Assessment of Mitochondrial Function and Oxygen Consumption Measured During Ex Vivo Normothermic Machine Perfusion of Injured Pig Kidneys Helps to Monitor Organ Viability

Abstract

Donor kidney assessment may improve organ utilisation. Normothermic Machine Perfusion (NMP) has the potential to facilitate this advance. The mechanism of action is not yet determined and we aimed to assess mitochondrial function during NMP. Anaesthetised pigs (n = 6) had one kidney clamped for 60 min. The healthy contralateral kidney was removed and underwent NMP for 8 h (healthy control (HC), n = 6). Following 60 min warm ischaemia the injured kidney underwent HMP for 24 h, followed by NMP for 8 h (n = 6). Mitochondria were extracted from fresh tissue for analysis. Injured kidneys were analysed as two separate groups (IMa, n = 3 and IMb, n = 3). Renal resistance was higher (0.39ï, ± 0.29 vs. 1.65ï, ± 0.85; p = 0.01) and flow was lower (55ï, ± 28 vs. 7ï, ± 4; p = 0.03) during HMP in IMb than IMa. NMP blood flow was higher in IMa versus IMb (2-way ANOVA; p < 0.001) After 60 min NMP, O₂ consumption was significantly lower in IMb versus IMa (p ≤ 0.002). State-3 respiration was significantly different between the groups (37ï, ± 19 vs. 24ï, ± 14 vs. 10ï, ± 8; nmolO₂/min/mg; p = 0.049). Lactate levels were significantly lower in IMa versus IMb (p = 0.028). Mitochondrial respiration levels during NMP may be suggestive of kidney viability. Oxygen consumption, renal blood flow and lactate can differentiate severity of kidney injury during NMP.

Keywords: ischemia/reperfusion injury; kidney; mitochondria; normothermic machine perfusion; preservation.

FIGURE 1

Schematic diagram of the study methods illustrating the differing injury sustained and preservation methods between the injury groups (n = 6) and the control group (n = 6).

FIGURE 2

(A) Histogram showing Intrarenal resistance (IRR) and (B) Renal blood flow (RBF) during Hypothermic Machine Perfusion (HMP) of Injury model kidney groups IMa and IMb (1-way ANOVA with = p < 0.05 *). (C) Line graph showing renal blood flow and (D) Cumulative Urine Output during Normothermic Machine Perfusion of Healthy and Injury model kidney groups IMa and IMb (2-way ANOVA analysed over 5 h NMP with = p < 0.05*).

FIGURE 3

(A) Line graph showing oxygen consumption (B) Perfusate lactate levels (C) Perfusate AST and (D) Perfusate LDH levels during Normothermic Machine Perfusion of Healthy and Injury model kidney groups IMa and IMb (2-way ANOVA analysed over 5 h NMP with = p < 0.05 *). AST, aspartate transaminase, LDH, lactate dehydrogenase (* = p < 0.05).

FIGURE 4

(A) Line graph showing urine albumin:creatinine ratio and (B) creatinine clearance during Normothermic Machine Perfusion of Healthy and Injury model kidney groups IMa and IMb (2-way ANOVA analysed over 5 h NMP with = p < 0.05 *). (C) Graph showing mean State-3 respiration and (D) mitochondrial aconitase activity during Normothermic Machine Perfusion of Healthy and Injury model kidney groups IMa and IMb (* = p < 0.05).

FIGURE 5

(A) Representative histological image of a healthy kidney section at 20x magnification after 8 h NMP showing significant cytoplasmic vacuolation. (B) histological image of an injury model kidney after 8 h NMP showing tubular dilatation.