O3 Ex vivo normothermic machine perfusion facilitates gymnotic delivery of RNA interference therapeutics in donor kidneys

Abstract Introduction Normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for delivery of novel therapeutics to optimize organ quality. This includes RNA interference (RNAi) therapeutics e.g. antisense oligonucleotides (ASO) that block detrimental microRNAs. The intracellular kinetics of RNAi therapeutics are crucial for their pharmacological effect, however, it remains poorly understood. NMP provides an ideal platform to investigate this further. Method During NMP, human kidneys (n = 12) were treated for 6 hours with a fluorescently-labelled ASO designed to block microRNA-24-3p activity. Biopsies were taken at 0, 2, 4, and 6 hours. Kidney sections were stained with antibodies against early endosomes (Rab5), late endosomes (Rab7), RNA-induced silencing complexes (GW182) and lysosomes (LAMP2). Confocal microscopy images were obtained and co-localisation quantified using Hugyens™ software following batch deconvolution. The global transcriptomic impact of ASO therapy was also assessed using RNA sequencing. Result Following 2 hours of NMP, ASO was primarily found in tubular epithelial cells. Co-localisation studies revealed ASO uptake via endocytosis and endosomal sorting occurring during NMP. This was followed by cytoplasmic escape and co-localisation of ASO with GW182 proteins. This pattern of co-localisation was not seen in scrambled sequence or cold perfusion controls. RNAseq analysis revealed a decrease in inflammatory pathways and upregulation of microRNA-24-3p targets. Discussion This is the first study to demonstrate NMP facilitates gymnotic ASO delivery directly into the RISC, whereby, it blocks microRNA-mediated mRNA silencing and increases bioavailability of protective targets. This study highlights the capacity of NMP to re-programme gene expression in donor kidneys using RNAi therapeutics. Take-home Message Ex vivo normothermic machine perfusion of donor kidneys provides a unique window of opportunity prior to transplantation when we can deliver therapies to improve the quality of the organ. Novel genetic therapies designed to protect kidneys against ischemia reperfusion injury could potentially increase organ utilisation and improve post-transplant outcomes for the many patients on the kidney transplant waiting list.

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Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys

Science Translational Medicine ◽

10.1126/scitranslmed.aam6764 ◽

2017 ◽

Vol 9 (418) ◽

pp. eaam6764 ◽

Cited By ~ 43

Author(s):

Gregory T. Tietjen ◽

Sarah A. Hosgood ◽

Jenna DiRito ◽

Jiajia Cui ◽

Deeksha Deep ◽

...

Keyword(s):

Ischemia Reperfusion Injury ◽

Therapeutic Potential ◽

Vascular Endothelial Cells ◽

Ex Vivo ◽

Polymeric Nanoparticles ◽

Ischemia Reperfusion ◽

Machine Perfusion ◽

Renal Vasculature ◽

Normothermic Machine Perfusion

Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient’s immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host’s alloimmune response. Using NMP to administer therapeutics directly to the graft avoids many of the limitations associated with systemic drug delivery. We have previously shown that polymeric nanoparticles (NPs) can serve as depots for long-term drug release, but ensuring robust NP accumulation within a target cell type (graft ECs in this case) remains a fundamental challenge of nanomedicine. We show that surface conjugation of an anti-CD31 antibody enhances targeting of NPs to graft ECs of human kidneys undergoing NMP. Using a two-color quantitative microscopy approach, we demonstrate that targeting can enhance EC accumulation by about 5- to 10-fold or higher in discrete regions of the renal vasculature. In addition, our studies reveal that NPs can also nonspecifically accumulate within obstructed regions of the vasculature that are poorly perfused. These quantitative preclinical human studies demonstrate the therapeutic potential for targeted nanomedicines delivered during ex vivo NMP.

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Metformin Preconditioning and Postconditioning to Reduce Ischemia Reperfusion Injury in an Isolated Ex Vivo Rat and Porcine Kidney Normothermic Machine Perfusion Model

Clinical and Translational Science ◽

10.1111/cts.12846 ◽

2020 ◽

Author(s):

Tobias M. Huijink ◽

Leonie H. Venema ◽

Rene A. Posma ◽

Nynke J. Vries ◽

Andrie C. Westerkamp ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Porcine Kidney ◽

Machine Perfusion ◽

Perfusion Model ◽

Normothermic Machine Perfusion

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Normothermic machine perfusion may prevent regulated cell death following renal ischemia‐reperfusion injury

American Journal of Transplantation ◽

10.1111/ajt.15189 ◽

2018 ◽

Vol 19 (4) ◽

pp. 1245-1245 ◽

Cited By ~ 1

Author(s):

Stephan Kemmner ◽

Quirin Bachmann

Keyword(s):

Cell Death ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Renal Ischemia ◽

Machine Perfusion ◽

Regulated Cell Death ◽

Renal Ischemia Reperfusion Injury ◽

Normothermic Machine Perfusion

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Restoring Mitochondrial Function While Avoiding Redox Stress: The Key to Preventing Ischemia/Reperfusion Injury in Machine Perfused Liver Grafts?

