Prompt Thrombo-Inflammatory Response to Ischemia-Reperfusion Injury and Kidney Transplant Outcomes.

Introduction: In kidney transplantation (KT), the role of the intravascular innate immune system (IIIS) in response to ischemia-reperfusion injury (IRI) is not well-understood. Here, we studied parallel changes in the generation of key activation products of the proteolytic cascade systems of the IIIS following living donor (LD) and deceased donor (DD) transplantation and evaluated potential associations with clinical outcomes. Methods: In a cohort study, 63 patients undergoing LD (n = 26) and DD (n = 37) transplantation were prospectively included. Fifteen DD kidneys were preserved with hypothermic machine perfusion (HMP), and the remaining were cold stored. Activation products of the kallikrein-kinin, coagulation, and complement systems were measured in blood samples obtained systemically at baseline and locally from the transplant renal vein at 1, 10, and 30 minutes after reperfusion. Results: DD kidneys exhibited a prompt and interlinked activation of all 3 cascade systems of IIIS postreperfusion, indicating a robust and local thrombo-inflammatory response to IRI. In this initial response, the complement activation product sC5b-9 exhibited a robust correlation with other IIIS activation markers and displayed a strong association with short-term and mid-term (24-month) graft dysfunction. In contrast, LD kidneys did not exhibit this thrombo-inflammatory response. The use of HMP was associated with reduced thromboinflammation and preserved mid-term kidney function. Conclusion: Kidneys from DD are vulnerable to a prompt thrombo-inflammatory response to IRI, which adversely affects both short-term and long-term allograft function. Strategies aimed at minimizing graft immunogenicity prior to reperfusion are crucial to mitigate the intricate inflammatory response to IRI.

Nursing and training of pigs used in renal transplantation studies.

The pig is commonly used in renal transplantation studies since the porcine kidney resembles the human kidney. To meet the requirements of intense caretaking and examination without stress, a 2-week socialisation and training programme was developed. Conventional cross-breed pigs (n = 36) with high health status were trained for 15 min/day in a four-step training programme before kidney transplantation. The systematic training resulted in calm animals, which allowed for ultrasound examination, blood sampling and urine sampling without restraint. When a 2-methacryloyloxyethyl phosphorylcholine polymer-coated jugular catheter introduced via the auricular vein was used for post-operative blood sampling, clotting was avoided. To assess renal function, urinary output was observed and creatinine and cystatin C were measured; the latter was not found to be useful in recently transplanted pigs. The results presented contribute to the 3Rs (refine, reduce, replace).

Standardizing the freeze-thaw preparation of growth factors from platelet lysate.

BACKGROUND: Over the past decades, the focus on the regenerative properties of platelets (PLTs) has intensified and many PLT-derived growth factors are readily used in medical settings. A general lack of standardization in the preparation of these growth factors remains, however, and this study therefore examines the dynamics of growth factors throughout the freeze-thaw procedure. STUDY DESIGN AND METHODS: Plateletpheresis (PA) and PLT-poor plasma (PPP) samples were collected from 10 healthy donors. PA was lysed to produce PLT lysate (PL) for 1, 3, 5, 10, and 30 freeze-thaw cycles. The resulting growth factor and cytokine concentrations from PPP, PA, and PL of different cycles were analyzed and compared using enzyme-linked immunosorbent assay and multiplex bead assays. RESULTS: PL produced by the freeze-thaw procedure resulted in approximately four- to 10-fold enrichment of transforming growth factor-ß1, epidermal growth factor, PLT-derived growth factor (PDGF)-AB/BB, PLT factor-4, and fibroblast growth factor-2. The increase in concentrations plateaued at Cycles 3 and 5 and in some cases declined with further cycles. The concentrations of insulin-like growth factor-1, hepatocyte growth factor, vascular endothelial growth factor, and bone morphogenetic protein-2 in PL were essentially comparable to those in PPP. CONCLUSION: Using the freeze-thaw method, optimal preparation of PL with regard to the concentration of growth factors was achieved at Cycles 3 to 5. Based on our findings, the clinical significance of using a greater number of cycles is likely limited.

Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

BACKGROUND: Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. MATERIALS AND METHODS: Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-ß isoform 1, IL-1ß, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. RESULTS: The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-ß, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p < 0.0001), 29.5% (p = 0.04) and 8.2% (p = 0.0004), respectively. A decrease was seen in the levels of IGF-1 and FGF-2 with 22% (p = 0.041) and 11% (p = 0.01), respectively. Cytokines were present only in very low concentrations and all other growth factors remained stable with time in storage. CONCLUSION: The composition of mediators in platelet lysate obtained from pathogen-inactivated platelet units differs when produced from fresh and stored platelet units, respectively. This underscores the need for further standardization and optimization of this important product, which potentially may influence the clinical effects.