Tom Blydt-Hansen

Despite advances in immunosuppression, kidney transplant success is limited by alloimmune mediated graft destruction and the gradual development of chronic rejection. Currently, we lack markers to predict the progression of the recipient alloimmune response at an early timepoint. Innate factors serve as potential targets to modulate the alloimmune environment and predict rejection risk. Metabolomics is a promising platform for identifying markers of alloimmune outcomes in kidney transplantation. Metabolite patterns reflect mechanisms occurring at the molecular, cellular, tissue, and organ level. Immune mechanisms persisting at the time of transplant produce a characteristic pattern of metabolites that may be detected in biospecimen. Additionally, cytokine profiling represents underlying immune signalling networks. By integrating patient metabolite and cytokine profiles, we develop a comprehensive assessment of recipient risk and identify targets for pre-transplant interventions. In the first study, we aimed to identify pre-transplant metabolite patterns related to i) early allograft inflammation (uCXCL10/Cr) and ii) early graft function (GFR) in pediatric renal transplant recipients. As part of this analysis, we applied a metabolite classifier that is predictive of chronic rejection in a cohort of adult kidney transplant recipients. In the third chapter, we summarized the existing literature on processing requirements for blood samples intended for use in metabolomics analyses. In the fourth chapter, we investigated the association between metabolome composition and age in a healthy pediatric cohort. We concluded by assessing differences in pre-transplant cytokine profiles associated with chronic rejection in adult kidney transplant recipients. We also compared the predictive utility of cytokine and metabolite profiling. We determined that pre-transplant metabolite patterns indicative of chronic rejection in adults are not associated with early graft inflammation or function in children. Furthermore, we report that age and body size are significant contributors to the measured pediatric metabolome and must be accounted for during biomarker identification. Our findings and literature review indicate the importance of standardized protocols to minimize sample processing-related metabolome perturbations. Finally, we identified differences in pre-transplant cytokine profiles related to chronic rejection and graft loss. We also report that the pre-transplant cytokine and metabolite profiles are correlated and improve in predictive capacity when combined.

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Improvements seen in short-term kidney transplant survival over the preceding three decades have not been reflected in long-term graft outcomes. This is particularly pertinent in the pediatric population, who experience high rates of graft failure, a large proportion of which are attributed to non-adherence. Extended-release tacrolimus (ER-Tac), taken once daily, is associated with improved adherence in adults but this has not been extensively studied in pediatric kidney transplant recipients. This study assessed 1) the clinical factors that influence conversion to ER-Tac 2) whether conversion to ER-Tac is associated with improved adherence and 3) whether conversion to ER-Tac is associated with improved allograft function and rejection outcomes. The first analysis showed that older age and female sex predicted conversion to ER-Tac. Adherence measures (medication adherence measure (MAM-MM) and tacrolimus trough variability (Tac CV%)), individual barriers to adherence, renal function, and rejection were not significant predictors of conversion. In the second analysis, we found that baseline adherence in this population was high and that ER-Tac was not subsequently associated with improved Tac CV% or self-reported adherence. Children were more likely to miss their morning medication and listed forgetfulness or schedule clashes as their most common reason to be non-adherent. Likewise, in the third analysis, ER-Tac was not superior to IR-Tac with regards to preventing rejection, decline in eGFR or graft loss. Older age and female sex have been associated in other studies with poorer allograft outcomes and perhaps act as a high-level risk assessment for conversion to ER-Tac based on perception of risk. We did not find that age or sex were strongly associated with adherence or outcomes in multivariable analyses in this study. The lack of association between ER-Tac and adherence may be explained by a high baseline adherence in this population and because patients were not selected for conversion based on adherence behaviour. The finding of stable late graft outcomes between IR-Tac and ER-Tac remains important, especially given patient preference for ER-Tac regarding convenience and quality of life reported in other studies.

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Transplantation has greatly improved the lives of people with end-stage kidney failure, increasing life expectancy by an average of 10 years. However, threats to extended transplant survival continue to pose legitimate concern. Furthermore, the demand for healthy viable organs greatly exceeds supply and ensuring maximal longevity is of utmost importance. Early allograft kidney injury may negatively impact long-term outcomes. Similarly, the emergence of chronic rejection presents a major obstacle for prolonged graft survival and signifies a fundamental failure to achieve stable immune adaptation. Metabolomics focuses on the global measurement of small molecules and is a promising tool in the setting of kidney transplantation. Metabolites reflect ongoing bodily changes occurring at multiple levels —molecule, cell, tissue, organ— and offer a unique perspective that may improve our understanding of the intricate processes involved in graft injury and rejection. This thesis examines metabolite concentrations in the serum before transplantation, and how they may influence immediate and long-term transplant outcomes. To begin, we measured the levels of one individual metabolite (oxythiamine) prior to kidney transplant surgery and tested for association with 1) signs of functional thiamine deficiency early post-transplant; and 2) level of uremia (dialysis adequacy) pre-transplant. Afterwards, we investigated if there are characterizable differences in the pre-transplant serum metabolome of kidney transplant recipients, and whether those differences are associated with chronic rejection outcomes. In the first study, we found that oxythiamine levels are associated with dialysis adequacy at transplant. Patients treated with peritoneal dialysis, who have no residual kidney function and low dialysis adequacy, are particularly vulnerable to manifesting high oxythiamine levels. This subset of patients may be at an increased risk for developing acute thiamine deficiency in the early post-transplant period. In the second study, we were able to demonstrate the presence in serum of innate metabolomic differences between patients, which were associated with chronic rejection outcomes, suggesting that, even before transplantation, the metabolite environment may be an important factor involved in the predisposition of alloimmune differentiation towards a rejection response.

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