livers were induced by feeding rats a high-fat high-fructose diet for 2 weeks. Hepatic triglyceride was approximately 9-fold higher in rats fed the high-fat high-fructose diet than those fed a low-fat low-fructose diet. Lean and fatty liver explants were reduced in size ex vivo to approximately one third, stored in the University of Wisconsin cold storage solution for 2 hr, and implanted.

Results: Posttransplantational hepatic iNOS expression and reactive nitrogen species (RNS) formation (nitrite and nitrate levels and 3-nitrotyrosine adducts) increased more profoundly in FPG than in lean partial grafts (LPG). Serum alanine aminotransferase and bilirubin were 2- and 5.5-fold higher after transplantation of FPG than LPG. 5-Bromo-2'-deoxyuridine incorporation was 25% in LPG but only 5% in FPG, and graft weight increased by 64% in LPG while remaining unchanged in FPG. All rats that received FPG died, whereas all those receiving LPG survived. N-(1-naphtyl)ethylendiamine dihydrochloride (5 microM), a specific iNOS inhibitor, largely blunted the production of RNS, prevented the increase of alanine aminotransferase and bilirubin, restored liver regeneration, and improved survival of FPG. Mitochondrial cytochrome c oxidase-IV, ATP synthase-beta, and NADH dehydrogenase-3 decreased markedly in FPG, and these effects were blocked by N-(1-naphtyl)ethylendiamine dihydrochloride.

Conclusion: Thus, hepatic steatosis causes failure of partial liver grafts, most likely by increasing RNS that leads to mitochondrial damage and dysfunction.

Fig. 1. High-Fat High-Fructose Diet Caused Hepatic Steatosis

Rats were fed a low-fat low-fructose control diet (Control) or a high-fat high-fructose diet (HFFr) for 2 weeks. Livers were harvested for Oil-Red-O staining to detect fat droplets (A) just before transplantation (Before Tx; upper panels) or at 48 h after transplantation of FPG from HFFr-fed rats (After Tx; lower panels). Bar is 10 μm. Hepatic triglycerides just before liver transplantation were detected colorimetrically (B). **, p< 0.01 vs the control group.

Fig. 2. Upregulation of iNOS and Increased RNS Production after Transplantation of FPG

Livers were collected 48 h after sham-operation (Sham) or transplantation of lean or fatty partial liver grafts. A: inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. Serum nitrite and nitrate values at 24 and 48 h after surgery are shown in B. a, p< 0.05 vs sham-operation; b, p< 0.05 vs LPG, and c, p< 0.05 vs FPG. Hepatic 3-NT was detected immunohistochemically (C). Panels are: upper left, liver from sham-operated rats; upper right, LPG; lower left, FPG; lower right, FPG treated with 1400W. Bar is 50 μm.

Fig. 3. 1400W Prevented Injury and Improved Function of FPG

Livers were collected 48 h after sham-operation (Sham) or transplantation of LPG and FPG. A shows the images of H+E stained liver slides. Panels are: upper left, liver from sham-operated rats; upper right, LPG; lower left, FPG; lower right, FPG pretreated with 1400W. Bar is 50 μm. Blood was collected at 24 h and 48 h after transplantation. ALT (B) and total bilirubin (C) in sera were measured. a, p< 0.05 vs sham-operation; b, p< 0.05 vs LPG, and c, p< 0.05 vs FPG.

Fig. 4. 1400W Improved Regeneration of FPG

Livers were collected 48 h after sham-operation (Sham) or transplantation of LPG and FPG. 5-Bromo-2′-deoxyuridine (BrdU) incorporation was detected immunohistochemically (A to D). BrdU-positive and –negative cells (E) as well as mitotic and non-mitotic cells (F) were counted in 10 randomly selected fields in a blinded manner. Partial grafts were weighed before implantation and 48 h later to calculate graft weight increases (G). a, p< 0.05 vs sham-operation; b, p< 0.05 vs LPG, and c, p< 0.05 vs FPG.

Fig. 5. 1400W Improved Survival of FPG

Conditions were as in Fig. 1. Rats were observed 7 days for survival after sham operation or transplantation of LPG and FPG. Difference is statistically significant as assessed by the Kaplan-Meier test (p<0.05) between FPG with and without 1400W-treatment.

Fig. 6. 1400W Prevented Decreases of Mitochondrial Oxidative Phosphorylation Proteins in FPG

Livers were harvested 48 h after transplantation. Representative images of cytochrome c oxidase IV (COX IV), ATP synthase-β (AS-β), and NADH dehydrogenase-3 (ND3) detected by immunoblotting are shown in A and quantification of images by densitometry is shown in B, C and D. a, p<0.05 vs sham; b, p<0.05 vs LPG and c, p<0.05 vs FPG.