Trevor L. Nydam, MD
University of Colorado School of Medicine

Defining the role of XIAP in renal cold ischemia and apoptosis

Synopsis: Failure of a kidney post-transplant is referred to as Delayed Graft Function (DGF). DGF occurs in up to 50% of cadaveric transplants and independently predicts reduced 1- and 5-year kidney transplant survival. Before transplantation, donor kidneys are cooled to 4°C (referred to as cold-ischemia-CI) to delay ex vivo cell death. Kidneys can only sustain limited CI, and DGF significantly increases when CI time exceeds 30 hours. Prolonged CI followed by warm reperfusion (WR) during implantation is a risk factor for DGF. Widespread apoptotic renal tubular epithelial cell (RTEC) death caused by caspase-3 is a feature of kidneys with prolonged CI/WR. CI/WR during hibernation is a unique natural model we have used to understand DGF. Hibernating ground squirrel (GS) kidneys survive CI for several days in torpor followed by WR in Arousal without kidney injury or RTEC apoptosis. Protection from apoptosis in hibernating GS is associated with increased X-linked inhibitor of apoptosis protein (XIAP). Our preliminary data indicates that CI/WR of non-hibernating mouse RTECS is associated with decreased XIAP, increased capsase-3 and apoptosis. XIAP is inhibited by a protease HTRA2, which can be chemically inhibited by UCF-101. Use of UCF-101 to prevent injury to donor kidneys subjected to prolonged CI/WR is a unique and untried approach derived from studies of hibernators. Specific Aim 1 investigates the protective effect of XIAP up-regulation (by UCF-101) in a mouse kidney transplant model. Specific Aim 2 investigates XIAP expression and RTEC apoptosis in biopsies from human kidney transplants with prompt graft function or DGF.             

Sang-Mo Kang, MD
University of California San Francisco

Qizhi Tang, PhD
University of California San Francisco

Charles C. Brinkman, PhD
University of Maryland School of Medicine

The role of lymphotoxin in Treg migration and suppressor function

Synopsis: Regulatory T cells (Treg) are specialized suppressive cells that are essential to maintain normal immune function and prevent autoimmunity. Treg are therapeutically interesting due to their potential for inducing and maintaining tolerance to organ transplants without systemic immune suppression. The ability to specifically control Treg migration into and out of transplants would be a big step in developing them as therapeutic tools. We have previously reported that in an islet allo-transplant model Treg must migrate sequentially to the graft and then to the draining lymph node via the lymphatics in order to mediate graft protection. We have preliminary evidence that Treg but not non-Treg T cells use the lymphotoxin system during migration from the graft to the graft-draining LN. Preliminary evidence suggests that Treg lymphotoxin rapidly modulates the cytoskeletal and membrane structure of the lymphatic endothelial cells that physically compose the lymphatic vessels. We propose to define the mechanism of this pathway and describe its effect on Treg mediated transplant protection.

Anthony M. Swatek, MD
University of Iowa Hospitals and Clinics

Epithelial Stem Cells in Lung Transplantation

Synopsis: Lung transplant is an excellent treatment option for end-stage lung diseases. Obliterative Bronchiolitis (OB), which is considered a form of chronic rejection of transplanted lung, prevents long-term survival as it affects almost half of the recipients by five years. Once OB sets in the allograft, it is progressively fatal and there is no effective treatment available. The mechanisms that cause OB are unclear and it has been proposed that epithelial stem cell depletion may be one of contributing factors that leads to development of OB. However, there is currently no research done to test this hypothesis. We recently have developed an orthotopic lung transplant model in the ferret that develops classic OB. We have also shown that calcitonin gene related peptide (CGRP), a known marker for the stem/progenitor celI niches in the distal airways, promotes proliferation and transformation of the stem cell into a Transient-amplifying cell with limited proliferative capacity. In this proposal we will address the following hypotheses: Epithelial stem/progenitor cells within the donor lung respond inappropriately to the injury following lung transplantation (Aim1) due to dysregulated CGRP signaling in the context of denervation (Aim2), and this leads to exhaustion of stem/progenitor-cell populations in the distal airways and leads to OB. Understanding the role of the epithelial stem cells in the injury following a lung transplantation will be a major step in understanding the pathogenesis of OB and provide an opportunity to prevent or delay the development of OB in the allografts.

William H. Kitchens, Jr., MD, PhD
Emory Transplant Center

Combined Costimulatory and OX40/OX40L Blockade to Prolong Allograft Survival in Murine and Non-Human Primate Transplant Systems

Synopsis: Costimulatory blockade is a novel transplant immunosuppression strategy that avoids the nephrotoxicity of calcineurin inhibitors such as tacrolimus. Recently, belatacept (a CD28 antagonist) became the first transplant immunosuppressant based on costimulatory blockade to gain clinical approval. Renal transplant patients treated with belatacept experienced mar ked improvements in long-term graft function, albeit at the cost of an increased incidence of acute rejection, a finding which might limit the clinical utility of this promising drug. This high incidence of breakthrough rejection may be attributed to the failure of belatacept to target various secondary costimulatory receptors. In our preliminary findings, we demonstrate that blockade of one of these secondary costimulatory pathways (OX40/OX40L) synergizes with conventional costimulatory blockade (with CTLA4-Ig and anti-CD40L) to prolong murine transplant survival.