International Journal of Molecular Sciences ◽

10.3390/ijms21093132 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3132 ◽

Cited By ~ 3

Author(s):

Julia Hofmann ◽

Giorgi Otarashvili ◽

Andras Meszaros ◽

Susanne Ebner ◽

Annemarie Weissenbacher ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Ros Production ◽

Machine Perfusion ◽

Redox Stress ◽

Molecular Events ◽

Regulate Cell Death

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

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Cold Preflush of Porcine Kidney Grafts Prior to Normothermic Machine Perfusion Aggravates Ischemia Reperfusion Injury

Scientific Reports ◽

10.1038/s41598-019-50101-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Gregor Fabry ◽

Benedict M. Doorschodt ◽

Tim Grzanna ◽

Peter Boor ◽

Aaron Elliott ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Warm Ischemia ◽

Total Output ◽

Machine Perfusion ◽

Buffer Solution ◽

Preservation Method ◽

Fractional Excretion Of Sodium ◽

Normothermic Machine Perfusion

Abstract Normothermic machine perfusion (NMP) of kidney grafts is a promising new preservation method to improve graft quality and clinical outcome. Routinely, kidneys are washed out of blood remnants and cooled using organ preservation solutions prior to NMP. Here we assessed the effect of cold preflush compared to direct NMP. After 30 min of warm ischemia, porcine kidneys were either preflushed with cold histidine-tryptophan-ketoglutarate solution (PFNMP group) prior to NMP or directly subjected to NMP (DNMP group) using a blood/buffer solution. NMP was performed at a perfusion pressure of 75 mmHg for 6 h. Functional parameters were assessed as well as histopathological and biochemical analyses. Renal function as expressed by creatinine clearance, fractional excretion of sodium and total output of urine was inferior in PFNMP. Urine protein and neutrophil gelatinase-associated lipocalin (NGAL) concentrations as markers for kidney damage were significantly higher in the PFNMP group. Additionally, increased osmotic nephropathy was found after PFNMP. This study demonstrated that cold preflush prior to NMP aggravates ischemia reperfusion injury in comparison to direct NMP of warm ischemia-damaged kidney grafts. With increasing use of NMP systems for kidneys and other organs, further research into graft flushing during retrieval is warranted.

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Ischemia-Reperfusion Injury in Marginal Liver Grafts and the Role of Hypothermic Machine Perfusion: Molecular Mechanisms and Clinical Implications

Journal of Clinical Medicine ◽

10.3390/jcm9030846 ◽

2020 ◽

Vol 9 (3) ◽

pp. 846 ◽

Cited By ~ 4

Author(s):

Zoltan Czigany ◽

Isabella Lurje ◽

Moritz Schmelzle ◽

Wenzel Schöning ◽

Robert Öllinger ◽

...

Keyword(s):

Reperfusion Injury ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Organ Shortage ◽

Machine Perfusion ◽

Hypothermic Machine Perfusion ◽

Critical Organ

Ischemia-reperfusion injury (IRI) constitutes a significant source of morbidity and mortality after orthotopic liver transplantation (OLT). The allograft is metabolically impaired during warm and cold ischemia and is further damaged by a paradox reperfusion injury after revascularization and reoxygenation. Short-term and long-term complications including post-reperfusion syndrome, delayed graft function, and immune activation have been associated with IRI. Due to the current critical organ shortage, extended criteria grafts are increasingly considered for transplantation, however, with an elevated risk to develop significant features of IRI. In recent years, ex vivo machine perfusion (MP) of the donor liver has witnessed significant advancements. Here, we describe the concept of hypothermic (oxygenated) machine perfusion (HMP/HOPE) approaches and highlight which allografts may benefit from this technology. This review also summarizes clinical applications and the main aspects of ongoing randomized controlled trials on hypothermic perfusion. The mechanistic aspects of IRI and hypothermic MP—which include tissue energy replenishment, optimization of mitochondrial function, and the reduction of oxidative and inflammatory damage following reperfusion—will be comprehensively discussed within the context of current preclinical and clinical evidence. Finally, we highlight novel trends and future perspectives in the field of hypothermic MP in the context of recent findings of basic and translational research.

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