Ex Vivo Normothermic Perfusion Induces Donor-Derived Leukocyte Mobilization and Removal Prior to Renal Transplantation

Abstract

Introduction: Ex vivo normothermic perfusion offers an alternative method of organ preservation, allowing donor kidneys to be reanimated and evaluated prior to transplantation. Beyond preservation, it can be used to characterize the immunological contribution of the donor kidney in isolation. Furthermore, it has the potential to be used as an immunomodulatory strategy to manipulate donor kidneys prior to transplantation.

Methods: Explanted porcine kidneys underwent 6 hours of perfusion. Sequential perfusate samples were collected and leukocytes characterized via flow cytometry. An inflammatory profile was generated via cytokine quantification. Cell-free DNA was also determined as markers of cell death.

Results: All kidneys functioned within normal parameters and met the criteria for transplantation at the end of perfusion. Throughout perfusion there were continuous increases in pro-inflammatory cytokines, including large concentrations of interferon-γ, suggesting that perfusion drives a significant inflammatory response. Increasing concentrations in cell-free DNA were also observed, suggesting cell death. During perfusion there was a marked cellular diapedesis of T cells, B cells, natural killer (NK) cells, and monocytes from the kidney into the circuit. Minor populations of granulocytes and macrophages were also detected.

Discussion: We demonstrate that ex vivo normothermic perfusion initiates an inflammatory cytokine storm and release of mitochondrial and genomic DNA. This is likely to be responsible for immune cell activation and mobilization into the circuit prior to transplantation. Interestingly this did not have an impact on renal function. These data therefore suggest that normothermic perfusion can be used to immunodeplete and to saturate the pro-inflammatory capacity of donor kidneys prior to transplantation.

Keywords: allorecognition; ex vivo normothermic perfusion; immune migration; kidney transplantation; passenger leukocytes.

Figure 1

Renal hemodynamics and oxygen consumption remain stable during perfusion. (a) Renal blood flow and (b) intrarenal resistance remained within acceptable ranges during perfusion. (c) Oxygen consumption was also maintained throughout the experiment.

Figure 2

Tubular function is restored and retained over 6 hours of perfusion. (a) Urine production began immediately following revascularization, peaking in the first hour of perfusion. (b) Creatinine is continually removed from the circuit by the kidney and excreted in the urine.

Figure 3

Cytokine secretion increases over time during ex vivo perfusion. (a−d) Serial perfusate samples were analyzed by Luminex to detect a range of cytokines and chemokines. After approximately 60 minutes on the circuit, EVNP is associated with a rapid increase in the secretion of IFN-γ, IL-1α, IL-1β, IL-1RA, IL-2, IL-6, CXCL-8, IL-10, and IL-18. The concentration of GM-CSF, IL-4, IL-12, and TNF-α remains unaffected. (CXCL, C-X-C motif chemokine ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.)

Figure 4

Ex vivo normothermic perfusion is associated with increasing concentrations of (a) cell-free genomic DNA and (b) mitochondrial DNA. Cell-free mitochondrial and genomic DNA concentrations from plasma samples were quantified using quantitative polymerase chain reaction. Increasing concentrations are detected during perfusion, suggesting cellular injury occurs.

Figure 5

Donor leukocytes migrate out of the kidney into the perfusate during ex vivo normothermic perfusion. Serial perfusate samples were analyzed by flow cytometry to identify migrating leukocytes. Large populations of T cells, B cells (a), and NK cells (b) were detected in increasing concentrations during the perfusion period. Populations of monocytes, macrophages (c), neutrophils and basophils and eosinophils (d) were also detected.