Colin Hugh Wilson

Introduction: Ischaemia reperfusion injury (IRI) is an unavoidable, significant consequence of renal transplantation. MicroRNAs are small, non-coding RNA molecules that regulate multiple downstream mRNA targets. MiRNA-21-5p and miRNA-24-3p have been previously implicated in IRI. Antisense oligonucleotides (ASOs) block specific microRNAs, with previous work by our group demonstrating their delivery to kidneys using normothermic machine perfusion. Imaging these kidneys revealed ASO localisation around proximal tubule epithelial cells (PTECs). This project aimed to characterise ASO blockade against miRNA-21-5p and miRNA-24-3p in PTECs. Method: HKC8 cells, a human PTEC cell line, were used throughout these experiments. Cells were placed in a hypoxic incubator for 24hrs, followed by 6hrs of reoxygenation to mimic IRI. HKC8s were transfected with ASOs using lipofectamine. RT-qPCR and Western Blots were used to evaluate expression of antioxidant targets, SOD2 and HMOX1. Result: MiRNA-21-5p and miRNA-24-3p levels were high throughout hypoxia and reoxygenation. Single blockade with anti-miRNA-21-5p resulted in a significant increase in its downstream target SOD2 (P<0.05). Anti-miRNA-24-3p treatment resulted in no change in either of its downstream targets, HMOX1 or SOD2. This was reflected in the failure of dual blockade to produce a synergistic effect on the shared target, SOD2. Conclusion: Anti-miRNA-21-5p results in a significant increase of SOD2, which is well characterised as protective during IRI. Anti-miRNA-24-3p appears to have no effect on PTECs, contrary to previous work in endothelial cells, perhaps suggesting a cell specific response of microRNAs. Normothermic machine perfusion could be used to deliver dual ASOs; allowing simultaneous targeting of different kidney cell types. Take-home message: The delivery of anti-miRNA-21-5p therapy pre-transplant, using normothermic machine perfusion, has the potential to reduce ischaemia reperfusion injury and improve kidney transplant outcomes.

Introduction: Normothermic machine perfusion (NMP) is a method of organ preservation that aims to replicate the physiological environment, achieved by perfusing the livers with a blood-based perfusate at physiological inflow pressures and temperature. NMP also permits viability assessment through evaluation of the perfusate flow rates through the portal vein and hepatic artery. In addition to this, biochemical assessment and perfusate gas analysis can be performed to provide insights into the metabolic activity of the liver. Method: Discarded human liver grafts (n=6), were perfused for 24 hours. Core biopsies and perfusate samples were taken from each liver at 5 distinct time intervals over 24 hours. Core biopsies were fixed and stained with periodic acid-Schiff and analysed with Leica software to provide a quantitative estimate of the hepatocellular glycogen content. Result: Hepatic glycogen concentration rose during the first hour, followed by a steady decline thereafter until the end of perfusion. Contrary to our initial hypothesis that glucose concentration within the circuit would show an inverse relationship to glycogen stores in the liver cells, we found that glucose concentration closely followed the same trend. Conclusion: Change in hepatocyte glycogen content provides an important insight into the synthetic function of a liver destined for transplant. Our research suggests that glucose concentration can be used as a surrogate marker for the synthetic function of a liver on NMP and provides valuable information on the glycogen-synthesising capability of the hepatocytes. In future, this could potentially aid the decision-making process with regards to liver graft transplant viability. Take-home message: Perfusate glucose concentration could provide an insight into the viability of liver transplants

Ex-vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimise organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC ^® ) possess potent immunomodulatory properties which could prove beneficial in minimising subsequent ischaemia reperfusion injury. We investigated the potential reconditioning capability of MAPC cells in kidney NMP. Methods: Pairs (5) of human kidneys from the same donor were simultaneously perfused for 7 hours. The right or left kidney was randomly allocated to receive MAPC treatment. Serial samples of perfusate, urine and tissue biopsies were taken for comparison with the control paired kidney. Results: MAPC-treated kidneys demonstrated improved urine output (p<0.01), decreased expression of the kidney injury biomarker NGAL (p<0.01), improved microvascular perfusion on contrast enhanced ultrasound (cortex p<0.05, medulla p<0.01), downregulation of IL-1β (p<0.05) and upregulation of IL-10 (p<0.05) and Indolamine-2, 3-dioxygenase (p<0.05). A mouse model of intraperitoneal chemotaxis demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (p<0.01). Immunofluorescence revealed pre-labelled MAPC cells home to the perivascular space in the kidneys during NMP. MAPC therapy was not associated with detrimental physiological or embolic events. Conclusion: We report the first successful delivery of cellular therapy to a kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant functional parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to clinical transplantation. One Sentence Summary: Ex-vivo reconditioning of human kidneys using Multipotent Adult Progenitor Cell therapy delivered during normothermic machine perfusion.

