Normothermic Machine Perfusion and Inflammatory Mediators Adsorption: First Kidney Transplant Experience

Abstract

Kidney transplantation faces challenges due to the shortage of donor organs, leading to the increased use of extended criteria donor (ECD) organs. Recent advancements in ex-situ organ perfusion technologies have facilitated the use of ECD kidneys by preserving organs in near-physiological conditions to tackle ischemia-reperfusion injury (IRI), a process that leads to long-term graft injury. This study focuses on the application of an inflammatory mediators' removal (IMR) integrated in a normothermic machine perfusion (NMP) for the recovery of an ECD kidney before transplantation. This IMR device, designed to adsorb inflammatory molecules, demonstrated effective removal of cytokines during the perfusion process. An ECD kidney underwent 320 minutes of NMP, allowing detailed organ viability assessments and cytokine modulation. A significant volume of urine output and successful post-transplantation outcomes, with no delayed graft function (DGF), highlight the efficacy of this approach. Additionally, the adsorption of inflammatory cytokines was characterized by concentration-dependent removal, suggesting a balanced modulation of both pro- and anti-inflammatory responses. The integration of IMR device into the perfusion process might offer a promising option for evaluating organ viability and mitigating IRI. Further studies are needed to explore the long-term clinical impact of this approach on graft survival and function.

Keywords: donor after brain death; ischemia/reperfusion injury; kidney transplantation; normothermic machine perfusion.

Graphical abstract

Figure 1.

Cytokine profiles. The plots represent the levels of RANTES, TNFα, IL-10, IL-6, and IL-8 in the perfusion liquid, expressed as means of pg/ml (± SE), at three different time points: 60 minutes after the perfusion start (T1) 120 minutes after the perfusion start (T2), 320 minutes after the perfusion start (tend). The major amount of specific cytokines, and particularly, the major concentration represents the pre‐IMR, while inferior curves represents the post‐IMR values. In the cytokine profile, description is reported the total and specific amount of adsorbed cytokines during the treatment. IL, interleukin; IMR, inflammatory mediators’ removal; RANTES, regulated on activation, normal T cell expressed and secreted; TNF-α, tumor necrosis factor alpha.

Figure 2.

Bubble plot of GOCC analysis. Each sample was analyzed in triplicate, and 50 µg of protein per sample processed with the iST-kit from PreOmics (PreOmics GmbH, Martinsried, Germany) following the manufacturer’s protocol. Samples were dissolved in 50 µl of LYSE buffer (PreOmics), with proteins reduced, alkylated, and then digested for 2 hours at 37°C using Lys-C and trypsin. The resulting peptides were washed, eluted from the cartridge, and vacuum-dried. Peptides were re-suspended in LC-LOAD buffer (PreOmics) and sonicated in a water bath. Peptide concentration was measured with a Nanodrop 2000 (Thermo Scientific, Wilmington, DE), and 1 µg of peptides per sample was separated on the Vanquish Neo UHPLC nanoLC system (Thermo Scientific), coupled online to an Exploris 480 mass spectrometer, using a 25 cm × 75 µm Acclaim PEPMap C18 column (Thermo Fisher Scientific). Protein quantification was performed using LFQ. Peptides were separated over a 132 minute binary gradient of water and acetonitrile, each containing 0.1% formic acid. DIA was performed with an MS1 full scan from 400 to 1,200 m/z, followed by 60 sequential DIA windows with 1 m/z overlap and optimized window placement. Full scans were acquired at a resolution of 120,000, with an automatic gain control (AGC) target of 3 × 106 and a maximum injection time of 50 ms. The 60 isolation windows were acquired at a resolution of 30,000, with an AGC target of 8 × 105 and the maximum injection time set to “auto” for optimal cycle time. Fragmentation was achieved with 30% normalized HCD collision energy. Data analysis was conducted using DIA-NN (version 1.8.1) with a predicted library generated from an in silico-digested human UniProt reference database (proteome ID UP000005640_9606), allowing for K* and R* cleavages, up to two missed cleavages, and a minimum peptide length of six amino acids. The FDR for peptide and protein identification was set at 0.01%. The plot highlights the 13 most significant GOCC terms derived from the 331 proteins identified through mass spectrometry analysis. The varying color intensities of the bubbles represent the different adjusted p values, while the bubble sizes indicate the number of genes associated with each term. B: Bubble plot of KEGG analysis. The plot highlights the 13 most significant KEGG pathways derived from the 331 proteins identified through mass spectrometry analysis. C–E: Volcano plot analysis showing the log₂ of protein ratio between perfusate after and before IMR device at time T1 (C), T2 (D), and Tf (E). The black hyperbolas indicate the FDR and proteins displayed above the FDR curves were considered significantly different between the two conditions (FDR < 0.05). DIA, data-independent acquisition; FDR, false discovery rate; GOCC, gene ontology-cellular component; HCD, higher-energy collisional dissociation; IMR, inflammatory mediators’ removal; LFQ, label-free quantification.