Impact of Red Blood Cells on Function and Metabolism of Porcine Deceased Donor Kidneys During Normothermic Machine Perfusion

Abstract

Background: Normothermic machine perfusion (NMP) protocols using blood-based solutions are commonly used in the assessment of kidneys before transplantation. This procedure is, nevertheless, limited by blood availability and warrants the search for alternatives. We compared a blood-based solution with a serum-like preservation solution (Aqix) enriched with colloids with and without red blood cells (RBCs).

Methods: Porcine kidneys retrieved from an abattoir were subjected to 30 min of warm ischemia, followed by 3 h of hypothermic oxygenated machine perfusion at 4 °C. Subsequently, kidneys (n = 6 per group) were evaluated with NMP for 4 h with 5 different solutions: diluted blood, Aqix with BSA ± RBCs, or Aqix with dextran 40 ± RBCs.

Results: Throughout NMP, markers of renal function and tubular metabolism were favorable in groups with RBCs. The addition of RBCs resulted in 4- to 6-fold higher oxygen consumption rates. Controls had significantly higher ATP levels post-NMP, exhibited decreased production of oxidative stress markers, and had the highest creatinine clearance. In conclusion, this study shows that the addition of RBCs during NMP reduced renal injury, improved function, and was associated with increased renal metabolism.

Conclusions: Although the RBC-BSA-supplemented Aqix solution was also able to support metabolism and renal function, a blood-based perfusion solution remains superior.

Graphical abstract

FIGURE 1.

Energy-demanding processes measured during NMP. Porcine kidneys were perfused for 4 h with blood (control) with Aqix supplemented with a colloid (2.2% BSA or 3.5% Dex) or Aqix with a colloid (BSA or Dex) and RBC. A, Oxygen consumption rates during 4 h normothermic perfusion, *P < 0.05 AUCs of all RBC-containing groups compared with acellular groups. B, Fractional sodium excretion levels during 4 h perfusion, *P < 0.02 Aqix-BSA, Aqix-Dex vs controls (Dunnett test). C, T_Na during 4 h perfusion, *P < 0.001 control vs Aqix-BSA/Dex and control vs Aqix-Dex-RBC group. D, ATP content in kidney cortex tissue after 30 min warm ischemia and before preservation (HMP0), 3 h preservation (HMP180), 30 min of NMP (NMP30), and at the end of 240 min of NMP (NMP240), #P < 0.03 vs controls. Every group contains 6 kidneys. Data are presented as mean ± SEM. Except for ATP, areas under the curves were compared to assess differences between experimental groups. ATP, adenosine triphosphate; BSA, bovine serum albumin; Dex, dextran 40; HMP, hypothermic machine perfusion; NMP, normothermic machine perfusion; RBC, red blood cell; T_Na, total sodium reabsorption.

FIGURE 2.

Renal function and the effects of colloids on the kidney. Porcine kidneys were perfused for 4 h with blood (control) with Aqix supplemented with a colloid (2.2% BSA or 3.5% Dex) or Aqix with a colloid (BSA or Dex) and RBC. A, Renal flow rates during 3 h of HMP and (B) during 4 h NMP; (C) creatinine clearance during 4 h NMP, *P = 0.02 control vs Aqix-Dex-RBC group; (D) total urine production and weight gain, respectively, during 4 h NMP, *P ≤ 0.05. Every group contains 6 kidneys. Data are presented as mean ± SEM. Except for urine production and weight gain, areas under the curves were compared to assess differences between experimental groups. BSA, bovine serum albumin; Dex, dextran 40; HMP, hypothermic machine perfusion; NMP, normothermic machine perfusion; RBC, red blood cell.

FIGURE 3.

Kidney injury and oxidative stress markers during NMP. Porcine kidneys were perfused for 4 h with blood (control) with Aqix supplemented with a colloid (2.2% BSA or 3.5% Dex) or with a colloid (BSA or Dex) and RBC. A, ASAT and LDH levels during 4 h NMP, *P < 0.001 Aqix + BSA vs all other experimental groups. B, uNAG levels during 4 h NMP. C, Histology damage scores from sections taken after 4 h NMP. Negative control samples are additional kidney tissue samples taken directly after death of the pig, **P < 0.05. D, Total TBARS production during 4 h perfusion, **P < 0.04. Every group contains 6 kidneys. Data are presented as mean ± SEM, except in Figure 3C, data are presented as median ± interquartile range. ASAT, aspartate aminotransferase; BSA, bovine serum albumin; Dex, dextran 40; LDH, lactate dehydrogenase; NMP, normothermic machine perfusion; RBC, red blood cell; TBARS, thiobarbituric acid-reactive substances; uNAG, urinary N-acetyl-beta-d-glucosaminidase.