Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation

Abstract

Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

Figure 1. iNO therapy and human liver transplantation.

(A) Experimental protocol for administering placebo or iNO to patients and sample (blood and liver biopsy) collection. (B) Methemoglobin (metHb) levels as a function of blood draw. ^#P ≤ 0.001 for corresponding placebo versus iNO measurements by unpaired t test. (C) Volume of platelets transfused during surgery ^#P ≤ 0.05. (D and E) Average percent decrease in PT and PTT after surgery. Data are normalized to coagulation times measured immediately (<1 hour) after surgery and were 26.7 ± 1.4 seconds (placebo) and 34.4 ± 2.5 seconds (iNO) for PT and 54.6 ± 7.3 seconds (placebo) and 70.2 ± 6.5 seconds (iNO) for PTT. (F and G) Average percent decrease in serum ALT and AST levels after surgery. Data were normalized to ALT and AST levels measured immediately (<1 hour) after surgery and were 601.8 ± 145.4 U/l (placebo) and 689.3 ± 149.5 U/l (iNO) for ALT and 922.1 ± 228.7 U/l (placebo) and 940.9 ± 211.3 U/l (iNO) for AST. For data in panels D–G, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 for corresponding placebo versus iNO measurements. (H) Cox analysis of patient hospital length of stay. P = 0.034 adjusted for sex and cold ischemic time. Filled squares: placebo; filled circles: iNO.

Figure 2. iNO decreases reperfusion-dependent hepatic cell death.

(A) Histopathologic scoring of hepatic tissue samples before (white bars) and 1 hour after reperfusion (black bars). P values represent significance calculated by paired t test. (B) Representative H&E-stained sections indicating increased injury in LB2. Original magnification, ×25. The circled area is shown at a higher magnification (×100) in the inset and shows increased PMN infiltration adjacent to the hepatic vein (zone 3). (C) Representative fluorescence micrographs showing changes in TUNEL-positive cells (green); blue staining: DAPI. Original magnification, ×25. (D) Paired changes in TUNEL-positive objects in liver biopsies before (LB1) and 1 hour after reperfusion (LB2). P values represent significance calculated by paired t test. (E) Average reperfusion-dependent increases in TUNEL-positive objects. *P ≤ 0.0005 relative to placebo.

Figure 3. Effects of iNO on ROS and RNS metabolism.

(A) Plasma XOR activities as a function of blood draws during surgery. ^†P ≤ 0.05, BD4 relative to BD1 for placebo; ^‡P = < 0.001, BD4 relative to BD1 for iNO by repeated-measures ANOVA with Tukey post-hoc analysis. (B) Changes in hepatocellular expression of XOR (normalized to β-actin) before and 1 hour after reperfusion. (C) Changes in hepatic expression of eNOS (normalized to β-actin) before and 1 hour after reperfusion. P values represent significance calculated by paired t test. n = 10, placebo; n = 9, iNO. Inset in D shows representative Western blots showing changes in eNOS in paired (LB1-LB2) samples from 2 patients in each group. (D) Average fold change in eNOS expression. ^#P ≤ 0.004 relative to placebo.

Figure 4. Changes in circulating NOx metabolites.

Plasma (A, C, E, and G) and rbc (B, D, F, H, and I) of nitrate (A and B), nitrite (C and D), SNO (E and F), XNO (G and H), and HbNO (I) are shown as a function of blood draw in placebo (filled squares) and iNO (filled circles) groups. Plasma NOx levels were normalized to protein. Absolute concentration ranges (minimum to maximal) were: nitrate (μM), placebo 47.9 ± 15.7 to 54.7 ± 19.2, iNO 84.6 ± 32.5 to 140.3 ± 24.5; nitrite (nM), placebo 202 ± 38.4 to 381.3 ± 49.3, iNO 462.2 ± 181.13 to 923.1 ± 240; SNO (nM), placebo –0.16 ± 0.17 to 1.3 ± 0.66, iNO 0.05 ± 0.2 to 2.3 ± 0.66; XNO (nM), placebo, 0.29 ± 0.2 to 3.5 ± 0.58, iNO 1.04 ± 0.35 to 4.7 ± 1.9. rbc NOx levels were normalized to heme. Absolute concentrations (in packed rbc) were: nitrate (μM), placebo 31.7 ± 12.5 to 41.5 ± 19.1, iNO 80.3 ± 48.3 to 196.2 ± 44.3; nitrite (nM), placebo 646.1 ± 80.8 to 1,219.5 ± 131.7, iNO 1,241.4 ± 447.7 to 2,533.3 ± 681.1; SNO (nM), placebo 4.9 ± 2.5 to 6.2 ± 2.2, iNO 29.1 ± 16.3 to 40.7 ± 18.2; XNO (nM), placebo 0.19 ± 0.15 to 1.8 ± 1.2, iNO 7.3 ± 4.9 to 13.4 ± 9.3; HbNO (nM), placebo 93.4 ± 4.4 to 242.8 ± 32.9, iNO- 222.9 ± 53.2 to 764.6 ± 108.1. Differences were assessed by 2-way repeated measures ANOVA and Bonferroni post-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 for placebo versus iNO at corresponding blood draw. (J) Plasma and rbc A-V gradients at BD3 for indicated NO intermediates. Concentration units for each NOx are as indicated in respective panels showing changes in individual NOx as a function of BD (A–I). *P ≤ 0.001 for placebo versus iNO for plasma nitrite. All values are mean ± SEM; n = 10, and n = 6–7 for HbNO.