In this proposal, we hope to illuminate the mechanisms that underpin the efficacy of combined costimulatory and OX40/OX40L blockade. We hypothesize that anti-OX40L primarily targets alloreactive memory T cells, a subset known to resist conventional costimulatory blockade. We will employ two different murine models of transplant rejection that is mediated by alloreactive memory T cells to assess this theory, including a novel transgenic system and a system that mimics alloreactive memory generated by heterologous immunity. Next, based on earlier findings concerning OX40/OX40L pathway disruption, we will examine whether combined blockade therapy augments graft-protective FoxP3+ Treg responses. Finally, we hope to translate this regimen into a clinically-relevant non-human primate kidney transplant system using combined belatacept and humanized pegylated anti-OX40 Fab, a reagent currently in clinical development.

Nikolai Arendovich
University of Illinois College of Medicine

Effects of combined stem cell and Low Level Light therapy on the mitigation of cold-ischemia induced tissue injury

Synopsis: Cold ischemia is a major contributor to delayed graft function and strongly correlates to the long-­‐term outcome of kidney transplantation. Low level light therapy of different wavelengths has been known to promote the healing of wounds and reduce pain and inflammation. Mesenchymal stem cells are capable of expressing immunomodulatory factors that can reduce inflammatory macrophage activity and ischemic kidney injury. The purpose of this study is to investigate whether low level light therapy plays a role in reducing apoptosis following ischemic reperfusion injury and whether this effect can synergize with MSC to reduce pro-­‐inflammatory, IFN-­‐gamma, secreting macrophages to pro-­‐regeneratory IL-­‐10 secreting macrophages, promote endothelial and epithelial proliferation and as a consequence, significantly reduce the effects of cold ischemia injury in the LEW autologous kidney transplant model.

Brian J. Boyarsky
Johns Hopkins School of Medicine

Hypertension attributable to living kidney donation

Synopsis: In light of a significant knowledge gap regarding long-term outcomes following kidney donation, we propose to estimate the incidence of hypertension following kidney donation, as well as the risk of hypertension attributable to donation, comparing African American donors to Caucasian American donors and to healthy non-donor controls. Data will be drawn from the ongoing Wellness and Health Outcomes in LivE DONORs (WHOLE-DONOR) retrospective cohort study at Johns Hopkins, which to date has enrolled more than 1,100 former donors. The ultimate objective of this proposal is to characterize the development of hypertension in living kidney donors, and to improve the long-term comparison group by identifying a matched cohort of healthy non-donors who have baseline characteristics similar to the donor cohort and are followed longitudinally. As hypertension is an important risk factor for cardiovascular and renal disease; it is important to characterize in a racially diverse sample of former donors from the United States. Furthermore, additional evidence about the safety of living donation may, in fact, encourage donation among those considering living donation, particularly among racial minorities.

Aakash Y. Shah
University of Maryland School of Medicine

Effect of EPCR and HLA-E expression by pig lung on coagulation and NK-cell related processes in a xenogenic lung-human blood perfusion model

Synopsis: Xenotransplantation, using pig organs, is a promising solution to the shortage of human donor organs. However, this approach is limited by hyperacute rejection initiated by the binding of natural antibodies to terminal galactosyl residues (Gal) and other surface proteins on pig cells, which trigger complement and coagulation activation. To bypass this, pigs have been genetically modified to lack galactosyltransferase, the key enzyme for Gal expression (GalKO) and to express human complement regulatory proteins, such as membrane co-factor protein (CD46). These genetic modifications significantly increased xenograft survival, by preventing most of antibody-complement-mediated rejection.

However, even GalTKO.hCD46 xenografts, particularly lungs, still get injured, demonstrating that other mechanisms must be involved. Recent work performed with transgenic pig organs points to dysregulated coagulation and inflammation caused by adhesion of platelets and white blood cell populations, including natural killer (NK) cells. During recent evaluation of pigs expressing various human transgenes, expression of human endothelial protein C receptor (EPCR) appeared to significantly improve lung outcome. However, the mechanisms of action remain unclear. Another recent genetic modification includes expression of a human histocompatibility antigen type E (HLA-E) by pig cells, which is designed to block human NK cell activation.

Our study will focus on the effects of EPCR and HLA-E expression by pig lung in a xenogenic lung-human blood perfusion model, by measuring changes in platelet/coagulation pathway activation, NK cell adhesion, and effects on inflammation. In particular, we will extend preliminary observations that pig cells migrate out from the organ and circulate in the human blood perfusate.

Sarah E. Sheppard
University of Massachusetts Medical School

MicroRNAs as biomarkers of viability in Cardiac Death Liver Grafts preserved by oxygenated normothermic machine perfusion

Synopsis: There are currently 15,696 candidates on the waiting list for liver transplants, and because of the high waiting time, mortality ranges from 10-20% on the wait list. The use of donation after cardiac death (DCD) livers has increased. Unfortunately, compared with brain-death donor livers, DCD livers have a greater risk of biliary complications secondary to warm ischemia time and ischemic reperfusion injury. Specifically, earlier studies demonstrated ATP depletion results in increased inflammation and subsequent bile duct fibrosis. In Paulo Martins lab, I will examine bile duct injury in human DCD livers after normothermic machine preservation or oxygenated hypothermic preservation in University of Wisconsin solution. These will be compared to the current to standard hypothermic preservation in UW solution. Biliary injury and viability will be assessed by ATP measurements, immunohistochemistry, electron microscopy, and gene expression analysis. MicroRNAs that are upregulated or downregulated in the experimental groups compared to control will be identified. Finally, we will correlate the gene expression and biomarker analysis with graft viability to create a biliary injury index that can be used to predict the outcome of a DCD liver. In summary, improving DCD liver graft viability and creating a predictive index for biliary injury may ultimately allow for decreased waiting time on the liver transplant list and decreased morbidity and mortality associated with DCD liver grafts.