Kidney transplantation is the treatment of choice for patients with end-stage kidney disease. In a previous review we concluded that the routine use of ureteric stents in kidney transplantation reduces the incidence of major urological complications (MUC). Unfortunately, this reduction appears to lead to a concomitant rise in urinary tract infections (UTI). For kidney recipients UTI is now the commonest post-transplant complication. This represents a considerable risk to the immunosuppressed transplant recipient, particularly in the era of increased immunologically challenging transplants. There are a number of different approaches taken when considering ureteric stenting and these are associated with differing degrees of morbidity and hospital cost. This review aimed to look at the benefits and harms of early versus late removal of the ureteric stent in kidney transplant recipients. We searched the Cochrane Kidney and Transplant Specialised Register up to 27 March 2017 through contact with the Information Specialist using search terms relevant to this review. Studies contained in the Specialised Register are identified through search strategies specifically designed for CENTRAL, MEDLINE, and EMBASE; handsearching conference proceedings; and searching the International Clinical Trials Register Search Portal and ClinicalTrials.gov. All RCTs and quasi-RCTs were included in our meta-analysis. We included recipients of kidney transplants regardless of demography (adults or children) or the type of stent used. Two authors reviewed the identified studies to ascertain if they met inclusion criteria. We designated removal of a ureteric stent before the third postoperative week (< day 15) or during the index transplant admission as "early" removal. The studies were assessed for quality using the risk of bias tool. The primary outcome of interest was the incidence of MUC. Further outcomes of interest were the incidence of UTI, idiosyncratic stent-related complications, hospital-related costs and adverse events. A subgroup analysis was performed examining the difference in complications reported depending on the type of ureteric stent used; bladder indwelling (BI) versus per-urethral (PU). Statistical analyses were performed using the random effects model and results expressed as relative risk (RR) with 95% confidence intervals (CI). Five studies (1127 patients) were included in our analysis. Generally the risk of bias of the included studies was judged low or unclear; they addressed the research question and utilised a prospective randomised design. It is uncertain whether early stent removal verus late stent removal improved the incidence of MUC (5 studies, 1127 participants: RR 1.87, 95% CI 0.61 to 5.71; I² = 21%; low certainty evidence). The incidence of UTI may be reduced in the early stent removal group (5 studies, 1127 participants: RR 0.49 95% CI 0.30 to 0.81; I² = 59%; moderate certainty evidence). This possible reduction in the UTI incidence was only apparent if a BI stent was used, (3 studies, 539 participants, RR 0.45 95% CI 0.29 to 0.70; I² = 13%; moderate certainty evidence). However, if an externalised PU stent was used there was no discernible difference in UTI incidence between the early and late group (2 studies, 588 participants: RR 0.60 95% CI 0.17, 2.03; I² = 83%; low certainty evidence). Data on health economics and quality of life outcomes were lacking. Early removal of ureteric stents following kidney transplantation may reduce the incidence of UTI while it uncertain if there is a higher risk of MUC. BI stents are the optimum method for achieving this benefit.

Introduction: Carcinoid crisis, as an entity is poorly defined, but can be seen in patients with small bowel NET tumours after open bowel surgery or tumour unrelated procedures as cardiovascular instability (CI). Recent evidence suggests that cardiovascular instability (CI) during NET surgery is more common than previously considered. Aims and methods: We audited the incidence of CI during procedures and determined the relationship to perioperative octreotide; considering the patient’s underlying disease burden. Patients were identified from the hospital pathology database having had their primary tumour resected in our institution between January 2011 and December 2015. Our prophylactic octreotide protocol is an intravenous infusion of 50 micrograms an hour for 24 hours prior to surgery. Results: About 54 patients underwent 65 procedures. About 24% (n=13) had carcinoid symptoms or syndrome prior to surgery. There was no postoperative mortality or direct morbidity related to CI in the 65 procedures. About 77% of patients had preoperative octreotide for at least 4 hours. Most procedures (67%) were complicated by CI not related to blood loss (Table 1). About 31 detailed anaesthetic charts were available for review. Table 1 Presence of CI in relation to Octreotide therapy. Cardiovascular Instability (Oct vs. No Oct) Chi χ² Bowel resection, no liver mets (n=13) 45.5% vs 50% P=0.9 Bowel resection, liver mets (n=9) 50% vs 100% P=0.35 Simultaneous liver/bowel resection (n=9) 87.5% vs 100% P=0.6 Conclusions: Octreotide infusion does reduce the incidence and severity of cardiovascular instability during surgery- but is not universal. Almost all patients undergoing bowel or liver resection in the presence of liver metastases will have some form of cardiovascular instability during the procedure. The postoperative effects are not directly discernible.