Oxygen carriers affect kidney immunogenicity during ex-vivo machine perfusion

Abstract

Normothermic ex-vivo machine perfusion provides a powerful tool to improve donor kidney preservation and a route for the delivery of pharmacological or gene therapeutic interventions prior to transplantation. However, perfusion at normothermic temperatures requires adequate tissue oxygenation to meet the physiological metabolic demand. For this purpose, the addition of appropriate oxygen carriers (OCs) to the perfusion solution is essential to ensure a sufficient oxygen supply and reduce the risk for tissue injury due to hypoxia. It is crucial that the selected OCs preserve the integrity and low immunogenicity of the graft. In this study, the effect of two OCs on the organ's integrity and immunogenicity was evaluated. Porcine kidneys were perfused ex-vivo for four hours using perfusion solutions supplemented with red blood cells (RBCs) as conventional OC, perfluorocarbon (PFC)-based OC, or Hemarina-M101 (M101), a lugworm hemoglobin-based OC named HEMO₂life®, recently approved in Europe (i.e., CE obtained in October 2022). Perfusions with all OCs led to decreased lactate levels. Additionally, none of the OCs negatively affected renal morphology as determined by histological analyses. Remarkably, all OCs improved the perfusion solution by reducing the expression of pro-inflammatory mediators (IL-6, IL-8, TNFα) and adhesion molecules (ICAM-1) on both transcript and protein level, suggesting a beneficial effect of the OCs in maintaining the low immunogenicity of the graft. Thus, PFC-based OCs and M101 may constitute a promising alternative to RBCs during normothermic ex-vivo kidney perfusion.

Keywords: HEMO2Life; hemarina-M101; kidney preservation; normothermic ex-vivo machine perfusion; organ immunogenicity; oxygen carriers; perfluorocarbon-based oxygen carriers; transplantation.

Figure 1

Normothermic ex-vivo kidney machine perfusion (EVKP) with perfluorocarbon (PFC)-based oxygen carriers (OCs) and M101. (A) Kidney connected to the Kidney Assist® perfusion system via the renal artery. (B) Kidney Assist® perfusion system. (C) Schematic representation of the EVKP system with its main components: perfusion reservoir, thermo unit, pump unit, and oxygenator. The oxygenated and deoxygenated perfusion solution circulates as red and blue line through the system, respectively. The following OCs are supplemented to the STEEN/Ringer-based solution: blood, PFC-based OC (PFC), and Hemarina-M101 (M101) (created with BioRender.com). (D) Graphs display flow rate, vascular resistance (VR), temperature, and oxygen partial pressure of kidneys perfused with different OCs over time. Graphs are represented as mean and standard deviation (n = 3).

Figure 2

Quality assessment of the kidney during normothermic EVKP using OCs. Lactate levels (A) and aspartate aminotransferase (AST) activity (B) were quantified in perfusates of control kidneys and kidneys exposed to OCs at different time points (0, 60, 120, 180, and 240 min). Graphs represent means and standard deviations (n = 3). Lactate levels differed significantly between the individual groups (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; two-way ANOVA).

Figure 3

Histological analyses of kidneys after normothermic EVKP using OCs. Representative images of hematoxylin and eosin stained renal cortex regions: Kidneys perfused with no OCs (A), blood (B), PFC-based OCs (C), or M101 (D) showed necrotic cell detritus within the tubular lumen (blue arrow) as well as a mild tubular vacuolization (black arrow), indicating a potentially reversible mild to moderate acute tubular injury and minor signs of tubular necrosis. However, overall renal morphology of the individual groups was intact (scale bar: 200 µm).

Figure 4

Cytokine secretion profile during normothermic EVKP using OCs. Concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF)α, IL-6, and IL-8 were measured in perfusates of control kidneys and kidneys exposed to OCs at different time points (0, 60, 120, 180, and 240 min). Graphs represent means and standard deviations (n = 3). IL-6 and IL-8 levels decreased significantly after addition of OCs (**p < 0.01; ***p < 0.001; ****p < 0.0001; two-way ANOVA).

Figure 5

Heatmap representing the gene expression profile of ex-vivo perfused kidneys using OCs. Relative quantification (RQ) of heat shock protein 70 (HSP70), IL-6, IL-8, TNFα, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), β2-microglobulin (β2 m), and swine leukocyte antigen-DRα (SLA-DRα) gene expression in renal tissues measured by qPCR. The data were normalized to the endogenous housekeeper gene GAPDH. Color intensity represents the up- and downregulation of mRNA expression values relative to the control tissue of non-perfused kidneys. Compared to the kidneys perfused without OCs, IL-6 and ICAM-1 gene expression levels significantly decreased after addition of OCs (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; one-way ANOVA).